Biology subjects

Trost, M.

Publications and source records attributed to Trost, M..

44 records · Page 3Linked to original sources

bioRxiv2020

Targeted Proteomic Analysis Reveals Enrichment of Atypical Ubiquitin Chains in Contractile Tissues

Ubiquitylation is an elaborate post-translational modification involved in all biological processes. Its pleotropic effect is driven by the ability to form complex polyubiquitin chain architectures that can influence biological functions. In this study, we optimised sample preparation and chromatographic separation of Ubiquitin peptides for Absolute Quantification by Parallel Reaction Monitoring (Ub-AQUA-PRM). Using this refined Ub-AQUA-PRM assay, we were able to quantify all ubiquitin chain types in 10-minute LC-MS/MS runs. We used this method to determine the ubiquitin chain-linkage composition in murine bone marrow-derived macrophages and different mouse tissues. We could show tissue-specific differences in ubiquitin levels in murine tissues, with polyubiquitin chain types contributing a small proportion to the total pool of ubiquitin. Interestingly, we observed enrichment of atypical (K33) ubiquitin chains in heart and muscle. Our approach enabled high-throughput screening of ubiquitin chain-linkage composition in different murine tissues and highlighted a possible role for atypical ubiquitylation in contractile tissues.

biochemistry↗

bioRxiv2020

Macrophage Scavenger Receptor 1 mediates lipid-induced inflammation in non-alcoholic fatty liver disease

Obesity-associated inflammation is a key player in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). However, the exact mechanisms remain incompletely understood. Here we demonstrate that macrophage scavenger receptor 1 (MSR1, CD204) expression is associated with the occurrence of hepatic lipid-laden foamy macrophages and correlates with the degree of steatosis and steatohepatitis in a cohort of 170 NAFLD patients. Mice lacking Msr1 are protected against high fat-cholesterol diet (HFD)-induced metabolic disorder, showing fewer hepatic lipid-laden foamy macrophages, less hepatic inflammation, improved dyslipidemia and glucose tolerance, while showing a change in hepatic lipid metabolism. We show that MSR1 induces a pro-inflammatory response via the JNK signaling pathway upon triggering by saturated fatty acids. In vitro blockade of the receptor prevented the accumulation of lipids in primary macrophages which inhibited the switch towards a pro-inflammatory phenotype and the release of cytokines such as TNFa. Targeting MSR1 using monoclonal antibody therapy in an obesity-associated NAFLD mouse model and ex vivo human liver slices resulted in the prevention of foamy macrophage formation and liver inflammation. Moreover, we identified that rs41505344, a polymorphism in the upstream transcriptional region of MSR1, was associated with altered serum triglycerides and aspartate transaminase levels in a cohort of over 400,000 patients. Taken together, our data suggest a critical role for MSR1 in lipid homeostasis and a potential therapeutic target for the treatment of NAFLD. One Sentence SummaryThe immunometabolic role of MSR1 in human NAFLD.

immunology↗

bioRxiv2019

Phosphoproteomics reveals that the hVPS34 regulated SGK3 kinase specifically phosphorylates endosomal proteins including Syntaxin-7, Syntaxin-12, RFIP4 and WDR44

The serum- and glucocorticoid-regulated kinase (SGK) isoforms contribute resistance to cancer therapies targeting the PI3K pathway. SGKs are homologous to Akt and these kinases display overlapping specificity and phosphorylate several substrates at the same residues, such as TSC2 to promote tumor growth by switching on the mTORC1 pathway. The SGK3 isoform is upregulated in breast cancer cells treated with PI3K or Akt inhibitors and recruited and activated at endosomes, through its phox homology domain binding to PtdIns(3)P. We undertook genetic and pharmacological phosphoproteomic screens to uncover novel SGK3 substrates. We identified 40 potential novel SGK3 substrates, including 4 endosomal proteins STX7 (Ser126) and STX12 (Ser139), RFIP4 (Ser527) and WDR44 (Ser346) that were efficiently phosphorylated in vitro by SGK3 at the sites identified in vivo, but poorly by Akt. We demonstrate that these substrates are poorly phosphorylated by Akt as they possess an n+1 residue from the phosphorylation site that is unfavorable for Akt phosphorylation. Phos-tag analysis revealed that stimulation of HEK293 cells with IGF1 to activate SGK3, promoted phosphorylation of a significant fraction of endogenous STX7 and STX12, in a manner that was blocked by knock-out of SGK3 or treatment with 14H inhibitor. SGK3 phosphorylation of STX12 enhanced interaction with the VAMP4/VTI1A/STX6 containing SNARE complex and promoted plasma membrane localization. Our data reveal novel substrates for SGK3 and suggest a mechanism by which STX7 and STX12 SNARE complexes are regulated by SGK3. They reveal new biomarkers for monitoring SGK3 pathway activity.

