WO2024030080A1 - Protéine et motifs spécifiques de l'adp-actine pour remodeler le cytosquelette de l'actine en biochimie de cellules vivantes et in vitro - Google Patents

Protéine et motifs spécifiques de l'adp-actine pour remodeler le cytosquelette de l'actine en biochimie de cellules vivantes et in vitro Download PDF

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WO2024030080A1
WO2024030080A1 PCT/SG2023/050540 SG2023050540W WO2024030080A1 WO 2024030080 A1 WO2024030080 A1 WO 2024030080A1 SG 2023050540 W SG2023050540 W SG 2023050540W WO 2024030080 A1 WO2024030080 A1 WO 2024030080A1
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actin
adp
spa2
seq
polypeptide
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PCT/SG2023/050540
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English (en)
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Yansong MIAO
Qianqian MA
Danxia HE
Cai Xin NG
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Nanyang Technological University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
    • C07K14/395—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts from Saccharomyces
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention generally relates to recombinant polypeptides capable of regulating actin remodelling.
  • the polypeptides disclosed herein are selective for ADP-actin and may modulate ADP-actin remodelling under energy starvation conditions.
  • the recombinant polypeptides of the present invention are also suitable for use in detecting ADP- actin activity in vitro or in living cells, in drug screening, in therapeutic approaches and for the treatment and prophylaxis of actin-associated diseases.
  • Actin is highly conserved and is one of the most abundant proteins on earth. Actin participates in a variety of biological activities such as cell division, intracellular transport, morphogenesis, and muscle contraction, and is essential for the survival of most cells by influencing eukaryotic cell shape and behaviour.
  • monomeric actins polymerize into filaments and is then assembled into filamentous networks by going through different steps of actin polymerization from initial nucleation, capping, elongation, crosslinking, and depolymerization (Pollard, T.D., and Cooper, J.A., Science 326, 1208-1212 (2009)).
  • Actin-binding proteins (ABPs) on the other hand, are known to regulate the organization and dynamics of the actin cytoskeleton.
  • ABPs function at different steps of actin polymerization and depolymerization to coordinate the appropriate assembly speed and network shapes.
  • ABPs regulate at almost every stage of the actin filament assembly and disassembly cycles, and control how the actin filaments are arranged into various three-dimensional configurations as well.
  • the remodelling of the actin cytoskeleton during diverse signaling events is sophisticated, and the underlying mechanisms remain elusive.
  • Dynamic treadmilling and network organization of the actin cytoskeleton rely on the orchestrated operation of different ABPs in an actin nucleotide-dependent fashion. While the structures of ATP- and ADP-actin are nearly identical, actin can also adopt dynamic conformations.
  • the nucleotide state is one of the major factors that can control actin conformation via allosteric communication between nucleotide binding sites and their spatially distinct regions (Ali, R. et al., Nature structural & molecular biology 29, 320-328 (2022)).
  • nucleotide state can modulate actin nucleation by creating a nucleation-potent conformation
  • nucleotide-specific binding by actin-binding proteins also plays critical roles in the rapid influence on actin treadmilling and the timely remodelling of the actin cytoskeleton.
  • nucleation, elongation and crosslinking were mostly studied only in the presence of ATP but remained unclear for ADP-actin, which could be a dominant state during cell signaling, such as under energy starvation (ES).
  • ES energy starvation
  • ES energy starvation
  • GS Glucose starvation
  • Actin cable treadmilling was orchestrated spatiotemporally by a series of ABPs to drive stepwise polymerization and depolymerization steps, coordinating with the nucleotide exchange of actin.
  • GS-induced ATP-depleting conditions interrupted the turnover rate of ATP hydrolysis and inorganic phosphate (Pi) release and increased the ADP:ATP ratio, thereby leading to faster aging of the actin filament from ATP to ADP subunits.
  • GS instead of being depolymerized by ADP-actin-based depolymerization factors, GS triggers actin cable crosslinking to stabilize F-actin and retain the mass of actin networks.
  • ADP-actin-specific regulators are poorly understood, particularly in cell signaling. Therefore, targeting ADP-actin specific modulators may provide a potential approach in the study of actin dynamics, in cell-based drug screenings and in the provision of therapeutics for the treatment of actin-associated diseases.
  • the present invention relates to the provision of recombinant polypeptides capable of modulating ADP-actin remodelling and preferably, regulating ADP-actin remodelling induced under energy starvation.
  • the recombinant polypeptides disclosed herein are also suitable for use in drug screening and for therapeutic applications in the treatment of actin-related diseases.
  • a recombinant polypeptide comprising an amino acid sequence of at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, or a functional fragment or a functional variant thereof, wherein the polypeptide, fragment or variant thereof specifically binds to ADP-actin and modulates ADP-actin remodelling.
  • the recombinant polypeptides disclosed herein preferably modulate ADP-actin remodelling by enhancing ADP-actin nucleation, elongation/polymerization, crosslinking, bundling, stabilization and/or inhibition of actin depolymerization.
  • the ADP-actin remodelling is induced under energy starvation conditions, such as glucose starvation conditions.
  • a polynucleic acid molecule wherein said polynucleic acid molecule has a sequence of at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the polynucleic acid sequence set forth in SEQ ID NO: 8 or in SEQ ID NO: 9, or a fragment thereof, which encodes the polypeptide of the first aspect or its functional fragment or functional variant thereof.
  • a recombinant cell transfected or transformed with the polynucleic acid molecule of the second aspect.
  • a method for the detection and/or modulation of ADP- actin remodelling, or in drug screening of candidate modulators of ADP-actin remodelling may comprise (i) expressing a tagged recombinant polypeptide of the first aspect in a cell; (ii) detecting whether the tagged polypeptide associates with ADP-actin or F-actin using total internal reflection (TIRF) microscopy; and for drug screening, exposing the cell before step (ii) to candidate modulators of ADP-actin remodelling.
  • the method is in vitro.
  • a pharmaceutical composition comprising a recombinant polypeptide of the first aspect and a pharmaceutically acceptable carrier.
  • a recombinant polypeptide of the first aspect a polynucleic acid molecule of the second aspect, or a recombinant cell of the third aspect for use in medicine.
  • a recombinant polypeptide of the first aspect a polynucleic acid molecule of the second aspect, or a recombinant cell of the third aspect, in cell-based screens for drugs or in the study of diseases associated with actin-remodelling, wherein the use is not in a human being.
  • a method of treating a disease associated with actin- remodelling comprising administering a therapeutically effective amount of a recombinant polypeptide of the first aspect or a pharmaceutical composition of the fifth aspect, to a patient in need thereof.
  • a recombinant peptide according to the first aspect, or a pharmaceutical composition of the fifth aspect in the manufacture of a medicament for the treatment of a disease associated with actin-remodelling.
  • a disease associated with actin-remodelling may include myopathies, familial thoracic aortic aneurysms and heart diseases, being related to actin mutation.
  • myopathies familial thoracic aortic aneurysms and heart diseases, being related to actin mutation.
  • Baraitser- Winter syndrome is caused by Met47 to Thr, and this amino acid is located in the actin D-loop.
  • the disease associated with actin-remodelling is a disease associated with ADP-actin remodelling.
  • polypeptides disclosed herein may target different stages in the actin remodelling cycle and act as multifunctional ADP-actin modulators.
  • FIG. 1 shows that Spa2 regulates actin cable remodelling upon energy starvation.
