Duodenases are a small subfamily of ruminant intestinal serine proteases that have undergone a remarkable diversification in cleavage specificity

Ruminants have a very complex digestive system adapted for the digestion of cellulose rich food. Gene duplications have been central in the process of adapting their digestive system for this complex food source. One of the new loci involved in food digestion is the lysozyme c locus where cows have ten active such genes compared to a single gene in humans and where four of the bovine copies are expressed in the abomasum, the real stomach. The second locus that has become part of the ruminant digestive system is the chymase locus. The chymase locus encodes several of the major hematopoietic granule proteases. In ruminants, genes within the chymase locus have duplicated and some of them are expressed in the duodenum and are therefore called duodenases. To obtain information on their specificities and functions we produced six recombinant proteolytically active duodenases (three from cows, two from sheep and one from pigs). Two of the sheep duodenases were found to be highly specific tryptases and one of the bovine duodenases was a highly specific asp-ase. The remaining two bovine duodenases were dual enzymes with potent tryptase and chymase activities. In contrast, the pig enzyme was a chymase with no tryptase or asp-ase activity. These results point to a remarkable flexibility in both the primary and extended specificities within a single chromosomal locus that most likely has originated from one or a few genes by several rounds of local gene duplications. Interestingly, using the consensus cleavage site for the bovine asp-ase to screen the entire bovine proteome, it revealed Mucin-5B as one of the potential targets. Using the same strategy for one of the sheep tryptases, this enzyme was found to have potential cleavage sites in two chemokine receptors, CCR3 and 7, suggesting a role for this enzyme to suppress intestinal inflammation.

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COX-2: A Pivotal Enzyme in Mucosal Protection and Resolution of Inflammation

The Scientific World JOURNAL ◽

10.1100/tsw.2006.122 ◽

2006 ◽

Vol 6 ◽

pp. 577-588 ◽

Cited By ~ 50

Author(s):

John L. Wallace

Keyword(s):

Digestive System ◽

Intestinal Inflammation ◽

Mucosal Protection ◽

Cox Inhibitors ◽

Gastrointestinal Injury ◽

Inflammatory Drugs ◽

Long Term Consequences ◽

Cox 2 ◽

Cox 2 Inhibitors

The discovery of a second form of cyclooxygenase, COX-2, led to a burst of research aimed at the development of nonsteroidal anti-inflammatory drugs that would not damage the gastrointestinal tract. In the years since, this promise has only been partially fulfilled. Selective COX-2 inhibitors cause less gastric damage than conventional, nonselective COX inhibitors, but their use is still associated with significant gastrointestinal injury, and with toxicity in the renal and cardiovascular systems. COX-2 is now recognized as a source of mediators that produce many beneficial and detrimental effects in the digestive system. In this review, the roles of COX-2 in mucosal defense and injury are discussed. Furthermore, contributions of COX-2derived products to the long-term consequences of intestinal inflammation, including cancer, are reviewed.

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Activities of Thrombin and Factor Xa Are Essential for Replication of Hepatitis E Virus and Are Possibly Implicated in ORF1 Polyprotein Processing

Journal of Virology ◽

10.1128/jvi.01853-17 ◽

2018 ◽

Vol 92 (6) ◽

Cited By ~ 15

Author(s):

Gayatri D. Kanade ◽

Kunal D. Pingale ◽

Yogesh A. Karpe

Keyword(s):

