WO2019210271A1

WO2019210271A1 - Degradation-activated polypeptides and methods of using the same

Info

Publication number: WO2019210271A1
Application number: PCT/US2019/029491
Authority: WO
Inventors: Russell Eaton VANCE; Patrick Samuel MITCHELL; Andrew Ronald SANDSTROM
Original assignee: The Regents Of The University Of California
Priority date: 2018-04-27
Filing date: 2019-04-26
Publication date: 2019-10-31

Abstract

Degradation-activated polypeptides, including chimeric forms thereof, as well as methods of modulating cellular activities using such degradation-activated polypeptides, are provided. Nucleic acids encoding degradation-activated polypeptides or chimeric forms thereof, as well as, vectors and cells containing such nucleic acids are also provided. Methods of screening that employ degradation-activated polypeptides, or polypeptides that include one or more domains thereof, are also provided. In addition, the present disclosure provides methods of modulating innate immune responses in a subject by modulating proteasome-mediated degradation of degradation-activated proteins. Kits for practising the methods of the present disclosure are also provided.

Description

DEGRADATION-ACTIVATED POLYPEPTIDES AND METHODS OF USING THE SAME

CROSS-REFERENCE TO RELATED APPLICATION

Pursuant to 35 U.S.C. § 1 19(e), this application claims priority to the filing dates of United States Provisional Patent Application Serial No. 62/663,970, filed April 27, 2018, and United States Provisional Patent Application Serial No. 62/694,904, filed July 6, 2018; the disclosures of which applications are incorporated herein by reference.

INTRODUCTION

Common in all classes of plant and animal, non-specific or innate immunity is an organism’s first line of defense against infection by a foreign pathogen. In contrast to the adaptive immune system, the innate immune system does not have memory and cannot specifically recognize a pathogen from a prior infection. However, innate immunity is

evolutionarily older and the dominant system in many organisms including plants, fungi and lower invertebrates, including arthropods (insects, crustaceans, and related forms).

While innate immunity does not provide antigen memory from one infection to the next, innate immune responses can be at least semi-specific, recognizing certain pathogens or classes of related pathogen-derived molecules. One strategy employed by the mammalian innate immune system involves pattern recognition receptors (PRRs) that are encoded in the germline to recognize molecular patterns expressed by invading pathogens, which may be either surface expressed, such as e.g. Toll-like receptors (TLRs) and C-type Lectin Receptors (CLRs), or cytoplasmic, such as e.g. Nod-like receptors (NLRs) and RIG-l-like receptors (RLRs).

A particular type of protein falling within the NLR family, referred to as NACHT, LRR and PYD domains-containing protein (NLRP), was found to be capable of assembly and

oligomerization to form structures that activate caspase-1 signaling. Activation of caspase-1 signaling results in pro-inflammatory cytokine production, including interleukins 1 b and 18. Upon its discovery, the complex formed from multiple NLRP1 subunits was named the

“inflammasome” to reflect its role in proinflammatory caspase activation (Martinon et al., Mol Cell. (2002) 10(2):417-26).

As summarized, inflammasomes are multi-protein platforms that initiate innate immunity by recruitment and activation of Caspase-1 in myeloid cells. Such activation can lead to a form of pro-inflammatory programmed cell death termed pyroptosis, and also to the processing of certain pro-inflammatory cytokines such as interleukin-1 beta and interleukin-18 into their bioactive forms. The NACFIT, leucine-rich repeat (LRR) and pyrin (PYD) domains-containing protein 1 b (NLRP1 B) inflammasome is activated in response to pathogens and other-damage associated signals and can lead to inflammasome formation and pyroptosis.

NLRP1 B is the sensor component of the NLRP1 inflammasome. Among other pathogens and cell damage signals, NLRP1 B may be activated upon direct cleavage by the anthrax lethal toxin protease (Chavarria-Smith & Vance, PLoS Pathog. 2013; 9(6):e1003452, and Levinsohn et al., PLoS Pathog. 2012;8(3):e1002638). The anthrax bacterium secretes a tripartite anthrax toxin. The protective antigen (PA) subunit of the toxin binds to cell surface receptors of target cells and allows cytosolic entry of the metalloprotease lethal factor (LF) protease and the adenylate cyclase edema factor (EF) subunits. The combination of PA and EF (edema toxin) induces edema, whereas the combined action of PA and LF is responsible for lethality in experimental animals. Expression of NLRP1 inflammasome components can confer susceptibility to anthrax lethal toxin to non-myeloid cells that would be otherwise insensitive. However, the mechanism by which anthrax LF cleavage results in NLRP1 B activation is unknown.

SUMMARY

Degradation-activated polypeptides, including chimeric forms thereof, as well as methods of modulating cellular activities using such degradation-activated polypeptides, are provided. Nucleic acids encoding degradation-activated polypeptides or chimeric forms thereof, as well as, vectors and cells containing such nucleic acids are also provided. Methods of screening that employ degradation-activated polypeptides, or polypeptides that include one or more domains thereof, are also provided. In addition, the present disclosure provides methods of modulating innate immune responses in a subject by modulating proteasome-mediated degradation of degradation-activated proteins. Kits for practicing the methods of the present disclosure are also provided.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides a schematic depiction of a degradation-activated polypeptide that includes an auto-proteolytic domain, a degradation-targeting domain and a latent activity domain.

FIG. 2 provides a schematic depiction of degradation-mediated activation of a latent activity domain of a degradation-activated polypeptide. FIG. 3 provides a multiple sequence alignment of portions of mouse and human

Nlrp1 b/NLRP1/Nalp1 b amino acid sequences that include FUND domains of the relevant proteins (from top to bottom, SEQ ID NOs: 01 , 13, 15, 18 and 23).

FIG. 4 provides a schematic depiction of degradation mediated activation of cell death by a degradation-activated polypeptide having a latent activity domain that includes a cell death domain

FIG. 5 provides a schematic depiction of degradation mediated expression of a desired coding sequence by a degradation-activated polypeptide having a latent activity domain that includes a transcriptional activator in the presence of the desired coding sequence operably linked to a regulatory sequence that is responsive to the transcriptional activator.

FIG. 6A-6G show that the N-terminal domain of NLRP1 B does not mediate auto inhibition. FIG. 6A provides a schematic representation of NLRP1 B with certain domains, and boundaries thereof, identified. FIG. 6B shows that replacing groups of three consecutive amino acids with alanines in the NLRP1 B N-terminus did not result in an auto-active mutant. FIG. 6C shows that replacing groups of five consecutive amino acids with a flexible motif in the NLRP1 B N-terminus did not result in an auto-active mutant. FIG. 6D shows that replacing the entire NLRP1 B N-terminus with a heterologous alpha-helical domain from bacterial flagellin did not result in an auto-active mutant. FIG. 6E and FIG. 6F show that cleavage of as few as 10 amino acids from the N-terminus was sufficient to activate NLRP1 B and that there was not a significant correlation between the position of cleavage and NLRP1 B activity. FIG. 6G shows that NLRP1 B degradation, but not the position of protease cleavage, positively correlates with IL1 b processing.

FIG. 7A-7C show that degradation of NLRP1 B is necessary and sufficient for NLRP1 B inflammasome activation. FIG. 7 A shows that the presence of proteasome inhibitors abrogates NLRP1 B activation and prevents loss of the cleaved NLRP1 B fragment. FIG. 7B shows that the amount of NLRP1 B decreases after cleavage and this decrease can be blocked by proteasome- blocker treatment. FIG. 7C shows that inducible-degradation of an NLRP1 B with a heterologous N-terminal fused degron results in activation of the inflammasome that can be blocked by proteasome-blocker treatment.

FIG. 8A-8E show that“functional degradation” of NLRP1 B liberates the FIIND(UPA)- CARD fragment, a highly potent inflammasome activator. FIG. 8A provides a schematic depiction of a functional degradation model where NLRP1 B degradation leads to its own activation and inflammasome assembly. FIG. 8B shows that the FIIND(UPA)-CARD fragment was sufficient to promote robust caspase activity. FIG. 8C demonstrates that the FIIND(UPA)- CARD fragment is a highly potent activator of inflammatory signaling. FIG. 8D shows that only the FIIND(UPA)-CARD fragment assembles into the inflammasome. FIG. 8E shows that the non-FIIND-CARD fragment portion of NLRP1 B is dispensable for inflammasome activation.

FIG. 9A-9G show that the secreted Shigella flexneri lpaH7.8 E3 ubiquitin ligase activates NLRP1 B. FIG. 9A shows that lpaH7.8, but not other IpaH family of E3 ubiquitin ligases, induced NLRP1 B-dependent inflammasome activation. FIG. 9B shows that lpaH7.8 selectively activates the 129S1 but not the C57BL/6 (B6) allele of NLRP1 B. FIG. 9C shows that FUND auto-processing is required for lpaH7.8-induced NLRP1 B activation. FIG. 9D shows that various modification of lpaH7.8 also abolishes lpaH7.8-mediated inflammasome activation. FIG. 9E shows the dependency of cell killing on lpaH7.8, using deletion and rescue mutants, as well as the effects of Caspl , Nlrpl b and Nlrc4 deletion. FIG. 9F shows correlations between lpaH7.8 sensitivity and decreased levels of NLRP1 B and caspase induction. FIG. 9G shows that immortalized 129 macrophages were also sensitive to lpaH7.8-dependent killing, which correlated with decreased levels of endogenous NLRP1 B and the induction of CASP1 maturation.

FIG. 10 shows that the 2A12 monoclonal antibody recognizes the CARD domain of NLRP1 B.

FIG. 1 1 shows that proteasome inhibitors do not block NAIP/NLRC4 inflammasome activation.

FIG. 12 shows that the FIIND(UPA)-CARD fragment is a potent activator of IL1 b processing.

FIG. 13 shows that P2' residue identity modulates TEV-induced proteasomal

degradation and activation of NLRP1 B.

FIG. 14 shows visualization of the simultaneous degradation of the N-terminal domains and the release of the FIIND(UPA)-CARD fragment upon NLRP1 B activation.

FIG. 15 shows that FUND auto-processing is required for release of the FIIND(UPA)- CARD and its colocalization to the ASC speck.

FIG. 16A-16D show that lpaH7.8 activates mouse NLRP1 B, but not human NLRP1 , independently of N-end rule ubiquitin ligases.

DEFINITIONS

The terms“polynucleotide” and“nucleic acid,” used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides.

Thus, this term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

"Operably linked" refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For instance, a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression. Operably linked nucleic acid sequences may but need not necessarily be adjacent. For example, in some instances a coding sequence operably linked to a promoter may be adjacent to the promoter. In some instances, a coding sequence operably linked to a promoter may be separated by one or more intervening sequences, including coding and non-coding sequences. Also, in some instances, more than two sequences may be operably linked including but not limited to e.g., where two or more coding sequences are operably linked to a single promoter.

A "vector" or "expression vector" is a replicon, such as plasmid, phage, virus, or cosmid, to which another DNA segment, i.e. an "insert", may be attached so as to bring about the replication of the attached segment in a cell.

"Fleterologous," as used herein, means a nucleotide or polypeptide sequence that is not found in the native (e.g., naturally-occurring) nucleic acid or protein, respectively. Fleterologous nucleic acids or polypeptide may be derived from a different species as the organism or cell within which the nucleic acid or polypeptide is present or is expressed. Accordingly, a heterologous nucleic acid or polypeptide is generally of unlike evolutionary origin as compared to the cell or organism in which it resides. Nucleic acids may also include domains that are heterologous to one another, i.e., where a first domain is derived from a nucleic acid that is different from the nucleic acid from which a second domain is derived. Polypeptides may also include domains that are heterologous to one another, i.e., where a first domain is derived from a polypeptide that is different from the polypeptide from which a second domain is derived.

The terms“synthetic”,“chimeric” and“engineered” as used herein generally refer to artificially derived polypeptides or polypeptide encoding nucleic acids that are not naturally occurring. Synthetic polypeptides and/or nucleic acids may be assembled de novo from basic subunits including, e.g., single amino acids, single nucleotides, etc., or may be derived from pre existing polypeptides or polynucleotides, whether naturally or artificially derived, e.g., as through recombinant methods. Chimeric and engineered polypeptides or polypeptide encoding nucleic acids will generally be constructed by the combination, joining or fusing of two or more different polypeptides or polypeptide encoding nucleic acids or polypeptide domains or polypeptide domain encoding nucleic acids. Chimeric and engineered polypeptides or polypeptide encoding nucleic acids include where two or more polypeptide or nucleic acid“parts” that are joined are derived from different proteins (or nucleic acids that encode different proteins) as well as where the joined parts include different regions of the same protein (or nucleic acid encoding a protein) but the parts are joined in a way that does not occur naturally.

The terms“domain” and“motif”, used interchangeably herein, refer to both structured domains having one or more particular functions and unstructured segments of a polypeptide that, although unstructured, retain one or more particular functions. For example, a structured domain may encompass but is not limited to a continuous or discontinuous plurality of amino acids, or portions thereof, in a folded polypeptide that comprise a three-dimensional structure which contributes to a particular function of the polypeptide. In other instances, a domain may include an unstructured segment of a polypeptide comprising a plurality of two or more amino acids, or portions thereof, that maintains a particular function of the polypeptide unfolded or disordered. Also encompassed within this definition are domains that may be disordered or unstructured but become structured or ordered upon association with a target or binding partner. Non-limiting examples of intrinsically unstructured domains and domains of intrinsically unstructured proteins are described, e.g., in Dyson & Wright. Nature Reviews Molecular Cell Biology 6:197 -208. The lengths of useful domains will vary and may range from 10 amino acid residues or less to 1000 amino acid residues or more, including but not limited to e.g., from 10 to 1000, from 10 to 900, from 10 to 800, from 10 to 700, from 10 to 600, from 10 to 500, from 10 to 400, from 10 to 300, from 10 to 200, from 10 to 100, from 10 to 50, from 20 to 1000, from 30 to 1000, from 40 to 1000, from 50 to 1000, from 100 to 1000, from 200 to 1000, from 300 to 1000, from 400 to 1000, from 500 to 1000, from 600 to 1000, from 700 to 1000, from 800 to 1000, from 900 to 1000, from 20 to 750, from 30 to 750, from 40 to 750, from 50 to 750, from 100 to 750, from 200 to 750, from 300 to 750, from 400 to 750, from 500 to 750, from 20 to 500, from 30 to 500, from 40 to 500, from 50 to 500, from 100 to 500, from 200 to 500, from 300 to 500, from 400 to 500, etc.

As used herein, the terms "treatment," "treating," and the like, refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease. "Treatment," as used herein, covers any treatment of a disease in a mammal, e.g., in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease. The terms“individual,”“subject,”“host,” and“patient,” used interchangeably herein, refer to a mammal, including, but not limited to, murines (e.g., rats, mice), lagomorphs (e.g., rabbits), non-human primates, humans, canines, felines, ungulates (e.g., equines, bovines, ovines, porcines, caprines), etc.

A“therapeutically effective amount” or“efficacious amount” refers to the amount of an agent, or combined amounts of two agents, that, when administered to a mammal or other subject for treating a disease, is sufficient to effect such treatment for the disease. The “therapeutically effective amount” will vary depending on the agent(s), the disease and its severity and the age, weight, etc., of the subject to be treated.

The term“binding” refers to a direct association between two molecules, due to, for example, covalent, electrostatic, hydrophobic, and ionic and/or hydrogen-bond interactions, including interactions such as salt bridges and water bridges. In some cases, a specific binding member present in the extracellular domain of a chimeric polypeptide of the present disclosure binds specifically to a peptide-major histocompatibility complex (peptide-MHC).“Specific binding” refers to binding with an affinity of at least about 10^-7 M or greater, e.g., 5x 10^-7 M, 10^-8 M, 5 x 10 ⁸ M, and greater.“Non-specific binding” refers to binding with an affinity of less than about 10 ⁷ M, e.g., binding with an affinity of 10 ⁶ M, 10 ⁵ M, 10 ⁴ M, etc.

As used herein, the term "affinity" refers to the equilibrium constant for the reversible binding of two agents (e.g., an antibody and an antigen) and is expressed as a dissociation constant (K_D). Affinity can be at least 1 -fold greater, at least 2-fold greater, at least 3-fold greater, at least 4-fold greater, at least 5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, at least 10-fold greater, at least 20-fold greater, at least 30-fold greater, at least 40-fold greater, at least 50-fold greater, at least 60-fold greater, at least 70-fold greater, at least 80-fold greater, at least 90-fold greater, at least 100-fold greater, or at least 1 ,000-fold greater, or more, than the affinity of an antibody for unrelated amino acid sequences. Affinity of an antibody to a target protein can be, for example, from about 100 nanomolar (nM) to about 0.1 nM, from about 100 nM to about 1 picomolar (pM), or from about 100 nM to about 1 femtomolar (fM) or more.

DETAILED DESCRIPTION

Degradation-activated polypeptides, including chimeric forms thereof, as well as methods of modulating cellular activities using such degradation-activated polypeptides, are provided. Aspects of the degradation-activated polypeptides include an auto-proteolytic domain, a degradation-targeting domain and a latent activity domain, whereby a non-covalent association between cleaved portions of the auto-proteolytic domain retains and/or inhibits the latent activity domain until degradation of the degradation-targeting domain. Nucleic acids encoding degradation-activated polypeptides or chimeric forms thereof, as well as, vectors and cells containing such nucleic acids are also provided. Methods of screening that employ degradation-activated polypeptides, or polypeptides that include one or more domains thereof, are also provided. In addition, the present disclosure provides methods of modulating innate immune responses in a subject by modulating proteasome-mediated degradation of

degradation-activated proteins. Kits for practicing the methods of the present disclosure are also provided.

Before the present invention is described in greater detail, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

Certain ranges are presented herein with numerical values being preceded by the term "about." The term "about" is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described. All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, the singular forms“a”,“an”, and“the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as“solely,”“only” and the like in connection with the recitation of claim elements, or use of a“negative” limitation.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

While the apparatus and method has or will be described for the sake of grammatical fluidity with functional explanations, it is to be expressly understood that the claims, unless expressly formulated under 35 U.S.C. §1 12, are not to be construed as necessarily limited in any way by the construction of "means" or "steps" limitations, but are to be accorded the full scope of the meaning and equivalents of the definition provided by the claims under the judicial doctrine of equivalents, and in the case where the claims are expressly formulated under 35 U.S.C. §1 12 are to be accorded full statutory equivalents under 35 U.S.C. §1 12.

As summarized above, the present disclosure includes degradation-activated polypeptides, including chimeric forms thereof. Degradation-activated polypeptides are activated to perform a function or otherwise modulate an activity in a cell upon degradation of a portion of the polypeptide. For example, a degradation-activated polypeptide may include a first portion that is non-covalently associated with a second portion. Thus, non-covalent association may render the first portion latent for a particular function of the first portion, i.e., the first portion may be inactive or otherwise incapable of modulating an activity in a cell when non-covalently associated with the second portion of the degradation-activated polypeptide. In such instances, upon degradation of the second portion, the first portion may disassociate from the second portion allowing the first portion to modulate the activity in the cell. Thus, degradation-mediated disassociation of a latent portion of a degradation-activated polypeptide may provide for activation of the latent portion, e.g., to modulate the activity of a cell that would not be modulated in the absence of degradation of the degradation-activated polypeptide.

Referring to the non-limiting schematic depiction provided in FIG. 1 , a degradation- activated polypeptide 100 may include an auto-proteolytic domain 101 that includes a C- terminal portion 102 and an N-terminal portion 103. A latent activity domain 104 is attached, directly or indirectly, to the C-terminal portion 102 of the auto-proteolytic domain 100 and a degradation-targeting domain 105 is attached, directly or indirectly, to the N-terminal portion 103 of the auto-proteolytic domain 100. Accordingly, first and second portions (e.g., a first portion that includes the latent activity domain and a second portion that is targeted for degradation) of the degradation-activated polypeptide may be defined, e.g., based on the C- and N-terminal sides of an autocleavage site within the auto-proteolytic domain.

As depicted in the non-limiting schematic depiction in FIG. 2, when the degradation targeting domain 105 is not degraded the C-terminal portion 102 and the N-terminal portion 103 of the auto-proteolytic domain 101 remain non-covalently associated such that the latent activity domain 104 is inactive, e.g., through sequestration. Upon degradation 206 of the degradation targeting domain, the non-covalent association between the C-terminal and N-terminal portions of the auto-proteolytic domain is disrupted 207. Correspondingly, a C-terminal portion of the degradation-activated polypeptide 208, which includes the C-terminal portion of the auto- proteolytic domain 102 and the latent activity domain 104, is released thereby activating the latent activity domain. An activated latent activity domain may modulate a cellular activity, where such cellular activities that are modulated may vary widely.

As summarized above, degradation-activated polypeptides may vary and may include various domains including but not limited to e.g., an auto-proteolytic domain, a degradation targeting domain, a latent activity domain, a detectable reporter domain, and the like.

An auto-proteolytic domain will generally include an autocleavage site that upon cleavage generates an N-terminal portion and a C-terminal portion of the auto-proteolytic domain. Following autocleavage, the N- and C-terminal portions of a cleaved auto-proteolytic domain will remain non-covalently associated such that the C-terminal portion of the

degradation-activated polypeptide remains associated with the N-terminal portion. The length of a subject auto-proteolytic domain may vary and may range from 100 amino acid residues or less to 800 amino acid residues or more, including but not limited to e.g., 100 to 800 residues, 150 to 800 residues, 200 to 800 residues, 250 to 800 residues, 300 to 800 residues, 350 to 800 residues, 400 to 800 residues, 100 to 750 residues, 100 to 700 residues, 100 to 650, residues, 100 to 600 residues, 100 to 550

A degradation-targeting domain will generally include at least a portion of which that is targeted for degradation by any one of various different degradation-targeting processes or mechanisms as described in more detail below. In some instances, modification of a

degradation-targeting domain or administration of an agent may be required to target the domain for degradation. In some instances, a useful degradation-targeting domain may be derived from a protein that is rapidly degraded. Latent activity domains generally include a domain that modulates at least one cellular activity, but is rendered latent or inactive due to association with the N-terminal portion(s) of the degradation-activated polypeptide. Detectable reporter domains may be directly or indirectly and/or conditionally or constitutively detectable. These domains are further described below.

In some instances, one or more domains of a degradation-activated polypeptide may be derived from one or more various proteins including e.g., proteins that include auto-proteolytic domains that, upon cleavage of an autocleavage site within the auto-proteolytic domains, generate two fragments that remain non-covalently associated with one another. Essentially any protein domain that undergoes an auto-proteolytic event to cleave an autocleavage site and generate C-terminal and N-terminal fragments that remain covalently associated may find use in a degradation-activated polypeptide of the present disclosure.

Examples of useful proteins from which domains, e.g., as listed above, of degradation- activated polypeptides may be derived include e.g., those proteins containing a function to find domain (FUND), similar domains with a function corresponding to a FUND domain, FIIND-like domain containing proteins (e.g., ankyrin-2 proteins, p53-induced death domain-containing protein 1 (PIDD1 ) proteins, Unc-5 family C-terminal like (UNC5C-like, UNC5CL) protein),

NLRP1 (and related homologs such as NLRP1 A, NLRP1 B, NLRP1 C), NUP98, CARD8, and the like.

