scholarly journals A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation

10.3791/58892 ◽  
2019 ◽  
Author(s):  
Javier A. Jaimes ◽  
Jean K. Millet ◽  
Monty E. Goldstein ◽  
Gary R. Whittaker ◽  
Marco R. Straus
Keyword(s):  
Proteolytic Activity ◽  
Spike Protein ◽  
Peptide Cleavage ◽  
Protein Activation ◽  
Cleavage Assay ◽  
Fluorogenic Peptide

P5449Neurohumoral regulation of the low-, medium- and high-renin HFrEF phenotypes

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
S Prausmueller ◽  
H Arfsten ◽  
G Spinka ◽  
J F Novak ◽  
A Cho ◽  
...  
Keyword(s):  
Heart Failure ◽  
Univariate Analysis ◽  
Renin Angiotensin System ◽  
Plasma Renin ◽  
Peptide Cleavage ◽  
Reduced Ejection Fraction ◽  
Ras Activation ◽  
Cleavage Assay ◽  
All Cause Mortality ◽  
Vasoactive Peptide

Abstract Background Previous investigations of plasma Renin-Angiotensin-System (RAS) fingerprints of patients with heart failure with reduced ejection fraction (HFrEF) revealed the existence of low, medium and high renin phenotypes independently of disease severity. Plasma renin serves as an excellent surrogate for angiotensin levels. The different renin phenotypes could not only hypothetically respond differently to RAS blockade but associated alterations of other vasoactive peptide systems could elucidate disease mechanisms and novel targets for heart failure therapy. The study aimed to investigate the relation between RAS regulation and pathophysiologically relevant vasoactive peptide systems based on different renin phenotypes. Methods We prospectively enrolled 369 patients with stable HFrEF. Laboratory markers including NT-proBNP and active renin concentration (ARC) were assessed. Plasma NEP levels (sNEP), bioactive adrenomedullin (bio-ADM) and big-endothelin1 (bigET-1) were measured by ELISA (R&D systems, UK; Sphingotec GmbH, Germany and Eagle Biosciences, Austria). NEP activity was determined by a fluorimetric peptide cleavage assay. The correlation between biomarkers and association with all-cause mortality was assessed. sNEP, bio-ADM and bigET-1 levels as well as NEP activity between the different renin phenotypes (i.e. <15. percentile, 15.-85. percentile and >85. percentile of ARC) was compared. Results Median age was 65 (IQR 53–73) years, 75% of patients were male. Median NT-proBNP levels were 1936 (IQR 855–4126) pg/mL. Median ARC was 155 (29–569) μIE/mL, the low, medium and high renin HFrEF phenotypes showed median ARC levels of 4.2μIE/mL (IQR 2.0–7.8), 155.1μIE/mL (IQR 43.3–353.5) and 2360μIE/mL (IQR 1483–3250) μIE/mL. Median bigET-1 was 0.62pmol/L (IQR 0.42–1.10), bio-ADM 26.0pg/mL (IQR 16.1–46.7), sNEP 413pg/mL (IQR 0–4111) and NEP activity 2.36nmol/mL/min (IQR 1.16–4.59). There was no correlation between sNEP and NEP activity [r=0.09, p=0.088]. ARC did not show a meaningful correlation with any of the four biomarkers [p=ns for sNEP, NEP activity and bigET-1; r=0.13, p=0.018 for bio-ADM]. In the univariate analysis ARC, bigET-1, bio-ADM but not sNEP and NEP activity, were associated with outcome. This association remained significant after adjustment for age, gender and kidney function for all three markers and for ARC after adding NT-proBNP [adj. HR per 1-IQR increase of ARC 1.27 (95% CI 1.04–1.22), p=0.003]. There were no differences in bigET-1, bio-ADM and sNEP or NEP activity stratified by the different renin phenotypes (Figure1). Figure 1 Conclusions ARC is a risk factor for mortality in HFrEF patients, independently of NT-proBNP. Plasma NEP levels and activity neither correlated with each other nor were associated with outcome. Bio-ADM and bigET-1 were strong risk factors for all-cause mortality. Interestingly, neither NEP nor bio-ADM or bigET-1 were related to RAS-activation, suggesting that there is no direct relationship with RAS regulation.

