US20100324267A1

US20100324267A1 - Novel traf family proteins

Info

Publication number: US20100324267A1
Application number: US12/642,723
Authority: US
Inventors: Juan M. Zapata; John C. Reed
Original assignee: Sanford Burnham Prebys Medical Discovery Institute
Current assignee: Sanford Burnham Prebys Medical Discovery Institute
Priority date: 1999-11-05
Filing date: 2009-12-18
Publication date: 2010-12-23
Also published as: US7666607B1; US7208581B1

Abstract

In accordance with the present invention, there are provided novel TRAF-Protein-Binding-Domain polypeptides (TPBDs). The invention also provides nucleic acid molecules encoding TPBDs, vectors containing these nucleic acid molecules and host cells containing the vectors. The invention also provides antibodies that can specifically bind to invention TPBDs. Such TPBDs and/or anti-TPBD antibodies are useful for discovery of drugs that suppress autoimmunity, inflammation, allergy, allograph rejection, sepsis, and other diseases.

Description

This application is a continuation of U.S. application Ser. No. 11/788,741, filed Apr. 20, 2007, which is a divisional of U.S. application Ser. No. 09/706,325, filed Nov. 3, 2000, now U.S. Patent No. 7,208,581, which claims the benefit of U.S. Provisional Application No. 60/287,568, filed Nov. 5, 1999, which was converted from U.S. Ser. No. 09/434,784, filed Nov. 5, 1999, each of which the entire contents are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Field of the Invention

This invention relates generally to the fields of molecular biology and molecular medicine and more specifically to proteins involved in the regulation of immunological response and cell death.
This invention was made with government support under grant number CA69381 awarded by the National Institute of Health. The United States Government has certain rights in this invention.
Tumor Necrosis Factor (TNF) family cytokines play an important role in a wide variety of immunological, allergic, and inflammatory responses. Several members of the TNF family have been identified, including TNFα, Lymphotoxin-α, Lymphotoxin-β, LIGHT, CD27 Ligand (CD27L), CD30L, CD40L, Fas-L, Trail, and others. These molecules are generally produced as Type-II integral membrane proteins on the surface of cells, undergoing subsequent release into the excellular milieu as a result of proteolytic cleavage. Many of the TNF-family cytokines however remain anchored in the plasma membrane, relying on interactions with receptor-bearing cells through cell-cell contact.
The receptors for TNF-family cytokines are equally diverse. All members of the family have a conserved arrangement of cysteines in their extracellular domains, which is one of the criteria for membership in this family. The intracellular cytosolic domain of TNF-family receptors are diverse in their amino acid sequences, but can be broadly classified into two types:

a) those that contain a protein-interaction module known as a Death-Domain (TNFR1, Fas,DR3, DR4, DR5) and those that do not (TNFR2, CD27, CD30, CD40, LTβR, 4B1 and others). Death Domains are responsible for interactions of a subgroup of the TNF-Receptor (TNFR) family with adapter proteins which bind in turn to caspase-family intracellular proteases involved in inducing apoptosis (programmed cell death). However, the Death Domains can also mediate binding to other types of adaptor molecules which bind kinases or other types of signaling molecules rather than proteases.

Those TNFR family members that do not contain a Death Domain in their cytosolic tail rely on a family of intracellular adapter proteins for transducing signals. This family of adaptor proteins is known as the “TNF Receptor Associated Factors” (TRAFs). TRAF-family proteins contain a protein interaction domain known as the TRAF-domain that mediates their binding to the cytosolic domains of TNF-family receptors. The TRAF domain also allows for interactions among TRAF-family members, creating opportunities for homo- and hetero-oligomerization that can have important functional consequences. In humans and mice, six members of the TRAF family have been described, including TRAF1, TRAF2, TRAF3, TRAF4, TRAF5, and TRAF6. The crystal structure of the TRAF domain of TRAF2 has been solved, revealing a trimeric assembly with 3-fold symmetry and demonstrating the presence of a surface pocket on each monomer that accounts for binding to discrete peptidyl motifs found within the cytosolic domains of many members of the TNF-family receptors.
Some TRAF family proteins physically associate with protein kinases and control the activation of these kinases. TRAF2, TRAF5, and TRAF6 for example have been reported to be capable of inducing the activation of kinases involved in activation of: (a) several members of the stress-kinase family, such as the Jun N-terminal kinase (JNK), and (b) phosphorylation of IκB, an inhibitor of the transcription factor NF-κB. In contrast, TRAF1, TRAF3, and TRAF4 do not activate these kinase, and in some contexts, may interfere with kinase activation by other TRAFs. All kinase-activating members of the TRAF family contain additional protein domains (besides the TRAF-domain) which are important for their function as kinase-activators, including a RING domain and sometimes zinc-finger domains.
Gene knock-out studies in mice have demonstrated critical roles for several of the TRAF-family proteins in signal transduction pathways stimulated by TNF-family receptors. Moreover, mutational analyses of the TRAF-binding sites within the cytosolic domains of TNF-family receptors have also provided evidence that interactions of TRAFs with these receptors are critical for many of the biochemical signal-transduction and cellular biological responses induced via TNF-family receptors.
In addition to their involvement in signaling by TNF-family receptors, at least one of the known TRAFs can also participate in signaling mediated by the Interleukin-1 Receptor/Toll family of receptors. TRAF6 interacts with a Death Domain-containing adapter protein (MyD88) and a Death Domain-containing protein kinase (IRAK) which are critical for NF-κB induction by IL-1 receptors. Thus, TRAFs may in some instances participate in signal transduction by other cytokine receptors beside those of the TNFR family. In addition, some TRAFs bind to viral proteins, suggesting that such protein interactions may play a role the mechanisms employed by viruses to either evade immune surveillance mechanisms or in virus-mediated malignant transformation.
A need exists, therefore, to identify novel TRAF family proteins or TRAF protein binding domains. The present invention satisfies this need and provides additional advantages as well.

SUMMARY OF THE INVENTION

In accordance with the present invention, there are provided novel TRAF-Protein-Binding-Domain polypeptides (TPBDs). The invention also provides nucleic acid molecules encoding TPBDs, vectors containing these nucleic acid molecules and host cells containing the vectors. The invention also provides antibodies that can specifically bind to invention TPBDs. Such TPBDs and/or anti-TPBD antibodies are useful for discovery of drugs that suppress autoimmunity, inflammation, allergy, allograph rejection, sepsis, and other diseases.
The present invention also provides a screening assay useful for identifying agents that can effectively alter the association of an invention TPBD with itself or with other proteins. By altering the self-association of TPBD or by altering their interactions with other proteins, an effective agent may increase or decrease the activation of kinases, or modulate cellular pathways that effect apoptosis, cell proliferation, cell adhesion, cell stress responses, B cell immunoglobulin class switching, and the like.
The invention also provides methods of altering the activity of TPBD in a cell, wherein such increased or decreased activity of TPBD can modulate the level of kinase activity or cellular pathways that effect apoptosis, cell proliferation, cell adhesion, cell stress responses, B cell immunoglobulin class switching, and the like. For example, the activity of TPBD in a cell can be increased by introducing into the cell and expressing a nucleic acid sequence encoding this polypeptide or proteins comprising such TPBD. In addition, the activity of TPBD or TPBD-comprising proteins in a cell can be decreased by introducing into the cell and expressing an antisense nucleotide sequence that is complementary to a portion of a nucleic acid molecule encoding the TPBD or TPBD-comprising proteins.
The invention also provides methods for using an agent that can specifically bind TPBD or a nucleotide sequence that can bind to a nucleic acid molecule encoding TPBD to diagnose a pathology that is characterized by an altered level of apoptosis, cell proliferation, cell adhesion, cell stress responses and B cell immunoglobulin class switching due to an increased or decreased level of TPBD in a cell.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a schematic representation of the structure of newly identified TRAF proteins referred to as HAUSP (SEQ ID NO:8)(also known as USP7), SPOP (SEQ ID NO:10) and TRAF 7 (SEQ ID NO:12); and additionally, TRAF 1 and TRAF 2, showing in shaded boxes the relative positions of the indicated protein domains: TRAF domain, protease domain, ring finger, Z-b box, zinc finger, poly-acidic domain, coiled coil, and nuclear localization signal.

FIG. 2 shows amino acid sequences of the TRAF domains of TRAF 7 (SEQ ID NO:25)(also known as KIAA), HAUSP (SEQ ID NO:23)(also known as USP7), and SPOP (SEQ ID NO:24) in alignment with TRAF domains of six other known human TRAF proteins (hT1td through hT6td, SEQ ID NOS:26-31). Dark boxes indicate identical residues between family members. Light boxes indicate structurally related residues.

FIG. 3 shows the predicted amino acid sequence of TRAF7 (SEQ ID NO:32, corresponding to amino acids 16-979 of SEQ ID NO:6). The different protein domains and regions of the protein are indicated: ring finger domain (amino acids 15-55); ZF-B Box domain (amino acids 90-132); coiled coil (amino acids 132-177); coiled coil (amino acids 195-231); two leucine zipper domains (amino acids 197-218 and 222-245); TRAF domain (amino acids 277-403); coiled coil (amino acids 427-446); and two poly-acidic regions (amino acids)(amino acids 868-964).

FIG. 4 shows Northern blot analysis of the mRNA levels of TRAF7, USP7 and SPOP in human tissues. Northern blot analyses were performed using the human 12 lane multiple tissue northern blot (MTN; Clontech; Palo Alto Calif.), as recommended by the manufacturer. The TRAF domains of TRAF7, USP7 and SPOP, as well as actin as a control, were labeled with ³²P-cytidine using a nick translation assay kit.

FIG. 5 shows an analysis of the binding of the invention TRAF-protein-binding-domains (TPBDs) to different members of the TNF receptor family. In vitro binding assay results of the cytosolic domains of selected members of the TNF-R family are presented. GST-Fas(ct), GST-TNF-R2(ct), GST-CD40(ct), GST-LTβR(ct), GST-NGFR(ct) and GST-DR4(ct) immobilized on glutathione-Sepharose were incubated with in vitro translated (³⁵)S-TRAF2,-SPOP, -HAUSP, and -TRAF7 TRAF domains. Alternatively, TRAF domains of TRAF7 or TRAF2 were expressed in 293 cells. Bound TRAF proteins were detected by SDS-PAGE and fluorography.

FIG. 6 shows an analysis of the binding of human TRAF proteins 1-6 and I-TRAF (Rothe et al., Proc. Natl. Acad. Sci. USA 93:8241-8246 (1996)) to TRAF-7, HAUSP and SPOP. GST-TRAF7 (282-435), GST HAUSP (1-213) and SPOP (1-180) proteins immobilized on glutathione-Sepharose were incubated with in vitro translated ³⁵S-TRAF proteins and I-TRAF, as indicated. Bound TRAF proteins were detected by SDS-PAGE and fluorography.

FIG. 7 shows that TPBDs of HAUSP, SPOP and TRAF7 can specifically inhibit the NF-κB activity induced by TRAF containing proteins. In upper panel relative NF-κB activities effected by different TRAF domains are shown. Cells were transfected with control plasmid, pcDNA3-myc-hTRAF6 alone or with Tug of either pcDNA3-myc-HAUSP(1-213) or pcDNA3-myc-SPOP(1-180), together with 0.5 μg pUC13-4xNFκB-luc plasmid and 1 μg pCMV-β-galactosidase plasmid, as indicated. Relative NF-κB activity was assessed by luciferase assays, with normalization for β-galactosidase activity. The results are presented as fold of activation relative to the control.

FIG. 8 shows the effect of TRAF7 and USP7 in regulating NFκB activity induced by TRAF2 and TRAF6 in a mammalian cell line.

FIG. 9 shows the effect of the TRAF domains of TRAF7, USP7 and SPOP in regulating the NFκB activity induced by TNFα or CD40 overexpression.

FIG. 10 shows regulation of NIK induced NFκB activation by TRAF7 and USP7.

FIG. 11 shows the subcellular localization of different TRAF7 deletion mutants.

