Methods and pharmaceutical compositions for treating tubulin carboxypeptidases associated diseases
Using chemical proteomics with a potent unique irreversible inhibitor, inventors found that major brain tubulin carboxypeptidase (TCP) is a complex of vasohibin-1 (VASH1) with the Small Vasohibin-Binding Protein (SVBP). VASH1 and its homologue vasohibin-2 (VASH2), when complexed with SVBP, exhibit robust and specific Tyr/Phe carboxypeptidase activity on microtubules. Accordingly inventors are the first to identify the enzymatic activity of vasohibin and vasohibin/SVBP complex. Knock down of vasohibins or SVBP in cultured neurons results in a marked reduction of tyrosinated α-tubulin levels and onset of severe differentiation defects. Furthermore, knock down of vasohibins disrupts neuronal migration in developing mouse neocortex. These results establish vasohibin/SVBP complexes as TCP enzymes. Accordingly, the present invention relates methods and pharmaceutical compositions for treating tubulin carboxypeptidases (TCP) associated diseases such as neurological disorders and cardiovascular diseases with an inhibitor of activity or expression of Vasohibin or Vasohibin/SVBP complex.
1. A method for identifying inhibitors of tubulin carboxypeptidase (TCP) comprising the steps of:
(i) incubating a candidate TCP inhibitor with a mouse or human vasohibin (VASH)/Small Vasohibin-Binding Protein (SVBP) complex, and the biotinylated peptide of sequence Biotinyl-V-D-S-V-E-G-E-G-E-E-E-D-E-E-Y (SEQ ID NO:13);
(ii) quantifying, by Mass Spectrometry, the biotinylated peptide Biotinyl-V-D-S-V-E-G-E-G-E-E-E-D-E-E-Y (SEQ ID NO:13) and/or the biotinylated peptide Biotinyl-V-D-S-V-E-G-E-G-E-E-E-D-E-E (SEQ ID NO:12); present in the mixture obtained at the end of step (i); and
(iii) determining whether the candidate TCP inhibitor is an inhibitor of VASH/SVBP complex activity from the results obtained in step (ii).
2. The method of claim 1 , further comprising a step of isolating the biotinylated peptide(s) of Biotinyl-V-D-S-V-E-G-E-G-E-E-E-D-E-E (SEQ ID NO: 12) and/or Biotinyl-V-D-S-V-E-G-E-G-E-E-E-D-E-E-Y (SEQ ID NO: 13) after step (i) and before step (ii).