METHODS FOR PURIFYING PROTEINS HAVING A TUBULIN CARBOXYPEPTIDASE ACTIVITY AND PEPTIDIC BASED INHIBITORS THEREOF

20200172598

16/631849

July 18, 2018

The invention relates to a method for purifying proteins having a tubulin carboxypeptidase activity from a biological extract, comprising polymerization/depolymerization cycle performed on a biological extract in presence of microtubules. The invention further relates to a peptidic based inhibitor for use in the treatment of a disorder involving altered microtubule detyrosination in an animal, wherein the peptidic based inhibitor comprises a peptidic moiety constituted of 1 to 20 amino acids, said peptidic moiety having an amino acid selected from Y or F at the C-terminal position, and wherein the peptidic based inhibitor inhibits at least partially a tubulin carboxypeptidase activity.

1-20. (canceled)

21. A method for purifying proteins having a tubulin carboxypeptidase activity from a biological extract, comprising: (a) centrifuging the biological extract at a temperature between 0 and 10° C.; (b) recovering the supernatant from step (a) and proceeding to a first microtubule polymerization cycle by adding GTP and incubating the mixture at a temperature between 35 and 40° C., then centrifuging; (c) recovering the pellets of step (b), resuspending in ice-cold buffer, incubating at 4° C.+/−1° C., and proceeding to a second microtubule polymerization cycle by adding GTP and incubating the mixture at 37° C., +/−2° C., then centrifuging; (d) recovering the pellets of step (c) resuspending in ice-cold buffer, incubating at 4° C.+1-1° C., and proceeding to a third microtubule polymerization cycle by adding GTP and incubating the mixture at 37° C., +/−2° C., then centrifuging; (e) resuspending the pellets of step (d) and submitting the mixture to an ion exchange chromatography and recovering the flow through; (f) precipitating the proteins of the flow through with a 60% saturated ammonium sulphate solution; and (g) submitting the precipitated fraction of step (f) to an hydrophobic chromatography and eluting by gradually decreasing ammonium sulphate concentration to zero to recover the fraction of proteins with a tubulin carboxypeptidase activity.

22. The method of claim 21, wherein the first polymerization cycle comprises (i) adding GTP and incubating the mixture at 37° C., +/−2° C., for 30 minutes, +/−10 minutes; and (ii) centrifuging at 22,000 g, +/−1,000 g, at 37° C., +/−2° C., for 45 minutes, +/−10 minutes; and the second polymerization cycle comprises: (i) incubating the mixture on ice for 30 minutes, +/−10 minutes; (ii) centrifuging at 150,000 g+/−10,000 g, 30 minutes, +/−10 minutes; (iii) recovering the supernatant and adding GTP; (iv) incubating the mixture at 37° C., +/−2° C., for at 30 minutes, +/−10 minutes; and (v) centrifuging at 50,000 g, +/−1,000 g at a temperature comprised between 30° C. and 37° C., for 30 minutes, +/−10 minutes; and the third polymerization cycle comprises: (i) incubating the mixture on ice for 30 minutes, +/−10 minutes; (ii) recovering the supernatant and adding GTP; (iii) incubating the mixture at 37° C., +/−2° C., for at 30 minutes, +/−10 minutes; and (iv) centrifuging at 50,000 g, +/−1,000 g at a temperature comprised between 30° C. and 37° C., for 30 minutes, +/−10 minutes.

23. The method of claim 21, further comprising a step of mass spectrometry characterization of the fraction of proteins of step (g).

24. The method of claim 21, further comprising a step of selecting proteins that contain a protease domain.

25. The method of claim 21, wherein the biological sample is selected from eukaryote organisms extracts.

26. The method of claim 21, wherein the biological sample is selected from mammal brain extract, mammal testis extract, and mammal lung extract.

27. The method of claim 21, wherein the fraction of proteins with a tubulin carboxypeptidase activity comprises at least one protein having at least 30% amino acid sequence identity with the amino acid sequence selected from SEQ ID N°1, SEQ ID N°2, SEQ ID N°3, SEQ ID N°4, SEQ ID N°5, SEQ ID N°6, SEQ ID N°7, SEQ ID N°8, SEQ ID N°9, SEQ ID N°10 and SEQ ID N°11.

28. The method of claim 21, wherein the fraction of native or recombinant proteins with a tubulin carboxypeptidase activity is further contacted with microtubules and the level of isolated tyrosine (Y) is measured, thereby confirming the tubulin carboxypeptidase activity of the fraction of proteins.

