Biology subjects

Rettel, M.

Publications and source records attributed to Rettel, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Granule microenvironment regulates the dual functions of FMR1

Fragile X Messenger Ribonucleoprotein 1 (FMR1) is an evolutionarily conserved RNA binding protein with important functions in cognition and female reproduction, and its disruption is associated with neurodevelopmental and reproductive disorders including the Fragile X syndrome. FMR1 is best known for its role as a translation repressor. However, several recent studies also suggest a role of FMR1 as a translation enhancer raising fundamental questions about the molecular regulation of these opposing functions. In this study, we identify FMR1 as part of the oskar mRNA-protein complex in the Drosophila oocyte and study the role of FMR1 as a translational enhancer of oskar. We provide the molecular mechanism for the dual functions of FMR1 and show that the two major RNA-binding domains of FMR1, the KH domains and the RGG box, play distinct separable roles in regulating translation. The KH domains enhance translation of mRNAs while the RGG box containing C-terminal domain (CTD) is required to repress translation. We further show that the condensation propensity of FMR1 containing granules regulates the two antagonistic functions, such that phase separation by FMR1-CTD creates the molecular microenvironment necessary for the repressive activity, whereas reduction in phase separation is associated with increased translation. Our findings highlight the importance of biomolecular condensates not just as a means of molecular compartmentalization but as a fundamental regulatory principle that dictates the functional output of modular protein domains.

molecular biology↗

bioRxiv2026

Intrinsic flexibility of Type VII secretion central pore is required for substrate translocation

Mycobacteria have up to five distinct type VII secretion pathways that play diverse roles in the cell ranging from iron uptake to virulence. To date, high-resolution structures of a hexameric ring-like pore complex have only been determined for closely related mycobacterial ESX-5 systems. The most significant difference between them is the arrangement and flexibility of the inner transmembrane helices of the EccC5 subunit within the central pore, leading to either a closed or to a semi-open conformation of the pore. In this work, we probed the functional roles of several central pore-forming residues in mediating secretion and demonstrate their crucial role in ESX-5 substrate translocation efficiency. Structural characterization of an ESX-5 variant with a conserved proline (P73A) surprisingly revealed rigidification of the pore-forming helices. In summary, our data demonstrate that maintaining the conformational flexibility of the central ESX-5 pore is essential for substrate secretion. Our findings reveal how the rigid structural ESX-5 scaffold with separate sections - facing the periplasm, the inner mycobacterial cell wall membrane and facing the cytosol - provides a complex framework for a high level of pore dynamics, restricted to the inner EccC5 subunit pore-forming helices. Their proper integration is required to generate a functional secretion system. The high level of conservation of several pore-forming residues across different type VII secretion systems indicates our findings to be general valid for these systems. SIGNIFICANCE STATEMENTA key virulence signature of the deadliest pathogenic mycobacteria is the presence of a number of highly specialized the type-VII secretion systems, which translocate distinct infection-promoting proteins through a central pore. Recent high-resolution structures of one of these systems (ESX-5) have revealed closed or partially closed pore states. This raises the question of how pore opening is mediated to allow substrate transport. Here, we demonstrate that this system is highly sensitive to even subtle changes within the central pore. Loss of conformational pore flexibility impairs secretion. A comprehensive understanding on the structure and associated dynamics of the central pore of type-VII secretion systems is essential to further unraveling mycobacterial virulence and developing future strategies for combatting it.

microbiology↗

bioRxiv2026

Decoding translational control by cis-regulatory elements and RNA binding proteins in human effector T cells

T cells are central players in killing virally infected and malignant cells. To achieve this, T cells depend on dynamic, tightly regulated alterations of the proteome. To decipher the rules instructing translation in T cells, we performed transcriptome and proteome analysis of polysome fractions from human effector CD8+ T cells. Transcriptome analysis informed on ribosomal occupancy of RNAs and uncovered the rapid and RNA-specific redistribution upon T cell activation. With machine learning, we identified RNA binding motifs that predict the RNA (re)distribution among the polysome fractions. Using matched proteome analysis, we identified polysome-associated RBPs and their swift shuttling across polysome fractions upon T cell activation. Integrating sequence feature analysis with polysome RBP localization uncovered PTBP1 as a positive translation regulator, through its interactions with cis-regulatory elements in 3'UTRs of target mRNAs. In conclusion, the multi-level analysis presented here identifies rules of selective translation control which shape the T cell proteome.

