Inhibition of Prolyl Oligopeptidase by Fmoc‐Aminoacylpyrrolidine‐2‐Nitriles

Li, Jingrong; Wilk, Elizabeth; Wilk, Sherwin

doi:10.1046/j.1471-4159.1996.66052105.x

Cited by 26 publications

(22 citation statements)

References 31 publications

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“…2 The inhibition of PRCP by Fmoc-Ala-Pyr-CN or Z-Pro-Pro-aldehyde-dimethyl acetate, two prolyl oligopeptidase inhibitors, provides evidence for its varied features. However, the relatively flat slope of inhibition produced by FmocAla-Pyr-CN probably reflects the fact that this inhibitor was developed against prolyl oligopeptidases and not prolylcarboxypeptidase (21). How PRCP cleaves PK to activate it to kallikrein is not completely known.…”

Section: Discussionmentioning

confidence: 99%

“…Further investigations determined whether increasing concentrations of angiotensin II, angiotensin II-(1-7), bradykinin, bradykinin-(1-5) (peptide RPPGF), or Fmoc-Ala-Pyr-CN inhibited PK activation by the PKA. Fmoc-AlaPyr-CN is a prolyl oligopeptidase inhibitor (generously provided by Dr. Sherwin Wilk, Mount Sinai Medical School, New York) (21). The ability of the partially purified PKA to activate FXI and to clot factor XII-deficient plasma (George King, Overland Park, KS) was performed by an amidolytic assay using 0.8 mM L-pyroglutamyl-L-prolyl-L-arginine-pNA (S2366, Dia-Pharm, Franklin, OH) and by factor XII coagulant assay, respectively (20).…”

Section: Materials-frozen Human Umbilical Vein Endothelial Cells (Huvec)mentioning

confidence: 99%

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Identification and Characterization of Prolylcarboxypeptidase as an Endothelial Cell Prekallikrein Activator

Shariat‐Madar

Mahdi

Schmaier

2002

Journal of Biological Chemistry

249

252

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Our recent investigations have postulated a human umbilical vein endothelial cell (HUVEC)-associated prekallikrein activator (PKA). When prekallikrein (PK) assembles on high molecular weight kininogen on HU-VEC, PK is activated to kallikrein. PKA was found in the 15,800 ؋ g pellet of HUVEC lysates using an assay that measures PK activation only when bound to high molecular weight kininogen linked to microtiter plates. Sequential DEAE, wheat germ lectin affinity, and hydroxyapatite chromatography resulted in four protein bands on SDS-PAGE. One protein in the 73-kDa band was identified by amino acid sequencing as prolylcarboxypeptidase (PRCP). On gel filtration, PKA activity was a single homogenous peak identical in migration to the 73-kDa immunoblot of PRCP. Anti-PRCP inhibits PKA activity and PK activation on HUVEC. Purified PKA was blocked by diisopropyl fluorophosphate (1 mM), phenylmethylsulfonyl fluoride (3 mM), leupeptin (100 M), antipain (IC 50 ‫؍‬ 2 M), HgCl 2 (IC 50 ‫؍‬ 500 M), Z-Pro-Pro-aldehydedimethyl acetate (IC 50 ‫؍‬ 1 M), and corn trypsin inhibitor (IC 50 ‫؍‬ 40 nM). PKA did not correct the coagulant defect in factor XII deficient plasma, was purified from HUVEC cultured in factor XII-deficient serum, was not detected by antibody to factor XII, did not activate FXI, and was not inhibited by a neutralizing antibody to FXII. Angiotensin II (IC 50 ‫؍‬ 2 M) or bradykinin (IC 50 ‫؍‬ 100 M), but not angiotensin II-(1-7) or bradykinin 1-5 , and the prolyl oligopeptidase inhibitor Fmoc-AlaPyr-CN (IC 50 ‫؍‬ 50 nM) also blocked purified PKA activation of PK. The K m of PK activation by PRCP is 6.7 nM. PRCP antigen is present on the membrane of fixed but not permeabilized HUVEC. PRCP appears to be a HU-VEC-associated PK activator.

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Section: Discussionmentioning

confidence: 99%

Section: Materials-frozen Human Umbilical Vein Endothelial Cells (Huvec)mentioning

confidence: 99%

Identification and Characterization of Prolylcarboxypeptidase as an Endothelial Cell Prekallikrein Activator

Shariat‐Madar

Mahdi

Schmaier

2002

Journal of Biological Chemistry

249

252

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“…Peptidyl ␣-keto heterocycles were designed to act as mechanism-based inhibitors of serine-proteases, including POPs, by interacting with both the serine hydroxyl group and the histidine imidazole ring of the catalytic triad (46). Nitrile derivatives have been reported to exert potent inhibitory effects toward POP (47). By associating both reactive groups with the substrate recognition sequence, highly selective inhibitors for POP Tc80 were obtained (Table I, selectivity ratio Ͼ3000).…”