Cell Culture ◽

Cell Line ◽

Liver Cell ◽

Serine Proteases ◽

Hepatitis E Virus ◽

Factor Xa ◽

Hepatitis E ◽

Cleavage Sites ◽

Polyprotein Processing ◽

Liver Cell Line

ABSTRACTHepatitis E virus (HEV) is a clinically important positive-sense RNA virus. The ORF1 of HEV encodes a nonstructural polyprotein of 1,693 amino acids. It is not clear whether the ORF1 polyprotein (pORF1) is processed into distinct enzymatic domains. Many researchers have attempted to understand the mechanisms of pORF1 processing. However, these studies gave various results and could never convincingly establish the mechanism of pORF1 processing. In this study, we demonstrated the possible role of thrombin and factor Xa in pORF1 processing. We observed that the HEV pORF1 polyprotein bears conserved cleavage sites of thrombin and factor Xa. Using a reverse genetics approach, we demonstrated that an HEV replicon having mutations in the cleavage sites of either thrombin or factor Xa could not replicate efficiently in cell culture. Further, we demonstratedin vitroprocessing when we incubated recombinant pORF1 fragments with thrombin, and we observed the processing of pORF1 polyprotein. The treatment of a liver cell line with a serine protease inhibitor as well as small interfering RNA (siRNA) knockdown of thrombin and factor Xa resulted in significant reduction in the replication of HEV. Thrombin and factor Xa have been well studied for their roles in blood clotting. Both of these proteins are believed to be present in the active form in the blood plasma. Interestingly, in this report, we demonstrated the presence of biologically active thrombin and factor Xa in a liver cell line. The results suggest that factor Xa and thrombin are essential for the replication of HEV and may be involved in pORF1 polyprotein processing of HEV.IMPORTANCEHepatitis E virus (HEV) causes a liver disorder called hepatitis in humans, which is mostly an acute and self-limiting infection in adults. A high mortality rate of about 30% is observed in HEV-infected pregnant women in developing countries. There is no convincing opinion about HEV ORF1 polyprotein processing owing to the variability of study results obtained so far. HEV pORF1 has cleavage sites for two host cellular serine proteases, thrombin and factor Xa, that are conserved among HEV genotypes. For the first time, this study demonstrated that thrombin and factor Xa cleavage sites on HEV pORF1 are obligatory for HEV replication. Intracellular biochemical activities of the said serine proteases are also essential for efficient HEV replication in cell culture and must be involved in pORF1 processing. This study sheds light on the presence and roles of clotting factors with respect to virus replication in the cells.

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A39 NOVEL GAIN OF FUNCTION NON-RECEPTOR TYROSINE KINASE MUTATIONS ARE LINKED TO THE PATHOGENESIS OF VEOIBD

Journal of the Canadian Association of Gastroenterology ◽

10.1093/jcag/gwz047.038 ◽

2020 ◽

Vol 3 (Supplement_1) ◽

pp. 46-48

Author(s):

M Mehta ◽

L Wang ◽

C Guo ◽

N Warner ◽

Q Li ◽

...

Keyword(s):

