Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.64 (proteinase K)
 4,071 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cleavage of cellular DNA into high molecular weight (predominantly 50 kb) fragments is an early event during apoptosis. We previously reported that this fragmentation was a Ca2+-independent process during apoptosis, which was induced by anticancer agents in human leukemia cells. The present study demonstrated that a high molecular weight DNA fragmentation activity (HDFA) was induced in the drug-treated cells and, upon fusion of the drug-treated cells with untreated target cells prelabeled with [14C]thymidine, caused fragmentation of the labeled DNA in the target cells. Furthermore, extracts of the drug-treated cells caused high molecular weight DNA fragmentation in nuclei isolated from untreated cells. Biochemical characterization of HDFA revealed the following properties: HDFA was proteinaceous in nature, as evidenced by its inactivation by heating or by digestion with proteinase K; HDFA required Mg2+ for optimal activity but was inhibited by Zn2+ and K+; HDFA was active in vitro at pH 6.0-8.0 and was inactive under more acidic conditions (pH < 6.0); addition of ATP (0.5-2 mM) substantially potentiated HDFA activity in isolated nuclei; and HDFA was not inhibited by actin (an inhibitor of DNase I) but was inhibited by the extracts from K562 cells, which were resistant to drug-induced apoptosis. The specific inhibitor of cysteine proteases (interleukin 1beta-converting enzyme protease family) blocked the generation of drug-induced high molecular weight DNA fragmentation in whole cells, whereas in isolated nuclei, the cysteine protease inhibitors did not prevent the cleavage of chromatin by exogenous HDFA. These results suggest that, once HDFA is activated during apoptosis, it does not require the presence of cysteine proteases for its endonucleolytic activity and that the cysteine proteases may be involved in the apoptotic process upstream of the activation of HDFA in whole cells.
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PMID:Biochemical characterization of the protein activity responsible for high molecular weight DNA fragmentation during drug-induced apoptosis. 927 6

Dendritic cells (DC) of the CD11c(+) myeloid phenotype have been implicated in the spread of scrapie in the host. Previously, we have shown that CD11c(+) DC can cause a rapid degradation of proteinase K-resistant prion proteins (PrP(Sc)) in vitro, indicating a possible role of these cells in the clearance of PrP(Sc). To determine the mechanisms of PrP(Sc) degradation, CD11c(+) DC that had been exposed to PrP(Sc) derived from a neuronal cell line (GT1-1) infected with scrapie (ScGT1-1) were treated with a battery of protease inhibitors. Following treatment with the cysteine protease inhibitors (2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methylbutane (E-64c), its ethyl ester (E-64d), and leupeptin, the degradation of PrP(Sc) was inhibited, while inhibitors of serine and aspartic and metalloproteases (aprotinin, pepstatin, and phosphoramidon) had no effect. An endogenous degradation of PrP(Sc) in ScGT1-1 cells was revealed by inhibiting the expression of cellular PrP (PrP(C)) by RNA interference, and this degradation could also be inhibited by the cysteine protease inhibitors. Our data show that PrP(Sc) is proteolytically cleaved preferentially by cysteine proteases in both CD11c(+) DC and ScGT1-1 cells and that the degradation of PrP(Sc) by proteases is different from that of PrP(C). Interference by protease inhibitors with DC-induced processing of PrP(Sc) has the potential to modify prion spread, clearance, and immunization in a host.
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PMID:Scrapie protein degradation by cysteine proteases in CD11c+ dendritic cells and GT1-1 neuronal cells. 1507 59

Calpain and calpastatin have been demonstrated to play many physiological roles in a variety of systems. It, therefore, appears important to study their localization and association in different suborganelles. Using immunoblot studies, we have identified 80 kDa m-calpain in both lumen and membrane of ER isolated from bovine pulmonary artery smooth muscle. Treatment of the ER with Na(2)CO(3) and proteinase K demonstrated that 80 kDa catalytic subunit and 28 kDa regulatory subunit (Rs) of m-calpain, and the 110-kDa and 70-kDa calpastatin (Cs) forms are localized in the cytosolic side of the ER membrane. Coimmunoprecipitation studies revealed that m-calpain is associated with calpastatin in the cytosolic face of the ER membrane. We have also identified m-calpain activity both in the ER membrane and lumen by casein-zymography. The casein-zymogram has also been utilized to demonstrate differential pattern of the effects of reversible and irreversible cysteine protease inhibitors on m-calpain activity. Thus, a potential site of Cs regulation of m-calpain activity is created by positioning Cs, 80 kDa and 28 kDa m-calpain in the cytosolic face of ER membrane. However, such is not the case for the 80-kDa m-calpain found within the lumen of the ER because of the conspicuous absence of 28 kDa Rs of m-calpain and Cs in this locale.
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PMID:Localization of m-calpain and calpastatin and studies of their association in pulmonary smooth muscle endoplasmic reticulum. 1765 25