Biology subjects

Trost, M.

Publications and source records attributed to Trost, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Single-cell proteomics resolves major cell populations and protein-defined chondrocyte states in the adult human osteoarthritic joint

Objective Single-cell RNA sequencing has transformed our understanding of cellular heterogeneity within osteoarthritic (OA) joint tissues, yet transcript abundance provides only an indirect measure of cellular phenotype and function. Recent advances in mass spectrometry now enable proteins to be measured directly at single-cell resolution. Here, we applied single-cell proteomics (SCP) to primary human OA cartilage and synovium to determine whether cellular heterogeneity within the diseased joint can be resolved directly at the proteome level. Design Cartilage and synovium were obtained from patients undergoing total knee arthroplasty for OA. Low-input proteomic profiling was first performed to establish proteome depth from limited cell numbers and validate biologically relevant clustering. SCP was then applied to individual cells, followed by unsupervised clustering, differential protein abundance analysis and iterative biological annotation. Results Low-input proteomics identified 7,800 proteins from 1,000 cells while retaining tissue- and cell-associated biological differences. Across the SCP dataset, ~2,300 unique proteins were detected, resolving chondrocyte, synovial fibroblast and immune-cell populations that closely paralleled those previously defined by single-cell transcriptomics. Higher-resolution analysis distinguished lining and sublining fibroblasts, discrete immune populations and multiple protein-defined chondrocyte states. Conclusions SCP enables direct characterisation of cellular heterogeneity within the human OA joint at the level of the proteome. By independently recovering established cellular populations while revealing protein-defined chondrocyte states, our findings establish SCP as a powerful complement to single-cell transcriptomics and open a new route to investigating functional cellular states and disease mechanisms that cannot be inferred from RNA abundance alone.

genetics↗

bioRxiv2026

Selective quality control of mistargeted mitochondrial proteins at the endoplasmic reticulum

In eukaryotic cells, the function of each organelle depends on its unique protein composition. Protein targeting errors threaten organelle identity and function, yet how mistargeting errors are detected and resolved remains poorly understood. Here, we show that mitochondrial import stress drives widespread rerouting of mitochondrial proteins to the endoplasmic reticulum (ER), with strong enrichment for hydrophobic oxidative phosphorylation (OXPHOS) components. Using proximity proteomics, a split-fluorescence reporter system, and genome-wide CRISPR screening, we find that mistargeted proteins partition into distinct classes with divergent fates, ranging from stable ER residence to rapid degradation by ER-associated degradation (ERAD). The clearance of these mislocalized proteins involves partially redundant ERAD branches, with the ubiquitin ligase MARCHF6 playing a central role. Together, these findings establish the ER as a key organelle for handling mistargeted mitochondrial proteins and reveal how ER quality control maintains proteostasis during mitochondrial dysfunction.

cell biology↗

bioRxiv2026

Pathogenic PTCD1 variants cause mitochondrial protein aggregation and cardiomyopathy

Disorders of mitochondrial oxidative phosphorylation affecting multiple respiratory chain complexes are among the most common causes of mitochondrial disease in humans. However, impaired energy metabolism alone does not fully account for tissue-specific vulnerability and disease progression, suggesting that additional molecular mechanisms contribute to disease pathology. We previously identified PTCD1 variants in a child with infantile cardiomyopathy associated with a combined respiratory chain deficiency. Here, we establish the pathogenicity of three PTCD1 (NM_015545.4) variants in which p.(Arg113Trp) and p.(Gly184Arg) segregate in cis whereas p.(Arg130*) is present in trans, demonstrating that disrupted mitochondrial proteostasis contributes to tissue damage in PTCD1 deficiency. PTCD1 patient cardiac tissue characterisation revealed impaired mitoribosome biogenesis, alongside increased aggregation of selective mitochondrial matrix proteins. Cell models expressing individual and combined PTCD1 missense variants, coupled with proteomics, recapitulated the protein aggregation, with the cis p.(Arg113Trp);p.(Gly184Arg) combination showing the most severe effect. Protein aggregation was accompanied by altered OPA1 processing and mitochondrial network remodelling. Our findings establish accumulating proteotoxic stress arising from impaired mitoribosome assembly as a pathogenic mechanism in post-mitotic tissues, driving PTCD1 cardiomyopathy.

cell biology↗

bioRxiv2026

A Context-Aware Single-Cell Proteomics Analysis pipeline.