  • n 312, 186, 209,194, 209, 186 cables from ⁇ 20 cells per condition.
  • H Dual-colour images of Spa2-GFP and Abp140-Tomato upon ES under the indicated conditions.
  • FIG. 2 depicts the results showing that Spa2-535 regulates actin cable remodelling upon ES.
  • A Intrinsically disordered region analysis.
  • B Representative fluorescent images of GFP- tagged Spa2 truncation variants upon ES under the indicated conditions.
  • E Localization of Abp140-3GFP in Spa2 truncating mutants, with or without ES at 5 min.
  • FIG. 3 depicts the results indicating that Spa2-535 nucleates and elongates ADP-actin.
  • A Fluorescence anisotropy profile of the indicated recombinant Spa2-N-terminal truncation variants (60 nM, Alexa488-labeled) that were titrated with increasing concentrations of ADP- G-actin. Measurements from three biological replicates were plotted and fit with the Hill equation.
  • (E) Comparison of nucleation efficiency at 5 min by measuring the seed number (from left to right: n 22, 28, 29, and 29 ROIs at 64x64 pm 2 ).
  • (F) Quantification of the actin elongation rates and kymograph in (E) (left to right, n 33, 35, 38, and 51 actin filaments). Bars for B and D, 5 pm; Bar for F, 2 pm. Error bars, mean ⁇ S.D.
  • FIG. 4 shows the results indicating that Spa2 promotes ADP-F-actin bundling.
  • A-C Sedimentation velocity profiles of Spa2-281-535 (18 pM), Spa2-535 (13 pM), and Spa2-281 (17 pM), containing the calculated apparent molecular weights (MW app ). The sedimentation coefficient and fiction ratio (f/fO) of the major species of each Spa2 variant are indicated. The predicted oligomerization status was indicated based on differential sedimentation coefficient distribution analysis.
  • D-F SPR sensorgrams of self-interacting Spa2-281-535, Spa2-535, and Spa2-281. The corresponding curves were fitted in the bivalent model.
  • FIG. 5 depicts the multifaceted remodelling of ADP-actin by Spa2 in a D-loop conformationdependent manner.
  • A Fluorescence micrographs of ADP-F-actin incubated with 5 pM Alexa 488-labeled Spa2-281 , Spa2-535, and Spa2-281-535 and labeled with Alexa 565-phalloidin before being imaged by spinning disk confocal microscope-coupled super-resolution imaging.
  • B Representative time-lapse TIRFM images of ADP- actin formed at the indicated time point in the presence of 3 pM ADP-actin (10% OG488- and 0.5% biotin-labeled) and 5 pM Spa2 variants.
  • FIG. 6 shows energy starvation-induced actin cable remodelling in yeast.
  • B Representative maximum Z-projection images of ABP140-3GFP- labeled actin cables in WT and glucose-relevant mutants hxk2 and cbp2l treated with 20 mM 2-DG for 5 min.
  • (D) The normalized QUEEN ratio (410 nm ex/480 nm ex) in WT, cbp2 , and hxk2 upon 5 min of 2-DG treatment. The ratio was calculated from the signal intensity of each pixel to generate the QUEEN ratio image of cells (from left to right: n 103, 103 and 80 cells).
  • F mean
  • G total fluorescence intensity
  • FIG. 7 depicts the screening of ES-triggered actin remodelling in yeast mutants.
  • A Representative maximum Z-projection images of ABP140-3GFP-labeled actin cables in WT actin-binding protein mutants upon 5 min ES.
  • B Quantification of the mean and total fluorescence intensity of the ABP140-3GFP-labeled actin cables in (A).
  • FIG. 8 shows Spa2-mediated actin remodelling upon ES is through the N-terminus.
  • A Domain illustration of the Spa2 truncating variants used in the study. Localization and pattern changes of GFP-tagged Spa2 truncating variants upon 5 min ES conditions.
  • B Representative fluorescence imaging of GFP-tagged Spa2-535 in the indicated mutants upon glucose starvation for 5 min.
  • FIG. 9 depicts nucleotide-specific actin polymerization by Spa2.
  • A SDS-PAGE of bacteria purified recombinant Spa2-281 , Spa2-535, and Spa2-281-535.
  • B Anisotropic measurements of 60 nM Alexa488-labeled Spa2-N titrated with increasing concentrations of ATP-G-actin. The average values with an error bar of ⁇ SD were calculated from three biological replicates and plotted with the Hill slope equation.
  • C Analysis of severing activities by measuring the cumulative severing events per micron of F-actin at the indicated time point. Left panel: Averaged values ⁇ SD from three independent experiments are shown and fit to an exponential association curve.
  • FIG. 10 shows that F-actin is bundled by Spa2 in a nucleotide-specific manner.
  • A Representative time-lapse images of Spa2-535 droplets in the FRAP assay in buffer with 50 mM KCI.
  • B Fluorescence micrographs of 3 pM ADP-F-actin filaments or 0.5 pM ATP-F-actin filaments that were incubated with a series of concentrations of Spa2-535 at 0, 0.25, 0.5, 1 , 2.5, 5, and 10 pM prior to Alexa565-phalloidin staining and imaging.
  • C Transmission light microscopic imaging of Spa2-535 phase separation at the indicated concentrations that were used in (B).
  • FIG. 11 depicts engineered Spa2-281-535 oligomers for ADP-actin polymerization and functional characterization of D-loop for Spa2-specific interaction.
  • A Cartoon depicting protein engineering of Spa2-281-535 using homotrimeric CC motifs.
  • B Comparison of Spa2- 281-535 and Spa2-281-535-trimer on SDS-PAGE.
  • C Sedimentation velocity analysis of the Spa2-281-535 trimer.
  • D Representative time-lapse TIRFM images of actin polymerization.
  • F-actin was polymerized using 3 pM ADP-actin or 0.5 pM ATP-actin (10% OG488- and 0.5% biotin-labeled), with or without 10 nM Spa2-281-535 or Spa2-281-535-trimer.
  • the box plot shows the mean ⁇ S.D.
  • FIG. 12 depicts the rational design of an ADP-actin specific binding motif based on structural predictions (A, B) Alphafold's predicted structure for the ADP-actin binding region Spa2-389- 535 and its sequences. (C, D) Detailed analysis of identified Helix 1 , responsible for ADP-actin specific binding, comprising a short and a long helix surrounded by flexible loops. New constructs expressing msfGFP fusions were designed for individual helices, labeled H1a and H1b.
  • FIG. 13 depicts the identification and characterization of an ADP-actin sensing peptide through in vitro biochemistry and in vivo cellular imaging using msfGFP-Spa2-H1b reporter.
  • A Domain diagram of Spa2 truncating variants and SDS-PAGE of bacterially purified recombinant Spa2-H1-H4 (residues 389-535), Spa2-H1 (residues 389-433), Spa2-H1a (residues 389-407), and Spa2-H1 b (residues 401-433).
  • D Representative LI2OS cells expressing Spa2-H1-GFP, exposed to DMEM with 0.45% glucose or DMEM with 0.45% 2-DG but no glucose for 10 min, then fixed and stained with phalloidin 565, scale bar 5 pm.
  • FIG. 14 shows the identification and characterization of the minimal helix required for ADP- actin sensing in vitro and in vivo.
  • A Domain diagram of Spa2 truncating variants.
