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

AbstractThe 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 (SYN1–120, SYN95–140), a synthetized peptide (SYN126–140) and a proteolytic fragment (SYN103–140) with TPPP/p25 to identify the SYN segment involved in the interaction. The binding of SYN103–140 to TPPP/p25 detected by ELISA suggested the involvement of a segment within the C-terminal of SYN. The studies performed with ELISA, Microscale Thermophoresis and affinity chromatography proved that SYN95–140 and SYN126–140 – in contrast to SYN1–120 – displayed significant binding to TPPP/p25. Fluorescence assay with ANS, a molten globule indicator, showed that SYN, but not SYN1–120 abolished the zinc-induced local folding of both the full length as well as the N- and C-terminal-free (core) TPPP/p25; SYN95–140 and SYN126–140 were effective as well. The aggregation-prone properties of the SYN species with full length or core TPPP/p25 visualized by immunofluorescent microscopy demonstrated that SYN95–140 and SYN126–140, but not SYN1–120, induced co-enrichment and massive intracellular aggregation after their premixing and uptake from the medium. These data with their innovative impact could contribute to the development of anti-Parkinson drugs with unique specificity by targeting the interface of the pathological TPPP/p25-SYN complex

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". Physiologically, the moonlighting TPPP/p25 modulates the dynamics and stability of the microtubule network by bundling microtubules and enhancing the tubulin acetylation due to the inhibition of tubulin deacetylases. The optimal endogenous TPPP/p25 level is crucial for its physiological functions, to the differentiation of oligodendrocytes, which are the major constituents of the myelin sheath. Pathologically, TPPP/p25 forms toxic oligomers/aggregates with α-synuclein in neurons and oligodendrocytes in Parkinson's disease and Multiple System Atrophy, respectively; and their complex is a potential therapeutic drug target. TPPP/p25-derived microtubule hyperacetylation counteracts uncontrolled cell division. All these issues reveal the anti-mitotic and α-synuclein aggregation-promoting potency of TPPP/p25, consistent with the finding that Parkinson's disease patients have reduced risk for certain cancers

Anti-aggregative effect of the antioxidant dj-1 on the tppp/p25-derived pathological associations of alpha-synuclein

DJ-1, a multi-functional protein with antioxidant properties, protects dopaminergic neurons against Parkinson’s disease (PD). The oligomerization/assembly of alpha-synuclein (SYN), promoted by Tubulin Polymerization Promoting Protein (TPPP/p25), is fatal in the early stage of PD. The pathological assembly of SYN with TPPP/p25 inhibits their proteolytic degradation. In this work, we identified DJ-1 as a new interactive partner of TPPP/p25, and revealed its influence on the association of TPPP/p25 with SYN. DJ-1 did not affect the TPPP/p25-derived tubulin polymerization; however, it did impede the toxic assembly of TPPP/p25 with SYN. The interaction of DJ-1 with TPPP/p25 was visualized in living human cells by fluorescence confocal microscopy coupled with Bifunctional Fluorescence Complementation (BiFC). While the transfected DJ-1 displayed homogeneous intracellular distribution, the TPPP/p25-DJ-1 complex was aligned along the microtubule network. The anti-aggregative effect of DJ-1 on the pathological TPPP/p25-SYN assemblies was established by the decrease in the intensity of their intracellular fluorescence (BiFC signal) and the increase in the proteolytic degradation of SYN complexed with TPPP/p25 due to the DJ-1-derived disassembly of SYN with TPPP/p25. These data obtained with HeLa and SH-SY5Y cells revealed the protective effect of DJ-1 against toxic SYN assemblies, which assigns a new function to the antioxidant sensor DJ-1. © 2021 by the authors. Licensee MDPI, Basel, Switzerland

A Potential Innovative Therapy for Parkinson’s Disease: Selective Destruction of the Pathological Assemblies of Alpha-Synuclein

With the aging of the population, Parkinson’s disease poses a serious socio-economic problem; there is no effective therapy that can arrest/revert the progression of the disease. The hallmarks of Parkinson’s disease and other synucleinopathies are the disordered alpha-synuclein and TPPP/p25. These proteins have neomorphic moonlighting characteristics by displaying both physiological and pathological functions. Physiologically TPPP/p25 regulates the dynamics/stability of the microtubules and is crucial for oligodendrocyte differentiation; while alpha-synuclein is involved in neuronal plasticity modulation and synaptic vesicle pool maintenance. In healthy brain, alpha-synuclein and TPPP/p25 occur predominantly in neurons and oligodendrocytes, respectively; however, they are co-enriched and co-localized in both cell types in brain inclusions in the cases of Parkinson’s disease and multiple system atrophy, respectively. The pathomechanisms of these diseases are largely unknown; the fatal species are the small, soluble homo- and hetero-associations of alpha-synuclein. These proteins with their high conformational plasticity and chameleon feature are challenging drug targets. Nevertheless, the contact surface of TPPP/p25-alpha-synuclein assemblies has been validated as a specific drug target. This new strategy with innovative impact, namely targeting the interface of the TPPP/p25-alpha-synuclein complex, could contribute to the development of anti-Parkinson drugs with unique specificity

Challenges in Discovering Drugs That Target the Protein–Protein Interactions of Disordered Proteins

Protein–protein interactions (PPIs) outnumber proteins and are crucial to many fundamental processes; in consequence, PPIs are associated with several pathological conditions including neurodegeneration and modulating them by drugs constitutes a potentially major class of therapy. Classically, however, the discovery of small molecules for use as drugs entails targeting individual proteins rather than targeting PPIs. This is largely because discovering small molecules to modulate PPIs has been seen as extremely challenging. Here, we review the difficulties and limitations of strategies to discover drugs that target PPIs directly or indirectly, taking as examples the disordered proteins involved in neurodegenerative diseases

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 elective 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. Therefore, the interactions of TPPP/p25 with tubulin and SYN were characterized which suggested the involvements of the 178–187 aa and 147–156 aa segments in the complexation of TPPP/p25 with tubulin and SYN, respectively. However, various truncated and deletion mutants reduced but did not abolish the interactions except one mutant; in addition synthetized fragments corresponding to the potential binding segments of TPPP/p25 failed to interact with SYN. In fact, the studies of the multiple interactions at molecular and cellular levels revealed the high conformational plasticity, chameleon feature, of TPPP/p25 that ensures exceptional functional resilience; the lack of previously identified binding segments could be replaced by other segments. The experimental results are underlined by distinct bioinformatics tools. All these data revealed that although targeting chameleon proteins is a challenging task, nevertheless, the validation of a drug target can be achieved by identifying the interface of complexes of the partner proteins existing at the given pathological conditions

Updated knowledge on the records for the endangered click beetle Limoniscus violaceus in Hungary (Coleoptera: Elateridae)

Faunistic data for the violet click beetle, Limoniscus violaceus(P. W. J. Müller, 1821), from Hungary and notes on its life history are given. With 7 figures

Modulation of microtubule acetylation by the interplay of TPPP/p25, SIRT2 and new anticancer agents with anti-SIRT2 potency

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