Characterisation of hydrolysate for identifying initial peptide cleavage site of κ-casein by milk coagulating Wrightia tinctoria serine proteases

2021 ◽  
Vol 115 ◽  
pp. 104934
Author(s):  
Anusha Rajagopalan ◽  
Vasuki Aluru ◽  
Bindhu Omana Sukumaran
Keyword(s):  
Cleavage Site ◽  
Serine Proteases ◽  
Peptide Cleavage ◽  
Wrightia Tinctoria

A reactive center loop–based prediction platform to enhance the design of therapeutic SERPINs

2021 ◽  
Vol 118 (45) ◽  
pp. e2108458118
Author(s):  
Wariya Sanrattana ◽  
Thibaud Sefiane ◽  
Simone Smits ◽  
Nadine D. van Kleef ◽  
Marcel H. Fens ◽  
...  
Keyword(s):  
Cleavage Site ◽  
Serine Proteases ◽  
Protein C ◽  
Activated Protein C ◽  
Reactive Center Loop ◽  
Physiological Processes ◽  
Naturally Occurring ◽  
Reactive Center ◽  
Insight Into

Serine proteases are essential for many physiological processes and require tight regulation by serine protease inhibitors (SERPINs). A disturbed SERPIN–protease balance may result in disease. The reactive center loop (RCL) contains an enzymatic cleavage site between the P1 through P1’ residues that controls SERPIN specificity. This RCL can be modified to improve SERPIN function; however, a lack of insight into sequence–function relationships limits SERPIN development. This is complicated by more than 25 billion mutants needed to screen the entire P4 to P4’ region. Here, we developed a platform to predict the effects of RCL mutagenesis by using α1-antitrypsin as a model SERPIN. We generated variants for each of the residues in P4 to P4’ region, mutating them into each of the 20 naturally occurring amino acids. Subsequently, we profiled the reactivity of the resulting 160 variants against seven proteases involved in coagulation. These profiles formed the basis of an in silico prediction platform for SERPIN inhibitory behavior with combined P4 to P4’ RCL mutations, which were validated experimentally. This prediction platform accurately predicted SERPIN behavior against five out of the seven screened proteases, one of which was activated protein C (APC). Using these findings, a next-generation APC-inhibiting α1-antitrypsin variant was designed (KMPR/RIRA; / indicates the cleavage site). This variant attenuates blood loss in an in vivo hemophilia A model at a lower dosage than the previously developed variant AIKR/KIPP because of improved potency and specificity. We propose that this SERPIN-based RCL mutagenesis approach improves our understanding of SERPIN behavior and will facilitate the design of therapeutic SERPINs.

Structure of the nisin leader peptidase NisP revealing a C-terminal autocleavage activity

2014 ◽  
Vol 70 (6) ◽  
pp. 1499-1505 ◽  
Author(s):  
Yueyang Xu ◽  
Xin Li ◽  
Ruiqing Li ◽  
Shanshan Li ◽  
Hongqian Ni ◽  
...  
Keyword(s):  
Calcium Ion ◽  
Cleavage Site ◽  
Biological Significance ◽  
Leader Peptide ◽  
Spectrometric Analysis ◽  
Peptide Cleavage ◽  
Biochemical Assays ◽  
Precursor Peptide ◽  
Leader Peptidase ◽  
Active Precursor

Nisin is a widely used antibacterial lantibiotic polypeptide produced byLactococcus lactis. NisP belongs to the subtilase family and functions in the last step of nisin maturation as the leader-peptide peptidase. Deletion of thenisPgene in LAC71 results in the production of a non-active precursor peptide with the leader peptide unremoved. Here, the 1.1 Å resolution crystal structure of NisP is reported. The structure shows similarity to other subtilases, which can bind varying numbers of Ca atoms. However, no calcium was found in this NisP structure, and the predicted calcium-chelating residues were placed so as to not allow NisP to bind a calcium ion in this conformation. Interestingly, a short peptide corresponding to its own 635–647 sequence was found to bind to the active site of NisP. Biochemical assays and native mass-spectrometric analysis confirmed that NisP possesses an auto-cleavage site between residues Arg647 and Ser648. Further, it was shown that NisP mutated at the auto-cleavage site (R647P/S648P) had full catalytic activity for nisin leader-peptide cleavage, although the C-terminal region of NisP was no longer cleaved. Expressing this mutant inL. lactisLAC71 did not affect the production of nisin but did decrease the proliferation rate of the bacteria, suggesting the biological significance of the C-terminal auto-cleavage of NisP.

scholarly journals Directed Evolution of Retrovirus Envelope Protein Cytoplasmic Tails Guided by Functional Incorporation into Lentivirus Particles

2005 ◽  
Vol 79 (2) ◽  
pp. 834-840 ◽  
Author(s):  
Christoph A. Merten ◽  
Jörn Stitz ◽  
Gundula Braun ◽  
Eric M. Poeschla ◽  
Klaus Cichutek ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Cleavage Site ◽  
Leukemia Virus ◽  
Cytoplasmic Tail ◽  
Hiv Protease ◽  
Evolution System ◽  
Peptide Cleavage ◽  
C Terminus ◽  
Hiv 1 ◽  
Vector Particles

ABSTRACT In contrast to most gammaretrovirus envelope proteins (Env), the Gibbon ape leukemia virus (GaLV) Env protein does not mediate the infectivity of human immunodeficiency virus type 1 (HIV-1) particles. We made use of this observation to set up a directed evolution system by creating a library of GaLV Env variants diversified at three critical amino acids, all located around the R-peptide cleavage site within the cytoplasmic tail. This library was screened for variants that were able to functionally pseudotype HIV-1 vector particles. All selected Env variants mediated the infectivity of HIV-1 vector particles and encoded novel cytoplasmic tail motifs. They were efficiently incorporated into HIV particles, and the R peptide was processed by the HIV protease. Interestingly, in some of the selected variants, the R-peptide cleavage site had shifted closer to the C terminus. These data demonstrate a valuable approach for the engineering of chimeric viruses and vector particles.