  • B Predicted structure of Saccharomyces cerevisiae's Spa2 corresponding to residues H1b, H1b1 , H1b2, and H1 b3.
  • C SDS-PAGE of bacterially purified recombinant Spa2-H1b, Spa2- H1b1 , Spa2-H1b2, and Spa2-H1b3.
  • the term “comprising” or “including” is to be interpreted as specifying the presence of the stated features, integers, steps or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps or components, or groups thereof.
  • the term “comprising” or “including” also includes “consisting of”.
  • the variations of the word “comprising”, such as “comprise” and “comprises”, and “including”, such as “include” and “includes”, have correspondingly varied meanings.
  • energy starvation or “energy starvation condition” may refer to a condition in which the energy status or level in a cell is lower than what is required for the cell to perform its function (e.g., maintaining the equilibrium between catabolism and anabolism).
  • the cell may be deprived of one or more nutrients, e.g., glucose etc., which may lead to a loss of ATP, which is the cellular energy currency.
  • energy starvation condition may also be interpreted to comprise any type of stress that would lead to a loss of ATP in the cell. It would be appreciated by a skilled person that cellular energy levels may be monitored by measuring ATP/ADP ratio in a cell.
  • the term “functional fragment” refers to a portion of a polypeptide/protein that retains some or all of the activity or function (e.g., biological activity or function, such as enzymatic activity) of the reference polypeptide/protein, such as, e.g., the ability to bind and/or interact with or modulate another protein or polynucleic acid.
  • the functional fragment of the polypeptide of the present invention may have the ability to bind to ADP-actin and modulate ADP-actin remodelling.
  • the functional fragment can be any size, provided that the fragment retains the activity/functionality of the reference polypeptide /protein.
  • polypeptide As used herein, the terms “peptide”, “polypeptide” and “protein” are used interchangeably to denote a polymer of at least two amino acids covalently linked by an amide bond. Whereas peptides are considered to be short amino acid chains, polypeptides are long amino acid chains and proteins tend to have a stable structure and may comprise modifications (e.g., glycosylation or phosphorylation).
  • polypeptide or “protein” may encompass a naturally-occurring as well as artificial (e.g., engineered or variant) full-length polypeptide/ protein as well as a functional fragment of the polypeptide/protein.
  • sequence "variant” refers to an amino acid sequence that is altered by one or more amino acids of the non-variant reference sequence, but retains the ability to recognize its target and effect its function.
  • a polypeptide variant of the present invention is altered by one or more amino acids of the non-variant polypeptide reference sequence, but retains the ability to bind to ADP-actin and modulate ADP-actin remodelling.
  • ADP-actin remodelling may include, for example, enhancing ADP-actin nucleation, elongation/polymerization, crosslinking, bundling, stabilization and/or inhibition of actin depolymerization.
  • the variant may have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties (e.g., replacement of leucine with isoleucine). More rarely, a variant may have "non-conservative" changes (e.g., replacement of glycine with tryptophan).
  • Analogous minor variations may also include amino acid deletions or insertions, or both. Guidance in determining which amino acid residues may be substituted, inserted, or deleted without abolishing biological activity may be found using computer programs well known in the art, for example, DNASTAR® software (DNASTAR, Inc. Madison, Wisconsin, USA). A homologous sequence from another species is considered to be a variant of the polypeptide of the invention.
  • nucleotide and “nucleic acid” refer to naturally occurring ribonucleotide or deoxyribonucleotide monomers, as well as non-naturally occurring derivatives and analogs thereof.
  • Nucleotides can include, for example, nucleotides comprising naturally occurring bases (e.g., adenosine, thymidine, guanosine, cytidine, uridine, inosine, deoxyadenosine, deoxythymidine, deoxyguanosine, or deoxycytidine) and nucleotides comprising modified bases known in the art.
  • polynucleotide and “polynucleic acid” relate in general to polyribonucleotides and polydeoxyribonucleotides, it being possible for these to be non-modified RNA or DNA or modified RNA or DNA.
  • recombinant as used herein, means that a molecule (e.g., a polynucleic acid or a polypeptide) has been artificially or synthetically (i.e., non-naturally) altered by human intervention. The alteration can be performed on the molecule within, or removed from, its natural environment or state.
  • the term "subject" is herein defined as a vertebrate, particularly a mammal, more particularly a human.
  • the subject may particularly be at least one animal model, e.g., a mouse, rat and the like.
  • the subject may be a human.
  • treatment refers to ameliorating, therapeutic or curative treatment.
  • the present invention is based, in part, on the development of a recombinant polypeptide capable of regulating actin remodelling.
  • the recombinant polypeptides disclosed herein are derived from the SPA2 gene of Saccharomyces cerevisiae and are highly selective for ADP- actin (as opposed to the ATP state of actin, ATP-actin for example).
  • the recombinant polypeptides disclosed herein are able to modulate ADP-actin remodelling induced under energy starvation conditions.
  • ADP-actin is a crucial component of the actin cytoskeleton network
  • selective targeting of ADP-actin may provide novel pathways for the development of actin cytoskeleton biosensors, the development of drugs and therapies for actin-associated diseases as well as conditions related to energy depletion-related cellular processes, including aging and stress adaptation.
  • a recombinant polypeptide comprising an amino acid sequence of at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 1 , or SEQ ID NO: 2, or a functional fragment or functional variant thereof, wherein the polypeptide, fragment or variant thereof specifically binds to ADP-actin and modulates ADP-actin remodelling.
  • actin remodelling is a coordinated biochemical process which plays an important role in governing the shape and movement of a biological cell.
  • the actin remodelling process may involve actin monomers elongating into polymers to form actin filaments and disassembling back into monomers in response to signalling cascades from environmental cues.
  • the actin remodelling cycle may comprise the nucleation stage, elongation stage/polymerization, termination stage, capping, crosslinking, bundling, stabilization, cargo motoring, and/or disassembly/depolymerization.
  • the actin remodelling may preferably refer to ADP-actin remodelling, and may comprise enhancing/upregulating ADP-actin nucleation, elongation/polymerization, crosslinking, bundling, stabilization and/or inhibiting actin depolymerization.
  • the modulating of ADP-actin remodelling is selected from one or more of the group comprising enhancing ADP-actin nucleation, elongation/polymerization, crosslinking, bundling, stabilization and/or inhibition of actin depolymerization.
  • the polypeptides disclosed herein may advantageously function as a nucleation factor to enhance ADP-actin nucleation, i.e. , initiating the formation of an ADP-actin nucleus from which an actin filament may elongate.
  • the polypeptides disclosed herein may function as the nucleation factor of ADP-G-actin.
  • the polypeptides disclosed herein may advantageously function as a elongation factor to enhance ADP-actin elongation, i.e., enhancing/upregulating the polymerization of ADP-actin filament at one end to elongate the actin filament.
  • the polypeptides disclosed herein may function as the elongation factor of ADP- G-actin.
  • polypeptides disclosed herein may enhance ADP-actin nucleation and/or elongation/polymerization.
  • the polypeptides disclosed herein may advantageously function as a crosslinking factor to enhance ADP-actin crosslinking and/or bundling, for example, enhancing the assembly of actin filaments into bundles and various architectures of actin networks to drive different critical cellular processes, such as but not limited to cell migration, membrane protrusion, endocytosis, vesicular transport, exocytosis, and the ABP function, e.g. motor activities.
  • the polypeptides disclosed herein may function as a crosslinking factor of ADP-F-actin.