FUND domains represent a family of related protein domains that undergo an autolytic cleavage event to generate two fragments that remain non-covalently associated. Accordingly, FUND domains include an auto-cleavage site and may include subdomains adjacent to and on either side of the auto-cleavage site, such as a ZU5 subdomain and a UPA subdomain. As such, in some instances, useful FUND domains may be derived from proteins that include an auto-cleavage site flanked by a ZU5 subdomain and a UPA subdomain. FUND domains include those domains identified by InterPro domain identifier IPR025307, Pfam family PF13553 and Prosite entry PS51830 as FUND domains. Useful FUND domain containing proteins include mammalian FUND domain containing proteins, such as but not limited to e.g., those from human, non-human primates, rodents (e.g., mouse, rat, etc.), etc.

A ZU5 subdomain (also referred to as a ZU5 domain for simplicity) may vary in length and may range from e.g., 90 amino acid residues or less to 240 amino acid residues more, including but not limited to e.g., from 90 to 240, from 90 to 200, from 90 to 150, from 90 to 1 10, etc. Non-limiting examples of proteins containing representative ZU5 domains include zona occludens 1 (ZO-1 ) protein, UNC5C-like proteins, ankyrins, and the like. ZU5 domains include those domains identified by InterPro domain identifier IPR000906, Pfam family PF00791 and Prosite entry PS51 145 as ZU5 domains. Useful ZU5 domain containing proteins include mammalian ZU5 domain containing proteins, such as but not limited to e.g., those from human, non-human primates, rodents (e.g., mouse, rat, etc.), etc.

A UPA subdomain (also referred to as a UPA domain for simplicity) may vary in length and may range from 90 amino acid residues or less to 200 amino acid residues or more, including but not limited to e.g., from 90 to 200, from 90 to 160, from 90 to 140, etc. Non-limiting examples of proteins containing representative UPA domains include UNC5, PIDD, ankyrins, and the like. UPA domains include those domains identified by InterPro domain identifier IPR033772 and Pfam family PF17217 as UPA domains. Useful UPA domain containing proteins include mammalian ZU5 domain containing proteins, such as but not limited to e.g., those from human, non-human primates, rodents (e.g., mouse, rat, etc.), etc.

Useful human FUND domain containing proteins may include but are not limited to e.g.: NACHT, LRR and PYD domains-containing protein 1 (NLRP1 ) (UniProt ID Q9C000 (SEQ ID NO:1 ) corresponding to UniGene ID FIs.652273 and RefSeq IDs NP 001028225.1 (SEQ ID NO:2), NP 055737.1 (SEQ ID NO:3), NP_127497.1 (SEQ ID NO:4), NP_127499.1 (SEQ ID NO:5), NP 127500.1 (SEQ ID NO:6), and the like; Caspase recruitment domain-containing protein 8 (CARD8) (UniProt ID Q9Y2G2 (SEQ ID NO:7) corresponding to UniGene IDs

Hs.446146, Hs.655940 and Hs.68846 and RefSeq IDs NP_001 171829.1 (SEQ ID NO:8), NP_001 171830.1 (SEQ ID NO:9), NP_001 171831 .1 (SEQ ID NO:10), NP_001 171832.1 (SEQ ID NO:1 1 ), NP 055774.2 (SEQ ID NO:12), and the like; NACHT, LRR and PYD domains- containing protein 1 (NLRP1 ) (UniProt ID I3L2G5 (SEQ ID NO:13) corresponding to UniGene ID Hs.652273 and RefSeq IDs NP_055737.1 (SEQ ID NO:14), and the like.

Useful non-human primate FUND domain containing proteins may include those of macaques (e.g., UniProt IDs B0FPF1 , F7H560, G7NI57, G7PTD3, G7PY16, G7PY17, H9H2X8, H9H2X9, I7GJ41 ), gorilla (e.g., UniProt IDs G3QDE2, G3R758, G3S2P9), chimpanzees (e.g., UniProt IDs H2QC06, H2R317, K7AMS9, K7B4Y0, K7BPM5, K7BQJ8, K7BVQ0, K7C5P0, K7D5D0), gibbons (e.g., UniProt ID G1 RD25), new world monkeys (e.g., UniProt IDs F6PN09, F6PXA2, F7GJ44, F7GJH8, F7GJK4), and the like.

Other useful mammalian FUND domain containing proteins may include those of cats (e.g., UniProt ID M3XBY4), canines (e.g., UniProt IDs F6Y5Q8, J9NS63, J9PB92), bovine (e.g., UniProt IDs E1 BNN6, L8HY41 , L8I1 Q9), sheep (e.g., UniProt IDs W5NVT5, W5NXB4,

W5NXB9), pigs (e.g., UniProt IDs I3LUZ4, K9IW94), horses (e.g., UniProt IDs F6RNB9, F6Y3F5), and the like.

Useful rodent FUND domain containing proteins may include those of hamsters (e.g., UniProt IDs G3GYE2, G3GYE3, G3GYE4, G3GYE6, G3I077), rats (e.g., UniProt IDs C0L7G5, D9I2F9, D9I2G1 , D9I2G3, D9I2G4, D9I2H0) and mice. Useful mouse FUND domain containing proteins may include but are not limited to e.g.: NACHT, LRR and PYD domains-containing protein 1 b allele 2 (Nlrpl b) (UniProt ID A1 Z198 (SEQ ID NO:15) corresponding to UniGene ID Mm.390402 and RefSeq IDs NP_001035786.1 (SEQ ID NO:16), NP_001 155886.1 (SEQ ID NO:17), and the like; NACHT, LRR and PYD domains-containing protein 1 b allele 5 (UniProt ID Q0GKD5 (SEQ ID NO:18) corresponding to UniGene ID Mm.390402; NACHT, LRR and PYD domains-containing protein 1 a (UniProt ID Q2LKU9 (SEQ ID NO:19) corresponding to UniGene ID Mm.240227 and RefSeq ID NP_001004142.2 (SEQ ID NO:20), and the like; (UniProt ID Q2LKV2 (SEQ ID NO:21 ) corresponding to UniGene ID Mm.390402; (UniProt ID Q2LKV5 (SEQ ID NO:22) corresponding to UniGene ID Mm.390402; (UniProt ID Q2LKW6 (SEQ ID NO:23) corresponding to UniGene ID Mm.390402, and the like.

Useful NLRP1A proteins include but are not limited to e.g., NLRP1A proteins containing FIIND-like domains, including e.g., mammalian NLRP1 A proteins, such as but not limited to e.g., those from rodents (e.g., mouse, rat, etc.), etc. Useful mouse NLRP1 A proteins may include but are not limited to e.g.: NLRP1 A (UniProt ID Q2LKU9 (SEQ ID NO:57) corresponding to

UniGene ID Mm.240227 and RefSeq ID NP_001004142.2 (SEQ ID NO:58), and the like.

Useful proteins, and useful domains thereof, also include proteins containing FIIND-like domains. Non-limiting examples of FIIND-like domain containing proteins include but are not limited to e.g., ankyrin-2 proteins, p53-induced death domain-containing protein 1 (PIDD1 ) proteins, nuclear pore NUP98 protein, CARD8 protein, UNC5-like proteins (e.g.., UNC5CL), and the like.

Useful ankyrin-2 proteins include but are not limited to e.g., proteins of the ankyrin-2 family containing FIIND-like domains, including e.g., mammalian ankyrin-2 proteins, such as but not limited to e.g., those from human, non-human primates, rodents (e.g., mouse, rat, etc.), etc. Useful human ankyrin-2 proteins may include but are not limited to e.g.: Ankyrin-2 (ANK-2) (UniProt ID Q01484 (SEQ ID NO:24) corresponding to UniGene ID Hs.620557 and RefSeq IDs NP_001 120965.1 (SEQ ID NO:25), NP_001 139.3 (SEQ ID NO:26), NP_066187.2 (SEQ ID NO:27), and the like. Useful mouse ankyrin-2 proteins may include but are not limited to e.g.: Ankyrin-2 (Ank2) (UniProt ID Q8C8R3 (SEQ ID NO:28) corresponding to UniGene IDs

Mm.220242, Mm.461850, Mm.484610 and RefSeq IDs NP_001029340.1 (SEQ ID NO:29),

NP 848770.2 (SEQ ID NO:30), and the like.

Useful PIDD1 proteins include but are not limited to e.g., proteins of the PIDD1 family containing FIIND-like domains, including e.g., mammalian PIDD1 , such as but not limited to e.g., those from human, non-human primates, rodents (e.g., mouse, rat, etc.), etc. Useful human PIDD1 proteins may include but are not limited to e.g.: p53-induced death domain- containing protein 1 (PIDD1 ) (UniProt ID Q9HB75 (SEQ ID NO:31 ) corresponding to UniGene ID Hs.592290 and RefSeq IDs NP_665893.2 (SEQ ID NO:32), NP_665894.2 (SEQ ID NO:33), and the like. Useful mouse PIDD1 proteins may include but are not limited to e.g.: p53-induced death domain-containing protein 1 (Piddl ) (UniProt ID Q9ERV7 (SEQ ID NO:34) corresponding to UniGene ID Mm.334321 and RefSeq ID NP_073145.1 (SEQ ID NO:35), and the like.

Useful UNC5-like proteins include but are not limited to e.g., UNC5C-like proteins containing FIIND-like domains, including e.g., mammalian UNC5CL proteins, such as but not limited to e.g., those from human, non-human primates, rodents (e.g., mouse, rat, etc.), etc. Useful human UNC5CL proteins may include but are not limited to e.g.: UNC5CL (UniProt ID Q8IV45 (SEQ ID NO:36) corresponding to UniGene ID Hs. 158357 and RefSeq NP 775832.2 (SEQ ID NO:37), and the like. Useful mouse UNC5CL proteins may include but are not limited to e.g.: UNC5CL (UniProt ID E9QLC2 (SEQ ID NO:38) corresponding to UniGene ID

Mm.31 1629 and RefSeq ID NP_690036.4 (SEQ ID NO:39), and the like. Useful rat UNC5CL proteins may include but are not limited to e.g.: Unc-5 family C-terminal-like (Unc5cl) (UniProt ID A0A096MKD8 (SEQ ID NO:40) corresponding to UniGene ID Rn.17973 and RefSeq ID

NP_001 100352.1 (SEQ ID NO:41 ), and the like.

Useful NUP98 proteins include but are not limited to e.g., NUP98 proteins containing FIIND-like domains, including e.g., mammalian NUP98 proteins, such as but not limited to e.g., those from human, non-human primates, rodents (e.g., mouse, rat, etc.), etc. Useful human NUP98 proteins may include but are not limited to e.g.: Nuclear pore complex protein Nup98- Nup96 (UniProt ID P52948 (SEQ ID NO:46) corresponding to UniGene ID Hs.524750 and RefSeq NP_005378.4 (SEQ ID NO:47), RefSeq NP_057404.2 (SEQ ID NO:48), RefSeq NP 624357.1 (SEQ ID NO:49), RefSeq NP_624358.2 (SEQ ID NO:50), and the like. Useful mouse NUP98 proteins may include but are not limited to e.g.: Nuclear pore complex protein Nup98-Nup96 (NUP98) (UniProt ID Q6PFD9 (SEQ ID NO:51 ) corresponding to UniGene IDs Mm.439800 and Mm.486276 and RefSeq ID NP_001274093.1 (SEQ ID NO:52), RefSeq ID NP_001274094.1 (SEQ ID NO:53), RefSeq ID NP_075355.1 (SEQ ID NO:54), and the like. Useful rat NUP98 proteins may include but are not limited to e.g.: Nup98 (UniProt ID P49793 (SEQ ID NO:55) corresponding to UniGene ID Rn.1 1324 and RefSeq ID NP_1 12336.2 (SEQ ID NO:56), and the like.

In some instances, FUND domain containing proteins or FIIND-like domain containing proteins (or one or more domains thereof) may be identified, determined and/or defined based on comparison and/or alignment with one or more other FUND domain (or FIIND-like domain) containing proteins, including but not limited to e.g., one or more of the above described proteins. For example, in some instances, one or more domains of a particular FUND (or FIIND- like) domain containing protein may be determined based on alignment with a mouse FUND domain containing protein, such as but not limited to e.g., the 1233 amino acid mouse Nlrpl b protein (UniProt ID Q2LKW6 (SEQ ID NO:23)), where e.g., the N-terminal side of the protein up to the FUND domain includes amino acid residues 1 -745, the FUND domain includes amino acid residues 745-1 142, the C-terminal portion of the protein (including the CARD domain) includes residues 1 142-1233, the LF cleavage site includes residues 44-45 and the FUND auto processing site includes residues 983-984). Other useful domains of the mouse Nlrpl b protein (UniProt ID Q2LKW6 (SEQ ID NO:23)) may include e.g., a NACHT domain (residues 126-435), a LRR 1 (residues 627-647), a LRR 2 (residues 684-704), a CARD (residues 1 143-1226), and the like. Useful subdomains and/or conserved regions or residues within FUND domain polypeptides also include e.g., those described in Finger et al., JBC (2012) 287(3): 25030- 25037; the disclosure of which is incorporated by reference herein in its entirety.

A multiple sequence alignment (Clustal O 1 .2.4, default settings) of mouse Nlrpl b protein (UniProt ID A1 Z198; SEQ ID NO:15), human NLRP1 protein (UniProt ID Q9C000; SEQ ID NO:01 ); human NLRP1 protein (UniProt ID I3L2G5;_SEQ ID NO:13); mouse Nlrpl b protein (UniProt ID Q0GKD5; SEQ ID NO:18); and mouse NACHT-, LRR-, and PYD-containing protein 1 paralog b (Nalpl b; Q2LKW6; SEQ ID NO:23) that includes the FUND domains of the proteins is provided in FIG. 3. The auto-processing/auto-cleavage site is indicated in bold underline. In some instances, a useful FUND domain, or a portion or subdomain thereof, may share 50% or greater sequence identity with one or more sequences of FIG. 3 over all or a portion of the length of sequence as shown in FIG. 3, including but not limited to e.g., 55% or greater, 60% or greater, 65% or greater, 70% or greater, 75% or greater, 80% or greater, 85% or greater, 86% or greater, 87% or greater, 88% or greater, 89% or greater, 90% or greater, 91 % or greater, 92% or greater, 93% or greater, 94% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, etc.

Any convenient method of protein comparison, depending on the similarity of the proteins to be compared, may be employed to identify domains of one protein relative to another, including but not limited to e.g., manual alignment, pairwise alignment, multiple sequence alignment, structural alignment, and the like. In some instances, a software tool for sequence alignment may be employed including e.g., ClustalW2, ClustalOmega, T-coffee, BLAST, etc. In some instances, a protein identified herein may be aligned with one or more other proteins to identify domains, or portions thereof, useful in the subject degradation- activated polypeptides of the present disclosure.

For example, a domain of a subject degradation-activated polypeptide, and/or a section of amino sequence of the degradation-activated polypeptide containing multiple domains, may share homology with a domain of a FIIND-containing protein or a FIIND-like domain-containing protein, including e.g., one or more of the FIIND-containing proteins or FIIND-like domain- containing proteins described herein.

In some instances, a domain of a degradation-activated polypeptide may share 100% sequence identity to a domain of a FIIND-containing protein or a FIIND-like domain-containing protein. In some instances, a domain of a degradation-activated polypeptide may share less than 100% sequence identity with a domain of a FIIND-containing protein or a FIIND-like domain-containing protein, including but not limited to where the domain of the degradation- activated polypeptide and the domain of the FIIND-containing or FIIND-like domain containing protein share e.g., at least 50% amino acid sequence identity, including e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, etc., amino acid sequence identity.

In some instances, a section of amino sequence of a degradation-activated polypeptide that may include multiple domains may share 100% sequence identity to a corresponding section of a FIIND-containing protein or a FIIND-like domain-containing protein. In some instances, a section of a degradation-activated polypeptide may share less than 100% sequence identity with a corresponding section of a FIIND-containing protein or a FIIND-like domain-containing protein, including but not limited to where the section of the degradation- activated polypeptide and the section of the FIIND-containing or FIIND-like domain containing protein share e.g., at least 50% amino acid sequence identity, including e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, etc., amino acid sequence identity.

AUTO-PROTEOLYTIC DOMAINS

Degradation-activated polypeptides may include an auto-proteolytic domain. Auto- proteolytic domains will generally include an autocleavage site, where such sites are

characterized by self-mediated cleavage without the necessity of a separate enzyme such as a protease. An autocleavage site present in an auto-proteolytic domain may define N-terminal and C-terminal sides of the auto-proteolytic domain where, following cleavage, the resultant protein may be divided into N-terminal and C-terminal fragments. As described herein, cleaved fragments of an auto-proteolytic domain-containing protein may remain associated with one another, including e.g., where the two fragments remain non-covalently bound to one another, including e.g., where the two fragments remain non-covalently bound by the cleaved portions of the auto-proteolytic domain, which non-covalently associate with one another.

In some instances, the N-terminal side of an auto-proteolytic domain may include a ZU5 subdomain of a function-to-find domain (FUND). In some instances, the C-terminal side of an auto-proteolytic domain may include a UPA subdomain of a FUND. In some instances, the auto- proteolytic domain may be a FUND. FUND domains may be derived from various proteins. In some instances, a FUND domain may be derived from a protein of the NLRP1 family, a CARD8 protein, an Ankyrin-2 protein, a PIDD1 protein, a NUP98 protein, or an UNC5CL protein. FUND domain-containing proteins, FIIND-like domain containing proteins, and the like, as well as non limiting examples thereof, are described above.

Useful auto-proteolytic domains may be derived from FUND domain (or FIIND-like domain) containing proteins and may consist of the entire FUND domain or entire FIIND-like domain of a FUND domain or FIIND-like domain containing protein or one or more portions of the FUND domain or FIIND-like domain of a FUND domain or FIIND-like domain containing protein, including but not limited to e.g., one or more subdomains thereof. In some instances, an auto-proteolytic domain may be derived from a NLR protein, such as but not limited to e.g., a mammalian NLR protein, such as but not limited to e.g., mammalian NLRP1/NLRP1 B proteins (e.g., rodent (e.g., mouse and rat) NLRP1 B, non-human primate, and human NLRP1 proteins), and the like. In some instances, an auto-proteolytic domain may be derived from a CARD8 protein, such as but not limited to e.g., a mammalian CARD8 protein, such as but not limited to e.g., rodent (e.g., mouse and rat), non-human primate, and human CARD8 proteins, and the like. In some instances, an auto-proteolytic domain may be derived from an Ankyrin-2 protein, such as but not limited to e.g., a mammalian Ankyrin-2 protein, such as but not limited to e.g., rodent (e.g., mouse and rat), non-human primate, and human Ankyrin-2 proteins, and the like.

In some instances, an auto-proteolytic domain may be derived from a PIDD1 protein, such as but not limited to e.g., a mammalian PIDD1 protein, such as but not limited to e.g., rodent (e.g., mouse and rat), non-human primate, and human PIDD1 proteins, and the like. In some instances, an auto-proteolytic domain may be derived from a UNC5CL protein, such as but not limited to e.g., a mammalian UNC5CL protein, such as but not limited to e.g., rodent (e.g., mouse and rat), non-human primate, and human UNC5CL proteins, and the like.

In some embodiments, the auto-proteolytic domain of a degradation-activated

polypeptide may include a FUND domain of a FUND domain containing protein or one or more portions thereof, such as one or more subdomains thereof. A FUND domain may also include subdomains, including e.g., a ZU5 subdomain and a UPA subdomain. For example, mouse NLRP1 B includes two separate sub-domains (ZU5 and UPA) with auto-processing occurring near the C-terminal end of the ZU5 domain (F983|S984). In some instances, a pre-processed or processed auto-proteolytic domain may be described as including an N-terminal portion, e.g., that may include a ZU5 domain or portion thereof. In some instances, a pre-processed or processed auto-proteolytic domain may be described as including a C-terminal portion, e.g., that may include a UPA domain or portion thereof.

DEGRADATION-TARGETING DOMAINS

Degradation-activated polypeptides may include a degradation-targeting domain. Such degradation-targeting domains may be linked to the N-terminal side of an auto-proteolytic domain. Degradation-targeting domains will generally be domains that are targeted for degradation through various mechanisms, including e.g., where the domain is tagged for degradation. Various mechanisms may be employed to tag a protein for degradation, including but not limited to e.g., ubiquitination of the domain, or other post-translational modification of the domain, including but not limited to proteolytic cleavage, phosphorylation, methylation, ADP- ribosylation, ampylation, lipidation, alkylation, nitrosylation, succinylation, sumoylation, neddylation, isgylation, etc. Various proteins and/or portions thereof may find use as a degradation-targeting domain in the subject polypeptides. In some instances, useful degradation-targeting domains may include polypeptides or fragments thereof that, when attached to a protein, increase the degradation rate of the protein, e.g., as compared to the degradation rate of the protein without the degradation-targeting domain attached. Such degradation-targeting domains may be ubiquitin-dependent or ubiquitin- independent. For example, in some instances, a protein may be targeted for ubiquitin- independent proteasomal degradation by attachment of an ornithine decarboxylase (ODC) domain. Fusion of a target protein to ODC can destabilize the protein increasing the rate of degradation of the protein, e.g., as described in Matsuzawa et al., PNAS (2005) 102(42): 14982- 7; the disclosure of which is incorporated herein by reference in its entirety. In some instances, a protein may be targeted for ubiquitin-independent proteasomal degradation by post- translational modifications (including but not limited to proteolytic cleavage, phosphorylation, methylation, ADP-ribosylation, ampylation, lipidation, alkylation, nitrosylation, succinylation, sumoylation, neddylation, isgylation, etc.) of the protein that leads to its partial or complete unfolding, or by other mechanisms that lead to the binding or recruitment of ubiquitin ligases.

Ubiquitin-dependent approaches include, but are not limited to, e.g., incorporating a PEST (proline (P), glutamic acid (E), serine (S), and threonine (T)) sequence, or employing an E2 or E3 ubiquitin ligase (or portion thereof) fused to a binding partner of a degradation targeting domain such that binding of the binding partner to the degradation-targeting domain results in ubiquitination of the polypeptide containing the degradation-targeting domain and its subsequent degradation. In some instances, such a fusion of a binding partner and an ubiquitin ligase may be referred to as an“ubiquibody” (see e.g., Portnoff et al., J Biol Chem. (2014)

289(1 1 ):7844-55; the disclosure of which is incorporated herein by reference in its entirety). Similar approaches include but are not limited to e.g., those described in Gosink & Vierstra PNAS (1995) 92:91 17-9121 ; and Neklesa et al., Nat Chem Biol. (201 1 ) 3;7(8):538-43; the disclosures of which are herein incorporated by reference in their entirety.

In some instances, degradation-targeted domains may be modified to enhance their delivery to cellular degradation machinery, such as the proteasome. Such modifications will vary and may include e.g., where the domain is unfolded, where the domain is disordered, where the domain is recruited to degradation machinery by adaptor protein, where the domain is cleaved (including e.g., an N-terminal cleavage event), where the domain is bound by an agent that promotes degradation tagging of the domain, combinations thereof, and the like.

In some instances, an inducible dimerization may be employed to regulate the degradation of a degradation-targeted domain. Useful artificial degradation systems employing inducible dimerization domains include e.g., those derived using FKBP and FRB, which bind the naturally-occurring small molecule rapamycin (or analogs thereof) such that the addition of rapamycin will cause any two fusion proteins containing these domains to dimerize.

Dimerization pairs, such as FKBP/FRB, may be employed to e.g., bind a protein to the proteasome upon dimerization, stabilize or destabilize a protein upon dimerization, associate a ubiquitin (or a portion of a split ubiquitin) with a protein upon dimerization, and the like.

