Biology subjects

Burlet-Schiltz, O.

Publications and source records attributed to Burlet-Schiltz, O..

13 recordsLinked to original sources

bioRxiv2025

Red bone marrow hosts metabolically active anucleate adipocytes that support hematopoiesis

Bone marrow adipocytes (BMAds) are a major component of the bone marrow (BM) that regulate bone turnover and hematopoiesis. In rodents, two distinct adipocyte populations exist: constitutive BMAds (cBMAds), located in areas devoid of hematopoietic cells and resistant to metabolic cues, and regulatory BMAds (rBMAds), interspersed within hematopoietic niches and responsive to energy stress. Despite their potential importance, rBMAds have remained poorly characterized due to their scarcity in rodents. Here, we used a high-yield method to isolate human rBMAds, enabling structural, proteomic, lipidomic, and functional analyses. Remarkably, human rBMAds are anucleate yet retain organelles and maintain lipid and glucose metabolism, but unlike their rodent counterparts, they lack lipolytic activity. They actively secrete factors that support hematopoiesis in vitro, implicating them as functional contributors to the BM niche. These findings suggest that human rBMAds may arise from cBMAds through terminal differentiation and define a novel adipocyte subtype critical for BM homeostasis.

cell biology↗

bioRxiv2025

Revealing the unexpected interplay between the Proteasome Activator PA200 and the immunoproteasome

The proteasome activator PA200 binds to the catalytic core of the proteasome, the 20S, and activates its proteolytic activities. The cellular function of PA200 is poorly understood and appears to be cell type and differentiation specific. Recent evidence suggests that PA200 not only binds to the standard 20S (s20S) proteasome but also to the specialized immunoproteasome (i20S) which plays a key role in anti-viral and anti-tumor immunity. We here investigated the interaction of PA200 and the immunoproteasome in detail. We show the very first cryo-EM structures of the singly- and doubly-capped i20S-PA200 complexes that revealed no major difference regarding the first binding event of PA200 to the i20S vs. the s20S. However, first PA200 binding triggered a subtle and long range allosteric bending of the i20S barrel which was not seen in the s20S-PA200 complexes. This resulted in major structural rearrangements in the opposite unbound ring - the displacement of atoms up to 5.4 [A] and the increase in its outer diameter - thereby increasing the occupancy of the second PA200 binding site. Mass photometry confirmed higher occupancy of PA200 to the i20S versus the s20S. Binding of PA200 to the i20S enhanced proteasomal activation compared to the s20S. Co-expression of PA200 and the i20S in cells and tissues, however, is restricted but their interaction is favored upon co-expression. The expression of PA200 and the catalytic subunits of the i20S is differentially regulated depending on the cellular context. Our data also suggest that PA200 has the potential to regulate i20S gene expression whereas the i20S has no effects on PA200 expression. Overall, this work sheds new light on the interaction of PA200 with the i20S from a structural, mechanistic and cellular point of view. Importantly, we identify PA200 as a key regulator of the i20S whenever PA200 and the catalytic subunits of the i20S are co-expressed in the same cell.

biochemistry↗

bioRxiv2024

The human and mouse dendritic cell receptor DCIR binds to LRP1 N-glycans containing terminal galactoses, including the α-Gal antigen

The dendritic cell immunoreceptor (DCIR) is a C-type lectin receptor expressed by myeloid cells that plays a key immunoregulatory role in a wide range of diseases, from inflammation to cancer. However, the ligand(s) of DCIR remain(s) unknown, hampering our understanding of the exact function of this immune receptor. Here, we found that both human DCIR and mouse DCIR1 bind specifically to the low-density lipoprotein receptor-related protein 1 (LRP1), a heavily glycosylated receptor mediating the clearance of various molecules from the extracellular matrix and apoptotic cells. This interaction is mediated by galactose-terminated biantennary complex-type N-glycans, including those carrying the immunogenic -Gal epitope. Our study provides a deeper understanding of the role of DCIR in immune regulation and its potential impact on a range of immune disorders, highlighting its specificity in ligand recognition which is crucial for developing therapeutic strategies.

immunology↗

bioRxiv2023

The intrinsically disordered region of the E3 ubiquitin ligase TRIP12 induces the formation of chromatin condensates and interferes with DNA damage response.