FIG. 12 shows the subcellular localization of different domains of USP7.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, there are provided novel TRAF-protein-binding-domains (TPBDs) of newly identified TRAF proteins, and fragments thereof. As used herein, an invention TPBD refers to a peptide region that binds to one or more TRAF proteins. Invention TPBDs share sequence homology with the C-TRAF domain of TRAF proteins, and have been found herein to have binding properties similar to those of other known TRAF proteins. This domain corresponds to the portion of the TRAF domain commonly referred to as the C-TRAF or TRAF-C domain (Arch et al., supra.; and Wajant et al., Cytokine Growth Factor Rev. 10:15-26 (1999), each of which is incorporated herein by reference). Previously characterized human TRAF domain proteins have also been found to have N-TRAF domains immediately amino-terminal of the C-TRAF domain.
TRAF proteins were initially identified as a family of proteins that are associated with members of the TNF-receptors family. TRAF proteins are known to influence a variety of cellular processes such as class switching in B cells, apoptosis, cell proliferation, and stress response (Arch et al., Genes Dev. 12:2821-2830 (1998)). These effects on cellular process are thought to occur by TRAF-mediated modulation of NF-κB and/or cJun N-terminal kinase (JNK) activity. TRAF proteins thus represent an important class of intracellular proteins that mediate the signal transduction of cell surface receptors such as TNFR-family members that ultimately influence a variety of cellular processes.
In addition to binding TNF-family receptors, TRAF domain proteins also interact with other TRAF domain proteins. For example, TRAF 1, TRAF 2 and TRAF 3 all have been shown to homo-oligomerize. TRAF 2 has further been demonstrated capable of hetero-oligomerization with TRAF 1 and TRAF 5. As shown herein, TRAF domains of the present invention have all been demonstrated as capable of interacting with other TRAF-domain proteins.
The functions of the TRAF domain containing proteins, generally, supports the role of invention TPBDs and invention TRAF domain proteins in cellular pathways that effect apoptosis, cell proliferation, cell adhesion, cell stress responses and B cell immunoglobulin class switching.
For example, invention TPBDs have been found to associate with other proteins, including proteins comprising TRAF domains. Exemplary TRAF proteins to which invention TPBDs bind are human TRAF1, TRAF2, TRAF3, TRAF4, TRAF5 or TRAF6. As used herein, the term “bind” or “binding” refers to the association of an invention TPBD with another protein relatively specifically and, therefore, can form a bound complex. In particular, the binding of a TPBD to a TRAF protein is sufficiently specific such that the bound complex can form in vivo in a cell or in vitro under suitable condition.
In one embodiment, it has been found that the invention HAUSP TPBD (SEQ ID NOS:8 or 23) binds TRAF1, TRAF2, TRAF3, TRAF4, TRAF5 and TRAF6, and can inhibit the NF-κB activation activity of TRAF2, TRAF5 and TRAF6. In another embodiment, it has been found that the invention SPOP TPBD domain (SEQ ID NOS:10 or 24) binds TRAF1 and TRAF6, and can inhibit the NF-κB activation activity of TRAF6. In yet another embodiment, it has been found that the invention TRAF-7 TPBD (SEQ ID NOS:12 or 25) binds TRAF1, TRAF2, TRAF3, TRAF4, TRAF5 and TRAF6, and can inhibit the NF-κB activation activity of TRAF2 and TRAF6, and can increase the NF-κB activation activity of TRAF5. Furthermore, it has been found that the TRAF domain of TRAF7 and USP7 can inhibit the NF-κB activation induced by TNFα and CD40 overexpression, whereas the TRAF domain of SPOP showed no inhibitory activity.
In an additional embodiment, it was found that TRAF7, the TRAF domain of TRAF7, and the TRAF domain of USP7 (HAUSP) inhibited NIK-induced NFκB activation. Therefore, the invention provides TPBDs that can be used to inhibit NIK-induced NFκB activation.
In another embodiment, invention TPBDs also bind to TNF receptor family proteins. For example, the SPOP TPBD of SEQ ID NO:10 binds TNF-R2, and the TRAF-7 TPBD (SEQ ID NOS:12 or 25) binds to TNF-R2, CD40, lymphotoxin-βR, NGFRp75 and DR4, and the TRAF domain of TRAF7 also binds with itself. Further, TRAF proteins generally, are known to bind to a wide variety of cell-surface receptors, and in particular, with cytokine receptors of the TNF receptor family. Exemplary receptors to which TRAF proteins are known to bind are: TNFR1, TNFR2, CD27, CD30, CD40, 4-1BB, Ox40, LT-βR, Fas, DR3, DR4, DR5, HVEM, LMP-1 and IL-1R. In addition, TRAF proteins have been observed to bind to all members of the TNF receptor family that do not comprise a death domain. Thus, it is contemplated herein that invention TPBDs also bind one or more receptors selected from TNFR1, TNFR2, CD27, CD30, CD40, 4-1BB, Ox40, LT-βR, Fas, DR3, DR4, DR5, HVEM, LMP-1, IL-1R, and members of the TNF receptor family that do not comprise a death domain.
In another embodiment of the invention, invention TPBDs have been found to bind to TRAF-associated proteins. For example, the TRAF-7 TPBD (SEQ ID NOS:12 or 25) binds I-TRAF. Further, TRAF proteins generally are known to interact with numerous TRAF-associated proteins, such as TRADD, FADD, I-TRAF, TRIP, A20, c-IAP1, c-IAP2, Casper, RIP, RIP2, NIK, Peg3, GCK, NIK, ASK1 and IRAK. Thus, it is contemplated herein that TPBDs also bind one or more TRAF-associated proteins selected from TRADD, FADD, I-TRAF, TRIP, A20, c-IAP1, c-IAP2, Casper, RIP, RIP2, NIK, Peg3, GCK, NIK, ASK1 and IRAK.
Structurally, an invention TPBD is characterized as having the sequence: E(X)_17-21LXW(X)₃VXP(X)_15-16L(X)_24-28K(X)_15-16W (SEQ ID NO:19), where X is any amino acid. Alternatively, an invention TRAF domain is characterized as having the sequence: LXWX(X′)XVXP (SEQ ID NO:20) where X is any amino acid and X′ is selected from L and I. Preferably, an invention TRAF domain has the sequence: E(X)_10-13S(X)_6-7LXW(X)₃VXP(x)_10-11S(X)4L(X)_24-28K(X)_9-10F(X)₅WG(X)₃F(X)₁₆D(X)_5-7V (SEQ ID NO:21), where X is any amino acid. More preferably, an invention TRAF domain has the following sequence: E(X)₄(X_A)(X)_5-8SX(X_B)(X)4-5LXWX(X_A)XVXP(X)_10-11S(X_A)(X)₃L(X)_16-18(X_A)(X)_4-6(X_C)(X)₂K(X)_9-10F(X)₅WG(X_D)(X)₂F(X)₅(X_A)X(X_C(X)₇(X_C) DX(X_A)(X)_2-4(X_C)V (SEQ ID NO:22), where X is any amino acid, X_Ais selected from V, L and I; X_Bis selected from P and G; X_Cis selected from D, E, N and Q; and X_Dis selected from Y and F. Most preferably, an invention TRAF domain comprises the sequence SEQ ID NOS:8, 10, 12, 23, 24 or 25.
TRAF domain proteins, generally, are well known in the art to modulate the activity of NF-κB and JNK. In accordance with another embodiment of the invention, invention TPBDs, HAUSP (preferably SEQ ID NOS:8 or 23), SPOP (SEQ ID NOS:10 or 24), and TRAF-7 (SEQ ID NOS:12 or 25), have been found to modulate the activity NF-κB or cJun N-terminal kinase (JNK).
It has also been found that invention TPBDs modulate a variety of cellular pathways. TRAF domain proteins, generally, are well known in the art as modulating the cellular pathways that effect apoptosis, cell proliferation, cell adhesion, cell stress responses and B cell immunoglobulin class switching, and NF-kB and JNK are further known to modulate these pathways. Thus, those of skill in the art will recognize that it is within the scope of the invention that TPBDs modulate one or more cellular pathways that effect apoptosis, cell proliferation, cell adhesion, cell stress responses and B cell immunoglobulin class switching.
Presently preferred TRAF domains of the invention include amino acid sequences that comprise substantially the same protein sequence set forth in SEQ ID NOS:8, 10, 12, 23, 24 and 25, as well as biologically active, modified forms thereof.
In another embodiment, invention TPBDs include proteins comprising TPBD fragments having the sequence SEQ ID NO:19 or SEQ ID NO:20, which retain at least one native biological TPBD activity, such as immunogenicity, the ability to bind to a TNF family receptor, the ability to bind a TRAF domain protein, the ability to bind a TRAF-associated protein, the ability to modulate NF-κB activity or JNK activity, the ability to modulate apoptosis, cell proliferation, cell adhesion, cell stress responses or B cell immunoglobulin class switching, provided the TRAF protein is no longer than 213 amino acids.
Use of the terms “isolated” and/or “purified” in the present specification and claims as a modifier of DNA, RNA, polypeptides or proteins means that the DNA, RNA, polypeptides or proteins so designated have been produced in such form by the hand of man, and thus are separated from their native in vivo cellular environment, and are substantially free of any other species of nucleic acid or protein. As a result of this human intervention, the recombinant DNAs, RNAs, polypeptides and proteins of the invention are useful in ways described herein that the DNAs, RNAs, polypeptides or proteins as they naturally occur are not.
As used herein, “eukaryotic” refers to the variety of species from which an invention TPBD is derived, e.g., yeast, slime mold, plant, insect, nematode, mammal, and the like. A preferred TPBD herein, is mammalian TRAF.
As used herein, “mammalian” refers to the variety of species from which a preferred invention TPBD is derived, e.g., human, rat, mouse, rabbit, monkey, baboon, bovine, porcine, ovine, canine, feline, and the like. A more preferred TPBD herein, is human TRAF.
The term “biologically active” or “functional”, when used herein as a modifier of invention TPBD(s), or polypeptide fragment thereof, refers to a polypeptide that exhibits functional characteristics similar to a TPBD. For example, one biological activity of a TPBD is the ability to bind, preferably in vivo, to a TRAF protein. Such TRAF binding activity can be assayed, for example, using the methods described in the Examples described herein.
Another biological activity of TRAF is the ability to act as an immunogen for the production of polyclonal and monoclonal antibodies that bind specifically to an invention TPBD. Thus, an invention TPBD will encode a polypeptide specifically recognized by an antibody that also specifically recognizes the TPBD having the amino acid sequence SEQ ID NO:19 or SEQ ID NO:20, preferably including an amino acid set forth in SEQ ID NOS:8, 10, 12, 23, 24 or 25. Such immunologic activity may be assayed by any method known to those of skill in the art. For example, a test-TPBD polypeptide can be used to produce antibodies, which are then assayed for their ability to bind to an invention TPBD comprising SEQ ID NO:19 or SEQ ID NO:20, preferably including a sequence set forth in SEQ ID NOS:8, 10, 12, 23, 24 or 25. If the antibody binds to the test-polypeptide and a protein including the sequence SEQ ID NO:19 or SEQ ID NO:20, and preferably also a protein including SEQ ID NOS:8, 10, 12, 23, 24 or 25 with substantially the same affinity, then the polypeptide possesses the requisite immunologic biological activity.
The TPBD containing protein set forth in SEQ ID NO:2 was initially identified as a herpes-associated ubiquitin-specific protease (HAUSP) associated with the nuclear domain of the PML protein and with Herpes simplex virus type 1 protein Vmw110 (Everett et al., EMBO J. 16:566-577 (1997)). The subject application represents the first identification of a portion of this protein as forming a TRAF domain.
The TPBD containing protein set forth in SEQ ID NO:4 was initially identified in serum from a scleroderma patient as having a speckled pattern in nuclei, and was termed a speckle-type POZ protein, or SPOP (Nagai et al., FEBS Lett. 418:23-26 (1997)). The POZ domain of SPOP has an undetermined function, but the protein was found to be widely expressed.
The TPBD containing protein set forth in SEQ ID NO:6 was identified as having a leucine-zipper region, a zinc finger region and a leucine zipper-like K-box region (Nagase et al., DNA Res. 5:355-364 (1998)). Initially named KIAA0898, this protein is also referred to herein as TRAF7.
In addition to the TRAF domain, TRAF7 was also found to have a number of previously unrecognized domains (see FIG. 3). The existence of a RING finger domain (amino acids 15-55) close to the N-terminus of TRAF7 was previously known. TRAF7 is KIAA0898, which was described in the database as a RING finger protein of unknown function. Besides this domain, several other new protein domains were identified in this molecule. The RING finger domain is followed by a ZF-BBox domain (amino acids 90-132) and a coiled coil (amino acids 132-177). These three protein domains form what is denominated the tripartite motif. The tripartite motif has been found in a very restricted list of proteins, including transcription factors, ribonucleoproteins, and oncoproteins and appears to be involved in protein-protein interaction (Borden, Biochem. Cell. Biol. 76:351-358 (1998)). After the tripartite motif, a second coiled coil (amino acids 195-231) is found. Two putative leucine zipper domains are also found in this region of the protein (amino acids 197-218 and 222-245). The TRAF domain (amino acids 277-403) is located after the leucine zippers. The TRAF domain is followed by another coiled coil (amino acids 427-446) and by two regions rich in acidic residues (amino acids 452-577 and 868-964). Therefore, in addition to TRAF domains, including the TRAF domain of TRAF7 (SEQ ID NOS:12 or 25), the invention also provides additional functional domains of TRAF7, including a ZF-BBox domain (amino acids 90-132 of SEQ ID NO:32); coiled coil domains (amino acids 132-177, 195-231 and 427-446 of SEQ ID NO:32); leucine zipper domains (amino acids 197-218 and 222-245 of SEQ ID NO:32); and poly-acidic domains (amino acids 452-577 and 868-964 of SEQ ID NO:32).
In accordance with one embodiment of the invention, it has been found that the invention TPBD (SEQ ID NOS:8 or 23) can bind all known human TRAF proteins (i.e., TRAF proteins 1 through 6), although the TRAF domain of HAUSP (SEQ ID NOS:8 or 23) binds TRAF 3 somewhat weakly, and binds TRAF 6 quite strongly. The invention TPBD of HAUSP (SEQ ID NOS:8 or 23) has also been found to inhibit NF-κB activation mediated by any of TRAF2, TRAF5 or TRAF6. The TPBD of TRAF7 (SEQ ID NOS:12 or 25) also was found to inhibit NFκB activation mediated by TRAF2 or TRAF6 (see Examples).
In another embodiment of the invention, the invention TPBD of SPOP (SEQ ID NOS:10 or 24) shows selectivity in TRAF protein interaction, binding human TRAF 1 and TRAF 6, but not human TRAF proteins 2, 3, 4 or 5. The invention TPBD of SPOP (SEQ ID NOS:10 or 24) has also been found to be specific in TNF-receptor interaction, binding TNF-receptor 2, but not FAS, CD40, lymphotoxin-β receptor, NGF receptor and DR4. The invention TPBD of SPOP (SEQ ID NOS:10 or 24) has also found to inhibit NF-κB activation mediated by TRAF6.
In a further embodiment of the invention, the invention TPBD of TRAF7 (SEQ ID NOS:12 or 25) binds all known human TRAF proteins, but binds TRAF 2 somewhat weakly, and binds TRAF 6 quite strongly. The invention TPBD of SPOP (SEQ ID NOS:10 or 24) also binds numerous members of the TNF-receptor family, including TNF-receptor 2, CD40 (albeit weakly), lymphotoxin-β receptor, NGF receptor and DR4, but not to FAS. The invention TPBD of SPOP (SEQ ID NOS:10 or 24) has also been demonstrated to inhibit NF-κB activation by either TRAF2 or TRAF6.
Those of skill in the art will recognize that numerous residues of the above-described sequences can be substituted with other, chemically, sterically and/or electronically similar residues without substantially altering the biological activity of the resulting receptor species. In addition, larger polypeptide sequences containing substantially the same sequence as amino acids set forth in SEQ ID NOS:8, 10, 12, 23, 24 and 25 therein (e.g., splice variants) are contemplated.
As employed herein, the term “substantially the same amino acid sequence” refers to amino acid sequences having at least about 70% identity with respect to the reference amino acid sequence, and retaining comparable functional and biological activity characteristic of the protein defined by the reference amino acid sequence. Preferably, proteins having “substantially the same amino acid sequence” will have at least about 80%, more preferably 90% amino acid identity with respect to the reference amino acid sequence; with greater than about 95% amino acid sequence identity being especially preferred. It is recognized, however, that polypeptides (or nucleic acids referred to hereinbefore) containing less than the described levels of sequence identity arising as splice variants or that are modified by conservative amino acid substitutions, or by substitution of degenerate codons are also encompassed within the scope of the present invention. Identity of any two amino acid sequences can be determined by those skilled in the art based, for example, on a BLAST 2.0 computer alignment, using default parameters.
The invention TPBDs can be isolated by a variety of methods well-known in the art, e.g., recombinant expression systems described herein, precipitation, gel filtration, ion-exchange, reverse-phase and affinity chromatography, and the like. Other well-known methods are described in Deutscher et al., Guide to Protein Purification: Methods in Enzymology Vol. 182, (Academic Press, (1990)), which is incorporated herein by reference. Alternatively, the isolated polypeptides of the present invention can be obtained using well-known recombinant methods as described, for example, in Sambrook et al., supra., 1989).
An example of the means for preparing the invention TPBD(s) is to express nucleic acids encoding the TPBD in a suitable host cell, such as a bacterial cell, a yeast cell, an amphibian cell (i.e., oocyte), or a mammalian cell, using methods well known in the art, and recovering the expressed polypeptide, again using well-known methods. Invention polypeptides can be isolated directly from cells that have been transformed with expression vectors as described below herein. The invention polypeptide, biologically functional fragments, and functional equivalents thereof can also be produced by chemical synthesis. For example, synthetic polypeptides can be produced using Applied Biosystems, Inc. Model 430A or 431A automatic peptide synthesizer (Foster City, Calif.) employing the chemistry provided by the manufacturer.