immunology↗

bioRxiv2026

Muscle LIM Protein is a Non-Canonical RNA-Binding Protein that Engages in RNA-Dependent Cytoskeletal Assemblies

Muscle LIM Protein (MLP) is a key component of the cardiomyocyte Z disc that is essential for sarcomere integrity and cardiac homeostasis. Accordingly, loss of MLP function results in (cardio)myopathy phenotypes in animal models and human patients. In this study, we present the first RNA-binding proteome (RBPome) of human cardiomyocytes using enhanced RNA interactome capture (eRIC). Data integration with existing RBPome datasets identified MLP as an evolutionary conserved and previously unrecognized non-canonical RNA-binding protein (RBP) in cardiomyocytes. Using complementary biochemical approaches, we confirmed direct RNA association of MLP. Moreover, RNA integrity was required for MLP interactions with cytoskeletal proteins and for the formation of higher-order MLP-containing assemblies, revealed by co-immunoprecipitation and RNA-dependent sedimentation profiling. Subcellular fractionation and imaging analyses indicated that MLPs association with cytoskeletal complexes may be weaker and more dynamic compared to core sarcomere proteins like -actinin. Domain mapping identified two glycine-rich regions within MLP as RNA contact sites, and RNA-binding-deficient MLP mutants showed impaired association with cytoskeletal complexes. Together, our findings suggest RNA binding as a regulated property of MLP and uncover an RNA-dependent layer of cytoskeletal organization in cardiomyocytes.

molecular biology↗

bioRxiv2026

An ancient monoaminergic signaling system coordinates contractility in a nerveless sponge

Chemical neurotransmission was a key animal innovation, enabling multicellular coordination of physiology and locomotion. Sponges are early-diverging animals that lack neurons and muscles, yet still coordinate contraction and relaxation of their filter-feeding water canals. Here, we show that Spongilla lacustris synthesizes the monoamines tryptamine, phenethylamine, and tyramine to elicit distinct canal behaviors. We identify previously uncharacterized decarboxylases and vesicular transporters coexpressed in secretory neuroid and metabolic cells. Using phosphoproteomics and label-free 3D imaging, we show that tryptamine activates GPCR signaling and Rho GTPases, remodeling adhesion and actomyosin networks in contractile canal epithelia to drive localized constrictions and whole-body deflations. Together, these findings define an ancestral monoaminergic system linking secretory and contractile cell types that predate neurons and was later elaborated for neuromodulation of synaptic transmission.

evolutionary biology↗

bioRxiv2024

Molecular dynamics of the matrisome across sea anemone life history

The evolutionary expansion of extracellular matrix (ECM) molecules has been crucial for the establishment of cell adhesion and the transition from unicellular to multicellular life. Members of the early diverging metazoan phylum cnidaria offer an exceptionally rich perspective into the metazoan core adhesome and its original function in developmental and morphogenetic processes. Here, we present the ensemble of ECM proteins and associated factors for the starlet sea anemone Nematostella vectensis based on in silico prediction and quantitative proteomic analysis of decellularized mesoglea from different life stages. The integration of the matrisome with single cell transcriptome atlases shows that gastrodermal cells are the primary producers of Nematostellas complex ECM, confirming the homology of the cnidarian inner cell layer with bilaterian mesoderm. The transition from larva to polyp is marked by an upregulation of metalloproteases and basement membrane components including all members of an unusually diversified SVEP1/Polydom family, suggesting massive epithelial remodeling. The enrichment of Wnt/PCP pathway factors during this process further indicates directed cell rearrangements as a key contributor to the polyps morphogenesis. Mesoglea maturation in adult polyps involves wound response proteins indicating shared molecular patterns in growth and regeneration. Our study identifies conserved matrisomal networks that coordinate transitions in Nematostellas life history.

