Biology subjects

Bader, V.

Publications and source records attributed to Bader, V..

12 recordsLinked to original sources

bioRxiv2026

Development and Characterisation of a Versatile Single-Domain Antibody Specific for M1-linked Ubiquitin Chains

Ubiquitin signalling is mediated by structurally distinct polyubiquitin chains that encode discrete cellular functions. Progress in deciphering this ubiquitin code, particularly for the less abundant atypical chain types, has been hindered by limited availability of versatile chain type-specific affinity reagents. Here, we demonstrate that human single-domain antibodies (sdAbs) provide a versatile scaffold for the generation of ubiquitin linkage-specific binders. Using phage display and synthetic human sdAb libraries, we identified 2A6, an sdAb that specifically recognises methionine-1 (M1)-linked ubiquitin chains. To our knowledge, 2A6 represents the first reported sdAb with specificity for a defined homotypic ubiquitin chain linkage. 2A6 bound M1-linked ubiquitin chains with nanomolar affinity and was specific for M1-linked chains at the level of both diubiquitin and long polyubiquitin chains. AlphaFold3 modelling, supported by saturation mutagenesis, predicted that 2A6 recognises the proximal and distal ubiquitin moieties together with the region near the M1 linkage. Functionally, 2A6 enabled specific detection and enrichment of M1-linked ubiquitin across multiple applications, including ELISA, immunoblotting, immunoprecipitation under semi-denaturing conditions, substrate ubiquitination analysis, and immunofluorescence microscopy. The sdAb can be readily produced in E. coli from a single expression plasmid, providing a tractable, cost-effective and versatile reagent for investigating M1-linked ubiquitin signalling. Our work establishes sdAbs as a versatile scaffold for ubiquitin linkage-specific affinity reagents, providing a framework for the development of analogous binders specifically targeting additional ubiquitin linkages or architectures.

biochemistry↗

bioRxiv2025

Modulation of repopulating microglia in multiple sclerosis models with implications for neuroprotection

Microglia play a critical role in central nervous system (CNS) pathologies including multiple sclerosis (MS), and their modulation offers therapeutic potential especially during progressive disease courses. Using cell culture and experimental autoimmune encephalomyelitis (EAE) models, we investigated microglial dynamics during depletion and repopulation (MGrepo) and their modulation using siponimod (sipo), an established CNS penetrating MS medication. Repopulating microglia exhibited a transient reactive state (CD86, MHC-II, Il1b, Tnf). Sipo modulated microglia populations, increasing CD163+, CD206+, and CX3CR1+ while reducing CD86+MHC-II+ cells accompanied by a reduction of neuronal damage. Proteomic spinal cord analysis revealed protein expression alterations by MGrepo and sipo linked to inflammation, myelination, and neuronal structural organization, supported by RNA sequencing of the spinal cord. The neuroinflammation attenuating role of sipo could be linked to cell maintenance and myelin formation associated processes. These findings highlight the capacity of pharmacological interventions to modulate microglia, offering new insights into therapeutic strategies targeting microglial activity in neuroinflammatory diseases.

neuroscience↗

bioRxiv2024

Topological confinement by a membrane anchor suppresses phase separation into protein aggregates: implications for prion diseases

Protein misfolding and aggregation are a hallmark of various neurodegenerative disorders. However, the underlying mechanisms driving protein misfolding in the cellular context are incompletely understood. Here we show that the restriction of conformational degrees of freedom by a membrane anchor stabilizes the native protein conformation and suppresses liquid-liquid phase separation and protein aggregation. Inherited prion diseases in humans and neurodegeneration in transgenic mice are linked to the expression of anchorless prion protein (PrP), suggesting that the C-terminal glycosylphosphatidylinositol (GPI) anchor of native PrP impedes spontaneous formation of neurotoxic and infectious PrP species. Combining novel in vitro and in vivo approaches, we show that anchoring to membranes prevents spontaneous aggregation of PrP. Upon release from the membrane, PrP undergoes a rapid conformational transition to detergent-insoluble aggregates. Our study supports an essential role of the GPI anchor in preventing spontaneous misfolding of PrPC.

