Biology subjects

Mertins, P.

Publications and source records attributed to Mertins, P..

46 records · Page 3Linked to original sources

bioRxiv2021

Implementing solid-phase-enhanced sample preparation for Co-Immunoprecipitation with Mass Spectrometry for C. elegans

Studying protein-protein interactions in vivo can reveal key molecular mechanisms of biological processes. Co-Immunoprecipitation followed by Mass Spectrometry (CoIP-MS) allows detection of protein-protein interactions in high-throughput. The nematode Caenorhabditis elegans (C. elegans) is a powerful genetic model organism for in vivo studies. Yet, its rigid cuticle and complex tissues require optimization for protein biochemistry applications to ensure robustness and reproducibility of experimental outcomes. Therefore, we optimized CoIP-MS application to C. elegans protein lysates by combining a native CoIP procedure with an efficient sample preparation method called single-pot, solid-phase-enhanced, sample preparation method (SP3). Our results based on the subunits of the conserved chromatin remodeler FACT demonstrate that our SP3-integrated CoIP-MS procedure for C. elegans samples is highly accurate and robust. Moreover, in a previous study (Baytek et al. 2021), we extended our technique to studying the chromodomain factor MRG-1 (MRG15 in human), which resulted in unprecedented findings. Method SummaryCombination of cryo-fracture with single-pot, solid-phase-enhanced, sample preparation (SP3) to perform Co-Immuno-Precipitation followed by Mass Spectrometry (CoIP-MS) provides robust assessments of protein-protein interaction using C. elegans whole animals.

biochemistry↗

bioRxiv2021

SUMOylation of the chromodomain factor MRG-1 in C. elegans affects chromatin-regulatory dynamics

Epigenetic mechanisms to control chromatin accessibility and structure is important for gene expression in eukaryotic cells. Chromatin regulation ensures proper development and cell fate specification but is also essential later in life. Modifications of histone proteins as an integral component of chromatin can promote either gene expression or repression, respectively. Proteins containing specific domains such as the chromodomain recognize mono-, di- or tri-methylated lysine residues on histone H3. The chromodomain protein MRG-1 in Caenorhabditis elegans is the ortholog of mammalian MRG15, which belongs to the MORF4 Related Gene (MRG) family in humans. In C. elegans MRG-1 predominantly binds methylated histone H3 lysine residues at position 36 (H3K36me3). MRG-1 is important during germline maturation and for safeguarding the germ cell identity. However, it lacks enzymatic activity and depends on protein-protein interaction to cooperate with other factors to regulate chromatin. To elucidate the variety of MRG-1 interaction partners we performed in-depth protein-protein interaction analysis using immunoprecipitations coupled with mass-spectrometry. Besides previously described and novel interactions with other proteins, we also detected a strong association with the Small Ubiquitin-like Modifier (SUMO). Since SUMO is known to be attached to proteins in order to modulate the target proteins activity we assessed whether MRG-1 is post-translationally modified by SUMOylation. Notably, we provide evidence that MRG-1 is indeed SUMOylated and that this post-translational modification influences the chromatin-binding profile of MRG-1 in the C. elegans genome. Our presented study hints towards an important role of SUMOylation in the context of epigenetic regulation via the chromodomain protein MRG-1, which may be a conserved phenomenon also in mammalian species.

biochemistry↗

bioRxiv2021

Protective immune trajectories in early viral containment of non-pneumonic SARS-CoV-2 infection

The immune system of most SARS-CoV-2 infected individuals limits viral spread to the upper airways without pulmonary involvement. This prevents the development of pneumonic COVID-19. However, the protective immunological responses causative of successful viral containment in the upper airways remain unclear. Here, we combine longitudinal single-cell RNA sequencing, proteomic profiling, multidimensional flow cytometry, RNA-Seq of FACS-sorted leukocyte subsets and multiplex plasma interferon profiling to uncover temporally resolved protective immune signatures in non-pneumonic and ambulatory SARS-CoV-2 infected patients. We compare host responses in a high-risk patient population infected with SARS-CoV-2 but without pulmonary involvement to patients with COVID-19 pneumonia. Our data reveal a distinct immunological signature of successful viral containment, characterized by an early prominent interferon stimulated gene (ISG) upregulation across immune cell subsets. In addition, reduced cytotoxic potential of Natural Killer (NK) and T cells, as well as a monocyte phenotype with immune-modulatory potential are hallmarks of protective immunity. Temporal resolution across disease trajectories highlights ISG upregulation as particularly prominent early in the disease and confirms increased expression also in comparison to healthy controls. We validate this distinct temporal ISG signature by in-depth RNA-seq of FACS-sorted leukocyte subsets in a large prospective ambulatory SARS-CoV-2 infected cohort confirming early and robust ISG upregulation particularly in monocytes and T cells. In conclusion, our data demonstrate a protective ISG phenotype in patients with successful containment of SARS-CoV-2 infection without progression to COVID-19. This early protective interferon response might be exploited as a therapeutic approach and for disease course prediction.

