Biology subjects

Stumpe, M.

Publications and source records attributed to Stumpe, M..

16 recordsLinked to original sources

bioRxiv2026

Proximity labeling at H3K9me3 reveals VRK-1 regulate global chromatin distribution in C. elegans

Heterochromatin marked by histone H3 lysine 9 di- or trimethylation (H3K9me2/3) underpins transcriptional silencing and nuclear organization, yet its full complement of associated proteins remains incompletely defined. Here, we apply ChromID proximity labelling with the mouse HP1{beta} chromodomains to map the H3K9me3-proximal proteome in Caenorhabditis elegans, recovering known heterochromatin factors alongside previously uncharacterized candidates. We pursued one such candidate, the vaccinia-related kinase VRK-1, because of its established but poorly understood links to chromatin organization. Intriguingly, VRK-1 dynamically relocates from a broad nuclear distribution to the nuclear periphery upon azide or heat stress. Following these stresses, bulk chromatin exhibits similarly increased peripheral enrichment and apparent compaction, as assessed by radial fluorescence profiles. Although VRK-1 is not necessary for stress-induced chromatin reorganization, decompaction and repositioning of chromatin away from the nuclear envelope during recovery requires VRK-1. VRK-1 depletion leads to persistent perinuclear chromatin retention and compromises post-stress survival. Furthermore, loss of VRK-1 catalytic activity results in over-retention of chromatin at the nuclear periphery under normal growth conditions; this phenotype can be reversed by depletion of a key VRK-1 substrate at the nuclear envelope BAF-1. Our findings identify VRK-1 as a key regulator that controls the interaction of chromatin with the nuclear lamina through regulation of BAF-1.

cell biology↗

bioRxiv2026

Sample preparation for mass spectrometry-based tissue (phospho)proteomics

This protocol describes the workflow for the preparation of tissue samples for proteome and phosphoproteome analyses using mass spectrometry. The tissue samples are cryogenically pulverized and homogenized in a sucrose-based buffer to ensure proper tissue disruption. For depletion of lipid contaminants, proteins are purified using chloroform-methanol precipitation, followed by a resuspension in a urea-based buffer for enzymatic digestion. Peptides are desalted and enriched for phosphopeptides prior LC-MS/MS analysis. The workflow was developed for skin biopsies but is compatible with a broad range of tissue types. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=51 SRC="FIGDIR/small/732915v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1bfe743org.highwire.dtl.DTLVardef@1e812deorg.highwire.dtl.DTLVardef@12ed3c0org.highwire.dtl.DTLVardef@1501c4d_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO C_FIG

biochemistry↗

bioRxiv2026

The transcription factor LSL-1 interacts with the chromatin factors HIM-17, XND-1 and BRA-2 to promote the germline-specific transcriptional repertoire and to safeguard germ cell fate in C. elegans

Germ cells are the only cells of an organism that pass onto the next generation and, hence perpetuate the species. To ensure this, germ cells need dedicated transcriptional repertoire, that ensure specification, proliferation, differentiation and fate maintenance. We previously characterized LSL-1, a conserved zinc-finger transcription factor that acts as a major direct transcriptional activator of genes involved in germ cell development, fate specification, meiosis and genome stability. Here, we show that LSL-1 interacts with the transcription factor HIM-17, the chromatin proteins BRA-2 and XND-1. These proteins are functionally related to LSL-1 and they colocalize at germline gene promoters, forming most likely a transcription-promoting complex. Furthermore, LSL-1 lies in close proximity to members of the COMPASS and the MOF complexes, corroborating the observation that HIM-17 and LSL-1 are required to maintain normal level of H3K4 methylation in the gonad. Finally, we show that LSL-1 interacting partners are necessary to maintain germ cell fate. Altogether, we propose that LSL-1 interacts with transcription regulators and chromatin modifiers to ensure the establishment of the transcriptional repertoire appropriate for germline function as well as for cell fate maintenance.

biochemistry↗

bioRxiv2026

Autophagy shapes the peptide repertoire of rheumatoid arthritis-associated HLA class II alleles