molecular biology↗

bioRxiv2023

Linear ubiquitination at damaged lysosomes induces local NF-κB activation and controls cell survival

Lysosomes are the major cellular organelles responsible for nutrient recycling and degradation of cellular material. Maintenance of lysosomal integrity is essential for cellular homeostasis and lysosomal membrane permeabilization (LMP), induced by lysosomotrophic agents, sensitizes towards cell death. Damaged lysosomes are repaired or degraded via lysophagy, during which glycans, exposed on ruptured lysosomal membranes, are recognized by galectins leading to K48- and K63-linked poly-ubiquitination (poly-Ub) of lysosomal proteins followed by recruitment of the autophagic machinery and degradation. Linear (M1) poly-Ub, catalyzed by the E3 ligase linear ubiquitin chain assembly complex (LUBAC) and removed by the OTU domain-containing deubiquitinase with linear linkage specificity (OTULIN) exerts important functions in immune signaling and cell survival, but the role of M1 poly-Ub in lysosomal homeostasis remains largely unexplored. Here, we demonstrate that damaged lysosomes are decorated with M1 poly-Ub in a LUBAC-, OTULIN- and K63-dependent manner. LMP-induced M1 poly-Ub at damaged lysosomes contributes to lysosome degradation, recruits nuclear factor {kappa}-B (NF-{kappa}B) essential modulator (NEMO) and locally activates inhibitor of NF-B kinase (IKK) to trigger NF-{kappa}B activation in a K63 poly-Ub-dependent manner. Inhibition of lysosomal degradation enhances LMP- and OTULIN-dependent cell death, indicating pro-survival functions of LMP and potentially lysophagy. Finally, we demonstrate that M1 poly-Ub occurs at L-leucyl-leucine methyl ester (LLOMe)-damaged lysosomes in primary mouse neurons and induced pluripotent stem cell (iPSC)-derived primary human dopaminergic neurons. Together, our results reveal novel functions of M1 poly-Ub during lysosomal homeostasis, LMP and degradation of damaged lysosomes, with important implications for NF-{kappa}B signaling, inflammation and cell death.

cell biology↗

bioRxiv2023

A novel PEX14/PEX5 interface links peroxisomal protein import and receptor recycling

Newly synthesized peroxisomal proteins are recognized in the cytosol by the cycling receptor PEX5 and directed to a docking complex comprising PEX14 and PEX13 at the peroxisomal membrane. After cargo translocation, the unloaded PEX5 is recycled in an ATP-dependent manner. Receptor docking involves the WxxxF-motifs in the N-terminal domain (NTD) of PEX5 that are recognized by the N-terminal domain of PEX14. Here, we combine biochemical methods and NMR spectroscopy to identify a novel binding interface between human PEX5 and PEX14. The interaction involves the PEX5 C-terminal cargo-binding TPR domain and a conserved IPSWQI peptide motif in the C-terminal intrinsically disordered region of PEX14. The three-dimensional structure of the PEX14 IPSWQI peptide bound the PEX5 TPR domain, shows the PEX14 interaction is non-overlapping with PTS1 binding to the TPR domain. Notably, PEX14 IPSWQI motif binding to a hinge region in the TPR domain shows a more open supercoil of the TPR fold that resembles the apo conformation in the absence of PTS1 peptide. Mutation of binding site residues in PEX5 or PEX14 leads to a partial protein import defect and decrease of the steady-state-concentration of PEX5. This resembles the mutant phenotype of cells affected in receptor recycling, suggesting a role in this process.

biochemistry↗

bioRxiv2023

Protein Assembly Modulation: A New Approach to ALS Therapeutics

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease with a complex, multifactorial pathophysiology, most commonly manifest as loss of motor neurons. We introduce a new mechanism of ALS pathogenesis via a novel drug-like small molecule series that targets protein disulfide isomerase (PDI) within a previously unappreciated transient and energy-dependent multi-protein complex. This novel drug was found to have activity in cellular models for both familial and sporadic ALS, as well as in transgenic worms, flies, and mice bearing a diversity of human genes with ALS-associated mutations. These compounds were initially identified as modulators of human immunodeficiency virus (HIV) capsid assembly in cell-free protein synthesis and assembly (CFPSA) systems, with demonstrated antiviral activity in cell culture. Their advancement as ALS-therapeutics, and the subsequent separation of activity against HIV and ALS in chemical subseries through structure-activity-relationship optimization, may provide insights into the molecular mechanisms governing pathophysiology of disordered homeostasis relevant to ALS.