Section: Discussionmentioning

confidence: 99%

Trypanosoma cruzi Prolyl Oligopeptidase Tc80 Is Involved in Nonphagocytic Mammalian Cell Invasion by Trypomastigotes

Grellier¹,

Vendeville²,

Joyeau³

et al. 2001

Journal of Biological Chemistry

116

107

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Trypanosoma cruzi is an intracellular protozoan parasite able to invade a wide variety of mammalian cells. To have access to the target organs/cells, the parasite must cross the basal laminae and the extracellular matrix (ECM). We previously characterized an 80-kDa proteinase (Tc80) secreted by the infective trypomastigotes that hydrolyzes native collagens and might be involved in infection by degrading ECM components. Here, we present evidence indicating a role for Tc80 in the invasion of nonphagocytic cells. Tc80 was classified as a member of the prolyl oligopeptidase (POP) family of serine proteases and was also found to hydrolyze fibronectin. Selective inhibitors for POP Tc80 were synthesized that blocked parasite entry into cells. Blockage occurred when trypomastigotes were preincubated with irreversible inhibitors but not after host cell preincubation, and the blockage correlated with inhibition of POP Tc80 activity in treated parasites. These data and the enzyme location inside a vesicular compartment close to the flagellar pocket, a specialized domain in endocytosis/exocytosis, strongly suggest a role for POP Tc80 in the maturation of parasite protein(s) and/or, after secretion, in a local action on parasite or host cell/ECM components required for invasion.

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“…10 Tissue was desegregated and diluted up to 150 g/mL total protein with cold phosphate-saline buffer; 100-L aliquots of the homogenate were incubated at 37°C with: (1) Safer, University of Pennsylvania, Philadelphia, Pa), 20 was dissolved in water. Three different POPi were used: Z-prolyl-prolinal (Z-ProPro, Biomol), Fmoc-prolyl-pyrrolidine-2-nitrile (Fmoc-Pro-PyrrCN, a gift of S. Wilk, Mount Sinai School of Medicine, NY), 21 and S17092 (a gift of P. Vanhoutte, Institut de Recherches Internationales Servier, France). 22 Stock solutions of POPi were made with 20% DMSO, then diluted to their final concentration with water.…”

Section: Effect Of Popi On Ac-sdkp Synthesis In Vitromentioning

confidence: 99%

Prolyl Oligopeptidase Is Involved in Release of the Antifibrotic Peptide Ac-SDKP

et al. 2004

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Abstract-N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a ubiquitous tetrapeptide hydrolyzed almost exclusively by angiotensin-converting enzyme (ACE). Chronic treatment with Ac-SDKP decreases cardiac and renal fibrosis and inflammatory cell infiltration in hypertensive rats. However, very little is known about endogenous synthesis of Ac-SDKP, except that thymosin-␤ 4 may be the most likely precursor. Two enzymes are potentially able to release Ac-SDKP from thymosin-␤ 4 : prolyl oligopeptidase (POP) and endoproteinase asp-N. POP is widely present and active in several tissues and biological fluids, whereas endoproteinase asp-N appears to be lacking in mammals. Therefore, we hypothesized that POP is the main enzyme involved in synthesizing the antifibrotic peptide Ac-SDKP. We investigated in vitro and in vivo production of Ac-SDKP. Using kidney cortex homogenates, we observed that Ac-SDKP was generated in a time-dependent manner in the presence of exogenous thymosin-␤ 4 , and this generation was significantly inhibited by several POP inhibitors (POPi), Z-prolyl-prolinal, Fmoc-prolyl-pyrrolidine-2-nitrile, and S17092. Long-term administration of S17092 in rats significantly decreased endogenous levels of Ac-SDKP in the plasma (from 1.76Ϯ0.2 to 1.01Ϯ0.1 nM), heart (from 2.31Ϯ0.21 to 0.83Ϯ0.09 pmol/mg protein), and kidneys (from 5.62Ϯ0.34 to 2.86Ϯ0.76 pmol/mg protein). As expected, ACE inhibitors significantly increased endogenous levels of Ac-SDKP in the plasma, heart, and kidney, whereas coadministration of POPi prevented this increase. We concluded that POP is the main enzyme responsible for synthesis of the antifibrotic peptide

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Inhibition of Prolyl Oligopeptidase by Fmoc‐Aminoacylpyrrolidine‐2‐Nitriles

Cited by 26 publications

References 31 publications

Identification and Characterization of Prolylcarboxypeptidase as an Endothelial Cell Prekallikrein Activator

Identification and Characterization of Prolylcarboxypeptidase as an Endothelial Cell Prekallikrein Activator

Trypanosoma cruzi Prolyl Oligopeptidase Tc80 Is Involved in Nonphagocytic Mammalian Cell Invasion by Trypomastigotes

Prolyl Oligopeptidase Is Involved in Release of the Antifibrotic Peptide Ac-SDKP

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