Browse
Submit Data
Databases
API
Help

Dataset Information

8 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Role of sequence and position of the cleavage sites in prothrombin activation.

ABSTRACT: In the penultimate step of the coagulation cascade, the multidomain vitamin-K-dependent zymogen prothrombin is converted to thrombin by the prothrombinase complex composed of factor Xa, cofactor Va, and phospholipids. Activation of prothrombin requires cleavage at two residues, R271 and R320, along two possible pathways generating either the intermediate prethrombin-2 (following initial cleavage at R271) or meizothrombin (following initial cleavage at R320). The former pathway is preferred in the absence of and the latter in the presence of cofactor Va. Several mechanisms have been proposed to explain this preference, but the role of the sequence and position of the sites of cleavage has not been thoroughly investigated. In this study, we engineered constructs where the sequences ²⁶¹DEDSDRAIEGRTATSEYQT²⁷⁹ and ³¹⁰RELLESYIDGRIVEGSDAE³²⁸ were swapped between the R271 and R320 sites. We found that in the absence of cofactor Va, the wild-type sequence at the R271 site is cleaved preferentially regardless of its position at the R271 or R320 site, whereas in the presence of cofactor Va, the R320 site is cleaved preferentially regardless of its sequence. Additional single-molecule FRET measurements revealed that the environment of R271 changes significantly upon cleavage at R320 due to the conformational transition from the closed form of prothrombin to the open form of meizothrombin. Detailed kinetics of cleavage at the R271 site were monitored by a newly developed assay based on loss of FRET. These findings show how sequence and position of the cleavage sites at R271 and R320 dictate the preferred pathway of prothrombin activation.

SUBMITTER: Stojanovski BM

PROVIDER: S-EPMC8348271 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Position of Deltaproteobacteria Cas12e nuclease cleavage sites depends on spacer length of guide RNA.

Project description:Cas12e proteins (formerly CasX) form a distinct subtype of Class II type V CRISPR-Cas effectors. Recently, it was shown that DpbCas12e from Deltaproteobacteria and PlmCas12e from Planctomycetes can introduce programmable double-stranded breaks in mammalian genomes. Thus, along with Cas9 and Cas12a Class II effectors, Cas12e could be harnessed for genome editing and engineering. The location of cleavage points in DNA targets is important for application of Cas nucleases in biotechnology. DpbCas12e was reported to produce extensive 5'-overhangs at cleaved targets, which can make it superior for some applications. Here, we used high throughput sequencing to precisely map the DNA cut site positions of DpbCas12e on several DNA targets. In contrast to previous observations, our results demonstrate that DNA cleavage pattern of Cas12e is very similar to that of Cas12a: DpbCas12e predominantly cleaves DNA after nucleotide position 17-19 downstream of PAM in the non-target DNA strand, and after the 22nd position of target strand, producing 3-5 nucleotide-long 5'-overhangs. We also show that reduction of spacer sgRNA sequence from 20nt to 16nt shifts Cas12e cleavage positions on the non-target DNA strand closer to the PAM, producing longer 6-8nt 5'-overhangs. Overall, these findings advance the understanding of Cas12e endonucleases and may be useful for developing of DpbCas12e-based biotechnology instruments.

| S-EPMC7549622 | biostudies-literature

The linker connecting the two kringles plays a key role in prothrombin activation.