Also encompassed by the term TPBD are functional fragments or polypeptide analogs thereof. The term “functional fragment” refers to a peptide fragment that is a portion of a full length TPBD, provided that the portion has a biological activity, as defined above, that is characteristic of the corresponding full length protein. For example, a functional fragment of an invention TPBD can have an activity such as the ability, for example, to bind a TNF-family receptor, to bind another TRAF protein, to bind a TRAF-associated protein, or to modulate NF-κB activity or JNK activity, or to modulate the level of cell proliferation, apoptosis, cell adhesion, cell stress responses, class switching, and the like. In addition, the characteristic of a functional fragment of invention TPBDs to elicit an immune response is useful for obtaining an anti-TPBD antibodies. Thus, the invention also provides functional fragments of invention TPBDs, which can be identified using the binding and routine methods, such as bioassays described herein.
The term “polypeptide analog” includes any polypeptide having an amino acid residue sequence substantially the same as a sequence specifically shown herein in which one or more residues have been conservatively substituted with a functionally similar residue and which displays the ability to functionally mimic a TPBD as described herein. Examples of conservative substitutions include the substitution of one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another, the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine, the substitution of one basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another.
The amino acid length of functional fragments or polypeptide analogs of the present invention can range from about 5 amino acids up to one residue less than a full-length protein sequence of an invention TPBD. In certain embodiments, the amino acid lengths include, for example, at least about 10 amino acids, at least about 20, at least about 30, at least about 40, at least about 50, at least about 75, at least about 100, at least about 150, at least about 200, at least 213, at least about 250, at least about 300, at least about 350 or more amino acids in length up to one residue less than a full-length TPBD-containing protein sequence, provided as sequence SEQ ID NO:2, 4 or 6.
Preferably, a fragment comprises a sequence selected from SEQ ID NO:19, 21 or 22. Such a fragment can also include at least about 10 residues at its amino-terminus, carboxy-terminus, or both; at least about 20 residues at its amino-terminus, carboxy-terminus, or both; at least about 30 residues at its amino-terminus, carboxy-terminus, or both; at least about 40 residues at its amino-terminus, carboxy-terminus, or both; at least about 50 residues at its amino-terminus, carboxy-terminus, or both; at least about 60 residues at its amino-terminus, carboxy-terminus, or both.
More preferably, a fragment comprises a sequence selected from SEQ ID NO:19, 21 or 22, further comprising one or more domains selected from N-TRAF, Ring Finger, Zinc finger, coiled-coil, POZ, and USP. Most preferably, a fragment has at least one fewer domains than the domains in proteins from SEQ ID NO:2, 4 or 6, wherein the domains are selected from N-TRAF, Ring Finger, Zinc finger, coiled-coil, POZ, and USP. Such domains are known in the art, as exemplified in Arch et al., supra.; and Wajant et al., supra. Identification of the domains in proteins from SEQ ID NO:2, 4 and 6 is carried out by reference to the publication reporting such proteins (i.e., Everett et al., supra., Nagai et al., supra., and Nagase et al., supra., for HAUSP (SEQ ID NO:2), SPOP (SEQ ID NO:4) and TRAF7 (SEQ ID NO:6), respectively).
As used herein the phrase “conservative substitution” also includes the use of a chemically derivatized residue in place of a non-derivatized residue, provided that such polypeptide displays the required binding activity. The phrase “chemical derivative” refers to a subject polypeptide having one or more residues chemically derivatized by reaction of a functional side group. Such derivatized molecules include, for example, those molecules in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides. Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine may be derivatized to form N-im-benzylhistidine. Also included as chemical derivatives are those peptides which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids. For example: 4-hydroxyproline may be substituted for proline; 5-hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted for serine; and ornithine may be substituted for lysine. Polypeptides of the present invention also include any polypeptide having one or more additions and/or deletions of residues, relative to the sequence of a polypeptide whose sequence is shown herein, so long as the required activity is maintained.
In accordance with another embodiment, novel TPBD-containing TRAF proteins are provided. Invention TPBD-containing TRAF proteins refer to a protein comprising an invention TPBD including SEQ ID NO:19 or including SEQ ID NO:20, or a recombinantly produced invention TPBD-containing TRAF protein, including naturally occurring allelic variants thereof encoded by mRNA generated by alternative splicing of a primary transcript, provided the TPBD-containing TRAF protein are not the sequence SEQ ID NO:2, 4 or 6. Preferably, a TPBD-containing TRAF protein comprises an invention TPBD with a sequence substantially the same as SEQ ID NO:8, 10, 12, 23, 24, or 25. More preferably, a TPBD-containing TRAF protein comprises an invention TPBD with the sequence of SEQ ID NO:8, 10, 12, 23, 24, or 25.
A TPBD-containing TRAF protein comprising an invention TPBD domain is further characterized as binding one or more members of the TNF-family of receptors, or binding to one or more TRAF domain proteins, or binding to one or more TRAF associated proteins; or modulating NF-kB activity, or modulating cJun N-terminal kinase (JNK) activity; or modulating apoptosis, cell proliferation, cell adhesion, cell stress responses or B cell immunoglobulin class switching; or any combination thereof.
In another embodiment of the invention, TPBD-containing chimeric proteins are provided comprising an invention TPBD, or fragments thereof, having the sequence of SEQ ID NO:19 or SEQ ID NO:20, and further comprising one or more sequences from a heterologous protein. Invention TPBD-containing chimeric proteins include, for example, polypeptides having the sequence SEQ ID NO:8, 10, 12, 23, 24 or 25. Sequences from heterologous proteins with which the TPBD or functional fragment thereof are fused will include, for example, glutathione-S-transferase, an antibody, or other proteins or functional fragments thereof which facilitate recovery of the chimera. Further proteins with which the TPBD or functional fragment thereof are fused will include, for example, luciferase, green fluorescent protein, an antibody, or other proteins or functional fragments thereof which facilitate identification of the chimera. Still further proteins with which the TPBD or functional fragment thereof are fused will include, for example, the LexA DNA binding domain, ricin, α-sarcin, an antibody, or other proteins which have therapeutic properties or other biological activity.
As such chimeric proteins include sequences from two different proteins, the resultant amino acid sequence of the chimeric protein will typically be a non-naturally occurring sequence. Thus, in accordance with this embodiment of the invention, there are provided chimeric proteins comprising an invention TPBD, or fragments thereof, having the sequence of SEQ ID NO:19 or SEQ ID NO:20, provided the sequence of the chimeric protein is not naturally occurring.
Further invention chimeric proteins contemplated herein are chimeric proteins wherein an invention TPBD is combined with one or more domains selected from a Ring-Finger domain, a Zinc-finger domain and an N-TRAF domain from a heterologous protein. For example, the TPBD of SEQ ID NO:8, 10, 12, 23, 24, or 25 can be fused with the Ring-Finger domain of TRAF proteins such as human TRAF 2, 3, 4, 5 or 6, and the like. Another example of such a chimera is a protein wherein the TPBD of SEQ ID NO:8, 10, 12, 23, 24, or 25 is fused with the N-TRAF domain from a TRAF protein such as human TRAF 1, 2, 3, 4, 5 or 6, and the like.
Another embodiment of the invention provides TPBD, or a functional fragment thereof, fused with a moiety to form a conjugate. As used herein, a “moiety” can be a physical, chemical or biological entity which contributes functionality to TPBD or a functional fragment thereof. Functionalities contributed by a moiety include therapeutic or other biological activity, or the ability to facilitate identification or recovery of TPBD. Therefore, a moiety will include molecules known in the art to be useful for detection of the conjugate by, for example, by fluorescence, magnetic imaging, detection of radioactive emission, and the like. A moiety may also be useful for recovery of the conjugate, for example a His tag or other known tags used for protein isolation/purification, or a physical substance such as a bead. A moiety can be a therapeutic compound, for example, a cytotoxic drug which can be useful to effect a biological change in cells to which the conjugate localizes.
In accordance with another embodiment of the invention there are provided oligomers comprising invention TPBDs and fragments thereof, invention TPBD-containing proteins, TPBD-containing chimeric proteins, or combinations thereof. It has been found that a TPBD such as the TPBD of TRAF 7 (SEQ ID NOS:12 or 25) can bind at least one other, equivalent TPBD in forming a homo-oligomer. Thus in one embodiment, the invention comprises homo-oligomers of invention TPBDs and fragments thereof, invention TPBD-containing proteins, TPBD-containing chimeric proteins, or combinations thereof.
In another embodiment of the invention, there are provided hetero-oligomers comprising invention TPBDs and fragments thereof, invention TPBD-containing proteins, TPBD-containing chimeric proteins, or combinations thereof. It was found that invention HAUSP-TPBD (SEQ ID NOS:8 or 23), SPOP-TPBD (SEQ ID NOS:10 or 24) and TRAF7-TPBD (SEQ ID NOS:12 or 25) all bind, for example, multiple TRAF proteins. Thus hetero-oligomers comprising invention TPBDs and fragments thereof, invention TPBD-containing proteins, TPBD-containing chimeric proteins, or combinations thereof, and further comprising TRAF proteins such as human TRAF1, TRAF2, TRAF3, TRAF4, TRAF5, TRAF6, or combinations thereof. For example, the HAUSP-TPBD (SEQ ID NOS:8 or 23) can form a hetero-oligomer with human TRAF1, TRAF2, TRAF3, TRAF4, TRAF5, TRAF6, or combinations thereof. In another example, the SPOP-TPBD (SEQ ID NOS:10 or 24) can form a hetero-oligomer with human TRAF1, TRAF6, or combinations thereof. In a further example, the TRAF7-TPBD (SEQ ID NOS:12 or 25) can form a hetero-oligomer with human TRAF1, TRAF2, TRAF3, TRAF4, TRAF5, TRAF6, or combinations thereof.
In accordance with another embodiment of the invention, there are provided isolated nucleic acids, which encode a novel TPBD, and fragments thereof, TPBD-containing TRAF proteins and TPBD-containing chimeric proteins. Nucleic acids that encode a TPBD are those that encode a protein with the ability to bind, preferably in vivo, to one or more members of the Tumor Necrosis Factor Receptor-family (TNFR-family), or to one or more members of the TRAF family, or to one or more TRAF-associated proteins, or any combination thereof, or have the ability to modulate NF-κB activity, JNK activity, apoptosis, cell proliferation, cell adhesion, cell stress responses or B cell immunoglobulin class switching. An invention nucleic acid encodes a TPBD having the sequence: E(X)_17-21L(X)₂W(X)₃VXP(X)_15-16L(X)_24-28K(X)_15-16W (SEQ ID NO:19), where X is any amino acid. Alternatively, an invention nucleic acid encodes a TPBD having the sequence: LXWX(X′)XVXP (SEQ ID NO:20) where X is any amino acid and X′ is selected from L and I. Preferably, an invention nucleic acid encodes a TPBD having the sequence: E(X)_10-13S(X)_6-7LXW(X)₃VXP(x)_10-11S(X)₄L(X)_24-28K(X)_9-10F(X)₅WG(X)₃F(X)₁₆D(X)_5-7V (SEQ ID NO:21), where X is any amino acid. More preferably, an invention nucleic acid encodes a TPBD having the sequence: E(X)₄(X_A)(X)_5-8SX(X_B)(X)_4-5LXWX(X_A)XVXP(X)_10-11S (X_A)(X)₃L(X)_16-18(X_A)(X)_4-6(X_C)(X)₂K(X)_9-10F(X)₅WG(X_A)(X)₂F(X)₅(X_A)X(X_C)(X)₇(X_C) DX(X_A)(X)_2-4(X_C)V (SEQ ID NO:22), where X is any amino acid, X_Ais selected from V, L and I; X_Bis selected from P and G; X_Cis selected from D, E, N and Q; and X_Dis selected from Y and F. Most preferably, an invention nucleic acid encodes a TPBD comprising the sequence SEQ ID NO:8, 10, 12, 23, 24 or 25.
The nucleic acid molecules described herein are useful for producing invention proteins, when such nucleic acids are incorporated into a variety of protein expression systems known to those of skill in the art. In addition, such nucleic acid molecules or fragments thereof can be labeled with a readily detectable substituent and used as hybridization probes for assaying for the presence and/or amount of an invention TPBD gene or mRNA transcript in a given sample. The nucleic acid molecules described herein, and fragments thereof, are also useful as primers and/or templates in a Polymerase Chain Reacion (PCR) for amplifying genes encoding invention proteins described herein.
The term “nucleic acid” (also referred to as polynucleotides) encompasses ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), probes, oligonucleotides, and primers. DNA can be either complementary DNA (cDNA) or genomic DNA, e.g. a gene encoding a TPBD. One means of isolating a nucleic acid encoding a TPBD is to probe a mammalian genomic library with a natural or artificially designed DNA probe using methods well known in the art. DNA probes derived from the TPBD gene are particularly useful for this purpose. DNA and cDNA molecules that encode TPBDs can be used to obtain complementary genomic DNA, cDNA or RNA from eukaryotic (e.g., human, primate, mammal, plant, nematode, insect, yeast, and the like), or mammalian sources, or to isolate related cDNA or genomic clones by the screening of cDNA or genomic libraries, by methods described in more detail below. Examples of nucleic acids are RNA, cDNA, or isolated genomic DNA encoding a TPBD, provided the nucleic acids do not comprise the nucleotide sequence set forth in SEQ ID NOs:1, 3, or 5. Such nucleic acids may include, but are not limited to, nucleic acids comprising substantially the same nucleotide sequence as set forth in SEQ ID NOs:7, 9, and 11.
In one embodiment of the present invention, cDNAs encoding the invention TPBD disclosed herein comprise substantially the same nucleotide sequence as set forth in SEQ ID NOs:19 or 20, provided they do not comprise the sequence set forth in SEQ ID NO: 1, 3 or 5. Preferably, cDNAs encoding the invention TPBDs disclosed herein comprise substantially the same nucleotide sequence as set forth in any of SEQ ID NOs:7, 9, and 11, provided they do not comprise the sequence set forth in SEQ ID NO: 1, 3 or 5. Preferred cDNA molecules encoding the invention proteins comprise the same nucleotide sequence as set forth in SEQ ID No: 7, 9 and 11.
In another embodiment of the present invention, cDNAs encoding the invention TPBDs disclosed herein comprise substantially the same nucleotide sequence as set forth in SEQ ID NOs:19 or 20, provided they are no longer than 639 bases in length. Preferably, cDNAs encoding the invention TPBDs disclosed herein comprise substantially the same nucleotide sequence as set forth in any of SEQ ID NOs:7, 9, and 11, provided they are not longer than 639 bases in length. Preferred cDNA molecules encoding the invention proteins comprise the same nucleotide sequence as set forth in SEQ ID No: 7, 9 and 11.
cDNA molecules SEQ ID NOs:7, 9 and 11 encoding the invention TRAF domains respectively represent the same nucleotide sequence as nucleotides 1-639 set forth in SEQ ID No:1; nucleotides 1-540 set forth in SEQ ID NO:3; and nucleotides 847-1305 set forth in SEQ ID NO:5.
As employed herein, the term “substantially the same nucleotide sequence” refers to DNA having sufficient identity to the reference polynucleotide, such that it will hybridize to the reference nucleotide under moderately stringent hybridization conditions. In one embodiment, DNA having substantially the same nucleotide sequence as the reference nucleotide sequence encodes substantially the same amino acid sequence as that set forth in any of SEQ ID Nos:8, 10, 12, 23, 24 and 25, provided the DNA does not encode the sequence set forth in SEQ ID NO: 2, 4 or 6. In another embodiment, DNA having “substantially the same nucleotide sequence” as the reference nucleotide sequence has at least 60% identity with respect to the reference nucleotide sequence. DNA having at least 70%, more preferably at least 90%, yet more preferably at least 95%, identity to the reference nucleotide sequence is preferred. Identity of any two nucleic acid sequences can be determined by those skilled in the art based, for example, on a BLAST 2.0 computer alignment, using default parameters. BLAST 2.0 searching is available at http://www.ncbi.nlm.nih.gov/gorf/b12.html., as described by Tatiana et al., FEMS Microbiol Lett. 174:247-250 (1999).
This invention also encompasses nucleic acids which differ from the nucleic acids shown in SEQ ID NOs:7, 9, and 11, but which have the same phenotype. Phenotypically similar nucleic acids are also referred to as “functionally equivalent nucleic acids”. As used herein, the phrase “functionally equivalent nucleic acids” encompasses nucleic acids characterized by slight and non-consequential sequence variations that will function in substantially the same manner to produce the same protein product(s) as the nucleic acids disclosed herein. In particular, functionally equivalent nucleic acids encode polypeptides that are the same as those encoded by the nucleic acids disclosed herein or that have conservative amino acid variations. For example, conservative variations include substitution of a non-polar residue with another non-polar residue, or substitution of a charged residue with a similarly charged residue. These variations include those recognized by skilled artisans as those that do not substantially alter the tertiary structure of the protein.
Further provided are nucleic acids encoding TPBDs that, by virtue of the degeneracy of the genetic code, do not necessarily hybridize to the invention nucleic acids under specified hybridization conditions. Preferred nucleic acids encoding the invention TPBDs are comprised of nucleotides that encode substantially the same amino acid sequence as set forth in SEQ ID NOs:8, 10, and 12, provided they do not encode the sequence set forth in SEQ ID NO: 2, 4 or 6.
Thus, an exemplary nucleic acid encoding an invention TPBD may be selected from:

