Biology subjects

Schiltz, O.

Publications and source records attributed to Schiltz, O..

9 recordsLinked to original sources

bioRxiv2026

ST8SIA4-mediated polysialylation is critical for CCR2-driven monocyte egress from the bone marrow

Polysialylation is a rare post-translational protein modification essential for brain development and synaptic plasticity, where it fine-tunes cell-cell and cell-matrix interactions to direct neuronal migration, neurite outgrowth and synaptogenesis. Although polysialic acid is also expressed on circulating leukocytes, its functions in the immune system remain largely unexplored. Guided by analysis of publicly available human genomic data showing that naturally occurring variants of ST8SIA4, which encodes one of the two enzymes that mediate polysialylation, associate with reduced circulating monocyte counts, we investigated the in vivo role of this enzyme in monocyte biology. Using St8sia4-deficient mice, we show that ST8SIA4-dependent polysialylation is essential for CCR2-mediated egress of inflammatory monocytes from the bone marrow at steady state and during Mycobacterium tuberculosis infection. We confirm NCAM1 as the principal polysialylated protein in inflammatory monocytes and demonstrate that Ncam1-deficient mice phenocopy the monocyte defects observed in St8sia4-deficient animals. Mechanistically, loss of ST8SIA4-dependent polysialylation impairs engagement and internalization of the CCR2 ligands CCL2 and CCL7, accompanied by disrupted CCR2 surface organization and extensive cytoskeletal remodeling. Together, these findings identify polysialylation as a previously unrecognized regulator of CCR2 function and monocyte mobilization, with broad implications for immune surveillance and inflammatory responses. Highlights- Human ST8SIA4 variants are associated with reduced blood monocyte and lymphocyte counts and increased risk of SLE. - St8sia4-/- mice exhibit monocytopenia with impaired CCR2-mediated egress of inflammatory monocytes from the bone marrow both at steady state and during Mycobacterium tuberculosis infection. - ST8SIA4 polysialylates NCAM1 (CD56) in inflammatory monocytes, and Ncam1-/- - mice phenocopy the monocytopenia observed in St8sia4-/- mice. - Loss of ST8SIA4 and polysialylation impairs CCR2-mediated binding and endocytosis of CCL2 and CCL7 by monocytes. - Polysialic acid itself does not function as a co-receptor for these chemokines, revealing a new paradigm for its role in immune cell trafficking. - Proper CCR2 surface distribution and function, as well as cytoskeletal organization in inflammatory monocytes, depend on ST8SIA4-mediated polysialylation.

immunology↗

bioRxiv2025

Drosophila melanogaster miPEP8 regulates cell size through its interaction with ref(2)P/p62

MiPEPs are microproteins encoded by primary transcripts of microRNAs (pri-miRNAs). Initially identified in plants, we recently characterized a miPEP in Drosophila melanogaster, named miPEP8, which is involved in the regulation of wing size. However, mechanisms at play are unknown. In the present study, we take advantage of the drosophila cell line Schneider 2 (S2) to further investigate miPEP8 function at the molecular level. Overexpressing miPEP8 in S2 cells induced a reduction of cell size as well as an increase of the proportion of cells in the G1 phase of the cell cycle and an increase of the autophagic flux. A proteomics analysis revealed that miPEP8 overexpression in S2 cells induces the upregulation of several proteins including the autophagosome cargo protein ref(2)P (the orthologue of the human p62/Sequestosome 1 protein). The interactome of miPEP8 was generated and revealed interactions between this miPEP8 and the mTORC1/autophagy pathway. Bioinformatics analysis identified a short linear motif (SLiM) on miPEP8 sequence. Mutation of this SLiM prevented the interaction between ref(2)P/p62 and miPEP8. Mutation of the SLiM also reverted the smaller cell size phenotype observed when overexpressing miPEP8 in S2 cells. Finally, the cell size phenotype was reversed when cells were treated with RNA interference targeting ref(2)P/p62, suggesting that this protein plays a role in regulating the cell size in Drosophila.

biochemistry↗

bioRxiv2024

Integrative in vivo analysis of the ethanolamine utilization bacterial microcompartment in Escherichia coli.