Chromatin compaction is crucial for the faithful expression and integrity of the genome. Although largely studied, proteins and mechanisms that control the chromatin compaction are not entirely discovered. We previously showed that the nuclear HECT-type E3 ubiquitin ligase Thyroid hormone Receptor Interacting Protein 12 (TRIP12) is tightly associated to chromatin. As TRIP12 is overexpressed in several types of cancers, we explored herein the consequences of a TRIP12 overexpression on chromatin homeostasis. First, we established the TRIP12 proxisome and unveiled its pleiotropic role in chromatin regulation. Second, we demonstrated that TRIP12 overexpression leads to the formation of chromatin condensates enriched in heterochromatin marks via its intrinsically disordered region (IDR). We further discovered that the formation of TRIP12-mediated chromatin condensates is highly dynamic and driven by a mechanism of phase separation. Chromatin condensate formation depends on the TRIP12 concentration, the length of the TRIP12-IDR and relies on electrostatic interactions. We found that the formation of TRIP12 mediated-condensates alters cell cycle progression, genome accessibility, transcription as well as DNA damage response by inhibiting the accumulation of Mediator of DNA Damage Checkpoint 1 (MDC1). Altogether, this study reveals a novel dynamic role for TRIP12 in chromatin compaction independently of its ubiquitin ligase activity with important consequences on cellular homeostasis. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=155 SRC="FIGDIR/small/556486v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@13b3d90org.highwire.dtl.DTLVardef@46b991org.highwire.dtl.DTLVardef@1410bdeorg.highwire.dtl.DTLVardef@1722197_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2023

Collagen remodeling leads to inflammation-free expansion of periprostatic adipose tissue and promotes prostate cancer progression

One of the most striking features of the adipose depot surrounding the prostate (periprostatic adipose tissue, PPAT) is that its accumulation is independent of body mass index. Its volume varies considerably between individual with some patients exhibiting abundant PPATs that have been correlated to occurrence of aggressive prostate cancer (PCa). However, abundant PPAT are not defined at biological levels. We used a new statistical approach to define abundant PPAT by normalizing PPAT volume to prostate volume in a cohort of 351 patients with a linear regression model. Applying this definition, we confirmed the link between abundant PPAT and PCa aggressiveness, therefore validating our approach. At biological levels, we showed that abundant PPAT exhibited extensive extracellular matrix remodeling, notably of the collagen network, decreasing the mechanical constraints in hypertrophic adipocytes leading to an inflammation free-expansion. Degradation of the most abundant collagen in AT, collagen VI was associated with increased production of endotrophin, a signaling peptide derived from AT, that was also elevated in the urine of patients with abundant PPAT confirming the clinical relevance of our results. These results highlight a unique mechanism of expansion of an adipose depot and open new mechanistic avenues to explain its role in prostate-related disorders.

cancer biology↗

bioRxiv2023

LptM promotes oxidative maturation of the lipopolysaccharide translocon by substrate binding mimicry.

Insertion of lipopolysaccharide (LPS) into the outer membrane (OM) of Gram-negative bacteria is mediated by a druggable OM translocon consisting of a {beta}-barrel membrane protein, LptD, and a lipoprotein, LptE. The {beta}-barrel assembly machinery (BAM) assembles LptD together with LptE to form a plug-and-barrel structure. In the enterobacterium Escherichia coli, formation of two native disulfide bonds in LptD controls LPS translocon activation. Here we report the discovery of LptM (formerly YifL), a conserved lipoprotein that assembles together with LptD and LptE at the BAM complex. We demonstrate that LptM stabilizes a conformation of LptD that can efficiently acquire native disulfide bonds and be released as mature LPS translocon by the BAM complex. Inactivation of LptM causes the accumulation of non-natively oxidized LptD, making disulfide bond isomerization by DsbC become essential for viability. Our structural prediction and biochemical analyses indicate that LptM binds to sites in both LptD and LptE that are proposed to coordinate LPS insertion into the OM. These results suggest that LptM facilitates oxidative maturation of LptD by mimicking LPS binding, thereby activating the LPS translocon.