neuroscience↗

bioRxiv2023

NEMO reshapes the protein aggregate interface and promotes aggrephagy by co-condensation with p62

NEMO is a ubiquitin-binding protein which regulates canonical NF-{kappa}B pathway activation in innate immune signaling, cell death regulation and host-pathogen interactions. Here we identified an NF-{kappa}B-independent function of NEMO in proteostasis regulation by promoting autophagosomal clearance of protein aggregates. NEMO-deficient cells accumulate misfolded proteins upon proteotoxic stress and are vulnerable to proteostasis challenges. Moreover, a patient with a mutation in the NEMO gene resulting in defective binding of NEMO to linear ubiquitin chains, developed a widespread mixed brain proteinopathy, including -synuclein, tau and TDP-43 pathology. NEMO amplifies linear ubiquitylation at -synuclein aggregates and promotes the local concentration of p62 into foci. In vitro, NEMO lowers the threshold concentrations required for ubiquitin-dependent phase transition of p62. In summary, NEMO reshapes the aggregate surface for efficient autophagosomal clearance by providing a mobile phase at the aggregate interphase favoring co-condensation with p62.

cell biology↗

bioRxiv2022

LUBAC assembles a signaling platform at mitochondria for signal amplification and shuttling of NF-ĸB to the nucleus

Mitochondria are increasingly recognized as cellular hubs to orchestrate signaling pathways that regulate metabolism, redox homeostasis, and cell fate decisions. Recent research revealed a role of mitochondria also in innate immune signaling, however, the mechanisms of how mitochondria affect signal transduction are poorly understood. Here we show that the NF-B pathway activated by TNF employs mitochondria as a platform for signal amplification and shuttling of activated NF-B to the nucleus. TNF induces the recruitment of HOIP, the catalytic component of the linear ubiquitin chain assembly complex (LUBAC), and its substrate NEMO to the outer mitochondrial membrane, where M1- and K63-linked ubiquitin chains are generated. NF-B is locally activated and transported to the nucleus by mitochondria, resulting in an increase in mitochondria-nucleus contact sites in a HOIP-dependent manner. Notably, TNF-induced stabilization of the mitochondrial kinase PINK1 contributes to signal amplification by antagonizing the M1-ubiquitin-specific deubiquitinase OTULIN.

cell biology↗

bioRxiv2022

Peroxisomal ATPase ATAD1 acts in quality control of the protein import machinery

ATAD1 is an AAA-ATPase which shows a dual localization at mitochondria and peroxisomes. While its peroxisomal function is not known, in mitochondria ATAD1 is part of a quality control mechanism extracting mislocalised tail-anchored and accumulated precursor proteins from the outer membrane. Here, we studied the peroxisomal interactome of ATAD1 and could show that human ATAD1 interacts with PEX5, a cytosolic receptor for peroxisomal matrix proteins which transiently inserts into peroxisomal membranes. Upon cargo-release, mono-ubiquitinated PEX5 is recycled into the cytosol by the AAA-peroxins PEX1 and PEX6. The accumulation of ubiquitinated PEX5 is known to trigger degradation of whole organelles called pexophagy. Here, we used ATAD1-, PEX1- and ATAD1/PEX1-CRISPR-Knockout cell lines to investigate the physiological role of an ATAD1-PEX5 interaction. We could show an influence of ATAD1 on the stability of accumulated PEX5 and hypothesize a role in a peroxisomal quality control mechanism enabling clearance of ubiquitinated receptor from the membrane.

biochemistry↗

bioRxiv2022

Brevican, Neurocan, Tenascin-C and Tenascin-R Act as Important Regulators of the Interplay between Perineuronal Nets, Synaptic Integrity, Inhibitory Interneurons and Otx2