29. A method for selecting a peptidic based inhibitor able to inhibit a tubulin carboxypeptidase activity among peptidic based inhibitor candidates that comprise a peptidic moiety of 1 to 20 amino acids, said peptidic moiety having at the C-terminal position an amino acid selected from Y or F, wherein the method comprises: (a) contacting the peptidic based inhibitor candidate with a mixture containing both a fraction of native or recombinant proteins with a tubulin carboxypeptidase activity and microtubules, with labeled C-terminal Y; and (b) measuring the level of isolated Y.

30. The method of claim 29, wherein the level of isolated Y in the sample is compared to the level of isolated Y in a control sample comprising solely a protein extract obtained with a method for purifying proteins comprising: (a) centrifuging the biological extract at a temperature between 0 and 10° C.; (b) recovering the supernatant from step (a) and proceeding to a first microtubule polymerization cycle by adding GTP and incubating the mixture at a temperature between 35 and 40° C., then centrifuging; (c) recovering the pellets of step (b), resuspending in ice-cold buffer, incubating at 4° C.+/−1° C., and proceeding to a second microtubule polymerization cycle by adding GTP and incubating the mixture at 37° C., +/−2° C., then centrifuging; (d) recovering the pellets of step (c) resuspending in ice-cold buffer, incubating at 4° C.+7-1° C., and proceeding to a third microtubule polymerization cycle by adding GTP and incubating the mixture at 37° C., +/−2° C., then centrifuging; (e) resuspending the pellets of step (d) and submitting the mixture to an ion exchange chromatography and recovering the flow through; (f) precipitating the proteins of the flow through with a 60% saturated ammonium sulphate solution; and (g) submitting the precipitated fraction of step (f) to an hydrophobic chromatography and eluting by gradually decreasing ammonium sulphate concentration to zero to recover the fraction of proteins with a tubulin carboxypeptidase activity.

31. The method of claim 29, wherein the peptidic moiety of the peptidic based inhibitor candidate is between 1 and 20 amino acids of the most C-terminal amino acids of an alpha-tubulin.

32. The method of claim 29, wherein the peptidic moiety of the peptidic based inhibitor candidate is between 1 and 16 of the most C-terminal amino acids of the amino acid sequence Nter-X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-Cter, wherein X1, X2, X5, X7, X9 and X13 are hydrophobic amino acids, selected from G, A or V, X3, X6, X8, X10, X11, X12, X14 and X15 are negatively charged amino acids, selected from E or D, X4 is a polar uncharged side chains, selected from S, T, N or Q, and X16 is a large hydrophobic amino acid, selected from Y or F.

33. The method of claim 29, wherein the peptidic moiety of the peptidic based inhibitor candidate has an amino acid sequence selected from Y, EDY, EAY and EEY.

34. The method of claim 29, wherein the peptidic based inhibitor candidate further comprises a reactive moiety selected from epoxysuccinyl, acyloxymethyl, aldehydes and ketones.

35. A method of treatment of a disorder involving altered microtubule detyrosination in an animal comprising administering to a subject in need thereof a peptidic based inhibitor comprising a peptidic moiety of 1 to 20 amino acids of the most C-terminal amino acids of an alpha-tubulin that has been chemically modified or not, said peptidic moiety having an amino acid selected from Y or F at the C-terminal position, and wherein the peptidic based inhibitor inhibits, at least partially, a tubulin carboxypeptidase activity.

36. The method of claim 35, wherein the peptidic moiety is 1 and 16 amino acids of the most C-terminal amino acids of the amino acid sequence Nter-X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-Cter, wherein X1, X2, X5, X7, X9 and X13 are hydrophobic amino acids, elected from G, A or V, X3, X6, X8, X10, X11, X12, X14 and X15 are negatively charged amino acids, selected from E or D, X4 is a polar uncharged side chains, selected from S, T, N or Q, and X16 is a large hydrophobic amino acid, selected from Y or F.

37. The method of claim 35, wherein the peptidic moiety has the amino acid sequence Y, EDY, EAY or EEY.

38. The method of claim 35, wherein the peptidic based inhibitor further comprises a reactive moiety, selected from epoxysuccinyl, acyloxymethyl, aldehydes and ketones.

39. The method of claim 35, wherein the disorder is selected from neurodegenerative diseases, selected from Alzheimer disease, Parkinson disease, psychiatric disorders, and neural disorders, cancers, selected from colon cancer and neuroblastoma, muscular dystrophies, heart diseases, vascular disorders, infertility, retinal degeneration and ciliopathies.

40. A pharmaceutical composition comprising a therapeutically effective amount of a peptidic based inhibitors comprising a peptidic moiety of 1 to 20 amino acids of the most C-terminal amino acids of an alpha-tubulin that has been chemically modified or not, said peptidic moiety having an amino acid selected from Y or F at the C-terminal position, and wherein the peptidic based inhibitor inhibits at least partially a tubulin carboxypeptidase activity.

07; 12; 61; 61; 07; 12; 61

edspap.20200172598