Implantation serine proteinase 2 is a monomeric enzyme with mixed serine proteolytic activity and can silence signalling via proteinase activated receptors

2013 ◽  
Vol 91 (6) ◽  
pp. 487-497
Author(s):  
Navneet Sharma ◽  
Jochen Fahr ◽  
Bernard Renaux ◽  
Mahmoud Saifeddine ◽  
Rajeev Kumar ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Expression System ◽  
Serine Proteinase ◽  
Receptor Activation ◽  
Peptide Sequence ◽  
Mouse Uterus ◽  
Peptide Cleavage ◽  
Pichia Pastoris Expression System ◽  
Proteinase Activated Receptors

Implantation serine proteinase 2 (ISP2), a S1 family serine proteinase, is known for its role in the critical processes of embryo hatching and implantation in the mouse uterus. Native implantation serine proteinases (ISPs) are co-expressed and co-exist as heterodimers in uterine and blastocyst tissues. The ISP1–ISP2 enzyme complex shows trypsin-like substrate specificity. In contrast, we found that ISP2, isolated as a 34 kDa monomer from a Pichia pastoris expression system, exhibited a mixed serine proteolytic substrate specificity, as determined by a phage display peptide cleavage approach and verified by the in vitro cleavage of synthetic peptides. Based upon the peptide sequence substrate selectivity, a database search identified many potential ISP2 targets of physiological relevance, including the proteinase activated receptor 2 (PAR2). The in vitro cleavage studies with PAR2-derived peptides confirmed the mixed substrate specificity of ISP2. Treatment of cell lines expressing proteinase-activated receptors (PARs) 1, 2, and 4 with ISP2 prevented receptor activation by either thrombin (PARs 1 and 4) or trypsin (PAR2). The disarming and silencing of PARs by ISP2 may play a role in successful embryo implantation.

scholarly journals Implantation Serine Proteinase 1 Exhibits Mixed Substrate Specificity that Silences Signaling via Proteinase-Activated Receptors

PLoS ONE ◽  
2011 ◽  
Vol 6 (11) ◽  
pp. e27888 ◽  
Author(s):  
Navneet Sharma ◽  
Rajeev Kumar ◽  
Bernard Renaux ◽  
Mahmoud Saifeddine ◽  
Sandra Nishikawa ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Serine Proteinase ◽  
Mixed Substrate ◽  
Proteinase Activated Receptors

scholarly journals Proteinase-activated Receptors, Targets for Kallikrein Signaling

2006 ◽  
Vol 281 (43) ◽  
pp. 32095-32112 ◽  
Author(s):  
Katerina Oikonomopoulou ◽  
Kristina K. Hansen ◽  
Mahmoud Saifeddine ◽  
Illa Tea ◽  
Michael Blaber ◽  
...  
Keyword(s):  
Calcium Signaling ◽  
Vascular Ring ◽  
Receptor Activation ◽  
Human Platelets ◽  
Aggregation Assay ◽  
Peptide Cleavage ◽  
Mass Spectral ◽  
Proteinase Activated Receptors ◽  
Platelet Aggregation Assay ◽  
Endogenous Regulators

Serine proteinases like thrombin can signal to cells by the cleavage/activation of proteinase-activated receptors (PARs). Although thrombin is a recognized physiological activator of PAR1 and PAR4, the endogenous enzymes responsible for activating PAR2 in settings other than the gastrointestinal system, where trypsin can activate PAR2, are unknown. We tested the hypothesis that the human tissue kallikrein (hK) family of proteinases regulates PAR signaling by using the following: 1) a high pressure liquid chromatography (HPLC)-mass spectral analysis of the cleavage products yielded upon incubation of hK5, -6, and -14 with synthetic PAR N-terminal peptide sequences representing the cleavage/activation motifs of PAR1, PAR2, and PAR4; 2) PAR-dependent calcium signaling responses in cells expressing PAR1, PAR2, and PAR4 and in human platelets; 3) a vascular ring vasorelaxation assay; and 4) a PAR4-dependent rat and human platelet aggregation assay. We found that hK5, -6, and -14 all yielded PAR peptide cleavage sequences consistent with either receptor activation or inactivation/disarming. Furthermore, hK14 was able to activate PAR1, PAR2, and PAR4 and to disarm/inhibit PAR1. Although hK5 and -6 were also able to activate PAR2, they failed to cause PAR4-dependent aggregation of rat and human platelets, although hK14 did. Furthermore, the relative potencies and maximum effects of hK14 and -6 to activate PAR2-mediated calcium signaling differed. Our data indicate that in physiological settings, hKs may represent important endogenous regulators of the PARs and that different hKs can have differential actions on PAR1, PAR2, and PAR4.

