Biology subjects

Loew, D.

Publications and source records attributed to Loew, D..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

Ku-binding motifs in RAG2, XLF, PAXX and MRI support functional redundancy during V(D)J recombination

The interaction of several partners with Ku through Ku-binding motifs (KBMs) in their sequences governs their enrolment in NHEJ repair complexes. Here, we first established more specifically the function of KBMs in V(D)J recombination as the molecular basis of functional redundancy between XLF and the NHEJ proteins MRI and PAXX. Then, given the functional redundancy between RAG2 and XLF, we explored the hypothesis of a KBM-mediated interaction between RAG2 and Ku. Through sequence alignment and biophysical methods, we identified a KBM at the C-terminus of RAG2 (R2CT) that mediates its interaction with Ku both in vitro and in cellulo. Notably, we showed that R2CT/Ku interaction is independent of the RAG nuclease activity. Finally, we demonstrated that the respective KBMs of RAG2 and XLF support their functional redundancy for V(D)J recombination.

molecular biology↗

bioRxiv2023

Inhibition of the macrophage demethylase LSD1 reverses Leishmania amazonensis-induced transcriptomic changes and causes a decrease in parasite load

Intracellular pathogens exploit host cell functions to favor their own survival. In recent years, the subversion of epigenetic regulation has emerged as a key microbial strategy to modify host cell gene expression and evade antimicrobial immune responses. Using the protozoan parasite Leishmania as a model system, we have recently demonstrated that infection causes histone H3 hypomethylation, which is associated with the establishment of an anti-inflammatory phenotype, suggesting that host cell demethylases may play a role in the intracellular survival of these parasites. In this study, we combined pharmacological inhibition with RNA sequencing and quantitative immune-precipitation analysis to investigate the role of the macrophage lysine demethylase LSD1 (KDM1a) in Leishmania intracellular infection in vitro. Treatment of infected macrophages with validated, LSD1-specific inhibitors resulted in a significant reduction in parasite burden. We confirmed the impact of these inhibitors on LSD1 activity within macrophage nuclear extracts using an in vitro demethylase assay and established their LSD1 target engagement in situ by cellular thermal shift assay. RNA-seq analysis of infected and inhibitor-treated macrophages linked parasite killing to a partial reversion of infection-dependent expression changes, restoring the macrophage anti-microbial response and limiting cholesterol biosynthesis. While we ruled out any impact of Leishmania on LSD1 expression or localization, we uncovered significant alterations in LSD1 complex formation within infected macrophages, involving unique interactions with host cell regulatory proteins such as Rcor-1. Our study sheds important new light on the epigenetic mechanisms of macrophage immuno-metabolic subversion by intracellular Leishmania and identifies LSD1 as a potential candidate for host-directed, anti-leishmanial therapy.

cell biology↗

bioRxiv2023

Growth factor-induced desialylation for the fast control of endocytosis

It is commonly assumed that the glycan makeup of glycoproteins that reach the cell surface is final and static. Here, we challenge this notion by the discovery of a molecular switch that induces acute and reversible changes of glycans on the plasma membrane. We demonstrate that within minutes, the epidermal growth factor triggers the galectin-driven endocytosis of cell surface glycoproteins, such as integrins, that are key regulators of cell adhesion and migration. The onset of this process, mediated by the Na+/H+ antiporter NHE-1 and the neuraminidases Neu1/3, requires the pH-triggered enzymatic removal of sialic acids whose presence otherwise prevents galectin binding. Desialylated glycoproteins are then retrogradely transported to the Golgi apparatus where their glycan makeup is reset, and their function is repurposed to regulate EGF-dependent invasive cell migration. Glycosylation at the cell surface thereby emerges as a dynamic and reversible regulatory post-translational modification that controls a highly adaptable trafficking pathway.