immunology↗

bioRxiv2020

A comprehensive motifs-based interactome of the C/EBPα transcription factor

The pioneering transcription factor C/EBP coordinates cell fate and cell differentiation. C/EBP represents an intrinsically disordered protein with multiple short linear motifs and extensive post-translational side chain modifications (PTM), reflecting its modularity and functional plasticity. Here, we combined arrayed peptide matrix screening (PRISMA) with biotin ligase proximity labeling proteomics (BioID) to generate a linear, isoform specific and PTM-dependent protein interaction map of C/EBP in myeloid cells. The C/EBP interactome comprises promiscuous and PTM-regulated interactions with protein machineries involved in gene expression, epigenetics, genome organization, DNA replication, RNA processing, and nuclear transport as the basis of functional C/EBP plasticity. Protein interaction hotspots were identified that coincide with homologous conserved regions of the C/EBP family and revealed interaction motifs that score as molecular recognition features (MoRF). PTMs alter the interaction spectrum of multi-valent C/EBP-motifs to configure a multimodal transcription factor hub that allows interaction with multiple co-regulatory components, including BAF/SWI-SNF or Mediator complexes. Combining PRISMA and BioID acts as a powerful strategy to systematically explore the interactomes of intrinsically disordered proteins and their PTM-regulated, multimodal capacity. Key pointsO_LIIntegration of proximity labeling and arrayed peptide screen proteomics refines the interactome of C/EBP isoforms C_LIO_LIHotspots of protein interactions in C/EBP mostly occur in conserved short linear motifs C_LIO_LIInteractions of the BAF/SWI-SNF complex with C/EBP are modulated by arginine methylation and isoform status C_LIO_LIThe integrated experimental strategy suits systematic interactome studies of intrinsically disordered proteins C_LI

molecular biology↗

bioRxiv2020

Exploiting a PAX3-FOXO1-induced synthetic lethal ATR dependency for rhabdomyosarcoma therapy

Pathognomonic PAX3-FOXO1 fusion oncogene expression is associated with poor outcome in rhabdomyosarcoma. Combining genome-wide CRISPR screening with cell- based functional genetic approaches, we here provide evidence that PAX3-FOXO1 induces replication stress, resulting in a synthetic lethal dependency to ATR-mediated DNA damage-response signaling in rhabdomyosarcoma. Expression of PAX3-FOXO1 in muscle progenitor cells was not only sufficient to induce hypersensitivity to ATR inhibition, but PAX3-FOXO1-expressing rhabdomyosarcoma cells also exhibited increased sensitivity to structurally diverse inhibitors of ATR, a dependency that could be validated genetically. Mechanistically, ATR inhibition led to replication stress exacerbation, decreased BRCA1 phosphorylation and reduced homologous recombination-mediated DNA repair pathway activity. Consequently, ATR inhibitor treatment increased sensitivity of rhabdomyosarcoma cells to PARP inhibition in vitro, and combined ATR and PARP inhibition induced regression of primary patient-derived alveolar rhabdomyosarcoma xenografts in vivo. Moreover, a genome-wide CRISPR activation screen (CRISPRa) identified FOS gene family members as inducers of resistance against ATR inhibitors. Mechanistically, FOS gene family members reduced replication stress in rhabdomyosarcoma cells. Lastly, compassionate use of ATR inhibitors in two pediatric patients suffering from relapsed PAX3-FOXO1-expressing alveolar rhabdomyosarcoma showed signs of tolerability, paving the way to clinically exploit this novel synthetic lethal dependency in rhabdomyosarcoma.

cancer biology↗

bioRxiv2020

C-terminal-dependent control of EAAT2 signaling, corticostriatal synaptic glutamate clearance and spontaneous motor activity in mice with hypokinesia

Rapid removal of glutamate from the sites of glutamate release is an essential step in excitatory synaptic transmission. Despite many years of research, the molecular mechanisms underlying the intracellular regulation of glutamate transport at tripartite synapses have remained unclear. This limits the options for pharmacological treatment of motor disorders associated with glutamate excitotoxicity. Therefore, using the Q175 mouse model of Huntingtons disease (HD), we explored the effects of structural changes in the astrocytic excitatory amino acid transporter type 2 (EAAT2). We report that expression of a C-terminal-modified variant of EAAT2 can alleviate the symptoms of hypokinesia in mice with already advanced HD. At a cellular level, this beneficial outcome correlated with faster synaptic glutamate clearance, higher astrocytic glutamate uptake and larger amounts of native EAAT2 protein. Proteomics data indicate a partial reversal of HD-induced changes in the EAAT2 interactor spectrum. Thus, astrocytic glutamate transport remains a target for therapeutic intervention.

