In vitro Screening Assay of TCPase Modulators

Abstract

The present invention concerns an in vitro screening assay for identification of modulators of tubulin carboxypeptidase (TCPase) activity comprising the steps of: (i) Contacting (a) a substrate of TCPase enzyme comprising an amino acids sequence having at least the last 4 amino acids residues of the C-terminal sequence of an α-tubulin and/or a Microtubule Associated Protein (MAP) and, as ultimate C-terminal amino acid residue, an aromatic amino acid residue, preferably a tyrosine (Y), and b) an isolated or recombinant TCPase enzyme; in the presence or absence (control) of the modulator compound to be tested, and under conditions for substrate cleavage, preferably detyrosination, and/or liberation of a C-terminal free aromatic amino acid residue, preferably a C-terminal free tyrosine; (ii) Using reagents for detecting and measuring the signal related to substrate cleavage, preferably detyrosination, and/or liberation of the C-terminal free aromatic amino acid residue, preferably the C-terminal free tyrosine; (iii) Measuring and comparing the level of substrate cleavage, preferably detyrosination and/or the level of the C-terminal free aromatic amino acid residue, preferably the C-terminal free tyrosine in presence and in absence (control) of the compound to be tested, and (iv) Selecting the modulators of TCPase for which the level of substrate cleavage, preferably detyrosination or liberation of the C-terminal aromatic amino acid residue, preferably C-terminal free tyrosine is increased in the presence of the compound to be tested (activators of TCPase) or decreased in the presence of the compound to be tested (inhibitors of TCPase). The present invention also concerns kits for performing such in vitro screening assay.

Claims

1.-15. (canceled)

16. An in vitro screening assay for identification of modulators of tubulin carboxypeptidase (TCPase) activity comprising the steps of: (i) contacting (a) a substrate of TCPase enzyme comprising an amino acid sequence having at least the last 4 amino acids residues of the C-terminal sequence of an α-tubulin and/or a Microtubule Associated Protein (MAP) and, as ultimate C-terminal amino acid residue, an aromatic amino acid residue, and (b) an isolated or recombinant TCPase enzyme; in the presence or absence (control) of the modulator compound to be tested, and under conditions for substrate cleavage, and/or liberation of a C-terminal free aromatic amino acid residue; (ii) using reagents for detecting and measuring the signal related to substrate cleavage, and/or liberation of the C-terminal free aromatic amino acid residue; (iii) measuring and comparing the level of substrate cleavage, and/or the level of the C-terminal free aromatic amino acid residue, in the presence and in the absence (control) of the compound to be tested, and (iv) identifying the modulators of TCPase for which the level of substrate cleavage, or liberation of the C-terminal free aromatic amino acid residue, is increased in the presence of the compound to be tested (activators of TCPase) or decreased in the presence of the compound to be tested (inhibitors of TCPase).

17. The in vitro screening assay of claim 16, wherein the substrate of TCPase enzyme comprising an amino acid sequence having at least the last 4 amino acids residues of the C-terminal sequence of an α-tubulin and/or a Microtubule Associated Protein (MAP) and, as ultimate C-terminal amino acid residue, an aromatic amino acid residue, is selected from the group consisting of: a) purified or recombinant α-tubulin, b) recombinant engineered telokin, c) isolated or recombinant peptide from 4 to 50 amino acid residues comprising an amino acid sequence having at least the last 4 amino acids residues of the C-terminal sequence of an α-tubulin and/or a Microtubule Associated Protein (MAP) and, as ultimate C-terminal amino acid residue, an aromatic amino acid residue, d) isolated or recombinant microtubules, e) isolated or recombinant tubulin dimers of alpha and beta tubulin, f) microtubule-associated proteins (MAP), and g) mixtures thereof.

18. The in vitro screening assay according to claim 16, wherein the substrate of TCPase enzyme is selected from recombinant engineered telokin having at least the last 4 amino acids residues of the C-terminal sequence of an α-tubulin and a tyrosine (Y) as ultimate C-terminal amino acid residue.

19. The in vitro screening assay according to claim 16, wherein the isolated or recombinant TCPase enzyme is selected from proteins having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% amino acid sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO:1 (Ubiquitin carboxyl-terminal hydrolase 14), SEQ ID NO:2 (Ubiquitin carboxyl-terminal hydrolase 5), SEQ ID NO:3 (Methionine aminopeptidase 2), SEQ ID NO:4 (Xaa-Pro aminopeptidase 1), SEQ ID NO:5 (Tripeptidyl-peptidase 2), SEQ ID NO:6 (Vasohibin-1), SEQ ID NO:7 (Dihydropyrimidinase-related protein 1, CRM P1), SEQ ID NO:8 (Dihydropyrimidinase-related protein 2), SEQ ID NO:9 (Dihydropyrimidinase-related protein 3), SEQ ID NO:10 (Dihydropyrimidinase-related protein 4), SEQ ID N0:11 (Dihydropyrimidinase-related protein 5), SEQ ID NO:12 (Vasohibin-2), and SEQ ID NO:14 (Uncharacterized protein/VASH of Trypanosoma bruce;), in the presence or absence of SEQ ID NO:13 (Small vasohibin-binding protein).

20. The in vitro screening assay according to claim 19, wherein (a) the substrate of TCPase enzyme is selected from purified or recombinant α-tubulin from parasites, and (b) the isolated or recombinant TCPase enzyme is selected from parasites proteins.

