Abstract
The microtubule network exerts multifarious functions controlled by its decoration with various proteins and post-translational modifications. The disordered microtubule associated Tubulin Polymerization Promoting Protein (TPPP/p25) and the NAD+-dependent tubulin deacetylase sirtuin-2 (SIRT2) play key roles in oligodendrocyte differentiation by acting as dominant factors in the organization of myelin proteome. Herein, we show that SIRT2 impedes the TPPP/p25-promoted microtubule assembly independently of NAD+; however, the TPPP/p25-assembled tubulin ultrastructures were resistant against SIRT2 activity. TPPP/p25 counteracts the SIRT2-derived tubulin deacetylation producing enhanced microtubule acetylation. The inhibition of the SIRT2 deacetylase activity by TPPP/p25 is evolved by the assembly of these tubulin binding proteins into a ternary complex, the concentration-dependent formation of which was quantified by experimental-based mathematical modelling. Co-localization of the SIRT2-TPPP/p25 complex on the microtubule network was visualized in HeLa cells by immunofluorescence microscopy using Bimolecular Fluorescence Complementation. We also revealed that a new potent SIRT2 inhibitor (MZ242) and its proteolysis targeting chimera (SH1) acting together with TPPP/p25 provoke microtubule hyperacetylation, which is coupled with process elongation only in the case of the degrader SH1. Both the structural and the functional effects manifesting themselves by this deacetylase proteome could lead to the fine-tuning of the regulation of microtubule dynamics and stability.
Highlights
Motives such as a zinc finger and a GTP consensus sequence[10,11]
The interaction of SIRT2 with TPPP/p25 as well as their associations to tubulin was characterized by circular dichroism (CD) and enzyme-linked immunosorbent assay (ELISA)
The pair-wise interactions were quantified by ELISA (Fig. 2a): SIRT2 was immobilized on the plate, TPPP/p25 or tubulin was added at various concentrations and their binding to SIRT2 was detected by specific TPPP/p25 or tubulin antibodies as described in the Materials and Methods
Summary
A number of in vitro and cellular studies with wild type and recombinant mutants as well as their fluorescently labelled variants have revealed that the structural changes of the disordered TPPP/p25 are mediated by its dimerization and heterologous interactions with the bivalent zinc cation, GTP, mitogen-activated protein kinase 1 and histone deacetylase 6 (HDAC6), which significantly affect its tubulin polymerization promoting potency[10,11,12,13,14,15,16] This feature of TPPP/p25 is tightly coupled with its physiological functions, namely, the modulation and coordination of the dynamics and stability of the MT network[7,9,17]. In comparison with their parental direct enzyme inhibitors, PROTACs can be very useful tools to distinguish between the effects that are linked to the catalytic activity of an enzyme and those that are associated with non-enzymatic protein-protein interactions
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