Biology subjects

Knobeloch, K.-P.

Publications and source records attributed to Knobeloch, K.-P..

12 recordsLinked to original sources

bioRxiv2026

Csf1r-mediated depletion of midbrain microglia prevents dopaminergic neuron loss during chronic colitis

Inflammatory bowel disease (IBD) predisposes to neuropsychiatric comorbidity and particularly increases the risk of Parkinsons Disease (PD) in later life. Although the gut-immune-brain axis was proposed as a link between IBD and PD and a driver of PD immunopathogenesis, the regional pattern and single-cell landscape of the brain immune response during colitis and its contribution to PD pathology remain poorly defined. Here, we observe a loss of dopaminergic neurons in the substantia nigra pars compacta of adult mice with chronic colitis. By confocal microscopy and integrated multi-omics, we reveal a complex midbrain-centered immune response to chronic colitis in comparison to the cortex, hippocampus, and striatum. Single-cell mapping of the midbrain immune landscape showed an inflammatory shift of microglial clusters including an expansion of interferon-response microglia, CD8+ T cell extravasation, and increased numbers of vessel-associated neutrophils. Selective myeloid cell depletion using a colony stimulating factor 1 receptor (Csf1r) inhibitor after colitis onset reduced midbrain microglia by 67% and led to a complete rescue of dopaminergic neuron loss, without affecting mucosal pathology or T cell and neutrophil migration to the midbrain. Collectively, within the complex innate and adaptive midbrain immune response to chronic colitis, we demonstrate a causal role of Csf1r-dependent microglia for dopaminergic neurodegeneration. Thus, Csf1r inhibition in IBD may not locally ameliorate colitis, but provide neuroprotection to dopaminergic neurons. These results reveal a novel cellular link between chronic gut-derived peripheral inflammation and midbrain vulnerability and thereby substantially enhance our understanding of the risk for PD related to the gut-immune-brain axis. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=174 SRC="FIGDIR/small/700559v1_ufig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@1bf7217org.highwire.dtl.DTLVardef@1db66c0org.highwire.dtl.DTLVardef@136ef2eorg.highwire.dtl.DTLVardef@190f641_HPS_FORMAT_FIGEXP M_FIG Graphical abstract. C_FIG

neuroscience↗

bioRxiv2025

Inflammation-enhanced synapse-specific phagocytosis by adult APP microglia in a microfluidic neuron-microglia co-culture model

Microglia play a critical role in synapse remodeling and neuroinflammation, both of which are dysregulated in Alzheimers disease (AD). However, most in vitro models rely on neonatal or immortalized microglia, limiting their relevance to adult pathophysiological context. Here, we present a compartmentalized microfluidic co-culture platform that enables spatially controlled interactions between primary cortical neurons and adult microglia from wild-type (WT) and APP-transgenic mice. This system allows precise functional analysis of microglia-synapse interactions under defined inflammatory conditions. Upon lipopolysaccharide (LPS) stimulation, APP microglia exhibited exaggerated morphological activation, elevated IL-1{beta} secretion, and selectively increased engulfment of synaptic material. In contrast, phagocytosis of non-specific substrates such as pHrodo Zymosan remained unchanged, suggesting a substrate-specific enhancement of microglial phagocytic activity. Blocking the complement receptor CD11b abolished the LPS-induced increase in synaptic uptake, confirming the role of complement-dependent pathways. Transcriptomic profiling revealed robust inflammatory responses in both genotypes, with selectively heightened expression of proinflammatory genes in APP microglia, consistent with a primed immune phenotype. Importantly, increased synaptic uptake occurred without measurable loss of global synaptic connectivity, highlighting the specificity and sensitivity of the system to detect microglial functional changes. This model captures genotype-dependent microglial reactivity (revealing phenotypes not fully captured by transcriptomic rofiling) and provides a physiologically relevant, tractable in vitro platform for dissecting microglial contributions to synaptic pathology in neurodegenerative disease.

neuroscience↗

bioRxiv2025

High-Throughput Synthesis and Screening of a Cyanimide Library Identifies Selective Inhibitors of ISG15-specific Protease USP18