21. The in vitro screening assay according to claim 19, wherein (a) the substrate of TCPase enzyme is a recombinant engineered telokin, and (b) the isolated or recombinant TCPase enzyme is selected from proteins having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% amino acid sequence identity with amino acid sequence SEQ ID NO:6 (Vasohibin-1) or SEQ ID NO:12 (Vasohibin-2) in the presence or absence of SEQ ID NO:13 (Small vasohibin-binding protein).

22. The in vitro screening assay according to claim 16, wherein step (ii) comprises detection of the C-terminal free aromatic amino-acid via a colorimetric assay or using fluorescent properties of the aromatic residues.

23. The in vitro screening assay according to claim 16, wherein the assay is an immuno-assay, in which the step (ii) comprises: adding an effective amount of specific labelled antibody raised against cleaved substrate, under conditions that favor the formation of a complex antibody-cleaved substrate; and means for revealing the labelled signal; or, alternatively, adding an effective amount of a primary specific antibody raised against cleaved substrate, under conditions that favor the formation of a complex antibody-cleaved substrate; adding an effective amount of a secondary labelled antibody specific of the primary antibody, under conditions that favor the formation of a complex primary antibody-cleaved substrate-labelled secondary antibody, and means for revealing the labelled signal, wherein the reaction occurs in soluble (fluid) phase or solid phase.

24. The in vitro screening assay according to claim 23, wherein the antibody is labelled with a marker selected in the group consisting of an enzyme, a fluorescent compound or fluorophore, a (chemo)luminescent compound, and a radioactive element.

25. The in vitro screening assay according to claim 23, wherein said immunoassay is an enzyme immunoassay, a fluoroimmunoassay, a luminescent immunoassay, or a radioimmunoassay.

26. The in vitro screening immunoassay according to claim 23, wherein the reaction occurs in solid phase and wherein: substrate of TCPase is coated on solid support and the TCPase enzyme, the compound(s) to be tested, and the antibodies raised against the cleaved substrate are added in the reaction solution, or the antibodies raised against cleaved substrate are coated on a solid support, and the TCPase enzyme, substrate of TCPase, the compound(s) to be tested, and secondary labelled antibodies raised against the cleaved substrate are added in the reaction solution, or the TCPase enzyme is coated on a solid support and the compound(s) to be tested and the antibodies raised against the cleaved substrate are added in the reaction solution.

27. The in vitro screening immuno-assay according to claim 23, wherein (a) the substrate of TCPase comprises dimers of alpha-tubulin extracted from SF9 cells (Spodoptera frugiperda origin), (b) the TCPase enzyme is vasohibin-2 (VASH2) in the presence or absence of SVBP, and the step (ii) of detecting and measuring the level of detyrosinated-substrate comprises at least an antibody raised against detyrosinated α-tubulin SF9 (dTyr-Ab SF9) coupled to horseradish peroxidase (HRP).

28. The in vitro screening assay according to claim 16, which is a High Throughput Screening (HTS) assay.

29. A kit for performing the in vitro screening immuno-assay of claim 23, comprising: (i) a TCPase enzyme substrate, and, as ultimate C-terminal amino acid residue, an aromatic amino acid residue, (ii) a TCPase enzyme, (iii) antibodies raised against cleaved substrate, (iv) a negative control, (v) a positive control, (vi) a fluid vessel or a solid support for coating or pre-coating either the substrate of TCPase or the TCPase enzyme, (vii) reagents for allowing contact of said substrate with TCPase enzyme in reaction conditions for substrate cleavage, (viii) reagents for detecting and measuring the level of substrate cleavage; and (ix) optionally, instructions for use.

30. A kit for performing the in vitro screening assay of claim 22 for detection of the free C-terminal aromatic amino-acid via a colorimetric assay or using fluorescent properties of the aromatic residues, comprising: (i) a TCPase enzyme substrate, and, as ultimate C-terminal amino acid residue, an aromatic amino acid residue, (ii) a TCPase enzyme, (iii) optionally, a tyrosinase for the detection of a C-terminal free-tyrosine via colorimetric assay, (iv) a fluid vessel or a solid support for coating or pre-coating either the substrate of TCPase or the TCPase enzyme, (v) reagents for allowing contact of said substrate with TCPase enzyme in reaction conditions for liberation of a C-terminal free-aromatic amino acid residue, (vi) reagents for detecting and measuring the level of the C-terminal free-aromatic amino acid residue; and (vii) optionally, instructions for use.

31. The in vitro screening assay of claim 16, wherein said substrate cleavage is detyrosination and said C-terminal free aromatic amino acid residue is a C-terminal free tyrosine.

32. The in vitro screening assay of claim 17, wherein: (a) said aromatic amino acid residue is a tyrosine (Y), (b) said purified or recombinant α-tubulin comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:19 to SEQ ID NO: 24 (Homo sapiens); SEQ ID NO:25 (Trypanosoma bruce;); SEQ ID NO:26 (Trypanosoma bruce;); SEQ ID NO:27 (Toxoplasma gondii); SEQ ID NO:28 (Spodoptera frugiperda); SEQ ID NO:29 (Drosophila melanogaster); and variants thereof, (c) said recombinant engineered telokin comprises the amino acid sequence of SEQ ID NO:17 or a variant thereof, (d) said isolated or recombinant peptide has 6 to 12 amino acid residues, and (e) said microtubule-associated protein is one from RP/EB family members 1-3.

33. The in vitro screening immunoassay of claim 26, wherein: (a) said solid support is a membrane or microplate, and (b) said antibodies raised against cleaved substrate are detyrosinated α-tubulin (dTyr-Ab), optionally labelled or combined with labelled secondary antibodies raised against primary antibodies.

