Biology subjects

Wanker, E. E.

Publications and source records attributed to Wanker, E. E..

12 recordsLinked to original sources

bioRxiv2026

HAP40 functions as a proteostasis regulator by controlling huntingtin interactions and its release into the extracellular space

Huntingtin-associated protein 40 (HAP40) forms a stable protein complex with huntingtin (HTT). Its cellular function and how HAP40 loss influences mutant HTT (mHTT) abundance and pathobiology are currently unclear. Here, using diverse cellular models and OMICs methods, we demonstrate that HAP40 is an obligate interaction partner of full-length HTT and through its binding controls the abundance of HTT-associated proteins, indicating that it functions as HTT interaction regulatory unit. Also, loss of HAP40 in mHTT-expressing striatal cells impairs autophagosome-lysosome flux, triggers massive transcriptional dysregulation, including the activation of the CLEAR network, demonstrating that it functions as proteostasis regulator that acts on quality control pathways. Finally, mHTT-expressing cells lacking HAP40 showed increased secretion of mHTT through the ER-to-Golgi route, indicating that striatal cells reduce intracellular mHTT-induced proteotoxicity through activation of secretory pathways. Together, these results establish HAP40 as a critical proteostasis regulator that through controlling HTT interactions maintains cellular homeostasis.

cell biology↗

bioRxiv2025

Chemical targeting of the ATXN1 aa99-163 interaction site suppresses polyQ-expanded protein dimerization

Spinocerebellar ataxia type 1 (SCA1) is a neurodegenerative disease caused by the expansion of a polyglutamine (polyQ) tract in the ATXN1 protein. This expansion is thought to be responsible for the gradual aggregation of the mutant protein, which is associated with increased cytotoxicity and neuronal cell death. Apart from the polyQ tract, other domains in ATXN1 are also involved in the initial events of protein aggregation such as a dimerization domain that promotes protein oligomerization. ATXN1 interacts with various proteins; among them, MED15 that significantly enhances the aggregation of the polyQ-expanded protein. Therefore, we set to identify the interaction site between ATXN1 and MED15 and assess whether its chemical targeting would affect polyQ protein aggregation. First, we predicted the structure of ATXN1 and MED15 and simulated their interaction. We experimentally validated that amino acids (aa) 99-163 of ATXN1 and aa548-665 of MED15 are critical for this protein-protein interaction (PPI). We also show that the aa99-163 domain in ATXN1 is involved in the dimerization of the mutant isoform. Targeting this domain with a chemical compound identified through virtual screening (Chembridge ID: 5755483) inhibited both the interaction of ATXN1 with MED15 and the dimerization of polyQ-expanded ATXN1. These results strengthen our assumption that the aa99-163 domain of ATXN1 may be involved in polyQ protein aggregation and highlight compound 5755483 as a potent first-in-class therapeutic agent for SCA1.

neuroscience↗

bioRxiv2025

Formation of amyloid-like HTTex1 aggregates in neurons, downregulation of synaptic proteins and early mortality of Huntington's disease flies are causally linked

Amyloidogenic mutant huntingtin exon-1 (mHTTex1) protein aggregates with pathogenic polyglutamine (polyQ) tracts are the potential root cause of Huntingtons disease (HD). Here, we assessed the gain-of-function toxicity of mHTTex1 aggregation in neurons of HD transgenic flies. We show that the rate of mHTTex1 aggregation in neurons and early mortality of HD transgenic flies are correlated. We observed sequestration of key synaptic proteins into amyloid-like mHTTex1 aggregates and a concomitant decrease of their transcript levels, suggesting that progressive mHTTex1 aggregate stress in neurons leads to an impairment of synaptic function. Machine learning-based data analysis revealed that the abundance of synaptic proteins such as the vesicular monoamine transporter Vmat in the brain is predictive of fly survival. RNAi knockdown of Vmat-encoding transcripts in neurons with pathogenic amyloid-like HTTex1Q97 aggregates further shortened the lifespan of HD flies, supporting the hypothesis that mHTTex1 aggregation drives impairment of synaptic processes and pathogenesis of HD.

molecular biology↗

bioRxiv2024

Molecular architecture of synaptic vesicles.