cell biology↗

bioRxiv2023

RNA polymerase III is involved in regulating Plasmodium falciparum virulence

While often undetected and untreated, persistent seasonal asymptomatic malaria infections remain a global public health problem. Despite the presence of parasites in the peripheral blood, no symptoms develop. Disease severity is correlated with the levels of infected red blood cells (iRBCs) adhering within blood vessels. Changes in iRBC adhesion capacity have been linked to seasonal asymptomatic malaria infections, however how this is occurring is still unknown. Here we present evidence that RNA polymerase III (RNA Pol III) transcription in Plasmodium falciparum is downregulated in field isolates obtained from asymptomatic individuals during the dry season. Through experiments with in vitro cultured parasites, we have uncovered an RNA Pol III-dependent mechanism that controls pathogen proliferation and expression of a major virulence factor in response to external stimuli. Our findings establish a connection between P. falciparum cytoadhesion and a non-coding RNA family transcribed by Pol III. Additionally, we have identified P. falciparum Maf1 as a pivotal regulator of Pol III transcription, both for maintaining cellular homeostasis and responding adaptively to external signals. These results introduce a novel perspective that contributes to our understanding of P. falciparum virulence. Furthermore, they establish a connection between this regulatory process and the occurrence of seasonal asymptomatic malaria infections.

microbiology↗

bioRxiv2023

Identification of the Intracellular Protein Targets of a Bio-active Clickable Half-Sandwich Iridium Complex by Chemical Proteomics

Identification of intracellular targets of anticancer drug candidates provides key information on their mechanism of action. Exploiting the ability of the anticancer (C^N)-chelated half-sandwich iridium(III) complexes to covalently bind proteins, click chemistry with a bioorthogonal azido probe was used to localize a phenyloxazoline-chelated iridium complex within cells and profile its interactome at the proteome-wide scale. Proteins involved in protein folding and actin cytoskeleton regulation were identified as high affinity targets. Upon iridium complex treatment, HSP90 folding activity was inhibited in vitro and major cytoskeleton disorganization was observed. We used a wide array of imaging and biochemical methods to validate selected targets and obtain a multiscale overview of the effects of this complex on live human cells. We demonstrate that it behaves as a dual agent, inducing both electrophilic and oxidative stresses in cells that account for its cytotoxicity.

biochemistry↗

bioRxiv2023

Polθ is phosphorylated by Polo-like kinase 1 (PLK1) to enable repair of DNA double strand breaks in mitosis

DNA double strand breaks (DSBs) are deleterious lesions that challenge genome integrity. To mitigate this threat, human cells rely on the activity of multiple DNA repair machineries that are tightly regulated throughout the cell cycle1. In interphase, DSBs are mainly repaired by non-homologous end joining (NHEJ) and homologous recombination (HR)2. However, these pathways are completely inhibited in mitosis3-5, leaving the fate of mitotic DSBs unknown. Here we show that DNA polymerase theta (Pol{theta})6 repairs mitotic DSBs and thereby maintains genome integrity. In contrast to other DSB repair factors, Pol{theta} function is activated in mitosis upon phosphorylation by the Polo-like kinase 1 (PLK1). Phosphorylated Pol{theta} is recruited to mitotic DSBs, where it mediates joining of broken DNA ends, while halting mitotic progression. The lack of Pol{theta} leads to a shortening of mitotic duration and defective repair of mitotic DSBs, resulting in a loss of genome integrity. In addition, we identify mitotic Pol{theta} repair as the underlying cause of the synthetic lethality between Pol{theta} and HR. Our findings reveal the critical importance of mitotic DSB repair for maintaining genome stability.

cell biology↗

bioRxiv2023

Structural characterization of stem cell factors Oct4, Sox2, Nanog and Esrrb disordered domains, and a method to identify their phospho-dependent binding partners.

The combined expression of a handful of pluripotency transcription factors (PluriTFs) in somatic cells can generate induced pluripotent stem cells (iPSCs). Here, we report the structural characterization of disordered regions contained in four important PluriTFs, namely Oct4, Sox2, Nanog and Esrrb. Moreover, many post-translational modifications (PTMs) have been detected on PluriTFs, whose roles are not yet characterized. To help in their study, we also present a method i) to produce well-characterized phosphorylation states of PluriTFs, using NMR analysis, and ii) to use them for pull-downs in stem cell extracts analyzed by quantitative proteomics to identify of Sox2 binders.

biochemistry↗

bioRxiv2023

Specific subcellular localization drives the different functions of CDC42 isoforms during migration