34. The kit according to claim 29, wherein: (a) said TCPase enzyme substrate is selected from (i1) a purified or recombinant tubulin from Spodoptera Frugiperda (Sf) cells, (i2) a recombinant engineered telokin as the substrate of TCPase enzyme, and (i3) recombinant peptides from 5 to 20 amino acid residues comprising an amino acid sequence having at least the last 4 amino acids residues of the C-terminal sequence of an α-tubulin and/or a Microtubule Associated Protein (MAP), (b) said aromatic amino acid residue is a tyrosine (Y), (c) said TCPase enzyme is an isolated or recombinant TCPase enzyme selected from proteins having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% amino acid sequence identity with the amino acid sequence of SEQ ID NO:12 (Vasohibin-2) in the presence or absence of SEQ ID NO:13 (Small vasohibin-binding protein), (d) said antibodies raised against cleaved substrate are raised against detyrosinated α-tubulin SF9 (dTyr-Ab SF9) and coupled to horseradish peroxidase (HRP), (e) said negative control is non-tyrosinated isolated or recombinant telokin, (f) said positive control is one comprising Epoxy-Y compound, (g) said solid support is a microplate, and (h) said substrate cleavage is detyrosination.

35. The kit according to claim 30, wherein: (a) said TCPase enzyme substrate is selected from (i1) a purified or recombinant tubulin from Spodoptera Frugiperda (Sf) cells, (i2) a recombinant engineered telokin as the substrate of TCPase enzyme, and (i3) recombinant peptides from 5 to 20 amino acid residues comprising an amino acid sequence having at least the last 4 amino acids residues of the C-terminal sequence of an α-tubulin and/or a Microtubule Associated Protein (MAP), (b) said aromatic amino acid residue is a tyrosine (Y), (c) said TCPase enzyme is an isolated or recombinant TCPase enzyme selected from proteins having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% amino acid sequence identity with the amino acid sequence of SEQ ID NO:12 (Vasohibin-2) in the presence or absence of SEQ ID NO:13 (Small vasohibin-binding protein), (d) said solid support is a microplate, and (e) said C-terminal free-aromatic amino acid residue is a C-terminal free-tyrosine.

Description

DESCRIPTION OF THE FIGURES

[0243] FIG. 1: Schematic representation of tubulin purification procedure from Spodoptera Frugiperda (SF) cells (Insect cells). Tubulin was mock- or carboxypeptidase treated and analyzed on by gel electrophoresis. Next tubulin was in-gel digested using trypsin (0.5 μg/band; Gold; Promega). Peptides were then analyzed online by nano-flow HPLC-nanoelectrospray ionization using a Qexactive Plus mass spectrometer (Thermo Fisher Scientific) coupled to a nano-LC system (U3000-RSLC, Thermo Fisher Scientific).

[0244] FIG. 2: Coomassie staining of samples collected at various steps during the purification protocol of bacterially produced CRMP1 recombinant protein. Purification consists of immobilized metal affinity chromatography (IMAC). Arrow indicates recombinant CRMP1 protein.

[0245] FIG. 3: Coomassie staining of samples collected at various steps during the two-step purification protocol of bacterially produced VASH1 recombinant protein. A first step consisting of immobilized metal affinity chromatography (IMAC) was followed by ion exchange (IEX) chromatography in order to further increase purity of recombinant protein. Arrow indicates recombinant VASH1 protein.

[0246] FIG. 4: Coomassie staining of samples collected at various steps during the two-step purification protocol of bacterially produced VASH2 recombinant protein. A first step consisting of immobilized metal affinity chromatography (IMAC) was followed by ion exchange (IEX) chromatography in order to further increase purity of recombinant protein. Arrow indicates recombinant VASH2 protein.

[0247] FIG. 5: Coomassie staining of samples collected at various steps during the two-step purification protocol of bacterially produced Trypanosoma brucei VASH (TbVASH) recombinant protein. A first step consisting of immobilized metal affinity chromatography (IMAC) was followed by ion exchange (IEX) chromatography in order to further increase purity of recombinant protein. Arrow indicates recombinant TbVASH protein.

[0248] FIG. 6: Characterization of the Spodoptera frugiperda (Sf) antibody. The antibody was generated by injection of the detyrosinated tubulin sequence in rabbits (Seq: C-EGEGAEE). Immunoblot analysis of HEK cells transfected with indicated tubulin constructs. Twenty-four hours post transfection samples were collected and analyzed by immunoblot with indicated antibodies. Immunoblot analysis showed that the mouse (Mm) antibody failed to recognize Spodoptera Frugiperda (Sf) detyrosinated tubulin and inversely

[0249] FIG. 7: Polymerization of tubulin into microtubules. Microscopy analysis of rhodamine labeled microtubules. Polymerization was achieved in presence of 20 μM Taxol at 37° C. during 30 minutes. Representative imaged of the obtained microtubules fibers.

[0250] FIG. 8: VASH2 acts as autonomous TCPase. In vitro detyrosination assay using recombinant VASH1 and VASH2 proteins alone or coexpressed with Small Vasohibin

[0251] Binding Protein (SVBP). In vitro detyrosination was performed using Spodoptera Frugiperda (Sf) purified tubulin.

[0252] FIG. 9: Activation of TCPase by SVBP. In cellulo (HEK) detyrosination assay using coexpression of VASH1 and VASH2 without or in presence of Small Vasohibin Binding Protein (SVBP). Coexpression of SVBP induces VASH1 and VASH2 detyrosination activity.