Synaptic vesicles (SVs) store and transport neurotransmitters to the presynaptic active zone for release by exocytosis. After release, SV proteins and excess membrane are recycled via endocytosis, and new SVs are formed in a clathrin-dependent manner. This process maintains the morphology and complex molecular composition of SVs through multiple recycling rounds. Previous studies explored the molecular composition of SVs through proteomic analysis and fluorescent microscopy, proposing a model for an average SV1,2. However, the structural heterogeneity and molecular architecture of individual SVs are not well described. Here we used cryo-electron tomography to visualize morphological and molecular details of SVs isolated from mouse brains and inside cultured neurons. We describe several classes of small proteins on the SV surface and long proteinaceous densities inside SVs. We identified V-ATPases, determined a structure using subtomogram average, and showed them forming a complex with the membrane-embedded protein synaptophysin. Our bioluminescence assay revealed pairwise interactions between VAMP2 and synaptophysin and V-ATPase Voe1 domains. Interestingly, V-ATPases were randomly distributed on the surface of SVs irrespective of vesicle sizes. A subpopulation of isolated vesicles and vesicles inside neurons contained a partially assembled clathrin coat with a soccer-ball symmetry. We observed a V-ATPase under clathrin cage in several isolated clathrin-coated vesicles. Additionally, from isolated SV preparations and within hippocampal neurons we identified clathrin baskets without vesicles. We determined their preferential location in proximity to the cell membrane. Our analysis advances the understanding of individual SVs diversity and their molecular architecture.

biophysics↗

bioRxiv2023

AI-guided pipeline for protein-protein interaction drug discovery identifies a SARS-CoV-2 inhibitor

Protein-protein interactions (PPIs) offer great opportunities to expand the druggable proteome and therapeutically tackle various diseases, but remain challenging targets for drug discovery. Here, we provide a comprehensive pipeline that combines experimental and computational tools to identify and validate PPI targets and perform early-stage drug discovery. We have developed a machine learning approach that prioritizes interactions by analyzing quantitative data from binary PPI assays and AlphaFold-Multimer predictions. Using the quantitative assay LuTHy together with our machine learning algorithm, we identified high-confidence interactions among SARS-CoV-2 proteins for which we predicted three-dimensional structures using AlphaFold Multimer. We employed VirtualFlow to target the contact interface of the NSP10-NSP16 SARS-CoV-2 methyltransferase complex by ultra-large virtual drug screening. Thereby, we identified a compound that binds to NSP10 and inhibits its interaction with NSP16, while also disrupting the methyltransferase activity of the complex, and SARS-CoV-2 replication. Overall, this pipeline will help to prioritize PPI targets to accelerate the discovery of early-stage drug candidates targeting protein complexes and pathways.

systems biology↗

bioRxiv2023

A proteomics analysis of 5xFAD mouse brain regions reveals the lysosome-associated protein Arl8b as a candidate biomarker for Alzheimer's disease

BackgroundAlzheimers disease (AD) is characterized by the accumulation of amyloid-{beta} (A{beta}) peptides in intra- and extracellular deposits. How A{beta} aggregates perturb the proteome in brains of patients and AD transgenic mouse models, however, remains largely unclear. State-of-the-art mass spectrometry (MS) methods can comprehensively detect proteomic alterations in neurodegenerative disorders, providing relevant insights unobtainable with transcriptomics investigations. Analyses of the relationship between progressive A{beta} aggregation and protein abundance changes in brains of 5xFAD transgenic mice have not been reported previously. MethodsWe quantified progressive A{beta} aggregation in hippocampus and cortex of 5xFAD mice and controls with immunohistochemistry and biochemical membrane filter assays. Protein changes in different mouse tissues were analysed by MS-based proteomics using label-free quantification (LFQ); resulting MS data were processed using an established pipeline. Results were contrasted with existing proteomic data sets from postmortem AD patient brains. Finally, abundance changes in the candidate marker Arl8b were validated in CSF from AD patients and controls using ELISAs. ResultsExperiments revealed a more rapid accumulation of A{beta}42 peptides in hippocampus than in cortex of 5xFAD mice, accompanied by many more protein abundance changes in hippocampus than in cortex, indicating that A{beta}42 aggregate deposition is associated with brain region-specific proteome perturbations. Generating time-resolved data sets, we defined A{beta} aggregate-correlated and anticorrelated proteome changes, a fraction of which was conserved in postmortem AD patient brain tissue, suggesting that proteome changes in 5xFAD mice mimic disease relevant changes in human AD. We detected a positive correlation between A{beta}42 aggregate deposition in the hippocampus of 5xFAD mice and the abundance of the lysosome-associated small GTPase Arl8b, which accumulated together with axonal lysosomal membranes in close proximity of extracellular A{beta} plaques in 5xFAD brains. Abnormal aggregation of Arl8b was observed in AD brain tissue. Arl8b protein levels were significantly increased in cerebrospinal fluid (CSF) of AD patients, a clinically accessible body fluid. ConclusionsWe report a comprehensive biochemical and proteomic investigation of hippocampal and cortical brain tissue derived from 5xFAD transgenic mice, providing a valuable resource to the neuroscientific community. We identified Arl8b, with significant abundance changes in 5xFAD and AD patient brains. Arl8b might enable the measurement of progressive lysosome accumulation in AD patients and have clinical utility as a candidate biomarker. Data are available via ProteomeXchange with identifier PXD030348.