microbiology↗

bioRxiv2022

In depth exploration of the alternative proteome of Drosophila melanogaster

Recent studies have shown that hundreds of small proteins were occulted when protein coding genes were annotated. These proteins, called alternative proteins, have failed to be annotated notably due to the short length of their open reading frame (less than 100 codons) or the enforced rule establishing that messenger RNAs (mRNAs) are monocystronic. Several alternative proteins were shown to be biologically active molecules and seem to be involved in a wide range of biological functions. However, genome wide exploration of the alternative proteome is still limited to a few species. In the present article we describe a deep peptidomics workflow which enabled the identification of 401 alternative proteins in Drosophila melanogaster. Subcellular localization, protein domains and short linear motifs were predicted for 235 of the alternative proteins identified and point towards specific functions of these small proteins. Several alternative proteins had approximated abundances higher than their canonical counterparts, suggesting that these alternative proteins are actually the main products of their corresponding genes. Finally, we observed fourteen alternative proteins with developmentally regulated expression patterns and ten induced upon heat shock treatment of embryos, demonstrating stage or stress specific production of alternative proteins.

biochemistry↗

bioRxiv2021

Human Cytomegalovirus modifies placental small extracellular vesicle secretion and composition towards a proviral phenotype to enhance infection of fetal recipient cells

Although placental small extracellular vesicles (sEVs) are extensively studied in the context of pregnancy, little is known about their role during human cytomegalovirus (hCMV) congenital infection, especially at the beginning of pregnancy. In this study, we examined the consequences of hCMV infection on sEVs production, composition and function using an immortalized human cytotrophoblast cell line derived from first trimester placenta. By combining complementary approaches of biochemistry, electron microscopy and quantitative proteomic analysis, we showed that hCMV infection increases the yield of sEVs produced by cytotrophoblasts and modifies their protein content towards a proviral phenotype. We further demonstrate that sEVs secreted by hCMV-infected cytotrophoblasts potentiate infection in naive recipient cells of fetal origin, including human neural stem cells. Importantly, these functional consequences are also observed with sEVs prepared from either an ex vivo model of infected histocultures from early placenta or from the amniotic fluid of patients naturally infected by hCMV at the beginning of pregnancy. Based on these findings, we propose that placental sEVs could be key actors favoring viral dissemination to the fetal brain during hCMV congenital infection. Significance StatementHuman cytomegalovirus (hCMV) infection is a major issue during pregnancy, affecting 1% of births in western countries. Despite extensive research, the pathophysiology of this congenital infection remains unclear. Recently, increasing evidence point to the key role of placental small extracellular vesicles (sEVs) in materno-fetal communication during pregnancy. Here, we examined the impact of hCMV infection on the protein composition and function of placental sEVs. We observe that hCMV infection leads to major changes in placental sEV protein content. Functional studies show the ability of sEVs produced by placental infected cells to facilitate further infection of naive recipient fetal cells, notably human neural stem cells. Our study demonstrates that placental sEVs are key players of hCMV pathophysiology during congenital infection.

cell biology↗

bioRxiv2021

SDR enzymes oxidize specific lipidic alkynylcarbinols into cytotoxic protein-reactive species

