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.

Tubulin Binding and Polymerization Promoting Properties of Tubulin Polymerization Promoting Proteins Are Evolutionarily Conserved.

Tubulin polymerization promoting proteins (TPPPs) constitute a eukaryotic protein family. There are three TPPP paralogs in the human genome, denoted as TPPP1-TPPP3. TPPP1 and TPPP3 are intrinsically unstructured proteins (IUPs) that bind and polymerize tubulin and stabilize microtubules, but TPPP2 does not. Vertebrate TPPPs originated from the ancient invertebrate TPPP by two-round whole-genome duplication; thus, whether the tubulin/microtubule binding function of TPPP1 and TPPP3 is a newly acquired property or was present in the invertebrate orthologs (generally one TPPP per species) has been an open question. To answer this question, we investigated a TPPP from a simple and early branching animal, the sponge Suberites domuncula. Bioinformatics, biochemical, immunochemical, spectroscopic, and electron microscopic data showed that the properties of the sponge protein correspond to those of TPPP1; namely, it is an IUP that strongly binds tubulin and induces its polymerization, proving that these features of animal TPPPs have been evolutionarily conserved.

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.

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. The binding of SYN(103-140) 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 SYN(95-140) and SYN(126-140) - in contrast to SYN(1-120) - displayed significant binding to TPPP/p25. Fluorescence assay with ANS, a molten globule indicator, showed that SYN, but not SYN(1-120) abolished the zinc-induced local folding of both the full length as well as the N- and C-terminal-free (core) TPPP/p25; SYN(95-140) and SYN(126-140) 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 SYN(95-140) and SYN(126-140), but not SYN(1-120), 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.

Extracellular deposition of matrilin-2 controls the timing of the myogenic program during muscle regeneration.

Here, we identify a role for the matrilin-2 (Matn2) extracellular matrix protein in controlling the early stages of myogenic differentiation. We observed Matn2 deposition around proliferating, differentiating and fusing myoblasts in culture and during muscle regeneration in vivo. Silencing of Matn2 delayed the expression of the Cdk inhibitor p21 and of the myogenic genes Nfix, MyoD and Myog, explaining the retarded cell cycle exit and myoblast differentiation. Rescue of Matn2 expression restored differentiation and the expression of p21 and of the myogenic genes. TGF-ß1 inhibited myogenic differentiation at least in part by repressing Matn2 expression, which inhibited the onset of a positive-feedback loop whereby Matn2 and Nfix activate the expression of one another and activate myoblast differentiation. In vivo, myoblast cell cycle arrest and muscle regeneration was delayed in Matn2(-/-) relative to wild-type mice. The expression levels of Trf3 and myogenic genes were robustly reduced in Matn2(-/-) fetal limbs and in differentiating primary myoblast cultures, establishing Matn2 as a key modulator of the regulatory cascade that initiates terminal myogenic differentiation. Our data thus identify Matn2 as a crucial component of a genetic switch that modulates the onset of tissue repair.

Hmgb1 can facilitate activation of the matrilin-1 gene promoter by Sox9 and L-Sox5/Sox6 in early steps of chondrogenesis.

The architectural high mobility group box 1 (Hmgb1) protein acts as both a nuclear and an extracellular regulator of various biological processes, including skeletogenesis. Here we report its contribution to the evolutionarily conserved, distinctive regulation of the matrilin-1 gene (Matn1) expression in amniotes. We previously demonstrated that uniquely assembled proximal promoter elements restrict Matn1 expression to specific growth plate cartilage zones by allowing varying doses of L-Sox5/Sox6 and Nfi proteins to fine-tune their Sox9-mediated transactivation. Here, we dissected the regulatory mechanisms underlying the activity of a conserved distal promoter element 1. We show that this element carries three Sox-binding sites, works as an enhancer in vivo, and allows promoter activation by the Sox5/6/9 chondrogenic trio. In early steps of chondrogenesis, declining Hmgb1 expression overlaps with the onset of Sox9 expression. Unlike repression in late steps, Hmgb1 overexpression in early chondrogenesis increases Matn1 promoter activation by the Sox trio, and forced Hmgb1 expression in COS-7 cells facilitates induction of Matn1 expression by the Sox trio. The conserved Matn1 control elements bind Hmgb1 and SOX9 with opposite efficiency in vitro. They show higher HMGB1 than SOX trio occupancy in established chondrogenic cell lines, and HMGB1 silencing greatly increases MATN1 and COL2A1 expression. Together, these data thus suggest a model whereby Hmgb1 helps recruit the Sox trio to the Matn1 promoter and thereby facilitates activation of the gene in early chondrogenesis. We anticipate that Hmgb1 may similarly affect transcription of other cartilage-specific genes.