Dimerization pairs may form a homodimer including two molecules of the same dimerizer or heterodimers including two different molecules that form the bound dimerization pair. Non limiting examples of useful dimerization pairs include but are not limited to e.g., FK506 binding protein (FKBP) and FKBP; FKBP and calcineurin catalytic subunit A (CnA); FKBP and cyclophilin; FKBP and FKBP-rapamycin associated protein (FRB); gyrase B (GyrB) and GyrB; dihydrofolate reductase (DHFR) and DHFR; DmrB and DmrB; PYL (pyrabactin resistance-like) and ABI (abscisic acid-insensitive); Cry2 (cryptochrome 2) and CIB1 (cryptochrome-interacting basic-helix-loop-helix); GAI (gibberellin insensitive) and GID1 (gibberellin insensitive dwarfl ); and the like. Dimerizer pairs may be dimerized by dimerizers (i.e., dimerizing agents), non limiting examples of which include but are not limited to e.g., rapamycin and analogs thereof (i.e., rapalogs), coumermycin and analogs thereof, methotrexate and analogs thereof, AP20187 and analogs thereof, abscisic acid and analogs thereof, gibberellin and analogs thereof, and the like.

In some instances, dimerization may be induced by light, including light of a particular wavelength or within a particular band of wavelengths, such as e.g., blue light. Useful systems also include but are not limited to e.g., those described in Wilmington & Matouschek PLoS One. (2016) 1 1 (4):e0152679; and Schrader et al., Nat Chem Biol. (2009) 5(1 1 ): 815-822; Spencer et al., Curr Biol. (1996) 6(7):839-47; Pruschy et al., Chem Biol. (1994) 1 (3):163-72; Zhou et al.,

Exp Hematol. (2016) 44(1 1 ):1013-1019; Kawano et al., Nat Chem Biol. (2016) 12(12):1059- 1064; Miyamoto et al., Nat Chem Biol. (2012) 8(5):465-70; U.S. Patent Nos.: 5,834,266;

6,984,635; 5,830,462; and 9,587,020; U.S. Patent Application Pub. Nos.: 20130158098,

201 10003385, 20020173474, 20170306303, 2017008141 1 , 2016031 1907, 2016031 1901 , and 20150266973; the disclosures of which are herein incorporated by reference in their entirety.

As summarized above, in some instances, the degradation-targeting domain may be modified, where useful methods of modification will vary and may include e.g., enzymatic modification, light-induced modification, temperature change-induced modification, ionic concentration change-induced modification, and the like. In some instances, the degradation targeting domain is enzymatically modified for degradation. Useful enzymatic modifications may include but are not limited to e.g., ubiquitination, proteolytic cleavage, phosphorylation, acetylation, methylation, ampylation, and/or combinations thereof. In some instances, the enzymatic modification may induce delivery of the degradation-activated chimeric polypeptide to the proteasome. In some instances, a degradation-targeting domain may be employed that is recruited for enzymatic modification by a proteolysis-targeting agent that specifically binds the degradation-targeting domain. As a non-limiting example, in some instances, the proteolysis targeting agent may be a proteolysis-targeting chimera (PROTAC). Methods of employing a PROTAC for targeted degradation which may be adapted for use in the herein described degradation-activated polypeptides and methods include but are not limited to e.g., those described in e.g., Raina & Crews, J Biol Chem. (2010) 285(15):1 1057-60; Raina & Crews, Curr Opin Chem Biol. (2017) 39:46-53; Coleman & Crews, Annual Review of Cancer Biology (2018) 2:41 -58; Bondeson et al., Cell Chemical Biology (2018) 25(1 ):78-87; Neklesa et al.,

Pharmacology & Therapeutics. (2017) 174:138-144; the disclosures of which are herein incorporated by reference in their entirety.

Enzymatic modifications that induce or otherwise enhance degradation of a degradation targeting domain may directly or indirectly modify the degradation-targeting domain. For example, in some instances, an enzymatic modification may directly modify the degradation targeting domain e.g., by ubiquitination of the degradation-targeting domain, cleaving a portion of the degradation-targeting domain (e.g., an N-terminal portion of the degradation-targeting domain), or the like. In some instances, an enzymatic modification may indirectly modify the degradation-targeting domain, e.g., by ubiquitination of a polypeptide attached to the degradation-targeting domain, by cleavage of a polypeptide attached to the degradation targeting domain, or the like. For example, in some instances, a dormant degron (i.e., a “degradation sequence” or a“destabilizing domain”) may be attached to the degradation targeting domain and degradation of the degradation-targeting domain may be induced by enzymatically modifying the dormant degron to become active. Useful dormant degrons include but are not limited to e.g., dormant N-degrons that, when attached to the N-terminus of a protein, may be deprotected by a site-specific protease (such as an tobacco etch virus (TEV) protease or a variant thereof). Examples of such include TEV based systems, including TEV protease induced protein inactivation (TIPI) of TIPI-degron (TDeg) system described in Taxis et al., Mol Syst Biol. (2009) 5:267 and the system described in Jungbluth et al., BMC Syst Biol. (2010) 4:176; the disclosures of which are incorporated herein by reference in their entirety.

In some instances, the degradation-targeting domain is modified for degradation using light-induced modification. For example, in some instances, a degradation-targeting domain may include a photosensitive degron whereby irradiation of the photosensitive degron with light modifies and induces proteasomal degradation of the degradation-targeting domain. Useful photosensitive degrons include e.g., various polypeptide fusions of a light-reactive domain with a degradation sequence, such as but not limited to e.g., the light-reactive LOV2 (light oxygen voltage 2) domain of Arabidopsis thaliana photl (phototropin 1 ) with the murine ornithine decarboxylase-like degradation sequence cODC1 , including e.g., those described in Renicke et al., Chemistry & Biology (2013) 20:619-626; Lutz et al., Methods Mol Biol. (2016) 1408:67-78; Usherenko et al., BMC Syst Biol. (2014) 8:128; and Hermann et al., Current Biology (2015) 25:R733-R752; the disclosures of which are incorporated herein by reference. In some instances, a photosensitizer miniature singlet oxygen generator (miniSOG) may be fused to degradation-targeting domain, such that modification of the miniSOG by exposure to light results in light-induced degradation of the degradation-targeting domain. Non-limiting examples of miniSOGs include but are not limited to e.g., those described in Ruiz-Gonzalez, et al., J. Am. Chem. Soc., (2013) 135 (26): 9564-9567; Lin, et al., Neuron. (2013) 79(2):241 -53 and Hermann et al., Current Biology (2015) 25:R733-R752; the disclosures of which are incorporated herein by reference.

Accordingly, various methods of tagging and/or targeting a degradation-targeted domain for degradation, as well as modifying the relative degradation rate of such a domain, may be employed in the herein described degradation-activated polypeptides and methods. Additional approaches include but are not limited to e.g., those reviewed in Schrader et al., Nat Chem Biol. (2009) 5(1 1 ): 815-822; and Banaszynski & Wandless Cell: Chemistry & Biology (2006) 13:1 1- 21 ; the disclosures of which are herein incorporated by reference in their entirety.

LATENT ACTIVITY DOMAINS

As summarized above, degradation-activated polypeptides may include a domain that performs a desired activity within a cell, but when associated (i.e., covalently associated prior to auto-proteolytic processing or non-covalently associated, e.g., after auto-cleavage) with other portion(s) of the degradation-activated polypeptide such domain remains in a latent state. Such domains may be referred to as latent activity domains. In a degradation-activated polypeptide, a latent activity domain may be linked to a C-terminal side of the auto-proteolytic domain. Upon degradation-mediated dissociation of the latent activity domain from the degradation-activated polypeptide, the latent activity domain may be rendered capable of modulating an activity of a cell within which the activity domain resides.

As summarized above, useful latent activity domains will vary. Useful latent activity domains may include but are not limited to e.g., cell death-like fold domains (including Caspase Activation and Recruitment domains (CARD), Death Domains (DD), Pyrin Domain (PYD) and Death Effector Domains (DED) domains), transcriptional modulatory domains (e.g.,

transcriptional activators, transcriptional repressors, and the like), enzymes or the catalytic portion(s) thereof (including kinases, ubiquitin ligases, etc), or split fluorescent or other reporter proteins (such as those described in Wehr & Rossner. Drug Discov Today. 2016; 21 (3):415-29; the disclosure of which is incorporated herein by reference in its entirety), and the like.

As depicted in FIG. 2, described above, when the degradation-targeting domain is not degraded then the C- and N-terminal portions of the auto-proteolytic domain remain non- covalently associated such that the latent activity domain remains in a latent state. However, upon degradation of the degradation-targeting domain, the non-covalent association between the C-terminal and N-terminal portions of the auto-proteolytic domain is disrupted and the latent activity domain is released thereby activating the latent activity domain. An activated latent activity domain may modulate a cellular activity, where such cellular activities that are modulated may vary widely.

Referring to the example depicted in FIG. 4, where, for example, the latent activity domain includes a cell death domain 400, such as but not limited to a CARD domain. When associated (covalently or non-covalently) with the other components of the degradation- activated polypeptide 401 , the cell death domain-containing latent activity domain remains inactive and does not induce cell death or otherwise activate cell death pathways. Upon degradation of the degradation targeting domain 402, the non-covalent association between the N-terminal and C-terminal portions of the degradation-activated polypeptide is disrupted. Thus, the latent activity domain is released 403. Once released, the cell death domain-containing latent activity domain may become active and, e.g., associate with other components of a cell death pathway, including but not limited to e.g., oligomerize or otherwise form a complex 404 with other free cell death domain-containing polypeptides to induce or otherwise enhance signaling through a cell death pathway. In some instances, such a death-inducing complex that is formed to include released latent activity domains is an inflammasome.

As summarized above, in some instances, a latent activity domain may include a cell death domain. Cell death domains generally include protein domains that, when active, modulate cell death (e.g., induce or otherwise promote cell death), including e.g., apoptotic cell death and non-apoptotic cell death (such as pyroptosis), and/or activate other signaling pathways, such as e.g., kinase pathways. Cell death domains may be derived from a protein that, when activated, induces or promotes cell death, and/or other signaling events, including e.g., apoptotic cell death, non-apoptotic cell death, kinase signaling, or other signaling events. For example, useful cell death domains may include those derived from a NLRP1 protein, a CARD8 protein, an Ankyrin-2 protein, a PIDD1 protein or an UNC5CL protein, including but not limited to e.g., those described herein. In some instances, useful cell death domains may be derived from a protein other than a NLRP1 B protein, a CARD8 protein, an Ankyrin-2 protein, a PIDD1 protein or an UNC5CL protein. A cell death domain employed in a degradation-activated polypeptide may be endogenous or heterologous to one or more domains of the degradation- activated polypeptide, such as the auto-proteolytic domain.

Useful death domains may include but are not limited to e.g., domains of the death domain (DD) superfamily (Pfam PF00531 ; InterPro IPR000488; PROSITE PDOC50017), which include a death fold motif which is formed by several protein-interaction domains, including six- seven tightly coiled alpha-helices arranged in a "Greek-key fold" motif. In some instances, a cell death domain of a subject polypeptide may include a death effector domain (DED). In some instances, a cell death domain of a subject polypeptide may include a caspase activation and recruitment domain (CARD). In some instances, a cell death domain of a subject polypeptide may include a pyrin domain (PYD). In some instances, a cell death domain of a subject polypeptide may include a death domain (DD), including but not limited to e.g., those found with other types of domains including but not limited to e.g., Ankyrin repeats, caspase-like folds, kinase domains, leucine zippers, leucine-rich repeats (LRR), TIR domains, and ZU5 domains.

In some instances, a latent activity domain may include a transcriptional activator or a transcriptional repressor. In some instances, a latent activity domain including a transcriptional activator may remain latent when associated with the degradation-activated polypeptide and become activated upon degradation of the degradation-activated polypeptide and/or and release of the activity domain from the degradation-activated polypeptide upon degradation. In some instances, a latent activity domain including a transcriptional repressor may remain latent when associated with the degradation-activated polypeptide and become activated to repress its target upon degradation of the degradation-activated polypeptide and/or and release of the activity domain from the degradation-activated polypeptide upon degradation.

Referring to the example depicted in FIG. 5, where, for example, the latent activity domain includes a transcriptional activator 500. When associated (covalently or non-covalently) with the other components of the degradation-activated polypeptide 501 , the transcriptional activator-containing latent activity domain remains inactive and does not activate or otherwise induce transcription. Upon degradation of the degradation targeting domain 502, the non- covalent association between the N-terminal and C-terminal portions of the degradation- activated polypeptide is disrupted. Thus, the latent activity domain is released 503. In the presence of a nucleic acid 504, that includes a coding sequence 505 operably linked to a regulatory sequence 506, the released transcriptional activator-containing latent activity domain may bind 507 the regulatory sequence (directly or indirectly through one or more transcriptional regulators bound to the regulatory sequence), thereby activating/inducing expression 508 of the coding sequence 505.

Any convenient transcriptional activator may find use in a chimeric polypeptide of the instant disclosure. Within a cell or system, a transcriptional activator may be paired with a transcriptional control element that is responsive to the transcriptional activator, e.g., to drive expression of a nucleic acid encoding a polypeptide of interest that is operably linked to the transcriptional control element. Useful transcriptional activators, transcriptional control elements, activator/control element pairs, and components of such systems may include but are not limited to e.g., those used in inducible expression systems including but not limited to e.g., those described in Goverdhana et al. Mol Ther. (2005) 12(2): 189-21 1 ; U.S. Patent Application Pub. Nos. 20160152701 , 20150376627, 20130212722, 20070077642, 20050164237, 20050066376, 20040235169, 20040038249, 20030220286, 20030199022, 20020106720; the disclosures of which are incorporated herein by reference in their entirety. Non-limiting examples of useful transcriptional activators include zinc finger (ZnF) proteins, tetracycline-controlled transcriptional activators (tTA), GAL4-VP16 transcriptional activators, VP64 Zip(+) transcriptional activators, TALE based DNA binding domain containing transcriptional activators, and the like.

In some instances, useful transcriptional activators may include a DNA binding domain such as, e.g., a ZnF DNA binding domain or a GAL4, fused to an effector domain such as e.g., VP16 or VP64. Upon binding its target, a transcriptional activator may induce expression of from a coding sequence operably linked to the target site. In contrast, a transcriptional repressor may include a DNA binding domain that inhibits the expression of one or more coding sequences, e.g., by binding to the operator or associated silencers. Transcriptional repressors may include a DNA binding domain such as, e.g., a Znf, a TALE, a deactivated Cas9, etc., fused to a transcriptional repression domain such as, e.g., a Tet repressor, and the like.

Useful components of tetracycline-regulated systems, such as tetracycline-responsive transcriptional activators and repressors as well as tetracycline response elements (TRE) and other components, include but are not limited to e.g., those of the Tet-On, Tet-Off, Tet-On Advanced, Tet-Off Advanced, Tet On-3G and Tet-One systems commercially available from Takara Bio USA (Mountain View, CA); those of the T-REx System commercially available from ThermoFisher Scientific (Waltham, MA); and the like. Where a tetracycline-regulated system is employed, a transactivator (e.g., tTA) or a Tet repressor (e.g., rtTA) may be present in the latent activity domain of a degradation-activated protein such that, upon degradation of the protein, the transactivator/repressor is released. Such a released transactivator or repressor may then control expression of a coding sequence linked to a TRE. Expression from such systems may be further controlled through the addition or withdrawal of tetracycline or an analogue thereof, such as doxycycline when the released latent domain is present.

Useful components UAS-GAL4-based systems include but are not limited to a GAL4 transcription factor, an upstream activation sequence (UAS) or a tandem repeat thereof (including but not limited to e.g., a 5X UAS, a 6X UAS, and the like), a transactivator (such as a viral trans-acting protein) or a portion thereof, and the like. Useful components include but are not limited to e.g., GAL4-transactivator fusions such as but not limited to e.g., GAL4-VP16 transcriptional activators, such as the following:

MKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTKRSPLTRAHLTEVES RLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNKDAVTDRLASVETDMPLTLRQ HRISATSSSEESSNKGQRQLTVSPEFPGIWAPPTDVSLGDELHLDGEDVAMAHADALDDFDLD MLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGGLERLEQLFLLIFPREDLD MILKMDSLQDIKALL (SEQ ID NO:42); GAL4-VP64 transcriptional activators, such as the following:

MKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTKRSPLTRAHLTEVES RLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNKDAVTDRLASVETDMPLTLRQ HRISATSSSEESSNKGQRQLTVSAAAGGSGGSGGSDALDDFDLDMLGSDALDDFDLDMLGSD ALDDFDLDMLGSDALDDFDLDMLGS (SEQ ID NO:43), and the like. Useful UAS sequences, which e.g., may be placed upstream of a coding sequence to be controlled by GAL4 or a GAL4- transactivator fusion (e.g., GAL4-VP16, GAL4-VP64, etc., include but are not limited to e.g., cggagtactgtcctccgag (SEQ ID NO:44), ggagcactgtcctccgaacgtc (SEQ ID NO:45, such as included in pHR_5x Gal4 UAS; e.g., as described in Morsut et al., Cell (2016) 164(4)780-91 ; the disclosure of which is incorporated herein by reference in its entirety), tandem repeats thereof, and the like. Useful vectors for employing and/or adapting components of a UAS/GAL4 system for use in the degradation-activated polypeptides and/or methods of the present disclosure are available from various sources including but not limited to e.g., by request from www(dot)addgene(dot)org.

Accordingly, transcriptional activation and transcriptional repression using a degradation- activated polypeptide of the present disclosure may be reversible, e.g., by employing a reversible expression modulating system such as e.g., a Tet or GAL4/UAS system as described above. In some instances, an irreversible expression modulating system may be employed including but not limited to e.g., a Cre recombinase system, a flippase recognition target (FRT) system, an ER (estrogen receptor) conditional gene expression system, and the like.

In some instances, a latent activity domain may include an enzyme or a catalytic portion thereof. Useful enzymes, or catalytic portions thereof, may include but are not limited to e.g., a nuclease (including e.g., site-specific nucleases), a recombinase, a SUMOylase, a de- SUMOylase, a ubiquitin ligase, a deubiquitinase, a protease, a kinase, a phosphatase, an acetyltransferase, a deacetylase, a methyltransferase, a demethylase, an AMPylator, a de- AMPylator, and the like.

Various domains having enzymatic activities may be employed in a subject latent activity domain, including but not limited to e.g., a domain from an oxidoreductase, a transferase, a hydrolase, a lyase, an isomerases, a ligase, etc. In some instances, a latent activity domain may include a domain derived from a nuclease (i.e., a nuclease domain), including but not limited to e.g., a site-specific nuclease domain, such as but not limited to e.g., a RNA guided nuclease (e.g., CRISPR/Cas9 site-specific nuclease and derivatives thereof (e.g., a nickase) domain, a non-Cas9 site-specific nuclease (e.g., a zinc-finger nuclease (ZFN), a TAL effector nucleases (TALEN), etc.) domain or the like. Also of use may be a Cas9 variant that lacks nuclease activity such as“dead Cas9” or“dCas9”. Examples of domains that may be employed include those described in PCT Pub. No. WO 2016/138034; the disclosure of which is incorporated herein by reference in its entirety.

Domains of degradation-activated polypeptides may be heterologous to one another. Degradation-activated polypeptides containing at least two domains that are heterologous to one another may be referred to as chimeric polypeptides, e.g., as degradation-activated chimeric polypeptides. In some instances, the degradation-targeting domain may be

heterologous to the auto-proteolytic domain. In some instances, the latent activity domain may be heterologous to the auto-proteolytic domain. In some instances, both the degradation targeting domain and the latent activity domain may be heterologous to the auto-proteolytic domain.

DETECTION AND BIOSENSORS

In some instances, degradation-activated polypeptides of the present disclosure may be employed for the detection and/or sensing of the presence of a biological agent and/or for the occurrence of a biological event, such as e.g., the degradation of a particular protein, cleavage of a protein, etc. For example, as summarized above, in some instances, degradation-activated polypeptides of the present disclosure may be employed in methods of screening, including e.g., screening for the presence of a particular agent, screening for a particular biological event or process, etc. Accordingly, in some instances, a degradation-activated polypeptide may be employed as a biosensor, e.g., to monitor for the presence of an agent, to detect or transmit information relating to a physiological or cellular process such as, e.g., protein degradation.

Degradation-activated polypeptides useful in detection and/or biosensor applications may include a detectable reporter. Detectable reporters may allow for visualization of all or a portion of a degradation-activated polypeptide. In some instances, a detectable reporter may allow for the visualization of a biological event, such as e.g., degradation of a degradation targeting domain of a degradation-activated polypeptide. A detectable reporter attached to or within a domain of a degradation-activated polypeptide may or may not be detectable (e.g., visible) when the degradation-activated polypeptide is in a non-degraded state. For example, in some instances, a fluorescent detectable reporter may be detectable, e.g., able to fluoresce, when the degradation-activated polypeptide is in a non-degraded state. In some instances, a fluorescent detectable reporter may not be detectable, e.g., unable to fluoresce, when the degradation-activated polypeptide is in a non-degraded state.

In some instances, a single detectable reporter may be included in a subject

degradation-activated polypeptide. In some instances, multiple, i.e., 2 or more, 3 or more, 4 or more, etc., detectable reporters may be included in a subject degradation-activated polypeptide. Where multiple reporters are employed the subject reporters may be attached to the same or different domains of the degradation-activated polypeptide. For example, in some instances, a first reporter may be attached to a domain on the N-terminal side of the auto-proteolytic domain and a second reporter may be attached to a domain on the C-terminal side of the auto- proteolytic domain. Accordingly, the use of detectable reporters in the subject degradation- activated polypeptides may or may not be limited to the latent activity domain.

As summarized above, in some instances, a subject polypeptide may include a detectable reporter or a portion thereof. In some instances, the detectable reporter or portion thereof may be linked to a domain of the polypeptide, including e.g., where the detectable reporter or portion thereof is linked to a latent activity domain. Accordingly, release of the latent activity domain may, e.g., allow for detection (e.g., visualization) of the released latent activity domain. A detectable reporter domain may be linked to any other appropriate domain of a degradation-activated polypeptide of the present disclosure where desired. For example, in some instances, a fluorescent reporter domain may be linked to, or in included within, a degradation-targeting domain such that, upon degradation of the degradation-targeting domain the fluorescent reporter domain may be no longer visible. Useful fluorescent reporter domains include but are not limited to e.g., fluorescent proteins, and portions thereof. Suitable fluorescent proteins include, but are not limited to, green fluorescent protein (GFP) or variants thereof, blue fluorescent variant of GFP (BFP), cyan fluorescent variant of GFP (CFP), yellow fluorescent variant of GFP (YFP), enhanced GFP (EGFP), enhanced CFP (ECFP), enhanced YFP (EYFP), GFPS65T, Emerald, Topaz (TYFP), Venus, Citrine, mCitrine, GFPuv, destabilised EGFP (dEGFP), destabilised ECFP (dECFP), destabilised EYFP (dEYFP), mCFPm, Cerulean, T-Sapphire, CyPet, YPet, mKO, FlcRed, t- HcRed, DsRed, DsRed2, DsRed-monomer, J-Red, dimer2, t-dimer2(12), mRFP1 , pocilloporin, Renilla GFP, Monster GFP, paGFP, Kaede protein and kindling protein, Phycobiliproteins and Phycobiliprotein conjugates including B-Phycoerythrin, R-Phycoerythrin and Allophycocyanin. Other examples of fluorescent proteins include mHoneydew, mBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry, mCherry, mGrapel , mRaspberry, mGrape2, mPlum (Shaner et al. (2005) Nat. Methods 2:905-909), and the like. Any of a variety of fluorescent and colored proteins from Anthozoan species, as described in, e.g., Matz et al. (1999) Nature Biotechnol. 17:969-973, or a derivative thereof is suitable for use.