scholarly journals Activation of GPR116/ADGRF5 by its tethered agonist requires key amino acids in extracellular loop 2 of the transmembrane domain

2021 ◽  
Author(s):  
James P Bridges ◽  
Caterina Safina ◽  
Bernard Picard ◽  
Kari Brown ◽  
Alyssa Filuta ◽  
...  
Keyword(s):  
Amino Acids ◽  
Transmembrane Domain ◽  
Receptor Activation ◽  
Site Directed Mutagenesis ◽  
Peptide Sequence ◽  
Knock Out ◽  
Autocatalytic Cleavage ◽  
Knock Out Mice

The mechanistic details of the tethered agonist mode of activation for adhesion GPCRs has not been completely deciphered. We set out to investigate the physiologic importance of autocatalytic cleavage upstream of the agonistic peptide sequence, an event necessary for NTF displacement and subsequent receptor activation. To examine this hypothesis, we characterized tethered agonist-mediated activation of GPR116 in vitro and in vivo. A knock-in mouse expressing a non-cleavable GPR116 mutant phenocopies the pulmonary phenotype of GPR116 knock-out mice, demonstrating that tethered agonist-mediated receptor activation is indispensable for function in vivo. Using site-directed mutagenesis and species swapping approaches we identified key conserved amino acids for GPR116 activation in the tethered agonist sequence and in extracellular loops 2/3 (ECL2/3). We further highlight residues in transmembrane7 (TM7) that mediate stronger signaling in mouse versus human GPR116 and recapitulate these findings in a model supporting tethered agonist:ECL2 interactions for GPR116 activation.

scholarly journals Mapping residues of SUMO precursors essential in differential maturation by SUMO-specific protease, SENP1

2005 ◽  
Vol 386 (2) ◽  
pp. 325-330 ◽  
Author(s):  
Zheng XU ◽  
Shannon W. N. AU
Keyword(s):  
Escherichia Coli ◽  
Substrate Specificity ◽  
Tissue Distribution ◽  
Catalytic Domain ◽  
Expression System ◽  
Proteolytic Processing ◽  
Maturation Process ◽  
Specific Protease ◽  
Escherichia Coli Expression

SUMO (small ubiquitin-related modifier) is a member of the ubiquitin-like protein family that regulates cellular function of a variety of target proteins. SUMO proteins are expressed as their precursor forms. Cleavage of the residues after the ‘GG’ region of these precursors by SUMO-specific proteases in maturation is a prerequisite for subsequent sumoylation. To understand further this proteolytic processing, we expressed and purified SENP1 (sentrin-specific protease 1), one of the SUMO-specific proteases, using an Escherichia coli expression system. We show that SENP1 is capable of processing all SUMO-1, -2 and -3 in vitro; however, the proteolytic efficiency of SUMO-1 is the highest followed by SUMO-2 and -3. We demonstrate further that the catalytic domain of SENP1 (SENP1C) alone can determine the substrate specificity towards SUMO-1, -2 and -3. Replacement of the C-terminal fragments after the ‘GG’ region of SUMO-1 and -2 precursors with that of the SUMO-3, indicates that the C-terminal fragment is essential for efficient maturation. In mutagenesis analysis, we further map two residues immediately after the ‘GG’ region, which determine the differential maturation. Distinct patterns of tissue distribution of SENP1, SUMO-1, -2 and -3 are characterized. Taken together, we suggest that the observed differential maturation process has its physiological significance in the regulation of the sumoylation pathway.

PAR-2 elicits afferent arteriolar vasodilation by NO-dependent and NO-independent actions

2002 ◽  
Vol 282 (5) ◽  
pp. F891-F897 ◽  
Author(s):  
Greg Trottier ◽  
Morley Hollenberg ◽  
Xuemei Wang ◽  
Yu Gui ◽  
Kathy Loutzenhiser ◽  
...  
Keyword(s):  
Rat Kidney ◽  
Peptide Sequence ◽  
Afferent Arteriole ◽  
Proteinase Activated Receptors ◽  
Normal Rat ◽  
Afferent Arterioles ◽  
Arteriolar Vasodilation ◽  
G Protein Coupled ◽  
Arteriolar Diameter

