Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.2.1.23 (beta-galactosidase)
 14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase is a key regulatory enzyme of cholesterol biosynthesis and is located in the endoplasmic reticulum (ER). A fusion protein, HMGal, consisting of the membrane domain of HMG-CoA reductase fused to Escherichia coli beta-galactosidase and expressed in Chinese hamster ovary (CHO) cells from the SV40 promoter, was previously constructed and was found to respond to regulatory signals for degradation in a similar fashion to the intact HMG-CoA reductase. Degradation of both HMG-CoA reductase and HMGal in CHO cells was enhanced by addition of mevalonate or low density lipoprotein (LDL). In this report we show that 2 cysteine protease inhibitors, N-acetyl-leucyl-leucyl-norleucinal (ALLN) and N-acetyl-leucyl-leucyl-methioninal (ALLM), completely inhibit the mevalonate- or LDL-accelerated degradation of HMG-CoA reductase and HMGal and also block the basal degradation of these enzymes. It has been shown that in vitro these protease inhibitors inhibit the activities of Ca(2+)-dependent neutral proteases as well as lysosomal proteases, including cathepsin L, cathepsin b, and cathepsin D. However, the mevalonate-accelerated degradation of HMG-CoA reductase and HMGal is not affected by lysosomotropic agents, suggesting that the site of action of these inhibitor peptides in preventing the degradation is not the cathepsins. In brefeldin A-treated cells, where protein export from the ER is blocked, ALLN is still effective in inhibiting the degradation of HMG-CoA reductase and HMGal. These results indicate the involvement of non-lysosomal Ca(2+)-dependent proteases in the basal and the accelerated degradation of HMG-CoA reductase and HMGal. Enzymatic assays in vitro and immunoblot analyses have revealed calpain- and calpastatin-like proteins in CHO cells. The activities and the amount of these proteins do not change under conditions of enhanced degradation, indicating that the levels of these proteins are not subject to mevalonate regulation.
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PMID:Inhibition of degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in vivo by cysteine protease inhibitors. 190 66

Sequence analysis of the BamHI F fragment of the genome of Bombyx mori nuclear polyhedrosis virus (BmNPV) revealed an open reading frame whose deduced amino acid sequence had homology to those of cysteine proteases of the papain superfamily. The putative cysteine protease sequence (BmNPV-CP) was 323 amino acids long and showed 35% identity to a cysteine proteinase precursor from Trypanosoma brucei. Of 36 residues conserved among cathepsins B, H, L, and S and papain, 31 were identical in BmNPV-CP. In order to determine the activity and function of the putative cysteine protease, a BmNPV mutant (BmCysPD) was constructed by homologous recombination of the protease gene with a beta-galactosidase gene cassette. BmCysPD-infected BmN cell extracts were significantly reduced in acid protease activity compared with wild-type virus-infected cell extracts. The cysteine protease inhibitor E-64 [trans-epoxysuccinylleucylamido-(4-guanidino)butane] inhibited wild-type virus-expressed protease activity. Deletion of the cysteine protease gene had no significant effect on viral growth or polyhedron production in BmN cells, indicating that the cysteine protease was not essential for viral replication in vitro. However, B. mori larvae infected with BmCysPD showed symptoms different from those of wild-type BmNPV-infected larvae, e.g., less degradation of the body, including fat body cells, white body surface color due presumably to undegraded epidermal cells, and an increase in the number of polyhedra released into the hemolymph. This is the first report of (i) a virus-encoded protease with activity on general substrates and (ii) evidence that a virus-encoded protease may play a role in degradation of infected larvae to facilitate horizontal transmission of the virus.
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PMID:A cysteine protease encoded by the baculovirus Bombyx mori nuclear polyhedrosis virus. 808 97

Lysosomal beta-galactosidase precursor is processed to a mature form and associated with protective protein in lysosomes. In this study we used two cysteine protease proinhibitors, E64-d for cathepsins B, S, H, and L, and CA074Me for cathepsin B. They are converted intracellularly to active forms, E-64c and CA074, respectively. Both active compounds inhibited maturation of the exogenous beta-galactosidase precursor, but E-64c did not inhibit further degradation to an inactive 50-kDa product. We concluded that cathepsin B participated exclusively in maturation of beta-galactosidase, and a non-cysteine protease was involved in further degradation and inactivation of the enzyme molecule.
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PMID:Maturation and degradation of beta-galactosidase in the post-Golgi compartment are regulated by cathepsin B and a non-cysteine protease. 942 40