The small G-protein CDC42 is an evolutionary conserved polarity protein and a key regulator of numerous polarized cell functions, including directed cell migration. In vertebrates, alternative splicing gives rise to two CDC42 proteins: the ubiquitously expressed isoform (CDC42u) and the brain isoform (CDC42b), whose specific roles are not fully elucidated. The two isoforms only differ in their carboxy-terminal sequence, which includes the CAAX motif essential for CDC42 interaction with membrane. Here we show that these divergent sequences do not directly affect the range of CDC42s potential binding partners, but indirectly influence CDC42-driven signaling by controlling the specific subcellular localization of the two isoforms. In astrocytes and neural precursors, which naturally express both variants, CDC42u is mainly cytosolic and associates with the leading-edge plasma membrane of migrating cells where it recruits the Par6-PKC{zeta} complex to fulfill its polarity function. In contrast, CDC42b mainly localizes to intracellular membrane compartments, where it interacts with N-WASP. CDC42b does not participate in cell polarization but embodies the major isoform regulating endocytosis. Both CDC42 isoforms act in concert by contributing their specific functions to promote chemotaxis of neural precursors, demonstrating that the expression pattern of the two isoforms is decisive for the tissue-specific behavior of cells.

cell biology↗

bioRxiv2023

The oncogenic role of Streptococcus gallolyticus subsp. gallolyticus is linked to activation of multiple cancer-related signaling pathways

Streptococcus gallolyticus subsp. gallolyticus (SGG), an opportunistic gram-positive pathogen responsible for septicemia and endocarditis in the elderly, is often associated with colon cancer (CRC). In this work, we investigated the oncogenic role of SGG strain UCN34 using the azoxymethane (AOM)-induced CRC model in vivo, organoid formation ex vivo and proteomic and phosphoproteomic analyses from murine colons. We showed that SGG UCN34 accelerates colon tumor development in the murine CRC model. Full proteome and phosphoproteome analysis of murine colons chronically colonized by SGG UCN34 or the closely related non-pathogenic S. gallolyticus subsp. macedonicus (SGM) revealed that 164 proteins and 725 phosphorylation sites were differentially regulated following colonization by SGG UCN34. Ingenuity Pathway Analysis (IPA) indicates a pro-tumoral shift specifically induced following colonization by SGG UCN34, as most proteins and phosphoproteins identified were associated with digestive cancer. Comprehensive analysis of the altered phosphoproteins using ROMA software revealed significantly elevated activities in several cancer hallmark pathways affecting tumoral cells and their microenvironment, i.e. MAPK (ERK, JNK and p38), mTOR and integrin/ILK/actin signaling, in SGG UCN34 colonized colon. Importantly, analysis of protein arrays of human colon tumors colonized with SGG showed up-regulation of PI3K/Akt/mTOR and MAPK pathways, providing clinical relevance to our findings. To test SGGs capacity to induce pre-cancerous transformation of the murine colonic epithelium, we grew ex vivo organoids which revealed unusual structures with compact morphology following exposure to SGG. Taken together, our results reveal that the oncogenic role of SGG UCN34 is associated with activation of multiple cancer-related signaling pathways. Author SummaryColorectal cancer is the third most common cause of cancer mortality worldwide. The colon is a very singular organ, colonized by a vast and complex community of microorganisms, known as the gut microbiota. Strong evidence supports a role of the microbiota in colon cancer development. Streptococcus gallolyticus subsp. gallolyticus (SGG), a gut commensal, was one of the first bacteria to be associated with colorectal cancer. A better understanding of the role of SGG in colon cancer development is critical to developing novel strategies to improve clinical diagnosis and treatment of this disease. Here, using a global proteomic analysis of mouse colonic tissue colonized by SGG, we show that over 90% of the proteins with altered levels are involved in cancer. SGG colonization promotes autocrine and paracrine pro-tumor signals contributing to transformation of the colonic epithelium but also modifying the stromal microenvironment, which in turn sustains tumor development. Importantly, two tumor hallmark pathways (PI3K/Akt/mTOR and MAPK) identified in our mouse model were also found in human colon tumor biopsies colonized by SGG, strengthening the clinical relevance to our study.

microbiology↗

bioRxiv2023

Atheroprone shear stress stimulates noxious endothelial extracellular vesicle uptake by MCAM and PECAM-1 cell adhesion molecules