Single-cell proteomics (SCP) by mass spectrometry can now quantify hundreds to thousands of proteins per cell, but the field still lacks standardised analytical pipelines that accommodate the diversity of instruments, sample preparation workflows and biological contexts encountered in practice. Existing workflows, largely adapted from single-cell transcriptomics, do not account for the informative missingness, pervasive ambient protein contamination and limited feature space that distinguish proteomic from transcriptomic data. In addition, cell type annotation remains a manual bottleneck that is subjective, difficult to reproduce and hard to scale. Here we present an end-to-end pipeline that integrates adaptive quality control, entropy-guided iterative batch correction, multi-modal marker discovery that exploits detection patterns unique to proteomics, and context-aware annotation by large language models (LLMs) coupled to structured contradiction reasoning and orthogonal data-driven validation. Benchmarking on published single-cell proteomic datasets from developing human brain and glioblastoma-associated neutrophils revealed systematic LLM failure modes, including context-insensitive marker vocabulary and misinterpretation of phagocytic or lytic cell states. We addressed these errors using a three-round prompt architecture that combines general biological principles with auto-generated dataset-specific constraints. In held-out validation on a skin tumour dataset acquired, the pipeline showed high concordance with FACS-sorted ground truth. In the caerulein-injured pancreas, orthogonal immunohistochemistry further supported annotations of macrophage, stellate and immune populations. The pipeline is fully automated under fixed settings, and available as Context-Aware Single-Cell Proteomics Analysis (CASPA), providing SCP laboratories and facilities with a reproducible workflow that delivers interpretable, confidence-quantified annotations suitable for downstream expert review.

bioinformatics↗

bioRxiv2026

Ubi-SCAPE enables deep exploration of the poly-ubiquitylome

Post-translational modification with chains of the 76-amino-acid protein ubiquitin ( poly-ubiquitylation) confers diverse fates to the targeted protein and non-protein substrates, including degradation, intracellular trafficking, and signal transduction. Despite being one of the most frequent modifications, the complexity of poly-ubiquitin chains adds methodological challenges to their characterization. Trypsin-resistant tandem-ubiquitin-binding entities (trTUBEs), engineered from natural ubiquitin-binding domains, can capture intact poly-ubiquitylated proteins with high cumulative avidity. However, such approaches have suffered from considerable co-eluting contaminants in proteomics applications. Here, we introduce an optimized trTUBE-based method for poly-ubiquitylated proteome purification, drastically depleting non-ubiquitylated protein contaminants and mono-ubiquitylated proteins. The method, termed Ubiquitylomics by Stringent, Cleavable, Affinity-based Proteome Extraction (Ubi-SCAPE), offers a streamlined and reproducible (median R2 > 0.98; CV < 8%) means of characterizing the poly-ubiquitylome. Over 7,800 poly-ubiquitylated proteins and 8,500 ubiquitin-modified peptides (diGly) were quantified with Ubi-SCAPE at a throughput of 40 samples per day, as well as over 6,000 from an equivalent of 33 g unstressed cell lysate. Upon acute stress by heat-shock, we identified 2,700 proteins and 8,000 diGly peptides with increased poly-ubiquitylation--offering similar biological insight as with far more material-, labor-, and cost-intensive peptide-based ubiquitin enrichment methods. Ubi-SCAPE therefore provides a simple and effective means of comprehensively quantifying a selective-enriched poly-ubiquitylome. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/703496v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@a28120org.highwire.dtl.DTLVardef@cb801aorg.highwire.dtl.DTLVardef@47577aorg.highwire.dtl.DTLVardef@1c030af_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2026