  • the polypeptides disclosed herein may function as a stabilization factor to enhance actin filament stabilization, for example, enhancing the filaments’ ability to maintain its structure and/or increasing the filaments’ resistance to depolymerization.
  • the polypeptides disclosed herein may function as a stabilization factor of ADP- F-actin.
  • polypeptides disclosed herein may also advantageously act as a multifunctional ADP-actin modulator.
  • actin remodelling may be induced under energy starvation conditions.
  • the ADP-actin remodelling may occur under an energy starvation condition.
  • the energy starvation condition may be glucose starvation.
  • sequences of the polypeptides of the present disclosure may be sufficiently varied so long as the peptides maintain their functionality and can exhibit the required activity (for example, the ability to bind to ADP-actin and modulate ADP-actin remodelling).
  • a minimal binding motif would be required for the peptide to bind to its target and exhibit its functionality.
  • methods of determining a protein sequence identity are known in the art.
  • the polypeptide may comprise an amino acid sequence of at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 1.
  • the polypeptide may comprise the amino acid sequence set forth in SEQ ID NO: 1 .
  • the polypeptide may consist of the amino acid sequence set forth in SEQ ID NO: 1.
  • the polypeptide may comprise an amino acid sequence of at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 2, which corresponds to amino acids 1-535 of SEQ ID NO: 1.
  • the polypeptide may comprise the amino acid sequence set forth in SEQ ID NO: 2.
  • the polypeptide may consist of the amino acid sequence set forth in SEQ ID NO: 2.
  • the polypeptide may comprise or consist of an amino acid sequence of at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to an amino acid sequence selected from the group comprising SEQ ID NO: 3, which corresponds to amino acids 281-535 of SEQ ID NO: 1 , SEQ ID NO: 4, which corresponds to amino acids 389-535 of SEQ ID NO: 1 , SEQ ID NO: 5, which corresponds to amino acids 389-433 of SEQ ID NO: 1 and SEQ ID NO: 6, which corresponds to amino acids 409-428 of SEQ ID NO: 1.
  • the polypeptide may consist of the amino acid sequence set forth in SEQ ID NO: 3. In certain embodiments, the polypeptide may consist of the amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments, the polypeptide may consist of the amino acid sequence set forth in SEQ ID NO: 5. In certain embodiments, the polypeptide may consist of the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, the polypeptide may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 and may enhance ADP-actin crosslinking and/or bundling.
  • the polypeptide may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 and may enhance ADP-actin nucleation and/or elongation/polymerization .
  • polypeptides disclosed herein are preferably capable of efficiently binding to actin, particularly to ADP-actin.
  • the ADP-actin may be ADP-G-actin and/or ADP-F-actin.
  • polypeptides of the present disclosure may also be monomeric or multimeric.
  • the polypeptides may be dimeric or trimeric.
  • the polypeptides may self-assemble to form multimers.
  • multimerization of the polypeptides may comprise the use of linkers and/or coiled-coil motif.
  • the multimer is a multimer of polypeptide Spa2-281-535.
  • the polypeptide may be coupled to at least one heterologous molecule.
  • the heterologous molecule may be a labelling group which allows for the detection of the polypeptides disclosed herein.
  • the heterologous molecule may be a labelling peptide such as the FLAG epitope, a labelling fluorescent polypeptide such as GFP, sfGFP, msfGFP or mRFPruby, or any variant thereof.
  • the heterologous molecule may also be a non-peptidic molecule, e.g. a fluorescent chemical labelling group such as fluorescein, and rhodamine.
  • heterologous molecule may be coupled to the polypeptides of the present disclosure without affecting its binding to ADP-actin and its ability to modulate actin remodelling.
  • a polypeptide of the disclosure may be expressed coupled to a label within a transformed cell to provide the location of the polypeptide in relation to actin undergoing remodelling and, for example, provide an indication of energy status within the cell and/or a relative amount of ADP-actin.
  • any other heterologous molecules comprising the desired labelling properties may be suitable for use in the practice of the present invention.
  • the polynucleic acid molecule encoding the recombinant polypeptide as described herein.
  • the polynucleic acid molecule may be a single- or double-stranded DNA or RNA molecule.
  • the polynucleic acid molecule may comprise a sequence of at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the polynucleic acid sequence set forth in SEQ ID NO: 8 or in SEQ ID NO: 9, or a fragment thereof.
  • the polynucleic acid sequence comprises or consists of the polynucleic acid sequence set forth in the group comprising SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12 and SEQ ID NO: 13.
  • the polynucleic acid molecule may be operatively linked to a promoter sequence.
  • the promoter sequence is suitably adapted to effectively control the gene expression in a suitable host cell, e.g. a prokaryotic or eukaryotic host cell.
  • the eukaryotic host cell may be a fungal cell, a plant cell or a mammalian cell.
  • the recombinant cell may be a prokaryotic cell such as an E. coli cell.
  • the recombinant cell may be a eukaryotic cell such as a fungal cell, a plant cell or an animal cell.
  • the animal cell may be a fish, insect, bird or mammalian cell.
  • standard methods of transfecting or transforming recombinant cells known in the art, such as calcium phosphate precipitation or electroporation may be applied suitably in the present invention.
  • the transgenic organism may be a fungus, a plant or an animal.
  • the transgenic organism may be a nematode (e.g., C. elegans), an insect (e.g., Drosophila), a fish (e.g., Danio rerd), a mammal (e.g., a rodent) or a plant (e.g., Arabidopsis thaliana).
  • nematode e.g., C. elegans
  • an insect e.g., Drosophila
  • a fish e.g., Danio rerd
  • a mammal e.g., a rodent
  • a plant e.g., Arabidopsis thaliana
  • a recombinant polypeptide, polynucleic acid molecule, or a recombinant cell as described in the present invention in methods for the detection and/or modulation of ADP-actin remodelling, or in drug screening of candidate modulators of ADP-actin remodelling.
  • the recombinant polypeptides, polynucleic acid molecules and cells disclosed herein may thus be suitably adapted for use as a biosensor for the detection of ADP-actin activity within a cell, preferably within a living cell.
  • polypeptides of the invention can be tagged with a fluorescence tag and developed to identify ADP-actin forms.
  • a cell may be transfected with a polynucleic acid expression construct for production of GFP-tagged recombinant polypeptide of the invention.
  • total internal reflection (TIRF) microscopy may be used to image dynamic ADP-actin remodelling.
  • the present disclosure also provides a method for the detection and/or modulation of ADP-actin remodelling, or in drug screening of candidate modulators of ADP-actin remodelling, wherein the method may comprise (i) expressing a tagged recombinant polypeptide of the first aspect in a cell; (ii) detecting whether the tagged polypeptide associates with ADP-actin or F-actin using total internal reflection (TIRF) microscopy; and for drug screening, exposing the cell before step (ii) to candidate modulators of ADP-actin remodelling.
  • TIRF total internal reflection
  • ADP-actin-associated cellular processes such as but not limited to, cell movement /migration, cell shape, cell-matrix interaction, cell polarity, muscle development, cell division and/or differentiation may also be detected by means of the present invention.
  • the present invention may also be suitably adapted to detect glucose-starvation or other energy depletion conditions-related cellular processes, including aging and stress adaptation.
  • the present invention may also be suitably applied for use in pharmaceutical research.