Atherosclerotic lesions mainly form in arterial areas exposed to low shear stress (LSS), where endothelial cells express a senescent and inflammatory phenotype. Conversely, high shear stress (HSS) has atheroprotective effects on the endothelium. Endothelial cell-derived extracellular vesicles have been shown to regulate inflammation, senescence and angiogenesis and therefore play a crucial role in vascular homeostasis and disease. While previous studies have shown links between hemodynamic forces and extracellular vesicle release, the exact consequences of shear stress on the release and uptake of endothelial EVs remains elusive. Our aim is therefore to decipher the interplay between these processes in endothelial cells exposed to atheroprone or atheroprotective shear stress. Confluent human umbilical vein endothelial cells (HUVEC) were exposed to either LSS or HSS for 24 hours. Large and small EVs were isolated from conditioned medium by sequential centrifugation and size exclusion chromatography. They were characterized by TEM, Western blot analysis of EV markers, tunable resistive pulse sensing, flow cytometry and proteomics. Uptake experiments were performed using fluorescently-labeled EVs and differences between groups were assessed by flow cytometry and confocal microscopy. We found that levels of large and small EVs in HUVEC conditioned media were fifty and five times higher in HSS than in LSS conditions, respectively. In vivo and in vitro uptake experiments revealed greater EV incorporation by cells exposed to LSS conditions compared to HSS. Additionally, endothelial LSS-EVs appeared to have a greater affinity for HUVECs than HSS-EVs or EVs derived from platelets, red blood cells, granulocytes and peripheral blood mononuclear cells. Proteomic analysis revealed that LSS-EVs were enriched in adhesion proteins such as PECAM1, MCAM, which were involved in EV uptake by endothelial cells. LSS-EVs also carried mitochondrial material, which may be involved in elevating reactive oxygen species levels in recipient cells. These findings suggest that endothelial shear stress has a significant impact during EV biogenesis and uptake. Given the major role of EVs and shear stress in vascular health, deciphering the relation between these processes may yield innovative strategies for the early detection and treatment of endothelial dysfunction.

cell biology↗

bioRxiv2022

A Legionella pneumophila effector impedes host gene silencing to promote virulence

RNA silencing is a gene silencing mechanism directed by small RNAs. Human miRNAs act as central regulators of host-bacteria interactions. However, it is unknown whether human pathogenic bacteria could impede RNA silencing to promote virulence. Here, we show that the Legionella pneumophila type IV-secreted effector LegK1 efficiently suppresses siRNA and miRNA activities in human cells. This effect depends on its known kinase activity, but also on its novel capacity, found here, to bind Argonaute (Ago) proteins. We further demonstrate that the ability of LegK1 to activate NF-{kappa}B signaling is required for RNA silencing suppression, establishing a link between effector-mediated NF-{kappa}B signaling and RNA silencing suppression. LegK1 also promotes L. pneumophila growth in both amoeba and human macrophages, supporting a role for this effector in virulence. Finally, we show that, in infected-macrophages, the latter activity relies, in part, on the genetic targeting of human Ago4. These findings indicate that a L. pneumophila effector has evolved to suppress RNA silencing to promote virulence. Significance StatementIt is now well established that mammalian viruses suppress RNAi to promote their replication in host cells. However, whether mammalian pathogenic bacteria use a similar virulence strategy remains unknown. Here, we show that the LegK1 effector from Legionella pneumophia, the causal agent of Legionnaires disease, efficiently suppresses RNAi in human cells. This effect depends on its ability to interact with Argonaute (Ago) proteins and to activate NF-{kappa}B signaling. In addition, LegK1 promotes virulence in infected-macrophages through the genetic targeting of human Ago4. Based on the lack of NF-{kappa}B-related factors in amoebae, and on the presence of canonical Ago proteins in these natural L. pneumophila hosts, we propose that the RNAi suppression activity of LegK1 represents its primary virulence function.

molecular biology↗

bioRxiv2022

CTNND2 moderates neuronal excitation and links human evolution to prolonged synaptic development in the neocortex

