PacL1-ALFA tag based proximity proteomics in Mycobacterium tuberculosis
Ontology highlight
ABSTRACT: Proteomic analysis of peptides in close association to PacL1 under replete metal conditions (Sauton’s media), in Mycobacterium Tuberculosis (Erdman strain).
Project description:The protein Yin-Yang 1 (YY1) is a ubiquitous multifunctional transcription factor. Interestingly, there are several cellular functions controlled by YY1 that could play a role in Leishmania pathogenesis. Leishmaniasis is a human disease caused by protozoan parasites of the genus Leishmania. This study examined the potential role of macrophage YY1 in promoting Leishmania intracellular survival. Knockdown of YY1 resulted in attenuated survival of Leishmania in infected macrophages, suggesting a role of YY1 in Leishmania persistence. Biochemical fractionation studies revealed Leishmania infection caused redistribution of YY1 to the cytoplasm from the nucleus where it is primarily located. Inhibition of nuclear transport by leptomycin B attenuates infection-mediated YY1 redistribution and reduces Leishmania survival. This suggests that Leishmania induces the translocation of YY1 from the nucleus to the cytoplasm of infected cells, where it may regulate host molecules to favour parasite survival. A label-free quantitative whole proteome approach showed that the expression of a large number of macrophage proteins was dependent on the YY1 level. Interestingly, several of these proteins were modulated in Leishmania-infected cells, revealing YY1-dependent host response and suggesting their potential role in Leishmania pathogenesis. Together, these findings identify YY1 as a novel and essential virulence factor by proxy that promotes Leishmania survival.
Project description:In this study we demonstrate the effect of oncolytic adenoviruses armed with CXCL9, CXCL10 and IL-15 to infect cancer cells, secrete the encoded protein and drive gradient-dependent T-cell attraction. Our in vivo validation demonstrated an increased intratumoral CD4+ and CD8+ T-cell infiltration following treatment with armed viruses compared to control groups. Finally, RCC cells were screened for tumor-specific peptides to validate their immunogenicity in a (personalized) oncolytic cancer vaccine approach, previously referred to as PeptiCRAd.
Project description:Bats have adapted to pathogens through diverse mechanisms, including increased resistance - rapid pathogen elimination, and tolerance - limiting tissue damage following infection. In the Egyptian fruit bat (an important model in comparative immunology) several mechanisms conferring disease tolerance were discovered, but mechanisms underpinning resistance remain poorly understood. Previous studies on other species suggested that elevated basal expression of innate immune genes may lead to increased resistance to infection. Here, we test whether such transcriptional patterns occur in Egyptian fruit bat tissues through single-cell and spatial transcriptomics of gut, lung and blood cells, comparing gene expression between bat, mouse and human. Despite numerous recent loss and expansion events of interferons in the bat genome, interferon expression and induction are remarkably similar to that of mouse. In contrast, central complement system genes are highly and uniquely expressed in key regions in bat lung and gut epithelium, unlike in human and mouse. These genes also evolve rapidly in their coding sequence across the bat lineage. Finally, the bat complement system displays strong hemolytic and inhibitory activities. Together, these results indicate a distinctive transcriptional divergence of the complement system, which may be linked to bat resistance, and highlight the intricate evolutionary landscape of bat immunity.
Project description:This part of the data submission of PXD046505. LD3 knock out cells were generated in HEK293T and HMC3 cells. Proteomics was done to test if they have any significant changes in the abundance of proteins that metabolize sugar lipids like gangliosides.
Project description:LD3 knock out cells were generated in HEK293T and HMC3 cells. Proteomics was done to test if they have any significant changes in the abundance of proteins that metabolize sugar lipids like gangliosides.
Project description:µ-proteins (≤ 70 amino acids) have important and often essential roles in all kingdoms of life, including cell motility, regulation of membrane transport and as transcription factors. In the halophilic archaeon and model system Haloferax volcanii a significant number of µ-proteins were predicted to be zinc finger proteins. Here we used mass spectrometry-based proteomics to systematically investigate the impact of single gene deletions of 19, previously uncharacterized, zinc finger µ-proteins on H. volcanii grown in glycerol media. We employed a state-of-the-art dia-PASEF acquisition strategy, detecting over 3400 proteins across the 19 deletion strains and the wild-type. The comprehensive proteome coverage enabled a systematic analysis of proteome remodeling. We found that in 11 out of the 19 mutants the proteome remodeling involved proteins annotated to play a role in cell motility, matching swarming and cell growth phenotypes we observed for these strains. Taken together, our data provide the most comprehensive proteome coverage of H. volcanii to date, and the effect of 19 different zinc-finger µ-proteins deletion strains on the proteome of this organism. The combined data provide a valuable resource for future research in the field.