- (a) DNA encoding the amino acid sequence set forth in SEQ ID NOs:19 or 20
- (b) DNA that hybridizes to the DNA of (a) under moderately stringent conditions, wherein said DNA encodes biologically active TPBD, or
- (c) DNA degenerate with (b), wherein said DNA encodes biologically active TPBD,
  wherein the nucleic acid sequence does not encode an amino acid sequence longer than 213 residues.

Another exemplary nucleic acid encoding an invention TPBD may be selected from:

- (a) DNA encoding the amino acid sequence set forth in SEQ ID NOS:8, 10, 12, 23, 24 and 25,
- (b) DNA that hybridizes to the DNA of (a) under moderately stringent conditions, wherein said DNA encodes biologically active TPBD, or
- (c) DNA degenerate with (b), wherein said DNA encodes biologically active TPBD,
  wherein the nucleic acid sequence does not encode the amino acid sequence set forth in SEQ ID Nos:2, 4 or 6.

Hybridization refers to the binding of complementary strands of nucleic acid (i.e., sense:antisense strands or probe:target-DNA) to each other through hydrogen bonds, similar to the bonds that naturally occur in chromosomal DNA. Stringency levels used to hybridize a given probe with target-DNA can be readily varied by those of skill in the art.
The phrase “stringent hybridization” is used herein to refer to conditions under which polynucleic acid hybrids are stable. As known to those of skill in the art, the stability of hybrids is reflected in the melting temperature (T_m) of the hybrids. In general, the stability of a hybrid is a function of sodium ion concentration and temperature. Typically, the hybridization reaction is performed under conditions of lower stringency, followed by washes of varying, but higher, stringency. Reference to hybridization stringency relates to such washing conditions.
As used herein, the phrase “moderately stringent hybridization” refers to conditions that permit target-DNA to bind a complementary nucleic acid that has about 60% identity, preferably about 75% identity, more preferably about 85% identity to the target DNA; with greater than about 90% identity to target-DNA being especially preferred. Preferably, moderately stringent conditions are conditions equivalent to hybridization in 50% formamide, 5× Denhart's solution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.2×SSPE, 0.2% SDS, at 42° C.
The phrase “high stringency hybridization” refers to conditions that permit hybridization of only those nucleic acid sequences that form stable hybrids in 0.018M NaCl at 65° C. (i.e., if a hybrid is not stable in 0.018M NaCl at 65° C., it will not be stable under high stringency conditions, as contemplated herein). High stringency conditions can be provided, for example, by hybridization in 50% formamide, 5× Denhart's solution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.1×SSPE, and 0.1% SDS at 65° C.
The phrase “low stringency hybridization” refers to conditions equivalent to hybridization in 10% formamide, 5× Denhart's solution, 6×SSPE, 0.2% SDS at 42° C., followed by washing in 1×SSPE, 0.2% SDS, at 50° C. Denhart's solution and SSPE (see, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989) are well known to those of skill in the art as are other suitable hybridization buffers.
As used herein, the term “degenerate” refers to codons that differ in at least one nucleotide from a reference nucleic acid, e.g., SEQ ID NOs:7, 9, and 11, but encode the same amino acids as the reference nucleic acid. For example, codons specified by the triplets “UCU”, “UCC”, “UCA”, and “UCG” are degenerate with respect to each other since all four of these codons encode the amino acid serine.
Preferred nucleic acids encoding the invention polypeptide(s) hybridize under moderately stringent, preferably high stringency, conditions to substantially the entire sequence, or substantial portions (i.e., typically at least 15-30 nucleotides) of the nucleic acid sequence set forth in SEQ ID NOs:7, 9, and 11, provided they do not comprise the sequence set forth in SEQ ID NO: 1, 3 or 5.
The invention nucleic acids can be produced by a variety of methods well-known in the art, e.g., the methods described herein, employing PCR amplification using oligonucleotide primers from various regions of SEQ ID NOs:7, 9, and 11, and the like.
In accordance with a further embodiment of the present invention, optionally labeled TRAF-encoding cDNAs, or fragments thereof, can be employed to probe library(ies) (e.g., cDNA, genomic, and the like) for additional nucleic acid sequences encoding novel eukaryotic TPBDs. Construction of suitable eukaryotic cDNA libraries is well-known in the art. Screening of such a cDNA library is initially carried out under low-stringency conditions, which comprise a temperature of less than about 42° C., a formamide concentration of less than about 50%, and a moderate to low salt concentration.
Presently preferred probe-based screening conditions comprise a temperature of about 37° C., a formamide concentration of about 20%, and a salt concentration of about 5× standard saline citrate (SSC; 20×SSC contains 3M sodium chloride, 0.3M sodium citrate, pH 7.0). Such conditions will allow the identification of sequences which have a substantial degree of similarity with the probe sequence, without requiring perfect homology. The phrase “substantial similarity” refers to sequences which share at least 50% homology. Preferably, hybridization conditions will be selected which allow the identification of sequences having at least 70% homology with the probe, while discriminating against sequences which have a lower degree of homology with the probe. As a result, nucleic acids having substantially the same nucleotide sequence as SEQ ID NOs:1, 3, and 5, are obtained.
As used herein, a nucleic acid “probe” is single-stranded DNA or RNA, or analogs thereof, that has a sequence of nucleotides that includes at least 14, at least 20, at least 50, at least 100, at least 200, at least 300, at least 400, or at least 500 contiguous bases that are the same as (or the complement of) any contiguous bases set forth in any of SEQ ID NOs:7, 9, and 11. Preferred regions from which to construct probes include 5′ and/or 3′ coding regions of SEQ ID NOs:7, 9, and 11. In addition, the entire cDNA encoding region of an invention TRAF, or the entire sequence corresponding to SEQ ID NOs:7, 9, and 11, may be used as a probe. Probes may be labeled by methods well-known in the art, as described hereinafter, and used in various diagnostic kits.
As used herein, the terms “label” and “indicating means” in their various grammatical forms refer to single atoms and molecules that are either directly or indirectly involved in the production of a detectable signal. Any label or indicating means can be linked to invention nucleic acid probes, expressed proteins, polypeptide fragments, or antibody molecules. These atoms or molecules can be used alone or in conjunction with additional reagents. Such labels are themselves well-known in clinical diagnostic chemistry.
The labeling means can be a fluorescent labeling agent that chemically binds to antibodies or antigens without denaturation to form a fluorochrome (dye) that is a useful immunofluorescent tracer. A description of immunofluorescent analytic techniques is found in DeLuca, “Immunofluorescence Analysis”, in Antibody As a Tool, Marchalonis et al., eds., John Wiley & Sons, Ltd., pp. 189-231 (1982), which is incorporated herein by reference.
In one embodiment, the indicating group is an enzyme, such as horseradish peroxidase (HRP), glucose oxidase, and the like. In another embodiment, radioactive elements are employed labeling agents. The linking of a label to a substrate, i.e., labeling of nucleic acid probes, antibodies, polypeptides, and proteins, is well known in the art. For instance, an invention antibody can be labeled by metabolic incorporation of radiolabeled amino acids provided in the culture medium. See, for example, Galfre et al., Meth. Enzymol., 73:3-46 (1981). Conventional means of protein conjugation or coupling by activated functional groups are particularly applicable. See, for example, Aurameas et al., Scand. J. Immunol., Vol. 8, Suppl. 7:7-23 (1978), Rodwell et al., Biotech., 3:889-894 (1984), and U.S. Pat. No. 4,493,795.
In another embodiment of the invention, nucleic acids are provided encoding chimeric proteins comprising an invention TPBD, or fragment thereof, having the sequence of SEQ ID NO:19 or SEQ ID NO:20, and further comprising one or more sequences from a heterologous protein. Functional fragments of TPBD include, for example, polypeptides having the sequence SEQ ID NO:8, 10, 12, 23, 24 or 25. Nucleic acids encoding proteins with which the TPBD or functional fragment thereof are fused will also encode, for example, glutathione-S-transferase, an antibody, or other proteins or functional fragments thereof which facilitate recovery of the chimera. Nucleic acids of the invention can also encode proteins with which the TPBD or functional fragment thereof are fused, for example, luciferase, green fluorescent protein, an antibody, or other proteins or functional fragments thereof which facilitate identification of the chimera. Still further nucleic acids of the invention encode proteins with which the TPBD or functional fragment thereof are fused including, for example, the LexA DNA binding domain, ricin, α-sarcin, an antibody, or other proteins which have therapeutic properties or other biological activity.
The present invention also provides compositions containing an acceptable carrier and any of an isolated, purified TPBD-containing protein or functional polypeptide fragments thereof, alone or in combination with each other. These polypeptides or proteins can be recombinantly derived, chemically synthesized or purified from native sources. As used herein, the term “acceptable carrier” encompasses any of the standard pharmaceutical carriers, such as phosphate buffered saline solution, water and emulsions such as an oil/water or water/oil emulsion, and various types of wetting agents.
The TPBD compositions described herein can be used, for example, in methods for modulating the activity of members of the TNFR family. TNF family receptor binding is well known in the art as mediating the signal transduction activity of the receptor, and it is demonstrated herein that invention TPBDs can bind TNF receptors. Thus it is within the scope of the present invention that a protein comprising the sequence SEQ ID NO:19 or 20, or a nucleic acid encoding a protein comprising the sequence SEQ ID NO:19 or 20, modulates the activity of one or more TNF family receptors.
In one embodiment, modulation of a member of the TNFR family will comprise the step of contacting a member of the TNFR family with a protein comprising the sequence SEQ ID NO:19 or 20. Preferably, the method comprises contacting a cell with a protein comprising the sequence of SEQ ID NO:8, 10, 12, 23, 24 or 25.
In another embodiment, modulation of a member of the TNFR family will comprise the step of contacting a member of the TNFR family with a nucleic acid encoding a protein comprising the sequence SEQ ID NO:19 or 20. Preferably, the method comprises contacting a cell with a nucleic acid encoding a protein comprising the sequence of SEQ ID NO:8, 10, 12, 23, 24 or 25.
In another embodiment, the TPBD compositions described herein can be used, for example, in methods for modulating the activity of TRAF domain containing proteins. Thus it is within the scope of the present invention that a protein comprising the sequence SEQ ID NO:19 or 20, or a nucleic acid encoding a protein comprising the sequence SEQ ID NO:19 or 20, modulates the activity of one or more TRAF domain containing proteins.
In one embodiment, modulation of a TRAF domain containing protein will comprise the step of contacting a TRAF domain containing protein with a protein comprising the sequence SEQ ID NO:19 or 20. Preferably, the method comprises contacting a cell with a protein comprising the sequence of SEQ ID NO:8, 10, 12, 23, 24 or 25.
In another embodiment, modulation of a TRAF domain containing protein will comprise the step of contacting a TRAF domain containing protein with a nucleic acid encoding a protein comprising the sequence SEQ ID NO:19 or 20. Preferably, the method comprises contacting a cell with a nucleic acid encoding a protein comprising the sequence of SEQ ID NO:8, 10, 12, 23, 24 or 25.
In another embodiment, a TPBD comprising the sequence SEQ ID NO:19 or 20, or a nucleic acid encoding a protein comprising the sequence SEQ ID NO:19 or 20, modulates the activity of one or more TRAF-associated proteins. While some TRAF-associated proteins are known to modulate NF-κB activity, others are known to modulate cJun N-terminal kinase (JNK) activity, and still others are known to modulate the activity of other proteins. For example, c-IAP1 and c-IAP2 modulate caspase activity and thus influence apoptosis. Thus it is within the scope of the invention that an invention TRAF domain protein can modulate the activity of TRAF-associated proteins any protein with which TRAF-associated proteins are known to interact.
In one embodiment, modulation of a TRAF-associated protein will comprise the step of contacting a TRAF-associated protein with a protein comprising the sequence SEQ ID NO:19 or 20. Preferably, the method comprises contacting a cell with a protein comprising the sequence of SEQ ID NO:8, 10, 12, 23, 24 or 25.
In another embodiment, modulation of a TRAF-associated protein will comprise the step of contacting a TRAF-associated protein with a nucleic acid encoding a protein comprising the sequence SEQ ID NO:19 or 20. Preferably, the method comprises contacting a cell with a nucleic acid encoding a protein comprising the sequence of SEQ ID NO:8, 10, 12, 23, 24 or 25.
TPBD compositions can also be used, for example, in methods for modulating the activity of NF-κB and cJun N-terminal kinase (JNK). Proteins homologous to invention TPBDs, for example, human TRAF domain proteins, are well known in the art as modulating the activity of NF-κB and JNK, and it is further shown herein that SEQ ID NOS:8, 10, 12, 23, 24 and 25 can modulate NF-κB activity. Thus, in accordance with another embodiment of the invention, a protein comprising the sequence SEQ ID NO:19 or 20, or a nucleic acid encoding a protein comprising the sequence SEQ ID NO:19 or 20, modulates the activity of NF-κB or JNK.
In one embodiment, modulation of NF-κB or JNK activity will comprise the step of contacting a cell containing NF-κB or JNK activity with a protein comprising the sequence SEQ ID NO:19 or 20. Preferably, the method comprises contacting a cell with a protein comprising the sequence of SEQ ID NO:8, 10, 12, 23, 24 or 25.
In another embodiment, modulation of NF-κB or JNK activity will comprise the step of contacting a cell containing NF-κB or JNK activity with a nucleic acid encoding a protein comprising the sequence SEQ ID NO:19 or 20. Preferably, the method comprises contacting a cell with a nucleic acid encoding a protein comprising the sequence of SEQ ID NO:8, 10, 12, 23, 24 or 25.
The functions of the invention TPBDs support the role of TRAFs in modulating cellular pathways that effect apoptosis, cell proliferation, cell adhesion, cell stress responses and B cell immunoglobulin class switching. Thus, in accordance with another embodiment of the invention, a protein comprising the sequence SEQ ID NO:19 or 20, or a nucleic acid encoding a protein comprising the sequence SEQ ID NO:19 or 20, modulates apoptosis, cell proliferation, cell adhesion, cell stress responses or B cell immunoglobulin class switching.
In one embodiment, modulation of apoptosis, cell proliferation, cell adhesion, cell stress responses or B cell immunoglobulin class switching will comprise the step of contacting a cell with a protein comprising the sequence SEQ ID NO:19 or 20, whereby apoptosis, cell proliferation, cell adhesion, cell stress responses or B cell immunoglobulin class switching is modulated. Preferably, the method comprises contacting a cell with a protein comprising the sequence of SEQ ID NO:8, 10, 12, 23, 24 or 25.
In another embodiment, modulation of apoptosis, cell proliferation, cell adhesion, cell stress responses or B cell immunoglobulin class switching will comprise the step of contacting a cell with a nucleic acid encoding a protein comprising the sequence SEQ ID NO:19 or 20, whereby apoptosis, cell proliferation, cell adhesion, cell stress responses or B cell immunoglobulin class switching is modulated. Preferably, the method comprises contacting a cell with a nucleic acid encoding a protein comprising the sequence of SEQ ID NO:8, 10, 12, 23, 24 or 25.
Also provided are antisense-nucleic acids having a sequence capable of binding specifically with full-length or any portion of an mRNA that encodes TPBD polypeptides so as to prevent translation of the mRNA. The antisense-nucleic acid may have a sequence capable of binding specifically with any portion of the sequence of the cDNA encoding TPBD polypeptides. As used herein, the phrase “binding specifically” encompasses the ability of a nucleic acid sequence to recognize a complementary nucleic acid sequence and to form double-helical segments therewith via the formation of hydrogen bonds between the complementary base pairs. An example of an antisense-nucleic acid is an antisense-nucleic acid comprising chemical analogs of nucleotides.
Compositions comprising an amount of the antisense-nucleic acid, described above, effective to reduce expression of TPBD polypeptides by passing through a cell membrane and binding specifically with mRNA encoding TPBD polypeptides so as to prevent translation and an acceptable hydrophobic carrier capable of passing through a cell membrane are also provided herein. Suitable hydrophobic carriers are described, for example, in U.S. Pat. Nos. 5,334,761; 4,889,953; 4,897,355, and the like. The acceptable hydrophobic carrier capable of passing through cell membranes may also comprise a structure which binds to a receptor specific for a selected cell type and is thereby taken up by cells of the selected cell type. The structure may be part of a protein known to bind to a cell-type specific receptor.
Antisense-nucleic acid compositions are useful to inhibit translation of mRNA encoding invention polypeptides. Synthetic oligonucleotides, or other antisense chemical structures are designed to bind to mRNA encoding TPBD polypeptides and inhibit translation of mRNA and are useful as compositions to inhibit expression of TPBD associated genes in a tissue sample or in a subject.
In accordance with another embodiment of the invention, kits for detecting mutations, duplications, deletions, rearrangements and aneuploidies in TPBD genes comprising at least one invention probe or antisense nucleotide.