  • actin cytoskeleton plays an important role in practically all types of cellular morphogenesis
  • ADP-actin may be thus used as a marker or target in cellbased screening methods and/or therapeutic approaches.
  • the present invention may be suitably applied for use in drug screening such as cell-based screens for drugs, or in the study of actin-associated diseases such as myopathies, familial thoracic aortic aneurysms, heart diseases and, more particularly, Baraitser-Winter syndrome.
  • the present invention may also be useful in the study and/or detection of cancer progression, aging progression, and microbial and/or viral infections in mammalian and/or plant hosts.
  • the use is in-vitro. In some embodiments, the use is not in a human being.
  • composition comprising the recombinant polypeptide of the present invention, and a pharmaceutically acceptable carrier.
  • Suitable pharmaceutical carriers typically will contain inert ingredients that do not interact with the agent or active ingredient.
  • Suitable pharmaceutical carriers for parenteral administration include, for example, sterile water, physiological saline, bacteriostatic saline (saline containing about 0.9% 10 mg/ml benzyl alcohol), phosphate-buffered saline, Hank’s solution, Ringer’s lactate and the like.
  • Formulations can also include small amounts of substances that enhance the effectiveness of the active ingredient (e.g., emulsifying agents, solubilizing agents, pH buffering agents, wetting agents). Methods of encapsulating compositions (such as in a coating of hard gelatin or cyclodextran) are known in the art.
  • the agent can be solubilized and loaded into a suitable dispenser for administration (e.g., anatomizer or nebulizer or pressurized aerosol dispenser).
  • a recombinant polypeptide in another aspect, there is provided a recombinant polypeptide, a polynucleic acid molecule, a recombinant cell or a pharmaceutical composition as disclosed in the present invention for use in medicine.
  • a recombinant polypeptide, a polynucleic acid molecule or a recombinant cell as disclosed herein in cell-based screens for drugs or in the study of diseases associated with actin-remodelling, wherein the use is not in a human being.
  • a method of treating a disease associated with actin-remodelling comprising administering to a subject in need thereof, a therapeutically effective amount of a recombinant polypeptide or a pharmaceutical composition as disclosed herein, to a patient in need thereof.
  • a recombinant polypeptide or a pharmaceutical composition of the present invention in the manufacture of a medicament for the treatment of a disease associated with actin-remodelling.
  • the disease associated with actin-remodelling may include but is not limited to the group comprising cancer, myopathies, familial thoracic aortic aneurysms, heart diseases such as hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), Baraitser- Winter syndrome and auto-inflammatory diseases.
  • the disease associated with actin-remodelling may be linked to energy depletion-related cellular processes, such as aging.
  • the composition of the present disclosure can be delivered to a subject in need thereof by a variety of routes of administration including, for example, oral, nasal, dietary, topical, transdermal, or parenteral (e.g., intra-arterial, intravenous, intramuscular, subcutaneous injection, intradermal injection) routes of administration. Administration can be local or systemic. Preferably, the medicament is formulated for subcutaneous or intravenous administration.
  • the actual dose of a therapeutic amount of the composition disclosed herein and treatment regimen can be determined by a skilled physician, taking into account the nature of the condition being treated, and patient characteristics.
  • Yeast strains in S288C background, primers, and plasmids used in this work are listed in Table 2.
  • Yeast strains were grown in standard-rich media or complete synthetic media as previously described (Miao et al., Nature comms 7, 11265 (2016); Xie, Y. et al., Nature comms 10, 1-18 (2019)).
  • Genomic C-terminal fluorescent fusion tagging at the endogenous locus was performed using the lithium acetate-based method by transforming PCR-fragments derived from pFA6a-GFP(S65T)-kanMX or pFA6a-GFP(S65T)-His3MX6 (Longtine et al., Yeast 14, 953-961 (1998); Miao et al., Nature comms 7, 11265 (2016); Xie, Y. et al., Nature comms 10, 1-18 (2019)).
  • the yeast strains expressing Spa2-truncating variants were generated from the integration vector pRS305 containing Spa2 genomic +500 bp and DNA fragment 741-1605bp.
  • Transformants were selected on the YPD agar plates containing 200 ug/ml G418 or synthetic complete media supplemented with appropriate amino acids.
  • the information for yeast strains and primers used are indicated in Tables 2 and 3.
  • Cells were immobilized onto Concanavalin A (ConA, 1 mg/ml)-coated coverslips and imaged by Spinning disc confocal (SDC) microscopy and super resolution SDC-structured illumination microscopy (SDC-SIM) were performed on a setup built around a Nikon Ti2 inverted microscope equipped with a Yokogawa CSLI-W1 confocal spinning head, a Plan-Apo objective (100x1.45-NA), a back-illuminated sCMOS camera (Prime95B; Teledyne Photometries), and a super resolution module (Live-SR; GATACA Systems) based on structured illumination with optical reassignment and image processing improvement (Roth and Heintzmann, Method
  • the method known as multifocal structured illumination microscopy (York et al., Nat Methods 9, 749-754 (2012)), allows combining the doubling of the resolution with the optical sectioning capability of confocal microscopy.
  • the maximum resolution is 128 nm with a pixel size in super resolution mode of 64 nm.
  • Excitation light was provided by 488-nm/150mW (Vortran) (for GFP), and all image acquisition and processing was controlled by MetaMorph (Molecular Device) software.
  • the images were acquired continuously at a 0.25-pm interval for a total range of 7.5 pm in the z-direction, using an exposure time of 200 ms for actin cable, 100 ms for Spa2 localization, and 2* binning.
  • Queen plasmid used was purchased from NBRP Yeast Genetic Resource Center (NBRP/YGRC) (Tanaka et al., Nat Commun 9, 1860 (2018)). Queen-expressing budding yeast strains were generated by linearizing the constructs by Pstl and then inserting them into the his3 1 locus of the YMY2012 and glucose relevant mutant strains hxk2 , gprl , snf3l rgt2l .
  • Budding yeast cells were immobilized on the glass cover slip-coated with concanavalin A at 1 mg/ml before imaging, the immobilized cells were imaged using a Spinning disc confocal (SDC) microscopy on a setup built around a Nikon Ti2 inverted microscope equipped with a Yokogawa CSLI-W1 confocal spinning head, a Plan-Apo objective (100x1 ,45-NA), a back-illuminated sCMOS camera (Prime95B; Teledyne Photometries), Imaging lasers were provided by 405nm/100mW (Vortran), 488nm /150mW (Vortran), combined in a laser launch (iLaunch, GATACA Systems).
  • SDC Spinning disc confocal
  • QUEEN ratio was calculated using Fiji software (Schindelin et al., Nat Methods 9, 676-682 (2012)) and was calculated as follows. First, both the QUEEN images were converted to signed 16-bit floating-pointed grayscale and the QUEEN signals in cells were corrected for background by subtracting the mean pixel values from the signal outside the cells, next The pixel values of the ex405 image were divided by those of the ex480 image to calculate the QUEEN ratio at each pixel. The mean ratio in pixels was used to represent the ATP level in cells.
  • coverslips (Marienfeld Superior) were cleaned with 20% sulfuric acid overnight and rinsed thoroughly with sterile water. The coverslips were then coated with 2 pg/ml biotin-PEG-silane (Laysan Bio Inc.) in 80% ethanol (pH 2.0, adjusted by HCI) and 2 mg/ml methoxy-PEG-silane at 70 °C for overnight. The next day, coverslips were rinsed thoroughly with sterile water and dried in nitrogen stream and kept at -80 °C before use.