The A16V signal peptide cleavage site mutation in the cationic trypsinogen gene and chronic pancreatitis

1999 ◽  
Vol 117 (6) ◽  
pp. 1508-1509 ◽  
Author(s):  
Jian-Min Chen ◽  
Odile Raguenes ◽  
Claude Ferec ◽  
Pierre H. Deprez ◽  
Christine Verellen-Dumoulin ◽  
...  
Keyword(s):  
Chronic Pancreatitis ◽  
Signal Peptide ◽  
Cleavage Site ◽  
Site Mutation ◽  
Peptide Cleavage ◽  
Signal Peptide Cleavage Site ◽  
Cationic Trypsinogen

scholarly journals Neutrophil Cathepsin G Proteolysis of Protease Activated Receptor 4 Generates a Novel, Functional Tethered Ligand

2021 ◽  
Author(s):  
Michelle L. Stoller ◽  
Indranil Basak ◽  
Frederik Denorme ◽  
Jesse W Rowley ◽  
James Alsobrooks ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Synthetic Peptide ◽  
Cleavage Site ◽  
Serine Proteases ◽  
Cathepsin G ◽  
Mass Spectrometry Analysis ◽  
Calcium Flux ◽  
Spectrometry Analysis ◽  
Thrombin Cleavage ◽  
Tethered Ligand

Platelet-neutrophil interactions regulate ischemic vascular injury. Platelets are activated by serine proteases that cleave protease activated receptor (PAR) amino-termini, resulting in an activating tethered ligand. Neutrophils release cathepsin G (CatG) at sites of injury and inflammation, which activates PAR4 but not PAR1, although the molecular mechanism of CatG-induced PAR4 activation is unknown. We show that blockade of the canonical PAR4 thrombin cleavage site did not alter CatG-induced platelet aggregation, suggesting CatG cleaves a different site than thrombin. Mass spectrometry analysis using PAR4 N-terminus peptides revealed CatG cleavage at Ser67-Arg68. A synthetic peptide, RALLLGWVPTR, representing the tethered ligand resulting from CatG proteolyzed PAR4, induced PAR4-dependent calcium flux and greater platelet aggregation than the thrombin-generated GYPGQV peptide. Mutating PAR4 Ser67 or Arg68 reduced CatG-induced calcium flux without affecting thrombin-induced calcium flux. Dog platelets, which contain a conserved CatG PAR4 Ser-Arg cleavage site, aggregated in response to human CatG and RALLLGWVPTR, while mouse (Ser-Gln) and rat (Ser-Glu) platelets, were unresponsive. Thus, CatG amputates the PAR4 thrombin cleavage site by cleavage at Ser67-Arg68 and activates PAR4 by generating a new functional tethered ligand. These findings support PAR4 as an important CatG signaling receptor and suggest a novel therapeutic approach for blocking platelet-neutrophil-mediated pathophysiologies.

scholarly journals Using serpins cysteine protease cross-specificity to possibly trap SARS-CoV-2 Mpro with reactive center loop chimera

2020 ◽  
Vol 134 (17) ◽  
pp. 2235-2241
Author(s):  
Mohamad Aman Jairajpuri ◽  
Shoyab Ansari
Keyword(s):  
Cysteine Protease ◽  
Active Site ◽  
Cleavage Site ◽  
Serine Proteases ◽  
Therapeutic Potential ◽  
Cysteine Proteases ◽  
Protease Inhibition ◽  
Reactive Center Loop ◽  
Main Protease ◽  
Reactive Center

Abstract Human serine protease inhibitors (serpins) are the main inhibitors of serine proteases, but some of them also have the capability to effectively inhibit cysteine proteases. Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) main protease (Mpro) is a chymotrypsin-type cysteine protease that is needed to produce functional proteins essential for virus replication and transcription. Serpin traps its target proteases by presenting a reactive center loop (RCL) as protease-specific cleavage site, resulting in protease inactivation. Mpro target sites with its active site serine and other flanking residues can possibly interact with serpins. Alternatively, RCL cleavage site of serpins with known evidence of inhibition of cysteine proteases can be replaced by Mpro target site to make chimeric proteins. Purified chimeric serpin can possibly inhibit Mpro that can be assessed indirectly by observing the decrease in ability of Mpro to cleave its chromogenic substrate. Chimeric serpins with best interaction and active site binding and with ability to form 1:1 serpin–Mpro complex in human plasma can be assessed by using SDS/PAGE and Western blot analysis with serpin antibody. Trapping SARS-CoV-2 Mpro cysteine protease using cross-class serpin cysteine protease inhibition activity is a novel idea with significant therapeutic potential.

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