The present invention provides means to modulate levels of expression of TPBD polypeptides by employing synthetic antisense-nucleic acid compositions (hereinafter SANC) which inhibit translation of mRNA encoding these polypeptides. Synthetic oligonucleotides, or other antisense-nucleic acid chemical structures designed to recognize and selectively bind to mRNA, are constructed to be complementary to full-length or portions of a TPBD coding strand, including nucleotide sequences set forth in SEQ ID NOs:7, 9, and 11. The SANC is designed to be stable in the blood stream for administration to a subject by injection, or in laboratory cell culture conditions. The SANC is designed to be capable of passing through the cell membrane in order to enter the cytoplasm of the cell by virtue of physical and chemical properties of the SANC which render it capable of passing through cell membranes, for example, by designing small, hydrophobic SANC chemical structures, or by virtue of specific transport systems in the cell which recognize and transport the SANC into the cell. In addition, the SANC can be designed for administration only to certain selected cell populations by targeting the SANC to be recognized by specific cellular uptake mechanisms which bind and take up the SANC only within select cell populations. In a particular embodiment the SANC is an antisense oligonucleotide.
For example, the SANC may be designed to bind to a receptor found only in a certain cell type, as discussed supra. The SANC is also designed to recognize and selectively bind to target mRNA sequence, which may correspond to a sequence contained within the sequences shown in SEQ ID NOs:7, 9, and 11. The SANC is designed to inactivate target mRNA sequence by either binding thereto and inducing degradation of the mRNA by, for example, RNase I digestion, or inhibiting translation of mRNA target sequence by interfering with the binding of translation-regulating factors or ribosomes, or inclusion of other chemical structures, such as ribozyme sequences or reactive chemical groups which either degrade or chemically modify the target mRNA. SANCs have been shown to be capable of such properties when directed against mRNA targets (see Cohen et al., TIPS, 10:435 (1989) and Weintraub, Sci. American, January (1990), pp.40; both incorporated herein by reference).
In accordance with yet another embodiment of the present invention, there is provided a method for the recombinant production of invention TPBDs by expressing the above-described nucleic acid sequences in suitable host cells. Recombinant DNA expression systems that are suitable to produce TPBDs described herein are well-known in the art. For example, the above-described nucleotide sequences can be incorporated into vectors for further manipulation. As used herein, vector (or plasmid) refers to discrete elements that are used to introduce heterologous DNA into cells for either expression or replication thereof.
Suitable expression vectors are well-known in the art, and include vectors capable of expressing DNA operatively linked to a regulatory sequence, such as a promoter region that is capable of regulating expression of such DNA. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector that, upon introduction into an appropriate host cell, results in expression of the inserted DNA. Appropriate expression vectors are well known to those of skill in the art and include those that are replicable in eukaryotic cells and/or prokaryotic cells and those that remain episomal or those which integrate into the host cell genome.
As used herein, a promoter region refers to a segment of DNA that controls transcription of DNA to which it is operatively linked. The promoter region includes specific sequences that are sufficient for RNA polymerase recognition, binding and transcription initiation. In addition, the promoter region includes sequences that modulate this recognition, binding and transcription initiation activity of RNA polymerase. These sequences may be cis acting or may be responsive to trans acting factors. Promoters, depending upon the nature of the regulation, may be constitutive or regulated. Exemplary promoters contemplated for use in the practice of the present invention include the SV40 early promoter, the cytomegalovirus (CMV) promoter, the mouse mammary tumor virus (MMTV) steroid-inducible promoter, Moloney murine leukemia virus (MMLV) promoter, and the like.
As used herein, the term “operatively linked” refers to the functional relationship of DNA with regulatory and effector nucleotide sequences, such as promoters, enhancers, transcriptional and translational stop sites, and other signal sequences. For example, operative linkage of DNA to a promoter refers to the physical and functional relationship between the DNA and the promoter such that the transcription of such DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds to and transcribes the DNA.
As used herein, expression refers to the process by which polynucleic acids are transcribed into mRNA and translated into peptides, polypeptides, or proteins. If the polynucleic acid is derived from genomic DNA, expression may, if an appropriate eukaryotic host cell or organism is selected, include splicing of the mRNA.
Prokaryotic transformation vectors are well-known in the art and include pBlueskript and phage Lambda ZAP vectors (Stratagene, La Jolla, Calif.), and the like. Other suitable vectors and promoters are disclosed in detail in U.S. Pat. No. 4,798,885, issued Jan. 17, 1989, the disclosure of which is incorporated herein by reference in its entirety.
Other suitable vectors for transformation of E. coli cells include the pET expression vectors (Novagen, see U.S. Pat. No. 4,952,496), e.g., pET11a, which contains the T7 promoter, T7 terminator, the inducible E. coli lac operator, and the lac repressor gene; and pET 12a-c, which contain the T7 promoter, T7 terminator, and the E. coli ompT secretion signal. Another suitable vector is the pIN-IIIompA2 (see Duffaud et al., Meth. in Enzymology, 153:492-507, 1987), which contains the 1 pp promoter, the lacUV5 promoter operator, the ompA secretion signal, and the lac repressor gene.
Exemplary, eukaryotic transformation vectors, include the cloned bovine papilloma virus genome, the cloned genomes of the murine retroviruses, and eukaryotic cassettes, such as the pSV-2 gpt system (described by Mulligan and Berg, Nature Vol. 277:108-114 (1979)) the Okayama-Berg cloning system (Mol. Cell Biol. 2:161-170 (1982)), and the expression cloning vector described by Genetics Institute (Wong et al., Science 228:810-815 (1985)), are available which provide substantial assurance of at least some expression of the protein of interest in the transformed eukaryotic cell line.
Particularly preferred base vectors which contain regulatory elements that can be linked to the invention TPBD-encoding DNAs for transfection of mammalian cells are cytomegalovirus (CMV) promoter-based vectors such as pcDNA1 (Invitrogen, San Diego, Calif.), MMTV promoter-based vectors such as pMAMNeo (Clontech, Palo Alto, Calif,.) and pMSG (Pharmacia, Piscataway, N.J.), and SV40 promoter-based vectors such as pSVβ (Clontech, Palo Alto, Calif.).
In accordance with another embodiment of the present invention, there are provided “recombinant cells” containing the nucleic acid molecules (i.e., DNA or mRNA) of the present invention. Methods of transforming suitable host cells, preferably bacterial cells, and more preferably E. coli cells, as well as methods applicable for culturing said cells containing a gene encoding a heterologous protein, are generally known in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2 ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (1989).
Exemplary methods of transformation include, e.g., transformation employing plasmids, viral, or bacterial phage vectors, transfection, electroporation, lipofection, and the like. The heterologous DNA can optionally include sequences which allow for its extrachromosomal maintenance, or said heterologous DNA can be caused to integrate into the genome of the host (as an alternative means to ensure stable maintenance in the host).
Host organisms contemplated for use in the practice of the present invention include those organisms in which recombinant production of heterologous proteins has been carried out. Examples of such host organisms include bacteria (e.g., E. coli), yeast (e.g., Saccharomyces cerevisiae, Candida tropicalis, Hansenula polymorpha and P. pastoris; see, e.g., U.S. Pat. Nos. 4,882,279, 4,837,148, 4,929,555 and 4,855,231), mammalian cells (e.g., HEK293, CHO and Ltk⁻ cells), insect cells, and the like. Presently preferred host organisms are bacteria. The most preferred bacteria is E. coli.
In one embodiment, nucleic acids encoding the invention TPBDs can be delivered into mammalian cells, either in vivo or in vitro using suitable viral vectors well-known in the art. Suitable retroviral vectors, designed specifically for “gene therapy” methods, are described, for example, in WIPO publications WO 9205266 and WO 9214829, which provide a description of methods for efficiently introducing nucleic acids into human cells. In addition, where it is desirable to limit or reduce the in vivo expression of the invention TRAF, the introduction of the antisense strand of the invention nucleic acid is contemplated.
Viral based systems provide the advantage of being able to introduce relatively high levels of the heterologous nucleic acid into a variety of cells. Suitable viral vectors for introducing invention nucleic acid encoding an TPBD into mammalian cells (e.g., vascular tissue segments) are well known in the art. These viral vectors include, for example, Herpes simplex virus vectors (e.g., Geller et al., Science, 241:1667-1669 (1988)), Vaccinia virus vectors (e.g., Piccini et al., Meth. in Enzymology, 153:545-563 (1987); Cytomegalovirus vectors (Mocarski et al., in Viral Vectors, Y. Gluzman and S. H. Hughes, Eds., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1988, pp. 78-84), Moloney murine leukemia virus vectors (Danos et al., PNAS, USA, 85:6469 (1980)), adenovirus vectors (e.g., Logan et al., PNAS, USA, 81:3655-3659 (1984); Jones et al., Cell, 17:683-689 (1979); Berkner, Biotechniques, 6:616-626 (1988); Cotten et al., PNAS, USA, 89:6094-6098 (1992); Graham et al., Meth. Mol. Biol., 7:109-127 (1991)), adeno-associated virus vectors, retrovirus vectors (see, e.g., U.S. Pat. Nos. 4,405,712 and 4,650,764), and the like. Especially preferred viral vectors are the adenovirus and retroviral vectors.
For example, in one embodiment of the present invention, adenovirus-transferrin/polylysine-DNA (TfAdpl-DNA) vector complexes (Wagner et al., PNAS, USA, 89:6099-6103 (1992); Curiel et al., Hum. Gene Ther., 3:147-154 (1992); Gao et al., Hum. Gene Ther., 4:14-24 (1993)) are employed to transduce mammalian cells with heterologous TPBD nucleic acid. Any of the plasmid expression vectors described herein may be employed in a TfAdpl-DNA complex.
As used herein, “retroviral vector” refers to the well-known gene transfer plasmids that have an expression cassette encoding an heterologous gene residing between two retroviral LTRs. Retroviral vectors typically contain appropriate packaging signals that enable the retroviral vector, or RNA transcribed using the retroviral vector as a template, to be packaged into a viral virion in an appropriate packaging cell line (see, e.g., U.S. Pat. No. 4,650,764).
Suitable retroviral vectors for use herein are described, for example, in U.S. Pat. No. 5,252,479, and in WIPO publications WO 92/07573, WO 90/06997, WO 89/05345, WO 92/05266 and WO 92/14829, incorporated herein by reference, which provide a description of methods for efficiently introducing nucleic acids into human cells using such retroviral vectors. Other retroviral vectors include, for example, the mouse mammary tumor virus vectors (e.g., Shackleford et al., PNAS, USA, 85:9655-9659 (1988)), and the like.
In accordance with yet another embodiment of the present invention, there are provided anti-TPBD antibodies having specific reactivity with one or more TPBD polypeptides of the present invention. Active fragments of antibodies are encompassed within the definition of “antibody”. Invention antibodies can be produced by methods known in the art using invention polypeptides, proteins or portions thereof as antigens. For example, polyclonal and monoclonal antibodies can be produced by methods well known in the art, as described, for example, in Harlow and Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory (1988)), which is incorporated herein by reference. Invention polypeptides can be used as immunogens in generating such antibodies. Alternatively, synthetic peptides can be prepared (using commercially available synthesizers) and used as immunogens. Amino acid sequences can be analyzed by methods well known in the art to determine whether they encode hydrophobic or hydrophilic domains of the corresponding polypeptide. Altered antibodies such as chimeric, humanized, CDR-grafted or bifunctional antibodies can also be produced by methods well known in the art. Such antibodies can also be produced by hybridoma, chemical synthesis or recombinant methods described, for example, in Sambrook et al., supra., and Harlow and Lane, supra. Both anti-peptide and anti-fusion protein antibodies can be used. (see, for example, Bahouth et al., Trends Pharmacol. Sci. 12:338 (1991); Ausubel et al., Current Protocols in Molecular Biology (John Wiley and Sons, NY (1989) which are incorporated herein by reference).
Antibody so produced can be used, inter alia, in diagnostic methods and systems to detect the level of TPBD present in a mammalian, preferably human, body sample, such as tissue or vascular fluid. Such antibodies can also be used for the immunoaffinity or affinity chromatography purification of the invention TRAF. In addition, methods are contemplated herein for detecting the presence of an invention TPBD either within a cell, or on the surface of a cell, comprising contacting the cell with an antibody that specifically binds to TPBD polypeptides, under conditions permitting binding of the antibody to the TPBD polypeptides, detecting the presence of the antibody bound to the TPBD polypeptide, and thereby detecting the presence of invention polypeptides on the surface of the cell. With respect to the detection of such polypeptides, the antibodies can be used for in vitro diagnostic or in vivo imaging methods.
Immunological procedures useful for in vitro detection of target TPBD polypeptides in a sample include immunoassays that employ a detectable antibody. Such immunoassays include, for example, ELISA, Pandex microfluorimetric assay, agglutination assays, flow cytometry, serum diagnostic assays and immunohistochemical staining procedures which are well known in the art. An antibody can be made detectable by various means well known in the art. For example, a detectable marker can be directly or indirectly attached to the antibody. Useful markers include, for example, radionucleotides, enzymes, fluorogens, chromogens and chemiluminescent labels.
Invention anti-TPBD antibodies are contemplated for use herein to modulate the activity of the TPBD polypeptide in living animals, in humans, or in biological tissues or fluids isolated therefrom. The term “modulate” refers to a compound's ability to increase (e.g., via an agonist), decrease (e.g., via an antagonist), or otherwise modify (e.g., increasing a first TPBD activity while decreasing a second TPBD activity) the biological activity of an invention TPBD protein, such as TNFR family-binding, TRAF protein binding activity, TRAF-associated protein binding activity, NF-κB modulating activity, JNK modulating activity, apoptosis modulating activity, cell proliferation modulating activity, cell adhesion, cell stress responses modulating activity, or B cell immunoglobulin class switching modulating activity, and the like. Accordingly, compositions comprising a carrier and an amount of an antibody having specificity for TPBD polypeptides effective to block naturally occurring ligands or other TPBD-associated proteins, and the like, from binding to invention TPBD polypeptides are contemplated herein. For example, a monoclonal antibody directed to an epitope of an invention TPBD polypeptide including an amino acid sequence set forth in SEQ ID NOs:8, 10, 12, 23, 24 and 25 can be useful for this purpose.
The present invention further provides transgenic non-human mammals that are capable of expressing exogenous nucleic acids encoding TPBDs. As employed herein, the phrase “exogenous nucleic acid” refers to nucleic acid sequence which is not native to the host, or which is present in the host in other than its native environment (e.g., as part of a genetically engineered DNA construct). In addition to naturally occurring levels of TPBD-containing proteins, invention TPBDs can either be overexpressed or underexpressed (such as in the well-known knock-out transgenics) in transgenic mammals.
Also provided are transgenic non-human mammals capable of expressing nucleic acids encoding TPBD polypeptides so mutated as to be incapable of normal activity, i.e., do not express native TPBD. The present invention also provides transgenic non-human mammals having a genome comprising antisense nucleic acids complementary to nucleic acids encoding TPBD polypeptides, placed so as to be transcribed into antisense mRNA complementary to mRNA encoding TPBD polypeptides, which hybridizes to the mRNA and, thereby, reduces the translation thereof. The nucleic acid may additionally comprise an inducible promoter and/or tissue specific regulatory elements, so that expression can be induced, or restricted to specific cell types. Examples of nucleic acids are DNA or cDNA having a coding sequence substantially the same as the coding sequence shown in SEQ ID NOs:1, 3, and 5. An example of a non-human transgenic mammal is a transgenic mouse. Examples of tissue specificity-determining elements are the metallothionein promoter and the L7 promoter.
Animal model systems which elucidate the physiological and behavioral roles of TPBD polypeptides are also provided, and are produced by creating transgenic animals in which the expression of the TPBD polypeptide is altered using a variety of techniques. Examples of such techniques include the insertion of normal or mutant versions of nucleic acids encoding an TRAF polypeptide by microinjection, retroviral infection or other means well known to those skilled in the art, into appropriate fertilized embryos to produce a transgenic animal. (See, for example, Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual (Cold Spring Harbor Laboratory, (1986)).