Democratised single cell proteomics uncovers tumor associated macrophage signature in CYLD cutaneous syndrome skin tumors

Single-cell proteomics (SCP) reveals cellular heterogeneity and biological insights inaccessible to bulk analysis. Existing limitations are cost, sample loss during processing, and accessibility to state-of-the-art instrumentation. We describe a label free SCP methodology in human tissue, combining fluorescence activated cell sorting (FACS), oil-immersion cell handling, mass-spectrometry, and neural-network derived spectral libraries which address these issues. We tested this methodology in a skin tumor syndrome, CYLD cutaneous syndrome (CCS), assessing tumor heterogeneity. Using a Bruker timsTOF HT platform we quantified > 4000 proteins, averaging [~]700 per cell, through a cost-effective pipeline without specialised liquid handling infrastructure. By utilising pre-existing bioinformatic tools from the scRNA-seq field we implemented a robust analysis methodology, discriminating between macrophages, dendritic cells and tumor keratinocytes, in an unbiased analysis of 419 CCS tumor cells. We validated the biological accuracy of cell annotations by cross referencing with each cells FACS markers. Furthermore, we identified a novel CCS tumor associated macrophage population which carried a tumor microenvironment remodelling signature. Our findings demonstrate an accessible SCP technology capable of yielding novel biological discoveries in clinical tissue. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=86 SRC="FIGDIR/small/704354v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@6fe6a3org.highwire.dtl.DTLVardef@8f0ff9org.highwire.dtl.DTLVardef@61e037org.highwire.dtl.DTLVardef@1c76a43_HPS_FORMAT_FIGEXP M_FIG C_FIG IMPORTANTO_LIManuscripts submitted to Review Commons are peer reviewed in a journal-agnostic way. C_LIO_LIUpon transfer of the peer reviewed preprint to a journal, the referee reports will be available in full to the handling editor. C_LIO_LIThe identity of the referees will NOT be communicated to the authors unless the reviewers choose to sign their report. C_LIO_LIThe identity of the referee will be confidentially disclosed to any affiliate journals to which the manuscript is transferred. C_LI GUIDELINESO_LIFor reviewers: https://www.reviewcommons.org/reviewers C_LIO_LIFor authors: https://www.reviewcommons.org/authors C_LI CONTACTThe Review Commons office can be contacted directly at: office@reviewcommons.org

biochemistry↗

bioRxiv2026

Empirical-Bayes and Bayesian Hierarchical Modelling forMissingness and Differential Expression in Proteomics

Mass spectrometry-based label-free proteomics data often suffer from missing values, especially for low-abundance proteins or when a protein is completely absent in one condition. This makes it challenging to estimate fold changes reliably and perform downstream analyses. Traditional imputation methods often show inconsistent performance across datasets and they typically treat imputed values as fixed rather than uncertain. This can lead to an underestimation of variability in downstream analyses. To address those problems, we present a hierarchical model that accounts for both observed protein intensities and patterns of missing data. Missing values are modelled as left-censored observations below protein-specific detection limits, reflecting the limited sensitivity of the instrument, or being missing with the probability of an intensity dependent manner. Our proposed model captures structure at multiple levels: intensity-level measurements, group-level effects (e.g., experimental conditions), and protein-level variation. To estimate model parameters, we employ an empirical Bayes framework to infer hyperparameters across proteins and use Markov Chain Monte Carlo (MCMC) methods to sample parameters from the posterior distribution. Our pipeline avoids the need for imputing missing values and is designed to produce more reliable fold-change estimates and uncertainty measures. We benchmark our method against existing approaches and demonstrate that it provides more accurate, stable, and robust estimates for differential expression analysis. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/699650v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@11da768org.highwire.dtl.DTLVardef@1d9de0aorg.highwire.dtl.DTLVardef@809df1org.highwire.dtl.DTLVardef@16cc3_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2025

Global identification of Chromobacterium violaceum T6SS effectors reveals an Rhs antibacterial toxin featuring FIX and ADP-ribosyltransferase domains

