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Identification C3 cleavage sites by alpha-Thrombin

ABSTRACT: Identification C3 cleavage sites by alpha-Thrombin. Goal of the experiment is the validation of predicted cleavage sites by HTPS

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Federico Uliana

LAB HEAD: Ruedi Aebersold

PROVIDER: PXD022971 | Pride | 2021-02-04

REPOSITORIES: Pride

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			Action	DRS
	200908_OE1_mvizovisek_073.raw	Raw
	200908_OE1_mvizovisek_074.raw	Raw
	200908_OE1_mvizovisek_083.raw	Raw
	200908_OE1_mvizovisek_084.raw	Raw
	200908_OE1_mvizovisek_093.raw	Raw

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1 - 5 of 11

Publications

Mapping specificity, cleavage entropy, allosteric changes and substrates of blood proteases in a high-throughput screen.

Uliana Federico F Vizovišek Matej M Acquasaliente Laura L Ciuffa Rodolfo R Fossati Andrea A Frommelt Fabian F Goetze Sandra S Wollscheid Bernd B Gstaiger Matthias M De Filippis Vincenzo V Auf dem Keller Ulrich U Aebersold Ruedi R

Nature communications 20210316 1

Proteases are among the largest protein families and critical regulators of biochemical processes like apoptosis and blood coagulation. Knowledge of proteases has been expanded by the development of proteomic approaches, however, technology for multiplexed screening of proteases within native environments is currently lacking behind. Here we introduce a simple method to profile protease activity based on isolation of protease products from native lysates using a 96FASP filter, their analysis in ...[more]

PMID: 33727531

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Quantitative analysis of design substrates

Project description:Proteases are crucial physiologic regulators of protein structure and function. While proteomic methods contributed extensively to protease characterization efforts, technical challenges remain in terms of throughput, scalability and large datasets of protease cleavages remain scarce. Here, we describe a high-throughput protease screen (HTPS) which allows simultaneous characterization of multiple proteases under various conditions on a microscale 96FASP format. After benchmarking the performance with Trypsin, LysC, GluC, AspN, Chymotrypsin, MMP-2 and MMP-3, we applied it to profile proteases of the blood coagulation cascade (Factors VIIa, IXa, Xa, XIa, Thrombin α, β and γ, Plasmin and Protein C). The large dataset enabled us to map activity, specificity, cleave entropy, allosteric changes of blood cascade proteases induced by Na+ and to predict potential substrates in a data-driven way. Here, we designed two octapeptides, GIPRAAGD (α Thrombin) and GIGRRIAE (Factor Xa), and evaluated their cleavage with the target proteases.

2021-02-04 | PXD020321 | Pride

Targeted quantification of octapeptide substrate of FII and FX

2021-02-04 | PXD020319 | Pride

Target quantification of octapeptide for Thrombin and FX

2021-02-04 | PXD020320 | Pride

Multiple experiments: Analysis of the HTPS FT; Kinetics of Thrombin and Chymotrypsin; Effect of temperature on Thrombin and Chymotrypsin

Project description:1) Analysis of HTPS FT (analysis of the background, without protease) 2) Analysis of specificity and activity of Thrombin and Chymotrypsin at multiple time point (0,5,15,30,60,120,240 min) 3) Analysis of specificity and activity of Thrombin and Chymotrpysin with 2 hr incubation at 20C, 25C and 37C

2021-02-04 | PXD022972 | Pride

Mapping specificity, entropy, allosteric changes and substrates in blood coagulation proteases by a high-throughput protease screen

2021-02-05 | PXD018976 | Pride

Paradigm of biased PAR1 activation and inhibition in endothelial cells dissected by phosphoproteomics

Project description:Objective: Thrombin is the key serine protease of the coagulation cascade and mediates cellular responses by activation of protease-activated receptors (PARs). The predominant thrombin receptor is PAR1 and in endothelial cells (ECs) thrombin dynamically regulates a plethora of phosphorylation events. However, it has remained unclear if thrombin signaling is exclusively mediated through PAR1. Furthermore, mechanistic insight into activation and inhibition of PAR1-mediated EC signaling is lacking. In addition, signaling networks of biased PAR1 activation after differential cleavage of the PAR1 N-terminus have remained an unresolved issue.Approach and Results: Here, we used a quantitative phosphoproteomics approach to show that ‘classical’ and ‘peptide’ activation of PAR1 induce highly similar signaling, that low thrombin concentrations initiate only limited phosphoregulation, and that the PAR1 inhibitors vorapaxar and parmodulin-2 demonstrate distinct antagonistic properties. Subsequent analysis of the thrombin-regulated phosphosites in presence of PAR1 inhibitors revealed that biased activation of PAR1 is not solely linked to a specific G-protein downstream of PAR1. In addition, we showed that only the canonical thrombin PAR1 tethered ligand induces extensive early phosphoregulation in ECs.Conclusions: Our study provides detailed insight in the signaling mechanisms downstream of PAR1. Our data demonstrates that thrombin-induced EC phosphoregulation is mediated exclusively through PAR1, that thrombin and thrombin-TL peptide induce similar phosphoregulation and that only canonical PAR1 cleavage by thrombin generates a tethered ligand that potently induces early signaling. Furthermore, platelet PAR1 inhibitors directly affect EC signaling, indicating it will be a challenge to design a PAR1 antagonist that will target only those pathways responsible for tissue pathology.