In some instances, a split fluorescent protein may be employed as a detectable reporter. For example, a latent activity domain may include or have attached thereto a portion of split fluorescent protein that, by itself, is incapable of generating fluorescence or capable of generating only minimal fluorescence. Upon release of such a latent activity domain, the split fluorescent reporter may associate with at least a second portion of the split fluorescent reporter such that the two or more associated portions are capable of generating fluorescence or capable of generating an increased amount of fluorescence. Various other approaches employing split fluorescent proteins or other methods of protein-fragment complementation may be employed that will be readily apparent and this example should not be construed as limiting.

Non-limiting examples of various approaches employing split fluorescent proteins that may be adapted to the herein described compositions and methods include but are not limited to e.g., Kaddoum et al., Biotechniques 49:727-736 (2010); Van Engelenburg et al., Nat.

Methods 7:325-330 (2010); Kamiyama et al., Nat. Commun. 7, 1 1046 (2016); Chun et al., J. Neurochem. 103:2529-2539 (2007); Feng et al., Nat Commun. (2017) 8(1 ):370; and U.S.

Patent Pub. Nos. 20180164324 and 20150099271 ; the disclosures of which are incorporated herein by reference in their entirety.

Detectable reporters used in a protein-fragment complementation strategy need not necessarily be limited to fluorescent proteins and essentially any protein-fragment

complementation assay (PCA) components may be adapted for use in the herein described methods. PCA are generally used to detect protein-protein interactions, where e.g., proteins of interest (sometimes referred to as“bait” and“prey”) are each covalently linked to fragments of a third protein (a“reporter”). Interaction between the bait and the prey proteins may bring the fragments of the reporter protein in close proximity to allow them to perform a reporter function (e.g., by forming a functional reporter protein). The reporter function can then be detected and/or measured. Whereas split fluorescent reporters may be employed in a PCA, essentially any protein with a detectable activity (including non-fluorescent proteins) such as but not limited to e.g., beta-lactamase, dihydrofolate reductase (DHFR), focal adhesion kinase (FAK), Gal4, horseradish peroxidase, LacZ (beta-galactosidase), luciferase, TEV (Tobacco etch virus protease), ubiquitin, and the like, may be similarly adapted and employed.

Fluorescent reporters need not necessarily be proteins and may e.g., also include in some instances, fluorophores including those commonly described as“fluorescent dyes”. A large number of dyes are commercially available from a variety of sources, such as, for example, Molecular Probes (a division of Thermo Fisher Scientific, Waltham, MA USA) and Exciton (Dayton, OH). Examples of fluorophores of interest include, but are not limited to, 4- acetamido-4'-isothiocyanatostilbene -2,2'disulfonic acid; acridine and derivatives such as acridine, acridine orange, acridine yellow, acridine red, and acridine isothiocyanate; 5-(2'- aminoethyl)aminonaphthalene-1 -sulfonic acid (EDANS); 4-amino-N-[3- vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS); N-(4-anilino-1 - naphthyl)maleimide; anthranilamide; Brilliant Yellow; coumarin and derivatives such as coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4-trifluoromethylcouluarin (Coumarin 151 ); cyanine and derivatives such as cyanosine, Cy3, Cy5, Cy5.5, and Cy7; 4', 6- diaminidino-2-phenylindole (DAPI); 5', 5"-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red); 7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin; diethylaminocoumarin; diethylenetriamine pentaacetate; 4,4'-diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid; 4,4'- diisothiocyanatostilbene-2,2'-disulfonic acid; 5-[dimethylamino]naphthalene-1 -sulfonyl chloride (DNS, dansyl chloride); 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL); 4- dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC); eosin and derivatives such as eosin and eosin isothiocyanate; erythrosin and derivatives such as erythrosin B and erythrosin isothiocyanate; ethidium; fluorescein and derivatives such as 5-carboxyfluorescein (FAM), 5- (4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF), 2'7'-dimethoxy-4^,5^,-dichloro-6- carboxyfluorescein (JOE), fluorescein isothiocyanate (FITC), fluorescein chlorotriazinyl, naphthofluorescein, and QFITC (XRITC); fluorescamine; I R 144 ; IR1446; Lissamine™;

Lissamine rhodamine, Lucifer yellow; Malachite Green isothiocyanate; 4-methylumbelliferone; ortho cresolphthalein; nitrotyrosine; pararosaniline; Nile Red; Oregon Green; Phenol Red; B- phycoerythrin; o-phthaldialdehyde; pyrene and derivatives such as pyrene, pyrene butyrate and succinimidyl 1 -pyrene butyrate; Reactive Red 4 (Cibacron™ Brilliant Red 3B-A); rhodamine and derivatives such as 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), 4,7- dichlororhodamine lissamine, rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101 , sulfonyl chloride derivative of sulforhodamine 101 (Texas Red), N,N,N',N'-tetramethyl-6- carboxyrhodamine (TAMRA), tetramethyl rhodamine, and tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid and terbium chelate derivatives; xanthene; or combinations thereof. Other fluorophores or combinations thereof known to those skilled in the art may also be used, for example those available from Molecular Probes (a division of Thermo Fisher Scientific, Waltham, MA USA) and Exciton (Dayton, OH).

Useful detectable reporters may also include, in some instances, enzymes that catalyze a reaction that generates a detectable signal as a product. Non-limiting examples of useful enzymes include but are not limited to e.g., horse radish peroxidase (HRP), alkaline

phosphatase (AP), beta-galactosidase (GAL), glucose-6-phosphate dehydrogenase, beta-N- acetylglucosaminidase, b-glucuronidase, invertase, Xanthine Oxidase, firefly luciferase, glucose oxidase (GO), and the like. In some instances, release of a latent activity domain containing, or having attached thereto, an enzyme that catalyzes a reaction that generates a detectable signal or having may allow the enzyme to act upon a substrate, thus generating a detectable signal. Various other approaches employing enzymes that catalyze a reaction that generates a detectable signal may be employed that will be readily apparent and this example should not be construed as limiting.

NUCLEIC ACIDS, VECTORS, CELLS AND LIBRARIES THEREOF

As summarized above, the present disclosure also includes nucleic acids that include a sequence that encodes a degradation-activated polypeptide, e.g., a degradation-activated chimeric polypeptide. In some instances, such nucleic acids encode a degradation-activated polypeptide or a degradation-activated chimeric polypeptide described above. In some instances, subject nucleic acids may include a promoter operably linked to the sequence encoding the degradation-activated chimeric polypeptide. Useful promoters may be constitutive or inducible.

As summarized above, the present disclosure also includes vectors that include nucleic acids that include a sequence that encodes a degradation-activated polypeptide, e.g., a degradation-activated chimeric polypeptide. In some instances, such vectors include nucleic acids that encode a degradation-activated polypeptide or a degradation-activated chimeric polypeptide described above. In some instances, subject vectors include nucleic acids that may include a promoter operably linked to the sequence encoding the degradation-activated chimeric polypeptide. Useful promoters may be constitutive or inducible.

In some instances, a cell may be genetically modified (e.g., by transfection) with nucleic acid, expression cassette, vector, etc. that contains a regulator sequence that is responsive to the activated latent activity domain of a degradation-activated polypeptide. For example, where a cell contains or expresses a degradation-activated polypeptide that includes a latent activity domain that includes a transcriptional activator, the cell may also be modified to contain a nucleic acid, expression cassette, vector containing a regulatory sequence that is responsive to the transcriptional activator. In some instances, such a regulator sequence may be operably linked to a coding sequence, e.g., encoding essentially any protein or peptide of interest (POI).

A POI may be essentially any peptide or polypeptide and may include but is not limited to polypeptides of research interest (e.g., reporter polypeptides, mutated polypeptides, novel synthetic polypeptides, etc.), polypeptides of therapeutic interest (e.g., naturally occurring therapeutic proteins, recombinant therapeutic polypeptides, etc.), polypeptides of industrial interest (e.g., polypeptides used in industrial applications such as e.g., manufacturing), and the like.

As summarized above, the present disclosure also includes cells that may include the degradation-activated polypeptide encoding nucleic acids and/or vectors. Such cells may be genetically modified, e.g., by transfection of nucleic acids and/or vectors, encoding a

degradation-activated polypeptide. Transfection may be stable or transient and may be achieved by any convenient means, including but not limited to e.g., viral transfection, electroporation, lipofection, bombardment, chemical transformation, use of a transducible carrier (e.g., a transducible carrier protein), and the like.

Various suitable cells may be employed, e.g., to express a degradation-activated polypeptide of the presence disclosure. Suitable cells include neural cells; liver cells; kidney cells; immune cells; cardiac cells; skeletal muscle cells; smooth muscle cells; lung cells; and the like. Suitable cells include a stem cell (e.g. an embryonic stem (ES) cell, an induced pluripotent stem (iPS) cell; a germ cell (e.g., an oocyte, a sperm, an oogonia, a spermatogonia, etc.); a somatic cell, e.g. a fibroblast, an oligodendrocyte, a glial cell, a hematopoietic cell, a neuron, a muscle cell, a bone cell, a hepatocyte, a pancreatic cell, etc. Suitable cells include human embryonic stem cells, fetal cardiomyocytes, myofibroblasts, mesenchymal stem cells, autotransplated expanded cardiomyocytes, adipocytes, totipotent cells, pluripotent cells, blood stem cells, myoblasts, adult stem cells, bone marrow cells, mesenchymal cells, embryonic stem cells, parenchymal cells, epithelial cells, endothelial cells, mesothelial cells, fibroblasts, osteoblasts, chondrocytes, exogenous cells, endogenous cells, stem cells, hematopoietic stem cells, bone-marrow derived progenitor cells, myocardial cells, skeletal cells, fetal cells, undifferentiated cells, multi-potent progenitor cells, unipotent progenitor cells, monocytes, cardiac myoblasts, skeletal myoblasts, macrophages, capillary endothelial cells, xenogenic cells, allogenic cells, and post-natal stem cells. Suitable immune cells include e.g., a T cell, a B cell, a monocyte, a natural killer cell, a dendritic cell, a macrophage, a cytotoxic T cell, a helper T cell, a regulatory T cell (Treg), and the like.

Introduced nucleic acids may be maintained within the cell or may be transiently present. As such, in some instances, an introduced nucleic acid may be maintained within the cell, e.g., integrated into the genome. Any convenient method of nucleic acid integration may find use in the subject methods, including but not limited to e.g., viral-based integration, transposon-based integration, homologous recombination-based integration, and the like. In some instance, an introduced nucleic acid may be transiently present, e.g., extrachromosomally present within the cell. Transiently present nucleic acids may persist, e.g., as part of any convenient transiently transfected vector.

Also included in the present disclosure are libraries. Useful libraries include libraries of nucleic acids, e.g., nucleic acids encoding a one or more, including a plurality of, degradation- activated polypeptides; nucleic acids encoding a one or more, including a plurality of, peptides/polypeptides to be screened in a screening assay; and the like. Useful libraries also include libraries of vectors e.g., vectors encoding a one or more, including a plurality of, degradation-activated polypeptides; vectors encoding a one or more, including a plurality of, peptides/polypeptides to be screened in a screening assay; vectors that contain sequences responsive to an activity of an activated latent activity domain; and the like. Useful libraries include libraries of cells, e.g., cells containing one or more degradation-activated polypeptide encoding nucleic acids or vectors. Also included are libraries of candidate molecules, including e.g., libraries of candidate molecules screened in a screening assay, e.g., for the ability to modulate the degradation of a target protein and the like.

In some instances, libraries may be barcoded, e.g., using a nucleic acid barcode, allowing for retroactive identification of individual library components, e.g., by nucleic acid sequencing of the barcode. MODULATING CELLULAR ACTIVITIES

As summarized above, the present disclosure includes methods of modulating an activity in a cell. Such methods may include expressing in a cell, a degradation-activated polypeptide or a degradation-activated chimeric polypeptide. An expressed degradation- activated polypeptide may be degraded to activate a latent activity domain and modulate an activity of the cell. The degrading of the degradation-activated chimeric polypeptide may occur in an N-terminal to C-terminal direction. Various cellular activities may be modulated including but not limited to e.g., cell death, expression of an endogenous protein, expression of a heterologous protein, expression of an endogenous protein, and the like.

In some instances, a latent activity domain that includes a transcriptional activator or transcriptional repressor may be employed. In such instances, the method may further include introducing into the cell a nucleic acid comprising a coding sequence operably linked to a regulatory sequence that is responsive to the transcriptional activator or transcriptional repressor. Various coding sequences may be employed. For example, in some instances, the coding sequence may encode a protein, including but not limited to e.g., a cell death protein, a therapeutic protein, a disease related protein, a reporter protein, a transcription factor, an enzyme, or a chimeric combination thereof. Given the diversity of proteins that may be encoded by a coding sequence in response to the activation of a degradation-activated polypeptide of the present disclosure, the cellular activities that may be modulated are diverse.

In some instances, cellular activities that may be modulated may include e.g., an immune response, e.g., an innate immune response. Useful modulations of an immune response include e.g., initiation, propagation or inhibition/repression of an immune response, including e.g., an innate immune response. Such activities, in some instances, modulation of an immune response may be employed to fight an infection, e.g., a Bacillus anthracis infection, or prevent an infection, e.g., an infection from exposure to Bacillus anthracis. In some instances, modulation of an immune response may be employed to treat cancer. In some instances, modulation of an immune response may be employed to boost production or activity of one or more cytokines, including but not limited to e.g., IL-1 b, IL-18 and the like.

METHODS OF SCREENING

As summarized above, the present disclosure also includes method of screening. For example, provided methods include methods of screening for an enzyme that enzymatically modifies a protein for degradation. Such methods may include expressing in a cell a polypeptide that includes: an auto-proteolytic domain comprising an autocleavage site between an N- terminal side and a C-terminal side of the auto-proteolytic domain; a latent activity domain linked to the C-terminal side of the auto-proteolytic domain; and the protein, or a portion thereof, that is modified for degradation linked to the N-terminal side of the auto-proteolytic domain. The cell expressing the polypeptide may be contacted with the enzyme to be screened and whether the enzyme enzymatically modifies the protein, or portion thereof, to target the protein, or portion thereof, for degradation may be detected. In some instances, such detection may be based on assaying for the activity of the latent activity domain and/or detecting a detectable reporter associated with the latent activity domain.

Various means of contacting may be employed. For example, in some instances, contacting the cell with the enzyme may include expressing the enzyme in the cell. In some instances, a cell is contacted with a nucleic acid encoding a protein, such as an enzyme. In some instances, contacting a cell with a protein, such as e.g., an enzyme, may include combining the protein, e.g., a transducible version of the protein, with the cell in solution.

In some instances, the latent activity domain may include a cell death domain. In some instances, the method may include assaying for cell death caused by activation of the latent activity domain. Any convenient method of assaying cell death may be employed including but not limited to e.g., viability assays or cell-death reporters, and the like. In some cases, cell death can be inferred by the depletion or induced-absence of specifically identifiable (e.g., barcoded) members of a genetically-encoded library of proteins that are expressed in the reporter cells.

In some instances, the latent activity domain may include a detectable reporter or a portion thereof. In some instances, the method may include where the detectable reporter is a fluorescent protein or a portion thereof. For example, in some instances, the latent activity domain may include a first portion of a split fluorescent protein and the method may include assaying for fluorescence resulting from association of a second portion of the split fluorescent protein with the first portion.

Various classes of enzymes may be screened in the subject methods, including but not limited to e.g., ubiquitin ligases, proteases, kinases, phosphatases, acetyltransf erases, deacetylases, methyltransferases, demethylases, AMPylators, a de-AMPylators, and the like.

The protein enzymatically modified for degradation may vary. Useful examples of such proteins include but are not limited to a nucleotide-binding domain leucine-rich repeat (NLR) protein or a portion thereof, a caspase recruitment domain-containing protein 8 (CARD8) or a portion thereof, an Ankyrin-2 protein or a portion thereof, a p53-induced death domain- containing protein 1 (PIDD1 ) or a portion thereof, a UNC5C-Like (UNC5CL) protein or a portion thereof, and the like. Useful NLR proteins, and/or portions thereof, include NLRP1 proteins or portions thereof, including but not limited to e.g., mammalian NLRP1 proteins, including rodent (e.g., mouse and rat NLRP1 A or NLRP1 B proteins), non-human primate, and human NLRP1 B proteins and/or portions thereof. The various domains of the protein may vary and may, in some instances, include where the auto-proteolytic domain is derived from NLRP1 B, CARD8, Ankyrin- 2, PIDD1 and/or UNC5CL, and/or the latent activity domain is derived from NLRP1 B, CARD8, Ankyrin-2, PIDD1 and/or UNC5CL. One or more domains of the protein may be heterologous. In some instances, the protein may be a disease related protein.

Methods of screening provided in the present disclosure also include a method of screening for a mutation that modifies the degradation of a protein. Such methods may include expressing in a mutagenized cell a polypeptide that includes: an auto-proteolytic domain comprising an autocleavage site between an N-terminal side and a C-terminal side of the auto- proteolytic domain; a latent activity domain linked to the C-terminal side of the auto-proteolytic domain; and the protein to be screened linked to the N-terminal side of the auto-proteolytic domain. Such methods may include assaying for the activity of a latent activity domain to detect whether an introduced mutation modifies degradation of the protein. In some instances, the method may further include mutagenizing a cell to produce the mutagenized cell. Useful methods of mutagenizing will vary and may include but are not limited to e.g., chemical mutagenesis, radiation induced mutagenesis, site-directed mutagenesis, combinations thereof, and the like.

Methods of screening provided in the present disclosure also include a method of screening for a degradation-modifying agent. Such methods may include expressing in a cell a polypeptide that includes: an auto-proteolytic domain comprising an autocleavage site between an N-terminal side and a C-terminal side of the auto-proteolytic domain; a latent activity domain linked to the C-terminal side of the auto-proteolytic domain; and a degradation-targeting domain linked to the N-terminal side of the auto-proteolytic domain. In such methods, a cell expressing such a polypeptide may be contacted with a candidate agent and the cell may be assayed for an activity of the latent activity domain. For example, in some instances, the activity of the latent activity domain may be employed to detect whether the candidate agent modifies degradation of the polypeptide.

Various candidate agents may be screened in the subject methods. Non-limiting examples of useful candidate agents include but are not limited to e.g., non-peptide small molecules, nucleic acids, peptides, proteins, and the like. In some instances, candidate agents may include chimeric peptides or chimeric proteins. For example, in some instances, candidate agents screened in such a methods may include proteolysis-targeting agents, where e.g., such agents include a motif that specifically binds the degradation-targeting domain and at least one other motif, e.g., a motif that targets the bound protein for degradation. Non-limiting examples of proteolysis-targeting agents include but are not limited to proteolysis-targeting chimeras (i.e., PROTACs).

In some instances, libraries may be employed in screening. Libraries may find use in screening a plurality of different enzymes. For example, in some instances, the method may include contacting a plurality of cells expressing the chimeric polypeptide with a plurality of different enzymes. In some instances, barcoding may be employed to retrospectively identify a component of the screen, e.g., a protein (e.g. an enzyme) expressed from a nucleic acid (e.g. a plasmid). In some instances, each different enzyme of a plurality of enzymes may be expressed from a barcoded nucleic acid sequence.

In some instances, the screening methods may employ one or more control cells and may include assaying the one or more control cells. Non-limiting examples of suitable control cells include but are not limited to e.g., a control cell that is defective for an activity of a latent activity domain, a control cell expressing a defective chimeric polypeptide comprising a defective auto-proteolytic domain, a control cell expressing a polypeptide comprising an auto- proteolytic domain and a latent activity domain linked to the C-terminal side of an auto- proteolytic domain where the polypeptide is not linked to the protein, and the like.

MODULATING AN INNATE IMMUNE RESPONSE

As summarized above, the present disclosure also includes methods of modulating an innate immune response in a subject. Useful methods include e.g., a method of modulating an innate immune response in a subject that includes modulating proteasome-mediated

degradation of a protein comprising a function-to-find domain (FUND) and a caspase activation and recruitment domain (CARD) to modulate the innate immune response in the subject. Such methods may initiate or increase the innate immune response in the subject or prevent or repress the innate immune response in the subject. Accordingly, subjects of the present methods include subjects in need of initiating or increasing an innate immune response as well as subjects in need of preventing or repressing an innate immune response.

In some instances, modulating of the method may include enhancing proteasome- mediated degradation of a protein to initiate or increase the innate immune response in the subject. In some instances, enhancing proteasome-mediated degradation of the protein comprises administering to the subject an agent that increases delivery of the protein to the proteasome. Useful agents will vary and may include but are not limited to e.g., agents that promote ubiquitination of the protein, including proteolysis-targeting agents comprising a motif that specifically binds the protein, including e.g., proteolysis-targeting chimeras (PROTACs).

Useful agents may also include, in some instances, agents that cause cleavage within the N-terminal region of the protein for which degradation is to be enhanced. In some instances, the cleavage removes at least 10 amino acids from the N-terminus of the protein. Useful agents may also include, in some instances, agents that enhance ubiquitin ligase activity.

As summarized above, in some instances, a subject treated in such a method may be a subject is in need of initiating or increasing the innate immune response. In some instances, the subject has an infection. In some instances, the subject has cancer. In some instances, the subject been exposed to an infectious agent, including but not limited to e.g., where the infectious agent includes Bacillus anthracis. In some instances, the treatment boosts production or activity of one or more cytokines in the subject, including but not limited to e.g., IL-1 b, IL-18 or both.

As summarized above, in some instances, modulating of the method may include inhibiting proteasome-mediated degradation of a protein to prevent or repress the innate immune response in the subject. For example, in some instances, inhibiting proteasome- mediated degradation of the protein may include administering to the subject an agent. Useful agents include but are not limited to e.g., proteasome inhibitors, ubiquitin ligase inhibitors, deubiquitinating agents, N-end rule inhibitors, combinations thereof, and the like.

As summarized above, in some instances, a subject treated in such a method may be a subject is in need of preventing or repressing an innate immune response. In some instances, the subject has an infection, including but not limited to e.g., where the infection includes a Bacillus anthracis infection. In some instances, the subject has inflammation, including but not limited to e.g., chronic inflammation, an autoimmune disease related inflammation,

combinations thereof, and the like. In some instances, the subject has cancer. In some instances, the treatment may repress production or activity of one or more cytokines in the subject, including e.g., IL-1 b, IL-18 or both.

Useful proteins, the degradation of which may be modulated in the present methods, may include e.g., nucleotide-binding domain leucine-rich repeat (NLR) proteins, caspase recruitment domain-containing protein 8 (CARD8) proteins, Ankyrin-2 proteins, p53-induced death domain-containing protein 1 (PIDD1 ) proteins, UNC5C-Like (UNC5CL) proteins, portions thereof, and the like. In some instances, the NLR protein or portion thereof may be a NLRP1 protein, including but not limited to e.g., mammalian NLRP1 proteins, including rodent (e.g., mouse and rat NLRPI A or NLRP1 B), non-human primate, and human NLRP1 proteins and/or portions thereof. In some instances, the protein is one that does not include a Bacillus anthracis lethal factor (LF) protease cleavage site.

Various subjects may be treated in the subject methods including but not limited to e.g., mammalian subjects, including rodents (rats, mice, etc.), non-human primates, humans, etc.