[0253] FIG. 10: Recombinant human VASH2 produced in bacteria detyrosinates polymerized microtubules (MT), tubulin dimers (TD) and recombinant alpha tubulin. Oppositely, VASH1 was much less effective on microtubules and tubulin dimers and almost inactive on recombinant alpha tubulin produced in bacteria.

[0254] FIG. 11: Recombinant human VASH2 and catalytically dead (VASH2D) produced in bacteria detyrosinates a 8 amino acid peptide consisting of GEEEGEEY. The samples were doted on nitrocellulose and assessed for detyrosination of the peptide using a specific antibody detecting detyrosination.

[0255] FIG. 12: Time course analysis of VASH2-mediated detyrosination. In vitro detyrosination assay of bacterially purified VASH2. Reactions were stopped at indicated time and analyzed by immunoblot and relative optical density was measured.

[0256] FIG. 13: Immuno-assay based detyrosination assay. In vitro detyrosination assay using recombinant VASH2 brought in contact with increasing dose (0.1; 1 and 10 μM) of previously described peptide-based inhibitor (Epoxy-Y). The assay is performed using Spodoptera Frugiperda (Sf) purified tubulin.

[0257] FIG. 14: Schematic representation of preferred HTS immuno-assay. The substrate coated 96 wells plate is first brought into contact, in absence (control) or presence of a compound to be tested, with the recombinant active bacterially produced VASH2 for 15 minutes. The enzymes is washed off using appropriate buffer (preferred is phosphate buffer), and HRP-coupled antibody specifically targeting detyrosinated tubulin is brought in contact with substrate. Excess antibody is washed off and detection is performed in plate reader.

[0258] FIG. 15: Colorimetric analysis of tyrosine concentration in the reaction mixture. (A) shows the ideal excitation wavelength (300 nm). (B) Linearity of colorimetric signal obtained when free tyrosine is measured at 305 nm. The concentration of tyrosine is displayed in molarity.

[0259] FIG. 16: Colorimetric analysis of free tyrosine released by VASH2 detyrosination of recombinant human alpha tubulin. OD305 was measured of control samples (filled histogram) and VASH2-mediated tyrosine release. [0260] A. control sample containing tyrosinase, [0261] B. control sample containing recombinant human alpha tubulin, [0262] C. control samples containing tyrosinase (Tyr) and VASH2 as detyrosinase, [0263] D reaction mixture containing recombinant human alpha tubulin, VASH2 mediating tyrosine release and tyrosinase to allow detection of free tyrosine. [0264] OD305 was measured 30 min after onset of reaction at room temperature.

[0265] FIG. 17: Detyrosination assay performed using tubulin purified from Sf9 cells and recombinant SVBP-VASH2 complex in absence or presence of putative inhibitors of detyrosinase. FIG. 17a: putative inhibitors of detyrosinase disclosed in Fonrose et al (2007). FIG. 17b: Western blot. FIG. 17c: Representation of relative optical density (detyrosination/tubulin).

[0266] FIG. 18: Detyrosination assay performed using human tubulin purified from HEK293 cells and recombinant SVBP-VASH2 complex. FIG. 18a: Western blot. FIG. 18b: Representation of relative optical density (detyrosination/tubulin).

[0267] FIG. 19: Detyrosination assay performed using Sf tubulin and recombinant Trypanosoma brucei VASH protein (Western blot).

[0268] The present invention will be illustrated by the following non-limitative examples.

EXAMPLES

[0269] Material and Methods

[0270] Preparation of Substrates of TCPase:

[0271] Preferred Substrate of TCPase:

[0272] In the context of the present invention, a recombinant tyrosinated telokin has been obtained as following: the C-terminal tail of alpha-tubulin (the 11 most C-terminal residues EGEGEEEGEEY=SEQ ID NO: 39) has been engineered at the C-terminal part of the Telokin isoform (Q6PDN3-4Q15746-8). Mouse cDNA was obtained using prime script RT-PCR kit (Takara) and telokin was amplified using adapted forward and reverse primers containing cloning sites. The amplicon was extracted and purified and the expression plasmid as well as the amplicon digested adapted restriction enzymes for cloning in bacterial expression vectors (pET-28a(+) and pGEX). In particular the both sequences are recombinantly expressed in a host cell, such as an E. coli. Obtained plasmid containing the last C-terminal amino acid sequence of human alpha tubulin 1A (N-EGEGEEEGEEY=SEQ ID NO: 39) at the very end of the telokin gene was sequence verified by double digestion diagnostics and by sequencing. Next, bacteria were transformed and selected on an antibiotic containing LB-dish. A single antibiotic-resistant colony was picked and grown to confluency as a starter overnight. Next day, fresh LB medium was inoculated with the bacteria and optical density (OD600) measured at regular interval. Once the bacteria OD600 reached between 0.4 and 0.6, IPTG was added for inducing protein production. After 4 hours, cells were harvested and telokin substrate purified using IMAC technology.

[0273] We obtained a recombinant tyrosinated telokin comprising the sequence SEQ ID NO:17 (Mus musculus, fully tyrosinated variant).