Project description:The zymogen prothrombin is proteolytically converted by factor Xa to the active protease thrombin in a reaction that is accelerated >3,000-fold by cofactor Va. This physiologically important effect is paradigmatic of analogous cofactor-dependent reactions in the coagulation and complement cascades, but its structural determinants remain poorly understood. Prothrombin has three linkers connecting the N-terminal Gla domain to kringle-1 (Lnk1), the two kringles (Lnk2), and kringle-2 to the C-terminal protease domain (Lnk3). Recent developments indicate that the linkers, and particularly Lnk2, confer on the zymogen significant flexibility in solution and enable prothrombin to sample alternative conformations. The role of this flexibility in the context of prothrombin activation was tested with several deletions. Removal of Lnk2 in almost its entirety (ProT?146-167) drastically reduces the enhancement of thrombin generation by cofactor Va from >3,000-fold to 60-fold because of a significant increase in the rate of activation in the absence of cofactor. Deletion of Lnk2 mimics the action of cofactor Va and offers insights into how prothrombin is activated at the molecular level. The crystal structure of ProT?146-167 reveals a contorted architecture where the domains are not vertically stacked, kringle-1 comes within 9 Å of the protease domain, and the Gla-domain primed for membrane binding comes in contact with kringle-2. These findings broaden our molecular understanding of a key reaction of the blood coagulation cascade where cofactor Va enhances activation of prothrombin by factor Xa by compressing Lnk2 and morphing prothrombin into a conformation similar to the structure of ProT?146-167.

| S-EPMC4040597 | biostudies-literature

Position-specific prediction of methylation sites from sequence conservation based on information theory.

Project description:Protein methylation plays vital roles in many biological processes and has been implicated in various human diseases. To fully understand the mechanisms underlying methylation for use in drug design and work in methylation-related diseases, an initial but crucial step is to identify methylation sites. The use of high-throughput bioinformatics methods has become imperative to predict methylation sites. In this study, we developed a novel method that is based only on sequence conservation to predict protein methylation sites. Conservation difference profiles between methylated and non-methylated peptides were constructed by the information entropy (IE) in a wider neighbor interval around the methylation sites that fully incorporated all of the environmental information. Then, the distinctive neighbor residues were identified by the importance scores of information gain (IG). The most representative model was constructed by support vector machine (SVM) for Arginine and Lysine methylation, respectively. This model yielded a promising result on both the benchmark dataset and independent test set. The model was used to screen the entire human proteome, and many unknown substrates were identified. These results indicate that our method can serve as a useful supplement to elucidate the mechanism of protein methylation and facilitate hypothesis-driven experimental design and validation.

| S-EPMC5378888 | biostudies-literature

Sequence features of Drosha and Dicer cleavage sites affect the complexity of isomiRs.

Project description:The deep-sequencing of small RNAs has revealed that different numbers and proportions of miRNA variants called isomiRs are formed from single miRNA genes and that this effect is attributable mainly to imprecise cleavage by Drosha and Dicer. Factors that influence the degree of cleavage precision of Drosha and Dicer are under investigation, and their identification may improve our understanding of the mechanisms by which cells modulate the regulatory potential of miRNAs. In this study, we focused on the sequences and structural determinants of Drosha and Dicer cleavage sites, which may explain the generation of homogeneous miRNAs (in which a single isomiR strongly predominates) as well as the generation of heterogeneous miRNAs. Using deep-sequencing data for small RNAs, we demonstrate that the generation of homogeneous miRNAs requires more sequence constraints at the cleavage sites than the formation of heterogeneous miRNAs. Additionally, our results indicate that specific Drosha cleavage sites have more sequence determinants in miRNA precursors than specific cleavage sites for Dicer and that secondary structural motifs in the miRNA precursors influence the precision of Dicer cleavage. Together, we present the sequence and structural features of Drosha and Dicer cleavage sites that influence the heterogeneity of the released miRNAs.

| S-EPMC4425070 | biostudies-literature

Procleave: Predicting Protease-specific Substrate Cleavage Sites by Combining Sequence and Structural Information.

