scholarly journals Role of sequence and position of the cleavage sites in prothrombin activation

2021 ◽  
pp. 100955
Author(s):  
Bosko M. Stojanovski ◽  
Enrico Di Cera
Keyword(s):  
Cleavage Sites ◽  
Prothrombin Activation

Rôle of Thrombin in Prothrombin Activation

1964 ◽  
Vol 12 (02) ◽  
pp. 484-488
Author(s):  
W. H Seegers ◽  
H Schröer ◽  
D Heene
Keyword(s):  
Partial Thromboplastin Time ◽  
Procoagulant Activity ◽  
Autoprothrombin C ◽  
Generation Test ◽  
Prothrombin Activation

SummaryThe partial thromboplastin time and purified thrombin were used to demonstrate the procoagulant power of thrombin. Only 0.007 μg of thrombin could be detected in prothrombin activation. Traces of thrombin and autoprothrombin C can fully account for the generation of procoagulant activity in the thromboplastin generation test. Inactivation of these two activities by antithrombin explains the disappearance of the procoagulant power in that test, so that there now remains no valid demonstration of the existence of plasma thromboplastin or of anti-plasma thromboplastin.

scholarly journals Proteolytic maturation of α2δ represents a checkpoint for activation and neuronal trafficking of latent calcium channels

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Ivan Kadurin ◽  
Laurent Ferron ◽  
Simon W Rothwell ◽  
James O Meyer ◽  
Leon R Douglas ◽  
...  
Keyword(s):  
Calcium Channels ◽  
Proteolytic Cleavage ◽  
Proteolytic Processing ◽  
Calcium Currents ◽  
Cleavage Sites ◽  
Voltage Dependent ◽  
Neuronal Processes ◽  
Associated Proteins ◽  
Synaptic Boutons

The auxiliary α2δ subunits of voltage-gated calcium channels are extracellular membrane-associated proteins, which are post-translationally cleaved into disulfide-linked polypeptides α2 and δ. We now show, using α2δ constructs containing artificial cleavage sites, that this processing is an essential step permitting voltage-dependent activation of plasma membrane N-type (CaV2.2) calcium channels. Indeed, uncleaved α2δ inhibits native calcium currents in mammalian neurons. By inducing acute cell-surface proteolytic cleavage of α2δ, voltage-dependent activation of channels is promoted, independent from the trafficking role of α2δ. Uncleaved α2δ does not support trafficking of CaV2.2 channel complexes into neuronal processes, and inhibits Ca2+ entry into synaptic boutons, and we can reverse this by controlled intracellular proteolytic cleavage. We propose a model whereby uncleaved α2δ subunits maintain immature calcium channels in an inhibited state. Proteolytic processing of α2δ then permits voltage-dependent activation of the channels, acting as a checkpoint allowing trafficking only of mature calcium channel complexes into neuronal processes.

Modulation of procaspase-7 self-activation by PEST amino acid residues of the N-terminal prodomain and intersubunit linker

2017 ◽  
Vol 95 (6) ◽  
pp. 634-643
Author(s):  
Juliano Alves ◽  
Miguel Garay-Malpartida ◽  
João M. Occhiucci ◽  
José E. Belizário
Keyword(s):  
Amino Acid ◽  
Large Subunit ◽  
Small Subunit ◽  
Cleavage Sites ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Caspase 7 ◽  
Caspase Family ◽  
The Impact

Procaspase-7 zymogen polypeptide is composed of a short prodomain, a large subunit (p20), and a small subunit (p10) connected to an intersubunit linker. Caspase-7 is activated by an initiator caspase-8 and -9, or by autocatalysis after specific cleavage at IQAD198↓S located at the intersubunit linker. Previously, we identified that PEST regions made of amino acid residues Pro (P), Glu (E), Asp (D), Ser (S), Thr (T), Asn (N), and Gln (Q) are conserved flanking amino acid residues in the cleavage sites within a prodomain and intersubunit linker of all caspase family members. Here we tested the impact of alanine substitution of PEST amino acid residues on procaspase-7 proteolytic self-activation directly in Escherichia coli. The p20 and p10 subunit cleavage were significantly delayed in double caspase-7 mutants in the prodomain (N18A/P26A) and intersubunit linker (S199A/P201A), compared with the wild-type caspase-7. The S199A/P201A mutants effectively inhibited the p10 small subunit cleavage. However, the mutations did not change the kinetic parameters (kcat/KM) and optimal tetrapeptide specificity (DEVD) of the purified mutant enzymes. The results suggest a role of PEST-amino acid residues in the molecular mechanism for prodomain and intersubunit cleavage and caspase-7 self-activation.

scholarly journals The plasmid system ofEscherichia colistrain UR12644: Identification and molecular characteristics of transposons involved in the generation of endogenous R-plasmids

1979 ◽  
Vol 34 (3) ◽  
pp. 287-301 ◽  
Author(s):  
F. Schöffl ◽  
A. Pühler
Keyword(s):  
Escherichia Coli ◽  
Molecular Characteristics ◽  
Multiple Drug ◽  
Restriction Map ◽  
Drug Resistant ◽  
Cleavage Sites ◽  
A Value ◽  
Multiple Drug Resistant ◽  
R Plasmids