  • biotin-PEG-silane Laysan Bio Inc.
  • the functionalized coverslip was attached to a plastic flow cell chamber (Ibidi, sticky-Slide VI 0.4), followed by a 30 s incubation with HBSA buffer (20 mM Hepes, pH 7.5, 1 mM EDTA, 50 mM KCI, and 1 % bovine serum albumin) and then 60 s incubation with 0.1 mg/ml streptavidin in HEKG10 (20 mM Hepes, pH 7.5, 1 mM EDTA, 50 mM KCI, 10% [vol/vol] glycerol).
  • HBSA buffer 20 mM Hepes, pH 7.5, 1 mM EDTA, 50 mM KCI, and 1 % bovine serum albumin
  • the flow cell chamber was washed by TIRF buffer (10 mM imidazole, 50 mM DTT, 15 mM glucose, 50 mM KCI, 1 mM MgCl2, 1 mM EGTA, 100 pg/ml glucose oxidase, and 0.5% methylcellulose [4000 cP], 0.3 mM ADP or ATP, pH 7.4).
  • Recombinant protein prepared in TIRF buffer were mixed with 3 pM G-ADP-actin or 0.5 pM G-ATP-actin (10% Oregon Green 488 labeled, 0.5% biotin labeled) before flowing into the chamber.
  • Time-lapse images were acquired at room temperature at 5-s intervals for 10 min or 60 min with the above-mentioned spinning-disc confocal system with TIRF module (iLasV2 Ring TIRF, GATACA Systems).
  • TIRF module iLasV2 Ring TIRF, GATACA Systems.
  • actin elongation rate quantification the fast elongation end of individual filament was traced by hand for a time period of 2 min each. We used the conversion factor of 370 subunits per micrometre of F-actin to calculate the elongation rate.
  • the immobilized cells were imaged by SDC-SIM with a Plan-Apo objective (100x1.45-NA), a back-illuminated sCMOS camera (Prime95B; Teledyne Photometries), and a super resolution module (Live-SR; GATACA Systems) based on structured illumination with optical reassignment and image processing improvement (Roth and Heintzmann, Methods Appl Fluoresc 4, 045005 (2016)).
  • the method known as multifocal structured illumination microscopy (York et al., Nat Methods 9, 749-754 (2012)), allows combining the doubling of the resolution with the optical sectioning capability of confocal microscopy.
  • the maximum resolution is 128 nm with a pixel size in super resolution mode of 64 nm.
  • Excitation light was provided by 488-nm/150mW (Vortran) (for GFP), 561-nm/100mW (Coherent) (for mCherry/mRFP/tagRFP) and all image acquisition and processing was controlled by MetaMorph (Molecular Device) software.
  • Each of condition cells were cropped, background was subtracted from average projections in Fiji. Individual cables in average projections of mother cells were traced in Fiji using a line that encompassed the entire width of the cable.
  • the mean fluorescence signal intensity per filament and total fluorescence actin cable intensity per cell was measured as previously described (Garabedian M. V. et al., J cell Biol. 217, (2016)). The mean intensity per filament and total intensity per cell after ES were normalized with glucose control.
  • Spa2 was flown in over the surface of the control and ligand for 60 s and dissociated with buffer (20 mM HEPES, 50 mM NaCI, pH 7.4) for 150 s at a rate of 30 pl/min. Spa2 was injected at gradient concentrations as 20, 10, 5, 2.5, 1.25, 0.625, 0.3125, and 0.15625 pM. The chip surface with left-over protein captured was regenerated by treating with 50 mM NaOH for 3 s at 100 pl/min after each cycle. The kinetics of binding was analyzed by the Biacore T200 Evaluation software (GE Healthcare). The sensorgram for the binding experiment was normalized with the reference cell and fitted to the bivalent analyte model using Biacore T200 Evaluation software (GE Healthcare). ii) Recombinant protein expression and purification
  • Spa2-281 , Spa2-281-535, Spa2-535 and Spa2-281-535-trimer proteins were expressed and purified from Escherichia coli (BL21(DE3) Rosetta T1 R).
  • Cells were cultured 2 L TB medium (24 g yeast extract per liter, 20 g tryptone per liter, 4 ml glycerol per liter, phosphate buffer pH 7.4) containing 50 pg/ml of Kanamycin at 37 °C to an GD600 of 0.6 before induction by 0.5 mM IPTG at 16 °C overnight.
  • the cells were harvested by centrifugation at 4 °C, 5000 x g (rotor JA10) for 15 min.
  • the pellet was resuspended in 20 mM Hepes PH 7.4 and 500 mM NaCI, 20mM Imidazole, 1 mM PMSF, protease inhibitor Cocktail Set III, EDTA free from Thermo Fisher and lysed by LM20 microfluidizer (20000 psi).
  • the lysate was clarified by centrifugation at 25,000 x g for 1 h using rotor JA25.5 (Beckman Coulter).
  • the supernatant was purified by FPLC AKTAxpress system (GE Healthcare) using Ni-NTA affinity chromatography.
  • the elution fractions containing targeted proteins from gradient elution with increasing imidazole concentration were pooled and further purified by size-exclusion chromatography using a HiLoad 16/600 Superdex75 column (GE Healthcare) in 20 mM Hepes and 500 mM NaCI. Proteins were flash-frozen in liquid N2 prior to storage at -80°C in small aliquots.
  • the filtration was repeated for three times and the actin-rich extracts were combined and subjected to centrifugation at 18,000 x g by rotor JA-25.5 (Beckman). Afterwards, the clear actin-rich supernant added 50 mM KCI and 2 mM MgCl2 to allow actin polymerization with slow stirring at 4 °C for 1 h. To remove F-actin binding proteins, KCI powder was used to add slowly until a final concentration of 0.8 M that was subsequently stirred slowly for an additional 30 min. The solution was subjected for 95,800 x g centrifugation for 3 h with rotor Ti45 (Beckman) at 4 °C to collect the polymerized F-actin.
  • F-actin pellet was rinsed with 1 ml G-buffer and all the F-actin pellets were transferred to the 10 ml homogenizer with 5 ml G-buffer and the pellet was homogenized by moving up and down. To further depolymerize the F-actin, 4 cycles of sonication with 3 s on and 10 s off were applied. F-actin were then dialyzed against 1 liter of G-buffer without DTT overnight. The next day, we changed to 1 liter of new G-buffer without DTT and kept on dialysis.
  • Recombinant Spa2 variants (10% Alexa488-labled) were incubated for 5 min at room temperature before applying 5 pl on the coverslip and being imaged by SDC-SIM. A serial dilution of protein concentration and ionic strength were performed by starting from the high concentration proteins examined at 500 mM NaCI after protein purification.
  • AUC Sedimentation Velocity (ALIC-SV) experiments were performed on Beckman Proteome Lab XL-I Analytical Ultracentrifuge using an 8-hole An-50 Ti analytical rotor. Samples were dialyzed overnight in buffer (20 mM Hepes, pH 7.4, 50 mM NaCI) and loaded into 2-sector cells fitted with 1.2 cm epon centerpiece and quartz windows. The samples were centrifuged at 30,000 rpm or 45,000 rpm at 20 °C and absorbance at 280 nm was recorded every 5-10 minutes during 15 hours centrifugation. The data were analyzed with SEDFIT software using c(s) and c(s,ffO) size distribution models, and plotted with GUSSI software. Sedimentation coefficients were standardized to s20,w using the partial specific volume of the proteins (calculated using SEDFIT software), solvent density, and viscosity (calculated using SEDNTERP software).