[0274] Negative control Telokin lacking the most C-terminal tyrosine (Y) residue can easily be implemented. In particular, we used an isolated (non-tyrosinated) telokin comprising the sequence SEQ ID NO:15 (Homo sapiens, Uniprot Q15746-8) or SEQ ID NO:16 (Mus musculus, Uniprot Q6PDN3-4) or a recombinant (detyrosinated V1 variant) telokin comprising the amino acid sequence SEQ ID NO: 18 (the C-terminal sequence of alpha-tubulin (Tubulin alpha-1A chain—human) without the ultimate Y, has been introduced in the Telokin sequence). Various substrates can be engineered to generate additional needed controls in the assay.

[0275] Additional Substrates of TCPase: [0276] Purified or recombinant tyrosinated tubulin from Spodoptera Frugiperda (Sf) cells (Sf9, Sf21 etc.): Tubulin was purified using a TOG column (Widlund et al. 2012 MBoC) and analyzed by mass spectrometry. In particular, we used a purified or recombinant tyrosinated tubulin comprising the amino acid sequence of SEQ ID NO:28 (Uniprot G3CKA7); Tubulin was polymerized to obtain fully purified microtubules. [0277] Recombinant alpha tubulin (α-tubulin) produced in bacteria according to known methods: in particular, we used a purified or recombinant α-tubulin from human in particular comprising the amino acid sequences SEQ ID NO: 19 to SEQ ID NO: 24), or from Trypanosoma brucei in particular comprising the amino acids sequences SEQ ID NO: 25 and SEQ ID NO: 26, or from Toxoplasma gondii in particular comprising the amino acids sequence SEQ ID NO: 27, or from Drosophila melanogaster in particular comprising the amino acids sequence (SEQ ID NO:29). [0278] Tubulin dimers purified from any eukaryotic cells. [0279] Isolated or recombinant peptides from 4 to 50 amino acid residues, in particular 5 to 20 amino acids, preferably 6 to 12 amino acid residues comprising an amino acid sequence having at least the last 4 amino acids residues of the C-terminal sequence of an α-tubulin and/or a Microtubule Associated Protein (MAP) and, as ultimate C-terminal amino acid residue, an aromatic amino acid residue, preferably a tyrosine (Y), in particular comprising an amino acid sequence selected from SEQ ID NO: 33 to SEQ ID NO: 39 or variant thereof.

[0280] Preparation of Recombinant TCPases (VASH1, VASH2, CRMP1):

[0281] Enzymes are produced in E. coli bacteria using expression vectors such as pET-28a(+), pGEX or any other adapted expression vectors. The different enzymes (human VASH1, VASH2, CRMP1, Trypanosoma brucei VASH) have been cloned and bacteria have been induced according to manufacturer's guidelines. Bacteria were grown until OD600 reached between 0.4-0.8, quickly cooled in ice water for 30 minutes. Protein production was induced using IPTG and incubated at 15° C. for 24 hours under rigorous shaking. The bacteria were pelleted by centrifugation and stored at −80° C. until further use. Bacteria were resuspended in disruption buffer and mechanically disrupted using a French press. Recombinant protein was purified by immobilized metal affinity chromatography (IMAC) and if needed purity was further increased by ion-exchange column (FIG. 2-5). The recombinant proteins have been assessed for their TCPase activity on various substrates. Importantly we found that VASH2 acted as an autonomous enzyme that did not require the small vasohibin binding protein as a chaperone to enzymatically be active (FIG. 8). Advantageously, recombinant purified VASH2 protein could be stored at 4° C. for at least 3 months without noticeable loss of activity. More advantageously, recombinant VASH2 protein showed detyrosination activity on polymerized microtubule, tubulin dimers, recombinant alpha tubulin, engineered Telokin protein containing C-terminal sequence of alpha tubulin and on synthetic peptides (FIG. 10). As such recombinant VASH2 presented unexpected advantageous characteristics allowing its use in various applications such as HTS assays for identification of detyrosinase modulating compounds.

[0282] Compounds to be Tested (Putative Modulators of TCPase)

[0283] The compounds to be tested may come from chemical libraries of compounds containing a large number of diverse and different compounds. Many of these libraries may be commercially available. Alternatively, smaller libraries may be tested on different substrates (microtubule versus tubulin dimer).

[0284] Reagents and Antibodies

[0285] For western blot or immunoblot, all antibodies were applied overnight at 4° C. in phosphate-buffered saline (PBS) containing 1% BSA and 0.1% Tween (Sigma). The antibodies used were directed against: Δ1-tubulin (Abcam)), GFP (Millipore), VASH2 (Millipore), beta-tubulin (E7-DSHB), SVBP (Sigma), YL1/2 (Synaptic Systems). The antibody directed against Sf9 Δ1-tubulin (deTyr-tub) were raised in rabbits against the peptide EGEGAEE (=SEQ ID NO: 40).

[0286] Specific antibodies directed against detyrosinated tubulin were generated. The antibody was raised by injecting rabbits with a peptide composed of the very C-terminal sequence of Spodoptera frugiperda (SF) tubulin. The sequence of the peptide contains the 7 most C-terminal sequence of the protein without the most C-terminal Y (DeTyr) and an additional cysteine at the N-terminal for practical reasons (Seq: C-EGEGAEE=(=SEQ ID NO: 40). Characterization of the antibody was performed by expression of full-length mouse or Spodoptera frugiperda alpha-tubulin or detyrosinated counterpart in HEK cells (FIG. 6).

[0287] Advantageously, the said antibody is labelled, ie coupled with a peroxidase, such as the horseradish peroxidase (HRP), and the chromogenic substrate is 3,3′,5,5′-tetramethylbenzidine TMB. For the colorimetric assay the tyrosinase enzyme was purchased at Sigma-Aldrich (Tyrosinase from mushroom) and spectrophotometric analysis was performed using Nanodrop (Thermo Fisher).

