Perspective Strategies for Interventions in Parkinsonism: Remedying the Neglected Role of TPPP.

Neurological disorders such as Parkinsonism cause serious socio-economic problems as there are, at present, only therapies that treat their symptoms. The well-established hallmark alpha-synuclein (SYN) is enriched in the inclusion bodies characteristic of Parkinsonism. We discovered a prominent partner of SYN, termed Tubulin Polymerization Promoting Protein (TPPP), which has important physiological and pathological activities such as the regulation of the microtubule network and the promotion of SYN aggregation. The role of TPPP in Parkinsonism is often neglected in research, which we here attempt to remedy. In the normal brain, SYN and TPPP are expressed endogenously in neurons and oligodendrocytes, respectively, whilst, at an early stage of Parkinsonism, soluble hetero-associations of these proteins are found in both cell types. The cell-to-cell transmission of these proteins, which is central to disease progression, provides a unique situation for specific drug targeting. Different strategies for intervention and for the discovery of biomarkers include (i) interface targeting of the SYN-TPPP hetero-complex; (ii) proteolytic degradation of SYN and/or TPPP using the PROTAC technology; and (iii) depletion of the proteins by miRNA technology. We also discuss the potential roles of SYN and TPPP in the phenotype stabilization of neurons and oligodendrocytes.

The Sherpa hypothesis: Phenotype-Preserving Disordered Proteins stabilize the phenotypes of neurons and oligodendrocytes.

Intrinsically disordered proteins (IDPs), which can interact with many partner proteins, are central to many physiological functions and to various pathologies that include neurodegeneration. Here, we introduce the Sherpa hypothesis, according to which a subset of stable IDPs that we term Phenotype-Preserving Disordered Proteins (PPDP) play a central role in protecting cell phenotypes from perturbations. To illustrate and test this hypothesis, we computer-simulate some salient features of how cells evolve and differentiate in the presence of either a single PPDP or two incompatible PPDPs. We relate this virtual experiment to the pathological interactions between two PPDPs, α-synuclein and Tubulin Polymerization Promoting Protein/p25, in neurodegenerative disorders. Finally, we discuss the implications of the Sherpa hypothesis for aptamer-based therapies of such disorders.

Modulatory Role of TPPP3 in Microtubule Organization and Its Impact on Alpha-Synuclein Pathology.

Parkinson's disease is characterized by locomotion deficits, dopaminergic neuronal loss and alpha-synuclein (SYN) aggregates; the Tubulin Polymerization Promoting Protein (TPPP/p25 or TPPP1) is also implicated in these processes. The moonlighting and chameleon TPPP1 modulates the dynamics/stability of the multifunctional microtubule network by promoting its acetylation and bundling. Previously, we identified the microtubule-associated TPPP3, a homologue of TPPP1 lacking its N-terminus; however, its involvement in physiological or pathological processes was not elucidated. In this work, we have shown the modulatory role of TPPP3, similarly to TPPP1, in microtubule organization, as well as its homo- and hetero-associations with TPPP1. TPPP3, in contrast to TPPP1, virtually does not bind to SYN; consequently, it does not promote SYN aggregation. Its anti-aggregative potency is achieved by counteracting the formation of the TPPP1-SYN pathological complex/aggregation leading to Parkinsonism. The interactions of TPPP3 have been determined and quantified in vitro with recombinant human proteins, cell extracts and in living human cells using different methods including bifunctional fluorescence complementation. The tight association of TPPP3 with TPPP1, but not with SYN, may ensure a unique mechanism for its inhibitory effect. TPPP3 or its selected fragments may become a leading agent for developing anti-Parkinson agents.

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.

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.

Role of Multifunctional Cytoskeletal Filaments in Coronaviridae Infections: Therapeutic Opportunities for COVID-19 in a Nutshell.