A full-length cDNA encoding a SUMO-1-specific protease, named SUSP1, was identified and cloned for the first time from the human brain. Nucleotide sequence analysis of the cDNA containing an open reading frame of 3336 base pairs revealed that the protease consists of 1112 amino acids with a calculated molecular mass of 126,116 Da. Like yeast Ulp1, SUSP1 is a cysteine protease containing the well conserved His/Asp/Cys catalytic triad. SUSP1 expressed in Escherichia coli cells efficiently released SUMO-1 from SUMO-1. beta-galactosidase fusion but not from other ubiquitin-like protein fusions, including Smt3.beta-galactosidase, suggesting its role in the generation of matured SUMO-1 specifically from its precursors. Interestingly, reproductive organs, such as testis, ovary, and prostate, contained much higher amounts of SUSP1 mRNA than colon and peripheral blood leukocyte, whereas other tissues, such as heart and spleen, had little or none. In addition, confocal microscopy using green fluorescent protein.SUSP1 fusion showed that SUSP1 is exclusively localized to the cytoplasm of NIH3T3 and HeLa cells. These results suggest that SUSP1 may play a role in the regulation of SUMO-1-mediated cellular processes particularly related to reproduction.
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PMID:A new SUMO-1-specific protease, SUSP1, that is highly expressed in reproductive organs. 1079 85

Calpain, a cytosolic cysteine protease, requires calcium ions for activity. It has been reported that calpain is involved in the degradation of myofibrillar and neurofilament proteins, and the activation of phosphorylase b kinase and protein kinase C. More recently, calpain was shown to participate in apoptosis. In order to understand the calpain-related signal transduction pathway and its changes during hypertrophy, and especially in hypertension, we screened a human heart cDNA library to find proteins that interact with calpain. 1) Using PCR we amplified the full-length, domain II, domain III and domain IV cDNA of calpain (calcium-activated neutral protease, CANP) I large subunit respectively. 2) Then the fragments were cloned into pGBKT7 vector, resulting in 4 bait expression constructs (pGBKT7-CANP, pGBKT7-CANP II, pGBKT7-CANP III, and pG BKT7-CANP IV). 3) After 4 bait vectors were transformed into AH109 by the lithium acetate-mediated method, AH109/pGBKT7-CANP, AH109/pGBKT7-CANP II, AH109/pGBKT7-CANP III, and AH109/pGBKT7-CANP IV were obtained, respectively. 4) After the human heart cDNA library was sequentially transformed into AH109/ pGBKT7-CANP, 1000-1200 positive clones were grown on SD/Trp-Leu-Ade-His-. Only 150 positive clones were obtained through a colony-lift filter assay to detect beta-galactosidase activity. 5) Total 105 clones among above 150 positive clones were eliminated through that the duplicate, pseudopositive and autoactive detection, respectively. 6) Finally, sequencing eliminated clones with a wrong open reading frame (ORF). Eight clones were cancelled with wrong ORF. The remaining 37 positive clones were analyzed using BLAST software available on the Internet and classified as follows: 1. enzymes or proteins related to signal transduction in the cell; 2. contraction proteins 3. matrix proteins 4. unknown proteins. 7) In order to determine which domain of the calpain I large subunit was involved in the interaction with these real clones, the 37 clones were transformed into AH109/pGBKT7-CANP II, AH109/pGBKT7-CANP III or AH109/pGBKT7-CANP IV. Among these 37 clones, 29 clones could interact with domain II, 5 clones could interact with domain III and 6 clones could interact with domain IV. Thus, we successfully constructed 4 bait expression vectors, pGBKT7-CANP, pGBKT7-CANP II, pGBKT7-CANP III and pGBKT7-CANP IV, and obtained 37 real positive clones that interacted with the calpain I large subunit by screening a human heart cDNA library using pGBKT7-CANP as bait. Among them, 29 clones could interact with domain II of the calpain I large subunit, where the active site of calpain is located. Additional studies will be needed to clarify the calpain-related signal transduction pathway in greater detail.
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PMID:Screening the proteins that interact with calpain in a human heart cDNA library using a yeast two-hybrid system. 1235 55