In some instances, the present methods may include a combination therapy, where e.g., a combination therapy may include administering to the subject at least an additional, second or supplement therapeutic agent. Useful therapeutic agents in combination therapies will vary and may include but are not limited to e.g., an antibiotic, an antibody, a vaccine, a receptor decoy, a competitive peptide, a small molecule (e.g., a small molecule inhibitor), combinations thereof and the like. In some instances, useful antibodies include those specific for a Bacillus anthracis protein. In some instances, useful small molecules include Bacillus anthracis protective antigen (PA) dominant negative mutants, small-molecule oligomerization inhibitors, Bacillus anthracis PA channel blockers, pore blocking agents, furin inhibitors, Bacillus anthracis edema factor (EF) inhibitors, Bacillus anthracis LF inhibitors, combinations thereof and the like.

KITS FOR USE AND METHODS OF MAKING

As summarized above, the present disclosure also includes kits for practicing the methods of the present disclosure. The kits may include, e.g., one or more of any of the polypeptides, nucleic acids, coding sequences, reagents, reaction mixtures (or components thereof) described herein with respect to the subject methods.

Kits of the present disclosure may include a degradation-activated chimeric polypeptide or a nucleic acid encoding a degradation-activated chimeric polypeptide. In some instances, a kit may include a degradation-activated chimeric polypeptide, or a nucleic acid encoding a degradation-activated chimeric polypeptide, which includes an auto-proteolytic domain, a degradation-targeting domain, and a latent activity domain. In some instances, a kit may include a degradation-activated chimeric polypeptide, or a nucleic acid encoding a degradation- activated chimeric polypeptide, that excludes one or more domains such as an auto-proteolytic domain, a degradation-targeting domain, and a latent activity domain. Such domains, and exemplary proteins from which they may be derived, are described in more detail above.

In some instances, a kit may include a nucleic acid for making a degradation-activated chimeric polypeptide or a library of different degradation-activated chimeric polypeptides. For example, a kit may include a nucleic acid that includes an expression cassette that includes a sequence encoding an auto-proteolytic domain. Such expression cassettes may or may not include other domains of a degradation-activated chimeric polypeptide such as a latent activity domain and/or a degradation-targeting domain.

In some instances, useful expression cassettes that include a sequence encoding an auto-proteolytic domain may include one or more cloning sites for introducing a domain of interest upstream, downstream or upstream and downstream of the sequence encoding an auto-proteolytic domain. Accordingly, latent activity domains of interest and/or degradation targeting domains of interest may be introduced into a subject degradation-activated polypeptide.

Cloning sites adjacent to a sequence encoding an auto-proteolytic domain may be configured such that a domain introduced into the cloning site is, when expressed, linked to the auto-proteolytic domain within an encoded degradation-activated polypeptide. Accordingly, coding sequences introduced into cloning sites will generally be in-frame with the sequence encoding an auto-proteolytic domain and may include or exclude any additional nucleic acids between the coding sequence and the sequence encoding the auto-proteolytic domain. For example, in some instances, a coding sequence may be directly linked to the sequence encoding the auto-proteolytic domain such that the two expressed polypeptide sequences are directly joined with no intervening amino acid residues. In some instances, a coding sequence may be indirectly linked to the sequence encoding the auto-proteolytic domain such that the two expressed polypeptide sequences are indirectly joined by one or more intervening amino acid residues. In some instances, a polypeptide of interest encoded by a coding sequence may be linked to the auto-proteolytic domain by one or more amino acid linkers.

Expression cassettes encoding a degradation-activated polypeptide may or may not express other polypeptides in addition to the degradation-activated polypeptide. For example, in some instances, the degradation-activated polypeptide may be the only polypeptide expressed from the expression cassette, including after the introduction of any coding sequences into one or more cloning sites present in the expression cassette. Where a single polypeptide is expressed from a subject expression cassette, the expression cassette may include only one stop codon and may exclude any additional cistrons, i.e., the expression cassette may be monocistronic (i.e., not polycistronic or bicistronic). Monocistronic sequences will generally exclude any polycistronic facilitating sequences such as e.g., furin cleavage sequences, T2A sequences, and V5 peptide tag sequences, internal ribosome entry site (IRES) sequences, and the like. Examples of polycistronic expression systems and the polycistronic facilitating sequences employed therein include but not limited to e.g., those described in Yang et al. (2008) Gene Therapy. 15(21 ):141 1 -1423; Martin et al. (2006) BMC Biotechnology. 6:4; the disclosures of which are incorporated herein by reference in their entirety.

In other instances, one or more polypeptides in addition to the degradation-activated polypeptide may be expressed from the expression cassette. Such expression cassettes may be polycistronic, including e.g., bicistronic. In some instances, polycistronic expression cassettes may include one or more polycistronic facilitating sequences, such as but not limited to one or more of the polycistronic facilitating sequences described above.

As summarized above, expression cassettes useful in making degradation-activated polypeptides of the present disclosure, may include one or more cloning sites for introducing a domain of interest adjacent (with or without a linker) to an auto-proteolytic domain, such as an auto-proteolytic domain comprising an autocleavage site between an N-terminal side and a C- terminal side of the auto-proteolytic domain.

Accordingly, a subject expression cassette may include sequence encoding the autocleavage site and a latent activity domain with a cloning site upstream of the autocleavage site sequence. Such an expression cassette may allow a user to insert essentially any degradation-targeting domain or candidate degradation targeting domain that, when expressed, will be linked on the N-terminal side of the autocleavage site. In some instances, such an expression cassette may include a latent activity domain that serves as a reporter, e.g., through the production of a detectable signal, through induction of cell death, etc. Such cassettes may facilitate the individual or multiplex screening of degradation-targeting domains or candidate degradation targeting domains by allowing a user to readily introduce sequence encoding essentially any domain of interest upstream of the auto-proteolytic domain, such that the domain of interest is, when expressed, linked to the auto-proteolytic domain and present within the encoded degradation-activated polypeptide.

In some embodiments, a subject expression cassette may include sequence encoding the autocleavage site and a degradation targeting domain with a cloning site downstream of the autocleavage site sequence. Such an expression cassette may allow a user to insert essentially any latent activity domain or candidate latent activity domain that, when expressed, will be linked on the C-terminal side of the autocleavage site. Such cassettes may facilitate the individual or multiplex screening of latent activity domains or candidate latent activity domains by allowing a user to readily introduce sequence encoding essentially any domain of interest downstream of the auto-proteolytic domain, such that the domain of interest is, when expressed, linked to the auto-proteolytic domain and present within the encoded degradation-activated polypeptide. Where present in a subject expression cassette, sequence encoding a latent activity domain and/or sequence encoding a degradation-targeting domain may be derived from the same or different gene and/or the same or different protein. In addition, where present in a subject expression cassette, sequence encoding a latent activity domain and/or sequence encoding a degradation-targeting domain may be derived from the same or different gene and/or the same or different protein as the auto-proteolytic domain.

Expression of encoding sequences of the expression cassettes described herein may be achieved through the use of a promoter, with or without additional regulatory sequences, within the expression cassette. Essentially any convenient and appropriate promoter may be employed and operably linked to one or more coding sequences including but not limited to e.g., constitutive promoters, inducible promoters, and the like.

As summarized above, the subject expression cassettes may be provided in a kit, where such kits may find use in generating degradation-activated polypeptides of a user’s choosing and/or libraries that include a plurality of different degradation-activated polypeptides of a user’s choosing. Expression cassettes of the subject kits may be provided as a nucleic acid that includes the expression cassette sequence, as a vector that includes the expression cassette sequence, or a cell that includes the expression cassette sequence (e.g., within a vector, integrated into the cellular genome, etc.).

Such kits may, in some instances, also include one or more cloning reagents. Cloning reagents may find use in e.g., introducing a desired cloning sequence into a cloning site of the subject expression cassette. In some instances, the cloning reagents provided with a subject kit may include all or a portion of the reagents necessary and/or sufficient to introduce an upstream or downstream coding sequence into the corresponding cloning site. Such coding sequence(s) may be introduced in-frame and when expressed, linked directly or indirectly, with the auto- proteolytic domain.

Useful cloning reagents may include but are not limited to e.g., enzymes, such as e.g., nucleases (e.g., restriction endonucleases, exonucleases, etc.), ligases, integrases,

recombinases, polymerases, and the like. Useful cloning strategies, and the reagents thereof, include ligation-based cloning, restriction cloning, amplification-based cloning, TA cloning, Gateway cloning, In-Fusion cloning, Gibson Assembly, and the like. In some instances, a subject cloning strategy may involve generating compatible ends (e.g., for ligation). Useful methods for generating compatible ends include but are not limited to, e.g., end-blunting, phosphorylation, dephosphorylation, etc. In addition, methods of generating a blunt end following digestion with a restriction endonuclease that does not generate blunt ends, i.e. “blunting”, may be utilized where appropriate, including but not limited to“end-filling” with a DNA polymerase, such as, e.g., DNA Polymerase I Large Fragment (i.e., Klenow), T4 DNA

Polymerase, Mung Bean Nuclease, etc., or terminal unpaired nucleotides may be removed by an enzyme with exonuclease activity. Corresponding reagents for such processes, e.g., cloning and end-modification processes, may be included in the subject kits.

In some instances, nucleic acids of interest, including expression cassettes, may be produced by non-cloning-based methods including but not limited toe.g., de novo sequence assembly, de novo nucleic acid synthesis and the like. In some instances, cloning strategies may be combined with non-cloning strategies such as, de novo sequence assembly, de novo nucleic acid synthesis, and the like. Appropriate reagents may therefore be included or excluded from the subject kits depending on the particular assembly strategy, or combination thereof, employed.

In some instances, a subject kit may include one or more detection reagents. For example, where a latent activity domain is employed that has an enzymatic activity when active, a subject kit may include one or more substrates for the enzyme. Non-limiting examples of substrates that may be provided in the subject kits include but are not limited to e.g., beta- lactamase substrates, DHFFt substrates, FAK substrates, horseradish peroxidase substrates, LacZ (beta-galactosidase) substrates, luciferase substrates, TEV (Tobacco etch virus protease) substrates, alkaline phosphatase substrates, 3,3'-diaminobenzidine substrates, tyramide substrates, and the like.

As described above, in some instances, a latent activity domain may be employed that includes a transcriptional activator or repressor. Kits that include sequence(s) encoding a transcriptional activator or repressor may further include one or more nucleic acids and/or expression cassettes and/or vectors that include a sequence that is responsive to the active transcriptional activator or repressor. For example, a subject kit may include a vector, such as a plasmid or virus, which includes a sequence responsive to a transcriptional activator that is operably linked to a reporter (such as a fluorescent, bioluminescent, chromogenic, or enzymatic reporter). Thus, upon release and activation of the latent activity domain, the reporter present on the vector may be activated.

In some instances, components of the subject kits may be presented as a“cocktail” where, as used herein, a cocktail refers to a collection or combination of two or more different but similar components in a single vessel. Components of the kits may be present in separate containers, or multiple components may be present in a single container, as desired. The subject compositions may be present in any suitable environment. According to one embodiment, the composition is present in a reaction tube (e.g., a 0.2 ml. tube, a 0.6 ml. tube, a 1.5 ml. tube, or the like) or a well or microfluidic chamber or droplet or other suitable container.

In certain aspects, the composition is present in two or more (e.g., a plurality of) reaction tubes or wells (e.g., a plate, such as a 96-well plate, a multi-well plate, e.g., containing about 1000, 5000, or 10,000 or more wells). The tubes and/or plates may be made of any suitable material, e.g., polypropylene, or the like, PDMS, or aluminum. The containers may also be treated to reduce adsorption of nucleic acids to the walls of the container.

In addition to the above-mentioned components, a subject kit may further include instructions for using the components of the kit, e.g., to practice the subject methods as described above. The instructions are generally recorded on a suitable recording medium. The instructions may be printed on a substrate, such as paper or plastic, etc. As such, the

instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or sub-packaging) etc. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, Hard Disk Drive (HDD) etc. In yet other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g. via the internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.

The following example(s) is/are offered by way of illustration and not by way of limitation.

EXAMPLES

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for.

Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

General methods in molecular and cellular biochemistry can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., HaRBor Laboratory Press 2001 ); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998), the disclosures of which are incorporated herein by reference.

Reagents, cloning vectors, and kits for genetic manipulation referred to in this disclosure are available from commercial vendors such as BioRad, Stratagene (Agilent Technologies), Invitrogen (Thermo Fisher Scientific), Sigma-Aldrich, and Clontech (Takara Bio USA, Inc.).

Example 1: Functional degradation: a mechanism of NLRP1 inflammasome activation by diverse pathogen enzymes

Here it is described that cleavage results in proteasome-mediated degradation of the N- terminal domains of NLRP1 B, liberating a C-terminal fragment that is a potent Caspase-1 activator. Proteasome-mediated degradation of NLRP1 B is both necessary and sufficient for NLRP1 B activation. Consistent with our new‘functional degradation’ model, lpaH7.8, a Shigella flexneri ubiquitin ligase secreted effector, was identified as an enzyme that induces NLRP1 B degradation and activation. These results provide a unified mechanism for NLRP1 B activation by diverse pathogen-encoded enzymatic activities.

In summary, in this example an innate immune sensor, NLRP1 B, is shown to be activated by two distinct pathogen enzymes by a mechanism that requires its own degradation.

In animals, pathogens are generally recognized by germline-encoded innate immune receptors that bind directly to conserved pathogen-associated molecular patterns (PAMPs) such as bacterial lipopolysaccharide or flagellin (Janeway, Cold Spring Harbor symposia on quantitative biology 54 Pt 1 , 1 (1989)). Recognition of PAMPs permits robust self-non-self discrimination, but since PAMPs are found on harmless as well as pathogenic microbes, PAMP receptors do not readily distinguish pathogens from non-pathogens. Plants also use germline- encoded receptors to detect PAMPs, but in addition, respond to infection by indirect detection of secreted pathogen enzymes called‘effectors’ (Jones & Dangl, Nature 444:323 (2006)). In this mode of recognition, called‘effector-triggered immunity’, intracellular proteins of the nucleotide binding domain leucine-rich repeat (NLR) superfamily sense effector-induced perturbation of host signaling pathways. Since harmless microbes do not deliver effectors into host cells, effector-triggered immunity is inherently pathogen-specific. It has been proposed that animals may also detect pathogen-encoded activities (Blander et al., Nature reviews. Immunology 12:215 (2012); Chung et al., Cell host & microbe 20:296 (2016); Keestra et al., Nature 496:233 (2013); Keestra-Gounder et al., Nature 532:394 (2016); Ratner et al., PLoS pathogens

12:e1006035 (2016); Vance et al., Cell host & microbe 6:10 (2009); Xu et al., Nature 513:237 (2014)).

How a particular mammalian NLR protein, mouse NLRP1 B, senses pathogen-encoded activities was investigated in this example. NLRP1 B belongs to a class of proteins that form inflammasomes, multi-protein platforms that initiate immune responses by recruiting and activating pro-inflammatory proteases, including Caspase-1 (CASP1 ) (Broz, Immunology 16:407 (2016); Chavarria-Smith et al., Immunological reviews 265:22 (2015); Rathinam et al., Cell 165:792 (2016)). CASP1 cleaves and activates specific cytokines (interleukins-1 b and -18) and a pore-forming protein called Gasdermin D, leading to a lytic host cell death called pyroptosis. In certain strains of mice and rats, NLRP1 B is activated via direct proteolysis of its N-terminus by the secreted Bacillus anthracis lethal factor (LF) protease (Boyden et al., Nature genetics 38:240 (2006); Chavarria-Smith et al., PLoS pathogens 9:e1003452 (2013); Hellmich et al.,

PloS one 7:e49741 (2012); Levinsohn et al., PLoS pathogens 8:e1002638 (2012)). Previous studies demonstrated that N-terminal proteolysis is sufficient to initiate NLRP1 B inflammasome activation (Chavarria-Smith et al., PLoS pathogens 9:e1003452 (2013)), but the molecular mechanism by which proteolysis activates NLRP1 B has been elusive.

Similar to other NLRs, NLRP1 B contains a nucleotide-binding domain and leucine-rich repeats (Fig. 6A). However, NLRP1 B also exhibits several unique features. First, the NLRP1 B caspase activation and recruitment domain (CARD) is C-terminal instead of N-terminal, as in other NLRs. Second, NLRP1 B contains a function-to-find domain (FUND) that constitutively undergoes an unusual auto-proteolytic event within the FUND that cleaves NLRP1 B into two separate polypeptides that remain non-covalently associated with each other (D'Osualdo et al., PloS one 6:e27396 (201 1 ); Finger et al., The Journal of biological chemistry 287:25030 (2012); Frew et al., PLoS pathogens 8:e1002659 (2012)). Mutations that abolish FUND auto-processing block inflammasome activation (Finger et al., The Journal of biological chemistry 287:25030 (2012); Frew et al., PLoS pathogens 8:e1002659 (2012)), but it remains unclear why FUND auto-processing is essential for NLRP1 B function. Lastly, NLRP1 B inflammasome activation is specifically blocked by proteasome inhibitors, but these inhibitors do not affect other

inflammasomes or inhibit LF protease (Fink et al., PNAS of the USA 105:4312 (2008); Squires et al., The Journal of biological chemistry 282:34260 (2007); Tang et al., Infection and immunity 67:3055 (1999); Wickliffe et al. Cellular microbiology 10:1352 (2008); Wickliffe et al. Cellular microbiology 10:332 (2008)). Cleavage of NLRP1 B by LF results in a loss of 45 amino acids from the N-terminus of NLRP1 B, an event that correlates with activation (Boyden et al., Nature genetics 38:240 (2006); Chavarria-Smith et al., PLoS pathogens 9:e1003452 (2013); Hellmich et al., PloS one 7:e49741 (2012); Levinsohn et al., PLoS pathogens 8:e1002638 (2012)). An‘auto-inhibition’ model has been previously proposed to explain NLRP1 B activation (Chavarria-Smith et al., Immunological reviews 265:22 (2015); Rathinam et al., Cell 165:792 (2016); Zhong et al., Cell 167:187 (2016)). In this model, the N-terminus of NLRP1 B functions as an auto-inhibitory domain that is lost after cleavage by LF. The NLRP1 B N-terminus might mediate auto-inhibition either through direct engagement with other NLRP1 B domains in cis, or by binding to an inhibitory co-factor. A clear prediction of the auto-inhibition model is that sequences within the N-terminus should be required to prevent spontaneous inflammasome activation. To identify such sequences, the NLRP1 B N-terminus was systematically mutated by replacing groups of three consecutive amino acids with alanine (Fig. 6B) or by replacing groups of five sequential amino acids with a flexible GGSGG motif (Fig. 6C). Mutants were also engineered to contain an N-terminal site for the TEV protease to permit their TEV-inducible activation (Chavarria-Smith et al., PLoS pathogens 9:e1003452 (2013); Chavarria-Smith et al., PLoS pathogens 12:e1006052 (2016)). Inflammasome activity was monitored by CASP1 -dependent processing of pro-IL1 b to p17 in a reconstituted inflammasome system in transfected 293T cells (Chavarria-Smith et al., PLoS pathogens 9:e1003452 (2013); Chavarria-Smith et al., PLoS pathogens 12:e1006052 (2016)). Surprisingly, none of the mutants was auto-active, even though all showed full activity upon cleavage. To test whether any N-terminal sequence mediated auto-inhibition, the entire N- terminus was replaced with a heterologous alpha-helical domain from bacterial flagellin. Again, surprisingly, the hybrid Fla-NLRP1 B protein was not auto-active, but was still functional after N- terminal cleavage (Fig. 6D). These results are consistent with prior experiments that also failed to show a role for specific N-terminal sequences in NLRP1 auto-inhibition (Chavarria-Smith et al., PLoS pathogens 12:e1006052 (2016); Neiman-Zenevich et al., Infection and immunity 82:3697 (2014)). Together, these findings prompted a reconsideration of the auto-inhibition model.

Whether the precise site of N-terminal proteolysis was a major determinant of NLRP1 B activation was then investigated. A series of NLRP1 B variants in which a TEV protease cleavage site was positioned at regular intervals from the N-terminus was generated. It was found that cleavage of as few as 10 amino acids from the N-terminus was sufficient to activate NLRP1 B, and there was not a significant correlation between the position of TEV cleavage and NLRP1 B activity (Fig. 6E, Fig. 6F). In contrast, a striking positive correlation between the extent of NLRP1 B protein loss following TEV cleavage and inflammasome activation was observed (Fig. 6E, Fig. 6F). This correlation, prompted the consideration of whether proteasome-mediated degradation of NLRP1 B could be an important step in its activation. Consistent with this idea, it was found that the proteasome inhibitors MG132 and Bortezomib not only abrogated NLRP1 B activation in the reconstituted 293T system, but also prevented loss of the cleaved NLRP1 B protein after LF treatment (Fig. 7A). To determine if this loss of cleaved NLRP1 B also occurred with the endogenous protein, a monoclonal antibody (2A12) against the C-terminal CARD domain of NLRP1 B was derived (Fig. 10). Using this antibody, endogenous NLRP1 B can be tracked in 129S1/SvimJ (129S1 ) bone marrow-derived macrophages (BMMs) after treatment with LF (Fig. 7B). As observed in the 293T system, LF treatment led to a decrease in NLRP1 B, which could be reversed by MG132 treatment. In contrast, proteasome inhibitors have no effect on NLRP3 (Squires et al., The Journal of biological chemistry 282:34260 (2007), Wickliffe et al. Cellular microbiology 10:332 (2008)) or NAIP5/NLRC4 inflammasome activation (Fig. 1 1 ), confirming that the proteasome functions uniquely in NLRP1 B inflammasome activation.

Cleaved proteins are known to be recognized by a quality control pathway called the N-end rule pathway (Bachmair et al., Science 234:179 (1986); Lucas et al., Current opinion in structural biology 44:101 (2017)), and N-end rule inhibitors block NLRP1 B activation (Wickliffe et al.

Cellular microbiology 10:1352 (2008)). Therefore, it is proposed that protease cleavage reveals a destabilizing neo-N-terminus that targets NLRP1 B for ubiquitylation by N-end rule E3 ubiquitin ligases. Consistent with this proposal, N-end rule ubiquitin ligases have recently been identified that play a role in NLRP1 B activation (see e.g., A. J. Chui et al. Science (2019) 364:82-85).

We note that the TEV-cleavable NLRP1 B variants that we examined generate a glycine residue at the neo-N terminus after TEV cleavage. According to the N-end rule, glycine is a stabilizing N-terminal amino acid, a prediction apparently at odds with the observation that TEV cleavage results in NLRP1 B destabilization. However, aminopeptidase inhibitors block the LF- mediated killing of RAW264.7 cells. Thus, it appears that the neo-N terminus generated by primary cleavage is further processed by aminopeptidases, resulting in the exposure of otherwise internal amino acids to N-end rule recognition. When the P2' residues were swapped between two differentially activated TEV-cleavable NLRP1 B variants, it was found that the P2' residue could also modulate activity (FIG. 13). Thus, the stability and activity of cleaved

NLRP1 B depends on more than just the identity of the neo-N-terminal amino acid.

In the above experiments, the inhibition of NLRP1 B by proteasome inhibitors might be due to stabilization of a negative regulator of NLRP1 B rather than to stabilization of NLRP1 B itself. Therefore, it was next investigated whether specific degradation of NLRP1 B is sufficient to induce its activation. To achieve selective degradation of NLRP1 B, the auxin-interacting degron (AID) (Holland et al., PNAS of the USA 109:E3350 (2012); Nishimura et al., Nature methods 6:917 (2009)) was fused to the N-terminus of NLRP1 B. Upon addition of the auxin hormone indole-3-acetic acid (IAA), the AID recruits a co-expressed TIR1 E3 ligase that specifically ubiquitylates AID-fusion proteins, targeting them to the proteasome. Indeed, IAA induced rapid degradation of the AID-NLRP1 B fusion protein, resulting in robust IL-1 b processing (Fig. 7C). Degradation and activation were both blocked with proteasome inhibitors (Fig. 7C)

demonstrating that proteasomal degradation of NLRP1 B itself is both necessary and sufficient for activation of the NLRP1 B inflammasome.