[0288] Mass Spectrometry

[0289] Proteins were separated on SDS-PAGE gels (10% polyacrylamide; Mini-Protean TGX Precast Gels; Bio-Rad) and stained with Page Blue Stain (Fermentas). Gel lanes were cut into several gel pieces and destained by three washes in 50% acetonitrile and 50 mM triethylammonium bicarbonate. Proteins were in-gel digested using trypsin (0.5 μg/band; Gold; Promega).

[0290] Peptides were then analyzed online by nano-flow HPLC-nanoelectrospray ionization using a Qexactive Plus mass spectrometer (Thermo Fisher Scientific) coupled to a nano-LC system (U3000-RSLC, Thermo Fisher Scientific). Desalting and preconcentration of samples were performed on-line on a Pepmap® precolumn (0.3×10 mm; Dionex). A gradient consisting of 0-40% B in A for 100 min (A: 0.1% formic acid, 2% acetonitrile in water, and B: 0.1% formic acid in 80% acetonitrile) at 300 nl/min, was used to elute peptides from the capillary reverse-phase column (0.075×150 mm, Pepmap®, Dionex). Data were acquired using the Xcalibur software (version 4.0). A cycle of one full-scan mass spectrum (375-1,500 m/z) at a resolution of 70,000 (at 200 m/z), followed by 12 data-dependent MS/MS spectra (at a resolution of 17,500, isolation window 1.2 m/z) was repeated continuously throughout the nanoLC separation.

[0291] Raw data analysis was performed using the MaxQuant software (version 1.5.5.1) with standard settings. Used database consist of Spodoptera frugiperda entries from Uniprot, G3CKA7 sequence (Uniprot) deleted from 1 to 6 amino acids in Cterm and 250 classical contaminants (MaxQuant contaminant database). Relative abundance of peptide was estimated using Skyline 3.6.0.

[0292] E. coli Bacterial and Sf Insect Cells Culture and Protein Production (VASH1, VASH2 and SVBP)

[0293] Both bacterial and insect cells (Sf9 and Sf21) were used for recombinant protein production. Insect cells Sf9 and Sf21 cells were grown in EX-CELL® 420 Serum-free medium (14420C, Sigma-Aldrich). Cells were maintained between 1 and 10 million cells/ml in shake flasks, at 28 degrees and 140 rpm agitation. For production of human VASH1, VASH2 and SVBP cDNAs were cloned into pFastBac vector MAX efficiency DH10Bac™ Next, competent bacteria (10361-012, Invitrogen) were transformed to produce bacmids. To generate baculoviruses, 12 million Sf21 cells (1 ml) were transfected with bacmid using Cellfectin™ (P/N 58760, Invitrogen). After 5 hours incubation, medium was added and cultures were incubated for 2.5 days. Supernatant (P1) was collected by centrifugation and used to infect 200 million Sf21 cells. Cells were incubated for 2 days and supernatant (P2) was collected by centrifugation. For infection, 40 million Sf21 cells (in 20 ml) were infected with combinations of P2 encoding VASH1, VASH2 and SVBP at 1:50 dilution. Coexpression of VASH1 and VASH2 with SVBP was performed by mixing 1:1 with respective P2 supernatants. After 48 hours, infected cells were collected by centrifugation and analyzed by immunoblotting. For bacterial production of recombinant protein, competent E. coli bacterial cells were transformed with plasmid expression vector containing the protein of interest (VASH1, VASH2, human alpha tubulin, engineered telokin, CRMP1, etc.) and grown on antibiotic containing LB medium. Bacteria were allowed to grow at 37° C. and a single colony was picked. A starter was allowed to grow to confluence in a small LB volume containing the antibiotic overnight. Next, the E. coli bacterial cells were inoculated in appropriate volume and OD600 was monitored regularly using a spectrometer. Once E. coli bacterial cell confluency reached between 0.4 and 0.8 at OD600, bacteria were IPTG induced at 16° C. for 24 hours and collected by centrifugation. Cell pellets were stored at −80° C. until further processed. Purification of recombinant protein was performed using IMAC. The purity of all recombinant proteins were assessed by Coomassie staining.

Example 1: In Vitro Immunoassay (Immunoblot and ELISA) for Identification of Detyrosinase (TCPase) Modulators Compounds

[0294] The proof of principle of an immunoassay such as ELISA assay has been obtained by using VASH2 recombinant protein for enzymatic description (FIG. 12). Advantageously the assay has been used in a dose-response analysis using a previously described Epoxy-Y inhibitor (Science 2017, Aillaud et al.). As expected increasing amounts of Epoxy-Y inhibitor resulted in a dose dependent inhibition of VASH2 detyrosinase activity. Oppositely SVBP induces tubulin carboxypeptidase activity of VASH2 (FIG. 9). Next, Tubulin purified from Sf9 insect cells (fully tyrosinated; FIG. 1) was polymerized in presence of Taxol at 37° C. for 30 min, washed and stored in −80° C. until further use. Recombinant VASH2 protein was pre-incubated 5 min at room temperature in presence or absence of Epoxy-Y. The previously prepared microtubules (MTs) (substrate of TCPase, ie VASH2 protein) were added to the reaction mixture in a phosphate buffer containing 100 mM NaCl at pH7.0. The reaction was performed for 10 minutes at room temperature, stopped using sample buffer and analyzed by immunoblotting using specific labelled antibodies directed against Sf9 tubulin (FIG. 13).

