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TAILS analysis of viral protease 3C substrates

ABSTRACT: Human Hela and murine HL1 cells were cultured and infected in vitro with C3 protease and inactive mutant protease. Cellular proteomes were collected, labeled by isotopic dimethylation and N-terminal peptides were enriched using TALS proteomics.

INSTRUMENT(S): impact II, Q Exactive

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

TISSUE(S): Cell Culture

SUBMITTER: Theo Klein

LAB HEAD: Christopher M. Overall

PROVIDER: PXD008718 | Pride | 2018-03-05

REPOSITORIES: Pride

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			Action	DRS
	OK20130920_J2C1_1.raw	Raw
	OK20130920_J3C2_2.raw	Raw
	OK20130920_J3C2_3.raw	Raw
	Orbi_Ndm_PEAKS.zip	Other
	Orbi_Ndm_SEARCH.zip	Other

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Publications

N-Terminomics TAILS Identifies Host Cell Substrates of Poliovirus and Coxsackievirus B3 3C Proteinases That Modulate Virus Infection.

Jagdeo Julienne M JM Dufour Antoine A Klein Theo T Solis Nestor N Kleifeld Oded O Kizhakkedathu Jayachandran J Luo Honglin H Overall Christopher M CM Jan Eric E

Journal of virology 20180328 8

Enteroviruses encode proteinases that are essential for processing of the translated viral polyprotein. In addition, viral proteinases also target host proteins to manipulate cellular processes and evade innate antiviral responses to promote replication and infection. Although some host protein substrates of enterovirus proteinases have been identified, the full repertoire of targets remains unknown. We used a novel quantitative <i>in vitro</i> proteomics-based approach, termed <u>t</u>erminal < ...[more]

PMID: 29437971

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TMT10-TAILS investigation of the proteolytic landscape in B cell activation

Project description:The N-terminome in EBV-B cells from a human patient homozygous for an W580S mutation in MALT1 were analyzed by TMT10-plex TAILS and compared to EBV-B cells from heterozygous subjects under resting and PMA/ionomycin stimulation for 2 and 4 h.

2018-10-03 | PXD006723 | Pride

mito-TAILS analysis of EPEC infection

Project description:Mito-TAILS N-terminomics analysis of proteolytic events during EPEC infection of human epithelial cells, on whole-cell level and mitochondria.

2020-05-21 | PXD016994 | Pride

MitoTAILS: N-terminomics profiling of mitochondrial proteolysis

Project description:Mitochandria were isolated from SILAC labeled HeLa cells, either naive or under early apoptosis-inducing conditions. Early proteolytic events were evaluated by enriching for N-terminal peptides through N-TAILS.

2018-10-15 | PXD009054 | Pride

Identification of novel natural substrates of fibroblast activation protein by differential degradomics and proteomics (TAILS dataset)

Project description:Fibroblast Activation Protein (FAP) is a cell-surface transmembrane-anchored anchored dimeric protease. This unique constitutively active serine protease has both dipeptidyl aminopeptidase and endopeptidase activities, and can hydrolyse post-proline bonds. FAP expression is very low in adult organs, but is greatly up regulated by activated fibroblasts in sites of tissue remodelling, including fibrosis, atherosclerosis, arthritis and tumours. Primary mouse embryonic fibroblasts (MEF) were isolated from FAP gene knockout (GKO) embryos, immortalised and then transduced to express either enzyme active or inactive FAP. The cell secretomes were analysed using degradomic and proteomic techniques. Terminal Amine Isotopic Labelling of Substrates based degradomics identified cleavage sites in collagens, many other extracellular matrix (ECM) and associated proteins, and lysyl oxidase-like-1, CCL2, IL13, C1qT6 and CSF-1 that were confirmed in vitro. In addition, differential metabolic labelling coupled with quantitative proteomic analysis also implicated FAP in ECM-cell interactions, as well as with coagulation and wound healing associated proteins. FAP GKO plasma exhibited slower that wildtype clotting times. This study greatly enhances the understanding of the roles of FAP through a significant expansion of the substrate repertoire of this protease and identifying downstream effects of its activity that support roles in pathological processes.

2018-12-19 | PXD004757 | Pride

Insight into the Human Pathodegradome of the V8 protease from Staphylococcus aureus

Project description:The major human pathogen Staphylococcus aureus possesses ten extracellular proteases that are central to the virulence process. While some host targets of these and other bacterial proteases are known, a major bottleneck to the study of their function is the low-throughout nature of substrate identification. Rapid identification of proteolytic cleavage events in complex substrates has, however, been rendered possible by recent developments in the proteomics sub discipline of N-terminomics. Thus, herein we perform the first study of bacterial protease-host interactions, combining the S. aureus V8 protease and the infection relevant substrate human serum. In so doing, we identified ~90 host-protein targets of V8, including previously known and novel targets. Amongst these were complement components (C1r, C1s, C3,C4b, C4BPA, C5, C6, C8G, C9, CfB, CfH, and ficolin), iron sequestration and transport proteins (ceruloplasmin, serotransferrin, hemopexin, haptoglobin), clotting cascade proteins (plasminogen, kallikrein proteins, prothrombin, factors X and XII), and multiple host protease inhibitors (α2M and members of the ITIH and SERPIN families). N-terminomics also provided insight into the disruption of host protein function by highlighting specific target regions of host proteins, where V8 cleaved within peptidase and sushi domains of complement proteins, and, in the case of host protease inhibitors, cleavage occurred predominantly outside their protease-trapping motifs. Collectively, our data highlight the potential for further application of N-terminomics for the discovery of bacterial protease substrates in other host niches, and provides unique insight into the role of the V8 protease in S. aureus pathogenesis.