neuroscience↗

bioRxiv2020

YAP and β-catenin co-operate to drive oncogenesis in basal breast cancer

Targeting cancer stem cells (CSCs) can serve as an effective approach toward limiting resistance to therapies and the development of metastases in many forms of cancer. While basal breast cancers encompass cells with CSC features, rational therapies remain poorly established. Here, we show that receptor tyrosine kinase Met signalling promotes the activity of the Hippo component YAP in basal breast cancer. Further analysis revealed enhanced YAP activity within the CSC population. Using both genetic and pharmaceutical approaches, we show that interfering with YAP activity delays basal cancer formation, prevents luminal to basal trans-differentiation and reduces CSC survival. Gene expression analysis of YAP knock-out mammary glands revealed a strong decrease in {beta}-catenin target genes in basal breast cancer, suggesting that YAP is required for nuclear {beta}-catenin activity. Mechanistically, we find that nuclear YAP interacts and overlaps with {beta}-catenin and TEAD4 at common gene regulatory elements. Analysis of proteomic data from primary breast cancer patients identified a significant upregulation of the YAP activity signature in basal compared to other breast cancers, suggesting that YAP activity is limited to basal types. Our findings demonstrate that in basal breast cancers, {beta}-catenin activity is dependent on YAP signalling and controls the CSC program. These findings suggest that targeting the YAP/TEAD4/{beta}-catenin complex offers a potential therapeutic strategy for eradicating CSCs in basal (triple-negative) breast cancers.

cancer biology↗

bioRxiv2020

Mapping the unique and shared functions of oncogenic KRAS and RIT1 with proteome and transcriptome profiling

Aberrant activation of RAS oncogenes is prevalent in lung adenocarcinoma, with somatic mutation of KRAS occurring in [~]30% of tumors. Recently, we identified somatic mutation of the RAS-family GTPase RIT1 in lung adenocarcinoma, but relatively little is known about the biological pathways regulated by RIT1 and how these relate to the oncogenic KRAS network. Here we present quantitative proteomic and transcriptomic profiles from KRAS-mutant and RIT1-mutant isogenic lung epithelial cells and globally characterize the signaling networks regulated by each oncogene. We find that both mutant KRAS and mutant RIT1 promote S6 kinase, AKT, and RAF/MEK signaling, and promote epithelial-to-mesenchymal transition and immune evasion via HLA protein loss. However, KRAS and RIT1 diverge in regulation of phosphorylation sites on EGFR, USO1, and AHNAK proteins. The majority of the proteome changes are related to altered transcriptional regulation, but a small subset of proteins are differentially regulated by both oncoproteins at the post-transcriptional level, including intermediate filament proteins, metallothioneins, and MHC Class I proteins. These data provide the first global, unbiased characterization of oncogenic RIT1 network and identify the shared and divergent functions of oncogenic RIT1 and KRAS GTPases in lung cancer.

cancer biology↗

bioRxiv2019

UbiFast, a rapid and deep-scale ubiquitylation profiling approach for biology and translational research

Protein ubiquitylation is involved in a plethora of cellular processes. Defects in the ubiquitin system are at the root of many acquired and hereditary diseases. While antibodies directed at ubiquitin remnants (K-{varepsilon}-GG) have improved the ability to monitor ubiquitylation using mass spectrometry, methods for highly-multiplexed measurement of ubiquitylation in tissues and primary cells using sub-milligram amounts of sample remains a challenge. Here we present a highly-sensitive, rapid and multiplexed protocol for quantifying [~]10,000 ubiquitylation sites from as little as 500 ug peptide per sample from cells or tissue in a TMT10 plex in ca. 5 hr. High-field Asymmetric Ion Mobility Spectrometry (FAIMS) is used to improve quantitative accuracy for posttranslational modification analysis. We use the approach to rediscover substrates of the E3 ligase targeting drug lenalidomide and to identify proteins modulated by ubiquitylation in models of basal and luminal human breast cancer. The sensitivity and speed of the UbiFast method makes it suitable for large-scale studies in primary tissue samples.

biochemistry↗

bioRxiv2018

Systematic Characterization of RhoGEF/RhoGAP Regulatory Proteins Reveals Organization Principles of Rho GTPase Signaling

Rho GTPases control cell morphogenesis and thus fundamental processes in all eukaryotes. They are regulated by 145 RhoGEF and RhoGAP multi-domain proteins in humans. How the Rho signaling system is organized to generate localized responses in cells and prevent their spreading is not understood. Here, we systematically characterized the substrate specificities, localization and interactome of the RhoGEFs/RhoGAPs and revealed their critical role in contextualizing and spatially delimiting Rho signaling. They localize to multiple compartments providing positional information, are extensively interconnected to jointly coordinate their signaling networks and are widely autoinhibited to remain sensitive to local activation. RhoGAPs exhibit lower substrate specificity than RhoGEFs and may contribute to preserving Rho activity gradients. Our approach led us to uncover a multi-RhoGEF complex downstream of G-protein-coupled receptors controlling a Cdc42/RhoA crosstalk. The spatial organization of Rho signaling thus differs from other small GTPases and expands the repertoire of mechanisms governing localized signaling activity.

cell biology↗