Tyrosine Kinase ◽

Western Blot ◽

Receptor Tyrosine Kinase ◽

Blot Analysis ◽

Western Blot Analysis ◽

Intestinal Inflammation ◽

De Novo ◽

Single Gene ◽

Gain Of Function

Abstract Background Very early-onset inflammatory bowel disease (VEOIBD) is an emerging global disease, that results in inflammation of the digestive tract. Severe forms of VEOIBD can be caused by mutations in a single gene (monogenic variants) and, can result in death. A candidate gene which codes for a non-receptor tyrosine kinase (nRTK) has recently been implicated as a monogenic cause of IBD (unpublished). Whole exome sequencing was performed in two unrelated children who presented with symptoms of IBD identifying two distinct de novo gain of function mutations (S550Y and P342T). Both mutations are located in the highly conserved region of the nRTK, and were predicted to have similar downstream effects. Furthermore, four other patients with a variety of adult-onset immune disorders have recently been identified with rare variants in the same gene (M450I, R42P, A353T, V433M, S550F) but, their potential gain of function status remains to be determined. Studies show that this nRTK is an essential mediator in inflammation. It is expressed in both intestinal epithelial and immune cells however, its role in infantile IBD is unclear. This protein is first activated by phosphorylation and is linked to activating downstream transcription factors such as ERK and JNK. All these target proteins play a meaningful role in intestinal inflammation in patients with IBD. Aims Since we identified P342T and S550Y to be gain of function, we wanted to determine if the new variants exhibit a similar downstream impact on target protein expression levels when compared with S550Y and P342T. We also wanted to identify if all variants can be rescued with a known nRTK inhibitor. It is hypothesized that the new variants are gain of function and that all variants can be rescued with the inhibitor. Methods Using western blot analysis, the activation of ERK, JNK and nRTK was compared between wildtype (WT) and mutants. This in vitro method helped identify the degree of activation. For the second part of the study, HEK293T cells were treated with inhibitor to test for a rescue of phenotypes via western blot analysis. Results Results show an increased activation of nRTK, ERK and JNK in all variants with S550Y and S550F having the highest activation. Furthermore, pharmacological inhibition using small molecular kinase inhibitors resulted in decreased activation of nRTK, ERK and JNK suggesting a rescue of phenotypes. Conclusions Characterizing the downstream functional impact of these nRTK variants is an important first step to determine if gain of function nRTK mutations drive IBD. With a rising prevalence of IBD worldwide, these findings may lead to the development of pharmacological nRTK inhibitors as a novel personalized therapeutic approach for these patients and possibly for the broader IBD population. Funding Agencies CIHR

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Targets of Neutrophil Influx and Weaponry: Therapeutic Opportunities for Chronic Obstructive Airway Disease

Journal of Immunology Research ◽

10.1155/2017/5273201 ◽

2017 ◽

Vol 2017 ◽

pp. 1-13 ◽

Cited By ~ 8

Author(s):

Carina Kärrman Mårdh ◽

James Root ◽

Mohib Uddin ◽

Kristina Stenvall ◽

Anna Malmgren ◽

...

Keyword(s):

Matrix Metalloproteinases ◽

Chemokine Receptors ◽

Serine Proteases ◽

Respiratory Diseases ◽

Tissue Injury ◽

Neutrophil Function ◽

Chronic Obstructive ◽

Primary Role ◽

Novel Therapy ◽

Neutrophil Influx

Neutrophils are important effector cells of antimicrobial immunity in an acute inflammatory response, with a primary role in the clearance of extracellular pathogens. However, in respiratory diseases such as asthma and chronic obstructive pulmonary disease (COPD), there is excessive infiltration and activation of neutrophils, subsequent production of reactive oxygen species, and release of serine proteases, matrix metalloproteinases, and myeloperoxidase—resulting in collateral damage as the cells infiltrate into the tissue. Increased neutrophil survival through dysregulated apoptosis facilitates continued release of neutrophil-derived mediators to perpetuate airway inflammation and tissue injury. Several target mechanisms have been investigated to address pathologic neutrophil biology and thereby provide a novel therapy for respiratory disease. These include neutrophil influx through inhibition of chemokine receptors CXCR2, CXCR1, and PI3Kγsignaling and neutrophil weaponry by protease inhibitors, targeting matrix metalloproteinases and neutrophil serine proteases. In addition, neutrophil function can be modulated using selective PI3Kδinhibitors. This review highlights the latest advances in targeting neutrophils and their function, discusses the opportunities and risks of neutrophil inhibition, and explores how we might better develop future strategies to regulate neutrophil influx and function for respiratory diseases in dire need of novel effective therapies.

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Effects of Humic Acids on Recovery of Salmonella Enterica Serovar Enteritidis

Annals of Animal Science ◽

10.1515/aoas-2017-0037 ◽

2018 ◽

Vol 18 (2) ◽

pp. 387-399 ◽

Cited By ~ 2

Author(s):

Jesus A. Maguey-Gonzalez ◽

Matias A. Michel ◽

Mikayla F.A. Baxter ◽

Bruno Solis-Cruz ◽

Daniel Hernandez-Patlan ◽

...