A novel coronavirus discovered in 2019 is a new strain of the Coronaviridae family (CoVs) that had not been previously identified in humans. It is known as SARS-CoV-2 for Severe Acute Respiratory Syndrome Coronavirus-2, whilst COVID-19 is the name of the disease associated with the virus. SARS-CoV-2 emerged over one year ago and still haunts the human community throughout the world, causing both healthcare and socioeconomic problems. SARS-CoV-2 is spreading with many uncertainties about treatment and prevention: the data available are limited and there are few randomized controlled trial data on the efficacy of antiviral or immunomodulatory agents. SARS-CoV-2 and its mutants are considered as unique within the Coronaviridae family insofar as they spread rapidly and can have severe effects on health. Although the scientific world has been succeeding in developing vaccines and medicines to combat COVID-19, the appearance and the spread of new, more aggressive mutants are posing extra problems for treatment. Nevertheless, our understanding of pandemics is increasing significantly due to this outbreak and is leading to the development of many different pharmacological, immunological and other treatments. This Review focuses on a subset of COVID-19 research, primarily the cytoskeleton-related physiological and pathological processes in which coronaviruses such as SARS-CoV-2 are intimately involved. The discovery of the exact mechanisms of the subversion of host cells by SARS-CoV-2 is critical to the validation of specific drug targets and effective treatments.

Co-Transmission of Alpha-Synuclein and TPPP/p25 Inhibits Their Proteolytic Degradation in Human Cell Models.

The pathological association of alpha-synuclein (SYN) and Tubulin Polymerization Promoting Protein (TPPP/p25) is a key factor in the etiology of synucleinopathies. In normal brains, the intrinsically disordered SYN and TPPP/p25 are not found together but exist separately in neurons and oligodendrocytes, respectively; in pathological states, however, they are found in both cell types due to their cell-to-cell transmission. The autophagy degradation of the accumulated/assembled SYN has been considered as a potential therapeutic target. We have shown that the hetero-association of SYN with TPPP/p25 after their uptake from the medium by human cells (which mimics cell-to-cell transmission) inhibits both their autophagy- and the ubiquitin-proteasome system-derived elimination. These results were obtained by ELISA, Western blot, FACS and immunofluorescence confocal microscopy using human recombinant proteins and living human cells; ANOVA statistical analysis confirmed that TPPP/p25 counteracts SYN degradation by hindering the autophagy maturation at the stage of LC3B-SQSTM1/p62-derived autophagosome formation and its fusion with lysosome. Recently, fragments of TPPP/p25 that bind to the interface between the two hallmark proteins have been shown to inhibit their pathological assembly. In this work, we show that the proteolytic degradation of SYN on its own is more effective than when it is complexed with TPPP/p25. The combined strategy of TPPP/p25 fragments and proteolysis may ensure prevention and/or elimination of pathological SYN assemblies.

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. Using this orthogonal approach for Sirt2 degradation, we show that other E3 ligases than cereblon, such as the E3 ubiquitin ligase parkin, can also be harnessed for small-molecule-induced Sirt2 degradation, thereby emphasizing the great potential of parkin to be used as an E3 ligase for new PROTACs approaches. Thus, our study provides new insights into targeted protein degradation in general and Sirt2 degradation in particular.

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.

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. In this paper, evidence is provided for the direct interaction of DYNLL/LC8 with TPPP/p25 and HDAC6 and their assembly into binary/ternary complexes with functional potency. The in vitro binding data was obtained with recombinant proteins and used for mathematical modelling. These data and visualization of their localizations by bimolecular fluorescence complementation technology and immunofluorescence microscopy in HeLa cells revealed the promoting effect of TPPP/p25 on the interaction of DYNLL/LC8 with both tubulin and HDAC6. Localization of the LC8-2-TPPP/p25 complex was observed on the MT network in contrast to the LC8-2-HDAC6 complex, which was partly translocated to the nucleus. LC8-2 did not influence directly the acetylation of the MT network. However, the binding of TPPP/p25 to a new binding site of DYNLL/LC8, outside the canonical binding groove, counteracted the TPPP/p25-derived hyperacetylation of the MT network. Our data suggest that multiple associations of the regulatory proteins of the MT network could ensure fine tuning in the regulation of the intracellular trafficking process either by the complexation of DYNLL/LC8 with new partners or indirectly by the modulation of the acetylation level of the MT network.

