Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.22.56 (caspase-3)
 35,750 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

MNEI (monocyte/neutrophil elastase inhibitor) is a 42 kDa serpin superfamily protein characterized initially as a fast-acting inhibitor of neutrophil elastase. Here we show that MNEI has a broader specificity, efficiently inhibiting proteases with elastase- and chymotrypsin-like specificities. Reaction of MNEI with neutrophil proteinase-3, an elastase-like protease, and porcine pancreatic elastase demonstrated rapid inhibition rate constants >10(7) M(-1) s(-1), similar to that observed for neutrophil elastase. Reactions of MNEI with chymotrypsin-like proteases were also rapid: cathepsin G from neutrophils (>10(6) M(-1) s(-1)), mast cell chymase (>10(5) M(-1) s(-1)), chymotrypsin (>10(6) M(-1) s(-1)), and prostate-specific antigen (PSA), which had the slowest rate constant at approximately 10(4) M(-1) s(-1). Inhibition of trypsin-like (plasmin, granzyme A, and thrombin) and caspase-like (granzyme B) serine proteases was not observed or highly inefficient (trypsin), nor was inhibition of proteases from the cysteine (caspase-1 and caspase-3) and metalloprotease (macrophage elastase, MMP-12) families. The stoichiometry of inhibition for all inhibitory reactions was near 1, and inhibitory complexes were resistant to dissociation by SDS, further indicating the specificity of MNEI for elastase- and chymotrypsin-like proteases. Determination of the reactive site of MNEI by N-terminal sequencing and mass analysis of reaction products identified two reactive sites, each with a different specificity. Cys(344), which corresponds to Met(358), the P(1) site of alpha1-antitrypsin, was the inhibitory site for elastase-like proteases and PSA, while the preceding residue, Phe(343), was the inhibitory site for chymotrypsin-like proteases. This study demonstrates that MNEI has two functional reactive sites corresponding to the predicted P(1) and P(2) positions of the reactive center loop. The data suggest that MNEI plays a regulatory role at extravascular sites to limit inflammatory damage due to proteases of cellular origin.
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PMID:The serpin MNEI inhibits elastase-like and chymotrypsin-like serine proteases through efficient reactions at two active sites. 1174 53

Apoptosis and necrosis are critical parameters of pancreatitis, the mechanisms of which remain unknown. Many characteristics of pancreatitis can be studied in vitro in pancreatic acini treated with high doses of cholecystokinin (CCK). We show here that CCK stimulates apoptosis and death signaling pathways in rat pancreatic acinar cells, including caspase activation, cytochrome c release, and mitochondrial depolarization. The mitochondrial dysfunction is mediated by upstream caspases (possibly caspase-8) and, in turn, leads to activation of caspase-3. CCK causes mitochondrial alterations through both permeability transition pore-dependent (cytochrome c release) and permeability transition pore-independent (mitochondrial depolarization) mechanisms. Caspase activation and mitochondrial alterations also occur in untreated pancreatic acinar cells; however, the underlying mechanisms are different. In particular, caspases protect untreated acinar cells from mitochondrial damage. We found that caspases not only mediate apoptosis but also regulate other parameters of CCK-induced acinar cell injury that are characteristic of pancreatitis; in particular, caspases negatively regulate necrosis and trypsin activation in acinar cells. The results suggest that the observed signaling pathways regulate parenchymal cell injury and death in CCK-induced pancreatitis. Protection against necrosis and trypsin activation by caspases can explain why the severity of pancreatitis in experimental models correlates inversely with the extent of apoptosis.
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PMID:Cholecystokinin induces caspase activation and mitochondrial dysfunction in pancreatic acinar cells. Roles in cell injury processes of pancreatitis. 1196 11