[0295] The same protocol is further reproduced with the compounds to be tested (putative modulators of TCPase, ie VASH2) in replacement to Epoxy-Y, previously used to validate the assay. So recombinant VASH2 protein was pre-incubated 5 min at room temperature in presence or absence of the compound to be tested. The previously prepared microtubules (MTs) (substrate of TCPase, ie VASH2 protein) were added to the reaction mixture in a phosphate buffer containing 100 mM NaCl at pH7.0. The reaction was performed for 10 minutes at room temperature, stopped using sample buffer and analyzed by immunoblotting using specific labelled antibodies directed against detyrosinated Sf9 tubulin (dTyr-tub). If the signal level (detyrosination) is lower than the control (in absence of the compound to be tested), it means that the compound to be tested is an inhibitor of VASH2, as Epoxy-Y, whereas if the signal level (detyrosination) is higher than the control (in absence of the compound to be tested), it means that the compound to be tested is an activator of VASH2. The man skilled in the art will adapt the concentration of enzyme, substrate and compounds to be tested, in order to favor the detyrosination of substrate (microtubules MT). In an alternative, other substrates of VASH2 are used instead of micrcotubules MT, in particular the ones disclosed in Material & Methods above, and preferably a recombinant tyrosinated telokin such as the one comprising the amino acids sequence SEQ ID NO: 17.

[0296] The method then allowed the development of an ELISA-based assay screening of chemical compounds (putative modulator compounds) in a microtiter plate. A schematic representation of an ELISA-based assay for HTS assay is shown in FIG. 14.

[0297] The substrate of TCPase is selected from the ones disclosed above in Material & Methods. In particular, we used a recombinant tyrosinated telokin comprising the sequence SEQ ID NO:17 (Mus musculus, fully tyrosinated variant).

[0298] As negative control, we used an isolated (non-tyrosinated) telokin comprising the sequence SEQ ID NO:16 Uniprot Q6PDN3-4) without the ultimate Y.

[0299] We use the reagents and instructions of commercial ELISA kit.

[0300] Standardized amount of recombinant Telokin protein containing the C-terminal sequence of alpha-tubulin as disclosed above is coated to a multiwell plate (96/384/1536).

[0301] The substrate coated 96 wells plate is first brought into contact, in absence (control) or presence of a compound to be tested (putative modulator of TCPase), with the recombinant active bacterially produced VASH2 for 15 minutes. The enzyme is washed off using appropriate buffer (preferred is phosphate buffer), and HRP-coupled antibody specifically targeting detyrosinated α-tubulin SF9 (dTyr-Ab SF9), is brought in contact with substrate in each well and excess appropriately washed off with a mild detergent solution to remove antibodies that are non-specifically bound. After the final wash step, the plate is developed by adding Chromogenic HRP substrate TMB is added and microplate measurement is performed accordingly.

Example 2: In Vitro Colorimetric Assay (Spectrophotometric Assay) for Identification of Detyrosinase (TCPase) Modulators Compounds

[0302] The assay relies on the well-document tyrosinase activity on free tyrosine. Free tyrosine can be converted by tyrosinase into a colorimetric detectable compound. This reaction has been described for almost a century (Lichtman, J B C 1929) and applied to measure free tyrosine in various samples.

[0303] Tyrosinase catalyzes tyrosine to DOPO and DOPA to DOPAquinone subsequently. During DOPAquinone formation, protons (H+) are produced and these protons could be monitored by substrate (ex: thymol blue) color change (Park et al., 2003).

[0304] Current method relies on the use of VASH2 and appropriate substrate of TCPase as disclosed in the above Material & Methods to release free tyrosine in the reaction mixture that can in a second step be used for tyrosinase-dependent analysis and spectrophotometric quantification.

[0305] A reaction mixture was prepared by pipetting a phosphate buffer, deionized water, VASH2 recombinant protein, TCPase substrate such as MTs or human alpha tubulin and tyrosinase. The TCPase activity is directly correlated to the increase in absorption due to tyrosinase-mediated generation of dopaquinone. The absorption may be measured with a SPECTRAmax PLUS spectrophotometer. The released C-terminal tyrosine residue by VASH2 becomes substrate of tyrosinase that catalyzes a colorimetric compound that can be measured using a spectrophotometer. We used recombinant tyrosinase (Sigma Aldrich) to determine the reaction kinetics by measuring optical density at 305 nm (FIG. 15). Increasing amounts of free tyrosine were mixed with recombinant tyrosinase in a phosphate buffer pH=7.0, incubated at room temperature for 15 min and analyzed by spectrophotometer at 305 nm (FIG. 15). Next, the proof of concept was performed using recombinant VASH2. Recombinant VASH2 was mixed with bacterially produced recombinant human alpha tubulin, in particular recombinant tubulin alpha-1A chain comprising the amino acid sequence SEQ ID NO: 19 (Uniprot Q71U36) and incubated for 15 min at room temperature in a phosphate buffer ph=7.0, 100 mM NaCl. Next, tyrosinase enzyme was added, mixed and incubated at room temperature for 30 min and analyzed using a spectrophotometer at 305 nm (FIG. 16).

[0306] All controls containing the individual components of the reaction were measured (samples A-C) as well. [0307] A. control sample containing tyrosinase, [0308] B. control sample containing recombinant human alpha tubulin, [0309] C. control samples containing tyrosinase (Tyr) and VASH2 as detyrosinase, [0310] D reaction mixture containing recombinant human alpha tubulin, VASH2 mediating tyrosine release and tyrosinase to allow detection of free tyrosine. [0311] OD305 was measured 30 min after onset of reaction at room temperature. [0312] Release of tyrosine by VASH2 could be observed (Sample D—FIG. 16.).