Keyword(s):

Humic Acids ◽

Broiler Chickens ◽

Salmonella Enteritidis ◽

Digestive System ◽

Intestinal Inflammation ◽

Gram Negative Bacteria ◽

Intestinal Colonization ◽

Gram Negative ◽

Intestinal Iga

AbstractTwo experiments were conducted to evaluate the effects of humic acids (HA) on recovery of Salmonella Enteritidis, in an in vitro digestive system and on intestinal colonization in neonate broiler chickens. In experiment 1, two runs using an in vitro digestion model with two sources of HA (commercial or natural extraction) at 0.1 or 0.2%, and inoculated with 107 CFU/tube of S. Enteritidis, were carried out. In experiment 2, one-day-old male broiler chickens were randomly allocated to one of two groups (n=25) with or without 0.2% of isolated HA from worm compost, and challenged with 106 CFU of S. Enteritidis per chicken at 10-d old. All chicks were euthanized 24-h post challenge, and were subjected to serum fluorescein isothiocyanate dextran (FITC-d) determination. A section of ileum was removed to obtain total concentration of IgA. Ceca-cecal tonsils were removed to evaluate Salmonella recovery, total lactic acid bacteria (LAB) and total Gram negative bacteria. In experiment 1, neither concentration of commercial nor natural HA were able to reduce the recovery of S. Enteritidis in any of the simulated compartments (P>0.05). Only the crop compartment showed significant differences in pH in both trials between control and treated groups. In experiment 2, no significant differences were observed in serum concentration of FITC-d, intestinal IgA, S. Enteritidis recovery, LAB or total Gram negative bacteria in the ceca between control and treated chickens. In conclusion, no effects of HA on recovery of Salmonella Enteritidis, in an in vitro digestive system and on intestinal colonization of Salmonella, bacterial counts in ceca, intestinal IgA and serum FITC-d in neonate broiler chickens were observed. Further studies to evaluate the effect of HA under feed restriction model as an inducer of intestinal inflammation are currently being conducted.

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Specificity of serine proteases for cleavage sites on proatrial natriuretic factor

Peptides ◽

10.1016/0196-9781(88)90192-1 ◽

1988 ◽

Vol 9 (6) ◽

pp. 1275-1283 ◽

Cited By ~ 7

Author(s):

A.J. Fischman ◽

G.M. Wildey ◽

G.R. Matsueda ◽

M.N. Margolies ◽

J.B. Zisfein ◽

...

Keyword(s):

Serine Proteases ◽

Cleavage Sites ◽

Natriuretic Factor

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Modulating hinge flexibility in the APP transmembrane domain alters γ-secretase cleavage

10.1101/375006 ◽

2018 ◽

Cited By ~ 2

Author(s):

Alexander Götz ◽

Nadine Mylonas ◽

Philipp Högel ◽

Mara Silber ◽

Hannes Heinel ◽

...

Keyword(s):

Transmembrane Domain ◽

Md Simulations ◽

Conformational Flexibility ◽

Hydrogen Deuterium Exchange ◽

Cleavage Sites ◽

Substrate Complex ◽

Cleavage Specificity ◽

Enzyme Substrate ◽

Hydrogen Deuterium ◽

Β Amyloid

ABSTRACTIntramembrane cleavage of the β-amyloid precursor protein C99 substrate by γ-secretase is implicated in Alzheimer’s disease pathogenesis. Since conformational flexibility of a di-glycine hinge in the C99 transmembrane domain (TMD) might be critical for γ-secretase cleavage, we mutated one of the glycine residues, G38, to a helix-stabilizing leucine and to a helix-distorting proline. CD, NMR and hydrogen/deuterium exchange measurements as well as MD simulations showed that the mutations distinctly altered the intrinsic structural and dynamical properties of the TMD. However, although helix destabilization/unfolding was not observed at the initial ε-cleavage sites of C99, both mutants impaired γ-secretase cleavage and altered its cleavage specificity. Moreover, helix flexibility enabled by the di-glycine hinge translated to motions of other helix parts. Our data suggest that both local helix stabilization and destabilization in the di-glycine hinge may decrease the occurrence of enzyme-substrate complex conformations required for normal catalysis and that hinge mobility can be conducive for productive substrate-enzyme interactions.