molecular biology↗

bioRxiv2022

Expanding the Huntingtons disease research toolbox; validated huntingtin subdomain constructs for biochemical and structural investigation of the huntingtin protein

Huntingtons disease is characterised by CAG expansion in the huntingtin gene above a critical threshold of ~35 repeats, resulting in polyglutamine expansion of the huntingtin protein (HTT). The biological role of wildtype HTT and the associated mechanisms of disease pathology caused by expanded HTT remain incompletely understood, in part, due to challenges characterising interactions between HTT and putative binding partners. Here we describe a biochemical toolkit of rationally designed, high-quality recombinant HTT subdomains; one spanning the N-terminal HEAT and bridge domains (NTD) and the second spanning the C-terminal HEAT domain (CTD). Using biophysical methods and cryo-electron microscopy, we show these smaller subdomains are natively folded and can associate to reconstitute a functional full-length HTT structure capable of forming a near native-like complex with 40 kDa HTT-associated protein (HAP40). We report biotin-tagged variants of these subdomains, as well as full-length HTT, that permit immobilisation of each protein for quantitative biophysical assays without impacting protein quality. We demonstrate the CTD alone can form a stable complex when co-expressed with HAP40, which can be structurally resolved. The CTD-HAP40 complex binds the NTD, with a dissociation constant of approximately 10 nM as measured by bio-layer interferometry. We validate the interaction between the CTD and HAP40 using a luciferase two-hybrid assay and use subdomain constructs to demonstrate their respective stabilization of HAP40 in cells. These open-source biochemical tools will enable the wider HD community to study fundamental HTT biology, discover new macromolecular or small-molecule binding partners and map interaction sites across this very large protein.

biochemistry↗

bioRxiv2021

A quantitative mapping approach to identify direct interactions within complexomes

Complementary methods are required to fully characterize all protein complexes, or the complexome, of a cell. Affinity purification coupled to mass-spectrometry (AP-MS) can identify the composition of complexes at proteome-scale. However, information on direct contacts between subunits is often lacking. In contrast, solving the 3D structure of protein complexes can provide this information, but structural biology techniques are not yet scalable for systematic, proteome-wide efforts. Here, we optimally combine two orthogonal high-throughput binary interaction assays, LuTHy and N2H, and demonstrate that their quantitative readouts can be used to differentiate direct interactions from indirect associations within multiprotein complexes. We also show that LuTHy allows accurate distance measurements between proteins in live cells and apply these findings to study the impact of the polyglutamine expansion mutation on the structurally unresolved N-terminal domain of Huntingtin. Thus, we present a new framework based on quantitative interaction assays to complement structural biology and AP-MS techniques, which should help to provide first-approximation contact maps of multiprotein complexes at proteome-scale. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC="FIGDIR/small/457734v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1cc7cf1org.highwire.dtl.DTLVardef@1eed9borg.highwire.dtl.DTLVardef@8197b2org.highwire.dtl.DTLVardef@e5cb8_HPS_FORMAT_FIGEXP M_FIG C_FIG

systems biology↗

bioRxiv2020

Assessment of ethanol-induced toxicity on iPSC-derived human dopaminergic neurons using a novel high-throughput mitochondrial neuronal health (MNH) assay

Excessive ethanol exposure can cause mitochondrial and cellular toxicity. In order to discover potential counteracting interventions, it is essential to develop assays capable of capturing the consequences of ethanol exposure in human dopaminergic (DA) neurons, which are crucial for the development and maintenance of alcohol use disorders (AUD). Here, we developed a novel high-throughput (HT) assay to quantify mitochondrial and neuronal toxicity in human DA neurons from induced pluripotent stem cells (iPSCs). The assay, dubbed mitochondrial neuronal health (MNH) assay, combines live-cell measurement of mitochondrial membrane potential (MMP) with quantification of neuronal branching complexity post-fixation. Using the MNH assay, we demonstrated that chronic ethanol exposure in human iPSC-derived DA neurons decreases MMP and branching complexity in a dose-dependent manner. The toxic effect of ethanol on DA neurons was already detectable after 1 hour of exposure, and occurred similarly in DA neurons derived from healthy individuals and from patients with AUD. We next used the MNH assay to carry out a proof-of-concept compound screening using FDA-approved drugs. We identified potential candidate drugs modulating acute ethanol toxicity in human DA neurons. Among these drugs, flavoxate and disulfiram influenced mitochondrial neuronal health independently from ethanol, leading to amelioration and worsening, respectively. Altogether, we developed an HT assay to probe human mitochondrial neuronal health and used it to assess ethanol neurotoxicity and to identify modulating agents. The MNH assay represents an effective new tool for discovering modulators of mitochondrial neuronal health and toxicity in live human neurons.

cell biology↗

bioRxiv2020

Megadalton-sized dityrosine aggregates of α-synuclein retain high degrees of structural disorder and internal dynamics.