Bacterial microcompartments (BMCs) are self-assembling protein megacomplexes that encapsulate metabolic pathways. Although approximately 20% of sequenced bacterial genomes contain operons encoding putative BMCs, few have been thoroughly characterized, nor any in the most studied Escherichia coli strains. We used an interdisciplinary approach to gain deep molecular and functional insights into the ethanolamine utilization (Eut) BMC system encoded by the eut operon in E. coli K-12. The eut genotype was linked with the ethanolamine utilization phenotype using deletion and overexpression mutants. The subcellular dynamics and morphology of the E. coli Eut BMC were characterized in cellula by fluorescence microscopy and electron (cryo)microscopy. The minimal proteome reorganization required for ethanolamine utilization and the in vivo stochiometric composition of the Eut BMC were determined by quantitative proteomics. Finally, the first flux map connecting the Eut BMC with central metabolism in cellula was obtained by genome scale modelling and 13C-fluxomics. Our results reveal that, contrary to previous suggestions, ethanolamine serves both as a nitrogen and a carbon source in E. coli K-12, while also contributing significant metabolic overflow. Overall, this study provides a quantitative molecular and functional understanding of the BMCs involved in ethanolamine assimilation by E. coli. ImportanceThe properties of BMCs make them an ideal tool to build orthogonal network structures with minimal interactions with native metabolic and regulatory networks. However, this requires an understanding of how BMCs work natively. In this study, we combined genetic manipulation, multi-omics, modelling and microscopy to address this issue for Eut BMCs. We show that the Eut BMC in E. coli turns ethanolamine into usable carbon and nitrogen substrates to sustain growth. These results improve our understanding of compartmentalization in a widely used bacterial chassis.

microbiology↗

bioRxiv2021

A comprehensive LFQ benchmark dataset on modern day acquisition strategies in proteomics

In the last decade, a revolution in liquid chromatography-mass spectrometry (LC-MS) based proteomics was unfolded with the introduction of dozens of novel instruments that incorporate additional data dimensions through innovative acquisition methodologies, in turn inspiring specialized data analysis pipelines. Simultaneously, a growing number of proteomics datasets have been made publicly available through data repositories such as ProteomeXchange, Zenodo and Skyline Panorama. However, developing algorithms to mine this data and assessing the performance on different platforms is currently hampered by the lack of a single benchmark experimental design. Therefore, we acquired a hybrid proteome mixture on different instrument platforms and in all currently available families of data acquisition. Here, we present a comprehensive Data-Dependent and Data-Independent Acquisition (DDA/DIA) dataset acquired using several of the most commonly used current day instrumental platforms. The dataset consists of over 700 LC-MS runs, including adequate replicates allowing robust statistics and covering over nearly 10 different data formats, including scanning quadrupole and ion mobility enabled acquisitions. Datasets are available via ProteomeXchange (PXD028735).

bioinformatics↗

bioRxiv2021

Pseudomonas aeruginosa infection reveals a Caspase-1-dependent neutrophil pyroptosis pathway that restrains damaging Histone release

Multiple neutrophil death programs contribute to host defense against infections. Although expressing all necessary components, neutrophils specifically fail to undergo pyroptosis, a lytic form of cell death triggered by the activation of the pro-inflammatory complex inflammasome. In the light of the arm race, we hypothesized that intrinsic neutrophil pyroptosis resistance might be bypassed in response to specific microbial species. We show that Pseudomonas aeruginosa (P. aeruginosa) stimulates Caspase-1-dependent pyroptosis in human and murine neutrophils. Mechanistically, activated NLRC4 inflammasome supports Caspase-1-driven Gasdermin-D (GSDMD) activation, IL-1{beta} cytokine release and neutrophil pyroptosis. Furthermore, GSDMD activates Peptidyl Arginine Deaminase-4 which drives an "incomplete NETosis" where neutrophil DNA fills the cell cytosol but fails crossing plasma membrane. Finally, we show that neutrophil Caspase-1 account for IL-1{beta} production and contributes to various P. aeruginosa strains spread in mice. Overall, we demonstrate that neutrophils are fully competent for Caspase-1-dependent pyroptosis, which drives an unsuspected "incomplete NETosis". SummaryNeutrophils play an essential roles against infections. Although multiple neutrophil death programs contribute to host defense against infections, they fail to undergo pyroptosis, a pro-inflammatory form of cell death. Upon Infections, pyroptosis can be induced in macrophages or epithelial cells upon activation of pro-inflammatory complexes, inflammasomes that trigger Caspase-1-driven Gasdermin dependent plasma membrane lysis. In the light of host-microbe interactions, we hypothesized that yet to find microbial species might hold the capacity to overcome neutrophil resistance to inflammasome-driven pyroptosis. Among several bacterial species, we describe that the bacterium Pseudomonas aeruginosa specifically engages the NLRC4 inflammasome, which promotes Caspase-1-dependent Gasdermin-D activation and subsequent neutrophil pyroptosis. Furthermore, inflammasome-driven pyroptosis leads to DNA decondensation and expansion into the host cell cytosol but not to the so called Neutrophil Extracellular Trap (NET) release as DNA fails breaching the plasma membrane. Finally, in vivo P. aeruginosa infections highlight that Caspase-1-driven neutrophil pyroptosis is functional and is detrimental upon P. aeruginosa infection. Altogether, our results unexpectedly underline neutrophil competence for Caspase-1-dependent pyroptosis, a process that contributes to host susceptibility to P. aeruginosa infection.

immunology↗

bioRxiv2021

Interactome analyses reveal profound proteasome structural and functional rearrangements throughout mammalian spermatogenesis