Alternative pathways of antigen presentation are crucial in different immunological contexts such as autoimmunity and anti-microbial defense. Among these pathways, autophagy has a central role in delivering cytosolic substrates to the MHC class II compartment. However, its contribution to endogenous MHC class II presentation was only demonstrated for a few antigens. Here we focused our study on the contribution of autophagy to the peptidome of one major allele of the HLA-DR shared epitope, HLA DRB1*04:01 conferring the greatest risk factor for the development of rheumatoid arthritis (RA). We provide an extensive qualitative and quantitative mass spectrometry analysis of the autophagy related MHC class II peptide repertoire of the human DRB1*04:01 allele. A fraction of peptides representing 30% of the repertoire differ profoundly between autophagy sufficient and deficient cells. Our analysis demonstrates that autophagy contributes to MHC class II presentation of peptides from seven described RA autoantigens, the majority of them being related to the ER folding and stress response (calreticulin, calnexin, the 78 kDa glucose-regulated protein (GRP78)-also known as binding immunoglobulin protein (BiP) and several protein from the heat-shock-protein 70 family). Our results correlate with an increased activation of autophagy, in situ, in synovial biopsies and synovial fibroblast (SF) of RA patients. We could further show that SF upregulate MHC class II and present peptides from autophagy related auto-antigens to CD4 T cells from RA patients. Our finding identifies autophagy as a potential process that could contribute to the break of peripheral tolerance during RA.

immunology↗

bioRxiv2025

O-mannosylation and protein maturation check-points represent therapeutic opportunities in BRAF fusion protein oncogenesis

Fusions between protein-coding genes are common oncogenic drivers across cancers, typically pairing a proto-oncogene with partner that does not independently drive cancer. In all therapeutically actionable fusions, the proto-oncogene is the drug target, the contributions to oncogenicity of the fusion partner have largely been ignored. We studied the role of BRAF fusion partners and found that they are necessary for transformation. In the setting of KIAA1549::BRAF, the most common fusion protein across brain tumors, we found that KIAA1549 is necessary for the oncogenicity of KIAA1549::BRAF and engenders a striking and specific dependency on the protein O-mannosyltransferase complex (POMT1/2). Specifically, we show that genetic silencing or pharmacologic inhibition of the protein O-mannosyltransferase complex (POMT1/2) reverses fusion-induced transformation, thereby representing a novel and MAPK independent therapeutic target. Furthermore, POMT1/2 is required to glycosylate and enable maturation of the K::B fusion protein. These findings represent a proof-of-concept for targeting the partners in oncogenic fusions as a potential cancer therapeutic strategy.

cancer biology↗

bioRxiv2025

The developing retina undergoes mitochondrial remodeling via PINK1/PRKN-dependent mitophagy

Mitophagy, the selective degradation of mitochondria, is essential for retinal ganglion cell (RGC) differentiation and retinal homeostasis. However, the specific mitophagy pathways involved and their temporal dynamics during retinal development and maturation remain poorly understood. Using proteomics analysis of isolated mouse retinas across developmental stages and the mitophagy reporter mouse line, mito-QC, we characterized mitophagy throughout retinogenesis. While mitolysosomes were more prevalent in the mature retina, we observed two distinct mitophagy peaks during embryonic development. The first, independent of PTEN-induced kinase 1 (PINK1) activation, was associated with RGCs. The second, PINK1-dependent peak was triggered after an increase in retinal oxidative stress. This PINK1-dependent, oxidative stress-induced mitophagy pathway is conserved in mice and zebrafish, providing the first evidence of programmed, PINK1-dependent mitophagy during development.

developmental biology↗

bioRxiv2025

Multi-omics analysis of keratinocytes reveals dermokine-dependent regulation of cell-cell adhesion via p120

Loss of keratinocyte differentiation is a leading cause in several skin diseases and needs to be controlled in adult homeostasis by for instance growth factors and proteases. Among them, we studied the role of isoform-rich dermokine - a wound- and tumour-related matrix metalloproteinase 10 substrate - via functional multi-omics. We generated dermokine isoform-dependent keratinocyte knockouts and three dimensional (3D) organotypic skin cultures and analyzed changes in their proteome and phosphoproteome by quantitative mass spectrometry. Through functional in vitro assays, we demonstrate that in the absence of dermokine-isoforms, p120 phosphorylation increases while cell-cell adhesion decreases in keratinocytes. Furthermore, we validate the link between decreased dermokine expression and phosphorylated p120-mediated adhesion in non-healing wounds samples derived from patients. Our data reveal a novel dermokine-p120-dependent cell-cell adhesion phenotype in keratinocytes and improve our understanding of wound-edge keratinocytes, expanding the hypothesis that dysregulated wounds resemble cancer.