Heterogeneous aggregates of the human protein -synuclein (Syn) are abundantly found in Lewy body inclusions of Parkinsons disease patients. While structural information on classical Syn amyloid fibrils is available, little is known about the conformational properties of disease-relevant, non-canonical aggregates. Here, we analyze the structural and dynamic properties of megadalton-sized dityrosine adducts of Syn that form in the presence of reactive oxygen species and cytochrome c, a proapoptotic peroxidase that is released from mitochondria during sustained oxidative stress. In contrast to canonical cross-{beta} amyloids, these aggregates retain high degrees of internal dynamics, which enables their characterization by solution-state NMR spectroscopy. We find that intermolecular dityrosine crosslinks restrict Syn motions only locally whereas large segments of concatenated molecules remain flexible and disordered. Indistinguishable aggregates form in crowded in vitro solutions and in complex environments of mammalian cell lysates, where relative amounts of free reactive oxygen species rather than cytochrome c are rate limiting. We further establish that dityrosine adducts inhibit classical amyloid formation by maintaining Syn in its monomeric form and that they are non-cytotoxic despite retaining basic membrane-binding properties. Our results suggest that oxidative Syn aggregation scavenges cytochrome cs activity into the formation of amorphous, high molecular-weight structures that may contribute to aggregate diversity in Lewy body deposits.

biophysics↗

bioRxiv2019

SURF1 mutations causative of Leigh syndrome impair human neurogenesis

Mutations in the mitochondrial complex IV assembly factor SURF1 represent a major cause of Leigh syndrome (LS), a rare fatal neurological disorder. SURF1-deficient animals have failed to recapitulate the neuronal pathology of human LS, hindering our understanding of the disease mechanisms. We generated induced pluripotent stem cells from LS patients carrying homozygous SURF1 mutations (SURF1 iPS) and performed biallelic correction via CRISPR/Cas9. In contrast to corrected cells, SURF1 iPS showed impaired neuronal differentiation. Aberrant bioenergetics in SURF1 iPS occurred already in neural progenitor cells (NPCs), disrupting their neurogenic potency. Cerebral organoids from SURF1 iPS were smaller and recapitulated the neurogenesis defects. Our data imply that SURF1 mutations cause a failure in the development of maturing neurons. Using NPC function as an interventional target, we identified SURF1 gene augmentation as a potential strategy for restoring neurogenesis in LS patients carrying SURF1 mutations.

neuroscience↗

bioRxiv2017

Fibril branching dominates self-assembly of mutant huntingtin exon-1 aggregates in vitro

Huntingtin (HTT) fragments with extended polyglutamine (polyQ) tracts self-assemble into amyloid-like fibrillar aggregates. Elucidating the fibril formation mechanism is critical for understanding Huntingtons disease pathology and for developing novel therapeutic strategies. Here, we performed systematic experimental and theoretical studies to examine the self-assembly of an aggregation-prone N-terminal HTT exon-1 fragment with 49 glutamines (Ex1Q49). Using high resolution imaging techniques such as electron microscopy and atomic force microscopy, we show that Ex1Q49 fragments in cell-free assays spontaneously convert into large, highly complex bundles of amyloid fibrils with multiple ends and fibril branching points. Furthermore, we present experimental evidence that two nucleation mechanisms control spontaneous Ex1Q49 fibrillogenesis: (1) a relatively slow primary fibril-independent nucleation process, which involves the spontaneous formation of aggregation-competent fibrillary structures, and (2) a fast secondary fibril-dependent nucleation process, which involves nucleated branching and promotes the rapid assembly of highly complex fibril bundles with multiple ends. The proposed aggregation mechanism is supported by studies with the small molecule O4, which perturbs early events in the aggregation cascade and delays Ex1Q49 fibril assembly, comprehensive mathematical and computational modelling studies, and seeding experiments with small, preformed fibrillar Ex1Q49 aggregates that promote the assembly of amyloid fibrils. Together, our results suggest that nucleated branching in vitro plays a critical role in the formation of complex fibrillar HTT exon-1 aggregates with multiple ends.

biochemistry↗