Biology subjects

Robert, A.

Publications and source records attributed to Robert, A..

14 recordsLinked to original sources

bioRxiv2026

The DGKK mRNA is a primary target of the Prader-Willi syndrome-associated snoRNA SNORD116

Box C/D small nucleolar RNAs (SNORDs) classically guide site-specific modification of ribosomal RNAs through antisense elements located upstream of their D[prime] and D boxes, termed ASE1 and ASE2. Yet many orphan SNORDs have no established primary targets, including the paternally expressed multicopy SNORD116 family implicated in Prader-Willi syndrome. Our previous comparative analysis identified the DGKK/Dgkk interaction as the most evolutionarily persistent candidate involving the highly conserved SNORD116 ASE1 sequence and showed that SNORD116 depletion increases DGKK RNA abundance in human cells. Here, we re-evaluated this interaction alongside a focused set of recently published candidates and asked whether evolutionary persistence was accompanied by independent evidence of regulation. DGKK/Dgkk showed reproducible increases following transient SNORD116 loss across human iPSC-derived neural cells and in brain tissues from Snord116-knockout mice. Its predicted SNORD116 interaction site combined a marked local depletion of human sequence variation with an overlapping cis-regulatory interval identified in mouse. SNORD116 deficiency was also associated with increased endogenous DGK[kappa]; protein across several mouse brain regions, including the hypothalamus. More broadly, comparison across the candidate set showed that evolutionary persistence, duplex stability and expression response did not systematically align, indicating that sequence-based properties alone do not allow reliable prediction of responses to SNORD116 loss. Together, these data support DGKK/Dgkk as the best-supported primary regulatory target of SNORD116 to date and establish it as a model for dissecting the regulatory mechanism of this orphan snoRNA family.

molecular biology↗

bioRxiv2026

P4HA1 and P4HA2 accept both shared and isoenzyme-specific substrate residues in type I collagen to directly and indirectly orchestrate its post-translational modification landscape

Type I collagen, the most abundant protein of vertebrate organisms, is polymerized into a macroscopic-scale fibrillar network, the detailed arrangement of which is controlled by numerous post-translational modifications (PTMs) to determine the biomechanics of connective tissues. Its most frequent post-translational modification, prolyl-4-hydroxylation (P4H), catalyzed by prolyl-4-hydroxylases (P4HAs), occupies about 100 residue positions per procollagen alpha chain and is crucial to stabilize its triple helical building block thermodynamically. Recently, the two main P4HAs, P4HA1 and P4HA2, were found to exhibit mutual substrate specificity based on the nature of the amino acid residue standing N-terminally to the considered substrate residue. Independently, the activities of both enzymes were suggested to influence lysyl residue hydroxylation and glycosylation. To clarify these findings, we performed here a proteome-, tripeptide- and amino acid-scale analysis of prolyl and lysyl modifications of collagens, both in the established ATDC5 cell line and in primary mouse skin fibroblasts after adenovirus-mediated Crispr/Cas9 invalidation of either or both P4ha1 and/or P4ha2 genes. We conclude that both enzymes have no more than 18 and 14 respective specific substrate positions among more than 200 potential P4H sites available on type I collagen alpha chains. Combining procollagen tryptic peptide and tripeptide analysis, we confirm that glutamate localized N-terminally to potential P4H sites determines the substrate specificity of P4HA2. P4HA1-specific sites are instead preceded by phenylalanine, lysine, arginine, asparagine or histidine. Furthermore, we evidence that P4H is not required for prolyl-3-hydroxylation at the so-called A1 site and demonstrate that it reduces lysyl glycosylation at multiple sites in a cell-type-dependent manner. These findings highlight the strategic molecular function of P4HA1 and P4HA2 within the collagen maturation machinery, for, together, steering subtle collagen post-translational modifications.

biochemistry↗

bioRxiv2026

LysoDCs in Peyer's patches program compartmentalized microbiota-specific Th17 responses