biochemistry↗

bioRxiv2019

Profiling embryonic stem cell differentiation by MALDI-MS: development of a reproducible and robust sample preparation workflow

MALDI-TOF mass spectrometry (MS) is widely used to characterize and biotype bacterial samples, but a complimentary method for profiling of mammalian cells is still underdeveloped. Current approaches vary dramatically in their sample preparation methods and are not suitable for high-throughput studies. In this work, we present a universal workflow for mammalian cell MALDI-TOF MS analysis and apply it to distinguish ground-state naive and differentiating mouse embryonic stem cells (mESCs), which can be used as a model for drug discovery. We employed a systematic approach testing many parameters to evaluate how efficiently and reproducibly each method extracted unique mass features from four different human cell lines. This data enabled us to develop a unique mammalian cell MALDI-TOF workflow involving a freeze-thaw cycle, methanol fixing and CHCA matrix to generate spectra that yield maximum information and are highly reproducible. We applied our optimized workflow to distinguish naive and differentiating populations using multivariate analysis and reproducibly identifying unique features. Consequently, our MALDI-TOF MS profiling method enables identification of unique biomarkers and robust phenotyping of mESC differentiation. This method can in the future be applied to profile other cell types and expanded towards cellular MALDI-TOF MS screening assays.

biochemistry↗

bioRxiv2018

A membrane-depolarising toxin substrate of the Staphylococcus aureus Type VII protein secretion system targets eukaryotes and bacteria

The type VII protein secretion system (T7SS) is conserved across Staphylococcus aureus strains and plays important roles in virulence and interbacterial competition. To date only one T7SS substrate protein, encoded in a subset of S. aureus genomes, has been functionally characterized. Here, using an unbiased proteomic approach, we identify TspA as a further T7SS substrate. TspA is encoded distantly from the T7SS gene cluster and is found across all S. aureus strains as well as in Listeria and Enterococci. Heterologous expression of TspA from S. aureus strain RN6390 indicates its C-terminal domain is toxic when targeted to the Escherichia coli periplasm and that it depolarizes the cytoplasmic membrane. The membrane depolarizing activity is alleviated by co-production of the membrane-bound TsaI immunity protein, which is encoded adjacent to tspA on the S. aureus chromosome. Using a zebrafish hindbrain ventricle infection model, we demonstrate that the T7SS of strain RN6390 promotes bacterial replication in vivo, and deletion of tspA leads to increased bacterial clearance. The toxin domain of TspA is highly polymorphic and S. aureus strains encode multiple tsaI homologues at the tspA locus, suggestive of additional roles in intra-species competition. In agreement, we demonstrate TspA-dependent growth inhibition of RN6390 by strain COL in the zebrafish infection model that is alleviated by the presence of TsaI homologues. Significance statementStaphylococcus aureus, a human commensal organism that asymptomatically colonizes the nares, is capable of causing serious disease following breach of the mucosal barrier. S. aureus strains encode a Type VII secretion system (T7SS) that is required for virulence in mouse infection models, and some strains also secrete a nuclease toxin by this route that has antibacterial activity. Here we identify TspA, widely found in Staphylococci and other pathogenic bacteria, as a T7 substrate. We show that TspA has membrane-depolarizing activity and that S. aureus uses TspA to inhibit the growth of a bacterial competitor in vivo.