cell biology↗

bioRxiv2024

M1-linked Ubiquitination by LUBAC Regulates AMPK Activity and the Response to Energetic Stress

Methionine-1 (M1)-linked ubiquitin chains, assembled by the ubiquitin ligase LUBAC and cleaved by the deubiquitinase OTULIN, are critical regulators of inflammation and immune homeostasis. Genetic loss of either LUBAC or OTULIN causes autoinflammatory syndromes, which are associated with defects in glycogen and lipid metabolism. However, how LUBAC and OTULIN regulate metabolic signalling remains unknown. Here, we demonstrate that LUBAC promotes, while OTULIN restricts, activation of the key metabolic regulator AMP-activated protein kinase (AMPK) in cells, mice, and human samples. LUBAC and OTULIN interact with AMPK, control its M1-ubiquitination, and regulate its activation in response to glucose starvation and allosteric activation. During starvation, LUBAC deficiency impairs autophagy induction and hinders the shift from oxidative phosphorylation to glycolysis. Strikingly, LUBAC-deficient Drosophila have a strongly reduced survival rate after starvation. Our work identifies LUBAC and OTULIN as physiological regulators of AMPK, providing the first mechanism by which M1-linked ubiquitin chains regulate metabolic signalling.

cell biology↗

bioRxiv2024

A CELL BIOLOGY STUDY REVEALS NEW INSIGHTS INTO THE TRANSPORT MECHANISMS OF OOMYCETE EFFECTORS

Oomycete pathogens use dedicated effector proteins (also known as virulence factors) that manipulate the host during infection to the benefit of the pathogen. Despite extensive efforts, the understanding of how oomycete effector proteins are transported and delivered to the host is limited. Here, we show that Phytophthora capsici infecting Nicotiana benthamiana secretes its effectors through secretory pockets, haustoria, and from hyphae. We provide evidence that Avr3, belonging to the class of RxLR effector proteins that can be secreted and translocated to the plant cell, is found to accumulate in a distinct ring-shaped zone around the neck region of haustoria. The association with the haustorial neck is determined by an RxLR-EER motif and mutating this motif redirects Avr3 to the extrahaustorial matrix enveloping the full haustoria body. Furthermore, mass spectrometry analysis of immunoprecipitated Avr3 indicates that the RXLR motif remains intact when Avr3 is secreted from the haustorium during plant infection. We thus propose that the RXLR-EER motif determines the entry in the host cell by guiding Avr3 to the haustorial neck region, which may be a structure analogous to the Biotrophic Interfacial Complex of the fungus Magnaporthe sp.

cell biology↗

bioRxiv2024

Sanctioning of bacterial cheaters by the host plant in nitrogen-fixing symbiosis between Medicago truncatula and Sinorhizobium meliloti

In plant-microbe interactions, the host plant invests considerable amounts of resources in the microbial partner until the symbiotic machinery is established. If the microbial partner does not reciprocate with a comparable symbiotic benefit, the interaction represents a parasitic relationship. This is thought to elicit a plants response to prevent the selective disadvantage of being parasitized by such microbial cheaters. Indeed, negative feedback against bad mutualists, known as sanctioning, has been observed in interactions such as the arbuscular mycorrhizal and legume-rhizobium symbioses. Here, to study sanctioning by the plant host, we manipulate the exchange of resources between the model legume Medicago truncatula and its bacterial partner Sinorhizobium meliloti by three ways: mutating the bacterial nitrogenase enzyme, replacing nitrogen in the atmosphere with argon gas, and supplying high nitrate to the host. Then, we follow the consequences for the interaction by examining the metabolome, proteome, and phosphoproteome of nodules. We find that such cheating conditions result in sanctioning of the bacterial partner, and observe characteristic shifts including induced defense markers, repressed symbiotic markers, and changes in central metabolism that may be relevant for microbial fitness and that could therefore contribute to sanctioning.