SUMMARYTwo spontaneously formed R-plasmids (pFS401 and pFS402) originating from the multiple drug-resistantEscherichia colistrain UR12644 were found to carry transposable drug-resistance elements. Incompatibility between these two plasmids was used to select for transposition. An ampicillin transposon (Tn1781) residing on pFS401 and a tetracycline transposon (Tn1771) present on pFS402 were independently translocated to the endogenous RTF-plasmid pFS2. Molecular weight determinations of pFS2::Tn1781(Ap) and pFS2::Tn1771(Tc) revealed a value of 2·9 Mdal for Tn1781 and 7·1 Mdal for Tn1771. The arrangement of 3PstI and 1BamHI restriction endonuclease sites was found to be characteristic for the ampicillin transposon whereas the restriction map of Tn1771 features a nearly symmetrical location of 3EcoRI cleavage sites, two of them close to the termini and one in the middle of the transposon. A model is presented suggesting the existence of repetitive DNA-segments at these positions which represent the structural preconditions for the genetic properties of Tn1771. The role of a cryptic plasmid involved in the generation of the endogenous R-plasmids pFS401 and pFS402 is discussed.

scholarly journals A phage parasite deploys a nicking nuclease effector to inhibit replication of its viral host

2021 ◽  
Author(s):  
Kristen LeGault ◽  
Zachary Barth ◽  
Peter DePaola ◽  
Kimberley Seed
Keyword(s):  
Genome Replication ◽  
Cleavage Sites ◽  
Structural Components ◽  
Rolling Circle Replication ◽  
Progeny Production ◽  
Unknown Mechanism ◽  
Rolling Circle ◽  
Putative Phage

PLEs are phage parasites integrated into the chromosome of epidemic Vibrio cholerae. In response to infection by its viral host ICP1, PLE excises, replicates and hijacks ICP1 structural components for transduction. Through an unknown mechanism PLE prevents ICP1 from transitioning to rolling circle replication (RCR), a prerequisite for efficient packaging of the viral genome. Here, we characterize a PLE-encoded nuclease, NixI, that blocks phage development likely by nicking ICP1s genome as it transitions to RCR. NixI-dependent cleavage sites appear in ICP1s genome during infection of PLE(+) V. cholerae. Purified NixI demonstrates in vitro specificity for sites in ICP1s genome and NixI activity is enhanced by a putative specificity determinant co-expressed with NixI during phage infection. Importantly, NixI is sufficient to limit ICP1 genome replication and eliminate progeny production. We identify distant NixI homologs in an expanded family of putative phage satellites in Vibrios that lack nucleotide homology to PLEs but nonetheless share genomic synteny with PLEs. More generally, our results reveal a previously unknown mechanism deployed by phage parasites to limit packaging of their viral hosts genome and highlight the prominent role of nuclease effectors as weapons in the arms race between antagonizing genomes.

Phospholipid-Specific Conformational Changes in Human Prothrombin upon Binding to Procoagulant Acidic Lipid Membranes

1994 ◽  
Vol 71 (05) ◽  
pp. 596-604 ◽  
Author(s):  
Jogin R Wu ◽  
Barry R Lentz
Keyword(s):  
Secondary Structure ◽  
Conformational Changes ◽  
Lipid Membranes ◽  
Membrane Vesicles ◽  
Scanning Calorimetry ◽  
Domain Organization ◽  
Α Helix ◽  
Β Sheet ◽  
Prothrombin Activation

SummaryThis paper provides evidence to demonstrate that human prothrombin undergoes conformational changes upon binding to procoagulant membranes specifically containing phosphatidylserine (PS). Fourier transform infrared spectroscopy was used to show a slight increase in ordered (α-helix, β-sheet, β-turns) secondary structure upon binding to PS-containing membranes. Thermograms representing prothrombin and prothrombin fragment 1 denaturation were obtained using differential scanning calorimetry. These were analyzed and interpreted in terms of changes in prothrombin domain organization associated with binding to PS-containing membranes. Changes in either secondary structure or domain organization upon binding to negatively-charged phosphatidylglycerol-containing membranes were, if they occurred at all, much less dramatic. The results paralleled results obtained previously with bovine prothrombin (1, 2). The implications of these results in terms of a possible molecular mechanism for the cofactor-like role of platelet membrane vesicles in prothrombin activation are discussed.