Hundreds of cytotoxic natural or synthetic lipidic compounds contain chiral alkynylcarbinol motifs, but the mechanism of action of those potential therapeutic agents remains unknown. Using a genetic screen in haploid human cells, we discovered that the enantiospecific cytotoxicity of numerous terminal alkynylcarbinols, including the highly cytotoxic dialkynylcarbinols, involves a bioactivation by HSD17B11, a short-chain dehydrogenase/reductase (SDR) known to oxidize the C-17 carbinol center of androstan-3-alpha,17-beta-diol to the corresponding ketone. A similar oxidation of dialkynylcarbinols generates dialkynylketones, that we characterize as highly protein-reactive electrophiles. We established that, once bioactivated in cells, the dialkynylcarbinols covalently modify several proteins involved in protein-quality control mechanisms, resulting in their lipoxidation on cysteines and lysines through Michael addition. For some proteins, this triggers their association to cellular membranes and results in endoplasmic reticulum stress, unfolded protein response activation, ubiquitin-proteasome system inhibition and cell death by apoptosis. Finally, as a proof-of-concept, we show that generic lipidic alkynylcarbinols can be devised to be bioactivated by other SDRs, including human RDH11 and HPGD/15-PGDH. Given that the SDR superfamily is one of the largest and most ubiquitous, this unique cytotoxic mechanism-of-action could be widely exploited to treat diseases, in particular cancer, through the design of tailored prodrugs. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/460423v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@12daae1org.highwire.dtl.DTLVardef@12db406org.highwire.dtl.DTLVardef@1863517org.highwire.dtl.DTLVardef@7b548_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG

biochemistry↗

bioRxiv2021

Long non-coding RNA Neat1 is a key translational regulator in hypoxia.

Internal ribosome entry sites (IRESs) drive translation initiation during stress. In response to hypoxia, (lymph)angiogenic factors responsible for tissue revascularization in ischemic diseases are induced by the IRES-dependent mechanism. Here we searched for IRES trans-acting factors (ITAFs) active in early hypoxia in mouse cardiomyocytes. Using knock-down and proteomics approaches, we show a link between a stressed-induced nuclear body, the paraspeckle, and IRES-dependent translation. Furthermore, smiFISH experiments demonstrate the recruitment of IRES-containing mRNA into paraspeckle during hypoxia. Our data reveal that the long non-coding RNA Neat1, an essential paraspeckle component, is a key translational regulator, active on IRESs of (lymph)angiogenic and cardioprotective factor mRNAs. In addition, paraspeckle proteins p54nrb and PSPC1 as well as nucleolin and Rps2, two p54nrb-interacting proteins identified by mass spectrometry, are ITAFs for IRES subgroups. Paraspeckle thus appears as a platform to recruit IRES-containing mRNAs and possibly host IRESome assembly. Polysome PCR array shows that Neat1 isoforms regulate IRES-dependent translation and, more widely, translation of mRNAs involved in stress response. HighlightsO_LIParaspeckle formation correlates with activation of translation via internal ribosome entry sites (IRES) in mouse hypoxic cardiomyocytes as well as in tumoral cells. C_LIO_LIThe long non-coding RNA Neat1, an essential paraspeckle component, is a key translational regulator of (lymph)angiogenic and cardioprotective factor expression in this process. C_LIO_LIIRES-containing mRNA is recruited into paraspeckles during hypoxia. C_LIO_LIParaspeckle proteins p54nrb and PSPC1 as well as two p54nrb-interacting proteins, nucleolin and RPS2, contribute to this process. C_LIO_LIParaspeckle appears as a platform for IRESome formation in the nucleus. C_LIO_LIThe Neat1 isoforms widely regulate the translation of mRNAs containing IRESs and of genes involved in the stress response. C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/430272v2_ufig1.gif" ALT="Figure 1"> View larger version (98K): org.highwire.dtl.DTLVardef@1741648org.highwire.dtl.DTLVardef@62e741org.highwire.dtl.DTLVardef@13c86a8org.highwire.dtl.DTLVardef@acae63_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2020

RIOK2 phosphorylation by RSK promotes synthesis of the human small ribosomal subunit

Ribosome biogenesis lies at the nexus of various signaling pathways coordinating protein synthesis with cell growth and proliferation. This process is regulated by well-described transcriptional mechanisms, but a growing body of evidence indicates that other levels of regulation exist. Here we show that the Ras/mitogen-activated protein kinase (MAPK) pathway stimulates post-transcriptional stages of human ribosome synthesis. We identify RIOK2, a pre-40S particle assembly factor, as a new target of the MAPK-activated kinase RSK. RIOK2 phosphorylation by RSK promotes cytoplasmic maturation of late pre-40S particles, which is required for optimal protein synthesis and cell proliferation. RIOK2 phosphorylation facilitates its release from pre-40S particles and its nuclear re-import, prior to completion of small ribosomal subunits. Our results bring a detailed mechanistic link between the Ras/MAPK pathway and the maturation of human pre-40S particles, which open a hitherto poorly explored area of ribosome biogenesis.