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Therapeutic targeting of coronavirus spike glycoprotein priming

10.21203/rs.3.rs-22208/v1 ◽

2020 ◽

Author(s):

Maurizio Pellecchia ◽

Elisa Barile ◽

Carlo Baggio ◽

Luca Gambini ◽

Sergey A. Shiryaev ◽

...

Keyword(s):

Serine Protease ◽

Protease Inhibitors ◽

Serine Proteases ◽

Viral Proteins ◽

Exact Nature ◽

Cleavage Sites ◽

Serine Protease Inhibitors ◽

Spike Glycoprotein ◽

Highly Pathogenic

Abstract Processing of certain viral proteins and bacterial toxins by host serine proteases is a frequent and critical step in virulence. The coronavirus spike glycoprotein contains three (S1, S2, and S2’) cleavage sites that are processed by human host proteases. The exact nature of these cleavage sites, and their respective processing proteases, can determine whether the virus can cross species, and the level of pathogenicity. Recent comparisons of the genomes of the highly pathogenic SARS-CoV2 and MERS-CoV, with less pathogenic strains (e.g., Bat-RaTG13, the bat homologue of SARS-CoV2) identified possible mutations in the receptor binding domain and in the S1 and S2’ cleavage sites of their spike glycoprotein. However there remains some confusion on the relative roles of the possible serine-proteases involved for priming. Using anthrax toxin as a model system, we show that in vivo inhibition of priming by pan-active serine protease inhibitors can be effective at suppressing virulence. Hence, our studies should encourage further efforts in developing either pan-serine protease inhibitors or inhibitor cocktails to target SARS-CoV2 and potentially ward off future pandemics that could develop because of the additional mutations in the S-protein priming sequence in coronaviruses.

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II. Modulating leukocyte recruitment to splanchnic organs to reduce inflammation

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00023.2003 ◽

2003 ◽

Vol 284 (5) ◽

pp. G729-G733 ◽

Cited By ~ 5

Author(s):

Claudine S. Bonder ◽

Paul Kubes

Keyword(s):

Chemokine Receptors ◽

Molecular Mechanisms ◽

Intestinal Inflammation ◽

Cell Types ◽

Neutrophil Recruitment ◽

Leukocyte Recruitment ◽

T Lymphocyte Subsets ◽

Growing Body ◽

Numerous Cell ◽

Inflammatory Bowel

A hallmark feature of intestinal inflammation is the recruitment and extravasation of numerous cell types from the blood to the afflicted site. Much of what we know about the mechanisms of leukocyte recruitment to splanchnic organs comes from an extensive series of studies on neutrophils in the mesenteric microvasculature. In this themes article, we highlight the important findings from these experiments but also emphasize some of the limitations. In fact, there is a growing body of evidence that neutrophil recruitment may be quite different in the mesentery than in other splanchnic organs. For example, the molecular mechanisms underlying neutrophil recruitment into the liver are quite different than the mesentery and are dependent on the type of inflammatory disease. We also discuss the effect of modulating leukocyte recruitment to splanchnic organs in chronic inflammation and emphasize that the approaches that have been successful in acute inflammation may be less effective in such conditions as inflammatory bowel disease (IBD). One obvious reason for this observation is the growing body of evidence to suggest that the initiation and maintenance of IBD is, in part, due to dysregulated or inappropriately activated populations of infiltrating T lymphocyte subsets. Therefore, we also discuss some interesting new approaches to limiting lymphocyte recruitment into the inflamed intestine either by targeting T helper (Th)1 vs. Th2 lymphocytes or perhaps by allowing the recruitment of regulatory T cells. Inhibiting specific adhesion molecules or specific chemokine receptors may work in this regard.