  • a 10 pM G-actin prepared in G-buffer was converted to Mg2+-ATP-actin or Mg2+-ADP-actin on ice for 5 min before being mixed with the examined recombinant proteins in the G buffer.
  • the actin polymerization was initiated by adding 10* KME (500 mM KCI, 10 mM MgCl2, and 10 mM EGTA) buffer mix in a total reaction volume of 50 pl for 30 min at room temperature.
  • acti-stain 488- or acti-stain 565- phal loidin (Cytoskeleton, Inc.) at a final concentration of 0.5 pM ATP-actin or 3 pM ADP-actin for 5 min before being diluted by F-Buffer (G-buffer plus 1x KME) and applied on the polylysine (0.01%)-coated coverglass for microscopic imaging using a *100 oil objective lens.
  • Spa2-535 sequence prediction and conservative analysis To identify the Spa2-535 homologs and perform conservative analysis, the query Spa2-535 sequence was submitted in FungiDB (https://fungidb.org/fungidb/) with default parameters. The top 100 hits of Aip5 homologs were chosen from the species. Their correspondent sequence alignment was performed in the online server Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/) and the phylogenetic tree was generated by the interactive tree of life (http://itol.embl.de/).
  • ANCHOR http://anchor.enzim.hu/
  • PHYRE2 protein fold recognition server http://www.sbg.bio.ic.ac.uk/ ⁇ phyre/
  • LI20S cell were used for Lipofectamine transfection.
  • Cells were grown in DMEM (high glucose, GlutaMAXTM, Gibco) supplemented with 10% fetal bovine serum (GE HyClone),100 units/ml penicillin and 100 pg/ml streptomycin at 37°C in a CO2 incubator.
  • DMEM high glucose, GlutaMAXTM, Gibco
  • GE HyClone 10% fetal bovine serum
  • streptomycin 100 pg/ml streptomycin at 37°C in a CO2 incubator.
  • sequences encoding yeast Spa2 were amplified by PCR using cDNA clones from yeast Spa2 as templates and inserted into the pEGF-N1 vector.
  • LI20S were transiently transfected with the plasmids described above using Lipofectamine 3000 (Invitrogen, USA) following the manufacturer’s protocol and grown overnight on around glass-bottom dish (Thermo Fisher) at 37°C in a CO2 incubator for protein expression. Transfected cells were treated with DMEM including 0.45% 2-DG but no glucose for 10 min, then fix cells and stained with phalloidin 565.
  • ATP:ADP decrease conditions were also examined by using the glucose analog 2- deoxyglucose (2-DG).
  • 2-DG diminishes the glycolytic supply of ATP and aerobic respirationregenerated ATP in all WT, hxk2A, and cbp2A strains, in which actin cables were all stabilized without recovery over time (FIGS. 6B to 6D).
  • the changes in the ATP:ADP ratio and actin cable bundling were similar to those in the WT (FIGS. 6E to 6H).
  • ABS potential regulatory actin-binding protein
  • Spa2 that are responsible for ES-triggered actin cable remodelling. Based on the prediction of structured and intrinsically disordered region (IDR) using IUPred2A (Meszaros et al., Nucleic Acids Res 46, W329-W337 (2016)), the inventors created GFP-tagged Spa2 variants with truncation at the C-terminus. In vivo localization experiments showed that Spa2-535 was the shortest examined version that retained ES-triggered filament formation (FIGS. 2A to 2C; FIG. 8A). Spa2-535 started to display a weak filamentous pattern on its own under normal growth conditions (FIG. 2B).
  • IDR intrinsically disordered region
  • Spa2-281-535-GFP and Spa2-281-GFP showed cytoplasmic puncta and diffusive patterns, respectively, where neither of them can form cables (FIG. 2B).
  • spa2-535 cells, but not spa2-281-535 and spa2-281 cells maintained the ability to crosslink and stabilize actin cables by ES (FIGS. 2E to 2F).
  • spa2-281-535, but not spa2-281 could still increase actin cable production upon ES (FIG. 2G; Fig. 8E), indicating that Spa2-281-535 functions in cable polymerization but not F-actin crosslinking.
  • Spa2-535-GFP filaments also showed ATP:ADP ratio-dependent bundling upon GS in the WT background and cbp2A, similar to the thickening of Spa2-GFP and Abp140-3xGFP cables, but showed little response in hxk2A (FIGS. 8B to 8D).
  • Spa2 is an ADP-specific actin nucleator that functions through Spa2-281-535
  • the inventors could not find a homologous region of known actin nucleators in Spa2-535, including the Arp2/3 complex, formins, or WH2-domain family proteins.
  • Spa2-535-mediated increase in actin polymerization the potential interactions between Spa2- 535 and G-actin in both ATP- and ADP-bound states were first examined.
  • Recombinant Spa2- 535, Spa2-281-535, and Spa2-281 proteins expressed and purified from the bacteria (FIG. 9A) were mixed with ADP-G-actin or ATP-G-actin, followed by binding affinity measurement using a fluorescence anisotropy assay.
  • Spa2-535 and Spa2-281-535 showed a similar affinity toward ADP-G-actin at Kd values of 0.36 ⁇ 0.11 pM and 0.24 ⁇ 0.04 pM, respectively, whereas no ATP-G-actin binding was detected (FIG. 3A; FIG. 9B).
  • Spa2-281 bound neither ADP-G- actin nor ATP-G-actin (FIG. 3A; FIG. 9B).
  • Actin nucleation and elongation activities were next examined via a total internal reflection fluorescence microscopy (TIRFM)-actin polymerization assay.
  • TIRFM total internal reflection fluorescence microscopy
  • Spa2-535 dimers While a trimerized Spa2-281-535 seems to support the ADP-G-actin nucleation well and the ⁇ 3-fold increase in elongation rate, a better understanding of the nucleation and elongation activity of Spa2-535 dimers is needed (FIGS. 3F and 3G).
  • SPR surface plasmon resonance
  • Spa2-535 displayed weak inter-dimer interactions, which is likely derived from the self-interactions between the IDR (Spa2-281 ; SEQ ID NO: 5).
  • Spa2- 281 but not Spa2-281-535 (SEQ ID NO: 2), showed obvious self-association (FIGS. 4D to 4F).
  • IDR-containing proteins Multivalent interactions of IDR-containing proteins (IDP) have been found to promote cytoskeleton nucleation by undergoing phase separation (Sun, H. et al., Nature comms 12, 4064 (2021); King, M.R., and Petry, S., Nature comms 11, 270 (2020); Case, L.B. et al., Science 363 (2019)).
  • IDR IDR-containing proteins
  • Spa2-535 proteins are immiscible with aqueous solvents and exhibit protein concentration- and ionic strength-dependent macromolecular condensation by forming spherical droplets (FIGS. 4G and 4H).
  • the homotypic assembly of Spa2-535 displayed LLPS properties with coalescence (FIG. 4I) and high fluidity with rapid recovery after fluorescence photo-bleaching (FIG. 4J; FIG. 10A).
  • Spa2-535 proteins were decorated along filamentous ADF-F-actin as puncta, whereas Spa2-535 proteins condensed on ADP-F-actin that associated and cross-linked actin filaments (FIG. 5A).