Also contemplated herein, is the use of homologous recombination of mutant or normal versions of TPBD genes with the native gene locus in transgenic animals, to alter the regulation of expression or the structure of TPBD polypeptides (see, Capecchi et al., Science 244:1288 (1989); Zimmer et al., Nature 338:150 (1989); which are incorporated herein by reference). Homologous recombination techniques are well known in the art. Homologous recombination replaces the native (endogenous) gene with a recombinant or mutated gene to produce an animal that cannot express native (endogenous) protein but can express, for example, a mutated protein which results in altered expression of TPBD polypeptides.
In contrast to homologous recombination, microinjection adds genes to the host genome, without removing host genes. Microinjection can produce a transgenic animal that is capable of expressing both endogenous and exogenous TPBDs. Inducible promoters can be linked to the coding region of nucleic acids to provide a means to regulate expression of the transgene. Tissue specific regulatory elements can be linked to the coding region to permit tissue-specific expression of the transgene. Transgenic animal model systems are useful for in vivo screening of compounds for identification of specific ligands, i.e., agonists and antagonists, which activate or inhibit protein responses.
Invention nucleic acids, oligonucleotides (including antisense), vectors containing same, transformed host cells, polypeptides and combinations thereof, as well as antibodies of the present invention, can be used to screen compounds in vitro to determine whether a compound functions as a potential agonist or antagonist to invention TPBDs. These in vitro screening assays provide information regarding the function and activity of invention TPBDs, which can lead to the identification and design of compounds that are capable of specific interaction with one or more types of polypeptides, peptides or proteins.
An invention TPBD, as used herein, contains the sequence E(X)_17-21L(X)2W(X)₃VXP(X)_15-16L(X)_24-28K(X)^15-16W (SEQ ID NO:19), where X is any amino acid. Alternatively, an invention TPBD is characterized as having the sequence: LXWX(X′)XVXP (SEQ ID NO:20) where X is any amino acid and X′ is selected from L and I. Preferably, an invention TPBD has the sequence: E(X)_10-13S(X)_6-7LXW(X)₃VXP(x)_10-11S(X)₄L(X)_24-28K(X)_9-10F(X)₅WG(X)₃F(X)₁₆D(X)_5-7V (SEQ ID NO:21), where X is any amino acid. More preferably, an invention TPBD has the following sequence: E(X)₄(X_A)(X)_5-8SX(X_B)(X)_4-5LXWX(X_A)XVXP(X)_10-11S(X_A)(X)₃L(X)_16-18(X_A)(X)_4-6(X_C)(X)_9-10F(X)5WG(X_A)(X)₂F(X)₅(X_A)X(X_C)(X)₇(X_C) DX(X_A)(X)_2-4(XC)V (SEQ ID NO:22), where X is any amino acid, X_Ais selected from V, L and I; X_Bis selected from P and G; X_Cis selected from D, E, N and Q; and X_Dis selected from Y and F. Most preferably, an invention TPBD comprises the sequence SEQ ID NO:8, 10, 12, 23, 24 or 25.
By the known homology of invention TPBDs to known TRAF domains, it is within the scope of the invention that invention TPBD also have a role in celluar pathways that effect apoptosis, cell proliferation, cell adhesion, cell stress responses and B cell immunoglobulin class switching. Thus, invention TPBDs also provide drug discovery targets for a broad variety of pathologies including autoimmunity, inflammation, allergy, allograph-rejection and sepsis, and for a broad variety of cancer pathologies, such as, gliomas, carcinomas, sarcomas, melanomas, hamartomas and the like. In certain aspects of the invention, invention TRAF proteins, agonist or antagonists thereto, are used to treat autoimmunity, inflammation, allergy, allograph-rejection, sepsis, keratinocyte hyperplasia, neoplasia, keloid, benign prostatic hypertrophy, inflammatory hyperplasia, fibrosis, smooth muscle cell proliferation in arteries following balloon angioplasty (restenosis), and the like. Exemplary cancer pathologies contemplated herein for treatment include, gliomas, carcinomas, adenocarcinomas, sarcomas, melanomas, hamartomas, leukemias, lymphomas, and the like.
Also provided herein are methods of treating pathologies, said method comprising administering an effective amount of an invention therapeutic composition. Such compositions are typically administered in a physiologically compatible composition.
Methods of treating pathologies of abnormal cell proliferation will include methods of modulating the activity of one or more oncogenic proteins, wherein the oncogenic proteins specifically interact with a TPBD. Methods of modulating the activity of such oncogenic proteins will include contacting the oncogenic protein with a substantially pure TPBD or an active fragment (i.e., oncogenic protein-binding fragment) thereof. This contacting will modulate the activity of the oncogenic protein, thereby providing a method of treating a pathology caused by the oncogenic protein. Further methods of modulating the activity of oncogenic proteins will include contacting the oncogenic protein with an agent, wherein the agent modulates the interactions between the TPBD and the oncogenic protein.
Methods of treating immune-based pathologies such as autoimmunity, inflammation, allergy, allograph-rejection, and sepsis will include modulating the activity of one or more proteins that modulate immune response, wherein the protein that modulates immune response specifically interact with a TPBD. Methods of modulating the activity of such protein that modulates immune response will include contacting the protein that modulates immune response with a substantially pure TPBD or an active fragment (i.e., protein-binding fragment) thereof. This contacting will modulate the activity of the protein that modulates immune response, thereby providing a method of treating a pathology caused by the protein that modulates immune response. Further methods of modulating the activity of a protein that modulates immune response will include contacting the protein that modulates immune response with an agent, wherein the agent modulates the interactions between the TPBD and the protein that modulates immune response.
Also contemplated herein, are therapeutic methods using invention pharmaceutical compositions for the treatment of pathological disorders in which there is too little cell division, such as, for example, bone marrow aplasias, immunodeficiencies due to a decreased number of lymphocytes, and the like. Methods of treating a variety of inflammatory diseases with invention therapeutic compositions are also contemplated herein, such as treatment of sepsis, fibrosis (e.g., scarring), arthritis, graft versus host disease, and the like.
The present invention also provides therapeutic compositions useful for practicing the therapeutic methods described herein. Therapeutic compositions of the present invention, such as pharmaceutical compositions, contain a physiologically compatible carrier together with an invention TPBD (or functional fragment thereof), a TPBD modulating agent, such as a compound (agonist or antagonist) identified by the methods described herein, or an anti-TPBD antibody, as described herein, dissolved or dispersed therein as an active ingredient. In a preferred embodiment, the therapeutic composition is not immunogenic when administered to a mammal or human patient for therapeutic purposes.
As used herein, the terms “pharmaceutically acceptable”, “physiologically compatible” and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to a mammal without the production of undesirable physiological effects such as nausea, dizziness, gastric upset, and the like.
The preparation of a pharmacological composition that contains active ingredients dissolved or dispersed therein is well known in the art. Typically such compositions are prepared as injectables either as liquid solutions or suspensions; however, solid forms suitable for solution, or suspension, in liquid prior to use can also be prepared. The preparation can also be emulsified.
The active ingredient can be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient in amounts suitable for use in the therapeutic methods described herein. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like, as well as combinations of any two or more thereof. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and the like, which enhance the effectiveness of the active ingredient.
The therapeutic composition of the present invention can include pharmaceutically acceptable salts of the components therein. Pharmaceutically acceptable nontoxic salts include the acid addition salts (formed with the free amino groups of the polypeptide) that are formed with inorganic acids such as, for example, hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, anthranilic acid, cinnamic acid, naphthalene sulfonic acid, sulfanilic acid, and the like.
Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium hydroxide, ammonium hydroxide, potassium hydroxide, and the like; and organic bases such as mono-, di-, and tri-alkyl and -aryl amines (e.g., triethylamine, diisopropyl amine, methyl amine, dimethyl amine, and the like) and optionally substituted ethanolamines (e.g., ethanolamine, diethanolamine, and the like).
Physiologically tolerable carriers are well known in the art. Exemplary liquid carriers are sterile aqueous solutions that contain no materials in addition to the active ingredients and water, or contain a buffer such as sodium phosphate at physiological pH, physiological saline or both, such as phosphate-buffered saline. Still further, aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, polyethylene glycol and other solutes.
Liquid compositions can also contain liquid phases in addition to and to the exclusion of water. Exemplary additional liquid phases include glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions.
As described herein, an “effective amount” is a predetermined amount calculated to achieve the desired therapeutic effect, e.g., to modulate activity of an invention TPBD. The required dosage will vary with the particular treatment and with the duration of desired treatment; however, it is anticipated that dosages between about 10 micrograms and about 1 milligram per kilogram of body weight per day will be used for therapeutic treatment. It may be particularly advantageous to administer such compounds in depot or long-lasting form as discussed hereinafter. A therapeutically effective amount is typically an amount of an TPBD-modulating agent or compound identified herein that, when administered in a physiologically acceptable composition, is sufficient to achieve a plasma concentration of from about 0.1 μg/ml to about 100 μg/ml, preferably from about 1.0 μg/ml to about 50 μg/ml, more preferably at least about 2 μg/ml and usually 5 to 10 μg/ml. Therapeutic invention anti-TPBD antibodies can be administered in proportionately appropriate amounts in accordance with known practices in this art.
In accordance with still another embodiment of the present invention, there are provided methods for identifying compounds which bind to TPBD polypeptides. The invention proteins may be employed in a competitive binding assay. Such an assay can accommodate the rapid screening of a large number of compounds to determine which compounds, if any, are capable of binding to TPBDs. Subsequently, more detailed assays can be carried out with those compounds found to bind, to further determine whether such compounds act as modulators, agonists or antagonists of invention TPBDs. Compounds that bind to and/or modulate invention TPBDs can be used to treat a variety of pathologies mediated by invention TPBDs.
In another embodiment of the invention, there is provided a bioassay for identifying compounds which modulate the activity of invention TPBD polypeptides. Invention TPBD polypeptides are known to influence the activities of, for example, NF-κB. Further, homologous TRAF polypeptides are known to influence the activities of, for example, NF-κB and cJun N-terminal kinase (JNK). Thus a reporter gene construct to assay for NF-κB or JNK activity can be used to test invention TPBD activity (see Examples). According to this method, invention TPBD polypeptides are contacted with an “unknown” or test substance, the activity of the invention TPBD polypeptide is monitored subsequent to the contact with the “unknown” or test substance, and those substances which effect a resultant modulation of, for example, NF-κB activity or JNK activity are identified as functional ligands for TPBD polypeptides.
Alternative bioassays for identifying compounds which modulate the activity of invention TPBD polypeptides can be used which routinely are used to test for protein:protein interactions. Such bioassays include yeast two-hybrid assays, glutathione-S-transferase fusion protein binding assays, co-immmunoprecipitation assays, and the like. Such assays are well known in the art and can be found in standard reference texts such as Sambrook et al., supra., and Current Protocols in Molecular Biology, supra.
In accordance with another embodiment of the present invention, transformed host cells that recombinantly express invention polypeptides can be contacted with a test compound, and the modulating effect(s) thereof can then be evaluated by comparing the TPBD-mediated response (e.g., via reporter gene expression) in the presence and absence of test compound, or by comparing the response of test cells or control cells (i.e., cells that do not express TPBD polypeptides), to the presence of the compound.
As used herein, a compound or a signal that “modulates the activity” of invention TPBD polypeptides refers to a compound or a signal that alters the activity of TRAF polypeptides so that the activity of the invention polypeptide is different in the presence of the compound or signal than in the absence of the compound or signal. In particular, such compounds or signals include agonists and antagonists. An agonist encompasses a compound or a signal that activates TPBD protein expression. Alternatively, an antagonist includes a compound or signal that interferes with TPBD expression. Typically, the effect of an antagonist is observed as a blocking of agonist-induced protein activation. Antagonists include competitive and non-competitive antagonists. A competitive antagonist (or competitive blocker) interacts with or near the site specific for agonist binding. A non-competitive antagonist or blocker inactivates the function of the polypeptide by interacting with a site other than the agonist interaction site.
As understood by those of skill in the art, assay methods for identifying compounds that modulate TPBD activity generally require comparison to a control. One type of a “control” is a cell or culture that is treated substantially the same as the test cell or test culture exposed to the compound, with the distinction that the “control” cell or culture is not exposed to the compound. For example, in methods that use voltage clamp electrophysiological procedures, the same cell can be tested in the presence or absence of compound, by merely changing the external solution bathing the cell. Another type of “control” cell or culture may be a cell or culture that is identical to the transfected cells, with the exception that the “control” cell or culture do not express native proteins. Accordingly, the response of the transfected cell to compound is compared to the response (or lack thereof) of the “control” cell or culture to the same compound under the same reaction conditions.
In yet another embodiment of the present invention, the activation of TPBD polypeptides can be modulated by contacting the polypeptides with an effective amount of at least one compound identified by the above-described bioassays.
In accordance with another embodiment of the present invention, there are provided methods for diagnosing cancer, said method comprising:
detecting, in said subject, a defective sequence or mutant of SEQ ID NOs:7, 9, and 11.
In accordance with another embodiment of the present invention, there are provided diagnostic systems, preferably in kit form, comprising at least one invention nucleic acid in a suitable packaging material. The diagnostic nucleic acids are derived from the TPBD-encoding nucleic acids described herein. In one embodiment, for example, the diagnostic nucleic acids are derived from any of SEQ ID NOs:7, 9, and 11. Invention diagnostic systems are useful for assaying for the presence or absence of nucleic acid encoding TPBD in either genomic DNA or in transcribed nucleic acid (such as mRNA or cDNA) encoding TRAF.
A suitable diagnostic system includes at least one invention nucleic acid, preferably two or more invention nucleic acids, as a separately packaged chemical reagent(s) in an amount sufficient for at least one assay. Instructions for use of the packaged reagent are also typically included. Those of skill in the art can readily incorporate invention nucleic probes and/or primers into kit form in combination with appropriate buffers and solutions for the practice of the invention methods as described herein.
As employed herein, the phrase “packaging material” refers to one or more physical structures used to house the contents of the kit, such as invention nucleic acid probes or primers, and the like. The packaging material is constructed by well known methods, preferably to provide a sterile, contaminant-free environment. The packaging material has a label which indicates that the invention nucleic acids can be used for detecting a particular sequence encoding TPBD including the nucleotide sequences set forth in SEQ ID NOs:7, 9, and 11 or mutations or deletions therein, thereby diagnosing the presence of, or a predisposition for, cancer. In addition, the packaging material contains instructions indicating how the materials within the kit are employed both to detect a particular sequence and diagnose the presence of, or a predisposition for, cancer.
The packaging materials employed herein in relation to diagnostic systems are those customarily utilized in nucleic acid-based diagnostic systems. As used herein, the term “package” refers to a solid matrix or material such as glass, plastic, paper, foil, and the like, capable of holding within fixed limits an isolated nucleic acid, oligonucleotide, or primer of the present invention. Thus, for example, a package can be a glass vial used to contain milligram quantities of a contemplated nucleic acid, oligonucleotide or primer, or it can be a microtiter plate well to which microgram quantities of a contemplated nucleic acid probe have been operatively affixed.
“Instructions for use” typically include a tangible expression describing the reagent concentration or at least one assay method parameter, such as the relative amounts of reagent and sample to be admixed, maintenance time periods for reagent/sample admixtures, temperature, buffer conditions, and the like.
All U.S. patents and all publications mentioned herein are incorporated in their entirety by reference thereto. The invention will now be described in greater detail by reference to the following non-limiting examples.