plant biology↗

bioRxiv2024

Targeted proteomics addresses selectivity and complexity of protein degradation by autophagy

Autophagy is a constitutively active catabolic lysosomal degradation pathway, often found dysregulated in human diseases. It is often considered to act in a cytoprotective manner and is commonly upregulated in cells undergoing stress. Its initiation is regulated at the protein level and does not require de novo protein synthesis. Historically, autophagy has been regarded as non-selective; however, it is now clear that different stimuli can lead to the selective degradation of cellular components via selective autophagy receptors (SARs). Due to its selective nature and the existence of multiple degradation pathways potentially acting in concert, monitoring of autophagy flux, i.e. selective autophagy-dependent protein degradation, should address this complexity. Here, we introduce a targeted proteomics approach monitoring abundance changes of 37 autophagy-relevant proteins covering process-relevant proteins such as the initiation complex and the ATG8 lipidation machinery, as well as most known SARs. We show that proteins involved in autophagosome biogenesis are upregulated and spared from degradation under autophagy inducing conditions in contrast to SARs. Classical bulk stimuli such as nutrient starvation mainly induce degradation of ubiquitin-dependent soluble SARs and not of ubiquitin-independent, membrane-bound SARs. In contrast, treatment with the iron chelator deferiprone leads to the degradation of ubiquitin-dependent and - independent SARs linked to mitophagy and reticulophagy/ER-phagy. Our approach is automatable and supports large-scale screening assays paving the way to (pre)clinical applications and monitoring of specific autophagy fluxes.

cell biology↗

bioRxiv2021

Dedicated chaperones coordinate co-translational regulation of ribosomal protein production with ribosome assembly to preserve proteostasis

The biogenesis of eukaryotic ribosomes involves the ordered assembly of around 80 ribosomal proteins. Supplying equimolar amounts of assembly-competent ribosomal proteins is complicated by their aggregation propensity and the spatial separation of their location of synthesis and pre-ribosome incorporation. Recent evidence has highlighted that dedicated chaperones protect individual, unassembled ribosomal proteins on their path to the pre-ribosomal assembly site. Here, we show that the co-translational recognition of Rpl3 and Rpl4 by their respective dedicated chaperone, Rrb1 or Acl4, prevents the degradation of the encoding RPL3 and RPL4 mRNAs in the yeast Saccharomyces cerevisiae. In both cases, negative regulation of mRNA levels occurs when the availability of the dedicated chaperone is limited and the nascent ribosomal protein is instead accessible to a regulatory machinery consisting of the nascent-polypeptide associated complex and the Caf130-associated Ccr4-Not complex. Notably, deregulated expression of Rpl3 and Rpl4 leads to their massive aggregation and a perturbation of overall proteostasis in cells lacking the E3 ubiquitin ligase Tom1. Taken together, we have uncovered an unprecedented regulatory mechanism that adjusts the de novo synthesis of Rpl3 and Rpl4 to their actual consumption during ribosome assembly and, thereby, protects cells from the potentially detrimental effects of their surplus production.

cell biology↗

bioRxiv2021

Androglobin, a chimeric mammalian globin, is required for male fertility

Spermatogenesis is a highly specialised process, involving multiple dedicated pathways and regulatory check-points. Defects ultimately lead to male sub-fertility or sterility, and numerous aspects of mammalian sperm formation remain unknown. The predominant expression of the latest globin family member, androglobin (Adgb) in mammalian testis tissue prompted us to assess its physiological function in spermatogenesis. Adgb knockout mice display male infertility, reduced testis weight, impaired maturation of elongating spermatids, abnormal sperm shape and ultrastructural defects in microtubule and mitochondrial organisation. Epididymal sperm from Adgb knockout animals display multiple flagellar malformations including coiled, bifide or shortened flagella, and erratic acrosomal development. Following immunoprecipitation and mass spectrometry, we could identify septin 10 (Sept10) as interactor of Adgb. The Sept10-Adgb interaction was confirmed both in vivo using testis lysates, and in vitro by reciprocal co-immunoprecipitation experiments. Furthermore, absence of Adgb leads to mislocalisation of Sept10 in sperm, indicating defective manchette and sperm annulus formation. Finally, in vitro data suggest that Adgb contributes to Sept10 proteolysis in a calmodulin (CaM)-dependent manner. Collectively, our results provide evidence that Adgb is essential for murine spermatogenesis and further suggest that interdependence between Adgb and Sept10 is required for sperm head shaping via the manchette and proper flagellum formation.