  • Spa2-535 mediated actin crosslinking in a concentration-dependent manner with a high correlation to Spa2 homotypic phase separation behaviour (FIG. 10B), suggesting Spa2 valency-dependent F-actin bundling.
  • Spa2-535-induced F-actin bundles did not develop into pearling and spindle-shaped anisotropic F-actin droplets, which can be generated by actin crosslinkers, such as mammalian filamin (Weirich, K.L. et al., Proc Natl Acad Sci USA 114, 2131-2136 (2017)) and phase-separated bacterial effector XopR (Sun, H. et al., Nature comms 12, 4064 (2021)).
  • actin crosslinkers such as mammalian filamin (Weirich, K.L. et al., Proc Natl Acad Sci USA 114, 2131-2136 (2017)) and phase-separated bacterial effector XopR (Sun, H. et al., Nature comms 12, 4064 (2021)).
  • actin crosslinkers such as mammalian filamin (Weirich, K.L. et al., Proc Natl
  • a multivalent Spa2-281-535 was created via protein engineering using a trimerization coiled-coil (CC) motif (Khairil Anuar, I.N.A. et al., Nature comms 10, 1734 (2019)) (Spa2-281-535-trimer, FIG. 11A).
  • the produced recombinant Spa2-281-535 trimer showed higher-order oligomers than the CC-defined trimeric state (FIGS. 11 B and 11C), likely due to mismatched intermolecular interactions in the combination of the trimeric CC motif and Spa2-281-535 regions.
  • Spa2-281-535 trimer showed potent ADP-G-actin- specific nucleation activity, which was even higher than that of trimeric Spa2-281-535 (FIGS. 11 D to 11 H).
  • Spa2-281-535 trimers are also capable of crosslinking ADP-F-actin with a slightly faster F-actin convergence than Spa2-535 over time at the same protein concentration (FIG. 5B), suggesting that the efficacy in crosslinking F-actin is dependent on the oligomerization degree.
  • no ATP-actin-dependent activities were detected for Spa2-281-535-trimer, such as nucleation (FIGS. 11 D, 11G and 11 H), binding (FIG. 111), or bundling (FIG. 11 J).
  • Spa2 structures were predicted by using Alphafold2 Software (FIGS.12A and 12B).
  • the Spa2 H1 domain spanning residues 389-433 was further subdivided into Spa2-H1a (residues 389- 407) and Spa2-H1b (residues 401-433) (FIGS. 12C and 12D).
  • Example 8 Identification and characterization of an ADP-actin sensing peptide using msfGFP- Spa2-H1b reporter
  • msfGFP was tagged into different variant Spa2 truncations (FIG. 13A), and were incubated with ADP-F-actin and ATP- F-actin. It is striking that Spa2-H1 and Spa2-H1b can specifically bind to ADP-F-actin but not ATP-F-actin (FIGS 13B, 13C).
  • GFP tagged Spa2 helix was expressed in LI2OS cell and energy starvation was performed, Spa2-H1-GFP and Spa2-H1b-GFP can colocalize with ADP-F-actin.
  • Example 9 Identification and characterization of the minimal helix reguired for ADP-actin sensing in vitro and in vivo
  • Spa2-H1 b was truncated into Spa2-H1 b1 , Spa2-H1 b2 and Spa2-H1b3 (FIGS. 14A, 14B, 14C).
  • in vitro actin binding assay was performed, and the data show that all of Spa2-H1b1 ,Spa2-H1b2 and Spa2-H1b3 can colocalize with ADP-F-actin but not ATP-F-actin (FIGS. 14D, 14E).
  • Spa2-H1 b3 was the minimum domain for ADP-F-actin binding after energy starvation (FIG. 14F - 14H).
  • the present disclosure relates to the application of SPA2-derived recombinant polypeptides in a novel mechanism of actin filament regulation under energy starvation conditions.
  • Actin polymerization a crucial cellular process typically driven by ATP, involves the transformation of globular actin (G-actin) into filamentous actin (F-actin). This transformation underpins various cellular activities, including cell motility, division, and intracellular transport.
  • G-actin globular actin
  • F-actin filamentous actin
  • the process involves ATP-actin monomers attaching to the growing end of the filament, which over time, hydrolyze to ADP.
  • the ADP-actin subunits then dissociate from the filament's other end, maintaining a dynamic equilibrium known as treadmilling.
  • the present invention introduces a significant departure from the traditional ATP-based actin polymerization process.
  • the key features of the invention include, inter alia:
  • polypeptides of the present invention may also suitably be adapted for use as biochemical reagents for ADP-actin assays, in sensing technology for ADP-actin-remodelling-related human diseases, and other biotechnological applications.
  • the present invention may be adapted to develop new diagnostic technologies for diseases that alter actin cytoskeleton polymerization and network formation.
  • the present invention could also be adapted for use in the biotech industry to engineer relevant recombinant proteins, organisms, or cells with specific sensitivity to environmental changes, such as energy starvation conditions.
  • Bound nucleotide can control the dynamic architecture of monomeric actin. Nature structural & molecular biology 29, 320-328.
  • TPX2 Phase separation of TPX2 enhances and spatially coordinates microtubule nucleation. Nature communications 11 , 270.
  • Drosophila Spire is an actin nucleation factor. Nature 433, 382- 388.
  • beta-Oxidation and autophagy are critical energy providers during acute glucose depletion in Saccharomyces cerevisiae. Proc Natl Acad Sci USA 117, 12239- 12248.
  • Polarisome assembly mediates actin remodeling during polarized yeast and fungal growth. J Cell Sci 134, jcs.247916.
  • Polarisome scaffolder Spa2-mediated macromolecular condensation of Aip5 for actin polymerization. Nature communications 10, 1-18.

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Abstract

La présente invention concerne de manière générale des polypeptides recombinants capables de réguler le remodelage de l'actine. En particulier, les polypeptides de l'invention sont sélectifs pour l'ADP-actine et peuvent moduler la remodélisation de l'ADP-actine dans des conditions de privation d'énergie. Les polypeptides recombinants de la présente invention sont également appropriés pour une utilisation dans la détection de l'activité de l'ADP-actine in vitro ou dans des cellules vivantes, dans le criblage de médicaments, dans des approches thérapeutiques et pour le traitement de maladies associées à l'actine.
PCT/SG2023/050540 2022-08-05 2023-08-04 Protéine et motifs spécifiques de l'adp-actine pour remodeler le cytosquelette de l'actine en biochimie de cellules vivantes et in vitro WO2024030080A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1258494A1 (fr) * 2001-05-15 2002-11-20 Cellzome Ag Complexe multiprotétique eucaryote
WO2003072602A2 (fr) * 2001-12-20 2003-09-04 Cellzome Ag Complexes de proteines et leurs procedes d'utilisation
US20150299720A1 (en) * 2002-02-21 2015-10-22 Monsanto Technology Llc Expression of microbial proteins in plants for production of plants with improved properties

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1258494A1 (fr) * 2001-05-15 2002-11-20 Cellzome Ag Complexe multiprotétique eucaryote
WO2003072602A2 (fr) * 2001-12-20 2003-09-04 Cellzome Ag Complexes de proteines et leurs procedes d'utilisation
US20150299720A1 (en) * 2002-02-21 2015-10-22 Monsanto Technology Llc Expression of microbial proteins in plants for production of plants with improved properties

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