A trypsin inhibitor (PDTI) was isolated from Peltophorum dubium seeds by affinity chromatography on a thyroglobulin-agarose or a trypsin-agarose column. In both cases, SDS-PAGE showed two bands of M(r) 20,000 and 22,000, which could not be resolved. Their amino-terminal sequences were identical and similar to that of Kunitz-type soybean trypsin inhibitor (SBTI). Mass spectrometry analysis of tryptic digests of both bands showed 16 coincident peaks, suggesting that they are closely related proteins. The K(i)s for trypsin and chymotrypsin inhibitory activity of PDTI were 1.6 x 10(-7) and 1.3 x 10(-5)M, respectively. Lectin-like activity of PDTI and SBTI, detected by hemagglutination of rabbit erythrocytes, was inhibited by sialic acid-containing compounds. PDTI and SBTI caused apoptosis of Nb2 rat lymphoma cells, demonstrated by decrease of viability, DNA hypodiploidy, DNA fragmentation, and caspase-3-like activity. They had no effect on normal mouse splenocytes or lymphocytes, whereas they caused apoptosis of concanavalin A-stimulated mouse lymphocytes.
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PMID:A novel trypsin inhibitor from Peltophorum dubium seeds, with lectin-like properties, triggers rat lymphoma cell apoptosis. 1259 Sep 27

Accumulating evidence suggests that human herpesvirus type 6 (HHV-6) plays a pathogenic role in diseases of the central nervous system including multiple sclerosis (MS). Recent studies have indicated that HHV-6 DNA is detected with high frequency in MS lesions compared to normal-appearing white matter, implicating a role for HHV-6 in MS pathogenesis. It appears that T cells, which infiltrate into the brain in MS patients, and resident oligodendrocytes harbor HHV-6 virus in MS lesions. Because T cells infected with HHV-6 have elevated proinflammatory gene expression, we hypothesized that HHV-6 could be indirectly cytotoxic to glial cells, including oligodendrocytes. Supernatants from SupT1 cells infected with HHV-6 variant A (GS or U1102) or variant B (Z29) significantly reduced MO3.1 cell proliferation by 75% +/- 10%, 78% +/- 8% or 51% +/- 9%, respectively. HHV-6 viral supernatants (GS or U1102 or Z29) significantly increased MO3.1 or primary human oligodendrocyte precursor cells (OPCs) cell death, whereas primary human fetal astrocytes were not affected. Removal of HHV-6 virions or proteins by trypsin treatment from culture supernatants did not reverse the loss in oligodendrocyte proliferation or viability. Supernatants from HHV-6 GS or U1102 cultures were significantly more cytotoxic to MO3.1 cells or OPCs compared to supernatants from T cells infected with Z29. Dying oligodendrocytes did not have an apoptotic-like phenotype and toxicity was not inhibited by general inhibitor of apoptosis, ZVAD. Further, oligodendrocytes had minimal caspase-3 activation even in the presence of staurosporine, suggesting that cell death followed caspase-independent pathways. These results indicate that HHV-6 is indirectly cytotoxic to oligodendrocytes and that cell death is driven primarily by caspase-independent pathways.
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PMID:Human herpesvirus type 6 indirectly enhances oligodendrocyte cell death. 1312 68

We have examined the enzymatic activity of an uncleavable procaspase-3 mutant (D9A/D28A/D175A), which contains the wild-type catalytic residues in the active site. The results are compared to those for the mature caspase-3. Although at pH 7.5 and 25 degrees C the K(m) values are similar, the catalytic efficiency (k(cat)) is approximately 130-fold lower in the zymogen. The mature caspase-3 demonstrates a maximum activity at pH 7.4, whereas the maximum activity of procaspase-3 occurs at pH 8.3. The pK(a) values of both catalytic groups, H121 and C163, are shifted to higher pH for procaspase-3. We developed limited proteolysis assays using trypsin and V8 proteases, and we show that these assays allow the examination of amino acids in three of five active site loops. In addition, we examined the fluorescence emission of the two tryptophanyl residues in the active site over the pH range of 2.5-9 as well as the response to several quenching agents. Overall, the data suggest that the major conformational change that occurs upon maturation results in formation of the loop bundle among loops L4, L2, and L2'. The pK(a) values of both catalytic groups decrease as a result of the loop movements. However, loop L3, which comprises the bulk of the substrate binding pocket, does not appear to be unraveled and solvent-exposed, even at lower pH.
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PMID:An uncleavable procaspase-3 mutant has a lower catalytic efficiency but an active site similar to that of mature caspase-3. 1456 91