Pharmacological targeting of α-synuclein and TPPP/p25 in Parkinson's disease: challenges and opportunities in a Nutshell.

With the aging of population, neurological disorders, and especially disorders involving defects in protein conformation (also known as proteopathies) pose a serious socio-economic problem. So far there is no effective treatment for most proteopathies, including Parkinson's disease (PD). The mechanism underlying PD pathogenesis is largely unknown, and the hallmark proteins, α-synuclein (SYN) and tubulin polymerization promoting protein (TPPP/p25) are challenging drug targets. These proteins are intrinsically disordered with high conformational plasticity, and have diverse physiological and pathological functions. In the healthy brain, SYN and TPPP/p25 occur in neurons and oligodendrocytes, respectively; however, in PD and multiple system atrophy, they are co-enriched and co-localized in both cell types, thereby marking pathogenesis. Although large inclusions appear at a late disease stage, small, soluble assemblies of SYN promoted by TPPP/p25 are pathogenic. In the light of these issues, we established a new innovative strategy for the validation of a specific drug target based upon the identification of contact surfaces of the pathological SYN-TPPP/p25 complex that may lead to the development of peptidomimetic foldamers suitable for pharmaceutical intervention.

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. Firstly, we report the development of a homogeneous fluorescence-based activity assay using unlabelled acylated peptides. Upon deacylation, the free lysine residue reacts with fluorescamine to form a fluorophore. Secondly, using click chemistry with a TAMRA-azide on a propargylated sirtuin inhibitor, we prepared the first fluorescently labelled small-molecule inhibitor of Sirt2. This is used in a binding assay, which is based on fluorescence polarization. We used it successfully to map potential inhibitor-binding sites and also to show cellular Sirt2 engagement. By means of these new assays, we were able to identify and characterize novel Sirt2 inhibitors out of a focused library screen. The binding of the identified Sirt2 inhibitors was rationalized by molecular docking studies. These new chemical tools thus can enhance further sirtuin research.This article is part of a discussion meeting issue 'Frontiers in epigenetic chemical biology'.

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. Localization of TPPP/p25 was established by confocal microscopy using calbindin and synaptophysin as markers of specific striations in the inner plexiform layer (IPL) and presynaptic terminals, respectively. Postsynaptic nerve terminals in striations S1, S3 and S5 in the IPL and a subset of amacrine cells show immunopositivity against TPPP/p25 both in mice and human eyes. The co-localization of TPPP/p25 with acetylated tubulin was detected in amacrine cells, oligodendrocyte cell bodies and in synapses in the IPL. Quantitative Western blot revealed that the TPPP/p25 level in the retina was 0.05-0.13 ng/µg protein, comparable to that in the brain. There was a central (from optic nerve head) to peripheral retinal gradient in TPPP/p25 protein levels. Our in vivo studies revealed that the oral zinc supplementation of mice significantly increased TPPP/p25 as well as acetylated tubulin levels in the IPL. These results suggest that TPPP/p25, a microtubule stabilizer can play a role in the organization and reorganization of synaptic connections and visual integration in the eye.

Chemically Induced Degradation of Sirtuin 2 (Sirt2) by a Proteolysis Targeting Chimera (PROTAC) Based on Sirtuin Rearranging Ligands (SirReals).

Here we report the development of a proteolysis targeting chimera (PROTAC) based on the combination of the unique features of the sirtuin rearranging ligands (SirReals) as highly potent and isotype-selective Sirt2 inhibitors with thalidomide, a bona fide cereblon ligand. For the first time, we report the formation of a PROTAC by Cu(I)-catalyzed cycloaddition of a thalidomide-derived azide to an alkynylated inhibitor. This thalidomide-derived azide as well as the highly versatile linking strategy can be readily adapted to alkynylated ligands of other targets. In HeLa cells, our SirReal-based PROTAC induced isotype-selective Sirt2 degradation that results in the hyperacetylation of the microtubule network coupled with enhanced process elongation. Thus, our SirReal-based PROTAC is the first example of a probe that is able to chemically induce the degradation of an epigenetic eraser protein.