Project description:Oral transmission of T. cruzi is probably the most frequent mechanism among animals in the wild. In this context, there is a high prevalence of human infections in regions where triatomine infection with T. cruzi is low. This led to the hypothesis that the consumption of raw or undercooked meat from animals infected with T. cruzi could be responsible for the transmission of the infection. Therefore, the general objective of this study was to demonstrate the role of meat consumption from infected animals in the oral transmission of T. cruzi infection. Groups of five female mice Balb/c were fed with muscles obtained from mice in the acute phase of infection by the clone H510 C8C3hvir of T. cruzi, and the infection of the fed mice was monitored by a parasitemia curve. Similarly, we assessed the infective capacity of T. cruzi trypomastigotes and amastigotes by infecting groups of five mice Balb/c females, which were infected orally using a nasogastric probe, and the infection was monitored by parasitemia curve. Finally, different trypomastigote and amastigote inoculums were used to determine their infective capacity. Adhesion assays of T. cruzi proteins to AGS stomach cells were performed and the adhered proteins were detected by western blotting using monoclonal or polyclonal antibodies. In all cases, 60–100% of animals were fed meat from mice infected in the acute phase or infected by means of a nasogastric probe with trypomastigotes or amastigotes. These animals developed high parasitemia, and 80% died around day 40 post-infection. The adhesion tests showed that cruzipain is a molecule of trypomastigotes and amastigotes that binds to AGS cells. LC-MS/MS also confirmed that transialidase may be involved in TCT attachment of TCT or invasion of stomach cells. It was concluded that the consumption of meat from infected animals in the acute phase of T. cruzi infection allows transmission of the infection. Similarly, trypomastigotes and amastigotes were able to infect mice when administered orally, while cruzipain was a relevant molecule in this infective process.
Project description:A microfluidics technology was implemented to the immunoaffinity purification process of MHC peptides in Ligandomics/Immunopeptidomics. The thus purified HLA peptides were analysed by LCMS with the nanoElute LC and TimsTOF Pro Mass Spectrometer from Bruker. The aim of the microfluidics implementation was to improve the sensitivity and robustness while also reducing antibody and other material requirements in the immunoaffinity purification protocol.
Project description:Bacteroidia ribosomes are “blind” to SD sequences, even though they contain the conserved anti-SD (ASD) element of 16S rRNA. A structure of the Flavobacterium johnsoniae ribosome shows that the 3’ tail of 16S rRNA is sequestered in a pocket formed by bS21, bS18, and bS6 on the 30S platform, explaining the basis of ASD inhibition. Interestingly, there is one gene of F. johnsoniae with a strong SD sequence—rpsU, which encodes bS21. F. johnsoniae ribosomes lacking bS21 exhibit a liberated ASD and translate rpsU at a higher rate, which sets up an autoregulatory cycle. Here, we targeted the ASD of each 16S rRNA gene in F. johnsoniae, ablating the core element (CCUCC to GAAGC). Consecutive replacement of each 16S gene with this quadruple-substituted (QS) allele had little effect on cell growth until the last gene was changed. The final strain, containing QS ribosomes only, grows very slowly. This growth defect can be largely rescued by replacing the native translation initiation region (TIR) with the SD-less TIR of tuf. Purified QS ribosomes are unable to translate native rpsU mRNA but are active on other mRNAs. We also selected suppressors of the ASD-ablated strain, many of which carried a single mutation in the SD of rpsU. Interestingly, wild-type ribosomes fail to initiate on these variant rpsU mRNAs, demonstrating that an extended SD-ASD duplex is normally needed for initiation on this message. These findings indicate that the main purpose of the ASD in F. johnsoniae is to facilitate translation of one gene, rpsU.
Project description:Protein ubiquitination controls diverse processes within eukaryotic cells, including protein degradation, and is often dysregulated in diseases1. Moreover, protein degraders that redirect ubiquitination activities toward disease targets are an emerging and promising therapeutic class2. Over 600 E3 ubiquitin ligases are expressed in humans3,4, but their substrates remain largely elusive due to a lack of robust methods to identify E3 ligase substrates. Here we report the development of E-STUB (E3 substrate tagging by ubiquitin biotinylation), a ubiquitin-specific proximity labeling method that biotinylates ubiquitinated substrates in proximity to an E3 ligase of interest. E-STUB accurately identifies the direct ubiquitinated targets of protein degraders, including collateral targets and ubiquitylation events that do not exhibit a degradative outcome. It also detects known substrates of E3 ligase cereblon (CRBN) and von Hippel-Lindau (VHL) with high precision. With the ability to elucidate proximal ubiquitination events, E-STUB may facilitate the development of proximity-inducing drugs and act as a generalizable method for E3 substrate mapping.