We report here that gene transfer using recombinant adenoviruses encoding interleukin (IL)-18 mutants induces potent antitumor activity in vivo. The precursor form of IL-18 (ProIL-18) is processed by caspase-1 to produce bioactive IL-18, but its cleavage by caspase-3 (CPP32) produces an inactive form. To prepare IL-18 molecules with an effective antitumor activity, a murine IL-18 mutant with the signal sequence of murine granulocyte-macrophage (GM)- colony stimulating factor (CSF) at the 5'-end of mature IL-18 cDNA (GMmIL-18) and human IL-18 mutant with the prepro leader sequence of trypsin (PPT), which is not cleaved by caspase-3 (PPThIL-18CPP32-), respectively, were constructed. Adenovirus vectors carrying GMmIL-18 or PPThIL-18CPP32- produced bioactive IL-18. Ad.GMmIL-18 had a more potent antitumor effect than Ad.mProIL-18 encoding immature IL-18 in renal cell adenocarcinoma (Renca) tumor-bearing mice. Tumor-specific cytotoxic T lymphocytes, the induction of Th1 cytokines, and an augmented natural killer (NK) cell activity were detected in Renca tumor-bearing mice treated with Ad.GMmIL-18. An immunohistological analysis revealed that CD4+ and CD8+ T cells abundantly infiltrated into tumors of mice treated with Ad.GMmIL-18. Huh-7 human hepatoma tumor growth in nude mice with a defect of T cell function was significantly inhibited by Ad.PPThIL-18CPP32- compared with Ad.hProIL-18 encoding immature IL-18. Nude mice treated with Ad.PPThIL-18CPP32- contained NK cells with increased cytotoxicity. The results suggest that the release of mature IL-18 in tumors is required for achieving an antitumor effect including tumor-specific cellular immunity and augmented NK cell-mediated cytotoxicity. These optimally designed IL-18 mutants could be useful for improving the antitumor effectiveness of wild-type IL-18.
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PMID:Adenovirus-mediated interleukin-18 mutant in vivo gene transfer inhibits tumor growth through the induction of T cell immunity and activation of natural killer cell cytotoxicity. 1504 62

Mutations in familial Parkinson's disease (PD) have been associated with the failure of protein degradation through the ubiquitin-proteasome system (UPS). Impairment of proteasome function has also been suggested to play a role in the pathogenesis of sporadic PD. We examined the proteasome activity in PC12 cells treated with 6-hydroxydopamine (6-OHDA), the dopamine synthetic derivate used in models of PD. We found that 6-OHDA treatment increased protein oxidation, as indicated by carbonyl group accumulation, and increased caspase-3 activity. In addition, there was an increase in trypsin-, chymotrypsin-, and postacidic-like proteasome activities in cells treated with 10-100 microM 6-OHDA, whereas higher doses caused a marked decline. 6-OHDA exposure also increased mRNA expression of the 19S regulatory subunit in a dose-dependent manner, whereas the expression of 20S- and 11S-subunit mRNAs did not change. Administration of the antioxidant N-acetylcysteine to 6-OHDA-treated cells prevented the alteration in proteasome functions. Moreover, reduction in cell viability owing to administration of proteasome inhibitor MG132 or lactacystin was partially prevented by the endogenous antioxidant-reduced glutathione. In conclusion, our data indicate that mild oxidative stress elevates proteasome activity in response to increase in protein damage. Severe oxidative insult might cause UPS failure, which leads to protein aggregation and cell death. Moreover, in the case of UPS inhibition or failure, the blockade of physiological reactive oxygen species production during normal aerobic metabolism is enough to ameliorate cell viability. Control of protein clearance by potent, brain-penetrating antioxidants might act to slow down the progression of PD.
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PMID:Oxidative stress, induced by 6-hydroxydopamine, reduces proteasome activities in PC12 cells: implications for the pathogenesis of Parkinson's disease. 1565 61