Fast-spiking parvalbumin interneurons are critical for the function of mature cortical inhibitory circuits. Most of these neurons are enwrapped by a specialized extracellular matrix structure (ECM) called perineuronal net (PNN), which can regulate their synaptic input. In this study, we investigated the relationship between PNNs, parvalbumin interneurons and synaptic distribution on these cells in the adult primary visual cortex (V1) of quadruple knockout mice deficient for the ECM molecules brevican, neurocan, tenascin-C and tenascin-R. We used super-resolution structured illumination microscopy (SIM) to analyze PNN structure and associated synapses. Additionally, we examined parvalbumin and calretinin interneuron populations. We observed a reduction in the number of PNN-enwrapped cells and a clear disorganization of the PNN structure in the quadruple knockout V1. This was accompanied by an imbalance of inhibitory and excitatory synapses with a reduction of inhibitory and an increase of excitatory synaptic elements along the PNNs. Also, the number of parvalbumin interneurons was reduced in the quadruple knockout, while calretinin interneurons, which do not wear PNNs did not display differences in number. Interestingly, we found the transcription factor Otx2 homeoprotein positive cell population also reduced. Otx2 is crucial for parvalbumin and PNN maturation and a positive feedback loop between these parameters has been described. Collectively, these data indicate an important role of brevican, neurocan, tenascin-C and tenascin-R in regulating the interplay between PNNs, inhibitory interneurons, synaptic distribution as well as Otx2 in the V1.

neuroscience↗

bioRxiv2021

Macrophage migration inhibitory factor is a valid drug target at the intersection of herpes simplex virus 1 replication and Alzheimer's disease-relevant cellular pathology

The viral life cycle usurps host cellular factors, redirecting them from physiological functions to viral needs thereby revealing their "moonlighting" functions, disturbing cellular proteostasis, and increasing risk of specific, virus-associated protein misfolding diseases (PMD). Identifying such virus-repurposed host proteins therefore allow study of fundamental cellular events leading to associated "sporadic" PMD. Here, we identified a small molecule with unprecedented activity against neurotropic herpes simplex virus 1 (HSV-1) modulating an allosteric site of Macrophage Migration Inhibitory Factor (MIF). The compound efficiently reduced HSV-1-mediated tau phosphorylation or aggregation in vitro and in vivo, even without HSV-1 infection. The lead compound specifically interacted with an oxidized conformer of MIF (oxMIF) from either recombinant MIF or post-mortem brain homogenates of patients with Alzheimers disease (AD). OxMIF thus participates in a host-viral interface connecting HSV-1 infection, and possibly other external stressors, with tau cellular pathology characteristic for PMD, including Alzheimes disease.

microbiology↗

bioRxiv2020

Amyloid precursor protein causes fusion of promyelocytic leukemia nuclear bodies in human hippocampal areas with high plaque load

The amyloid precursor protein (APP) is a type I transmembrane protein with unknown physiological function but potential impact in neurodegeneration. The current study demonstrates that APP signals to the nucleus causing the generation of aggregates comprising its adapter protein FE65 and the tumour suppressor proteins p53 and PML. The PML nuclear body generation, known to be of relevance in virus defence and cell division, is induced and fusion occurs over time depending on APP signalling. We further show that the nuclear aggregates of APP C-terminal (APP-CT) fragments together with PML and FE65 are present in the aged human brain but not in cerebral organoids differentiated from iPS cells. Notably, human Alzheimers disease brains reveal a highly significant loss of these nuclear aggregates in areas with high plaque load compared to plaque-free areas of the same individual. Based on these results we conclude that APP-CT signalling to the nucleus takes place in the aged human brain and is potentially involved in the pathophysiology of AD. Taken the current knowledge on PML bodies into account, we hypothesize a new role for APP as a twofold virus response protein. The APP-dependent defence strategy includes A{beta}-virus interaction at the extracellular matrix and APP-CT driven PML aggregation in the nucleus to encapsulate the viral nucleic acid. This defence strategy preferentially occurs in high-plaque regions of the human brain and overstimulation of this pathway results in a pyrrhic victory.

neuroscience↗