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SARS_CoV-2 substrate profiling in interferon-treated BEAS-2B cell proteomes

ABSTRACT: To profile the human substrate degradome of the SARS-CoV-2 main viral protease and to investigate whether 3CLpro cleavage of cellular substrates dampens host antiviral responses induced by type I interferons, we treated BEAS-2B cells with interferon-alpha (N = 3), interferon-beta (N = 3), or vehicle (N = 3), retrieve and exposed native proteome extracts from human embryonic kidney to 3CLpro cleavage. Then, N Terminal Amine Labeling of Substates (TAILS) was used to identify the substrates. Here, heavy [+34] isotope dimethyl labeling is performed at the whole protein level in the 3CLpro-exposed samples, and the P1' side of substrates substrates was revealed by comparing to the samples exposed to inactive mutant labeled with ligh [+28 Da] dimethyl.

INSTRUMENT(S): maXis

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

SUBMITTER: Yoan Machado

LAB HEAD: Christopher M. Overall

PROVIDER: PXD026815 | Pride | 2021-11-03

REPOSITORIES: Pride

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SARS_CoV-2 substrate profiling in HEK-293 cell proteomes

Project description:To profile the human substrate degradome of the SARS-CoV-2 main viral protease by exposingnative proteome extracts from human embryonic kidney (HEK-293) cells (N = 3) to 3CLpro cleavage. Then, N Terminal Amine Labeling of Substates (TAILS) was used to identify the substrates. Here, heavy [+34] isotope dimethyl labeling is performed at the whole protein level in the 3CLpro-exposed samples, and the P1' side of substrates substrates was revealed by comparing to the samples exposed to inactive mutant labeled with ligh [+28 Da] dimethyl.

2021-11-03 | PXD026797 | Pride

Mouse mitochondrial CLPP substrate identification

Project description:Identification ofputative ClpXP substrates by combination of proteome analysis and TAILS N termini profiling from wt and ClpP-deficient mouse heart mitochondria, combined with substrate trapping using catalytically inactive ClpP expressed in MEF cell culture. This dataset provides the preTAILS proteomedata

2020-06-01 | PXD018194 | Pride

Stunted Procoagulant Remodelling in Montreal Platelet Syndrome Kindred with p.V1316M mutation

Project description:The Montreal Platelet Syndrome Kindred (MPS) with p.V1316M mutation (2B-VWDMPS) has been associated with chronic macrothrombocytopenia, spontaneous platelet clumping, and mucocutaneous and other bleeding which can be largely prevented by von Willebrand factor concentrate infusion. However, supplemental platelet transfusion has been required on occasions, particularly for severe gastro-intestinal bleeds. This raises the question of whether a previously uncharacterized platelet dysfunction contributes to bleeding diathesis in 2B-VWDMPS patients. We studied the 2 members of the MPS kindred with the most severe bleeding phenotype and addressed this question by coupling quantitative platelet shotgun proteomics and validating biochemical assays, with the systematic analysis of platelet procoagulant membrane dynamics (PMD). Previously, we showed that membrane ballooning, a major procoagulant response of the human platelet after exposure to collagen, is driven by the influx of Na+ and Cl-, followed by the entry of osmotically obliged water. Here, we report in 2B-VWDMPS platelets, loss of the transmembrane chloride channel CLIC1, and reduced chloride ion influx after collagen stimulation. This was associated with diminished membrane ballooning and a distinct phenotypic composition of platelets over fibrillar collagen. Also, 2B-VWDMPS platelets showed marked loss of regulatory, cytoskeletal, and contractile proteins that may account for structure disorganization, giant platelet formation and further reduce the haemostatic response.

2023-08-22 | PXD035713 | Pride

N-terminomics/TAILS profiling of proteases and their substrates in ulcerative colitis

Project description:Dysregulated protease activity is often implicated in the initiation of inflammation and immune cell recruitment in gastrointestinal inflammatory diseases. Using N-terminomics/TAILS (terminal amine isotopic labeling of substrates), we compared proteases, along with their substrates and inhibitors, between colonic mucosal biopsies of healthy patients and those with ulcerative colitis (UC). Among the 1,642 N-termini enriched using TAILS, increased endogenous processing of proteins was identified in UC compared to healthy patients. Changes in the reactome pathways for proteins associated with metabolism, adherens junction proteins (E-cadherin, liver-intestinal cadherin, catenin alpha-1 and catenin delta-1) and neutrophil degranulation were identified between the two groups. Increased neutrophil infiltration and distinct proteases observed in ulcerative colitis may result in extensive break down, altered processing or increased remodeling of adherens junctions and other cellular functions. Analysis of the proteolytic preferred cleavage sites indicated that the majority of proteolytic activity and processing comes from host proteases, but that key microbial proteases may also play a role in maintaining homeostasis. Thus, the identification of distinct proteases and processing of their substrates improves the understanding of dysregulated proteolysis in normal intestinal physiology and ulcerative colitis.