To reconcile the seemingly paradoxical observation that NLRP1 B degradation leads to its own activation, the following‘functional degradation’ model is proposed (Fig. 8A). This model relies on the observation that FUND auto-processing is required for activation of NLRP1 B by LF. Of note, it was also found that FUND auto-processing is also required for IAA-induced activation of AID-NLRP1 B (Fig. 7C). The FUND domain comprises two separate subdomains, termed ZU5 and UPA, with auto-processing occurring nearly between them (D'Osualdo et al., PloS one 6:e27396 (201 1 ); Finger et al., The Journal of biological chemistry 287:25030 (2012); Frew et al., PLoS pathogens 8:e1002659 (2012)). After auto-processing, the C-terminal fragment of NLRP1 B thus consists of the UPA domain fused to a CARD that is required for CASP1 recruitment and activation. Prior to activation, the FIIND(UPA)-CARD fragment is non-covalently associated with the rest of NLRP1 B (Fig. 8A). After cleavage by LF, NLRP1 B is targeted to the proteasome, a processive protease that degrades polypeptides by feeding them through a central barrel (Nyquist et al., Trends in biochemical sciences 39:53 (2014)). Critically, however, directional (N- to C-terminus) and processive degradation of NLRP1 B by the proteasome can proceed only until it encounters the covalent break within the auto-processed FUND domain. At this point, the C-terminal FIIND(UPA)-CARD fragment is released. Once liberated, the bioactive FIIND(UPA)-CARD fragment would then seed inflammasome assembly (Fig. 8A).

This new‘functional degradation’ model of NLRP1 B inflammasome activation has several virtues. First, the model explains how N-terminal cleavage results in proteasome- dependent NLRP1 B activation without a requirement for specific N-terminal‘auto-inhibitory’ sequences. Second, the model explains why the NLRP1 B CARD must be C-terminal, rather than N-terminal, as only the C-terminus of NLRP1 B remains after proteasome-mediated degradation. Lastly, the model explains why FUND domain auto-processing is required for NLRP1 B activity: an unprocessed FUND mutant would be fully degraded and would not release a C-terminal CARD-containing fragment. A strong prediction of the‘functional degradation’ model is that the C-terminal FIIND(UPA)-CARD fragment possesses inflammasome activity. Studies showed that the FIIND(UPA)-CARD fragment was indeed sufficient to promote robust CASP1 activity in the 293T reconstituted inflammasome assay, whereas the full-length FIIND(ZU5+UPA)-CARD remained inactive despite auto-processing (Fig. 8B). Likewise, the isolated CARD domain (lacking any portion of the FUND) was also inactive, implying that the FIIND(UPA) contributes to inflammasome formation. This pattern was observed across a range of expression levels (Fig. 12). Importantly, the FIIND(UPA)-CARD fragment appears to be a highly potent activator of inflammatory signaling. By titrating the amount of expression construct, it was found that the FIIND(UPA)-CARD fragment was ~150X more potent than TEV-cleavable full-length NLRP1 B (Fig. 8C). These results imply that only a tiny fraction of the total NLRP1 B in a cell may need to be degraded to liberate sufficient amounts of the FIIND(UPA)-CARD fragment for robust inflammasome activation. Thus, even if NLRP1 B was only degraded half the time in the ‘productive’ N- to C-terminal direction, this would still likely be sufficient to produce robust inflammasome activation.

This‘functional degradation’ model predicts that the mature/assembled NLRP1 B inflammasome consists solely of the FIIND(UPA)-CARD fragment and CASP1 . In support of that hypothesis, only the UPA-containing FUND-CARD fragment was found to co- immunoprecipitate with CASP1 (Fig. 8D) and assemble into higher order oligomers when subjected to non-denaturing PAGE. As such, the model predicts that the non-FIIND-CARD fragment portion of NLRP1 B is dispensable for inflammasome activation and these results show that the NBD and LRR domain of NLRP1 B are dispensable for inflammasome activation.

Consistent with this hypothesis, it was observed that IAA-induced degradation of an AID- FIIND(ZU5-UPA)-CARD fragment is sufficient to induce CASP1 activity (Fig. 8E).

To visualize the simultaneous degradation of the N-terminal domains and the release of the FIIND(UPA)-CARD fragment upon NLRP1 B activation, a variant of NLRP1 B with a C- terminal FLAG tag and an HA tag following the N-terminal TEV cleavage site was engineered.

In unstimulated cells expressing this variant, FLAG and HA co-stained within the cytosol (Fig. 14). By contrast, cells co-transfected with TEV protease showed inflammasome activation, as indicated by formation of“specks” containing the ASC adaptor protein. Notably, it was found that the FLAG-tagged UPA-CARD fragment, but not the HA-tagged N-terminal domains, robustly formed puncta colocalized at the ASC speck (Fig. 14). Moreover, a near-complete loss of the HA signal was also observed, consistent with N-terminal degradation upon TEV cleavage. The FLAG signal was also lost upon TEV cleavage when FUND processing was disrupted (Fig. 15). Thus, the C-terminal UPA-CARD appears to be released to seed inflammasome formation upon proteolytic cleavage and the subsequent proteasomal degradation of the NLRP1 B N- terminal domains.

A major implication of the‘functional degradation’ model is that in addition to detecting pathogen-encoded proteases, NLRP1 B can potentially sense any enzymatic activity that results in NLRP1 B degradation. For example, several pathogens encode E3 ubiquitin ligases that promote virulence through degradation of host target proteins (Maculins et al., Cell research 26:499 (2016)). Thus, it was tested whether the T3SS-secreted IpaH family of E3 ubiquitin ligases, encoded by the intracellular bacterial pathogen Shigella flexneri (Rohde et al., Cell host & microbe 1 :77 (2007); Singer et al., Nature structural & molecular biology 15:1293 (2008); Y. Zhu et al., Nature structural & molecular biology 15:1302 (2008)), are detected by NLRP1 B. Using the reconstituted 293T cell system, it was found that lpaH7.8, but not IpaFU .4, 4.5 or 9.8, markedly reduced NLRP1 B protein levels and induced IL-1 b processing in an NLRP1 B- dependent manner (Fig. 9A). lpaH7.8 selectively activates the 129S1 but not the C57BL/6 (B6) allele of NLRP1 B (Fig. 9B). FUND auto-processing is required for lpaH7.8-induced NLRP1 B activation (Fig. 9C). Truncation of either the lpaH7.8 LRR or E3 domains, as well as mutation of the catalytic cysteine residue (CA) required for E3 ligase activity, also abolishes lpaH7.8- mediated inflammasome activation (Fig. 9D). Using recombinant proteins it was found that lpaH7.8 but not the CA mutant can directly ubiquitylate 129 but not B6 NLRP1 B in vitro. Thus, lpaH7.8/ubiquitylation-dependent degradation of NLRP1 B can result in its activation.

S. flexneri robustly activates multiple inflammasomes (Hermansson et al., Current topics in microbiology and immunology 397:91 (2016)) and has previously been reported to cause macrophage cell death in an lpaH7.8- and NLRP1 B-dependent manner (Fernandez-Prada et al., Infection and immunity 68:3608 (2000); Neiman-Zenevich et al., Infection and immunity 85 ( 2017); Suzuki et al., PNAS of the USA 1 1 1 :E4254 (2014)), but a connection between lpaH7.8 and NLRP1 B has not been established. Consistent with prior studies (Fernandez-Prada et al., Infection and immunity 68:3608 (2000); Suzuki et al., PNAS of the USA 1 1 1 :E4254 (2014)), it was found that wild-type S. flexneri induces robust LDH release from infected RAW264.7 macrophages, which is reduced in infections with a AlpaH7.8 mutant (Fig. 9E). Cell killing by the AlpaH7.8 strain complemented with a plasmid expressing lpaH7.8 strain is greater than even that observed by wild-type, likely due to effector over-expression. Using CRISPR/Cas9- engineered RAW264.7 cells (Okondo et al., Cell chemical biology 25:262 (2018)), it was found that lpaH7.8-dependent cell death is completely abrogated by the loss of CASP1 or NLRP1 B (Fig. 9F). The NLRC4 inflammasome also recognizes S. flexneri (Ruiz et al., PNAS of the USA 1 14:13242 (2017); Suzuki et al., PLoS pathogens 10:e1003926 (2014); Yang et al., PNAS of the USA 1 10, 14408 (2013).), but A//rc4^_/_ cells exhibit an even more robust differential response to lpaH7.fr and AlpaH7.8 strains, suggesting that NLRC4 inflammasome activation obscures lpaH7.8-induced responses to some extent. Although the N-end rule ubiquitin ligase Ubr2 is required for LF-mediated NLRP1 B activation, this requirement was circumvented by the direct ubiquitylation of NLRP1 B by S. flexneri (Fig. 16A-16D). Immortalized 129 macrophages were also sensitive to lpaH7.8-dependent killing, and lpaH7.8 sensitivity correlates with decreased levels of endogenous NLRP1 B and the induction of CASP1 maturation (Fig. 9G). S. flexneri is not a natural pathogen of mice; this may be due in part to species-specific NLRP1 B recognition, since human NLRP1 does not appear to detect lpaH7.8 (FIG. 16D). Nevertheless, by leveraging our new mechanistic understanding of NLRP1 B, these results identify ubiquitin ligases as a new category of pathogen-encoded enzyme that activates NLRP1 B.

Prior work in Arabidopsis has shown that an NLR called RPS5 detects proteolytic cleavage of the host PBS1 kinase by the translocated Pseudomonas syringae effector AvrPphB (Shao et al., Science 301 :1230 (2003)). In this system, RPS5 appears to directly detect the cleavage products of PBS1 (DeYoung et al. Cellular microbiology 14:1071 (2012)). Although NLRP1 B also appears to detect a pathogen-encoded protease, these results suggest that the underlying mechanism is very different than that of RPS5. Instead of proteolysis generating a specific ligand, it appears that NLRP1 B is itself the target of proteolysis, leading to its proteasomal degradation, and release of a functional inflammasome fragment. Thus, NLRP1 B is in essence a sensor of its own stability, permitting detection of diverse pathogen-encoded enzymes, potentially including those of viruses or parasites (Cirelli et al., PLoS pathogens 10:e1003927 (2014); Ewald et al., Infection and immunity 82:460 (2014); Gorfu et al., mBio 5 (2014)). Although lpaH7.8 and LF protease both activate NLRP1 B, a scenario is favored in which the‘intended’ targets of these pathogen-encoded enzymes are other host proteins, and that NLRP1 B has evolved as a‘decoy’ target of these proteins, a pathogen-sensing strategy also seen in some plant NLRs (Jones & Dangl, Nature 444:323 (2006)).

NLRP1 was the first protein shown to form an inflammasome (Martinon et al., Molecular cell 10:417 (2002)). These results provide a long-sought mechanism that finally explains how NLRP1 is activated. In addition to explaining how NLRP1 senses pathogens, our new mechanistic understanding also provides an explanation for why naturally occurring mutations that destabilize human NLRP1 (Zhong et al., Cell 167:187 (2016)) also result in its activation. Our results lay the foundation for identifying pathogen-encoded activators of human NLRP1 and provide a conceptual basis for designing therapeutic interventions that target NLRP1 . FIG. 6. The N-terminal domain of NLRP1 B does not mediate auto-inhibition.

(FIG. 6A) Schematic of mouse NLRP1 B domain architecture. Nt, N-terminus; NBD, nucleotide-binding domain; LRR, leucine-rich repeat; FUND, function-to-find domain; CARD, caspase activation and recruitment domain. FUND auto-processing results in two non-covalently associated proteins, which appears as a doublet [(FIG. 6B to FIG. 6D), upper blot] when probing with an antibody directed against the N-terminal MBP tag. The location of lethal factor (LF) cleavage (triangle) and FUND auto-processing (open triangle, diagonal line) is shown. (FIG. 6B to FIG. 6C) Mutagenesis of the Nt does not disrupt auto-inhibition. The effect of sequential, non overlapping replacement of Nt amino acids 1 -39 by triple alanine [(FIG. 6B), AAA] or glycine- serine-glycine [(FIG. 6C), GGSGG] scanning mutagenesis on NLRP1 B auto-inhibition was assessed. In (FIG. 6B to FIG. 6E) inflammasome activation was induced by tobacco-etch virus (TEV) protease cleavage of the indicated TEV site-containing MBP-NLRP1 B variant. Activation was monitored by CASP1 processing of pro-IL1 b to p17. (FIG. 6D) Replacement of the Nt of NLRP1 B with a heterologous sequence from flagellin does not affect auto-inhibition or protease- dependent activation of NLRP1 B. (FIG. 6E to FIG. 6G) NLRP1 B degradation, but not the position of protease cleavage, positively correlates with IL1 b processing. The TEV site was scanned sequentially along the NLRP1 B Nt and detected by probing for a C-terminal HA tag [(FIG. 6E), upper blot]. IL1 b processing was plotted relative to the position of the TEV site (FIG. 6F) or the protein level of cleaved-NLRP1 B (FIG. 6G) [band signal intensity was quantified using Image Studio Lite software v5.2.5.]. IB, immunoblot.

FIG. 7. Degradation of NLRP1 B is necessary and sufficient for NLRP1 B inflammasome activation.

(FIG 7A to FIG. 7B) The proteasome is required for NLRP1 B inflammasome activation. Proteasome inhibitors MG132 (10 nM) and Bortezomib block LF-induced NLRP1 B degradation and IL1 b processing in transfected 293T cells as in Fig. 6 (FIG. 7A) and immortalized 129S1 bone-marrow-derived macrophages (FIG. 7B). (FIG. 7C) Proteasomal degradation of NLRP1 B is sufficient for inflammasome activation. In (FIG. 7C) the plant auxin-interacting degron (AID) was fused to the N-terminus of indicated GFP-NLRP1 B variants; specific degradation was induced with lndole-3-acetic acid (IAA) in TIR1 -expressing 293T cells. IAA-induced NLRP1 B degradation induced IL1 b processing, which is dependent on FUND auto-processing and is blocked by proteasome inhibitors. C, cysteine. A, alanine. FIG. 8.‘Functional degradation’ of NLRP1 B liberates the FIIND(UPA)-CARD fragment, a highly potent inflammasome activator.

(FIG. 8A) A model for NLRP1 B activation via‘functional degradation.’ (/) constitutive auto-processing of the NLRP1 B FUND domain, resulting in two, non-covalently associated peptides: FIIND(ZU5) and FIIND(UPA)-CARD. (//) Lethal factor (LF) protease cleavage of the NLRP1 B Nt, yielding an unstable neo-Nt. (///) N-end rule factor recognition and ubiquitylation of the neo-Nt. (/V) NLRP1 B degraded by the proteasome. (v) Proteasomal degradation of NLRP1 B releases the FIIND(UPA)-CARD fragment (vi) The FIIND(UPA)-CARD fragment self-assembles into a high molecular weight oligomer (vii) The assembled FIIND(UPA)-CARD serves as a platform for CASP1 maturation and downstream inflammasome signaling. (FIG. 8B) The FIIND(UPA)-CARD fragment has inflammasome activity. The activity of C-terminal HA-tagged variants corresponding to the full length FIIND(ZU5+UPA)-CARD [(1 ), 745-1233), FIIND(UPA)- CARD fragment [(2), 986-1233), FIIND(UPA)-CARD truncation [(3), 1 124-1233) or the CARD domain [(4), 1 143-1233) was tested in 293T cells as in Fig. 6. Only the FIIND(UPA)-CARD fragment exhibited auto-activity. (FIG. 8C) The FIIND(UPA)-CARD fragment is a potent activator of IL1 b processing. Decreasing amounts (ng) of plasmid encoding the FIIND(UPA)-CARD fragment (right) compared to full-length NLRP1 B activated by TEV (left). (FIG. 8D) The

FIIND(UPA)-CARD fragment, but not the full length FIIND(ZU5+UPA)-CARD, truncated

FIIND(UPA)-CARD fragment or the CARD domain only, co-immunoprecipitates with CASP1 .

The FIIND(UPA)-CARD fragment, but not the full length FIIND(ZU5+UPA)-CARD, truncated FIIND(UPA)-CARD fragment or the CARD domain only, forms high molecular weight oligomers. For (FIG. 8D) lanes and FUND-CARD variants are labeled as in (FIG. 8C). (FIG. 8E) IAA- induced degradation of AID-FIIND-CARD is sufficient for inflammasome activation. An AID- FIIND(ZU5+UPA)-CARD is activated by TEV cleavage or IAA, which is dependent on FUND auto-processing and is blocked by MG132.

Fig. 9. The secreted Shigella flexneri lpaH7.8 E3 ubiquitin ligase activates NLRP1 B.

(FIG. 9A) lpaH7.8 induces the degradation and activation of NLRP1 B. lpaH7.8, but not 1.4, 4.5 or 9.8, induces NLRP1 B degradation and inflammasome activation in 293T cells, as described in Fig. 6. lpaH7.8 induction of IL1 b processing is dependent on the presence of NLRP1 B. (FIG. 9B) lpaH7.8 selectively activates the 129S1 but not the B6 allele of NLRP1 B. (FIG. 9C) Activation of NLRP1 B by lpaH7.8 requires FUND auto-processing. S, serine. A, alanine. (FIG. 10D) Activation of NLRP1 B by lpaH7.8 requires the catalytic cysteine of lpaH7.8. The activity of the mCherry-tagged lpaH7.8 variants: catalytic mutant [(CA), CXXXA], LRR (DE3) or E3 (ALRR), respectively, was assessed 293T cells, as described in Fig. 6. Only the full- length lpaH7.8 induced IL1 b processing, despite constitutive ubiquitin ligase activity of the ALRR variant. Recombinant lpaH7.8 ubiquitylates NLRP1 B in vitro. (FIG. 9E) lpaH7.8-mediated macrophage cell death requires NLRP1 B. Infection (MOI 30) of WT, Caspl^, Nlrplb^ or Nlrc4 ^~ RAW264.7 cells with the WT Shigella flexneri strain 2457T (black circle), or the mutants strains BS103, virulence plasmid-cured (white box); A7.8, lpaH7.8 deletion (triangle); A7.8+7.8, A7.8 strain complemented with pCMD136 lpaH7.8 (inverted triangle); D7.8+n, A7.8 strain

complemented with pCMD136 empty vector (diamond). Cell death was monitored by assaying for lactate dehydrogenase (LDH) activity in culture supernatants 30m post-infection. (FIG. 9G) lpaH7.8 activates the NLRP1 B inflammasome in macrophages. Immortalized 129S1 (M 29) bone-marrow-derived macrophages were infected with S. flexneri strains. lpaH7.8-expressing strains induce NLRP1 B degradation and CASP1 processing. Cell death was measured by LDH.

FIG. 10 The 2A12 monoclonal antibody recognizes the CARD domain of NLRP1 B.

The full length FIIND(ZU5+UPA)-CARD [(1 ), 745-1233), FIIND(UPA)-CARD fragment [(2), 986-1233), FIIND(UPA)-CARD truncation [(3), 1 124-1233) or the CARD domain [(4), 1 143- 1233) were transfected into 293T cells. NLRP1 B was detected either by a C-terminal HA-tag [(left), reproduced from FIG. 8B], or the anti-NLRP1 B monoclonal Ab 2A12 (right). 2A12 detects each FUND-CARD variant, indicating that 2A12 binds to the CARD.

FIG. 11 Proteasome inhibitors do not block NAIP/NLRC4 inflammasome activation.

The proteasome is specifically required for NLRP1 B inflammasome activation. The proteasome inhibitor MG132 (10 nM) does not block ligand-induced NAIP-NLRC4

inflammasome activation in reconstituted 293T cells. FlaTox, PA + LFn-FlaA.

FIG. 12 The FIIND(UPA)-CARD fragment is a potent activator of IL1 b processing.

The activity of C-terminal HA-tagged variants corresponding to the full length

FIIND(ZU5+UPA)-CARD [(1 ), 745-1233), FIIND(UPA)-CARD fragment [(2), 986-1233),

FIIND(UPA)-CARD truncation [(3), 1 124-1233) or the CARD domain [(4), 1 143-1233) were transfected across a range of ng inputs in 293T cells, as described for FIG. 8B. Only the FIIND(UPA)-CARD fragment exhibited auto-activity. FIG. 13 P2' residue identity modulates TEV-induced proteasomal degradation and activation of NLRP1 B.

The amino acid from the P2' position from a relatively inactive TEV cleavage site (site 7 (see FIG. 6E), P2'=K) was introduced into the P2' position of a relatively active TEV cleavage site (site 19, P2'=Q). The activity of the resulting C-terminal HA-tagged constructs was tested in 293T cells as described with respect to FIG. 6A-6G. The P2' lysine is sufficient to both stabilize and prevent NLRP1 B activation via cleavage at site 19, suggesting that residues beyond the P1 ' position are important for determining the stability (and subsequent activation) of NLRP1 B. Gel images are representative of experiments performed at least three times.

FIG. 14 Visualization of the simultaneous degradation of the N-terminal domains and the release of the FIIND(UPA)-CARD fragment upon NLRP1 B activation

293T cells were transfected with expression constructs for ASC and TEV-cleavable NLRP1 B encoding a C-terminal FLAG tag and HA tag (inserted after the TEV site, as shown). The number of ASC specks per field (± SD) was quantified for TEV-treated samples and compared with that of cells expressing a S984A FUND auto-processing NLRP1 B mutant. Representative images depict cytosolic FLAG and HA signal in untreated samples, with FLAG colocalization with ASC specks (white arrowheads) and concomitant loss of the HA signal in TEV- expressing cells. The total number of ASC specks or ASC specks positive for both FLAG and HA (n = 7) or only FLAG (n = 32) or HA (n = 0) in TEV-treated samples is quantified from 12 total fields representative of three independent experiments. Scale bars, 10 mm. Significance was determined by Student’s t test; ^***P < 0.001 .

FIG. 15 FUND auto-processing is required for release of the FIIND(UPA)-CARD and its colocalization to the ASC speck.

293T cells were transfected with constructs producing ASC (blue) and an NLRP1 B FUND mutant (S984A) variant marked with a C-terminal FLAG (green) and an N-terminal HA (magenta). As schematized, the HA is inserted directly after the TEV cleavage site, allowing detection of the TEV-cleaved protein. Representative images depict cytosolic FLAG and HA signal in untreated and TEV-expressing cells. Unlike in Fig. 3G, where ASC colocalizes specifically with FLAG and not HA, no specific FLAG-ASC staining was observed for the FUND mutant protein. Scale bar, 10 pm. Images are representative of 12 total fields of ~30 cells/field from three independent experiments. FIG. 16A-16D lpaH7.8 activates mouse NLRP1 B, but not human NLRP1 , independently of N-end rule ubiquitin ligases.

(FIG. 16A) NAIP2 inflammasome ligand (LFn-PrgJ) or varying amounts of NLRP1 B agonist (lethal factor, LF) (pg/ml) was delivered into Nlrc4-/- or Ubr2-/ RAW264.7 cells via the PA channel. Inflammasome activation was measured by assessing LDH-release following CASP1 -dependent pyroptosis. LF-mediated cell death remains saturated in Nlrc4-/~ cells at all LF concentrations, but is reduced in two Ubr2-/ cell lines at lower concentrations. (FIG. 16B) RAW264.7 cells of the indicated genotypes were infected (MOI 10) with WT Shigella flexneri strain 2457T (circle) or mutant strains. BS103, virulence plasmid-cured (box); vec, A7.8 strain complemented with pCMD136 empty vector (diamond); A7.8 strain complemented with pCMD136 ipaH7.8 (inverted triangle).