Segmented filamentous bacteria (SFB) are a canonical model of microbiota-driven Th17 immunity, but how distinct gut-associated lymphoid tissues shape the quality and effector potential of commensal-specific T-cell responses remains unclear. Here we show that Peyers patches (PPs) and mesenteric lymph nodes (MLNs) generate transcriptionally, clonally, and functionally distinct SFB-reactive CD4 T-cell programs. In PPs, CCR2-dependent monocyte-derived LysoDCs capture luminal SFB and locally prime antigen-specific CD4 T cells. PP priming drives robust T cell activation, Th17 differentiation with type 1 regulatory (Tr1)-like features, preferential clonal expansion within Th17-Tfh17 lineages, and tissue-retention programs. In contrast, CCR2-independent MLN priming induces a less differentiated, recirculating profile dominated by non-expanded clonotypes. Notably, these distinct programs carry functional consequences. Upon transfer into Citrobacter rodentium-infected lymphopenic mice, PP-primed T cells preserve barrier integrity and limit pathology, whereas MLN-primed cells from the same donors fail to provide equivalent protection. Together, these findings establish PP LysoDCs as specialized orchestrators of compartmentalized microbiota-specific immunity and identify the anatomical site of commensal priming as a key determinant of T-cell functional diversification and mucosal immune outcome. HIGHLIGHTSSFB colonization drives compartmentalized effector and regulatory CD4 T-cell programs in PPs versus MLNs. Embigin LysoDCs in PPs directly sample SFB antigens and prime SFB-reactive CD4 T cells locally. PP priming induces Tr1-like Th17 differentiation, preferential Th17-Tfh17 clonal expansion, and tissue residency. PP-primed CD4 T cells ameliorate colitis-associated pathology during enteric infection, whereas MLN-primed cells do not. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/734008v1_ufig1.gif" ALT="Figure 1000"> View larger version (44K): org.highwire.dtl.DTLVardef@8d59dborg.highwire.dtl.DTLVardef@6643borg.highwire.dtl.DTLVardef@19e63a7org.highwire.dtl.DTLVardef@1a0a64b_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2024

Single Cell Calcium profiling for the discrimination of cancer cell lines.

Calcium (Ca2+) is an essential second messenger that controls numerous cellular functions. Characteristics of intracellular Ca2+ oscillations define Ca2+ signatures representatives of the phenotype of a cell. Oncogenic functions such as migration, proliferation or resistance to chemotherapy have been associated with aberrant Ca2+ fluxes. However, the identification of Ca2+ signatures representatives of the oncogenic properties of cancer cells remains to be addressed. To characterize oncogenic Ca2+ signatures, we proposed an unbiased scalable method that combines single cell calcium imaging with unsupervised and supervised machine learning tools. Here, we applied this method to investigate the heterogeneity of Ca 2+ signatures in 16 cancer cell lines and the remodeling of oncogenic Ca2+ signatures induced by acquired docetaxel resistance and in the course of the interaction of cancer cells with fibroblasts. Finally, our method demonstrates the potential of Ca2+ profiling for discriminating cancer cells and predict their phenotypic characteristics at single cell level, and provides a framework for researchers to investigate the remodeling of the Ca2+ signature during cancer development.

cancer biology↗

bioRxiv2023

Encoding of the colorectal cancer metabolic program through MICU2

The mitochondrial Ca2+ uniporter (MCU) plays crucial role in intramitochondrial Ca2+ uptake, allowing Ca2+-dependent activation of oxidative metabolism. In recent decades, the role of MCU pore-forming proteins has been highlighted in cancer. However, the contribution of MCU-associated regulatory proteins mitochondrial calcium uptake 1 and 2 (MICU1 and MICU2) to pathophysiological conditions has been poorly investigated. Here, we describe the role of MICU2 in cell proliferation and migration using in vitro and in vivo models of colorectal cancer (CRC). Transcriptomic analysis demonstrated an increase in MICU2 expression and the MICU2/MICU1 ratio in advanced CRC and CRC-derived metastases. We report that expression of MICU2 is necessary for mitochondrial Ca2+ uptake and quality of the mitochondrial network. Our data reveal the interplay between MICU2 and MICU1 in the metabolic flexibility between anaerobic glycolysis and OXPHOS. Overall, our study sheds light on the potential role of the MICUs in diseases associated with metabolic reprogramming. HighlightsO_LIMICU2 plays a pivotal role in the balance between anaerobic glycolysis and oxidative phosphorylation C_LIO_LIMICU2 stabilizes the mitochondrial network and endoplasmic reticulum-mitochondrial calcium flux C_LIO_LIMICU2 expression and the MICU2/MICU1 ratio control proliferation and metastasis formation in colorectal cancer C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/564893v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@6bb307org.highwire.dtl.DTLVardef@b6a966org.highwire.dtl.DTLVardef@2ba053org.highwire.dtl.DTLVardef@2ab691_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2023