Bacteria coexist in polymicrobial communities where they engage in complex interactions, including interbacterial antagonism. We recently reported that the environmental bacterial pathogen Chromobacterium violaceum has an active type VI secretion system (T6SS), which plays a crucial role in interbacterial competition. However, the arsenal of toxic effectors delivered by this T6SS remains unknown. Here, we identify the repertoire of C. violaceum T6SS effectors and characterize a novel antibacterial Rhs-family effector, RhsF (Rhs with a FIX domain), and its cognate immunity protein, RhsFi. Using mass spectrometry analyses of secreted proteins and proteins co-immunoprecipitated with VgrG3, we identified six novel effector candidates, namely four phospholipases, a protein of unknown function, and the previously-uncharacterized Rhs protein, RhsF (CV_1431). RhsF contains an N-terminal FIX domain and was shown to intoxicate susceptible bacteria in a T6SS-dependent manner. The action of the C-terminal toxin domain of RhsF (RhsF-CT) is prevented in the presence of RhsFi (CV_1430), confirming that RhsF-RhsFi comprises an effector-immunity pair. The structure of the RhsF-CT/RhsFi complex determined by X-ray crystallography (1.85 [A] resolution) revealed that RhsF-CT shares structural similarity with ADP-ribosyltransferase toxins and that RhsFi inhibits toxicity via direct occlusion of the RhsF-CT catalytic site. Functional assays confirmed that RhsF toxicity requires a catalytic triad composed of R1403, Y1456, and E1497 residues. Overall, our findings reveal the effectors secreted by the T6SS of C. violaceum, establish RhsF as a potent antibacterial toxin, and confirm T6SS-dependent delivery of a FIX-containing Rhs protein, expanding the known repertoire of bacterial arms involved in microbial competition.

biochemistry↗

bioRxiv2025

MALDI-TOF mass spectrometry and proteomics as phenotypic screening tools for anti-inflammatory drugs

Phenotypic screening is a powerful technology to discover drug candidates in physiologically relevant systems without prior knowledge of molecular targets; however, mass spectrometry (MS) remains underutilised as readout strategy. In this proof-of-concept study, we developed and evaluated two complementary MS-based phenotypic screening approaches to identify anti-inflammatory compounds in human induced pluripotent stem cell-derived macrophages and compared them to a conventional targeted cytokine profiling assay. First, we established a novel MALDI-TOF MS fingerprinting strategy that effectively distinguished macrophage phenotypes, identified phenotype-specific biomarkers, and maintained high-throughput capabilities while reducing cost. Secondly, we performed an in-depth LC-MS proteomic analysis using low cell input on an Evosep-timsTOF HT setup, providing rich molecular detail. Both MS-based approaches demonstrated large comparability with the cytokine assay, with a large proportion of hits overlapping. Notably, the proteomics workflow uniquely enabled deeper insight into inflammation pathway engagement, off-target effects, compound potency, and cytotoxicity. Together, these findings highlight the potential of MS-driven phenotypic screening to enhance early drug discovery by enabling efficient, informative, and cost-effective hit selection. O_FIG O_LINKSMALLFIG WIDTH=196 HEIGHT=200 SRC="FIGDIR/small/691706v1_ufig1.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@c83cb7org.highwire.dtl.DTLVardef@a14d4org.highwire.dtl.DTLVardef@1dda6d7org.highwire.dtl.DTLVardef@f47564_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Comparative ubiquitinomics of human skin reveals insulin receptor ubiquitination as a regulator of collagen secretion.

