Biology subjects

Winklhofer, K. F.

Publications and source records attributed to Winklhofer, K. F..

15 recordsLinked to original sources

bioRxiv2026

PINK1 stabilizes RMDN3-VAPB interaction without altering the landscape of mito-ER contact sites

Mitochondria-ER contact sites (MERCS) have emerged as central hubs of organellar biology, participating in the exchange of lipids, Ca2+ and even protein precursors. During mitochondrial dysfunction, the contact between the ER-resident protein VAPB and the outer mitochondrial membrane protein RMDN3 (PTPIP51) increases, yet the mechanism stabilizing this connection despite ongoing mitophagy remains to be determined. Here we develop a splitAPEX2 based proximity biotinylation tool to assess the interaction of VAPB and RMDN3. We determine that activation of the mitochondrial depolarization-induced kinase PINK1 leads to phosphorylation of RMDN3 at T160, which is necessary to increase the interaction with VAPB in the presence of mitochondrial depolarization. Mass Spectrometric analysis of the RMDN3-VAPB MERCS proteome reveals that these contacts also include peroxisomal proteins and that the proteomic composition remains stable during mitochondrial depolarization.

cell biology↗

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↗

bioRxiv2025

Novel function of Contactin associated protein 1 (Caspr 1)/ Paranodin in embryonic cortical neurons: hypoxia modulated neurite development.

Hypoxia, a condition of inadequate oxygen supply, is a common phenomenon affecting neurons and brain tissue, leading to significant implications for neuronal health and function. The prevalence of hypoxia in the brain is associated with various neurological conditions, making it a critical area of study. Neuritogenesis, the process of neurite outgrowth, is an essential aspect of neuronal development and connectivity and is particularly sensitive to hypoxic stress. Investigating how hypoxia affects neurite outgrowth is vital for understanding neuronal response and adaptation under low oxygen conditions. This study explores how hypoxic stress affects neurite regulation mediated by Contactin Associated Protein-1 (Caspr1) in primary mouse embryonic cortical neurons. Hypoxia, induced by culturing neurons in a 2% oxygen environment, significantly reduced neurite length and induced notable changes in growth cone morphology. Concurrently, we observed an upregulation in the expression of Caspr1 and its transcriptional regulator C/EBP, suggesting a compensatory role for Caspr1 in neurite extension under low oxygen conditions. Shorter hypoxia exposure periods revealed a dynamic biphasic response in Caspr1 levels, with an initial decrease followed by a substantial increase, correlating with corresponding changes in neurite length. This pattern emphasizes the critical involvement of Caspr1 in adapting neurite growth to fluctuating hypoxia duration. Furthermore, comparative analyses using wild-type and Caspr1 knockout Neuro2a cells demonstrated that the absence of Caspr1 mitigates hypoxia-induced neurite shortening, indicating a potential protective role against hypoxic stress. Additionally, hypoxia profoundly impacted mitochondrial morphology and function. Under hypoxic conditions, mitochondria transitioned to a more spherical shape. Mitochondrial respiration analysis revealed significant reductions in oxygen consumption rates (OCR), highlighting compromised mitochondrial function during hypoxia. These findings underscore the multifaceted role of Caspr1 in neurite regulation and mitochondrial adaptation to hypoxic stress. The study provides insights into the molecular mechanisms underpinning hypoxia-induced changes in neuronal morphology and function. Understanding these processes opens avenues for therapeutic strategies targeting Caspr1 in treating neurological disorders characterized by hypoxic stress. Future research will benefit from extending these investigations to more complex models, such as brain organoids, to further elucidate the metabolic and structural changes under hypoxia and their implications for neurodegenerative diseases.

neuroscience↗

bioRxiv2025

Resting calcium ion fluxes protect cells from fast mitochondrial fragmentation, cell stress responses, and immediate transcriptional reprogramming

Homeostatic calcium ion (Ca2+) fluxes between the endoplasmic reticulum, cytosol, and extracellular space occur not only in response to cell stimulation but also in unstimulated cells. Using murine astrocytes as a model, we asked whether there is a signaling function of these resting Ca2+-fluxes. The data showed that endoplasmic reticulum (ER) Ca{superscript 2} depletion, induced by sarcoplasmic/endoplasmic reticulum Ca{superscript 2}-ATPase (SERCA) inhibition, resulted to prolonged Ca{superscript 2} influx and mitochondrial fragmentation within 10 to 30 minutes. This mitochondrial fragmentation could be prevented in Ca2+- free medium or by inhibiting store-operated Ca2+ entry (SOCE). Similarly, attenuation of STIM proteins, which are vital ER Ca2+ sensors, protected mitochondrial morphology. On the molecular level, ER Ca2+ depletion, achieved either by removing extracellular Ca2+ or through acute SERCA inhibition, led to changes in gene expression of about 13% and 41% of the transcriptome within an hour, respectively. Transcriptome changes were associated with universal biological processes such as transcription, differentiation, or cell stress. Strong increase in expression was observed for the transcription factor ATF4, which is under control of the kinase PERK (EIF2AK3), a key protein involved in ER stress. Corroborating these findings, PERK was rapidly phosphorylated in Ca2+-free medium or after acute pharmacological inhibition of SOCE. In summary, resting, homeostatic Ca2+ fluxes prevent immediate- early cell stress and transcriptional reprogramming.

physiology↗

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↗

bioRxiv2024

Antibody-dependent Intracellular neutralisation by TRIM21 is potentiated by HOIP and linear ubiquitin chains