Project description:Proteases are enzymes that cleave and hydrolyse the peptide bonds between two specific amino acid residues of target substrate proteins. Protease-controlled proteolysis plays a key role in the degradation and recycling of proteins, which is essential for various physiological processes. Thus, solving the substrate identification problem will have important implications for the precise understanding of functions and physiological roles of proteases, as well as for therapeutic target identification and pharmaceutical applicability. Consequently, there is a great demand for bioinformatics methods that can predict novel substrate cleavage events with high accuracy by utilizing both sequence and structural information. In this study, we present Procleave, a novel bioinformatics approach for predicting protease-specific substrates and specific cleavage sites by taking into account both their sequence and 3D structural information. Structural features of known cleavage sites were represented by discrete values using a LOWESS data-smoothing optimization method, which turned out to be critical for the performance of Procleave. The optimal approximations of all structural parameter values were encoded in a conditional random field (CRF) computational framework, alongside sequence and chemical group-based features. Here, we demonstrate the outstanding performance of Procleave through extensive benchmarking and independent tests. Procleave is capable of correctly identifying most cleavage sites in the case study. Importantly, when applied to the human structural proteome encompassing 17,628 protein structures, Procleave suggests a number of potential novel target substrates and their corresponding cleavage sites of different proteases. Procleave is implemented as a webserver and is freely accessible at http://procleave.erc.monash.edu/.

| S-EPMC7393547 | biostudies-literature

Fibroblast activation protein peptide substrates identified from human collagen I derived gelatin cleavage sites.

Project description:A highly consistent trait of tumor stromal fibroblasts is the induction of the membrane-bound serine protease fibroblast activation protein-alpha (FAP), which is overexpressed on the surface of reactive stromal fibroblasts present within the stroma of the majority of human epithelial tumors. In contrast, FAP is not expressed by tumor epithelial cells or by fibroblasts or other cell types in normal tissues. The proteolytic activity of FAP, therefore, represents a potential pan-tumor target that can be exploited for the release of potent cytotoxins from inactive prodrugs consisting of an FAP peptide substrate coupled to a cytotoxin. To identify FAP peptide substrates, we used liquid chromatography tandem mass spectroscopy based sequencing to generate a complete map of the FAP cleavage sites within human collagen I derived gelatin. Positional analysis of the frequency of each amino acid at each position within the cleavage sites revealed FAP consensus sequences PPGP and (D/E)-(R/K)-G-(E/D)-(T/S)-G-P. These studies further demonstrated that ranking cleavage sites based on the magnitude of the LC/MS/MS extracted ion current predicted FAP substrates that were cleaved with highest efficiency. Fluorescence-quenched peptides were synthesized on the basis of the cleavage sites with the highest ion current rankings, and kinetic parameters for FAP hydrolysis were determined. The substrate DRGETGP, which corresponded to the consensus sequence, had the lowest Km of 21 microM. Overall the Km values were relatively similar for both high and low ranked substrates, whereas the kcat values differed by up to 100-fold. On the basis of these results, the FAP consensus sequences are currently being evaluated as FAP-selective peptide carriers for incorporation into FAP-activated prodrugs.

| S-EPMC4696028 | biostudies-literature

Sequence dependencies and biophysical features both govern cleavage of diverse cut-sites by HIV protease.

Project description:The infectivity of HIV-1 requires its protease (PR) cleave multiple cut-sites with low sequence similarity. The diversity of cleavage sites has made it challenging to investigate the underlying sequence properties that determine binding and turnover of substrates by PR. We engineered a mutational scanning approach utilizing yeast display, flow cytometry, and deep sequencing to systematically measure the impacts of all individual amino acid changes at 12 positions in three different cut-sites (MA/CA, NC/p1, and p1/p6). The resulting fitness landscapes revealed common physical features that underlie cutting of all three cut-sites at the amino acid positions closest to the scissile bond. In contrast, positions more than two amino acids away from the scissile bond exhibited a strong dependence on the sequence background of the rest of the cut-site. We observed multiple amino acid changes in cut-sites that led to faster cleavage rates, including a preference for negative charge five and six amino acids away from the scissile bond at locations where the surface of protease is positively charged. Analysis of individual cut sites using full-length matrix-capsid proteins indicate that long-distance sequence context can contribute to cutting efficiency such that analyses of peptides or shorter engineered constructs including those in this work should be considered carefully. This work provides a framework for understanding how diverse substrates interact with HIV-1 PR and can be extended to investigate other viral PRs with similar properties.

| S-EPMC9207908 | biostudies-literature

Sequence selectivity of the cleavage sites induced by topoisomerase I inhibitors: a molecular dynamics study.