Ubiquitination is central to skin homeostasis and disease, however the cutaneous ubiquitinome remains poorly characterised. We investigated spatial patterns of ubiquitination in human skin, discovering specific ubiquitin linkages located within the hair follicle and epidermis. To create a comprehensive physiological profile of the ubiquitinated proteins within the skin, we profiled healthy skin and CYLD cutaneous syndrome (CCS) skin tumours through di-glycine remnant immunopurification proteomics, enabling identification of 1605 ubiquitin sites across 731 proteins. The healthy cutaneous ubiquitinome comprised substrate classes including keratins, S100 proteins and histones. Predictive upstream enzyme analysis ranked the most frequent E3 ligase as NEDD4, and the most frequent deubiquitinases included USP7 and CYLD. Analysis of the CCS tumor ubiquitinome, characterized by catalytically inactivated CYLD somatic mutations and prominent extracellular matrix (ECM) secretion, identified 251 differentially ubiquitinated sites compared to healthy skin. The CCS ubiquitinome was enriched for cell division and differentiation proteins, including predicted CYLD substrate insulin receptor (INSR). Secretome analysis of INSR knockdown CCS primary cells revealed reduced secretion of basement membrane proteins, particularly COL7A1. By defining how ubiquitination modulates pathophysiological ECM secretory output, our study highlights the ubiquitin post-translational system as a broader determinant of tissue architecture. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=86 SRC="FIGDIR/small/691868v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@565e59org.highwire.dtl.DTLVardef@4c3084org.highwire.dtl.DTLVardef@52a43org.highwire.dtl.DTLVardef@11999d8_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2025

The Type VI Secretion System Antifungal Effector Tfe2 Inhibits Protein Translation and Drives Hyperactivation of TORC1.

Type VI Secretion Systems (T6SS) are utilised by many bacteria to deliver toxic effectors into neighbouring bacterial, fungal or host cells. Whilst many antibacterial effectors are well characterised, much less is known regarding the identity or mode-of-action of antifungal effectors. Here we combine structural modelling with proteomics and in vivo approaches, to show that the Serratia marcescens antifungal effector Tfe2 adopts a novel fold and functions as a potent inhibitor of protein translation leading to hyperactivation of the TORC1 kinase. We show that Tfe2 expression in Saccharomyces cerevisiae, or treatment with the protein translation inhibitor cycloheximide, drive identical increases in free intracellular amino acids and hyperactivation of TORC1. This, in turn, triggers the Tfe2 and cycloheximide-mediated rapid turnover of amino acid transporters through stimulating substrate-independent endocytosis. Polysome profiling, however, revealed differences in Tfe2 and cycloheximide-mediated protein translation inhibition, with Tfe2 inhibiting initiation of translation. Tfe2-mediated hyperactivation of TORC1 may also underpin adaptive responses to this effector which include significant remodelling of the lipidome and notable alterations in organelle and cell wall structures. Collectively this study has provided new insight into the mode-of-action of a structurally novel antifungal effector Tfe2.

microbiology↗

bioRxiv2025

Recommendations for Quantitative Data-Independent Acquisition (DIA) Proteomics using Controlled Quantitative Experiments (CQEs)

Advances in mass spectrometry and associated computational tools have resulted in the wide-spread adoption of Data-Independent Acquisition (DIA) for proteomics. Experiments using state-of-the-art instrumentation and specialised modes of acquisition report ever increasing throughput and protein identification rates. However, experiments to test the efficacy of protein quantitation in these novel methods is surprisingly rare -especially in novel methods where low sample input or high-throughput is considered. Here, we performed a series of controlled quantitative experiments (CQEs) on two commonly used mass spectrometry platforms (Exploris480 and timsTOF HT), using defined mixtures of human, yeast, and bacterial proteomes. Peptide spectrum matching and initial quantification was performed using DIA-NN, with a series of post-processing options also compared. Overall, we identified >10,000 unique proteins and >160,000 precursors across samples with excellent accuracy and precision. Additional post-processing was found to have drastic impacts on ID rates, quantitative precision and accuracy, and false quantitative rate (FQR). These impacts were particularly notable in experiments using low amounts of sample. In optimal loading conditions, data processing could reduce FQR from [~]0.1 to 0 % with minimal losses in ID rates. Ultimately, we present a series of recommendations for proteomics data processing, and advice on the use of CQEs and resulting metrics when assessing quantitative efficacy in novel modes of acquisition.

biochemistry↗

bioRxiv2025

Flow cytometry-based isolation combined with ultra-sensitive proteomics of Salmonella-containing phagosomes reveals novel insights into host-pathogen interactions