Interactions between ß-arrestin proteins and the cytoskeletal system, and their relevance to neurodegenerative disorders.

ß-arrestins, which have multiple cellular functions, were initially described as proteins that desensitize rhodopsin and other G protein-coupled receptors. The cytoskeletal system plays a role in various cellular processes, including intracellular transport, cell division, organization of organelles, and cell cycle. The interactome of ß-arrestins includes the major proteins of the three main cytoskeletal systems: tubulins for microtubules, actins for the actin filaments, and vimentin for intermediate filaments. ß-arrestins bind to microtubules and regulate their activity by recruiting signaling proteins and interacting with assembly proteins that regulate the actin cytoskeleton and the intermediate filaments. Altered regulation of the cytoskeletal system plays an essential role in the development of Alzheimer's, Parkinson's and other neurodegenerative diseases. Thus, ß-arrestins, which interact with the cytoskeleton, were implicated in the pathogenesis progression of these diseases and are potential targets for the treatment of neurodegenerative disorders in the future.

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.

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.

Multiple Levels of Immunological Memory and Their Association with Vaccination.

Immunological memory is divided into many levels to counteract the provocations of diverse and ever-changing infections. Fast functions of effector memory and the superposition of both quantitatively and qualitatively plastic anticipatory memory responses together form the walls of protection against pathogens. Here we provide an overview of the role of different B and T cell subsets and their interplay, the parallel and independent functions of the B1, marginal zone B cells, T-independent- and T-dependent B cell responses, as well as functions of central and effector memory T cells, tissue-resident and follicular helper T cells in the memory responses. Age-related limitations in the immunological memory of these cell types in neonates and the elderly are also discussed. We review how certain aspects of immunological memory and the interactions of components can affect the efficacy of vaccines, in order to link our knowledge of immunological memory with the practical application of vaccination.

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.

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.

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.

The Curcumin Analog C-150, Influencing NF-κB, UPR and Akt/Notch Pathways Has Potent Anticancer Activity In Vitro and In Vivo.

C-150 a Mannich-type curcumin derivative, exhibited pronounced cytotoxic effects against eight glioma cell lines at micromolar concentrations. Inhibition of cell proliferation by C-150 was mediated by affecting multiple targets as confirmed at transcription and protein level. C-150 effectively reduced the transcription activation of NFkB, inhibited PKC-alpha which are constitutively over-expressed in glioblastoma. The effects of C-150 on the Akt/ Notch signaling were also demonstrated in a Drosophila tumorigenesis model. C-150 reduced the number of tumors in Drosophila with similar efficacy to mitoxantrone. In an in vivo orthotopic glioma model, C-150 significantly increased the median survival of treated nude rats compared to control animals. The multi-target action of C-150, and its preliminary in vivo efficacy would render this curcumin analogue as a potent clinical candidate against glioblastoma.

Evolutionarily conserved, growth plate zone-specific regulation of the matrilin-1 promoter: L-Sox5/Sox6 and Nfi factors bound near TATA finely tune activation by Sox9.

To help uncover the mechanisms underlying the staggered expression of cartilage-specific genes in the growth plate, we dissected the transcriptional mechanisms driving expression of the matrilin-1 gene (Matn1). We show that a unique assembly of evolutionarily conserved cis-acting elements in the Matn1 proximal promoter restricts expression to the proliferative and prehypertrophic zones of the growth plate. These elements functionally interact with distal elements and likewise are capable of restricting the domain of activity of a pancartilaginous Col2a1 enhancer. The proximal elements include a Pe1 element binding the chondrogenic L-Sox5, Sox6, and Sox9 proteins, a SI element binding Nfi proteins, and an initiator Ine element binding the Sox trio and other factors. Sox9 binding to Pe1 is indispensable for functional interaction with the distal promoter. Binding of L-Sox5/Sox6 to Ine and Nfib to SI modulates Sox9 transactivation in a protein dose-dependent manner, possibly to enhance Sox9 activity in early stages of chondrogenesis and repress it at later stages. Hence, our data suggest a novel model whereby Sox and Nfi proteins bind to conserved Matn1 proximal elements and functionally interact with each other to finely tune gene expression in specific zones of the cartilage growth plate.