High-throughput screening (HTS) of (large) compound collections is a critical early step in many drug discovery programs, enabling the rapid identification of lead molecules with desirable biological activity. The success of HTS depends heavily on the quality of the compound libraries used, and as such, the development of targeted libraries has emerged as a promising approach to enhance the effectiveness of HTS efforts. However, the acquisition and synthesis of such libraries remain costly and labor-intensive, often yielding compound quantities far exceeding the small amounts required for screening. To address these challenges, we present a high-throughput synthesis-to-screening method for the efficient inplate generation and immediate HTS of a deubiquitinase (DUB)-focused compound library. Central to our approach is the use of an Echo acoustic liquid handler, which enables precise nanoliter-scale transfers of DMSO-based solutions, facilitating efficient and miniaturized synthesis directly in 1,536-well plates. Using this platform, we constructed a library of 7,536 compounds featuring a DUB-privileged cyanimide warhead and screened it against a panel of twelve DUBs and ubiquitin-like proteases. This identified two structurally related molecules with selective inhibitory activity against the interferon-stimulated gene 15 (ISG15) protease USP18, which we further developed into a first-in-class USP18 inhibitor with 35 nM potency. This compound, BB07CA902, demonstrated exceptional specificity for USP18 across a panel of 41 DUBs and effectively increased ISGylation levels in cells by inhibiting USP18 activity. With our technology, we enabled the efficient preparation of large DUB-targeted cyanimide-based libraries, which will accelerate future DUB inhibitor development.

biochemistry↗

bioRxiv2025

A NanoBRET-based assay monitoring interactions in the USP18 signaling hub identifies the first cell-penetrant small molecule compromising USP18/ISG15 binding

Protein modification by interferon-stimulated gene 15 (ISG15), termed ISGylation, exhibits antiviral properties and influences tumorigenesis, genome stability and metabolic processes. ISGylation is counteracted by the specific protease USP18. Likewise, viral proteases such as the papain-like protease (PLpro) from SARS-CoV-2 cleave ISG15 to undermine the host immune response. Beyond its role as a deISGylating enzyme, USP18 acts as a major negative regulator of the IFN signaling pathway in a STAT2-dependent manner. In humans, unconjugated ISG15 secures USP18 stability and the absence of USP18 or impaired STAT2/USP18 binding cause fatal interferonopathies. Thus, the USP18 signaling hub represents a critical checkpoint for type I IFN signaling and ISGylation, qualifying it as a promising immune and cancer drug target. However, suitable assays to monitor protein-protein interactions (PPIs) within the USP18/ISG15/STAT2 signaling hub and to screen for PPI modulators are missing and no specific inhibitors targeting USP18 interactions are available. To address this gap, we developed a method based on the NanoLuc luciferase (NLuc) Bioluminescence Energy Transfer (NanoBRET) assay system to study PPIs. Firstly, we generated stable cell lines suitable to monitor USP18/ISG15 and USP18/STAT2 interactions, providing a semi high-throughput screening (HTS)-compatible platform. In combination with a virtual pre-screen of 60,000 compounds against USP18 in silico, this assay allowed us to identify a first small molecule (ZHAWOC8655) that compromises cellular USP18/ISG15 binding and inhibits USP18 protease activity in vitro. To further explore the potential of using the NanoBRET system for testing PPI modulators, we evaluated the effect of GRL0617, a compound which was shown to disrupt the interaction between SARS-CoV-2 PLpro/ISG15 as well as SARS-CoV-2 PLpro/ubiquitin. NanoBRET based stable cell lines as presented here will be suitable for monitoring PPIs in other multiprotein complexes after various stimuli, mutations or small molecule administration and can be challenged with siRNA or CRISPR/Cas9 libraries to identify previously unrecognized regulators.

molecular biology↗

bioRxiv2024

USP24 is an ISG15 cross-reactive deubiquitinasethat mediates IFN-I production by de-ISGylatingthe RNA helicase MOV10

The interferon-stimulated gene 15 (ISG15) is a ubiquitin-like modifier induced by type I Interferon (IFN-I) and plays a crucial role in the innate immune response against viral infections. ISG15 is conjugated to target proteins by an enzymatic cascade through a process called ISGylation. While ubiquitin-specific protease 18 (USP18) is a well-defined deISGylase counteracting ISG15 conjugation, ISG15 cross-reactive deubiquitylating enzymes (DUBs) have also been reported. Our study reports USP24 as a novel ISG15 cross-reactive DUB identified through activity-based protein profiling (ABPP). We demonstrate that recombinant USP24 processed pro-ISG15 and ISG15-linked synthetic substrates in vitro. Moreover, the depletion of USP24 significantly increased the accumulation of ISG15 conjugates upon IFN-{beta} stimulation. An extensive proteomic analysis of the USP24-dependent ISGylome, integrating total proteome, GG-peptidome, and ISG15 interactome data, identified the helicase Moloney leukemia virus 10 (MOV10) as a specific target of USP24 for deISGylation. Further validation in cells revealed that ISGylated MOV10 enhances IFN-{beta} production/secretion, whereas USP24 deISGylates MOV10 to negatively regulate the innate immune response. This study showcases USP24s novel roles in modulating ISGylation and modulation of the IFN-I-dependent immune responses, with potential therapeutic implications in infectious diseases, cancer, autoimmunity, and neuroinflammation.