Human-specific genes are potential drivers of brain evolution. Among them, SRGAP2C has contributed to the emergence of features characterizing human cortical synapses, including their extended period of maturation. SRGAP2C inhibits its ancestral copy, the postsynaptic protein SRGAP2A; yet the synaptic molecular pathways differentially regulated in humans by SRGAP2 proteins remain largely unknown. Here, we identify CTNND2, a protein implicated in severe intellectual disability (ID) in the Cri-du-Chat syndrome, as an SRGAP2 effector. We demonstrate that CTNND2 slows down synaptic maturation and promotes neuronal homeostasis. During postnatal development, CTNND2 moderates neuronal excitation and excitability. In adults, it supports synapse maintenance. While CTNND2 deficiency is deleterious and results in the synaptic loss of SYNGAP1, another major ID-associated protein, the human-specific protein SRGAP2C enhances CTNND2 synaptic accumulation in human neurons. Our findings reveal that CTNND2 regulation by SRGAP2C contributes to synaptic neoteny in humans, and link human-specific and ID genes at the synapse.

neuroscience↗

bioRxiv2022

Fibroblasts generate topographical cues that steer cancer cell migration

Fibroblasts play a fundamental role in tumor development. Among other functions, they regulate cancer cells migration through rearranging the extracellular matrix, secreting soluble factors and establishing direct physical contacts with cancer cells. Here, we report that migrating fibroblasts deposit on the substrate a network of tubular structures that serves as guidance cue for cancer cell migration. Such membranous tubular network, hereafter called tracks, is stably anchored to the substrate in a {beta}5 integrin-dependent manner. We found that cancer cells specifically adhere to tracks by using clathrin-coated structures that pinch and engulf tracks. Tracks represent thus a spatial memory of fibroblast migration paths that is read and erased by cancer cells directionally migrating along them. We propose that fibroblast tracks represent a topography-based intercellular communication system capable of steering cancer cells migration. TEASERThe migration path of fibroblasts is marked by tubules that act as railways to direct following cancer cell migration.

cancer biology↗

bioRxiv2022

Translational regulation by RACK1 in astrocytes represses KIR4.1 expression and regulates neuronal activity

The regulation of translation in astrocytes, the main glial cells in the brain, remains poorly characterized. We developed a high-throughput proteomic screen for polysome-associated proteins in astrocytes and focused on the ribosomal protein receptor of activated protein C kinase 1 (RACK1), a critical factor in translational regulation. In astrocyte somata and perisynaptic astrocytic processes (PAPs), RACK1 preferentially bound to a number of mRNAs, including Kcnj10, encoding the inward rectifying potassium (K+) channel KIR4.1, a critical astrocytic regulator of neurotransmission. By developing an astrocyte-specific, conditional RACK1 knock-out mouse model, we showed that RACK1 repressed the production of KIR4.1 in hippocampal astrocytes and PAPs. Reporter-based assays revealed that RACK1 controlled Kcnj10 translation through the transcripts 5 untranslated region. Upregulation of KIR4.1 in the absence of RACK1 modified the astrocyte territory volume and neuronal activity attenuatin burst frequency and duration in the hippocampus. Hence, astrocytic RACK1 represses KIR4.1 translation and influences neuronal activity.

neuroscience↗

bioRxiv2022

Microglial TNFα orchestrates brain phosphorylation during the sleep period and controls homeostatic sleep.

The time we spend asleep is adjusted to previous time spent awake, and therefore believed to be under tight homeostatic control. Here, we establish microglia as a new cellular component of the sleep homeostat circuit. By using quantitative phosphoproteomics we demonstrate that microglia-derived TNF controls thousands of phosphorylation sites during the sleep period. Substrates of microglial TNF comprise sleep-promoting kinases and numerous synaptic proteins, including a subset whose phosphorylation status codes sleep need and determines sleep duration. As a result, lack of microglial TNF attenuates the build-up of sleep need, as measured by slow wave activity, and prevents immediate compensation for loss of sleep. Together, we propose that microglia control sleep homeostasis by releasing TNF that acts at the neuronal circuitry through dynamic control of phosphorylation.

neuroscience↗

bioRxiv2021

SILAKin: A novel high throughput SILAC and mass spectrometry-based assay to identify the substratome of kinases secreted by pathogens