Example 3: Dot-Blot Assay for Identification of Detyrosinase (TCPase) Modulators Compounds

[0313] In this technique, 2 μl of the reaction mixture containing the substrate (8 amino acid peptide consisting of GEEEGEEY) and the enzyme (Recombinant human VASH2) in absence or in presence of the compound were doted on nitrocellulose and assessed for detyrosination of the peptide using a specific antibody detecting detyrosination.

[0314] The filter is allowed to dry and the membrane is incubated in blocking buffer to prevent non-specific binding. Similarly to ELISA, HRP-coupled antibody directed against the detyrosinated Telokin engineered substrate (as disclosed above) is incubated over the membrane, excess is washed-off and the membrane is developed using e.g. chromogenic HRP substrate. Intensities of the signals on autoradiograms were quantified by densitometric scanning. Alternatively, we designed a method using a slot blot approach.

[0315] The steps are identical as for the dot blot approach, this is the samples are applied to a nitrocellulose or PVDF membrane with a vacuum manifold to produce an orderly grid of samples. Once dry, dot blots and slot blots are subjected to the same immunodetection steps used for Western blotting (FIG. 11).

Example 4: Detyrosination Assay Performed Using Tubulin Purified from Sf9 Cells and Recombinant SVBP-VASH2 Complex in Absence or Presence of Putative Inhibitors of Detyrosinase

[0316] In order to test previously identified compounds as inhibitors of tubulin detyrosinase (Fonrose et al. 2007), we selected the three most promising compounds (Parthenolide, Alantolactone and Costunolide) (FIG. 17a) and assayed their activity in the in vitro detyrosination assay of the present invention using Sf9 tubulin. Using SVBP-VASH2 complex as detyrosinase, two different concentrations (0.1 μM and 10 μM) of these compounds together with Eps-Y (Epoxy Y) were incubated for 15 min at 37° C. in presence of microtubules polymerised using Sf9 cells purified tubulin (substrate of detyrosinase). A negative control sample consisting of the tubulin in the reaction buffer in absence of the detyrosinase (SVBP-VASH2 complex) was also analysed by western blotting (FIG. 17b). Surprisingly, while SVBP-VASH2 complex strongly induced detyrosination in the assay, the in cellulo identified putative inhibitors of detyrosinase including parthenolide, alantolactone and costunolide in human HEK293 cells, did not result in any significant reduction of the detyrosinating activity (FIG. 17c). This suggests that the effect of these compounds in human cells on the level of detyrosination likely reflects an indirect adaptive mechanism that alters microtubule detyrosination more than direct binding and inhibition of the VASH detyrosinating enzymes.

Example 5: Detyrosination Assay Performed Using Human Tubulin Purified from HEK293 Cells and Recombinant SVBP-VASH2 Complex

[0317] Human HEK293 cells were lysed and used for detyrosination assay in presence of parthenolide and Eps-Y compounds. Briefly, HEK293 cells were grown and harvested for further testing the detyrosinating activity of purified recombinant SVBP-VASH2 complex on human microtubules. Using SVBP-VASH2 complex as detyrosinase, extracted human tubulin from HEK293 cells and two different compounds, we performed a detyrosination assay. Samples were incubated for 15 min at 37° C. and analysed by western blot (FIG. 18a). Consistent with previous results and as opposed to Eps-Y, parthenolide did not result in inhibition of detyrosination activity in this assay (FIG. 18b). Moreover, the fold induction of the tubulin detyrosination resulting from SVBP-VASH2 complex was significantly lower with human tubulin purified from HEK293 cells in comparison to the one obtained using Sf9 cells extracted tubulin. This is likely to be the result of the numerous modifications tubulin is exposed to in human cells. The surprising observation that Sf cells extracted tubulin do not bare significant amount of tubulin modification is further exemplified by this observation. As expected, Eps-Y reduced detyrosination to background levels.

[0318] These results showed that the Sf cells extracted tubulin is of particular interest, as substrate of TCPase, in the in vitro assay of the present invention, because it does not bare significant amount of tubulin modification.

Example 6: Detvrosination Assay Performed Using Sf Tubulin and Recombinant Trypanosoma brucei VASH Protein

[0319] To test the use of the in vitro detyrosination system to other detyrosinase, we cloned the VASH gene of Trypanosoma brucei, a protozoan parasite that is responsible for neglected tropical diseases such as Human African Trypanosomiasis. Trypanosoma brucei cells were lysed and using a reverse transcription kit (Takara), we produced cDNA. Next, by PCR we specifically amplified the VASH gene and cloned into a bacterial expression vector. Purification protocols were optimized and recombinant Tb-VASH protein (SEQ ID NO: 14 (Uncharacterized protein/VASH of Trypanosoma brucei), was assayed using the in vitro detyrosination assay of the present invention. Using Tb-VASH as detyrosinase, samples were incubated for 15 min at 37° C. in presence of microtubules polymerised using Sf9 cells purified tubulin. A negative control sample consisting of the tubulin in the reaction buffer in absence of the detyrosinase was also analysed by western blotting. As expected, Tb-VASH was efficient in removing the C-terminal Y residue contained by the α-tubulin sequence (FIG. 19). As such, we provided evidence that the assay can be adapted to test any tubulin detyrosinase to identify compounds that directly modulate its activity.

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