Project description:A quantitative label-free secretome analysis protocol using a click chemistry-based approach for the enrichment of secreted glycoproteins was adapted for and applied to a T cell model. There, Jurkat cells were activated via PMA/ionomycin and the dynamic modulation of the T cell secretome was investigated and compared to the dynamic modulation of the T cell proteome of the same model.
Project description:Clear cell renal cell carcinoma (ccRCC) represents the most common form of kidney cancer and is typified by biallelic inactivation of the von Hippel-Lindau (VHL) tumour suppressor gene. Here, we undertake genome-wide CRISPR/Cas9 screening to reveal synthetic lethal interactors of VHL, and uncover that loss of Core Binding Factor β (CBF-β) causes cell death in VHL-null ccRCC cell lines and impairs tumour establishment and growth in vivo. This synthetic relationship is independent of the elevated activity of hypoxia inducible factors (HIFs) in VHL-null cells, but does involve the RUNX transcription factors that are known binding partners of CBF-β. Mechanistically, CBF-β loss leads to upregulation of type I interferon signalling, and we uncover a direct inhibitory role for CBF-β at the STING locus controlling Interferon Stimulated Gene expression. Targeting CBF-β in kidney cancer both selectively induces tumour cell lethality and promotes activation of type I interferon signalling.
Project description:In recent years, arboviral infections have surged dramatically due to the geographic expansion of Aedes and Culex mosquitoes, their main vector mosquitoes. Despite significant efforts to uncover arbovirus–host interactions and viral protein effector functions in mammals, systematic studies aiming to characterize virus–vector interactions in arthropods are largely missing, and the functions and cellular targets of many arboviral proteins in mosquitoes remain elusive. Here, we applied a multi-omic approach to systematically evaluate the ability of arboviral capsids to interact with the Ae. aegypti proteome. This extensive multi-modal atlas across 12 pathogenic arboviral species spanning three viral genera revealed shared and distinct host factor specificities, uncovering species-, genus- and vector preference-specific patterns of host usage in mosquitoes. Functional phenotypic screening of 110 newly discovered host proteins across three prototypic arboviruses (La Crosse virus, dengue virus and West Nile virus) identified several novel host dependency factors, including a new role for the chromatin-remodeling Brahma complex in orthoflavivirus replication. Using a combination of biochemical and sequencing approaches, we characterized the cellular determinants of these interactions and profiled their functional consequences on the chromatin landscape. Altogether, this study provides a multi-layered repository to categorize and characterize arboviral capsid effector functions in invertebrates, providing important cues on novel mechanisms of transcriptional regulation via capsid-mediated modulation of chromatin accessibility in insects.
Project description:A differential label-fee proteomics approach was performed using 27 biopsies from patients with HCV-associated hepatic fibrosis. For statistical analysis the patients were grouped into a low and a high fibrosis group. The low fibrosis group contained 13 patients of fibrosis stages 0, 1 and 2, whereas the high fibrosis group contained 14 patients of fibrosis stages 3 and 4 (fibrosis stages according to Batts-Ludwig classification).
Project description:Hepatitis B virus (HBV) uses e antigen (HBe) which is dispensable for virus infectivity to modulate host immune responses and to achieve viral persistence in human hepatocytes. The HBe precursor (p25) is directed to the endoplasmic reticulum (ER), where cleavage of the signal peptide (sp) gives rise to the first processing product, p22. The p22 can be retro-translocated back to the cytosol or it can enter the secretory pathway and undergo a second cleavage event resulting in secreted p17 (HBe). Here, we report that translocation of p25 precursor is promoted by translocon-associated protein complex (TRAP). We found that p25 is not completely translocated into the ER since a fraction of p25 is phosphorylated and remains present in the cytoplasm and the nucleus. Within the sp sequence of p25 we identified three cysteine residues which control the efficiency of sp cleavage and correct subcellular distribution of the precore pool. Highlights • A fraction of the HBV e antigen precursor p25 is not translocated into the ER and is phosphorylated in cytosol. • Cysteine residues in the signal peptide sequence regulate the p25 N-terminal processing and resulting p22 intracellular level. • Mutations of all three Cys residues within the signal peptide lead to a mislocalization of intracellular precore protein. • P25 translocation requires assistance of TRAP complex for an efficient mature HBe secretion. • The depletion of individual TRAP subunits results in a change of precore subcellular distribution.
Project description:The lysine acetylation of proteins is a major post-translational modification that plays an important regulatory role in almost every aspect of cells, both eukaryotes and prokaryotes. Vibrio parahaemolyticus, a model marine bacterium, is a worldwide cause of bacterial seafood-borne illness. Here, we conducted the first lysine acetylome in the bacterium through combination of highly sensitive immune-affinity purification and high-resolution LC-MS/MS. Overall, we identified 1413 lysine acetylation sites in 656 proteins, which account for 13.6% of the total proteins in the cells and is the highest ratio of acetyl proteins that has so far been identified in bacteria. The bioinformatics analysis of the acetylome showed that the acetylated proteins are involved in a wide range of cellular functions and exhibit diverse subcellular localizations. More specifically, proteins related to protein biosynthesis and carbon metabolism are the preferential targets of lysine acetylation. Moreover, two types of acetylation motifs, a lysine or arginine at the +4/+5 position and a tyrosine, histidine, or phenylalanine at the +1/+2 position, were revealed from the analysis of the acetylome. Additionally, the protein interaction network analysis demonstrates that a wide range of interactions are modulated by protein acetylation. This study provides a significant beginning for the in-depth exploration of the physiological role of lysine acetylation in V. parahaemolyticus.