developmental biology↗

bioRxiv2024

Dynamic multi-omics and mechanistic modeling approach uncovers novel mechanisms of kidney fibrosis progression

Kidney fibrosis, characterized by excessive extracellular matrix deposition, is a progressive disease that, despite affecting 10% of the population, lacks specific treatments and suitable biomarkers. This study presents a comprehensive, time-resolved multi-omics analysis of kidney fibrosis using an in vitro model system based on human kidney PDGFR{beta}+ mesenchymal cells aimed at unraveling disease mechanisms. Using transcriptomics, proteomics, phosphoproteomics, and secretomics we quantified over 14,000 biomolecules across seven time points following TGF-{beta} stimulation. This revealed distinct temporal patterns in the expression and activity of known and potential kidney fibrosis markers and modulators. Data integration resulted in time-resolved multi-omic network models which allowed us to propose mechanisms related to fibrosis progression through early transcriptional reprogramming. Using siRNA knockdowns and phenotypic assays, we validated predictions and regulatory mechanisms underlying kidney fibrosis. In particular, we show that several early-activated transcription factors, including FLI1 and E2F1, act as negative regulators of collagen deposition and propose underlying molecular mechanisms. This work advances our understanding of the pathogenesis of kidney fibrosis and provides a resource to be further leveraged by the community. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=71 SRC="FIGDIR/small/618507v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@d2bfaborg.highwire.dtl.DTLVardef@257d34org.highwire.dtl.DTLVardef@13f06f3org.highwire.dtl.DTLVardef@e16e6c_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG

systems biology↗

bioRxiv2024

A multi-year longitudinal harmonization of quality controls in mass spectrometry proteomics core facilities

Quality control procedures play a pivotal role in ensuring the reliability and consistency of data generated in mass spectrometry-based proteomics laboratories. However, the lack of standardized quality control practices across laboratories poses challenges for data comparability and reproducibility. In response, we conducted a harmonization study within proteomics laboratories of the Core for Life alliance with the aim to establish a common quality control framework, which facilitates comprehensive quality assessment and identification of potential sources of performance drift. Through collaborative efforts, we developed a consensus quality control standard for longitudinal assessment and we adopted a common processing software. We generated a 4-year longitudinal dataset from multiple instruments and laboratories, which enabled us to assess intra- and inter-laboratory variability, to identify causes of performance drift, and to establish community reference values for several quality control parameters. Our study enhances data comparability and reliability and fosters a culture of collaboration and continuous improvement within the proteomics community to ensure the integrity of proteomics data.

biochemistry↗

bioRxiv2024

Revealing the Arabidopsis AtGRP7 mRNA binding proteome by specific enhanced RNA interactome capture

BackgroundThe interaction of proteins with RNA in the cell is crucial to orchestrate all steps of RNA processing. RNA interactome capture (RIC) techniques have been implemented to catalogue RNA-binding proteins in the cell. In RIC, RNA-protein complexes are stabilized by UV crosslinking in vivo. Polyadenylated RNAs and associated proteins are pulled down from cell lysates using oligo(dT) beads and the RNA-binding proteome is identified by quantitative mass spectrometry. However, insights into the RNA-binding proteome of a single RNA that would yield mechanistic information on how RNA expression patterns are orchestrated, are scarce. ResultsHere, we explored RIC in Arabidopsis to identify proteins interacting with a single mRNA, using the circadian clock-regulated Arabidopsis thaliana GLYCINE-RICH RNA-BINDING PROTEIN 7 (AtGRP7) transcript, one of the most abundant transcripts in Arabidopsis, as a showcase. Seedlings were treated with UV light to covalently crosslink RNA and proteins. The AtGRP7 transcript was captured from cell lysates with antisense oligonucleotides directed against the 5untranslated region (UTR). The efficiency of RNA capture was greatly enhanced by using locked nucleic acid (LNA)/DNA oligonucleotides, as done in the enhanced RIC protocol. Furthermore, performing a tandem capture with two rounds of pulldown with the 5UTR oligonucleotide increased the yield. In total, we identified 356 proteins enriched relative to a pulldown from atgrp7 mutant plants. These were benchmarked against proteins pulled down from nuclear lysates by AtGRP7 in vitro transcripts immobilized on beads. Among the proteins validated by in vitro interaction we found the family of Acetylation Lowers Binding Affinity (ALBA) proteins. Interaction of ALBA4 with the AtGRP7 RNA was independently validated via individual-nucleotide resolution crosslinking and immunoprecipitation (iCLIP). The expression of the AtGRP7 transcript in an alba loss-of-function mutant was slightly changed compared to wild-type, demonstrating the functional relevance of the interaction. ConclusionWe adapted specific RNA interactome capture with LNA/DNA oligonucleotides for use in plants using AtGRP7 as a showcase. We anticipate that with further optimization and up-scaling the protocol should be applicable for less abundant transcripts.