Targeting the dependence on PIK3C3-mTORC1 signaling in dormancy-prone breast cancer cells blunts metastasis initiation

Halting breast cancer metastatic relapses following primary tumor removal and the clinical dormant phase, remains challenging, due to a lack of specific vulnerabilities to target during dormancy. To address this, we conducted genome-wide CRISPR screens on two breast cancer cell lines with distinct dormancy properties: 4T1 (short-term dormancy) and 4T07 (prolonged dormancy). We discovered that loss of class-III PI3K, Pik3c3, revealed a unique vulnerability in 4T07 cells. Surprisingly, dormancy-prone 4T07 cells exhibited higher mTORC1 activity than 4T1 cells, due to lysosome-dependent signaling occurring at the cell periphery. Pharmacological inhibition of Pik3c3 counteracted this phenotype in 4T07 cells, and selectively reduced metastasis burden only in the 4T07 dormancy-prone model. This mechanism was also detected in human breast cancer cell lines in addition to a breast cancer patient-derived xenograft supporting that it may be relevant in humans. Our findings suggest dormant cancer cell-initiated metastasis may be prevented in patients carrying tumor cells that display PIK3C3-peripheral lysosomal signaling to mTORC1. Statement of SignificanceWe reveal that dormancy-prone breast cancer cells depend on the class III PI3K to mediate a constant peripheral lysosomal positioning and mTORC1 hyperactivity. Targeting this pathway might blunt breast cancer metastasis.

cancer biology↗

bioRxiv2023

Mapping the MOB proteins' proximity network reveals a unique interaction between human MOB3C and the RNase P complex

Distinct functions mediated by members of the monopolar spindle-one-binder (MOB) family of proteins remain elusive beyond the evolutionary conserved and well-established roles of MOB1A and B in regulating the Hippo pathway. Since MOB proteins are adaptors, understanding how they engage in protein-protein interactions and complexes assembly is essential to define the full scope of their biological functions. To address this, we undertook a proximity-dependent biotin identification (BioID) approach to define the interactomes of all seven human MOB proteins in HeLa and HEK293 cell lines. We uncovered > 200 interactions, of which at least 70% are unreported on BioGrid. The generated dataset reliably recalled the bona fide interactors of the well-studied MOBs. We further defined the common and differential interactome between different MOBs on a subfamily and an individual level. We discovered a unique interaction between MOB3C and 7 out of 10 protein subunits of the RNase P complex, an endonuclease that catalyzes tRNA 5 maturation. As a proof-of-principle for the robustness of the generated dataset, we validated the specific interaction of MOB3C with catalytically active RNase P by using affinity purification-mass spectrometry and pre-tRNA cleavage assays of MOB3C pulldowns. In summary, our data provide novel insights into the biology of MOB proteins and reveal the first interactors of MOB3C, components of the RNase P complex, and hence an exciting nexus with RNA biology.

biochemistry↗

bioRxiv2023

ARF-family global interactome mapping uncovers spatial organization of cellular signaling pathways

The ADP-ribosylation factors (ARFs) and ARF-like (ARLs) GTPases serve as essential molecular switches governing a wide array of cellular processes. In this study, we utilized proximity-dependent biotin identification (BioID) to comprehensively map the interactome of 28 out of 29 ARF and ARL proteins in two cellular models. Through this approach, we identified [~]3000 high-confidence proximal interactors, enabling us to assign subcellular localizations to the family members. Notably, we uncovered previously undefined localizations for ARL4D and ARL10. Clustering analyses further exposed the distinctiveness of the interactors identified with these two GTPases. We also reveal that the expression of the understudied member ARL14 is confined to the stomach and intestines. We identified phospholipase D1 (PLD1) and the ESCPE-1 complex, more precisely SNX1, as proximity interactors. Functional assays demonstrated that ARL14 can activate PLD1 in cellulo and is involved in cargo trafficking via the ESCPE-1 complex. Overall, the BioID data generated in this study provide a valuable resource for dissecting the complexities of ARF and ARL spatial organization and signaling. SUMMARY STATEMENTGeneration of the ARF family interactome allowed the attribution of potential localizations and functions to previously understudied members. We found that ARL14 activates PLD1 and contributes to ESCPE-1-mediated trafficking.