Macrophages engulf pathogens into dynamic phagosomes, which many bacteria manipulate for survival. However, isolating pure pathogen-containing phagosomes remains challenging. Here, we developed a novel flow cytometry-based isolation and ultrasensitive proteomics approach to analyse phagosomal and bacterial proteomes from macrophages infected with wild-type (WT) Salmonella enterica serovar Typhimurium (STM) or a {Delta}phoP mutant at 30 min and 4 hrs post-infection. Our approach provides higher throughput, requires lower cell numbers and quantifies more proteins than previous techniques. Our data reveals key host-pathogen interactions, showing induction of PhoP-dependent virulence factors and novel putative proteins that shape STMs intracellular niche. Moreover, our data indicates that bacteria-containing phagosomes recruit mitochondrial membrane for production of reactive oxygen species. These findings provide new insights into Salmonellas manipulation of phagosomal maturation and intracellular niche formation.

microbiology↗

bioRxiv2025

Targeting non-canonical NF-κB signalling in CYLD cutaneous syndrome by selective inhibition of IκB kinase alpha.

CYLD cutaneous syndrome (CCS) skin tumors develop from puberty onwards, can number in the hundreds and progressively grow over time. CCS patients lack medical therapies and require repeated surgery to control tumor burden. CYLD loss of heterozygosity (LOH) drives tumor growth, and CCS tumors have previously been shown to demonstrate increased canonical NF-{kappa}B and Wnt signalling. Here, we demonstrate evidence of non-canonical NF-{kappa}B signalling in CCS tumor keratinocytes, with increased p100 to p52 processing and RelB protein expression compared to normal skin. Utilizing complementary transcriptomics and proteomics on patient derived CCS tumor cell fractions, we identify I{kappa}B kinase alpha (IKK) as a candidate target in the non-canonical NF-{kappa}B signalling pathway. A novel, highly selective, IKK inhibitor (SU1644) used in patient derived CCS tumor spheroid cultures demonstrated that IKK inhibition reduced tumor spheroid viability. These data provide the pre-clinical rationale for the assessment of topical IKK inhibitors as a novel preventative treatment for CCS. TeaserTopical IKK inhibition emerges as a potential therapy for CYLD cutaneous syndrome by targeting non-canonical NF-{kappa}B signalling Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=125 SRC="FIGDIR/small/635629v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@829218org.highwire.dtl.DTLVardef@459f5dorg.highwire.dtl.DTLVardef@e198f6org.highwire.dtl.DTLVardef@1015685_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2025

Discovery of a distinct BAM complex in the Bacteroidetes

The BAM ({beta}-barrel assembly machinery) complex is an evolutionarily conserved, multiprotein machine that catalyses the folding and membrane insertion of newly synthesised {beta}-barrel outer membrane (OM) proteins in Gram-negative bacteria. Based on Proteobacteria, bacterial BAM is also structurally conserved, with an essential BamAD core and up to three auxiliary periplasmic lipoproteins of poorly defined function. Here we show, using structural biology, quantitative proteomics and functional assays, that the BAM complex is radically different within the Bacteroidetes, a large and important phylum widely distributed within the environment and animal microbiomes. Cryogenic electron microscopy (cryo-EM) structures of BAM complexes from the human gut symbiont Bacteroides thetaiotaomicron and the human oral pathogen Porphyromonas gingivalis show similar, seven-component complexes of [~]325 kDa in size with most of the mass in the extracellular space. In addition to canonical BamA and BamD, the complexes contain an integral OM protein named BamF that is essential and intimately associated with BamA, as well as four surface-exposed lipoproteins (SLPs) named BamG-J. Together, BamF-J form a large, extracellular dome that likely serves as an assembly cage for the {beta}-barrel-SLP complexes that are a hallmark of the Bacteroidetes. Our data suggest that BAM functionality in Bacteroidetes is substantially expanded from that in Proteobacteria and underscores the importance of studying other phyla for a more complete understanding of fundamental biological processes.