Spodoptera frugiperda Sf-9 insect cells were infected with recombinant Autographa californica nuclear polyhedrosis virus at a low multiplicity of infection (MOI) (0.1), and the effect of dissolved oxygen (DO) on the production of a polyhedrin promoter-driven recombinant protein (beta-galactosidase), intrinsic proteases (carboxyl and cysteine proteases), and the virus was determined. The DO concentrations used in the present study were 45%, 25%, 5%, and 1.3% of air saturation. At 5% DO the cell growth following viral infection was greatest and beta-galactosidase was about 5-fold increased in volumetric yield compared to that at 45% and 25% DO, whereas the growth at 1.3% DO was extremely poor. The virus titer in the medium at 4-8 d post-infection (dpi) was also highest at 5% DO, but the titer was significantly decreased by further increasing the culture time. This was in part attributed to the fact that baculovirus is susceptible to oxidative inactivation under aerobic conditions. The DO dependency of the specific oxygen consumption rate of virus-infected and uninfected Sf-9 cells was expressed by a Monod-type equation. A critical DO, above which the rate of oxygen utilization is not limited by DO, was estimated to be 3.5% of air saturation for virus-infected Sf-9 cells. These results indicated that for a baculovirus-infected Sf-9 insect cell culture of low MOI, the optimal DO was likely to be approximately 5% of air saturation, which is above the critical DO for the infected Sf-9 cells but sufficiently low to reduce the possibility of the oxidative inactivation of virus. For the production of carboxyl and cysteine proteases, the accumulation behavior and concentrations did not significantly vary with DO, except that a peak of cysteine protease activity was observed intracellularly only at 5% DO, coinciding with beta-galactosidase production.
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PMID:Significant increase in recombinant protein production of a virus-infected Sf-9 insect cell culture of low MOI under low dissolved oxygen conditions. 1623 29

p94/calpain 3 is a skeletal muscle-specific member of the Ca(2+)-regulated cytosolic cysteine protease family, the calpains. Defective p94 protease activity originating from gene mutations causes a muscular dystrophy called calpainopathy, indicating the indispensability of p94 for muscle survival. Because of the existence of the p94-specific regions IS1 and IS2, p94 undergoes very rapid and exhaustive autolysis. To elucidate the physiological relevance of this unique activity, the autolytic profiles of p94 and the effect of the p94 binding protein, connectin/titin, on this process were investigated. In vitro analysis of p94 autolysis showed that autolysis in IS1 proceeds without immediate disassembly into fragments and that the newly identified cryptic autolytic site in IS2 is critical for disassembling autolyzed fragments. As a genetic system to assay p94 autolysis semiquantitatively, p94 was expressed in yeast as a hybrid protein between the DNA binding and activation domains of the yeast transcriptional activator Gal4. Transcriptional activation by the Gal4-p94:WT hybrid protein is precluded by p94 autolysis. Complete or partial loss of autolytic activity by C129S active site mutation, limb girdle muscular dystrophy type 2A pathogenic missense mutations, or PCR-based random mutagenesis could be detected by semiquantitative restoration of Gal4-dependent beta-galactosidase gene expression. Using this system, the N2A connectin fragment that binds to p94 was shown to suppress p94 autolytic disassembly. The proximity of the IS2 autolytic and connectin-binding sites in p94 suggested that N2A connectin suppresses IS2 autolysis. These data indicate the importance of p94-connectin interaction in the control of p94 functions by regulating autolytic decay of p94.
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PMID:Suppressed disassembly of autolyzing p94/CAPN3 by N2A connectin/titin in a genetic reporter system. 1662 76

The efficient degradation of internalized particulate matter is a principal objective of the macrophage's phagosome. Assessment of the true hydrolytic capacity within the phagosomal lumen is often difficult as it is subject to many factors beyond recruitment of lysosomal hydrolases. Here we outline three assays that allow quantitative measurements of serine-cysteine protease, triglyceride lipase, and beta-galactosidase activities within the phagosomes of macrophages, in real time. The assays utilize ratio fluorometry between particle-associated fluorogenic substrates and calibration fluorochromes to yield internally controlled values that record rates of substrate hydrolysis. The methods described utilize a spectrofluorometer for fluorometric measurements from a population of macrophages. These assays, however, can be expanded to high-throughput or single cell formats. In addition, this approach can be applied to measure a wide variety of phagosomal hydrolytic properties with the design of suitable fluorogenic substrates.
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PMID:Recording phagosome maturation through the real-time, spectrofluorometric measurement of hydrolytic activities. 1934 17