Proteolytic activity of Co(III) complex of 1-oxa-4,7,10-triazacyclododecane: a new catalytic center for peptide-cleavage agents

2008 ◽  
Vol 14 (1) ◽  
pp. 151-157 ◽  
Author(s):  
Hye Mi Kim ◽  
Boonjae Jang ◽  
Young Eun Cheon ◽  
Myunghyun Paik Suh ◽  
Junghun Suh
Keyword(s):  
Proteolytic Activity ◽  
Catalytic Center ◽  
Peptide Cleavage

scholarly journals A Review on Expression, Pathological Roles, and Inhibition of TMPRSS2, the Serine Protease Responsible for SARS-CoV-2 Spike Protein Activation

Scientifica ◽  
2021 ◽  
Vol 2021 ◽  
pp. 1-9
Author(s):  
Jyotirmoy Sarker ◽  
Pritha Das ◽  
Sabarni Sarker ◽  
Apurba Kumar Roy ◽  
A. Z. M. Ruhul Momen
Keyword(s):  
Host Cell ◽  
Serine Protease ◽  
Infection Rate ◽  
Spike Protein ◽  
Host Cell Membrane ◽  
Protein Activation ◽  
Host Membrane ◽  
Expression Rate ◽  
Transmembrane Serine Protease ◽  
Potential Inhibitors

SARS-CoV-2, the coronavirus responsible for the COVID-19 pandemic, uses the host cell membrane receptor angiotensin-converting enzyme 2 (ACE2) for anchoring its spike protein, and the subsequent membrane fusion process is facilitated by host membrane proteases. Recent studies have shown that transmembrane serine protease 2 (TMPRSS2), a protease known for similar role in previous coronavirus infections, severe acute respiratory syndrome (SARS), and Middle East respiratory syndrome (MERS), is responsible for the proteolytic cleavage of the SARS-CoV-2 spike protein, enabling host cell fusion of the virus. TMPRSS2 is known to be expressed in the epithelial cells of different sites including gastrointestinal, respiratory, and genitourinary system. The infection site of the SARS-CoV-2 correlates with the coexpression sites of ACE2 and TMPRSS2. Besides, age-, sex-, and comorbidity-associated variation in infection rate correlates with the expression rate of TMPRSS2 in those groups. These findings provide valid reasons for the assumption that inhibiting TMPRSS2 can have a beneficial effect in reducing the cellular entry of the virus, ultimately affecting the infection rate and case severity. Several drug development studies are going on to develop potential inhibitors of the protease, using both conventional and computational approaches. Complete understanding of the biological roles of TMPRSS2 is necessary before such therapies are applied.

Detection of botulinum neurotoxin-A activity in food by peptide cleavage assay

2008 ◽  
Vol 126 (1-2) ◽  
pp. 135-139 ◽  
Author(s):  
R RASOOLY ◽  
L STANKER ◽  
J CARTER ◽  
P DO ◽  
L CHENG ◽  
...  
Keyword(s):  
Botulinum Neurotoxin ◽  
Botulinum Neurotoxin A ◽  
Peptide Cleavage ◽  
Cleavage Assay

scholarly journals Enzymatic Activities of Human Cytomegalovirus Maturational Protease Assemblin and Its Precursor (pPR, pUL80a): Maximal Activity of pPR Requires Self-Interaction through Its Scaffolding Domain

2007 ◽  
Vol 81 (8) ◽  
pp. 4091-4103 ◽  
Author(s):  
Edward J. Brignole ◽  
Wade Gibson
Keyword(s):  
Catalytic Domain ◽  
Covalent Binding ◽  
Point Mutations ◽  
Maximal Activity ◽  
Enzymatic Activities ◽  
Unexpected Finding ◽  
Peptide Cleavage ◽  
Metal Affinity ◽  
Cleavage Assay ◽  
Catalytic Sites

ABSTRACT Herpesviruses encode an essential, maturational serine protease whose catalytic domain, assemblin (28 kDa), is released by self-cleavage from a 74-kDa precursor (pPR, pUL80a). Although there is considerable information about the structure and enzymatic characteristics of assemblin, a potential pharmacologic target, comparatively little is known about these features of the precursor. To begin studying pPR, we introduced five point mutations that stabilize it against self-cleavage at its internal (I), cryptic (C), release (R), and maturational (M) sites and at a newly discovered “tail” (T) site. The resulting mutants, called ICRM-pPR and ICRMT-pPR, were expressed in bacteria, denatured in urea, purified by immobilized metal affinity chromatography, and renatured by a two-step dialysis procedure and by a new method of sedimentation into glycerol gradients. The enzymatic activities of the pPR mutants were indistinguishable from that of IC-assemblin prepared in parallel for comparison, as determined by using a fluorogenic peptide cleavage assay, and approximated rates previously reported for purified assemblin. The percentage of active enzyme in the preparations was also comparable, as determined by using a covalent-binding suicide substrate. An unexpected finding was that, in the absence of the kosmotrope Na2SO4, optimal activity of pPR requires interaction through its scaffolding domain. We conclude that although the enzymatic activities of assemblin and its precursor are comparable, there may be differences in how their catalytic sites become fully activated.