microbiology↗

bioRxiv2024

Suppression of TGF-β/SMAD signaling by an inner nuclear membrane phosphatase complex

Cytokines of the TGF-{beta} superfamily control essential cell fate decisions via receptor regulated SMAD (R-SMAD) transcription factors. Ligand-induced R-SMAD phosphorylation in the cytosol triggers their activation and nuclear accumulation. We determined how R-SMADs are inactivated by dephosphorylation in the cell nucleus to counteract signaling by TGF-{beta} superfamily ligands. We showed that R-SMAD dephosphorylation is mediated by an inner nuclear membrane associated complex containing the scaffold protein MAN1 and the CTDNEP1-NEP1R1 phosphatase. Structural prediction, domain mapping and mutagenesis revealed that MAN1 binds independently to the CTDNEP1-NEP1R1 phosphatase and R-SMADs to promote their inactivation by dephosphorylation. Disruption of this complex led to nuclear accumulation of R-SMADs and aberrant signaling, even in the absence of TGF-{beta} ligands. These findings establish CTDNEP1-NEP1R1 as the elusive R-SMAD phosphatase and reveal the mechanistic basis for TGF-{beta} signaling inactivation and how this process is disrupted by disease-associated MAN1 mutations.

cell biology↗

bioRxiv2024

Roles of PknB and CslA in cell wall morphogenesis of Streptomyces

The bacterial cell wall is essential for maintaining cellular integrity and defining the mode of growth, with different species adopting distinct strategies for cell wall synthesis and remodelling. Streptomyces are filamentous bacteria predominantly found in soil and renowned for producing specialised metabolites, including antibiotics. They grow through tip extension and branching hyphal filaments, forming a multicellular mycelium. New branches are established by forming a new growth zone on the lateral cell wall. Proteins involved in this process are organised into complexes called polarisomes, with DivIVA being the most well-characterised component. To investigate the tip growth requirements in Streptomyces albus we developed a genetic screen utilising toxic DivIVA overproduction and searched for suppressors of its lethality, reasoning that such suppressors would likely encode components functionally linked to DivIVA or the tip growth machinery. Among the identified genes was pknB, encoding a serine/threonine protein kinase implicated in the regulation of cell growth and morphogenesis. We confirmed that deletion of pknB restored the growth phenotype of S. albus following DivIVA overproduction. The phosphoproteome analysis revealed that the absence of PknB alters the phosphorylation state of CslA, a cellulose synthase-like protein. We demonstrate that a phosphoablative mutant of CslA impairs {beta}-glucan synthesis and causes hypersensitivity to lysozyme. Overproduction of CslA restored colony growth defects arising from DivIVA-induced hyperbranching, without however suppressing the hyperbranching phenotype. These findings collectively identify PknB-dependent phosphorylation of CslA as a central regulatory point in Streptomyces cell envelope construction, revealing how modulation of {beta}-glucan synthesis can mitigate the cellular consequences of DivIVA dysregulation.

microbiology↗

bioRxiv2024

Mass spectrometry-based proteomic exploration of diverse murine macrophage cellular models

Immortalised cell lines analogous to their primary cell counterparts are fundamental to research, particularly when large cell numbers are required. Here we report that immortalisation of bone marrow-derived macrophages using the J2-virus resulted in the loss of a protein of interest, MSR1, in wild-type cells by an unknown mechanism. This led us to perform an in-depth mass spectrometry-based proteomic characterisation of common murine macrophage cell lines (J774A.1, RAW264.7, and BMA3.1A7), with comparison to the immortalised bone marrow-derived macrophages (iBMDMs), as well as primary BMDMs. This revealed striking differences in protein profiles associated with macrophage polarisation, phagocytosis, pathogen recognition, and IFN signalling. J774A.1 cells were determined to be the most similar to the gold standard primary BMDM model, with BMA3.1A7 cells the least similar due to the reduction in abundance of several proteins related closely to macrophage function. This comprehensive proteomic data offers valuable insights into the selection of specific macrophage cell lines for cell signalling and inflammation research.

immunology↗