Project description:Telomere end-protection by the shelterin complex prevents DNA damage signalling and promiscuous repair at chromosome ends. Evidence suggests that the 3’ single-stranded telomere end can assemble into a lasso-like t-loop configuration, which has been proposed to safeguard chromosome ends from being recognized as DNA double strand breaks. Mechanisms must also exist to transiently disassemble t-loops to allow faithful telomere replication and to permit telomerase access to the 3’-end to solve the end replication problem. However, the regulation and physiological importance of t-loops in end-protection remains uncertain. Here, we identify a CDK phosphorylation site in the shelterin subunit, TRF2 (Ser365), whose dephosphorylation in S-phase by the PP6C/R3 phosphatase provides a narrow window during which the helicase RTEL1 is able to transiently access and unwind t-loops to facilitate telomere replication. Re-phosphorylation of TRF2 on Ser365 outside of S-phase is required to release RTEL1 from telomeres, which not only protects t-loops from promiscuous unwinding and inappropriate ATM activation, but also counteracts replication conflicts at DNA secondary structures arising within telomeres and across the genome. Hence, a phospho-switch in TRF2 coordinates assembly and disassembly of t-loops during the cell cycle, which protects telomeres from replication stress and an unscheduled DNA damage response.
Project description:Viral dsRNA binds to Retinoic acid Inducible Gene I (RIG-I) Like Receptors (RLRs), promoting the production of Interferon (IFN). Interferon then stimulates the innate and adaptive immune system in an autocrine and paracrine manner. Outside of conical pathways, regulators of the interferon (IFN) activation/response system are poorly characterized. In this study, we used a discovery-biased approach to identify Kinases that are part of the interferon system. Differential changes in phosphorylation sites, in the context of dsRNA RIG-I stimulation, were identified with unbiased mass-spec biased phospho-proteomics. We then computationally identified several Kinases upregulated after RIG-I stimulation from phospho-proteomics data. A Chemoproteomics screen was then used to characterize the altered interferon response in the presence of Kinases inhibitors for the upregulated kinases. Combining unbiased phosphoproteomics with a chemoproteomics screen, we identified several potentially novel regulators of the Interferon system whose inhibition blocked the production of Interferon Stimulated Genes.
Project description:The mitochondrial deubiquitylase USP30 negatively regulates the selective autophagy of damaged mitochondria. It has been proposed as an actionable target to alleviate the loss of function of the mitophagy pathway governed by the Parkinson’s Disease associated genes PINK1 and PRKN. We present the characterisation of a N-cyano pyrrolidine derived compound, FT3967385, with high selectivity for USP30. The compound is well tolerated with no loss of total mitochondrial mass. We demonstrate that ubiquitylation of TOM20, a component of the outer mitochondrial membrane import machinery that directly interacts with USP30, represents a robust biomarker for both USP30 loss and inhibition. We have conducted proteomics analyses on a SHSY5Y neuroblastoma cell line model to directly compare the effects of genetic loss of USP30 with selective inhibition in an unbiased fashion. We have thereby identified a subset of ubiquitylation events consequent to mitochondrial depolarisation, that are USP30 sensitive. Within responsive elements of the ubiquitylome, several components of the outer mitochondrial membrane transport (TOM) complex are most prominent. Thus, our data support a model whereby USP30 can regulate the availability of ubiquitin at the specific site of mitochondrial PINK1 accumulation following membrane depolarisation. In this model, USP30 deubiquitylation of TOM complex components dampens the trigger that unleashes the Parkin-dependent amplification of mitochondrial ubiquitylation leading to mitophagy. Accordingly PINK1 generation of phospho-Ser65 Ubiquitin proceeds more rapidly and to a greater extent in cells either lacking USP30 or subject to USP30 inhibition.
Project description:In clinical settings, the intensive use of antibiotics, particularly in intensive care settings, leads to a significant increase in the number of bacterial species that are resistant to treatment. In this context, biofilm is a crucial virulence factor that enable bacteria to persist within the host, often resulting in the need for extensive antibiotic treatment. Staphylococcus epidermidis, a notable nosocomial pathogen, poses a risk to vulnerable patients due to its ability to form biofilms on indwelling medical devices and its high resistance to antibiotic therapy. For this purpose, investigating alternative strategies that target the virulence of pathogens could offer a promising alternative strategy. In this study, we analyzed innovative polymeric materials, such as polysaccharide-based nanohydrogels, for their potential application contrasting S. epidermidis monospecies biofilm on the surfaces of materials most employed in medical devices. These nanohydrogels were found to be effective in eradicating the biofilm matrix and preventing bacterial adhesion. Additionally, the treatment with hyaluronan-based nanohydrogels altered the surface protein profile of S. epidermidis, leading to the disappearance of AtlE, the primary autolysin involved in biofilm formation, suggesting a potential mechanism of action for these nanogels.