2019-09-08 | PXD014479 | Pride

Translational microarray analysis of liver samples treated with PredTox hepatotoxic compound FP014SC

Project description:Liver samples were lysed (15 mM Tris-HCl (pH 8.0), 300 mM NaCl and 15 mM MgCl2, plus inhibitors (1 mg/ml heparin 100 Âµg/ml cycloheximide and 80 U RNAsin)), added to a 10-50% sucrose gradient and ultracentrifuged at 182,000x g for 2 hours. The gradient was aliquotted into 16 1ml fractions and added to Tri reagent (Sigma). RNA was extracted as per the manufacturer's instructions and then sub-pooled into fractions corresponding to monosomes, light polysomes, medium polysomes and heavy polysomes, according to ribosomal density (more ribosomal occupancy = higher translational activity = heavier, and therefore, located in the more dense fractions). Microarray analysis was performed by hybridising control (vehicle treated) monosomes against test (high-dose treated) monosomes on one microarray, control light polysomes against test light polysomes on a second microarray, and so forth for each sub-pool of fractions. Following microarray analysis there were a maximum of four values for each mRNA, corresponding to the proportional representation of that mRNA within each sub-pool of fractions. By calculating the change in values, i.e. degree of slope, across the monosomal region, the light polysomal region, the medium polysomal region and the dense polysomal region it was possible to determine any translational change in activity. In addition, the overall transcriptional change could be analysed for each mRNA. Dual colour microarrays performed on n = 3 samples, implementing a dye swap design. Control samples were treated with vehicle only. The treated samples had 1120mg/kg of the compound (Tetraethyl[(3-hydroxy-2-pyridyl)amino]-methanediphosphonate) for 15 days.

2012-06-20 | E-GEOD-38807 | biostudies-arrayexpress

Arabidopsis chloroplast N-terminome

Project description:Protein N-termini are prone to post translational modification and are major determinants of protein stability in bacteria, eukaryotes, and perhaps also in chloroplasts. Most chloroplast proteins undergo N-terminal maturation, but this is poorly understood due to insufficient experimental information and the N-termini of mature chloroplast proteins cannot be accurately predicted. This motivated an extensive characterization of chloroplast protein N-termini using terminal amine isotopic labeling of substrates (TAILS). Many nuclear-encoded plastid proteins accumulated with two or three different N-termini; we evaluated the significance of these different proteoforms. Ala, Val, Thr (often in N-α acetylated form) and Ser were the most frequently observed N-terminal residues, even after normalization for their frequency in the plastid proteome, while other residues were absent or highly under-represented. Plastid-encoded proteins showed a similar distribution of N-terminal residues, but with a higher frequency of Met. Infrequent residues such as Ile, Arg, Cys, Pro, Asp and Glu were observed for several abundant proteins likely reflecting functional regulation through their N-termini. In contrast, the thylakoid lumenal proteome showed a wide diversity of N-terminal residues, including those typically associated with instability (Asp, Glu, Leu, Phe). We propose that after cleavage of the chloroplast transit peptide by stromal processing peptidase, additional processing by unidentified peptidases occurs to avoid unstable or otherwise unfavorable N-terminal residues. The possibility of a chloroplast N-end rule is discussed. This work provides a baseline for understanding N-terminal processing and maturation of chloroplast proteins.

2015-10-22 | PXD002476 | Pride

Proteomic profiling of the proteolytic events in the secretome of the transformed phenotype of melanocyte-derived cells using Terminal Amine Isotopic Labeling of Substrates

Project description:The comprehensive profiling of the repertoire of secreted proteins from cancer cells and/or tissue samples provides information on the signaling events that take place during oncogenesis as well as on the cross-talk between normal and tumoral cells. Moreover, the analysis of post translational modifications in secreted proteins may unravel biological circuits regulated by irreversible modifications such as, for example, proteolytic processing. In this context, we used Terminal Amine Isotopic Labeling of Substrates (TAILS) to perform a system-wide investigation on the N-terminome of the secretomes derived from a paired set of mouse melanocyte cell lines: Melan-a (a normal melanocyte) and Tm1 (its transformed phenotype). TAILS analysis allowed the profiling of co-translational modifications such as acetylated N-termini as well as proteolytic events in both secretomes. Although no significant difference has been found in the proportion of acetylated natural N-termini in both cell line secretomes, when evaluating amino acid identities at the scissile bond in internal peptides it was possible to detect significant differences, suggesting distinct proteolytic processes acting in the normal and tumoral secretomes. The mapping and annotation of cleavage sites in the tumoral secretome suggested functional roles of active proteases in central biological processes related to oncogenesis, such as the processing of growth factors, cleavage of extracellular matrix proteins and the shedding of ectopic domains from the cell surface, some of which may represent novel processed forms of the corresponding proteins. In the context of the tumor microenvironment, these results suggest important biological roles of proteolytic processing in murine melanoma secreted proteins.

2018-10-02 | PXD010169 | Pride

Rat podocyte degradomics - N-Degradomic Analysis Reveals a Proteolytic Network Processing the Podocyte Cytoskeleton

Project description:Regulated intracellular proteolysis is essential in maintaining the integrity of podocytes and the glomerular filtration barrier of the kidney. Altered proteolytic substrate turnover has been associated with various glomerular diseases ranging from diabetic nephropathy to focal and segmental glomerulosclerosis. However, thus far it has not been possible to systematically identify proteolytically cleaved proteins although some of the proteases have been characterized. Here we applied TAILS to identify N-termini in rat glomeruli and their changes on PAN-induced injury.

2017-07-20 | PXD006356 | Pride

Human podocyte cell culture degradomics

Project description:Regulated intracellular proteolysis is essential in maintaining the integrity of podocytes and the glomerular filtration barrier of the kidney. Altered proteolytic substrate turnover has been associated with various glomerular diseases ranging from diabetic nephropathy to focal and segmental glomerulosclerosis. However, thus far it has not been possible to systematically identify proteolytically cleaved proteins although some of the proteases have been characterized. Here we applied TAILS to map N-termini in the mouse glomeruli proteome and to determine and quantify N termini in podocyte cell cultures challenged with PAN.

2017-07-20 | PXD005140 | Pride

AtLEGbeta / gamma protease specificity and putative substrate characterization by HUNTER

Project description:Arabidopsis thaliana Legumain (aka VPE) beta / gamma were characterized for their cleavage specificity and putative substrates were identified by the N-termini enrichment method HUNTER.

2020-07-29 | PXD019276 | Pride

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