molecular biology↗

bioRxiv2022

Curcumin and NCLX Inhibitors Share Anti-Tumoral Mechanisms in Microsatellite-Instability-Driven Colorectal Cancer

Colorectal cancer (CRC) is associated with high mortality worldwide and new targets are needed to overcome treatment resistance. Recent evidences highlight a role of the mitochondria calcium homeostasis in the development of CRC. In this context, we aimed to evaluate the role of the mitochondrial sodium-calcium-lithium exchanger (NCLX) and its targeting in CRC. We also identified curcumin as a new potential inhibitor of NCLX. In vitro, curcumin exerted strong anti-tumoral activity through its action on NCLX with mtCa2+ and reactive oxygen species overload associated with a mitochondrial membrane depolarization, leading to reduced ATP production and apoptosis through mitochondrial permeability transition pore opening concomitant with G2/M cell cycle arrest. NCLX inhibition with either CGP37157 (a benzodiazepine derivative), small interfering RNA-mediated knock-down or knockout approaches reproduced the effects of curcumin. Altered mitochondrial respiration, cellular aerobic glycolysis and endoplasmic reticulum-mitochondria membrane perturbations participated in these mechanisms. In a xenograft mouse model, NCLX inhibitors decreased CRC tumor growth. Both transcriptomic analysis of The Cancer Genome Atlas dataset and immunohistochemical analysis of tissue microarrays from 381 patients with microsatellite instability (MSI)-driven CRC demonstrated that higher NCLX expression was associated with MSI status and for the first time NCLX expression was significantly associated with recurrence-free survival in MSI CRC patients. Our findings provide strong evidence that blocking NCLX inhibits CRC in vitro and in vivo. We highlight a novel anti-tumoral mechanism of curcumin through its action on NCLX and mitochondria calcium overload that could benefit for therapeutic treatment of patients with MSI CRC.

cancer biology↗

bioRxiv2021

Extreme longevity of highly fecund termite queens achieved by mitochondrial and insulin upregulation without harmful lipid signatures or accumulation

Kings and queens of eusocial termites can live for decades, while queens sustain a nearly maximal fertility. To investigate the molecular mechanisms underlying their long lifespan, we carried out transcriptomics, lipidomics and metabolomics in Macrotermes natalensis on sterile short-lived workers, long-lived kings and five stages spanning twenty years of adult queen maturation. Reproductives share gene expression differences from workers in agreement with a reduction of several aging-related processes, involving upregulation of DNA damage repair and mitochondrial functions. Anti-oxidant gene expression is downregulated, while peroxidability of membranes in queens decreases. Against expectations, we observed an upregulated gene expression in fat bodies of reproductives of several components of the IIS pathway, including an insulin-like peptide, Ilp9. This pattern does not lead to deleterious fat storage in physogastric queens, while simple sugars dominate in their hemolymph and large amounts of resources are allocated towards oogenesis. Our findings support the notion that all processes causing aging need to be addressed simultaneously in order to prevent it.

molecular biology↗

bioRxiv2021

Identifying drivers of spatio-temporal variation in survival in four blue tit populations

In a context of rapid climate change, the influence of large-scale and local climate on population demography is increasingly scrutinized, yet studies are usually focused on one population. Demographic parameters, including survival, can vary significantly across populations of a given species, depending on global or local climatic fluctuations but also on many other population-specific parameters such as breeding density, habitat naturalness, predation or parasitism. Such ecological differences between populations could lead to different paces-of-life (POL), whereby populations where individuals display higher reproductive investment and bolder behaviours would have lower survival probabilities. We use here long-term (19 to 38 years) monitoring datasets from four Mediterranean populations of blue tits (Cyanistes caeruleus) to investigate the effects of sex, age class, large-scale and local climate temporal variation and population breeding density, on adult survival, using Capture-Mark-Recapture modelling. Environment heterogeneity in these four populations (two in evergreen and two in deciduous forests) has been linked to strong multi-trait phenotypic variation, suggesting blue tits in deciduous forests display faster POL compared to their conspecifics in evergreen habitats. The present results show heterogeneity in average survival probabilities across the four populations, with, as predicted, lower survival in the fast blue tits occupying deciduous habitats. Interestingly, the year-to-year variation in survival probabilities was synchronous among populations. This suggests that regional environmental conditions could drive survival fluctuations across populations. However, breeding densities were not correlated across populations, and we found no evidence that adult survival is correlated with either large-scale or local, climate temporal variation in these four blue tit populations. Finally, two of the focal populations displayed a linear temporal decrease in adult survival over the study period, calling for further investigation to explain this decline. Overall, this multi-site study shows that blue tit parental survival from one spring to the next can vary substantially across years, in a synchronous way across populations, yet the climate indices we used are not correlated with the temporal variation. This calls for further investigations in other potential drivers such as resource (in particular insect) abundance, predation or parasitism.