During spermatogenesis, spermatogonia undergo a series of mitotic and meiotic divisions on their path to spermatozoa. To achieve this, a succession of processes requiring high proteolytic activity are in part orchestrated by the proteasome. The spermatoproteasome (s20S) is specific to the developing gametes, in which the gamete-specific 4s subunit replaces the 4 isoform found in the constitutive proteasome (c20S). Although the s20S is conserved across species and was shown to be crucial for germ cell development, its mechanism, function and structure remain incompletely characterized. Here, we used advanced mass spectrometry (MS) methods to map the composition of proteasome complexes and their interactomes throughout spermatogenesis. We observed that the s20S becomes highly activated as germ cells enter meiosis, mainly through a particularly extensive 19S activation and, to a lesser extent, PA200 binding. Additionally, the proteasome population shifts from c20S (98%) to s20S (>82-92%) during differentiation, presumably due to the shift from 4 to 4s expression. We demonstrated that s20S, but not c20S, interacts with components of the meiotic synaptonemal complex, where it may localize via association with the PI31 adaptor protein. In vitro, s20S preferentially binds to 19S, and displays higher trypsin- and chymotrypsin-like activities, both with and without PA200 activation. Moreover, using MS methods to monitor protein dynamics, we identified significant differences in domain flexibility between 4 and 4s. We propose that these differences induced by 4s incorporation result in significant changes in the way the s20S interacts with its partners, and dictate its role in germ cell differentiation.

biochemistry↗

bioRxiv2020

Irgm2 and Gate-16 cooperatively dampen targeting of caspase-11 to Gram-negative bacterial products

Inflammatory caspase-11 (rodent) and caspases-4 and -5 (human) detect gram-negative bacterial component LPS in the host cell cytosol, which promotes activation of the non-canonical inflammasome. Although non-canonical inflammasome-induced pyroptosis and IL-1 related cytokine release is of importance to mount an efficient immune response against various bacteria, its unrestrained activation drives sepsis. This suggests that cellular components might tightly control the threshold level of the non-canonical inflammasome in order to ensure efficient but not deleterious inflammatory response. Here we show that the IFN-inducible protein Irgm2 and the ATG8 family member Gate-16 cooperatively slow down non-canonical inflammasome activation both in macrophages and in vivo. Specifically, the Irgm2/Gate-16 axis dampens caspase-11 targeting to intracellular bacteria, which lower caspase-11-mediated pyroptosis and cytokine release. Specifically, deficiency in Irgm2 or Gate16 opens an alternative road for caspase-11 targeting to intracellular bacteria, independently of the classical pathway driven by the Guanylate Binding Proteins (GBPs). Thus, our findings provide new molecular effectors involved at fine-tuning the optimal non-canonical inflammasome response and add novel insights in the understanding of the immune pathways they control.

immunology↗

bioRxiv2019

Adipocyte vesicles: ‘all-in-one’ packages that stimulate tumor mitochondrial metabolism and dynamics

Extracellular vesicles are emerging key actors in adipocyte communication. Notably, small extracellular vesicles shed by adipocytes promote melanoma aggressiveness through fatty acid oxidation, with a heightened effect in obesity. However, the vesicular actors and cellular processes involved remain largely unknown. Here, we elucidate the mechanisms linking adipocyte extracellular vesicles to metabolic remodeling and cell migration. We show that adipocyte vesicles stimulate melanoma fatty acid oxidation by providing both enzymes and substrates. In obesity, the heightened effect of extracellular vesicles depends on increased transport of fatty acids, not fatty acid oxidation-related enzymes. These fatty acids, stored within lipid droplets in cancer cells, drive fatty acid oxidation after release through lipophagy. This increase in mitochondrial activity redistributes mitochondria to membrane protrusions of migrating cells, which is necessary to increase cell migration in the presence of adipocyte vesicles. Our results provide key insights into the role of extracellular vesicles in the metabolic cooperation that takes place between adipocytes and tumors with particular relevance in obesity.

cancer biology↗

bioRxiv2019

Yellow adipocytes comprise a new adipocyte sub-type present in human bone marrow

During energy demanding conditions, white adipocytes store triglycerides and release fatty acids through lipolysis. In contrast, bone marrow adipocytes (BM-Ad) increase in size during caloric restriction, suggesting this fat depot exhibits precise metabolic specificity. We found subcutaneous adipocytes (SC-Ad) and BM-Ad share morphological features, but possess distinct lipid metabolism. BM-Ad show enrichment in cholesterol-oriented metabolism that correlates with increased free cholesterol content, while proteins involved in lipolysis were downregulated. A strong down-regulation in expression of monoacylglycerol (MG) lipase was observed leading to an accumulation of major MG species and accordingly the basal and induced lipolytic responses were absent in BM-Ad. These features are not recapitulated in vitro using differentiated bone marrow mesenchymal stem cells. Since our data demonstrate that BM-Ad comprise a distinct class of adipocytes, we propose renaming them yellow adipocytes.

physiology↗