Protein phosphorylation is one of the most important reversible post-translational modifications. It affects every cellular process including differentiation, metabolism and cell cycle. Eukaryotic protein kinases (ePK) catalyse the transfer of a phosphate from ATP onto proteins, which regulates fast changes in protein activity, structure or subcellular localisation. The systematic identification of substrates is thus crucial to characterise the functions of kinases and determine the pathways they regulate, and even more so when studying the impact of pathogens-excreted kinases on the host cell signal transduction. Several strategies and approaches have been used to identify substrates, but all show important limitations thus calling for the development of new efficient and more convenient approaches for kinase substrate identification. Herein, we present SILAkin, a novel and easy method to identify substrates that is applicable to most kinases. It combines phosphatase treatment, pulse heating, in vitro kinase assay (IVKA) and SILAC (Stable Isotope Labeling with Amino acids in Cell culture)-based quantitative mass spectrometry (MS). We developed SILAkin using the Leishmania casein kinase 1 (L-CK1.2) as experimental model. Leishmania, an intracellular parasite causing Leishmaniasis, releases L-CK1.2 in its host cell. Applying this novel assay allowed us to gain unprecedented insight into host-pathogen interactions through the identification of host substrates phosphorylated by pathogen-excreted kinases. We identified 225 substrates, including 85% previously unknown that represent novel mammalian CK1 targets, and defined a novel CK1 phosphorylation motif. The substratome was validated experimentally by L-CK1.2 and human CK1{delta}, demonstrating the efficiency of SILAkin to identify new substrates and revealing novel regulatory pathways. Finally, SILAkin was instrumental in highlighting host pathways potentially regulated by L-CK1.2 in Leishmania-infected host cells, described by the GO terms viral & symbiotic interaction, apoptosis, actin cytoskeleton organisation, and RNA processing and splicing. SILAkin thus can generate important mechanistic insights into the signalling of host subversion by these parasites and other microbial pathogen adapted for intracellular survival.

biochemistry↗

bioRxiv2021

OTX2 homeoprotein functions in adult choroid plexus

Choroid plexus secretes cerebrospinal fluid important for brain development and homeostasis. The OTX2 homeoprotein is critical for choroid plexus development and remains highly expressed in adult choroid plexus. Through RNA sequencing analyses of constitutive and conditional knockdown adult mouse models, we reveal putative roles for OTX2 in choroid plexus function, including cell signaling and adhesion, and show that it regulates the expression of factors secreted into cerebrospinal fluid, notably transthyretin. We show that Otx2 expression impacts choroid plexus immune and stress responses, and also affects splicing which leads to changes in mRNA isoforms of proteins implicated in oxidative stress response and DNA repair. Through mass spectrometry analysis of OTX2 protein partners in the choroid plexus, and in known non-cell autonomous target regions such as visual cortex and ventricular-subventricular zone, we identified putative targets involved in cell adhesion, chromatin structure and RNA processing. Thus, OTX2 retains important roles in choroid plexus function and brain homeostasis throughout life.

neuroscience↗

bioRxiv2021

The Pif1 helicase is actively inhibited during meiotic recombination which restrains gene conversion tract length

Meiotic recombination ensures proper chromosome segregation to form viable gametes and results in gene conversions events between homologs. Conversion tracts are shorter in meiosis than in mitotically dividing cells. This results at least in part from the binding of a complex, containing the Mer3 helicase and the MutL{beta} heterodimer, to meiotic recombination intermediates. The molecular actors inhibited by this complex are elusive. The Pif1 DNA helicase is known to stimulate DNA polymerase delta (Pol {delta}) -mediated DNA synthesis from D-loops, allowing long synthesis required for break-induced replication. We show that Pif1 is also recruited genome wide to meiotic DNA double-strand break (DSB) sites. We further show that Pif1, through its interaction with PCNA, is required for the long gene conversions observed in the absence of MutL{beta} recruitment to recombination sites. In vivo, Mer3 interacts with the PCNA clamp loader RFC, and in vitro, Mer3-MutL{beta} ensemble inhibits Pif1-stimulated D-loop extension by Pol {delta} and RFC-PCNA. Mechanistically, our results suggest that Mer3-MutL{beta} may compete with Pif1 for binding to RFC-PCNA. Taken together, our data show that Pif1s activity that promotes meiotic DNA repair synthesis is restrained by the Mer3-MutL{beta} ensemble which in turn prevents long gene conversion tracts and possibly associated mutagenesis.

molecular biology↗