ecology↗

bioRxiv2021

Is earlier reproduction associated with higher or lower survival? Antagonistic results between individual and population scales in the blue tit

Although it has been shown that phenology can respond to temporal environmental variation in free ranging populations of several species, little is known about the mechanisms of these responses and their effects on demography, and in particular on survival. Exploring phenological responses and their associated consequences on survival can be achieved at two distinct scales: the population scale, which focusses on a set of common responses to environmental conditions, and the individual scale, focusing on the relative position of each individual in the distribution of survival and phenology under particular conditions. In this study, we apply capture-mark-recapture multistate modelling on a 38-year monitoring dataset of blue tits (Cyanistes caeruleus) to investigate the effects of breeding phenology and some demographic covariates (breeding density, average and individual breeding success) on adult survival, at both population and individual scales. Our analysis revealed that (i) at the population scale, early breeding years are followed by lower average adult survival. (ii) At the individual level, earlier breeders within the population have higher subsequent survival than later breeders, although this relationship is reversed in years with very harsh conditions, e.g. warm spring and high breeding density. (iii) High individual relative breeding success is also associated with higher subsequent survival and explains more survival variation than relative phenology. Overall, our study indicates that, although earlier breeding is associated with a survival cost at the population level, substantial intrapopulation hererogeneity shapes a positive association between earlier breeding, breeding success and survival at the individual level.

ecology↗

bioRxiv2018

The universal mechanism of intermediate filament transport

Intermediate filaments (IFs) are a major component of the cytoskeleton that regulates a wide range of physiological properties in eukaryotic cells. In motile cells, the IF network has to adapt to constant changes of cell shape and tension. In this study, we used two cell lines that express vimentin and keratins 8/18 to study the dynamic behavior of these IFs. We demonstrated that both IF types undergo extensive transport along microtubules. This was an unexpected result as keratin filament remodeling has been described to depend on actin dynamics. We established the role of kinesin-1 in vimentin and keratin IF transport by knocking out KIF5B, the ubiquitous isoform of kinesin-1. Futhermore, we demonstrated that unlike typical membrane cargoes, transport of both types of IFs does not involve kinesin light chains, but requires the presence of the same region of the kinesin-1 tail, suggesting a unified mechanism of IF transport.

cell biology↗

bioRxiv2017

Vaccination of health care workers to control Ebola virus disease

BackgroundHealth care workers (HCW) are at risk of infection during Ebola virus disease outbreaks and therefore may be targeted for vaccination before or during outbreaks. The effect of these strategies depends on the role of HCW in transmission which is understudied.\n\nMethodsTo evaluate the effect of HCW-targeted or community vaccination strategies, we used a transmission model to explore the relative contribution of HCW and the community to transmission. We calibrated the model to data from multiple Ebola outbreaks. We quantified the impact of ahead-of-time HCW-targeted strategies, and reactive HCW and community vaccination.\n\nResultsWe found that for some outbreaks (we call \"type 1\") HCW amplified transmission both to other HCW and the community, and in these outbreaks prophylactic vaccination of HCW decreased outbreak size. Reactive vaccination strategies had little effect because type 1 outbreaks ended quickly. However, in outbreaks with longer time courses (\"type 2 outbreaks\"), reactive community vaccination decreased the number of cases, with or without prophylactic HCW-targeted vaccination. For both outbreak types, we found that ahead-of-time HCW-targeted strategies had an impact at coverage of 30%.\n\nConclusionsThe optimal vaccine strategy depends on the dynamics of the outbreak and the impact of other interventions on transmission. Although we will not know the characteristics of a new outbreak, ahead-of-time HCW-targeted vaccination can decrease the total outbreak size, even at low vaccine coverage.\n\nsummaryTargeting health care workers for Ebola virus disease vaccination can decrease the size of outbreaks, and the number of health care workers infected. The impact of these strategies decrease depends on timing, coverage, and the dynamics of the outbreak.

epidemiology↗