plant biology↗

bioRxiv2024

Transverse Aortic COnstriction Multi-omics Analysis (TACOMA) uncovers pathophysiological cardiac molecular mechanisms

Time-course multi-omics data of a murine model of progressive heart failure induced by transverse aortic constriction (TAC) provide insights into the molecular mechanisms that are causatively involved in contractile failure and structural cardiac remodelling. We employ Illumina-based transcriptomics, Nanopore sequencing, and mass spectrometry-based proteomics on samples from the left ventricle (LV) and right ventricle (RV, RNA only) of the heart at 1, 7, 21, and 56 days following TAC and Sham surgery. Here, we present TACOMA, as an interactive web-application that integrates and visualizes transcriptomics and proteomics data collected in a TAC time-course experiment. TACOMA enables users to visualize the expression profile of known and novel genes and protein products thereof. Importantly, we capture alternative splicing events by assessing differential transcript and exon usage as well. Co-expression-based clustering algorithms and functional enrichment analysis revealed overrepresented annotations of biological processes and molecular functions at the protein and gene levels. To enhance data integration, TACOMA synchronizes transcriptomics and proteomics profiles, enabling cross-omics comparisons. With TACOMA (https://shiny.dieterichlab.org/app/tacoma), we offer a rich web-based resource to uncover molecular events and biological processes implicated in contractile failure and cardiac hypertrophy. For example, we highlight: (i) changes in metabolic genes and proteins in the time course of hypertrophic growth and contractile impairment; (ii) identification of RNA splicing changes in the expression of Tpm2 isoforms between RV and LV; and (iii) novel transcripts and genes likely contributing to the pathogenesis of heart failure. We plan to extend these data with additional environmental and genetic models of heart failure to decipher common and distinct molecular changes in heart diseases of different aetiologies. Database URLhttps://shiny.dieterichlab.org/app/tacoma

bioinformatics↗

bioRxiv2023

Structural Insights into The Role of MAGEA4 in RAD18 Regulation: Implications for Ubiquitin Ligase-Binding across the MAGE Protein Family

Melanoma associated antigen 4 (MAGEA4) is a cancer-testis antigen (CTA) that is primarily expressed in the testes of healthy adults but is aberrantly overexpressed and also a poor prognostic marker in several human cancers. In its cancer-specific role, MAGEA4 interacts with RAD18 and activates trans-lesion DNA synthesis (TLS), potentially favouring tumour evolution. However, the precise mode of interaction between RAD18 and MAGEA4 and its implications on the ubiquitination activity of RAD18 are unknown. Here, we employed NMR and AlphaFold2 (AF) to reveal that the ubiquitin-conjugating enzyme RAD6-binding domain (R6BD) of RAD18 interacts with a groove in the C-terminal winged-helix subdomain (WH) of MAGEA4. Using cross-linking mass spectrometry (XL-MS), we found that MAGEA4 displaces RAD6 from the R6BD of RAD18 and inhibits degradative autoubiquitination of RAD18, which could be countered by a competing short peptide of the RAD18 R6B region. AF and XL-MS also revealed an evolutionary invariant intramolecular interaction within RAD18 between the catalytic RING and the DNA-binding SAP domains that is essential for the ubiquitination of PCNA. Using interaction proteomics, we revealed that another Type-I MAGE, MAGE-C2, interacts with the RING ubiquitin ligase TRIM28 in a similar fashion as the MAGEA4/RAD18 complex. We propose that the peptide-binding groove identified in the C-terminal WH of MAGEA4 exists in other type-I MAGE proteins and serves as a ligase-binding cleft. Our data reveal crucial insights into RAD18-mediated ubiquitination of PCNA and its regulation by MAGE-A4.