Catalytic Properties of Intramembrane Aspartyl Protease Substrate Hydrolysis Evaluated Using a FRET Peptide Cleavage Assay

2015 ◽  
Vol 10 (9) ◽  
pp. 2166-2174 ◽  
Author(s):  
Swe-Htet Naing ◽  
Krishna M. Vukoti ◽  
Jason E. Drury ◽  
Jennifer L. Johnson ◽  
Sibel Kalyoncu ◽  
...  
Keyword(s):  
Catalytic Properties ◽  
Aspartyl Protease ◽  
Peptide Cleavage ◽  
Cleavage Assay ◽  
Protease Substrate ◽  
Substrate Hydrolysis

scholarly journals Ara12 subtilisin-like protease from Arabidopsis thaliana: purification, substrate specificity and tissue localization

2003 ◽  
Vol 370 (1) ◽  
pp. 57-67 ◽  
Author(s):  
John M.U. HAMILTON ◽  
David J. SIMPSON ◽  
Stefan C. HYMAN ◽  
Bongani K. NDIMBA ◽  
Antoni R. SLABAS
Keyword(s):  
Arabidopsis Thaliana ◽  
Substrate Specificity ◽  
Proteolytic Activity ◽  
Hydrophobic Interaction Chromatography ◽  
Cell Suspension Cultures ◽  
Immunogold Labelling ◽  
Peptide Substrates ◽  
Hydrophobic Residues ◽  
Acidic Conditions ◽  
Fluorogenic Peptide

A C-terminal portion of Ara12 subtilisin-like protease (residues 542—757) was expressed in Escherichia coli cells as a fusion protein bound to maltose binding protein. Polyclonal antisera raised against the expressed protein were used to examine the tissue specificity and subcellular localization of Ara12. The protease was found predominantly in the silique and stem of plants, but was hardly detectable in leaf and not seen in root tissue. The distribution observed using immunological techniques is different from that seen by an RNA analysis study, which demonstrated similar mRNA abundance in the stem and leaves. Using immunogold labelling, Ara12 was shown to have an extracellular localization and was found in the intercellular spaces in stem tissue. Ara12 protease was purified to homogeneity from Arabidopsis thaliana cell suspension cultures by anion exchange and hydrophobic interaction chromatography. Proteolytic activity of Ara12 was inhibited by a number of serine protease inhibitors, but was almost unaffected by inhibitors of other catalytic classes of proteases. Optimal proteolytic activity was displayed under acidic conditions (pH5.0). Ara12 activity was relatively thermostable and was stimulated in the presence of Ca2+ ions. Substrate specificity studies were conducted using a series of internally quenched fluorogenic peptide substrates. At the P1 position of substrates, hydrophobic residues, such as Phe and Ala, were preferred to Arg, whilst at the P1′ position, Asp, Leu and Ala were most favoured. Possible functions of Ara12 are discussed in the light of the involvement of a number of plant subtilisin-like proteases in morphogenesis.

Sem Observations in Gastric Mucosa Exposed to Progressive Enzymatic Etching

1980 ◽  
Vol 38 ◽  
pp. 570-571
Author(s):  
C.A.E. Lemmi ◽  
D. Booth ◽  
G.E. Adomian
Keyword(s):  
Gastric Mucosa ◽  
Critical Point ◽  
Proteolytic Activity ◽  
Etching Process ◽  
Cellular Types

In order to enrich populations of homogeneous cellular types we dissociated gastric mucosa by enzymatic techniques. In addition, we used SEM to monitor the progressive etching of the mucosa. Two enzymes were tested: collagenase III with minimum proteolytic activity and Pronase with broader proteolytic effects. The gastric mucosa was exposed to the effect of the enzymes using everted stomach preparations. In this way the digestive action occured progressively from the lumen of the stomach toward the base of the glands. This “etching” process could be monitored conveniently by SEM. After incubation for periods varying from 30 to 210 minutes the tissues were stretched on dental wax, fixed in 2 % glutaralheyde, post-fixed in osmium, dehydrated, critical point dryed and coated with gold. A model MSM-5 “Mini-SEM” was used for observation. Gentle uncurling of the preparation before coating with gold produced fractures which revealed the structure of the gastric glandsin more detail.

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.

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