2017-08-22 | PXD004264 | Pride

Cleavage Factor Im as a key regulator of 3’ UTR length

Project description:In eukaryotes, the 3' ends of RNA polymerase II-generated transcripts are made in the majority of cases by site-specific endonucleolytic cleavage, followed by the addition of a poly(A) tail. By alternative polyadenylation, a gene can give rise to multiple mRNA isoforms that differ in the length of their 3' UTRs and hence in their susceptibility to post-transcriptional regulatory factors such as microRNAs. A series of recently conducted high-throughput studies of poly(A) site usage revealed an extensive tissue-specific control of 3’ UTR length and drastic changes in 3’ UTR length of mRNAs upon induction of proliferation in resting cells. To understand the dynamics of polyadenylation site usage, we recently identified binding sites of the major pre-mRNA 3’ end processing factors - cleavage and polyadenylation specificity factor (CPSF), cleavage stimulation factor (CstF), and cleavage factor Im (CF Im) - and mapped cleaved polyadenylation sites in HEK293 cells. Our present study extends previous findings on the role of CF Im in alternative polyadenylation and reveals that subunits of the CF Im complex generally control 3’ UTR length. More specifically, we demonstrate that the loss-of-function of CF Im68 and CF Im25 but not of CF Im59 leads to a transcriptome-wide increase of the use of proximal polyadenylation sites. 3' ends of transcripts were profiled by high-throughput sequencing in HEK 293 cells under normal conditions, and in HEK 293 cells depleted of 3' end processing factors CF Im25, CF Im59, and CF Im68.

2012-10-30 | E-GEOD-40137 | biostudies-arrayexpress

Genome-wide analysis of pre-mRNA 3' end processing reveals a decisive role of human cleavage factor I in the regulation of 3' UTR length

Project description:This SuperSeries is composed of the following subset Series: GSE37037: Genome-wide analysis of pre-mRNA 3' end processing reveals a decisive role of human cleavage factor I in the regulation of 3' UTR length: A-seq GSE37398: Genome-wide analysis of pre-mRNA 3' end processing reveals a decisive role of human cleavage factor I in the regulation of 3' UTR length: CLIP Refer to individual Series

2012-05-14 | E-GEOD-37401 | biostudies-arrayexpress

Characterization of legumain degradation of intact proteins

Project description:Legumain or asparagine endopeptidase is a unique cysteine endopeptidase with a distinctive specificity for the hydrolysis of peptide bonds after asparagine and to a lesser extent after aspartate. In order to improve our understanding of legumain substrate recognition we have performed proteomic profiling of legumain specificity on native proteins using two different N-terminal labelling methodologies (FPPS and ISIL). Our data revealed a strict P1 specificity of legumain with clear cleavage preference for unstructured secondary regions in the substrate proteins. No extended cleavage specificity on native proteins was observed. Moreover, a limited number of identified cleavages on individual substrates suggest its primary role in precision proteolysis and regulatory proteolytic events.

2018-11-29 | PXD010466 | Pride

Proteomic QSAR analysis of calpain substrate specificity

Project description:Calpains are intracellular Ca2+-regulated cysteine proteases that are essential for various cellular functions. Mammalian conventional calpains (calpain-1 and calpain-2) modulate the structure and function of their substrates by limited proteolysis; however, their substrate specificity remains unclear because the amino acid (aa) sequences around their cleavage sites are very diverse. To clarify calpains’ substrate specificities, 84 20-mer oligopeptides, corresponding to P10-P10’ of reported cleavage site sequences, were proteolyzed by calpains, and the catalytic efficiencies (kcat/Km) were globally determined by LC/MS. This analysis revealed 483 cleavage site sequences, including 360 novel ones. The kcat/Kms for 119 sites ranged from 12.5~1,710 M-1s-1. Most sites were cleaved by both calpain-1 and -2 with a similar kcat/Km. The aa compositions of the novel sites were not significantly different from the 420 previously reported sites, suggesting calpains have a strict implicit rule for sequence specificity, and that the limited proteolysis of intact substrates is due to the substrates’ higher-order structures. Cleavage position frequencies indicated that longer sequences N-terminal to the cleavage site (P-sites) than C-terminal (P’-sites) were preferred for proteolysis. Quantitative structure-activity relationship (QSAR) analyses using partial least-squares regression and >1,300 aa descriptors achieved kcat/Km prediction with r=0.834, and binary-QSAR modeling attained 64.8% prediction accuracy for 132 reported calpain cleavage sites independent of our model construction. These results outperformed previous calpain cleavage predictors, and revealed the importance of the P2, P3’, P4’, and P1-P2 contexts. This study increases our understanding of calpain substrate specificities, and opens calpains to “next-generation,” i.e., activity-related quantitative and context-dependent analyses.

2016-07-19 | PXD002532 | Pride

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