biochemistry↗

bioRxiv2024

Ubiquitin-specific protease 8 controls B cell proteostasis and cell survival in multiple myeloma

Ubiquitin-specific protease 8 (USP8) is a multifunctional deubiquitinating enzyme that plays a pivotal role in the regulation of endosomal and lysosomal trafficking. Several studies showed that USP8 is critically involved in the pathogenesis of various tumor entities. Recently, USP8 emerged as a vulnerability gene in multiple myeloma (MM), suggesting a functional role in the B- and plasma cell compartment. Here we have comprehensively analyzed mice with deletion of Usp8 at different stages of B-cell development. Furthermore, we evaluated the function of USP8 in proteasome inhibition susceptible and Bortezomib (BTZ) resistant patient derived MM cells using depletion of USP8 and treatment with DUB-IN-2, a widely used inhibitor published to target USP8. Usp8 depletion in Usp8f/fCd19-Cre mice affected B-cell survival and development favoring immature, innate-like B cells, and germinal center and plasma cells, while also elevating immune-responses and causing Roquin depletion. Cells expressing catalytically inactive USP8 accumulated proteins modified with mixed ubiquitin/NEDD8 chains, indicating proteotoxic stress. Moreover, we identified these modifications as preferred substrates of USP8. In MM cells, efficient USP8 knockdown reduced survival by inducing lysosomal dysfunction. In contrast, DUB-IN-2 induced an enhanced ER stress response to treatment with BTZ. Our results highlight the potential of targeting USP8 and the combination of DUB-IN-2 and BTZ in treating BTZ-resistant MM. However, our biochemical and cellular analyses raise fundamental concerns about the function of DUB-IN-2 as a USP8 inhibitor.

molecular biology↗

bioRxiv2024

Rapid turnover of CTLA4 is associated with a complex architecture of reversible ubiquitylation.

The immune checkpoint regulator CTLA4 is an unusually short-lived membrane protein. Here we show that its lysosomal degradation is dependent on ubiquitylation at Lysine residues 203 and 213. Inhibition of the v-ATPase partially restores CTLA4 levels following cycloheximide treatment, but also reveals a fraction that is secreted in exosomes. The endosomal deubiquitylase, USP8, interacts with CTLA4 and its loss enhances CTLA4 ubiquitylation in cancer cells, mouse CD4+ T cells and in cancer cell-derived exosomes. Depletion of the USP8 adapter protein, HD-PTP, but not ESCRT-0 recapitulates this cellular phenotype, but shows distinct properties vis-a-vis exosome incorporation. Re-expression of wild-type USP8, but neither a catalytically inactive, nor a localisation-compromised {Delta}MIT domain mutant can rescue delayed degradation of CTLA4, or counteract its accumulation in clustered endosomes. UbiCRest analysis of CTLA4-associated ubiquitin chain linkages identifies a complex mixture of conventional Lys63- and more unusual Lys27- and Lys29-linked polyubiquitin chains that may underly the rapidity of protein turnover.

cell biology↗

bioRxiv2023

USP16 is an ISG15 cross-reactive deubiquitinase targeting a subset of metabolic pathway-related proteins

The ubiquitin-like modifier ISG15 can modulate host and viral proteins to restrict viral and microbial infections, and act as a cytokine. Its expression and conjugation are strongly up-regulated by type I interferons. Here we identify the deubiquitinating enzyme USP16 as an ISG15 cross-reactive protease. Ubiquitin-specific protease 16 (USP16) was found to react with an ISG15 activity-based probe in pull-down experiments using chronic myeloid leukaemia-derived human cells (HAP1). Supporting this finding, recombinant USP16 cleaved pro-ISG15 and ISG15 iso-peptide linked model substrates in vitro, as well as ISGylated substrates present in cell lysates. Moreover, the interferon-induced stimulation of ISGylation in human HAP1 cells was increased by knockdown or knockout of USP16. Depletion of USP16 did not affect interferon signaling, and interferon treatment did not affect USP16 expression or enzymatic activity either. A USP16-dependent ISG15 interactome was established by anti-ISG15 immunoprecipitation mass spectrometry (IP-MS), which indicated that the deISGylating function of USP16 may regulate metabolic pathways involving GOT1, ALDOA, SOD1 and MDH1, all of which were further confirmed to be deISGylated by USP16 in HEK293T cells. Together, our results indicate that USP16 may contribute to regulating the ISGylation status of a subset of proteins related to metabolism during type I interferon responses.

biochemistry↗

bioRxiv2021

Ring Finger Protein 213 Assembles into a Sensor for ISGylated Proteins with Antimicrobial Activity