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. 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.

Role of the microtubule-associated TPPP/p25 in Parkinson's and related diseases and its therapeutic potential.

INTRODUCTION: The discovery and development of therapeutic strategies for the treatments of Parkinson's disease (PD) and other synucleinopathies are limited by a lack of understanding of the pathomechanisms and their connection with different diseases such as cancers. Areas covered: The hallmarks of these diseases are frequently multifunctional disordered proteins displaying moonlighting and/or chameleon features, which are challenging drug targets. A representative of these proteins is the disordered Tubulin Polymerization Promoting Protein (TPPP/p25) expressed specifically in oligodendrocytes (OLGs) in normal brain. Its non-physiological level is tightly related to the etiology of PD and Multiple System Atrophy (TPPP/p25 enrichment in inclusions of neurons and OLGs, respectively), multiple sclerosis (TPPP/p25-positive OLG destruction), as well as glioma (loss of TPPP/p25 expression). The established anti-proliferative potency of TPPP/p25 may raise its influence in cancer development. The recognition that whereas too much TPPP/p25 could kill neurons in PD, but its loss keeps cells alive in cancer could contribute to our understanding of the interrelationship of 'TPPP/p25 diseases'. Expert commentary: The knowledge accumulated so far underlines the key roles of the multifunctional TPPP/p25 in both physiological and diverse pathological processes, consequently its validation as drug target sorely needs a new innovative strategy that is briefly reviewed here.

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. 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.

Structure-Based Development of an Affinity Probe for Sirtuin†2.

Sirtuins are NAD(+)-dependent protein deacylases that cleave off acetyl groups, as well as other acyl groups, from the ɛ-amino group of lysines in histones and other substrate proteins. Dysregulation of human Sirt2 activity has been associated with the pathogenesis of cancer, inflammation, and neurodegeneration, thus making Sirt2 a promising target for pharmaceutical intervention. Here, based on a crystal structure of Sirt2 in complex with an optimized sirtuin rearranging ligand (SirReal) that shows improved potency, water solubility, and cellular efficacy, we present the development of the first Sirt2-selective affinity probe. A slow dissociation of the probe/enzyme complex offers new applications for SirReals, such as biophysical characterization, fragment-based screening, and affinity pull-down assays. This possibility makes the SirReal probe an important tool for studying sirtuin biology.

Cross metathesis with hydroxamate and benzamide BOC-protected alkenes to access HDAC inhibitors and their biological evaluation highlighted intrinsic activity of BOC-protected dihydroxamates.

Conditions for the metathesis of alkenes in the convergent synthesis of HDAC inhibitors have been improved by continuous catalyst flow injection in the reaction media. Intermediate and target compounds obtained were tested for their ability to induce HDAC inhibition and tubulin acetylation, revealing the key role of the tert-butyloxycarbonyl (BOC) group for more HDAC6 selectivity. Molecular modelling added rationale for this BOC effect.

Aminothiazoles as Potent and Selective Sirt2 Inhibitors: A Structure-Activity Relationship Study.

Sirtuins are NAD(+)-dependent protein deacylases that cleave off acetyl but also other acyl groups from the ε-amino group of lysines in histones and other substrate proteins. Dysregulation of human Sirt2 (hSirt2) activity has been associated with the pathogenesis of cancer, inflammation, and neurodegeneration, which makes the modulation of hSirt2 activity a promising strategy for pharmaceutical intervention. The sirtuin rearranging ligands (SirReals) have recently been discovered by us as highly potent and isotype-selective hSirt2 inhibitors. Here, we present a well-defined structure-activity relationship study, which rationalizes the unique features of the SirReals and probes the limits of modifications on this scaffold regarding inhibitor potency. Moreover, we present a crystal structure of hSirt2 in complex with an optimized SirReal derivative that exhibits an improved in vitro activity. Lastly, we show cellular hyperacetylation of the hSirt2 targeted tubulin caused by our improved lead structure.