molecular biology↗

bioRxiv2020

Phosphoproteomics identifies PI3K inhibitor-selective adaptive responses in pancreatic cancer cell therapy and resistance

The PI3K pathway is highly active in human cancers. The four class I isoforms of PI3K are activated by distinct mechanisms leading to a common downstream signaling. Their downstream redundancy is thought to be responsible for treatment failures of PI3K inhibitors. We challenged this concept, by mapping the differential phosphoproteome evolution in response to PI3K inhibitors with different isoform selectivity patterns in pancreatic cancer, a disease currently without effective therapy. In this cancer, the PI3K signal was shown to control cell proliferation. We compared the effects of LY294002 that inhibit with equal potency all class I isoenzymes and downstream mTOR with the action of inhibitors with higher isoform-selectivity towards PI3K, PI3K{beta} or PI3K{gamma} (namely A66, TGX-221 and AS-252424). A bioinformatics global pathway analysis of phosphoproteomics data allowed us to identify common and specific signals activated by PI3K inhibitors supported by the biological data. AS-252424 was the most effective treatment and induced apoptotic pathway activation as well as the highest changes in global phosphorylation-regulated cell signal. However, AS-252424 treatment induced re-activation of Akt, therefore decreasing the treatment outcome on cell survival. Reversely, AS-252424 and A66 combination treatment prevented p-Akt reactivation and led to synergistic action in cell lines and patient organoids. The combination of clinically approved -selective BYL-719 with {gamma}-selective IPI-549 was more efficient than single molecule treatment on xenograft growth. Mapping unique adaptive signaling responses to isoform-selective PI3K inhibition will help to design better combinative treatments that prevent the induction of selective compensatory signals. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/313833v3_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@32004eorg.highwire.dtl.DTLVardef@b22f25org.highwire.dtl.DTLVardef@352a98org.highwire.dtl.DTLVardef@7f97a1_HPS_FORMAT_FIGEXP M_FIG C_FIG SignificanceHalf of all human cancers show increased PI3K activity but the mutational pattern of the PI3K pathway is not sufficient to predict sensitivity to PI3K inhibitors. By identifying for the first time specific signaling induced by PI3K inhibitors with different isoform-selectivity patterns, we provide insight in how to handle heterogeneity of PI3K expression in tumor samples for the choice of available PI3K-targetting drugs. Our work provides a roadmap to target the PI3K pathway, balancing the use of isoform-selective PI3K inhibitors with personalized information extending beyond the specific mutational status.

cancer biology↗

bioRxiv2020

Conformational maps of human 20S proteasomes reveal PA28- and immuno-dependant inter-ring crosstalks

Here, we used for the first time Hydrogen-Deuterium eXchange coupled to Mass Spectrometry (HDX-MS) to investigate conformational differences between the human standard 20S (std20S) and immuno 20S (i20s) proteasomes alone or in complex with PA28{beta} or PA28{gamma} activators. Their solvent accessibility was analyzed through a dedicated bioinformatic pipeline including stringent statistical analysis and 3D visualization. These data confirmed the existence of allosteric differences between the std20S and i20S at the surface of the -ring triggered from inside the catalytic {beta}-ring. Additionally, binding of the PA28 regulators to the 20S proteasomes modified solvent accessibility due to conformational changes of the {beta}-rings. This work is not only a proof-of-concept that HDX-MS can be used to get structural insights on large multi-protein complexes in solution, it also demonstrates that the binding of the std20S or i20S subtype to any of its PA28 activator triggers allosteric changes that are specific to this 20S/PA28 pair.

biophysics↗