biochemistry↗

bioRxiv2022

Transient inhibition of translation improves long-term cardiac function after ischemia/reperfusion by attenuating the inflammatory response

RationaleRapid reperfusion is the most effective treatment for attenuating cardiac injury caused by myocardial ischemia. Yet, reperfusion itself elicits damage to the myocardium through incompletely understood mechanisms, known as ischemia/reperfusion (I/R) injury. The myocardium adapts to I/R by changes in gene expression, which determines the cellular response to reperfusion. Protein translation is a key component of gene expression. However, it is unknown how regulation of translation contributes to cardiac gene expression in response to reperfusion and whether it can be targeted to mitigate I/R injury. MethodsTo examine translation and its impact on gene expression in response to I/R we assessed protein synthesis at different timepoints after ischemia and reperfusion in vitro and in vivo. Pharmacological inhibitors were used to dissect the underlying molecular mechanisms of translational control. Transient inhibition of protein synthesis was undertaken to decipher the effects of the translational response to reperfusion on cardiac function and inflammation. Cell-type-specific ribosome profiling was performed in mice subjected to I/R to determine the impact of translation on the regulation of gene expression in cardiomyocytes. ResultsReperfusion increased translation rates from a previously suppressed state during ischemia in cardiomyocytes, which was associated with the induction of cell death. In vivo, I/R resulted in strong activation of translation in the myocardial border zone. Detailed analysis revealed that the upregulation of translation is mediated by eIF4F complex formation, which was specifically mediated by the mTORC1-4EBP1-eIF4F axis. Short-term pharmacological inhibition of eIF4F complex formation by 4EGI-1 or rapamycin, respectively, attenuated translation, reduced infarct size and improved long-term cardiac function after myocardial infarction. Cardiomyocyte-specific ribosome profiling identified that reperfusion damage increased translation of mRNA networks in cardiomyocytes associated with cardiac inflammation and cell infiltration. Transient inhibition of the mTORC1-4EBP1-eIF4F axis decreased the expression of proinflammatory transcripts such as Ccl2, thereby reducing Ly6Chi monocyte infiltration and myocardial inflammation. ConclusionsMyocardial reperfusion induces protein synthesis in the border zone which contributes to I/R injury by rapidly translating a specific maladaptive mRNA network that mediates immune cell infiltration and inflammation. Transient inhibition of the mTORC1-4EBP1-eIF4F signaling axis during reperfusion attenuates this proinflammatory translational response, protects against I/R injury and improves long-term cardiac function after myocardial infarction. Clinical PerspectiveO_ST_ABSWhat Is New?C_ST_ABSO_LIThis is the first study to investigate the impact of translational regulation on cardiomyocyte gene expression in response to myocardial ischemia/reperfusion. C_LIO_LIWe show that translation regulates approximately two-thirds of differentially expressed genes in cardiomyocytes after ischemia/reperfusion, including many involved in inflammation and immune cell infiltration. C_LIO_LIThe translational response to ischemia/reperfusion is regulated by the mTORC1-4EBP1-eIF4F axis, which determines pro-inflammatory monocyte infiltration via control of the expression of the chemokine Ccl2. C_LI What Are the Clinical Implications?O_LICurrently, there are no specific therapies to prevent ischemia/reperfusion injury, which is mediated, at least in part, by a maladaptive inflammatory response. C_LIO_LIA translationally controlled network regulated by the mTORC1-4EBP1-eIF4F axis can be targeted by a short-term pharmacological intervention to attenuate the inflammatory response and improve cardiac function after ischemia/reperfusion in mice. C_LIO_LIThis study supports the emerging concept of selectively inhibiting maladaptive elements of the inflammatory response to improve outcome in patients after myocardial infarction; in addition, it provides a mechanistic basis for the currently ongoing CLEVER-ACS trial. C_LI

molecular biology↗

bioRxiv2022

The RNA-binding protein landscapes differ between mammalian organs and cultured cells