2021-03-29 | PXD014797 | Pride

Validation of N-terminal Labeling Mass Spectrometry for Characterization of Partially Hydrolyzed Gluten Proteins

Project description:Gluten, a group of proteins found in wheat, barley, and rye, is the trigger of celiac disease, an immune disorder which affects about 1% of people worldwide. The toxicity of partially hydrolyzed gluten (PHG) in fermented products such as beer is unknown due to the significant analytical challenges in PHG detection. In this project, an N-terminal labeling mass spectrometry method, terminal amine isotopic labeling of substrates (TAILS), was optimized for the in-depth analysis of PHG and validated using a test protease (trypsin) with known cleavage specificities. Gluten N-termini in test and control groups were labeled with heavy and light formaldehyde, respectively. Trypsin-generated and native N-termini were confirmed by showing an MS1 peak area Log2 H:L with and without a significant difference (p < 0.01) from zero, respectively. Using the strategy, all abundant theoretical protease generated peptides in alpha/beta gliadins and gamma gliadins were identified. The validation indicated that the strategy can identify multiple protease cleavage sites in gluten and be used in subsequent analysis of yeast cleavage patterns in beer throughout brewing. This strategy can be further applied to characterize a broader partially hydrolyzed allergens in foods and provide reference for their safety assessment to both industry and regulatory authorities.

2019-11-20 | PXD012296 | Pride

A human endogenous retrovirus encoded protease potentially cleaves numerous cellular proteins

Project description:Background: The human genome consists of considerable portions derived from retroviruses inherited for millions of years. So-called human endogenous retroviruses (HERVs) are usually severely mutated, yet some coding-competent HERVs exist. The HERV-K(HML-2) group includes evolutionarily young proviruses that still encode typical retroviral proteins. HERV-K(HML-2) has been implicated in various human diseases because transcription is often upregulated and some of the encoded proteins are known to affect cell biology. HERV-K(HML-2) Protease (Pro) has received little attention so far, although it appears expressed in some disease contexts and other retroviral proteases are known to process cellular proteins. Results: We set out to identify human cellular proteins being substrates of HERV-K(HML-2) Pro employing a modified Terminal Amine Isotopic Labeling of Substrates (TAILS) procedure. Thousands of human proteins were identified as significantly processed by HERV-K(HML-2) Pro. Identified proteins locate to various cellular compartments and participate in diverse, often disease-relevant cellular processes. We verified cleavage of a majority of selected human proteins in vitro and in vivo. Conclusions: Hundreds, if not thousands of cellular proteins are potential substrates of HERV-K(HML-2) Pro. It is conceivable that even low-level expression of HERV-K(HML-2) Pro has a functional impact on cell biology and thus relevance for human diseases. Specific studies will be required to elucidate effects of HERV-K(HML-2) Pro expression regarding human substrate proteins, cell biology and disease. Endogenous retrovirus-encoded Pro activity may also be relevant for disease development in species other than human.

2019-09-02 | PXD013296 | Pride

Transcription profiling of mouse testicular response to recombinant follicle stimulating hormone (rFSH) in hypogonadal males

Project description:In the normal mouse the pituitary gonadotrophins determine development, maturation and physiological regulation of the testis with follicle-stimulating hormone (FSH) activating the Sertoli cell and luteinising hormone (LH) the Leydig cell. To look at the potential interaction of cell types within the testis following hormone stimulation we have investigated the effect of rFSH on testicular gene expression in the hypogonadal (hpg) male mouse. Due to a deletion in the gene encoding gonadotrophin-releasing hormone (GnRH), FSH and LH levels are at the lower limit of detection in the circulation and mice remain in a pre-pubertal state throughout life unless given exogenous hormone. Here we have addressed important question of changes in the global gene expression profile of testicular tissue following hormone stimulation (rFSH) for 12, 24 and 72h following injections compared to control untreated hpg males of the same age. Experiment Overall Design: Adult 10 week old hpg males were injected sc with 8 I.U. of rFSH (Gonal F, Serono Pharmaceuticals) in 0.2ml of PBS (Sigma,UK) every 12 h for 12, 24 and 72 h. Mice were killed one hour after the last injection by cervical dislocation, testes dissected out, weighed and snap frozen in liquid nitrogen. Tissue was stored at -70oC until use. RNA was extracted using Qiagen RNeasy Lipid Tissue Mini kit (Qiagen, Valencia, CA) according to the manufacturerâs instructions and quantified on a NanoDrop ND-1000 (NanoDrop Wilmington, DE). RNA quality was checked using the Agilent bioanalyzer 2100 (Agilent, Santa Clara, CA). Testicular gene expression was examined in four individual mice at 12 and 72 h and 3 mice at 24h using high-density oligonucleotide mouse MOE430A arrays (Affymetrix, Santa Clara, CA), comparison was made with a group of 7 untreated hpg males of the same age.

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