Project description:Topoisomerase IB (Top1) inhibitors, such as camptothecin (CPT), stabilize the Top1-DNA cleavage complex in a DNA sequence-dependent manner. The sequence selectivity of Top1 inhibitors is important for targeting specific genomic sequences of therapeutic value. However, the molecular mechanisms underlying this selectivity remain largely unknown. We performed molecular dynamics simulations to delineate structural, dynamic and energetic features that contribute to the differential sequence selectivity of the Top1 inhibitors. We found the sequence selectivity of CPT to be highly correlated with the drug binding energies, dynamic and structural properties of the linker domain. Chemical insights, gained by per-residue binding energy analysis revealed that the non-polar interaction between CPT and nucleotide at the +1 position of the cleavage site was the major (favorable) contributor to the total binding energy. Mechanistic insights gained by a potential of mean force analysis implicated that the drug dissociation step was associated with the sequence selectivity. Pharmaceutical insights gained by our molecular dynamics analyses explained why LMP-776, an indenoisoquinoline derivative under clinical development at the National Institutes of Health, displays different sequence selectivity when compared with camptothecin and its clinical derivatives.

| S-EPMC3905861 | biostudies-literature

Identification of co-occurring transcription factor binding sites from DNA sequence using clustered position weight matrices.

Project description:Accurate prediction of transcription factor binding sites (TFBSs) is a prerequisite for identifying cis-regulatory modules that underlie transcriptional regulatory circuits encoded in the genome. Here, we present a computational framework for detecting TFBSs, when multiple position weight matrices (PWMs) for a transcription factor are available. Grouping multiple PWMs of a transcription factor (TF) based on their sequence similarity improves the specificity of TFBS prediction, which was evaluated using multiple genome-wide ChIP-Seq data sets from 26 TFs. The Z-scores of the area under a receiver operating characteristic curve (AUC) values of 368 TFs were calculated and used to statistically identify co-occurring regulatory motifs in the TF bound ChIP loci. Motifs that are co-occurring along with the empirical bindings of E2F, JUN or MYC have been evaluated, in the basal or stimulated condition. Results prove our method can be useful to systematically identify the co-occurring motifs of the TF for the given conditions.

| S-EPMC3300004 | biostudies-literature

Activation of the SARS coronavirus spike protein via sequential proteolytic cleavage at two distinct sites.

Project description:The coronavirus spike protein (S) plays a key role in the early steps of viral infection, with the S1 domain responsible for receptor binding and the S2 domain mediating membrane fusion. In some cases, the S protein is proteolytically cleaved at the S1-S2 boundary. In the case of the severe acute respiratory syndrome coronavirus (SARS-CoV), it has been shown that virus entry requires the endosomal protease cathepsin L; however, it was also found that infection of SARS-CoV could be strongly induced by trypsin treatment. Overall, in terms of how cleavage might activate membrane fusion, proteolytic processing of the SARS-CoV S protein remains unclear. Here, we identify a proteolytic cleavage site within the SARS-CoV S2 domain (S2', R797). Mutation of R797 specifically inhibited trypsin-dependent fusion in both cell-cell fusion and pseudovirion entry assays. We also introduced a furin cleavage site at both the S2' cleavage site within S2 793-KPTKR-797 (S2'), as well as at the junction of S1 and S2. Introduction of a furin cleavage site at the S2' position allowed trypsin-independent cell-cell fusion, which was strongly increased by the presence of a second furin cleavage site at the S1-S2 position. Taken together, these data suggest a novel priming mechanism for a viral fusion protein, with a critical proteolytic cleavage event on the SARS-CoV S protein at position 797 (S2'), acting in concert with the S1-S2 cleavage site to mediate membrane fusion and virus infectivity.

| S-EPMC2660061 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data