Mutations in the human cationic trypsinogen are associated with hereditary pancreatitis. The cDNA coding for human cationic trypsinogen was subcloned into the expression vector pcDNA3. The mutations R122H, N29I, A16V, D22G, and K23R were introduced by site directed mutagenesis. We constructed an expression vector coding for active trypsin by subcloning the cDNA of trypsin lacking the coding region for the trypsin activating peptide behind an appropriate signal peptide. Expression of protein was verified by Western blot and measurement of enzymatic activity. AR4-2J cells were transiently transfected with the different expression vectors and cell viability and intracellular caspase-3 activity were quantified. In contrast to wild-type trypsinogen, expression of active trypsin and mutated trypsinogens reduced cell viability of AR4-2J cells. Expression of trypsin and R122H trypsinogen induced caspase-3 activity. Acinar cells might react to intracellular trypsin activity by triggering apoptosis.
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PMID:Expression of mutated cationic trypsinogen reduces cellular viability in AR4-2J cells. 1603 33

Expression of isolated beta integrin cytoplasmic domains in cultured endothelial cells was reported to induce cell detachment and death. To test whether cell death was the cause or the consequence of cell detachment, we expressed isolated integrin beta1 cytoplasmic and transmembrane domains (CH1) in cultured human umbilical vein endothelial cells (HUVEC), and monitored detachment, viability, caspase activation and signaling. CH1 expression induced dose-dependent cell detachment. At 24 h over 90% of CH1-expressing HUVEC were detached but largely viable (>85%). No evidence of pro-caspase-8,-3, and PARP cleavage or suppression of phosphorylation of ERK, PKB and Ikappa-B was observed. The caspase inhibitor z-VAD did not prevent cell detachment. At 48 h, however, CH1-expressing cells were over 50% dead. As a comparison trypsin-mediated detachment resulted in a time-dependent cell death, paralleled by caspase-3 activation and suppression of ERK, PKB and Ikappa-B phosphoyrylation at 24 h or later after detachment. HUVEC stimulation with agents that strengthen integrin-mediated adhesion (i.e. PMA, the Src inhibitor PP2 and COMP-Ang1) did not prevent CH1-induced detachment. Expression of CH1 in rat carotid artery endothelial cells in vivo caused endothelial cell detachment and increased nuclear DNA fragmentation among detached cells. A construct lacking the integrin cytoplasmic domain (CH2) had no effect on adhesion and cell viability in vitro and in vivo. These results demonstrate that isolated beta1 cytoplasmic domain expression induces caspase-independent detachment of viable endothelial cells and that death is secondary to detachment (i.e. anoikis). They also reveal an essential role for integrins in the adhesion and survival of quiescent endothelial cells in vivo.
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PMID:Expressed isolated integrin beta1 subunit cytodomain induces endothelial cell death secondary to detachment. 1636 50

Endoplasmic reticulum (ER) stress mechanisms have been found to play critical roles in a number of diseases states, such as diabetes mellitus and Alzheimer disease, but whether they are involved in acute pancreatitis is unknown. Here we show for the first time that all major ER stress sensing and signaling mechanisms are present in exocrine acini and are activated early in the arginine model of experimental acute pancreatitis. Pancreatitis was induced in rats by intraperitoneal injection of 4.0 g/kg body wt arginine. Pancreatitis severity was assessed by analysis of serum amylase, pancreatic trypsin activity, water content, and histology. ER stress-related molecules PERK, eIF2alpha, ATF6, XBP-1, BiP, CHOP, and caspase-12 were analyzed. Arginine treatment induced rapid and severe pancreatitis, as indicated by increased serum amylase, pancreatic tissue edema, and acinar cell damage within 4 h. Arginine treatment also caused an early activation of ER stress, as indicated by phosphorylation of PERK and its downstream target eIF2alpha, ATF6 translocation into the nucleus (within 1 h), and upregulation of BiP (within 4 h). XBP-1 splicing and CHOP expression were observed within 8 h. After 24 h, increased activation of the ER stress-related proapoptotic molecule caspase-12 was observed along with an increase in caspase-3 activity and TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labeling (TUNEL) staining in exocrine acini. These results indicate that ER stress is an important early acinar cell event that likely contributes to the development of acute pancreatitis in the arginine model.
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PMID:Early activation of endoplasmic reticulum stress is associated with arginine-induced acute pancreatitis. 1657 87


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