ISG15 is an interferon-stimulated, ubiquitin-like protein that can conjugate to substrate proteins (ISGylation) to counteract microbial infection, but the underlying mechanisms remain elusive. Here, we used a viral-like particle trapping technology to identify ISG15-binding proteins and discovered Ring Finger Protein 213 (RNF213) as an ISG15 interactor and cellular sensor of ISGylated proteins. RNF213 is a poorly-characterized, interferon-induced megaprotein that is frequently mutated in Moyamoya disease, a rare cerebrovascular disorder. We found that interferon induces ISGylation and oligomerization of RNF213 on lipid droplets, where it acts as a sensor for ISGylated proteins. We showed that RNF213 has broad antimicrobial activity in vitro and in vivo, counteracting infection with Listeria monocytogenes, herpes simplex virus 1 (HSV-1), human respiratory syncytial virus (RSV) and coxsackievirus B3 (CVB3), and we observed a striking co-localization of RNF213 with intracellular bacteria. Together, our findings provide novel molecular insights into the ISGylation pathway and reveal RNF213 as a key antimicrobial effector.

cell biology↗

bioRxiv2021

SCFFbxw5 targets MCAK in G2/M to facilitate ciliogenesis in the following cell cycle

The microtubule depolymerase Kif2C/MCAK plays important roles in various cellular processes and is frequently overexpressed in different cancer types. Despite the importance of its correct abundance, remarkably little is known about how MCAK levels are regulated in cells. Using comprehensive screening on protein microarrays, we identified 161 candidate substrates of the multi-subunit ubiquitin E3 ligase SCFFbxw5, including MCAK. In vitro reconstitution assays demonstrate that MCAK and its closely related orthologs Kif2A and Kif2B become efficiently polyubiquitylated by neddylated SCFFbxw5 and Cdc34, without requiring preceding modifications. In cells, SCFFbxw5 targets MCAK for proteasomal degradation specifically during G2/M. While this seems largely dispensable for mitotic progression, loss of Fbxw5 leads to increased MCAK levels at basal bodies, which impair formation of primary cilia in the following G1. We have thus identified a novel regulatory event of ciliogenesis that occurs already within the G2/M phase of the preceding cell cycle.

cell biology↗

bioRxiv2020

ISG15 drives immune pathology and respiratory failure during viral infection

Cytokine storm during respiratory viral infection is an indicator of disease severity and poor prognosis. Type 1 interferon (IFN-I) production and signaling has been reported to be causal in cytokine storm-associated pathology in several respiratory viral infections, however, the mechanisms by which IFN-I promotes disease pathogenesis remain poorly understood. Here, using Usp18-deficient, USP18 enzymatic-inactive and Isg15-deficient mouse models, we report that lack of deISGylation during persistent viral infection leads to severe immune pathology characterized by hematological disruptions, cytokine amplification, lung vascular leakage and death. This pathology requires T cells but not T cell-intrinsic deletion of Usp18. However, lack of Usp18 in myeloid cells mimicked the pathological manifestations observed in Usp18-/- or Usp18C61A mice which were dependent on Isg15. We further mechanistically demonstrate that interrupting the ISGylation/deISGylation circuit increases extracellular levels of ISG15 which is accompanied by inflammatory neutrophil accumulation to the lung. Importantly, neutrophil depletion reversed morbidity and mortality in Usp18C61A mice. In summary, we reveal that the enzymatic function of Usp18 is crucial for regulating extracellular release of ISG15. This is accompanied by altered neutrophil differentiation, cytokine amplification and mortality following persistent viral infection. Moreover, our results suggest that extracellular ISG15 may drive the inflammatory pathology observed and could be both a prospective predictor of disease outcome and a therapeutic target during severe respiratory viral infections.

immunology↗

bioRxiv2017

Free ISG15 as a dimer generates IL-1β-producing CD8α+ dendritic cells at the site of infection

ISG15 is strongly induced after type I IFN stimulation producing a protein comprised of two ubiquitin-like domains. Intracellularly, ISG15 can be covalently linked and modify the function of target proteins (ISGylation). In addition, free unconjugated ISG15 can be released from cells. We found that ISG15 is released in the serum of Toxoplasma gondii infected mice early after infection in a type-I IFN independent manner. Once in the extracellular space, free ISG15 forms dimers and enhances the release of key cytokines involved in the immune response to the parasite: IL-12, IFN-{gamma}, and IL-1{beta}. Its action is dependent on an actively invading and replicating live parasite. ISG15 induces an increase of IL-1{beta} later during infection by leading to increased IL-1{beta} producing CD8+ dendritic cells at the site of infection. Here, we define for the first time the molecular determinants of active free ISG15 and link ISG15 to IL-1{beta} production by CD8+ dendritic cells. Thus we define ISG15 as a novel secreted modulator of the cytokine response during Toxoplasma infection.

immunology↗