developmental biology↗

bioRxiv2021

Dual control of MAPK activities by AP2C1 and MKP1 MAPK phosphatases regulates defence responses in Arabidopsis

Mitogen-activated protein kinase (MAPK) cascades transmit environmental signals and induce stress and defence responses in plants. These signalling cascades are negatively controlled by specific phosphatases of the type 2C Ser/Thr protein phosphatase (PP2C) and dual-specificity phosphatase (DSP) families that inactivate stress-induced MAPKs; however, the interplay between phosphatases of these different types has remained unknown. Our work reveals that different Arabidopsis MAPK phosphatases, the PP2C-type AP2C1 and the DSP-type MKP1, exhibit both specific and overlapping functions in plant stress responses. Each single mutant and ap2c1 mkp1 double mutant displayed enhanced wound-induced activation of MAPKs MPK3, MPK4, and MPK6, as well as induction of a set of transcription factors. Moreover, ap2c1 mkp1 double mutants show an autoimmune-like response, associated with elevated levels the stress hormones salicylic acid and ethylene, and of the phytoalexin camalexin. Interestingly, this phenotype is reduced in ap2c1 mkp1 mpk6 triple mutants, suggesting that the autoimmune-like response is due to MPK6 misregulation. We conclude that the evolutionarily distant MAPK phosphatases AP2C1 and MKP1 contribute crucially to the tight control of MPK6 activity, ensuring appropriately balanced stress signalling and suppression of autoimmune-like responses during plant growth and development. HighlightDouble MAPK phosphatase mutant plants ap2c1 mkp1 exhibit constitutive, autoimmune-like stress responses, dependent on their substrate MAPK MPK6.

plant biology↗

bioRxiv2021

Dynamic variation in a socially exchanged fluid indicates a role in the regulation of ant colony development and maturation

Socially exchanged fluids, like seminal fluid and milk, allow individuals to molecularly influence conspecifics. Many social insects have a social circulatory system, where food and endogenously produced molecules are transferred mouth-to-mouth (stomodeal trophallaxis), connecting all the individuals in the society. To understand how these endogenous molecules relate to colony life, we used quantitative proteomics to investigate the trophallactic fluid within colonies of the carpenter ant Camponotus floridanus. We show that different stages of the colony life cycle circulate different types of proteins: young colonies prioritize direct carbohydrate processing; mature colonies prioritize accumulation and transmission of stored resources. Further, colonies circulate proteins implicated in oxidative stress, ageing, and social insect caste determination, potentially acting as superorganismal hormones. Brood-caring individuals that are also closer to the queen in the social network (nurses) showed higher abundance of oxidative stress-related proteins. Thus, trophallaxis behavior provides a mechanism for distributed metabolism in social insect societies.

evolutionary biology↗

bioRxiv2020

Deletion of the Clock Gene Period2 (Per2) in Glial Cells Alters Mood-Related Behavior in Mice

The circadian clock regulates many biochemical and physiological pathways, and lack of clock genes, such as Period (Per) 2, do not only affect circadian activity rhythms, but can also modulate food-anticipatory and mood-related behaviors. However, it is not known how cell-type specific expression of Per2 contributes to these behaviors. In this study, we find that Per2 in glial cells is important for balancing mood-related behaviors. Genetic and adeno-associated virus-mediated deletion of Per2 in glial cells of mice leads to a depression-resistant phenotype, as manifested in reduced despair and anxiety. This is paralleled by an increase of the GABA transporter 3 (Gat3) mRNA and a reduction of glutamate levels in the nucleus accumbens (NAc). Exclusive deletion of Per2 in glia of the NAc reduced despair, but had no influence on anxiety. Our data provide strong evidence for an important role of glial Per2 in regulating mood-related behavior.

neuroscience↗