Data in (FIG. 16A, 16B) are representative of at least three independent experiments. Data sets (FIG. 16A, 16B) were analyzed using one-way ANOVA. P-values were determined by Dunnet’s multiple comparison post-hoc test. ^*, P < 0.05; ^**, P < 0.01 ; ^***, P < 0.001 . (FIG. 16C) The NLRP1 B inflammasome was reconstituted in wild-type (WT) or UBR2-/- 293T cells as described above and was activated by co-transfection with expression constructs for lethal factor (LF) or lpaH7.8. Inflammasome activation was assessed by immunoblotting for CASP1 - dependent processing of IL-1 b to p17. UT, untransfected. (FIG. 16D) The human NLRP1 inflammasome (NLRP1 , ASC, CASP1 ) was reconstituted in 293T cells, and inflammasome activation was monitored by immunoblotting for the processing of human pro-IL-1 b to p17 by co transfected human CASP1 . A TEV-cleavable human NLRP1 variant was used to allow for TEV- mediated inflammasome activation. Images are representative of experiments performed at least three times.

Materials and Methods

Plasmids and constructs: The coding sequence of Nlrpl b allele 1 : 129S1/ SvimJ (129)

N I rp 1 b (DQ1 17584.1 ), allele 2: C57BL/ 6J (B6) Nlrpl b (BC141354) or allele 3: AKR/J, and variants thereof, were cloned into either pCMSCVIRES- GFP or pAcSG2, with an N-terminal maltose binding protein (MBP) tag followed by a 3C protease cleavage site and a C-terminal HA or FLAG tag, respectively, or into pQCXIP with N-terminal green fluorescent protein (GFP) and C-terminal HA tags. The Fla-NLRP1 B hybrid was constructed by replacing the first 45 N- terminal amino acids of NLRP1 B with residues 431 to 475 of Legionella pneumophila flagellin (ANN95373) followed by the TEV cleavage sequence. CASP1 , IL-1 b, TEV, and LF producing constructs were described previously (Chavarria-Smith et al., PLOS Pathog. 9, e1003452 (2013)). The Nlrpl b coding sequence was subcloned in-frame with AID-GFP (Addgene 80076). GFP-fused IpaH producing plasmids were constructed as follows: the ipaH coding sequences from the S. flexneri 2a str. 2457T virulence plasmid were transferred using the Gateway vector conversion system (Thermo- Fisher) from Gateway entry clones (Schmitz, et al. Nat. Methods 6, 500-502 (2009)) into the Smal restriction site of the Gateway-compatible destination vector pC1 -eGFP (Clontech) via LR reactions. The ipaH7.8 coding sequence was also subcloned into pQCXIP with an N-terminal mCherry tag. For protein expression in Escherichia coli, the ipaH coding sequences were subcloned into pET28a with a C-terminal 6X-HIS tag. Mutations were engineered by overlapping PCR.

Cell culture: 293T and RAW264.7 cells were grown in DMEM supplemented with 10% FBS, 100 U/ml penicillin, 100 mg/ml streptomycin, and 2 mM L-glutamine. Primary BMDMs were cultured in RPMI supplemented with 5% FBS, 5% mCSF, 100 U/ml penicillin, 100 mg/ml streptomycin, and 2 mM L-glutamine. BMDM immortalization was performed as previously described.

Bacterial strains and infections: 2457T S. flexneri-derived ipaH deletion strains were constructed using the I red recombinase-mediated recombination system. To construct complemented strains, the coding sequence of ipaH7.8 and 407 base pairs upstream, representing the endogenous promoter, were Gateway cloned into the pCMD136 plasmid and transformed into the DipaH7.8 mutant strain. S. flexneri was grown at 37°C on tryptic soy agar plates containing 0.01% Congo red, supplemented with 100 mg/ml spectinomycin for growth of complemented strains. For infections, 5 ml of tryptic soy broth (TSB) was inoculated with a single Congo red positive colony and grown overnight shaking at 37°C. Saturated cultures were back-diluted 1 :100 in 5 ml of fresh TSB and incubated for 2 to 3 hours shaking at 37°C. Bacteria were washed in cell culture medium and spun onto cells for 10 min at 300xg. Infected cells were incubated at 37°C for 20 min and then washed twice with cell culture medium containing 25 mg/ml gentamicin, then returned to 37°C for further incubation (30 min to 2 hours). Cells were infected at an MOI of 30 unless otherwise specified. Cell death was assessed by LDH activity in clarified culture supernatants as previously described. Protein in supernatants was TCA precipitated for anti-CASP1 immunoblotting.

Reconstituted NLRP1 B activity assays: To reconstitute inflammasome activity in 293T cells, constructs producing NLRP1 B (or mutants), CASP1 , and IL-1 b were co-transfected with constructs producing TEV, LF, IpaHs, or empty vector (MSCV2.2 or pcDNA3) using

Lipofectamine 2000 (Invitrogen) following the manufacturer’s protocol. For experiments using recombinant proteins, fresh media containing 10 mg/ml PA and 2.5 mg/ml LF, supplemented with or without 10 mMMG132, 1 mM bortezomib, or 0.5 mM NMS- 873, was added to cells for 2 to 4 hours. For auxin-inducible degradation, AID-NLRP1 B and TIR1 -producing constructs (TIR1 , Addgene 80073) were co-transfected and treated with 500 mM indole-3-acetic acid sodium salt (IAA) (Sigma) for 3 to 6 hours in the presence or absence of 10 mM MG132. In all experiments, cells were lysed in RIPA buffer with protease inhibitor cocktail (Roche) 24 hours post transfection.

Endogenous NLRP1 B activity assays: Immortalized 129 (M 29) BMDMs (2.5 c 106) were plated in six-well plates. Two hours before challenge, cells were primed with 1 .0 mg/ml

Pam3CSK4 (Invivogen). Cells were washed with PBS and media was replaced with 0.5 ml of Opti-MEM (Gibco) with or without 20 mg/ml PA, 10 mg/ml LF, and/or 10 mMMG132. Cells and media were lysed by addition of 120 ml of 10XRIPAbufferwith protease inhibitor cocktail 2.5 hours posttreatment.

Immunoblotting and antibodies: Lysates were clarified by spinning at ~16,000xg for 10 min at 4°C. Clarified lysates were denatured in SDS loading buffer. Samples were separated on NuPAGE Bis-Tris 4% to 12% gradient gels (ThermoFisher) following the manufacturer’s protocol. Gels were transferred onto Immobilon- FL PVDF membranes at 35 V for 90 min and blocked with Odyssey blocking buffer (Li-Cor). Proteins were detected on a Li-Cor Odyssey Blot Imager using the following primary and secondary antibodies: 100 ng/ml anti-HA clone 3F10 (Sigma), 200 ng/ml anti-IL-1 b (R&D systems, AF- 401 -NA), 1 mg/ml anti-GFP (Clontech, JL8), 2 mg/ml anti-mCherry (ThermoFisher, 16D7), 1 mg/ml anti-CASP1 (Adipogen, AG-20B-0042- C100). Anti- MBP (NEB, E8032S) and anti-ubiquitin (Cell Signaling, P4D1 ) antibodies were used at 1 :1 ,000 dilution of manufacturer’s stock. Alexa Fluor 680- conjugated secondary antibodies (Invitrogen) were used at 0.4 mg/ml. Band intensities were quantified with Image Studio Lite software v5.2.5.

To produce the 2A12 mouse anti-NLRP1 B monoclonal antibody, the pAcSG2-Nlrp1 b construct was co-transfected with BestBac linearized baculovirus DNA (Expression Systems) into SF9 cells following the manufacturer’s protocol to generate NLRP1 B expressing

baculovirus. Primary virus was amplified in SF9 cells. NLRP1 B was produced by infecting 4 liters of High Five cells with 1 ml of amplified virus per 1 liter of cells. Cells were harvested 48 hours after infection by centrifugation at 300xg for 15 min. Cell pellets were resuspended in lysis buffer (50 mM Hepes pH 7.5, 150 mM NaCI, 1 % NP-40, and 5% glycerol) and lysed on ice using a dounce homogenizer. Homogenized samples were clarified at 24,000xg for 30 min, and supernatants were batch bound to 1 ml of amylose resin for 2 hours at 4°C. Samples were column purified by gravity. Resin was washed with 50 ml of wash buffer (20 mM Hepes pH 7.4, 150 mM NaCI, 0.02% NP-50, 5% glycerol). Samples were eluted with 1 ml of elution buffer (20 mM Hepes pH 7.4, 150 mM NaCI, 0.02% NP-50, 5% glycerol, 20mM maltose) fractions. Peak elutions were pooled and MBP was cleaved by treatment overnight with 3C protease. Free MBP was removed by passing the sample over amylose resin. BALB/c mice were immunized with 10 mg of NLRP1 B in 100 ml of Sigma adjuvant on days 0, 21 , and 42, and with 10 mg of NLRP1 B without adjuvant on day 60. Mice were sacrificed on day 63. Splenocytes were fused the with the P3X63-Ag8.653 parental line. Clones were screened via ELISA against recombinant NLRP1 B protein or recombinant FLAG-tagged MBP protein to identify clones specifically reactive to NLRP1 B. Clarified supernatant from the hybridoma clone 2A12 was used for immunoblotting.

In vitro ubiquitylation assay: Recombinant 129 or B6 NLRP1 B was produced in insect cells and purified as described above before 3C treatment. Recombinant lpaH7.8, lpaH7.8 C357A (catalytic mutant), and lpaH9.8 were expressed in BL21 E. coli. Cells (1 liter) were grown to ~0.7 OD600 and induced with 1 M IPTG (Sigma) for 4 hours at 37°C. Pellets were resuspended in 50 mM Tris pH 7.4, 150 mM NaCI, 1% NP-40 and sonicated to lyse. Samples were clarified at 24,000xg for 30 min. The NaCI concentration of the supernatants was increased to 400 mM and 20 mM imidazole pH8.0 was then added to samples. Supernatants were batch bound to 1 ml of Ni resin (Qiagen) at 4°C for 2 hours. Samples were purified by gravity, washed with 50 ml of 20 mM Tris pH 7.4, 400 mM NaCI, and 20 mM imidazole pH 8.0. Protein was eluted in 1 -ml fractions of 20 mM Tris pH 7.4, 150 mM NaCI, and 250mM imidazole pH8.0. Elution peaks were pooled and desalted into 20 mM HEPES pH 7.4, 150 mM NaCI, and 2 mM DTT.

In vitro ubiquitylation assays were performed in 25 mM Tris pH 7.4, 50 mM NaCI, 10 mM MgCI2, 5 mM ATP, O.l mMDTTwith 60 nM Ubiquitin E1 (Boston Biochemistry), 200 nM UbcH5c (Boston Biochemistry), 10 mM Ubiquitin (Boston Biochemistry), 300 nM IpaH, and 270 nM NLRP1 B. The reaction was run for 1 hour at 37°C. Solutions were then batch bound to anti- FLAGM2 agarose gel (Sigma) at 4°C for 2 hours. Bound samples were column-purified and washed with 5 ml of HBS (20 mM Hepes, 150 mM NaCI). Final samples were eluted in 150 mlHBS+150mg/ml of FLAG peptide.

Native gel oligomerization assay: Samples were transfected into 293T cells with constructs as above in a six-well plate. After 24 hours, samples were harvested by removing media and washing cells off plate with cold PBS. Harvested cells were centrifuged at 300xg for 10 min at 4°C. Cells were lysed in lysis buffer (50mM Hepes pH 7.5, 150mM NaCI, 1 % NP-40, 5% glycerol), and samples were clarified by spinning at 16,000xg for 10min at 4°C. Samples were run on NativePAGE Bis-Tris gels (ThermoFisher) according to manufacturer’s protocols.

Detection of UPA-CARD and ASC speck formation by immunofluorescence: 293T cells were grown on fibronectin-coated coverslips. Constructs producing NLRP1 B and ASC were co transfected with constructs producing TEV or an empty vector. The NLRP1 B construct was designed with a C-terminal FLAG and N-terminal HA tag, where the HA sequence was inserted following the P1’ position of the TEV cleavage site. Twenty hours post-transfection, cells were fixed with 4% PFA/PBS (20 min) and permeabilized in 0.5% saponin in PBS (5 min). Blocking and antibody staining was performed at room temperature in 5% BSA/0.1% saponin in PBS. Primary antibodies: 0.5 mg/ml of rabbit anti-ASC (Santa Cruz, N-15), 0.5 mg/ml of rat anti- HA (Roche, 3F10), and 1 mg/ml of mouse anti- FLAG (Sigma, M2). Secondary antibodies: 3 mg/ml of AMCA-labeled goat anti-rabbit IgG (Jackson Laboratories), 4 mg/ml of Alexa Fluor 647- labeled goat anti-rat IgG, and 4 mg/ml of Alexa Fluor 555-labeled goat anti-mouse (Molecular Probes). Coverslips were mounted onto slides using Vectashield medium (Vector Laboratories, Inc., H-1000), and imaged on a ZEISS LSM 710 with a W Plan-Apochromat 40x/1.0 DIC oil immersion objective. Fluorophores were excited at 405, 543, and 633 nM. To quantify the presence of FLAG or HA per ASC speck, the Imaris imaging software (Bitplane) was used to first identify ASC specks as objects, then to count FLAG- and / or HA-positive objects based on a fluorescence intensity threshold set manually for each channel. Thresholds were determined manually using a training set of samples and controls (i.e., no primary antibody) and were applied in batch to all samples.

Example 2: FUND-CARD Biosensors and Screening

FUND-CARD proteins may be employed as biosensors and in screening applications. The preceding example demonstrates that the FUND-CARD module can be activated upon proteasome-mediated degradation. After activation by proteasome-mediated degradation, an active FUND-CARD fragment is released. The native form of this fragment recruits and activates a potent cell death response.

In biosensor and screening applications cell death is employed as a readout of FUND- CARD fragment release and/or the FUND-CARD is engineered to activate alternative/additional reporter activities (e.g., fluorescence). The FUND-CARD module is covalently attached or tethered to bait proteins of interest. Upstream activators that target one or more of the bait proteins for degradation are then sensitively screened by detecting the relevant reporter activity. Upstream activators of interest include E3 ligases or proteases or other molecular entities that result in destabilization of the bait protein. In one example, this system is deployed as a platform to screen PROTAC small molecules that initiate degradation of host target proteins. These examples demonstrate that the FUND-CARD module is a flexible and widely adaptable cell screening platform for the discovery of novel modulators of cellular enzymatic activities.

Functional Degradation

As described above, NLRP1 is an immune sensor that detects a specific bacterial protein called lethal factor (produced by Bacillus anthracis, the causative agent of anthrax). Lethal factor is a protease that activates NLRP1 by cleaving the N-terminus of NLRP1. The basic molecular mechanism that explains how N-terminal cleavage of NLRP1 by LF results in its activation has been termed“Functional Degradation”. This mechanism is depicted in FIG. 8A as described above.

The critical protein domains of NLRP1 that allow it to function as a sensor are its FUND and CARD domains. The FUND domain includes two subdomains called the ZU5 and UPA domains. The FUND domain undergoes a spontaneous autoprocessing event that results in cleavage of the polypeptide chain at a site located near the boundary between the ZU5 and UPA domain. Thus, after autoprocessing, the C-terminus of NLRP1 includes a UPA-CARD fragment that remains non-covalently associated with the rest of the NLPR1 via an interaction with the ZU5 domain. Note that the FUND autoprocessing cleavage event is distinct from cleavage mediated by LF that results in NLRP1 activation. FUND autoprocessing is a maturation step that is necessary by not sufficient for NLRP1 activation.

After FUND auto-processing is complete, NLRP1 can be activated by cleavage at its N terminus by the lethal factor protease. The mechanism is as follows: (1 ) cleavage of NLRP1 results in destabilization/unfolding of NLRP1 ; (2) this results in its recognition by machinery in cells that ubiquitylates destabilized proteins; (3) ubiquitylation of NLRP1 results in its targeting to the proteasome; (4) the proteasome then degrades NLRP1 ; (5) degradation of NLRP1 proceeds processively from its N-terminus to its C-terminus; (6) when the proteasome encounters the covalent break in the polypeptide chain it can no longer continue to processively degrade the rest of the polypeptide and the C-terminal UPA-CARD fragment is released; (7) once released, the UPA-CARD fragment can oligomerize; (8) the oligomerized UPA-CARD fragment is functionally active and recruits and activates a downstream protease called Caspase-1 ; (9) active Caspase-1 has previously been shown to cleave and activate substrates in cells, including a substrate called Gasdermin D that causes cell death. Another bacterial protein, a secreted ubiquitin ligase from Shigella flexneh called lpaH7.8, can also activate NLRP1 as described above. In this case, the bacterial ligase appears to ubiquitylate NLRP1 directly, resulting in targeting to the proteasome and activation of NLRP1 following steps 3-9 above.

NLRP1 FUND-CARD domain module Biosensors

Based on the mechanism employed by NLRP1 to detect LF and lpaH7.8, NLRP1 has been engineered (or‘re-wired’) to serve as a detector of diverse cellular events in cells:

Sensors are constructed of the following general architecture: BAIT-FIIND-CARD. BAIT proteins that are cleaved or modified (or potentially cleaved or modified in screening

applications) by a post-translational modification that leads to thier degradation are employed. Exemplary BAIT proteins employed are those proteins ubiquitylated by an E3 ligase. Other BAIT proteins where proteolytic cleavage, phosphorylation, acetylation, methylation, ampylation or other enzymatic modifications of the BAIT result in its ubiquitylation (or other modification) that results in targeting of the BAIT-FIIND-CARD protein to the proteasome are also suitable.

The UPA-CARD fragment is shown to be a potent activator of the Caspase-1 protease. When Capsase-1 is activated, a variety of cellular outcomes can occur. Most commonly, Caspase-1 cleaves and thereby activates a pore-forming protein called Gasdermin D, resulting in a lytic form of cell death called pyroptosis. Thus, in some embodiments, targeting of BAIT protein to the proteasome is detected by assays that report on cell death. By quantifying or comparing the death that occurs in Caspase-1 ⁺ vs Caspase-1 cells, the BAIT-FIIND-CARD sensor is used to report on specific degradation of the BAIT protein.

Screening to identify E3 liaases that ubiquitylate a specific BAIT protein.

In this example, the BAIT-FIIND-CARD protein is expressed in cells with a functional Caspase-1 pathway, or alternatively, in Caspase-1 cells as a control. The cells are transfected with plasmids for expressing different E3 ligases. E3 ligases that ubiquitylate the BAIT target the BAIT to the proteasome, releasing the UPA-CARD fragment that activates Capsase-1 , killing the target cell. Target cell death is assayed by a standard cell death assay. Specific cell death in cells expressing the BAIT-FIIND-CARD fragment identifies the specific E3 ligase as an E3 ligase that ubiquitylates the BAIT.

The Capsase-1 cells do not undergo cell death and thus serve as a control to ensure that the ligase itself is not toxic to the cell. Additional controls employed include cells expressing a BAIT-FIIND-CARD protein with a mutated FUND domain such that the FUND no longer undergoes autoprocessing. Degradation of this control does not result in release of an active UPA-CARD fragment, Caspase-1 activation, or cell death. An additional negative control employed includes cells expressing the FUND-CARD fragment not fused to a BAIT protein.

Plasmids expressing individual E3 ligases are screened one by one or in multiplex using a library of barcoded E3 ligase expression plasmids, barcoding allows determination of the quantitative presence of an individual E3 ligase in the library by quantitative sequencing. The barcoded library of plasmids is pooled and expressed in the Caspase-1 + or Caspase-1- cells. An individual E3 ligase that activates the BAIT-FIIND-CARD reporter results in death of the cell transfected with that individual E3 ligase and specific depletion of the barcoded plasmid encoding the E3 ligase from the population of Caspase-1 + cells. Thus, the relative quantitative presence of a barcode in Caspase-1 ⁺ versus Capsase-1 cells identifies plasmids that encode an enzyme that induces BAIT degradation and activation of the BAIT-FIIND-CARD reporter.

This system allows for the rapid screening of E3 ligases against BAIT proteins and

quantification of the extent to which each E3 ligase targets each BAIT for degradation.

Screening to identify enzymatic activities that modify a BAIT protein of interest.

The above described screening method is modified by substituting expression and screening of a library of ubiquitin E3 ligases with the expression and screening of a library that encodes proteins harboring enzymatic activities that result in targeting of the BAIT protein to the proteasome for degradation. Non-limiting examples of proteins of interest that are screening include proteins that phosphorylate or acetylate the BAIT protein. Enzymatic activities of proteins screened in this example result in ubiquitylation of the BAIT by endogenous E3 ligases. With the substitution of the library and any necessary modifications related directly thereto, the screening procedure performed is essentially as described in the example above.

Loss of function screening

BAIT-FIIND-CARD proteins constitutively targeted to the proteasome and/or inducibly targeted to the proteasome upon application of some stimulus allow for loss-of-function screening by positively selecting for loss-of-function mutations that prevent BAIT-FIIND-CARD induced cell death. In this example, cells are mutagenized, and then induced to undergo cell death by expression of the BAIT-FIIND-CARD protein (or by inducible activation of the BAIT- FIIND-CARD protein). Surviving cells are identified as harboring a mutation that impairs degradation of the BAIT-FIIND-CARD fragment. As a control, the mutation is introduced into a different BAIT-FIIND-CARD reporter and degradation/activation is evaluated. Mutations specifically affecting the initial BAIT-FIIND-CARD protein in a loss-of-function manner do not impair the degradation/activation of the control BAIT-FIIND-CARD reporter.

Small molecule screening

The above described screening methods, screening for proteins that activate or inhibit activation of a BAIT-FIIND-CARD reporter, are modified for screening molecules that either induce degradation of the BAIT (and thereby activate the reporter) or inhibit degradation of the BAIT (and thereby prevent activation of the reporter). The method employed is similar to the screening examples described above, however, in this example the screened cells are treated with small molecules, e.g., instead of expressing or mutating specific activator proteins within the cells. Individual small molecules or libraries of candidates for binding the BAIT protein and inducing its degradation (thereby activating the BAIT-FIIND-CARD reporter) are employed. Small molecule candidates for inhibiting an E3 ligase or other enzyme that activates a specific BAIT-FIIND-CARD reporter are also employed. An exemplary class of candidate small molecules screened in this assay is PROTACS, which are small molecules that bridge a target (BAIT) protein to a specific E3 ligase.

Screening using alternative readouts of BAIT-FIIND-CARD activity

In the above examples, activation of the BAIT-FIIND-CARD protein results in Capsase- 1 -dependent cell death. Accordingly, cell death, e.g., measured by a cell death assay, provides the readout for BAIT-FIIND-CARD activity. In this example, the above screens are modified to activate alternative reporter activities. Reporters that are activated upon CARD oligomerization are utilized. In one example, split fluorescent protein (FP) fragments that assemble to become fluorescent upon heterodimerization are fused to the C-terminus of the BAIT-FIIND-CARD reporter. Upon BAIT degradation, the UPA-CARD-split FP fragment is released, allowing association of the split FP fragments and the production of a fluorescent signal by the cell. Detection and/or quantification of the emitted fluorescent signal is employed as the readout of BAIT-FIIND-CARD activity.