Examples

I. Isolation of the TPBDs of TRAF7, HAUSP and SPOP

Nucleic acids encoding the TRAF protein binding domain fragments (TPBDs) of TRAF7 (residues 282-435 of SEQ ID NO:6), HAUSP (residues 1-213 of SEQ ID NO:2) and SPOP (residues 1-180 of SEQ ID NO:4) were isolated by polymerase chain reaction (PCR) using 2 μg of cDNA from Jurkat cells (The method for RNA extraction and cDNA synthesis was provided by the manufacturer (Pharmingen). The PCR was performed for 1 cycle of 94° C. for 2 min followed by 35 cycles of 94° C. for 15 s, 60° C. for 20 s and 72° C. for 100 s, followed by a final cycle of 72° C. for 5 min, using the following primers HAUSP: 5′ GCGAATTCCAGGCCGCG 3′ (SEQ ID NO:13) and 5′ TTCCTCGAGCCGACTTAGCCTGTGTGC 3′ (SEQ ID NO:14); SPOP: 5′ CTTCGAATTCGCGATGTCAAGGGTTCC 3′ (SEQ ID NO:15) and 5′ CCATGCTCGAGGTATTCTAGCCAGAAATG 3′ (SEQ ID NO:16); TRAF 7: 5′ CCAGAATTCACCAGTGAATTAGTGCC 3′ (SEQ ID NO:17) and 5′ CCACTCGAGTAATGTACCAATGCTAGTCC 3′ (SEQ ID NO:18). The amplified fragments were purified, digested with EcoR1 and Xho 1 restriction enzymes and subcloned into pCDNA-3-myc tag and into pGEX4T (Pharmacia).

II. Expression of TPBCs TRAF7, USP7 and SPOP

To determine mRNA expression of genes encoding various TRAF domain proteins, Northern blot analysis of the mRNA levels of TRAF7, USP7 and SPOP was performed in human tissues. ³²P-labeled cDNA fragments of TRAF7, USP7 or SPOP corresponding to the TRAF domains were sequentially hybridized, in the order of TRAF7, USP7 and SPOP, to filter-immobilized poly-A⁺ RNA from various tissues (1 μg/lane) (Clontech; Palo Alto Calif.). The tissues tested were brain, heart, skeletal muscle, colon, thymus, spleen, kidney, liver, small intestine, placenta, lung and peripheral blood leukocytes (PBL). Hybridized fragments were visualized by autoradiography. The same RNA blot was finally hybridized with a human β-actin cDNA probe to controll for RNA loading (bottom panel).
As shown in FIG. 4, the TRAF7 probe hybridized to mRNA of about 5 kb. TRAF7 mRNA was most highly expressed in brain, heart, skeletal muscle, kidney, liver and placenta. TRAF7 was expressed at a lower level in thymus, small intestine, lung, spleen, PBL and colon.
USP7, also known as HAUSP, was expressed as a doublet of about 7 and 5.5 kb. USP7 was most highly expressed in heart, skeletal muscle, and kidney, with lower expression in brain, thymus, liver, placenta and PBLs, and lowest expression, barely detectable, in colon, spleen, small intestine and lung.
SPOP was expressed as mRNA of about 2.6 kb. SPOP was most highly expressed in heart and skeletal muscle, with lower expression in kidney and liver, and even lower expression in brain, skeletal muscle, colon, thymus, small intestine, placenta, lung, and PBLs.

III. Binding to the TNF-Receptor Family

For GST-fusion protein production, pGEX-plasmids were transformed into competent XL-1 blue bacteria cells and grown in LB medium and induced at an A₆₀₀=1.0 with 1 mM isopropyl-1-thio-β-D-galactopyranoside for 4 h at 25° C. Cells were then recovered and resuspended in PBS containing 1 mM dithiothreitol, 1 mM phenyl-methyl-sulphonyl-fluoride and 100 μg/ml lysozyme and lysed by sonication. The GST-TRAF2 (263-501), (GST-HAUSP (SEQ ID NO: 8), GST-SPOP (SEQ ID NO: 10) and GST-TRAF7 (SEQ ID NO: 12) proteins were purified from bacterial lysates by affinity chromatography using glutathione-Sepharose (Amersham Pharmacia Biotech). The resins were then washed with PBS containing 1mM dithiothreitol until the OD_{280 nm}reached <0.01.
Plasmids pGEX-TRAF2(263-501), pGEX-CD40(ct), pGEX-Fas(ct), pGEX-LT13R(ct), pGEX-DR4(ct) and pGEX-NGFR(ct) have been previously described (Leo et al., 1999, JBC 274, 22414; Sato et al., 1995, FEBS lett.358 113; Crowe et al., J Exp Med 1995,181,1205; McFarlane et al., J Biol Chem 1997, 272,25417; Rabizadeh et al., Proc Natl Acad Sci USA 1994, 91,10703). Plasmid pGEX-TNF-R2(ct) was kindly provided by Dr. C. Ware (La Jolla Institute for Alergy and Immunology).
In vitro GST-protein binding assays were performed as previously described (Hanada, M., Aime-Sempe, C., Sato, T. and Reed, J C. 1995, JBC 270, 11962-11968; Sato, T., Irie, S., Kitada, S. and Reed, J C. 1995, Science 268, 411-415; Takayama, S., Sato, T., Krajewski, S., Kochel, C., Irie, S., Millan, J. and Reed, J C. 1995, Cell 80, 279-284, Leo et al., 1999, JBC 274, 22414). Briefly, (³⁵S)-methionine labeled GST-fused TNF family receptors FAS(ct), TNF-R2(ct), CD40(ct), LTβR(ct), NGFRp75(ct) and DR4(ct) were produced by in vitro translation using the TnT coupled reticulocyte system following the manufacturer indications (Promega Inc.). Equal amounts of each labeled protein (2-6 μl lysate) were then diluted with 250 μl of binding buffer (142 mM KCl, 5 mM MgCl12, 10 mM Hepes pH 7.4, 0.2% Nonidet-P40, 0.5 mM dithiothreitol, 1 mM EGTA, 0.5 mM phenyl-methyl-sulphonyl-fluoride and a mixture of other protease inhibitors (Boehringer 1697498) and incubated with the GST-protein resins (0.25 μg protein) at 4° C. for 2 h. The resins were then extensively washed with binding buffer and the GST-protein binding complexes were eluted with buffer containing 50 mM Tris-HCl pH 8, 1 mM dithiothreitol and 100 mM glutathione and analyzed by SDS-PAGE and fluorography.
The results of the binding assay are shown in FIG. 5. In vitro protein binding assays were performed by in vitro translating TRAF2 or the TRAF domain of TRAF7 in the presence of ³⁵S-L-methionine in reticulocyte lysates, as described above, or following in vivo overexpression of TRAF2 or the TRAF domain of TRAF7 by transfection of 293T cells. Equal volumes of the in vitro translation mixtures (10 μl) were incubated with GST-fusion proteins containing the cytosolic domains of different members of the TNF-R family (1 μg)(FAS, TNF-R2, CD40, LTβR, NGFRp75, and DR4) immobilized on glutathione-Sepharose. For TRAF domain protein expression in vivo, 293T lysates (50 μl) were incubated with GST-fusion proteins (1 μg) containing the cytosolic domains of the TNF-R family members DR4, HVEM, TNF-R2, LTβR, NGFRp75, CD40, and TRAF7 and then immobilized on glutathione-Sepharose. Control GST and other GST control proteins were included in all assays After washing, bound proteins were analyzed by SDS-PAGE followed by fluorography for binding assays using in vitro translated proteins. For transfected 293 cells, samples were immunoblotted using anti-TRFA2 or anti-Myc antibodies to detect TRAF2 or TRAF7 TD binding, respectively. Bound proteins were then detected by standard chemiluminescence assay (ECL; Amersham; Piscataway N.J.).
As shown in FIG. 5, three TNF family receptors interacted with the GST-fused TRAF domain of TRAF2: TNF-R2, CD40 and LTβR. However, FAS, NGFRp75 and DR4 did not appreciably interact with the TRAF domain of TRAF2. In contrast, the GST-SPOP fusion protein showed significant interaction with only one of these six receptors, TNF-R2; and GST-HAUSP did not significantly interact with any of the six receptors. In contrast, GST-TRAF7 binds TNF-R2, CD40 (albeit weakly), LTβR, NRFRp75 and DR4. This result shows that invention TPBDs SPOP (SEQ ID NO: 10) and HAUSP (SEQ ID NO: 8) can demonstrate higher selectivity in receptor binding than the TRAF domain of TRAF2 and the invention TPBD of TRAF7 (SEQ ID NO:12).
In cell lysates of 293 cells transfected with TRAF2 or the TRAF domain of TRAF7, binding was observed with several TNF-R family members. TRAF2 interacted with HVEM, TNF-R2, LTβR, CD40 and TRAF7, with low binding activity also observed with NGFRp75. The TRAF domain of TRAF7 interacted with DR4, LTβR, and NGFRp75, with strongest binding observed with TRAF7. A faint band of TRAF7 was also observed to interact with TNF-R2 under these conditions, but no binding of TRAF7 to CD40 or HVEM was detected. Therefore, TRAF7 has strong binding activity with itself.