Antibody-dependent intracellular neutralisation (ADIN) promotes the rapid proteasomal degradation of viruses and other large substrates in the cytosol. It is dependent on detection of intracellular virus-bound antibodies by the Fc receptor and E3 ligase TRIM21, followed by disassembly of the virus by the unfoldase VCP/p97. It is not known how VCP is recruited to TRIM21. We performed a limited siRNA knock-down screen of known VCP adaptors to determine their involvement in ADIN. Knock-down of HOIP, the only ubiquitin E3 ligase capable of generating linear ubiquitin chains, resulted in impaired virus neutralisation. HOIPIN-8, a HOIP inhibitor, showed concentration-dependent reduction in virus neutralisation. We found that the activity of HOIP in ADIN is dependent on its ubiquitin binding domains and its PUB domain which recruits VCP. Knock-down of OTULIN, the only deubiquitinating enzyme that exclusively cleaves linear ubiquitin chains, potentiated neutralisation of the virus. Our results expand the role of HOIP and linear ubiquitin chains in proteostasis.

immunology↗

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

Nuclear quality control of non-imported secretory proteins attenuates proteostasis decline in the cytosol

Mistargeting of secretory proteins to the cytosol can induce formation of aggregation-prone conformers and subsequent proteostasis decline. We have identified a quality control pathway that redirects non-ER-imported prion protein (PrP) to proteasomal degradation in the nucleus to prevent formation of toxic aggregates in the cytosol. Upon aborted ER import, PrP sequentially interacted with VCP/p97 and importins, which kept PrP soluble and promoted its nuclear import. In the nucleus, RNA buffered aggregation of PrP to facilitate ubiquitin-dependent proteasomal degradation. Notably, the cytosolic interaction of PrP with VCP/p97 and its nuclear import were independent of ubiquitination but required the intrinsically unstructured N-terminal domain of PrP. Transient proteotoxic stress promoted the formation of self-perpetuating PrP aggregates in the cytosol, which disrupted further nuclear targeting of PrP and compromised cellular proteostasis. Our study delineates a VCP/p97-dependent nucleus-based quality control pathway of non-ER-imported secretory proteins and emphasizes the important role of the nuclear milieu for the degradation of aggregation-prone proteins.

cell biology↗

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

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

Increased levels of the mitochondrial import factor Mia40 prevent the aggregation of polyQ proteins in the cytosol

The formation of protein aggregates is a hallmark of neurodegenerative diseases. Observations on patient material and model systems demonstrated links between aggregate formation and declining mitochondrial functionality, but the causalities remained unclear. We used yeast as model system to analyze the relevance of mitochondrial processes for the behavior of an aggregation-prone polyQ protein derived from human huntingtin. Induction of Q97-GFP rapidly leads to insoluble cytosolic aggregates and cell death. Although this aggregation impairs mitochondrial respiration only slightly, it interferes with efficient import of mitochondrial precursor proteins. Mutants in the import component Mia40 are hypersensitive to Q97-GFP. Even more surprisingly, Mia40 overexpression strongly suppresses the formation of toxic Q97-GFP aggregates both in yeast and in human cells. Based on these observations, we propose that the posttranslational import into mitochondria competes with aggregation-prone cytosolic proteins for chaperones and proteasome capacity. Owing to its rate-limiting role for mitochondrial protein import, Mia40 acts as a regulatory component in this competition. This role of Mia40 as dynamic regulator in mitochondrial biogenesis can apparently be exploited to stabilize cytosolic proteostasis. (174/175 words)

biochemistry↗

bioRxiv2020

Optogenetic delivery of trophic signals in a genetic model of Parkinson's disease

Optogenetics has been harnessed to shed new mechanistic light on current therapies and to develop future treatment strategies. This has been to date achieved by the correction of electrical signals in neuronal cells and neural circuits that are affected by disease. In contrast, the optogenetic delivery of trophic biochemical signals, which support cell survival and thereby may modify progression of degenerative disorders, has never been demonstrated in an animal disease model. Here, we reengineered the human and Drosophila melanogaster REarranged during Transfection (hRET and dRET) receptors to be activated by light, creating one-component optogenetic tools termed Opto-hRET and Opto-dRET. Upon blue light stimulation, these receptors robustly induced the MAPK/ERK proliferative signaling pathway in cultured cells. In PINK1B9 flies that exhibit loss of PTEN-induced putative kinase 1 (PINK1), a kinase associated with familial Parkinsons disease (PD), light activation of Opto-dRET suppressed mitochondrial defects, tissue degeneration and behavioral deficits. In human cells with PINK1 loss-of-function, mitochondrial fragmentation was rescued using Opto-dRET via the PI3K/NF-[kcy]B pathway. Our results demonstrate that a light-activated receptor can ameliorate disease hallmarks in a genetic model of PD. The optogenetic delivery of trophic signals is cell type-specific and reversible and thus has the potential to overcome limitations of current strategies towards a spatio-temporal regulation of tissue repair. Significance StatementThe death of physiologically important cell populations underlies of a wide range of degenerative disorders, including Parkinsons disease (PD). Two major strategies to counter cell degeneration, soluble growth factor injection and growth factor gene therapy, can lead to the undesired activation of bystander cells and non-natural permanent signaling responses. Here, we employed optogenetics to deliver cell type-specific pro-survival signals in a genetic model of PD. In Drosophila and human cells exhibiting loss of the PINK1 kinase, akin to autosomal recessive PD, we efficiently suppressed disease phenotypes using a light-activated tyrosine kinase receptor. This work demonstrates a spatio-temporally precise strategy to interfere with degeneration and may open new avenues towards tissue repair in disease models.

neuroscience↗