microbiology↗

bioRxiv2018

Triggering MSR1 promotes JNK-mediated inflammation in IL-4 activated macrophages

Alternatively activated M2 macrophages play an important role in maintenance of tissue homeostasis by scavenging dead cells, cell debris and lipoprotein aggregates via phagocytosis. Using proteomics, we investigated how alternative activation, driven by IL-4, modulated the phagosomal proteome to control macrophage function. Our data indicate that alternative activation enhances homeostatic functions such as proteolysis, lipolysis and nutrient transport. Intriguingly, we identified the enhanced recruitment of the TAK1/MKK7/JNK signalling complex to phagosomes of IL-4 activated macrophages. The recruitment of this signalling complex was mediated through K63-polyubiquitylation of the macrophage scavenger receptor 1 (MSR1). Triggering of MSR1 in IL-4 activated macrophages leads to enhanced JNK activation, thereby promoting a phenotypic switch from an anti-inflammatory to a pro-inflammatory state, which was abolished upon MSR1 deletion or JNK inhibition. Moreover, MSR1 K63-polyubiquitylation correlated with the activation of JNK signalling in ovarian cancer tissue from human patients, suggesting that it may be relevant for macrophage phenotypic shift in vivo. Altogether, we identified that MSR1 signals through JNK via K63-polyubiquitylation and provide evidence for the receptors involvement in macrophage polarization.

immunology↗

bioRxiv2017

The MALDI TOF E2/E3 ligase assay as an universal tool for drug discovery in the ubiquitin pathway

In many diseases, components of the ubiquitin system - such as E2/E3 ligases and deubiquitylases - are dysregulated. The ubiquitin system has therefore become an emergent target for the treatment of a number of diseases, including cancer, neurodegeneration and autoimmunity. Despite of the efforts in this field, primary screenings of compound libraries to individuate new potential therapeutic molecules targeting the ubiquitin pathway have been strongly limited by the lack of robust and fast high-throughput assays. Here we report the first label-free high-throughput screening (HTS) assay for ubiquitin E2 conjugating enzymes and E3 ligases based on Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight (MALDI TOF) mass spectrometry. The MALDI TOF E2/E3 assay allows us to test E2 conjugating enzymes and E3 ligases for their ubiquitin transfer activity, to identify E2/E3 active pairs, inhibitor potency and specificity and to screen compound libraries in vitro without synthesis of chemical or fluorescent probes. We demonstrate that the MALDI TOF E2/E3 assay is a universal tool for drug discovery screening in the ubiquitin pathway as it is suitable for working with all E3 ligase families and requires a reduced amount of reagents, compared to standard biochemical assays.

biochemistry↗

bioRxiv2017

Thermal proteome profiling of breast cancer cells reveals proteasomal activation by CDK4/6 inhibitor palbociclib

Palbociclib is a CDK4/6 inhibitor approved for metastatic estrogen receptor positive breast cancer. In addition to G1 cell cycle arrest, palbociclib treatment results in cell senescence, a phenotype that are not readily explained by CDK4/6 inhibition. In order to identify a molecular mechanism responsible for palbociclib-induced senescence, we performed thermal proteome profiling of MCF7 breast cancer cells. In addition to affecting known CDK4/6 targets, palbociclib induces a thermal stabilization of the 20S proteasome, despite not directly binding to it. We further show that palbociclib treatment increases proteasome activity independently of the ubiquitin pathway. This leads to cellular senescence which can be counteracted by proteasome inhibitors. Palbociclib-induced proteasome activation and senescence is mediated by reduced proteasomal association of ECM29. Loss of ECM29 activates the proteasome, blocks cell proliferation and induces senescence. Finally, we find that ECM29 mRNA levels are predictive of relapse-free survival in breast cancer patients treated with endocrine therapy. In conclusion, thermal proteome profiling identifies the proteasome and ECM29 protein as mediators of palbociclib activity in breast cancer cells.

cancer biology↗