Project description:The aging process is characterized by cellular functional decline and increased susceptibility to infections. Understanding the association between virus infection and aging is crucial for developing effective strategies against viral infections in older individuals. Kaposi's sarcoma-associated herpesvirus (KSHV) infection increases the risk of Kaposi's sarcoma, a vascular cancer prevalent among the elderly without HIV infection. However, the relationship between KSHV pathogenesis and cellular senescence remains unknown. Here, we demonstrate that KSHV infectivity is significantly increased in senescent human endothelial cells due to enhanced binding of virions to cell surface. Proteomic analysis identified caveolin-1 and CD109 that promote KSHV infection and were significantly upregulated in senescent cells. In particular, CD109 is expressed on cell surface and directly interacts with KSHV virions to enhance KSHV infection. Knockout of CD109 abolished while overexpression of CD109 promote KSHV binding to cell surface, and infectivity. These results identify CD109 as a novel KSHV entry receptor that enhances KSHV infection in senescent cells, which might in part explain the higher sensitivity of elder subjects to KSHV infection and Kaposi's sarcoma.
Project description:Diverse classes of regulatory small (s)RNAs operate via ARGONAUTE-family proteins within RNA-induced-silencing-complexes (RISCs). Based the conserved biochemical properties intrinsic to all ARGONAUTEs, we have developed a universal, 15-min benchtop extraction procedure allowing simultaneous purification of all classes of RISC-associated sRNAs, without prior knowledge of the sample’s -intrinsic ARGONAUTE repertoires. Optimized as a user-friendly kit, the method –coined “TraPR” for Trans-kingdom, rapid, affordable Purification of RISCs– operates irrespectively of the organism, tissue, cell type or bio-fluid of interest, and scales to minute amounts of input material. The method is highly suited for direct sRNA deep-sequencing, with TraPR-generated libraries being qualitatively at least on-par with those obtained via gold-standard procedures that require immunoprecipitations and/or lengthy polyacrylamide gel excisions. TraPR considerably improves the quality and consistency of sRNA sample preparation including from notoriously difficult-to-handle tissues/bio-fluids such as starchy storage roots and mammalian plasma, and regardless of RNA contaminants or samples’ RNA-degradation status.
Project description:Amino acids (AA) are essential molecules for life because they are precursors for the synthesis of proteins and other organic compounds. Their metabolism in plants involves more than one cellular compartment, among which the chloroplast plays a key role. From the 20 essential AA, ten (Arg, Lys, Thr, Leu, Ile, Val, Trp, Phe, Tyr and His) are only synthesized in the plastids and seven (Asp, Cys, Gln, Glu, Gly, Ser and Met) can be synthesized in both chloroplast and other parts of the cell. Despite knowing the roles of AA in plant physiology, little has been assessed in plants with albino phenotypes, lacking chloroplasts. To assess the effect of chloroplast biogenesis disturbances on AA metabolism in albino and variegated somaclonal variations of Agave angustifolia Haw., a comparative proteomic based TMT- synchronous precursor selection (SPS)-MS3 strategy was used to determine the status of AA biosynthetic pathways. Besides the 20 essential AA was quantified to underpin proteomics finding. A total of 2,442 different proteins were identified in the proteome of the somaclonal variants, from which 82 correspond to enzymes participating in AA biosynthesis. From these 82, 32 proteins were differentially accumulated. On the other hand, the concentration of 16 and 12 of the AA synthesized by the chloroplast were over-accumulated in the variegated and albino somaclonal variant, respectively. In plantlets devoid of functional chloroplasts, there is a surprising increase of AA and key enzymes of the AA biosyntethic pathways, compared to green plantlets. This could suggest que la activation of AA biosynthesis is key to sustaining albino and variegated plantlets survival that lack chloroplasts.
Project description:we applied RNA-seq to detect novel expressed transcripts in 12 tissues of giant pandas, using a transcriptome reconstruction strategy combining reference-based and de novo methods. Then we used mass spectrometry method to identify proteomes of five selected tissues, aiming at validating these novel full-length genes we identified.