IV. Binding to TRAF Family Proteins

The above described GST-HAUSP and GST-SPOP, and GST-TRAF7 were produced and purified using the above described procedure.
Plasmids containing cDNAs encompasing the complete open reading frames of hTRAF 1 (pSG5-TRAF 1), hTRAF 2 (pcDNA3-HA-TRAF2), hTRAF3 (pbluscriptKS-TRAF3b), hTRAF4 (pcDNA3-HA-TRAF4), hTRAF5 (pcDNA3-Flag-TRAF5) and hTRAF6 (pcDNA3-myc-TRAF6) have been previously described (Sato et al., 1995, FEBS lett.358, 113; Nakano et al., 1996, JBC 271, 14661; Mosialos et al., 1995, Cell 80, 389; Krajewska et al., 1998, Am. J. Pathol. 152, 1549; Leo et al., 1999, JBC 274, 22414; Song and Donner.1995, Biochem. J. 309, 825; Rothe et al., 1995, Cell 78, 281).
In vitro translated TRAF1, TRAF2, TRAF3, TRAF4, TRAF5, TRAF6, full-length I-TRAF (Rothe et al., Proc. Natl. Acad. Sci. USA 93:8241-8246 (1996)), HAUSP (1-213, SEQ ID NO:8), SPOP (1-180, SEQ ID NO:10) and TRAF7 (282-435, SEQ ID NO:12) were incubated with the GST-protein resins using the previously described method.
The resultant images showed that the GST-fused TPBD of TRAF7 (SEQ ID NO: 12) interacted with all previously identified TRAF proteins, TRAFS1-6, and with I-TRAF (see FIG. 6). Additionally, the TPBD of TRAF7 showed an ability to self-associate. The GST-fused TPBD of HAUSP (SEQ ID NO: 8) also was able to interact with all previously known TRAF proteins, although somewhat weakly with TRAF3, and weakly with I-TRAF. However, HAUSP did not demonstrate an ability to self-associate. In contrast, the GST-fused TPBD of SPOP (SEQ ID NO: 10) demonstrated a higher selectivity, having significant interactions with only TRAF1 and TRAF6, and no ability to self-associate. Interestingly, no heterologous interaction between invention TPBDs (i.e., HAUSP:SPOP, HAUSP:TRAF7 and SPOP:TRAF7) were observed.

V. Reporter Gene Assays

293T cells were obtained from ATCC (Rockville, Mass.) and cultured in Dulbecco modified Eagle's-high glucose medium (Life technology, Inc) supplemented with 10% FCS (Hyclone, Utah), 1 mM glutamine and antibiotics. The promoter-containing reporter gene plasmids pUC13-4xNFkB-luc (containing 4 tandem HIV-NFkB response elements and the minimal fos promoter) and pCMV-β-galactosidase has been previously described (Miyashita and Reed, 1995, Cell 80, 293 ; Lin and Stavnezer, 1996, MCB 16, 4591).
For NF-κB reporter gene assays, 293T cells were calcium phosphate-transfected with 12 μg DNA, including 3.5 μg of either pcDNA3-myc-hTRAF2, pcDNA3-myc-hTRAF5 or pcDNA3-myc-hTRAF6, in combination with either 3.5 or 7 μg of control plasmid, pcDNA3-myc-HAUSP (1-213, SEQ ID NO: 8), pcDNA3-myc-SPOP (1-180, SEQ ID NO: 10) or pcDNA3-myc-TRAF7 (282-435, SEQ ID NO: 12) at 60% confluency in 6-well plates in duplicate, except in the case of TRAF5 in which only 7 μg of pcDNA3-myc-HAUSP or pcDNA3-myc-TRAF7 were transfected. In the case of SPOP, only 3.5 μg of either pcDNA3-myc-hTRAF6 was used. Also transfected were a total of 0.5 μg pUC13-4xNFκB-luc plasmid and 1 μg pCMV-β-galactosidase plasmid. After 36 h, cells were lysed with 0.5 ml of Promega lysis buffer. The luciferase activity from 10 μl of each cell lysate was determined using the Luciferase assay system from Promega, following the manufacturer protocol, and read using a luminometer (EG&G Berthold). The luciferase activity was normalized relative to B-galactosidase activity (Miyashita and Reed, 1995, Cell 80, 293).
Immunobloting was performed as described (Krajewski et al., 1996, Anal. Biochem. 236, 221). Briefly, 5 μl of each cell lysate was analyzed by immunoblotting using anti-TRAF6 or anti-myc antibodies and standard chemiluminiscence methods (Amersham) for detection.
The results show that TPBDs of HAUSP (SEQ ID NO: 8) and SPOP (SEQ ID NO: 10) and TRAF 7 (SEQ ID NO: 12)can inhibit NF-κB activitation induced by TRAF6 (see FIG. 7). In particular, the TPBD of HAUSP is capable of canceling nearly all of the TRAF6-induced increase in NF-κB activitation. Further, the TPBD of HAUSP also strongly inhibits NF-κB activation induced by TRAF 2 and TRAF 5. The TPBD of TRAF 7 also demonstrates the ability to inhibit TRAF 2-mediated NF-κB activation, but actually increases TRAF 5 mediated NF-κB activation.
The activity of various domains of the TRAF proteins USP7 and TRAF7 were further characterized with respect to regulating NFκB activity. The effect of TRAF7 and USP7 in regulating the NFκB activity induced by TRAF2 and TRAF6 in a mammalian cell line was tested. Briefly, 293T cells were transfected in 6 well plates with approximately 10 μg of pcDNA3 control plasmid, 3.5 μg of either pcDNA3-myc-hTRAF6 or pcDNA3-hTRAF2, and 7 μg of any of several TRAF7 or USP7 plasmid constructs containing various domains (see FIG. 8, bottom). The transfections also included 0.5 μg pUC13-4xNFκB-luc plasmid and 1 μg pCMV-β-galactosidase plasmid. Relative NFκB activity was assessed by luciferase assays using 10 μof each of the cell lysates prepared 36 h after transfection, with normalization for β-galactosidase activity. At least three to eight independent experiments were performed. The results are presented as fold of activation, with one representative experiment shown (FIG. 8).
As shown in FIG. 8, full length USP7 and a USP7 construct lacking the TRAF domain had a slight inhibitory effect on NFκB activity induced by TRAF2 or TRAF6. The USP7 TRAF domain alone strongly inhibited the TRAF2 and TRAF6-induced increase in NFκB activation. In contrast to USP7, full length TRAF7, as well as the TRAF domain of TRAF7 alone, had inhibitory activity for TRAF2 and TRAF6-induced increase in NFκB activation. A TRAF7 mutant construct lacking the TRAF domain did not show any effect on inhibiting the NFκB activation mediated by TRAF2 or TRAF6.
The effect of the TRAF domains of TRAF7, USP7 and SPOP in regulating the NFkB activity induced by TNFα or CD40 overexpression was also tested. 293T cells were transfected in 6 well plates with approximately 10 μg of pcDNA3 control plasmid, 7 μg of either TRAF7 TD, USP7 TD or SPOP TD plasmids, and either no plasmid (TNFα in FIG. 9) or 3.5 μg of pcDNA3-myc-CD40 (CD40 in FIG. 9), together with 0.5 μg pUC13-4xNFkB-luc plasmid and 1 μg pCMV-β-galactosidase plasmid. TNFα_Ctreatment was performed by adding 100 ng/ml of TNFα to the cells 12 h before harvesting. Relative NFκB activity was assessed by luciferase assays using 10 μl of each of the cell lysates prepared 36 h after transfection, with normalization for β-galactosidase activity. At least three independent experiments were performed. The results of a representative experiment are presented as fold of activation.
As shown in FIG. 9, the TRAF domain of TRAF7 had some inhibitory activity for activation of NFκB induced by both TNFα and CD40 overexpression. The TRAF domain of USP7 had even stronger inhibitory activity than the TRAF domain of TRAF7. In contrast, the TRAF domain of SPOP showed essentially no inhibitory activity of NFκB activation by either TNFα or CD40 overexpression.
Regulation by TRAF7 and USP7 of NFκB activation induced by the TRAF-associated protein NIK was also examined. 293T cells were transfected in 6 well plates with approximately 10 μg of pcDNA3 control plasmid, 7 μg of either empty pcDNA3, or pcDNA3-myc-containing TRAF7, TRAF7 TD, USP7 TD or SPOP TD plasmids, and 3.5 μg of pcDNA3-Flag-NIK, together with 0.5 μg pUC13-4xNFκB-luc plasmid and 1 μg pCMV-β-galactosidase plasmid. Relative NFκB activity was assessed by luciferase assays using 10 μl of each of the cell lysates prepared 36 h after transfection, with normalization for β-galactosidase activity. At least three independent experiments were performed. The results of a representative experiment are presented as fold of activation (FIG. 10).
As shown in FIG. 10, NIK induced NFκB activation can be partially regulated by TRAF7 and USP7. Both TRAF7 and the TRAF domain of TRAF7 partially inhibited NIK-induced NFκB activation. USP7 TRAF domain also partially inhibited NIK induced NFκB activation, although the full length USP7 did not inhibit NIK-induced NFκB activation (FIG. 10)

VI. Subcellular Localization of Domains of TPBDs

To test the function of various domains in subcellular localization, TRAF7 and USP7 deletion mutants were constructed. For TRAF7 deletion mutants, Cos7 cells were transfected with Lipofectamin plus (Life Technologies; Rockville Md.) and a total of 3 μg of plasmid. The TRAF7 deletion mutants (shown in FIG. 11) were constructed as Myc fusion proteins. 24 hours after transfection, cells were plated onto poly-lysinated cover-glasses and allowed to settle for 24 h more. Cells were fixed with methanol-acetone (50% each). After blocking, cells were stained with anti-myc mAb (Santa Cruz Biotechnology; Santa Cruz Calif.) and a secondary FITC-labelled rabbit anti-mouse Ig (Dako; Carpinteria Calif.). Analysis was performed using a Bio-Rad confocal microscope (BioRad; Hercules Calif.).
FIG. 11 shows the subcellular localization of TRAF7 and different deletion mutants of the molecule. A scheme of the different TRAF7 mutants analyzed is also shown. TRAF7 localizes in cytosolic corpuscles, which can appear as individual dots (panel A) or as large aggregates (A, small panel). Deletion mutants of TRAF7 lacking the RING finger domain (B), containing only the tripartite domain (C), lacking the poly-acidic region (D), or lacking the TRAF domain (E) have a similar subcellular localization as the full length TRAF7. In contrast, the TRAF domain alone (F) or the TRAF domain containing also the leucine zippers (G) did not show this particular subcellular localization, having a diffuse cytosolic localization. The C-terminal region of the molecule containing the TRAF domain and the poly-acidic region (H) or the poly-acidic region alone (I) have a similar diffuse cytosolic localization, although in this case they appear to be also located in the nucleus. These results indicate that the region of TRAF7 which is determinant for its subcellular localization can be mapped to the Z-b box and the first coiled coil.
For characterizing the localization of USP7 deletion mutants, Cos7 cells were transfected with Lipofectamin plus (Life Technologies) and a total of 3 μg of plasmid. USP7 full length and deletion mutants were constructed as Myc fusion proteins. The USP7 deletion mutants tested were a mutant lacking the TRAF domain and a mutant containing USP7 TRAF domain. 24 hours after transfection, cells were plated onto poly-lysinated cover-glasses and allowed to settle for 24 h more. Cells were fixed with methanol-acetone (50% each). After blocking, cells were stained with anti-myc mAb (Santa Cruz Biothech.) and a secondary FITC-labelled rabbit anti-mouse Ig (Dako). Analysis was performed using a Bio-Rad confocal microscope. Two representative examples of each construct are shown.
FIG. 12 shows the subcellular localization of USP7 full length (A and B), a deletion mutant of USP7 lacking the TRAF domain (C and D) and USP7 TRAF domain (E and F). USP7 is mainly located in the nucleus, although some cytosolic staining can also be observed in some cells. The deletion mutant lacking the TRAF domain is completely excluded from the nucleus, even when it still contains the nuclear localization signal. Finally, the TRAF domain of USP7 appears to have a diffuse localization, with a larger concentration in the perinuclear region. These results indicate that the TRAF domain of USP7 is necessary, but not sufficient, to target this molecule to the nucleus.
Although the invention has been described with reference to the examples above, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims.

Claims

1. A TRAF-Protein-Binding-Domain (TPBD) polypeptide comprising SEQ ID NO:19, provided said polypeptide is no longer than 213 amino acids.

2. The TPBD polypeptide of claim 1, further comprising SEQ ID NO:20.

3. The TPBD polypeptide of claim 1, further comprising SEQ ID NO:21.

4. The TPBD of claim 1, wherein the amino acid sequence of said protein comprises substantially the same sequence as any of SEQ ID NOS:8, 10, 12, 23, 24 or 25.

5. The TPBD of claim 1, comprising the same amino acid sequence as set forth in any of SEQ ID NOS:8, 10, 12, 23, 24 or 25.

6. A TPBD according to claim 4, wherein said polypeptide is encoded by a nucleotide sequence comprising substantially the same nucleotide sequence as set forth in SEQ ID NOs:7, 9 or 11.

7. A TPBD according to claim 1, wherein said polypeptide is encoded by a nucleotide sequence comprising the same sequence as set forth in SEQ ID NOs:7, 9 or 11.

8-11. (canceled)

12. Isolated nucleic acid encoding a TRAF protein binding domain (TPBD), or functional fragments thereof, selected from:

(a) DNA encoding the amino acid sequence set forth in SEQ ID NOS:8, 10 or 12, 23, 24 or 25, or

(b) DNA that hybridizes to the DNA of (a) under moderately stringent conditions, wherein said DNA encodes biologically active TPBD, or

(c) DNA degenerate with (b), wherein said DNA encodes biologically active TPBD.

13. A nucleic acid according to claim 12, wherein said nucleic acid hybridizes under high stringency conditions to the TPBD coding portion of any of SEQ ID NOs:7, 9 and 11.

14. A nucleic acid according to claim 12, wherein the nucleotide sequence of said nucleic acid is substantially the same as set forth in any of SEQ ID NO:7, 9 and 11.

15-50. (canceled)

51. A chimeric protein comprising the TPBD of claim 1.

52. A chimeric TPBD-containing protein, comprising the sequence SEQ ID NO:19, provided said chimeric protein is not naturally occurring.

53. The chimeric TPBD-containing protein of claim 52, comprising:

(a) the sequence SEQ ID NO:19; and

(b) a sequence from a heterologous protein.

54. The chimeric TPBD-containing protein of claim 53, further comprising SEQ ID NO:20.

55. The chimeric TPBD-containing protein of claim 53, further comprising SEQ ID NO:21.

56. The chimeric protein of claim 53 further comprising a RING finger domain.

57. An isolated TPBD-containing TRAF protein, or fragment thereof, comprising the sequence SEQ ID NO:19, provided said TRAF domain containing protein does not consist of the sequence SEQ ID NOs:2, 4 or 6.

58. The TPBD-containing TRAF protein of claim 57, further comprising the sequence SEQ ID NO:20.

59. The TPBD-containing TRAF protein of claim 57, further comprising the sequence SEQ ID NO:21.

60-73. (canceled)