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Cofactor Directed Catalysis.

Blood ◽

10.1182/blood.v104.11.1715.1715 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1715-1715

Author(s):

Michael A. Bukys ◽

Paul Kim ◽

Melissa A. Blum ◽

Michael E. Nesheim ◽

Michael Kalafatis

Keyword(s):

Heavy Chain ◽

Serine Proteases ◽

Membrane Surface ◽

Limited Proteolysis ◽

Factor Xa ◽

Fold Increase ◽

Cleavage Sites ◽

Wild Type ◽

Factor Va ◽

Initial Cleavage

Abstract Blood coagulation involves specific serine proteases that are activated by limited proteolysis. The process results in the conversion of prothrombin to thrombin which in turn cleaves fibrinogen to produce the insoluble fibrin mesh. Prothrombin is activated physiologically by the prothrombinase complex, which is composed of the non-enzymatic cofactor, factor Va, the enzyme, factor Xa, and the substrate prothrombin associated on a cell membrane-surface in the presence of Ca2+. Membrane-bound factor Xa alone can activate prothrombin by two sequential cleavages at R271 and R320, however the incorporation of Factor Va into prothrombinase results in the reversal of the order of cleavages, different intermediates being generated, and a 300,000-fold increase in the overall rate of catalysis. Initial cleavage at R271 will produce fragment 1•2 and prethrombin-2 while initial cleavage at R320 results in the formation of meizothrombin which has optimum esterase activity and diminished clotting activity. While the existence of these pathways and the kinetics of the rates of the cleavages have long been established, the consequences of the interaction of the cofactor with the components of prothrombinase and the molecular mechanism by which factor Va reverses the order of cleavages and increases the rate of the overall catalysis is unknown. We used recombinant factor Va molecules mutated at specific sites representing the binding domains of factor Va heavy chain for factor Xa (factor Va with the mutations E323 → F, Y324 → F, E330 → M, and V331 → I, factor VaFF/MI) and prothrombin (factor Va with the mutations D695 → K, Y696 → F, D697 → K, and Y698 → F, factor Va2K2F) in combination with plasma-derived prothrombin and mutant prothrombin molecules rMZ-II (prothrombin with the substitution R155 → A, R284 → A, and R271 → A) and rP2-II (prothrombin with the substitutions R155 → A, R284 → A, and R320 → A) to determine the molecular contribution of factor Va to each of the prothrombin-activating cleavage sites separately. The rate of cleavage of plasma-derived prothrombin at R320/R271 by prothrombinase assembled with factor VaFF/MI was 17-fold slower compared to prothrombinase assembled with the wild type cofactor. The incorporation of factor Va2K2F into prothrombinase resulted in an enzymatic complex that was both unable to activate plasma-derived prothrombin following initial cleavages at R320, and impaired in its ability to accelerate prothrombin activation through initial cleavage R271. Similarly, while the rates of cleavage of rMZ-II and rP2-II by prothrombinase assembled with factor VaFF/MI were 18- and 9-fold respectively slower compared to prothrombinase assembled with wild type factor Va, cleavage of both molecules by prothrombinase assembled with factor Va2K2F was considerly impaired. These data demonstrate that while the interaction of factor Va heavy chain with factor Xa is necessary to achieve optimal rates for thrombin formation, the interaction of factor Va with prothrombin is required because it promotes both initial cleavage at R320 and accelerates the rate of the cleavage at R271. The data presented herein dissects the cofactor’s contribution to the rate of each of the two prothrombin-activating cleavage sites, demonstrates that the interaction of factor Va heavy chain with prothrombin is responsible for the reversal of cleavage order, and strongly suggest that factor Va directs catalysis by factor Xa within prothrombinase at two spatially distinct sites.

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