Project description:LC-MS/MS analysis of O-GlcNAcylated ChREBP proteins, which were overexpressed in HEK293T cells and purified via affinity purification using Ni-NTA agarose
Project description:Magnaporthe oryzae snodprot1 homologous protein (MSP1) has been shown to act as a pathogen-associated molecular pattern (PAMPs) and trigger PAMP-triggered immunity (PTI) response involving programmed cell death and expression of various defense-related genes in rice. The involvement of several post-translational modifications (PTMs) in the regulation of plant immune response, especially PTI, during pathogen infection is well established, however, the information on the regulatory roles of these PTMs in response to MSP1-induced signaling in rice is currently elusive. Here, we report the phosphoproteome, ubiquitinome, and acetylproteome to investigate the MSP1-induced PTMs alterations in MSP1 overexpressed rice. Our analysis identified a total of 4,666 PTM modified sites in rice leaves including 4,292 phosphosites, 189 ubiquitin sites, and 185 acetylation sites. Among these, PTM status of 437 phosphorylated, 53 ubiquitinated, and 68 acetylated peptides were significantly changed by MSP1. Functional annotation of MSP1 modulated peptides by MapMan analysis revealed that these were majorly associated with cellular immune responses such as signaling, transcription factors, DNA and RNA regulation, and protein metabolism, among others. Taken together, this study uncovers the MSP1-induced PTMs changes in rice proteins and identified several novel components of rice-MSP1 interaction.
Project description:Homozygosity for the Z allele of α1-antitrypsin (ZAAT) predisposes affected individuals to developing liver disease as the serpin misfolds and forms insoluble polymers that accumulate in the endoplasmic reticulum (ER) of hepatocytes, resulting in gain-of-function hepatotoxicity. This prevents secretion of ZAAT leading to serum insufficiency. A zebrafish model expressing human ZAAT in the liver shows no signs of hepatic accumulation despite displaying serum insufficiency, suggesting defect in ZAAT secretion occurs independently of its tendency to accumulate in hepatocytes. In this study, proteomic and transcriptomic analysis of the ZAAT-expressing zebrafish liver provided strong evidence of suppressed Srebp2-mediated cholesterol biosynthesis. qPCR confirms this observation in the human liver cell line stably expressing ZAAT. We proposed that the engagement of misfolded ZAAT by the ER-associated degradation (ERAD) system inhibits the turnover of Srebp2-repressing elements therefore hindering the activation of Srebp2. Protein quality control factors was manipulated to dissect the intracellular processing of ZAAT further mechanistically. Ablation of erlec1 resulted in a further suppression in the cholesterol biosynthesis pathway, confirming a role of this ER lectin in targeting misfolded ZAAT to ERAD. Deletion of the two ER mannosidase I homologs in zebrafish enhanced ZAAT secretion without inducing hepatic accumulation. Together, the present study provides novel insights into hepatic ZAAT processing and suggest potential therapeutic targets to improve ZAAT secretion and alleviate serum insufficiency in this form of α1-antitrypsin disease.
Project description:Non-ribosomal peptide synthetases are important enzymes for the assembly of complex peptide natural products. Within these multi-modular assembly lines, condensation domains perform the central function of chain assembly, typically by forming a peptide bond between two peptidyl carrier protein (PCP)-bound substrates. In this work, we report the first structural snapshots of a condensation domain in complex with an aminoacyl-PCP acceptor substrate. These structures allow the identification of a mechanism that controls access of acceptor substrates to the active site in condensation domains. The structures of this previously uncharacterized complex also allow us to demonstrate that condensation domain active sites do not contain a distinct pocket to select the side chain of the acceptor substrate during peptide assembly but that residues within the active site motif can instead serve to tune the selectivity of these central biosynthetic domains.
Project description:Trypanosoma cruzi is a protozoan parasite that causes Chagas’ disease in humans and throughout its life cycle faces different environment changes. Protein methylation is an important post-translational modification by which cells respond and adapt to the environment. To understand the importance of protein methylation in T.cruzi biology, we applied mass spectrometry-based proteomics and report the first proteomic analysis of both arginine and lysine methylproteome in T. cruzi.
Project description:The biological process termed Epithelial-to-Mesenchymal Transition (EMT) plays a central role in cancer cell invasion, metastasis, self-renewal and resistance to therapy(1,2). Here, using western blot technique, we show that H3K9me2 decreases when MDA-MB-468 breast cancer cells undergo EMT upon EGF. We validate this decrease by performing high-resolution MS/MS spectrum. Interestingly, we find that H3K9me2 is associated with mesenchymal genes regulation. 1. Nieto, M. A., Huang, R. Y., Jackson, R. A. & Thiery, J. P. EMT: 2016. Cell 166, 21–45 (2016). 2. Puisieux, A. & Brabletz, T. & Caramel, J. Oncogenic roles of EMT-inducing transcription factors. Nat. Cell Biol. 16, 488–494 (2014).