System-wide approaches have unveiled an unexpected breadth of the RNA-bound proteomes of cultured cells. Corresponding information regarding RNA-binding proteins (RBPs) of mammalian organs is still missing, largely due to technical challenges. Here, we describe ex vivo eRIC (enhanced RNA interactome capture) to characterize the poly(A)RNA-bound proteomes of three different mouse organs. The resulting organ atlases encompass more than 1300 RBPs active in brain, kidney or liver. Nearly a quarter (291) of these had formerly not been identified in cultured cells, with more than 100 being metabolic enzymes. Remarkably, RBP activity differs between organs independent of RBP abundance, suggesting organ-specific levels of control. Similarly, we identify systematic differences in RNA binding between animal organs and cultured cells. The pervasive RNA binding of enzymes of intermediary metabolism in organs points to tightly knit connections between gene expression and metabolism, and displays a particular enrichment for enzymes that use nucleotide cofactors. We describe a generically applicable refinement of the eRIC technology and provide an instructive resource of RBPs active in intact mammalian organs, including the brain.

molecular biology↗

bioRxiv2020

Structure of the mycobacterial ESX-5 Type VII Secretion System hexameric pore complex

To establish an infection, pathogenic mycobacteria use the Type VII secretion or ESX system to secrete virulence proteins across their cell envelope. The five ESX systems (ESX-1 to ESX-5) have evolved diverse functions in the cell, with the ESX-5 found almost exclusively in pathogens. Here we present a high-resolution cryo-electron microscopy structure of the hexameric ESX-5 Type VII secretion system. This 2.1 MDa membrane protein complex is built by a total of 30 subunits from six protomeric units, which are composed of the core components EccB5, EccC5, two copies of EccD5, and EccE5. The hexameric assembly of the overall ESX-5 complex is defined by specific inter-protomer interactions mediated by EccB5 and EccC5. The central transmembrane pore is formed by six pairs of EccC5 transmembrane helices that adopt a closed conformation in the absence of substrate in our structure. On the periplasmic face of the ESX-5 complex, we observe an extended arrangement of the six EccB5 subunits around a central cleft. Our structural findings provide molecular details of ESX-5 assembly and observations of the central secretion pore, which reveal insights into possible gating mechanisms used to regulate the transport of substrates.

microbiology↗

bioRxiv2020

Pharmacological inhibition of nonsense-mediated RNA decay augments HLA class I-mediated presentation of neoepitopes in MSI CRC

Microsatellite-unstable (MSI) colorectal cancer is characterized by the accumulation of somatic insertion/deletion (InDel) mutations potentially generating tumor-specific, frameshifted protein sequences. Such mutations typically generate premature translation termination codons targeting the affected mRNAs to degradation by nonsense-mediated RNA decay (NMD), limiting the synthesis and HLA class I-mediated presentation of tumor-specific InDel neoepitopes. We reasoned that the NMD inhibitor 5-azacytidine (5AZA) could serve to increase the expression of NMD-sensitive neoepitopes and analyzed the immunopeptidome of MSI HCT-116 cells using a proteogenomic approach. After immunoprecipitation of HLA:peptide complexes, we identified more than 10,000 HLA class I-presented peptides by LC-MS/MS including five InDel neoepitopes. The InDel neoepitopes were verified on the genomic, transcriptomic, and peptidomic level. Treatment with 5AZA increased the expression of the corresponding frameshift- and premature termination codon-bearing mRNAs and enhanced the presentation of peptides originating from known NMD targets and one of the identified InDel neoepitopes. By analyzing an array of MSI colorectal cancer cell lines and patient samples, we found the underlying frameshift mutation to be highly recurrent and immunization with the corresponding neoepitope induced strong CD8+ T cell responses in an HLA-A*02:01 transgenic mouse model. Our data directly show that peptides originating from frameshifted open reading frames due to InDel mutations in mismatch repair-deficient cells are presented on the cell surface via HLA class I. Moreover, we demonstrate the utility of NMD inhibitor-enhanced HLA class I-mediated presentation of InDel neoepitopes as well as their immunogenicity, uncovering the clinical potential of NMD inhibition in anti-cancer immunotherapy strategies. One Sentence SummaryImmunopeptidomics identified increased HLA class I-mediated presentation of immunogenic, frameshift-derived neoepitopes following NMD inhibition.