Proteinase-activated receptors (PARs) are a novel class of G protein-coupled receptors that respond to signals through endogenous proteinases. PAR activation involves enzymatic cleavage of the extracellular NH2-terminal domain and unmasking of a new NH2 terminus, which serves as an anchored ligand to activate the receptor. At least four PAR subtypes have been identified. In the present study, we used the in vitro perfused hydronephrotic rat kidney to examine the effects of activating PAR-2 on the afferent arteriole. The synthetic peptide SLIGRL-NH2, which corresponds to the exposed ligand sequence and selectively activates PAR-2, did not alter basal afferent arteriolar diameter but caused a concentration-dependent vasodilation (3–30 μM) of arterioles preconstricted by angiotensin II (0.1 nM). A modified peptide sequence (LSIGRL-NH2, inactive at PAR-2) had no effect. This vasodilation was characterized by an initial transient component followed by a smaller sustained response. A similar pattern of vasodilation was seen when SLIGRL-NH2 was administered to isolated perfused normal rat kidney. The sustained component of the PAR-2-induced afferent arteriolar vasodilation was eliminated by nitric oxide (NO) synthase inhibition (100 μM nitro-l-arginine methyl ester). In contrast, the transient vasodilation persisted under these conditions. This transient response was not observed when afferent arterioles were preconstricted with elevated KCl, suggesting involvement of an endothelium-derived hyperpolarizing factor. Finally, RT-PCR revealed the presence of PAR-2 mRNA in isolated afferent arterioles. These findings indicate that PAR-2 is expressed in the afferent arteriole and that its activation elicits afferent arteriolar vasodilation by NO-dependent and NO-independent mechanisms.

scholarly journals Optimization of Pichia pastoris Expression System for High-Level Production of Margatoxin

2021 ◽  
Vol 12 ◽  
Author(s):  
Muhammad Umair Naseem ◽  
Gabor Tajti ◽  
Attila Gaspar ◽  
Tibor G. Szanto ◽  
Jesús Borrego ◽  
...  
Keyword(s):  
Ion Channels ◽  
Pichia Pastoris ◽  
Expression System ◽  
Scorpion Venom ◽  
Dissociation Rate ◽  
Binding Kinetics ◽  
Functional Assay ◽  
Activation Markers ◽  
Pichia Pastoris Expression System

Margatoxin (MgTx) is a high-affinity blocker of voltage-gated potassium (Kv) channels. It inhibits Kv1.1–Kv1.3 ion channels in picomolar concentrations. This toxin is widely used to study physiological function of Kv ion channels in various cell types, including immune cells. Isolation of native MgTx in large quantities from scorpion venom is not affordable. Chemical synthesis and recombinant production in Escherichia coli need in vitro oxidative refolding for proper disulfide bond formation, resulting in a very low yield of peptide production. The Pichia pastoris expression system offers an economical approach to overcome all these limitations and gives a higher yield of correctly refolded recombinant peptides. In this study, improved heterologous expression of recombinant MgTx (rMgTx) in P. pastoris was obtained by using preferential codons, selecting the hyper-resistant clone against Zeocin, and optimizing the culturing conditions. About 36 ± 4 mg/L of >98% pure His-tagged rMgTx (TrMgTx) was produced, which is a threefold higher yield than has been previously reported. Proteolytic digestion of TrMgTx with factor Xa generated untagged rMgTx (UrMgTx). Both TrMgTx and UrMgTx blocked the Kv1.2 and Kv1.3 currents (patch-clamp) (Kd for Kv1.2 were 64 and 14 pM, and for Kv1.3, 86 and 50 pM, respectively) with comparable potency to the native MgTx. The analysis of the binding kinetics showed that TrMgTx had a lower association rate than UrMgTx for both Kv1.2 and Kv1.3. The dissociation rate of both the analogues was the same for Kv1.3. However, in the case of Kv1.2, TrMgTx showed a much higher dissociation rate with full recovery of the block than UrMgTx. Moreover, in a biological functional assay, both peptides significantly downregulated the expression of early activation markers IL2R and CD40L in activated CD4+ TEM lymphocytes whose activation was Kv1.3 dependent. In conclusion, the authors report that the Pichia expression system is a powerful method to produce disulfide-rich peptides, the overexpression of which could be enhanced noticeably through optimization strategies, making it more cost-effective. Since the presence of the His-tag on rMgTx only mildly altered the block equilibrium and binding kinetics, recombinant toxins could be used in ion channel research without removing the tag and could thus reduce the cost and time demand for toxin production.