Screening using alternatives to NLRP1

NLRP1 is not the only FUND-CARD containing protein. For example, human protein CARD8 also contains FUND-CARD domains and may be similarly activated by proteasomal degradation. In addition, other proteins contain FIIND-like domains fused to a CARD-related domain called a death domain. These proteins, including ankyrin-B, PIDD, CARD8, and UNC5CL, may be activated by a similar mechanism. In this example, the FIIND(ZU5+UPA)- CARD or FIIND(ZU5+UPA)-DD of these various alternative proteins are fused to a BAIT target protein. The signaling pathway activated by each of these proteins, according to their respective latent activity domains, is then used as a readout of BAIT targeting to the proteasome.

Alternatively, the employed protein is modified to activate alternative reporter activities, such as those described above.

Notwithstanding the appended claims, the disclosure set forth herein is also defined by the following clauses:

1. A degradation-activated chimeric polypeptide, the polypeptide comprising:

an auto-proteolytic domain comprising an autocleavage site between an N-terminal side and a C-terminal side of the auto-proteolytic domain;

a degradation-targeting domain linked to the N-terminal side of the auto-proteolytic domain; and

a latent activity domain linked to the C-terminal side of the auto-proteolytic domain, wherein the degradation-targeting domain, the latent activity domain or both are heterologous to the auto-proteolytic domain.

2. The polypeptide according to Clause 1 , wherein the N-terminal side of the auto- proteolytic domain comprises a ZU5 subdomain.

3. The polypeptide according to Clauses 1 or 2, wherein the C-terminal side of the auto- proteolytic domain comprises a UPA subdomain.

4. The polypeptide according to any of the preceding clauses, wherein the auto-proteolytic domain is a function-to-find domain (FUND).

5. The polypeptide according to Clause 4, wherein the FUND domain is derived from a protein of the NLRP1 family, a CARD8 protein, an Ankyrin-2 protein, a PIDD1 protein or an UNC5CL protein.

6. The polypeptide according to any of the preceding clauses, wherein the latent activity domain comprises a cell death domain.

7. The polypeptide according to Clause 6, wherein the cell death domain is derived from a NLRP1 family protein, a CARD8 protein, an Ankyrin-2 protein, a PIDD1 protein or an UNC5CL protein.

8. The polypeptide according to Clauses 6 or 7, wherein the cell death domain comprises a caspase activation and recruitment domain (CARD). 9. The polypeptide according to any of the preceding clauses, wherein the latent activity domain comprises a detectable reporter or a portion thereof.

10. The polypeptide according to any of Clauses 1 to 5, wherein the latent activity domain comprises a transcriptional activator or a transcriptional repressor.

1 1 . The polypeptide according to any of Clauses 1 to 5, wherein the latent activity domain comprises an enzyme or a catalytic portion thereof.

12. The polypeptide according to Clause 1 1 , wherein the enzyme is selected from the group consisting of: a nuclease, a recombinase, a SUMOylase, a de-SUMOylase, a ubiquitin ligase, a deubiquitinase, a protease, a kinase, a phosphatase, an acetyltransferase, a deacetylase, a methyltransferase, a demethylase, an AMPylator and a de-AMPylator.

13. The polypeptide according to Clause 12, wherein the nuclease is a site-specific nuclease.

14. The polypeptide according to any of the preceding clauses, wherein the degradation targeting domain is enzymatically modified for degradation.

15. The polypeptide according to Clause 14, wherein the enzymatic modification comprises ubiquitination, proteolytic cleavage, phosphorylation, acetylation, methylation, ampylation, or a combination thereof.

16. The polypeptide according to Clauses 14 or 15, wherein the enzymatic modification induces delivery of the degradation-activated chimeric polypeptide to the proteasome.

17. The polypeptide according to any of the preceding clauses, wherein the degradation targeting domain is recruited for enzymatic modification by a proteolysis-targeting agent comprising a motif that specifically binds the degradation-targeting domain.

18. The polypeptide according to Clause 17, wherein the proteolysis-targeting agent is a proteolysis-targeting chimera (PROTAC).

19. A nucleic acid comprising a sequence encoding the degradation-activated chimeric polypeptide according to any of Clauses 1 to 18.

20. The nucleic acid according to Clause 19, further comprising a promoter operably linked to the sequence encoding the degradation-activated chimeric polypeptide.

21 . The nucleic acid according to Clause 20, wherein the promoter is a constitutive promoter.

22. The nucleic acid according to Clause 20, wherein the promoter is an inducible promoter.

23. A cell comprising the nucleic acid according to any of Clauses 19 to 22.

24. A vector comprising the nucleic acid according to any of Clauses 19 to 22.

25. A cell comprising the vector of Clause 24. 26. A method of modulating an activity in a cell, the method comprising:

expressing in the cell a degradation-activated chimeric polypeptide according to any of Clauses 1 to 18; and

degrading the degradation-activated chimeric polypeptide in an N-terminal to C-terminal direction to activate the latent activity domain, thereby modulating the activity of the cell.

27. The method according to Clause 26, wherein the activity is cell death.

28. The method according to Clause 26, wherein the activity is expression of an

endogenous protein.

29. The method according to Clause 26, wherein the activity is expression of an

heterologous protein.

30. The method according to any of Clauses 26 to 29, wherein the latent activity domain comprises a transcriptional activator or transcriptional repressor and the method further comprises introducing into the cell a nucleic acid comprising a coding sequence operably linked to a regulatory sequence that is responsive to the transcriptional activator or transcriptional repressor.

31 . The method according to Clause 30, wherein the coding sequence encodes a protein selected from the group consisting of: a cell death protein, a therapeutic protein, a disease related protein, a reporter protein, a transcription factor, an enzyme or a combination thereof.

32. A method of screening for an enzyme that enzymatically modifies a protein for degradation, the method comprising:

expressing in a cell a polypeptide comprising:

an auto-proteolytic domain comprising an autocleavage site between an N- terminal side and a C-terminal side of the auto-proteolytic domain;

a latent activity domain linked to the C-terminal side of the auto-proteolytic domain; and

the protein, or a portion thereof, linked to the N-terminal side of the auto- proteolytic domain;

contacting the cell with the enzyme; and

detecting whether the enzyme enzymatically modifies the protein, or portion thereof, to target the protein, or portion thereof, for degradation based on assaying for the activity of the latent activity domain.

33. The method according to Clause 32, wherein contacting the cell with the enzyme comprises expressing the enzyme in the cell. 34. The method according to Clause 32 or 33, wherein the latent activity domain comprises a cell death domain.

35. The method according to Clause 34, wherein the method comprises assaying for cell death caused by activation of the latent activity domain.

36. The method according to any of Clauses 32 to 35, wherein the latent activity domain comprises a detectable reporter or a portion thereof.

37. The method according to Clause 36, wherein the detectable reporter is a fluorescent protein.

38. The method according to Clause 37, wherein the latent activity domain comprises a first portion of a split fluorescent protein and the method comprises assaying for fluorescence resulting from association of a second portion of the split fluorescent protein with the first portion.

39. The method according to any of Clauses 32 to 38, wherein the enzyme is selected from the group consisting of: a ubiquitin ligase, a protease, a kinase, a phosphatase,

acetyltransferase, deacetylase, a methyltransferase, a demethylase, an AMPylator and a de- AMPylator.

40. The method according to any of Clauses 32 to 39, wherein the protein is selected from the group consisting of: a nucleotide-binding domain leucine-rich repeat (NLR) protein or a portion thereof, a caspase recruitment domain-containing protein 8 (CARD8) or a portion thereof, an Ankyrin-2 protein or a portion thereof, a p53-induced death domain-containing protein 1 (PIDD1 ) or a portion thereof, and UNC5C-Like (UNC5CL) protein or a portion thereof.

41 . The method according to Clause 40, wherein the NLR protein or portion thereof is a NLRP1 family protein or a portion thereof.

42. The method according to any of Clauses 32 to 41 , wherein the auto-proteolytic domain is derived from NLRP1 , CARD8, Ankyrin-2, PIDD1 or UNC5CL.

43. The method according to any of Clauses 32 to 42, wherein the latent activity domain is derived from NLRP1 , CARD8, Ankyrin-2, PIDD1 or UNC5CL.

44. The method according to any of Clauses 32 to 43, wherein the protein is heterologous to the auto-proteolytic domain, the latent activity domain or both.

45. The method according to any of Clauses 32 to 44, wherein the protein is a disease related protein.

46. The method according to any of Clauses 32 to 45, wherein the method comprises contacting a plurality of cells expressing the chimeric polypeptide with a plurality of different enzymes. 47. The method according to Clause 46, wherein each different enzyme of the plurality is expressed from a barcoded nucleic acid sequence.

48. The method according to any of Clauses 32 to 47, wherein the method further comprises assaying one or more control cells.

49. The method according to Clause 48, wherein the one or more control cells comprise a control cell that is defective for the activity of the latent activity domain.

50. The method according to Clauses 48 or 49, wherein the one or more control cells comprise a control cell expressing a defective chimeric polypeptide comprising a defective auto- proteolytic domain.

51 . The method according to any of Clauses 48 to 50, wherein the one or more control cells comprise a control cell expressing a polypeptide comprising the auto-proteolytic domain and the latent activity domain linked to the C-terminal side of the auto-proteolytic domain, wherein the polypeptide is not linked to the protein.

52. A method of screening for a mutation that modifies the degradation of a protein, the method comprising:

expressing in a mutagenized cell a polypeptide comprising:

the protein linked to the N-terminal side of the auto-proteolytic domain; and assaying for the activity of the latent activity domain to detect whether the mutation modifies degradation of the protein.

53. The method according to Clause 52, further comprising mutagenizing a cell to produce the mutagenized cell.

54. The method according to Clause 53, wherein the mutagenizing comprises chemical mutagenesis, radiation induced mutagenesis, site-directed mutagenesis or a combination thereof.

55. A method of screening for a degradation-modifying agent, the method comprising:

expressing in a cell a polypeptide comprising:

a latent activity domain linked to the C-terminal side of the auto-proteolytic domain; and a degradation-targeting domain linked to the N-terminal side of the auto- proteolytic domain; and

contacting the cell with a candidate agent; and

assaying for the activity of the latent activity domain to detect whether the candidate agent modifies degradation of the polypeptide.

56. The method according to Clause 55, wherein the candidate agent is selected from the group consisting of: a non-peptide small molecule, a nucleic acid, a peptide and a protein.

57. The method according to Clause 56, wherein the agent is a chimeric peptide or a chimeric protein.

58. The method according to Clause 57, wherein the candidate agent is a proteolysis targeting agent comprising a motif that specifically binds the degradation-targeting domain.

59. The method according to Clause 58, wherein the proteolysis-targeting agent is a proteolysis-targeting chimera (PROTAC).

60. A method of modulating an innate immune response in a subject, the method comprising:

modulating proteasome-mediated degradation of a protein comprising a function-to-find domain (FUND) and a caspase activation and recruitment domain (CARD) to modulate the innate immune response in the subject.

61 . The method according to Clause 60, wherein the modulating comprises enhancing proteasome-mediated degradation of the protein to initiate or increase the innate immune response in the subject.

62. The method according to Clause 61 , wherein enhancing proteasome-mediated degradation of the protein comprises administering to the subject an agent that increases delivery of the protein to the proteasome.

63. The method according to Clause 62, wherein the agent promotes ubiquitination of the protein.

64. The method according to Clauses 62 or 63, wherein the agent is a proteolysis-targeting agent comprising a motif that specifically binds the protein.

65. The method according to Clause 64, wherein the proteolysis-targeting agent is a proteolysis-targeting chimera (PROTAC).

66. The method according to Clauses 62 or 63, wherein the agent causes cleavage within the N-terminal region of the protein.

67. The method according to Clause 66, wherein the cleavage removes at least 10 amino acids 10 from the N-terminus of the protein. 68. The method according to Clauses 62 or 63, wherein the agent enhances ubiquitin ligase activity.

69. The method according to any of Clauses 60 to 68, wherein the subject is in need of initiating or increasing the innate immune response.

70. The method according to Clause 69, wherein the subject has an infection.

71 . The method according to Clauses 69 or 70, wherein the subject has cancer.

72. The method according to any of Clauses 69 to 71 , wherein the subject been exposed to an infectious agent.

73. The method according to Clause 72, wherein the infectious agent comprises Bacillus anthracis.

74. The method according to any of Clauses 61 to 73, wherein the method boosts production or activity of one or more cytokines in the subject.

75. The method according to Clause 74, wherein the one or more cytokines comprise IL-1 b, IL-18 or both.

76. The method according to Clause 60, wherein the modulating comprises inhibiting proteasome-mediated degradation of the protein to prevent or repress the innate immune response in the subject.

77. The method according to Clause 76, wherein inhibiting proteasome-mediated

degradation of the protein comprises administering to the subject a proteasome inhibitor, a ubiquitin ligase inhibitor, a deubiquitinating agent, a N-end rule inhibitor, or a combination thereof.

78. The method according to Clauses 76 or 77, wherein the subject is in need of preventing or repressing the innate immune response.

79. The method according to Clause 78, wherein the subject has an infection.

80. The method according to Clause 79, wherein the infection comprises Bacillus anthracis infection.

81 . The method according to any of Clauses 78 to 80, wherein the subject has inflammation.

82. The method according to Clause 81 , wherein the inflammation comprises chronic inflammation, an autoimmune disease, or both.

83. The method according to any of Clauses 78 to 82, wherein the subject has cancer.

84. The method according to any of Clauses 76 to 83, wherein the method represses production or activity of one or more cytokines in the subject.

85. The method according to Clause 84, wherein the one or more cytokines comprise IL-1 b, IL-18 or both. 86. The method according to any of Clauses 60 to 85, wherein the protein is selected from the group consisting of: a nucleotide-binding domain leucine-rich repeat (NLR) protein, a caspase recruitment domain-containing protein 8 (CARD8), an Ankyrin-2 protein, a p53-induced death domain-containing protein 1 (PIDD1 ), and a UNC5C-like (UNC5CL) protein.

87. The method according to Clause 86, wherein the NLR protein or portion thereof is a protein of the NLRP1 protein family.

88. The method according to any of Clauses 60 to 87, wherein the protein does not comprise a Bacillus anthracis lethal factor (LF) protease cleavage site.

89. The method according to any of Clauses 60 to 88, wherein the subject is a mammal.

90. The method according to any of Clauses 60 to 89, wherein the method further comprises a combination therapy.

91 . The method according to Clause 90, wherein the combination therapy comprises administering to the subject a therapeutic selected from the group consisting of: an antibiotic, an antibody, a vaccine, a receptor decoy, a competitive peptide, a small molecule inhibitor and a combination thereof.

92. The method according to Clause 91 , wherein the antibody is specific for a Bacillus anthracis protein.

93. The method according to Clauses 91 or 92, wherein the small molecule inhibitor is selected from the group consisting of: a Bacillus anthracis protective antigen (PA) dominant negative mutant, a small-molecule oligomerization inhibitor, a Bacillus anthracis PA channel blocker, a pore blocking agent, a furin inhibitor, a Bacillus anthracis edema factor (EF) inhibitor, and a Bacillus anthracis LF inhibitor.

94. A nucleic acid comprising:

an expression cassette comprising:

a sequence encoding an auto-proteolytic domain comprising an autocleavage site between an N-terminal side and a C-terminal side of the auto-proteolytic domain; a promoter operably linked to the sequence encoding the auto-proteolytic domain; and

an upstream cloning site interposed between the promoter and the sequence encoding the auto-proteolytic domain.

95. The nucleic acid according to Clause 94, wherein the nucleic acid further comprises a linker-encoding sequence interposed between the upstream cloning site and the sequence encoding the auto-proteolytic domain. 96. The nucleic acid according to Clauses 94 or 95, wherein the nucleic acid further comprises a sequence encoding a latent activity domain linked to the C-terminal side of the auto-proteolytic domain.

97. The nucleic acid according to Clause 96, wherein the latent activity domain and the auto- proteolytic domain are derived from the same protein.

98. The nucleic acid according to Clause 96, wherein the latent activity domain is

heterologous to the auto-proteolytic domain.

99. The nucleic acid according to any of Clauses 96 to 98, wherein the latent activity domain comprises a cell death domain.

100. The nucleic acid according to any of Clauses 96 to 99, wherein the latent activity domain comprises a detectable reporter or a portion thereof.

101 . The nucleic acid according to any of Clauses 94 to 100, wherein the nucleic acid further comprises a downstream cloning site.

102. The nucleic acid according to Clause 101 , wherein the wherein the nucleic acid further comprises a linker-encoding sequence interposed between the sequence encoding an auto- proteolytic domain and the downstream cloning site.

103. The nucleic acid according to any of Clauses 94 to 102, wherein the expression cassette encodes a single polypeptide.

104. The nucleic acid according to any of Clauses 94 to 103, wherein the expression cassette comprises a single stop codon.

105. The nucleic acid according to any of Clauses 94 to 104, wherein the expression cassette does not comprise a polycistronic facilitating sequence.

106. The nucleic acid according to any of Clauses 94 to 105, wherein the N-terminal side of the auto-proteolytic domain comprises a ZU5 subdomain.

107. The nucleic acid according to any of Clauses 94 to 106, wherein the C-terminal side of the auto-proteolytic domain comprises a UPA subdomain.

108. The nucleic acid according to any of Clauses 94 to 107, wherein the auto-proteolytic domain is a function-to-find domain (FUND).

109. The nucleic acid according to Clause 108, wherein the FUND domain is derived from a NLRP1 B protein, a CARD8 protein, an Ankyrin-2 protein, a PIDD1 protein or an UNC5CL protein.

1 10. The nucleic acid according to any of Clauses 94 to 109, wherein the promoter is a constitutive promoter. 1 1 1. The nucleic acid according to any of Clauses 94 to 109, wherein the promoter is an inducible promoter.

1 12. The nucleic acid according to any of Clauses 94 to 1 1 1 , wherein the upstream cloning site comprises a sequence encoding a degradation-targeting domain.

1 13. A vector comprising the expression cassette according to any of Clauses 94 to 1 12.

1 14. A cell comprising the expression cassette according to any of Clauses 94 to 1 12.

1 15. The cell according to Clause 1 14, wherein the expression cassette is present in a vector.

1 16. A kit comprising the expression cassette according to any of Clauses 94 to 1 12.

1 17. The kit according to Clause 1 16, wherein the expression cassette is present in a vector.

1 18. The kit according to Clauses 1 16 or 1 17, wherein the expression cassette is present in a cell.

1 19. The kit according to any of Clauses 1 16 to 1 18, wherein the kit further comprises one or more cloning reagents.

120. The kit according to Clause 1 19, wherein the one or more cloning reagents are sufficient to introduce an upstream coding sequence into the upstream cloning site in-frame with the sequence encoding the auto-proteolytic domain.

121. The kit according to Clauses 1 19 or 120, wherein the one or more cloning reagents comprises a restriction endonuclease that cleaves within the upstream cloning site.

122. The kit according to any of Clauses 1 19 to 121 , wherein the upstream cloning site comprises an integration site and the one or more cloning reagents comprises an integrase.

123. The kit according to any of Clauses 1 19 to 122, wherein the one or more cloning reagents comprises a ligase.

124. The kit according to any of Clauses 1 19 to 123, wherein the one or more cloning reagents comprises a polymerase.

125. The kit according to any of Clauses 1 16 to 124, wherein the kit further comprises one or more detection reagents.

126. The kit according to Clause 125, wherein the expression cassette comprises a sequence encoding a latent activity domain comprising an enzymatic activity and the one or more detection reagents comprises a substrate for the enzymatic activity.

127. The kit according to Clauses 125 or 126, wherein the expression cassette comprises a sequence encoding a latent activity domain comprising a transcriptional activator or repressor and the one or more detection reagents comprises an encoded reporter responsive to the transcriptional activator or repressor. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Accordingly, the preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions.

Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Moreover, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the claims.

The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims. In the claims, 35 U.S.C. §1 12(f) or 35 U.S.C.

§1 12(6) is expressly defined as being invoked for a limitation in the claim only when the exact phrase "means for" or the exact phrase "step for" is recited at the beginning of such limitation in the claim; if such exact phrase is not used in a limitation in the claim, then 35 U.S.C. § 1 12 (f) or 35 U.S.C. §1 12(6) is not invoked.

Claims

WHAT IS CLAIMED IS:

1. A degradation-activated chimeric polypeptide, the polypeptide comprising:

a latent activity domain linked to the C-terminal side of the auto-proteolytic domain, wherein the degradation-targeting domain, the latent activity domain or both are

heterologous to the auto-proteolytic domain.

2. The polypeptide according to Claim 1 , wherein the N-terminal side of the auto- proteolytic domain comprises a ZU5 subdomain, the C-terminal side of the auto-proteolytic domain comprises a UPA subdomain, or the N-terminal side of the auto-proteolytic domain comprises a ZU5 subdomain and the C-terminal side of the auto-proteolytic domain comprises a UPA subdomain.

3. The polypeptide according to any of the preceding claims, wherein the auto- proteolytic domain is a function-to-find domain (FUND), wherein the latent activity domain comprises a cell death domain, or the auto-proteolytic domain is a function-to-find domain (FUND) and the latent activity domain comprises a cell death domain.

4. The polypeptide according to Claim 3, wherein the FUND domain, the cell death domain, or both are derived from a NLRP1 family protein, a CARD8 protein, an Ankyrin-2 protein, a PIDD1 protein or an UNC5CL protein.

5. The polypeptide according to any of the preceding claims, wherein the latent activity domain comprises a detectable reporter or a portion thereof, a transcriptional activator or a transcriptional repressor, an enzyme or a catalytic portion thereof, or a combination thereof.

6. The polypeptide according to any of the preceding claims, wherein the degradation targeting domain is enzymatically modified for degradation.

7. A nucleic acid, or a cell comprising a nucleic acid, comprising a sequence encoding the degradation-activated chimeric polypeptide according to any of the preceding claims.

8. A method of modulating an activity in a cell, the method comprising: expressing in the cell a degradation-activated chimeric polypeptide according to any of Claims 1 to 6; and

degrading the degradation-activated chimeric polypeptide in an N-terminal to C- terminal direction to activate the latent activity domain, thereby modulating the activity of the cell.

9. The method according to Claim 8, wherein the activity comprises cell death, expression of an endogenous protein, expression of a heterologous protein, or a combination thereof.

10. The method according to Claim 8 or 9, wherein the latent activity domain comprises a transcriptional activator or transcriptional repressor and the method further comprises introducing into the cell a nucleic acid comprising a coding sequence operably linked to a regulatory sequence that is responsive to the transcriptional activator or transcriptional repressor.

1 1 . A method of screening for an enzyme that enzymatically modifies a protein for degradation, the method comprising:

expressing in a cell a polypeptide comprising:

contacting the cell with the enzyme; and

12. A method of screening for a mutation that modifies the degradation of a protein, the method comprising:

expressing in a mutagenized cell a polypeptide comprising: an auto-proteolytic domain comprising an autocleavage site between an N- terminal side and a C-terminal side of the auto-proteolytic domain;

13. A method of screening for a degradation-modifying agent, the method comprising: expressing in a cell a polypeptide comprising:

a degradation-targeting domain linked to the N-terminal side of the auto- proteolytic domain; and

contacting the cell with a candidate agent; and

14. A method of modulating an innate immune response in a subject, the method comprising:

modulating proteasome-mediated degradation of a protein comprising a function-to- find domain (FUND) and a caspase activation and recruitment domain (CARD) to modulate the innate immune response in the subject.

15. A nucleic acid comprising:

an expression cassette comprising:

16. A vector, a cell, or a kit comprising the nucleic acid according to Claim 15.