HaloTag-Targeted Sirtuin-Rearranging Ligand (SirReal) for the Development of Proteolysis-Targeting Chimeras (PROTACs) against the Lysine Deacetylase Sirtuin 2 (Sirt2)*.

We have discovered the sirtuin-rearranging ligands (SirReals) as a novel class of highly potent and selective inhibitors of the NAD -dependent lysine deacetylase sirtuin 2 (Sirt2). In previous studies, conjugation of a SirReal with a ligand for the E3 ubiquitin ligase cereblon to form a so-called proteolysis-targeting chimera (PROTAC) enabled small-molecule-induced degradation of Sirt2. Herein, we report the structure-based development of a chloroalkylated SirReal that induces the degradation of Sirt2 mediated by Halo-tagged E3 ubiquitin ligases.

Microtubule-Associated Proteins with Regulatory Functions by Day and Pathological Potency at Night.

The sensing, integrating, and coordinating features of the eukaryotic cells are achieved by the complex ultrastructural arrays and multifarious functions of the cytoskeleton, including the microtubule network. Microtubules play crucial roles achieved by their decoration with proteins/enzymes as well as by posttranslational modifications. This review focuses on the Tubulin Polymerization Promoting Protein (TPPP/p25), a new microtubule associated protein, on its "regulatory functions by day and pathological functions at night".

Interactions between two regulatory proteins of microtubule dynamics, HDAC6, TPPP/p25, and the hub protein, DYNLL/LC8.

Degradation of unwanted proteins is important in protein quality control cooperating with the dynein/dynactin-mediated trafficking along the acetylated microtubule (MT) network. Proteins associated directly/indirectly with tubulin/MTs play crucial roles in both physiological and pathological processes. Our studies focus on the interrelationship of the tubulin deacetylase HDAC6, the MT-associated TPPP/p25 with its deacetylase inhibitory potency and the hub dynein light chain DYNLL/LC8, constituent of dynein and numerous other protein complexes.

New chemical tools for probing activity and inhibition of the NAD-dependent lysine deacylase sirtuin 2.

Sirtuins are NAD-dependent protein deacylases capable of cleaving off acetyl as well as other acyl groups from the ɛ-amino group of lysines in histones and other substrate proteins. They have been reported as promising drug targets, and thus modulators of their activity are needed as molecular tools to uncover their biological function and as potential therapeutics. Here, we present new assay formats that complement existing assays for sirtuin biochemistry and cellular target engagement.

Localization of the zinc binding tubulin polymerization promoting protein in the mice and human eye.

Tubulin Polymerization Promoting Protein (TPPP/p25) modulates the dynamics and stability of the microtubule network by its bundling and acetylation enhancing activities that can be modulated by the binding of zinc to TPPP/p25. Its expression is essential for the differentiation of oligodendrocytes, the major constituents of the myelin sheath, and has been associated with neuronal inclusions. In this paper, evidence is provided for the expression and localization of TPPP/p25 in the zinc-rich retina and in the oligodendrocytes in the optic nerve.

Modulation Of Microtubule Acetylation By The Interplay Of TPPP/p25, SIRT2 And New Anticancer Agents With Anti-SIRT2 Potency.

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.

Challenging drug target for Parkinson's disease: Pathological complex of the chameleon TPPP/p25 and alpha-synuclein proteins.

The hallmarks of Parkinson's disease and other synucleinopathies, Tubulin Polymerization Promoting Protein (TPPP/p25) and α-synuclein (SYN) have two key features: they are disordered and co-enriched/co-localized in brain inclusions. These Neomorphic Moonlighting Proteins display both physiological and pathological functions due to their interactions with distinct partners. To achieve the selective targeting of the pathological TPPP/p25-SYN but not the physiological TPPP/p25-tubulin complex, their interfaces were identified as a specific innovative strategy for the development of anti-Parkinson drugs.

Targeting the interface of the pathological complex of α-synuclein and TPPP/p25.

The pathological interaction of intrinsically disordered proteins, such as α-synuclein (SYN) and Tubulin Polymerization Promoting Protein (TPPP/p25), is often associated with neurodegenerative disorders. These hallmark proteins are co-enriched and co-localized in brain inclusions of Parkinson's disease and other synucleinopathies; yet, their successful targeting does not provide adequate effect due to their multiple functions. Here we characterized the interactions of the human recombinant wild type SYN, its truncated forms (SYN(1-120), SYN(95-140)), a synthetized peptide (SYN(126-140)) and a proteolytic fragment (SYN(103-140)) with TPPP/p25 to identify the SYN segment involved in the interaction.

Zinc-induced structural changes of the disordered tppp/p25 inhibits its degradation by the proteasome.

Tubulin Polymerization Promoting Protein/p25 (TPPP/p25), a neomorphic moonlighting protein displaying both physiological and pathological functions, plays a crucial role in the differentiation of the zinc-rich oligodendrocytes, the major constituent of myelin sheath; and it is enriched and co-localizes with α-synuclein in brain inclusions hallmarking Parkinson's disease and other synucleinopathies. In this work we showed that the binding of Zn(2+) to TPPP/p25 promotes its dimerization resulting in increased tubulin polymerization promoting activity. We also demonstrated that the Zn(2+) increases the intracellular TPPP/p25 level resulting in a more decorated microtubule network in CHO10 and CG-4 cells expressing TPPP/p25 ectopically and endogenously, respectively.

Identification of motives mediating alternative functions of the neomorphic moonlighting TPPP/p25.

The disordered Tubulin Polymerization Promoting Protein (TPPP/p25), a prototype of neomorphic moonlighting proteins, displays physiological and pathological functions by interacting with distinct partners. Here the role of the disordered N- and C-termini straddling a middle flexible segment in the distinct functions of TPPP/p25 was established, and the binding motives responsible for its heteroassociations with tubulin and α-synuclein, its physiological and pathological interacting partner, respectively, were identified. We showed that the truncation of the disordered termini altered the folding state of the middle segment and has functional consequences concerning its physiological function.

Microtubule assembly-derived by dimerization of TPPP/p25. Evaluation of thermodynamic parameters for multiple equilibrium system from ITC data.

Background: The disordered Tubulin Polymerization Promoting Protein/p25 (TPPP/p25) modulates the dynamics and stability of the microtubule system. In this paper the role of dimerization in its microtubule-related functions is established, and an approach is proposed to evaluate thermodynamic constants for multiple equilibrium systems from ITC measurements.

Methods: For structural studies size exclusion chromatography, SDS-PAGE, chemical cross-linking, circular dichroism, fluorescence spectroscopy and isothermal titration calorimetry were used; the functional effect was analyzed by tubulin polymerization assay.