Insights into the biochemical and functional characterization of sortase E transpeptidase of Corynebacterium glutamicum

2019 ◽  
Vol 476 (24) ◽  
pp. 3835-3847 ◽  
Author(s):  
Aliyath Susmitha ◽  
Kesavan Madhavan Nampoothiri ◽  
Harsha Bajaj
Keyword(s):  
Protein Engineering ◽  
Cell Attachment ◽  
Functional Characterization ◽  
Protein Structures ◽  
Structural Similarity ◽  
Surface Proteins ◽  
Sortase A ◽  
Peptide Cleavage ◽  
New Findings

Most Gram-positive bacteria contain a membrane-bound transpeptidase known as sortase which covalently incorporates the surface proteins on to the cell wall. The sortase-displayed protein structures are involved in cell attachment, nutrient uptake and aerial hyphae formation. Among the six classes of sortase (A–F), sortase A of S. aureus is the well-characterized housekeeping enzyme considered as an ideal drug target and a valuable biochemical reagent for protein engineering. Similar to SrtA, class E sortase in GC rich bacteria plays a housekeeping role which is not studied extensively. However, C. glutamicum ATCC 13032, an industrially important organism known for amino acid production, carries a single putative sortase (NCgl2838) gene but neither in vitro peptide cleavage activity nor biochemical characterizations have been investigated. Here, we identified that the gene is having a sortase activity and analyzed its structural similarity with Cd-SrtF. The purified enzyme showed a greater affinity toward LAXTG substrate with a calculated KM of 12 ± 1 µM, one of the highest affinities reported for this class of enzyme. Moreover, site-directed mutation studies were carried to ascertain the structure functional relationship of Cg-SrtE and all these are new findings which will enable us to perceive exciting protein engineering applications with this class of enzyme from a non-pathogenic microbe.

Cloning and Expression of Recombinant Human Insulin Gene in Pichia pastoris

2019 ◽  
Vol 10 (01) ◽  
Author(s):  
Rafid A. Abdulkareem
Keyword(s):  
Pichia Pastoris ◽  
Human Insulin ◽  
Expression Vector ◽  
Expression System ◽  
Insulin Gene ◽  
Cloning And Expression ◽  
Pcr Analysis ◽  
Major Band ◽  
Human Insulin Gene ◽  
Pichia Pastoris Expression System

The main goal of the current study was cloning and expression of the human insulin gene in Pichia pastoris expression system, using genetic engineering techniques and its treatment application. Total RNA was purified from fresh normal human pancreatic tissue. RNA of good quality was chosen to obtain a first single strand cDNA. Human preproinsulin gene was amplified from cDNA strand, by using two sets of specific primers contain EcoR1 and Notl restriction sites. The amplified preproinsulin gene fragment was double digested with EcoRI and Not 1 restriction enzymes, then inserted into pPIC9K expression vector. The new pPIC9K-hpi constructive expression vector was transformed by the heat-shock method into the E.coli DH5α competent cells. pPic9k –hpi, which was propagated in the positive transformant E. coli cells, was isolated from cells and then linearised by restriction enzyme SalI, then transformed into Pichia pastoris GS115 using electroporation method. Genomic DNA of His+ transformants cell was extracted and used as a template for PCR analysis. The results showed, that the pPic9k – hpi was successfully integrated into the P. pastoris genome, for selected His+ transformants clones on the anticipated band at 330 bp, which is corresponded to the theoretical molecular size of the human insulin gene. To follow the insulin expression in transformans, Tricine–SDS gel electrophoresis and Western blot analysis were conducted. The results showed a successful expression of recombinant protein was detected by the presence of a single major band with about (5.8 KDa) on the gel. These bands correspond well with the size of human insulin with the theoretical molecular weight (5.8 KDa).

Prospective Application of Aptamer-based Assays and Therapeutics in Bloodstream Infections

2020 ◽  
Vol 20 (10) ◽  
pp. 831-840
Author(s):  
Weibin Li
Keyword(s):  
Pathogenic Bacteria ◽  
Genetic Diagnosis ◽  
Bloodstream Infections ◽  
High Specificity ◽  
Peptide Sequence ◽  
High Morbidity ◽  
Specificity And Sensitivity ◽  
Prospective Application ◽  
Small Molecule Therapeutics

Sepsis is still a severe health problem worldwide with high morbidity and mortality. Blood bacterial culture remains the gold standard for the detection of pathogenic bacteria in bloodstream infections, but it is time-consuming, and both the sophisticated equipment and well-trained personnel are required. Immunoassays and genetic diagnosis are expensive and limited to specificity and sensitivity. Aptamers are single-stranded deoxyribonucleic acid (ssDNA) and ribonucleic acid (RNA) oligonucleotide or peptide sequence generated in vitro based on the binding affinity of aptamer-target by a process known as Systematic Evolution of Ligands by Exponential Enrichment (SELEX). By taking several advantages over monoclonal antibodies and other conventional small-molecule therapeutics, such as high specificity and affinity, negligible batch-to-batch variation, flexible modification and production, thermal stability, low immunogenicity and lack of toxicity, aptamers are presently becoming promising novel diagnostic and therapeutic agents. This review describes the prospective application of aptamerbased laboratory diagnostic assays and therapeutics for pathogenic bacteria and toxins in bloodstream infections.

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