Role of exosite binding modulators in the inhibition of Fxa by TFPI

2016 ◽  
Vol 115 (03) ◽  
pp. 580-590 ◽  
Author(s):  
Alexandra Heinzmann ◽  
Tilman M. Hackeng ◽  
Rudolf Hartmann ◽  
Friedrich Scheiflinger ◽  
Michael Dockal ◽  
...  
Keyword(s):  
Plasma Proteins ◽  
Tissue Factor Pathway Inhibitor ◽  
Anticoagulant Activity ◽  
Protein S ◽  
Low Concentrations ◽  
Tissue Factor Pathway ◽  
Coagulation Pathway ◽  
Prothrombin Activation

SummaryTissue factor pathway inhibitor (TFPI) down-regulates the extrinsic coagulation pathway by inhibiting FXa and FVIIa. Both TFPI and FXa interact with several plasma proteins (e. g. prothrombin, FV/FVa, protein S) and non-proteinaceous compounds (e. g. phospholipids, heparin). It was our aim to investigate effects of ligands that bind to FXa and TFPI on FXa inhibition by full-length TFPI (designated TFPI) and truncated TFPI (TFPI1-150). Inhibition of FXa by TFPI and TFPI1-150 and effects of phospholipids, heparin, prothrombin, FV, FVa, and protein S thereon was quantified from progress curves of conversion of the FXa-specific chromogenic substrate CS11-(65). Low concentrations negatively charged phospholipids (~10 μM) already maximally stimulated (up to 5- to 6-fold) FXa inhibition by TFPI. Unfractionated heparin at concentrations (0.2–1 U/ml) enhanced FXa inhibition by TFPI ~8-fold, but impaired inhibition at concentrations > 1 U/ml. Physiological protein S and FV concentrations both enhanced FXa inhibition by TFPI 2- to 3-fold. In contrast, thrombin-activated FV (FVa) impaired the ability of TFPI to inhibit FXa. FXa inhibition by TFPI1–150 was not affected by FV, FVa, protein S, phospholipids and heparin. TFPI potently inhibited FXa-catalysed prothrombin activation in the absence of FVa, but hardly inhibited prothrombin activation in the presence of thrombin-activated FVa. In conclusion, physiological concentrations TFPI (0.25–0.5 nM TFPI) inhibit FXa with a t1/2 between 3–15 minutes. Direct FXa inhibition by TFPI is modulated by physiological concentrations prothrombin, FV, FVa, protein S, phospholipids and heparin indicating the importance of these modulators for the in vivo anticoagulant activity of TFPI.

scholarly journals Plum Pox Virus6K1 Protein Is Required for Viral Replication and Targets the Viral Replication Complex at the Early Stage of Infection

2016 ◽  
Vol 90 (10) ◽  
pp. 5119-5131 ◽  
Author(s):  
Hongguang Cui ◽  
Aiming Wang
Keyword(s):  
Viral Replication ◽  
Functional Role ◽  
Early Infection ◽  
Plum Pox Virus ◽  
Cdna Clones ◽  
Cleavage Sites ◽  
Replication Complex ◽  
Viral Replication Complex ◽  
Infection Stage

ABSTRACTThe potyviral RNA genome encodes two polyproteins that are proteolytically processed by three viral protease domains into 11 mature proteins. Extensive molecular studies have identified functions for the majority of the viral proteins. For example, 6K2, one of the two smallest potyviral proteins, is an integral membrane protein and induces the endoplasmic reticulum (ER)-originated replication vesicles that target the chloroplast for robust viral replication. However, the functional role of 6K1, the other smallest protein, remains uncharacterized. In this study, we developed a series of recombinant full-length viral cDNA clones derived from a CanadianPlum pox virus(PPV) isolate. We found that deletion of any of the short motifs of 6K1 (each of which ranged from 5 to 13 amino acids), most of the 6K1 sequence (but with the conserved sequence of the cleavage sites being retained), or all of the 6K1 sequence in the PPV infectious clone abolished viral replication. Thetransexpression of 6K1 or thecisexpression of a dislocated 6K1 failed to rescue the loss-of-replication phenotype, suggesting the temporal and spatial requirement of 6K1 for viral replication. Disruption of the N- or C-terminal cleavage site of 6K1, which prevented the release of 6K1 from the polyprotein, either partially or completely inhibited viral replication, suggesting the functional importance of the mature 6K1. We further found that green fluorescent protein-tagged 6K1 formed punctate inclusions at the viral early infection stage and colocalized with chloroplast-bound viral replicase elements 6K2 and NIb. Taken together, our results suggest that 6K1 is required for viral replication and is an important viral element of the viral replication complex at the early infection stage.IMPORTANCEPotyviruses account for more than 30% of known plant viruses and consist of many agriculturally important viruses. The genomes of potyviruses encode two polyproteins that are proteolytically processed into 11 mature proteins, with the majority of them having been at least partially functionally characterized. However, the functional role of a small protein named 6K1 remains obscure. In this study, we showed that deletion of 6K1 or a short motif/region of 6K1 in the full-length cDNA clones of plum pox virus abolishes viral replication and that mutation of the N- or C-terminal cleavage sites of 6K1 to prevent its release from the polyprotein greatly attenuates or completely inhibits viral replication, suggesting its important role in potyviral infection. We report that 6K1 forms punctate structures and targets the replication vesicles in PPV-infected plant leaf cells at the early infection stage. Our data reveal that 6K1 is an important viral protein of the potyviral replication complex.

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