cancer biology↗

bioRxiv2020

Structure and dynamics of the quaternary hunchback mRNA translation repression complex

A key regulatory process during Drosophila development is the localized suppression of the hunchback mRNA translation at the posterior, which gives rise to a hunchback gradient governing the formation of the anterior-posterior body axis. The suppression of the RNA is achieved by a concerted action of Brain Tumour (Brat), Pumilio (Pum) and Nanos. Each protein is necessary for proper Drosophila development. The RNA contacts have been elucidated for the proteins individually in several atomic-resolution structures. However, the interplay of all three proteins in the RNA suppression remains a long-standing open question. We characterize the quaternary complex of the RNA-binding domains of Brat, Pum and Nanos with hunchback mRNA by combining NMR spectroscopy, SANS/SAXS, XL/MS with MD simulations and ITC assays. The quaternary hunchback mRNA suppression complex is flexible with the unoccupied nucleotides of the RNA functioning as a flexible linker between the Brat and Pum-Nanos moieties of the complex. Moreover, Brat and Pum with Nanos bind the RNA completely independently. In accordance with previous studies, showing that Brat can suppress hunchback mRNA independently and is distributed uniformly throughout the embryo, this suggests that hunchback mRNA suppression by Brat is functionally separate from the suppression by Pumilio and Nanos.

molecular biology↗

bioRxiv2020

Global mapping of Salmonella enterica-host protein-protein interactions during infection

Intracellular bacterial pathogens inject effector proteins into host cells to hijack diverse cellular processes and promote their survival and proliferation. To systematically map effector-host protein-protein interactions (PPIs) during infection, we generated a library of 32 Salmonella enterica serovar Typhimurium (STm) strains expressing chromosomally encoded affinity-tagged effector proteins, and quantified PPIs in macrophages and epithelial cells by Affinity-Purification Quantitative Mass-Spectrometry. Thereby, we identified 25 previously described and 421 novel effector-host PPIs. While effectors converged on the same host cellular processes, most had multiple targets, which often differed between cell types. Using reciprocal co-immunoprecipitations, we validated 13 out of 22 new PPIs. We then used this host-pathogen physical interactome resource to demonstrate that SseJ and SseL collaborate in redirecting cholesterol to the Salmonella Containing Vacuole (SCV) via NPC1, PipB directly recruits the organelle contact site protein PDZD8 to the SCV, and SteC promotes actin bundling by directly phosphorylating formin-like proteins.

microbiology↗

bioRxiv2020

Aggregation and Disaggregation Features of the Human Proteome

Protein aggregates have negative implications in disease. While reductionist experiments have increased our understanding of aggregation processes, the systemic view in biological context is still limited. To extend this understanding, we used mass spectrometry-based proteomics to characterize aggregation and disaggregation in human cells after non-lethal heat shock. Aggregation-prone proteins were enriched in nuclear proteins, high proportion of intrinsically disordered regions, high molecular mass, high isoelectric point and hydrophilic amino acids. During recovery, most aggregating proteins disaggregated with a rate proportional to the aggregation propensity: larger loss in solubility was counteracted by faster disaggregation. High amount of intrinsically disordered regions also resulted in faster disaggregation. However, other characteristics enriched in aggregating proteins did not correlate with the disaggregation rates. In addition, we analyzed changes in protein thermal stability after heat shock. Soluble remnants of aggregated proteins were more thermally stable compared to control condition. Our results provide a rich resource of heat stress-related protein solubility data, propose novel roles for intrinsically disordered regions in protein quality control and reveal a protection mechanism to repress protein aggregation in heat stress.

systems biology↗