Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.24.27 (thermolysin)
 1,894 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We reported that several aquaporin-2 (AQP2) point mutants that cause nephrogenic diabetes insipidus (NDI) are retained in the endoplasmic reticulum (ER) of transfected mammalian cells and degraded but can be rescued by chemical chaperones to function as plasma membrane water channels (Tamarappoo, B. K., and Verkman, A. S. (1998) J. Clin. Invest. 101, 2257-2267). To test whether mutant AQP2 proteins are misfolded, AQP2 folding was assessed by comparative detergent extractability and limited proteolysis, and AQP2 degradation kinetics was measured by label-pulse-chase and immunoprecipitation. In ER membranes from transfected CHO cells containing [(35)S]methionine-labeled AQP2, mutants T126M and A147T were remarkably detergent-resistant; for example wild-type AQP2 was >95% solubilized by 0.5% CHAPS whereas T126M was <10% solubilized. E258K, an NDI-causing AQP2 mutant which is retained in the Golgi, is highly detergent soluble like wild-type AQP2. The mutants and wild-type AQP2 were equally susceptible to digestion by trypsin, thermolysin, and proteinase K. Stopped-flow light scattering measurements indicated that T126M AQP2 at the ER was fully functional as a water channel. Pulse-chase studies indicated that the increased degradation rates for T126M (t((1)/(2)) 2.5 h) and A147T (2 h) compared with wild-type AQP2 (4 h) involve a brefeldin A-resistant, ER-dependent degradation mechanism. After growth of cells for 48 h in the chemical chaperone glycerol, AQP2 mutants T126M and A147T became properly targeted and relatively detergent-soluble. These results provide evidence that NDI-causing mutant AQP2 proteins are misfolded, but functional, and that chemical chaperones both correct the trafficking and folding defects. Strategies to facilitate protein folding might thus have therapeutic efficacy in NDI.
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PMID:Misfolding of mutant aquaporin-2 water channels in nephrogenic diabetes insipidus. 1057 54

Engineered extremely thermostable variants of the thermolysin-like protease from Bacillus stearothermophilus possessing an introduced disulfide bond G8C/N60C (double mutant, DM) and six additional amino acid substitutions in the exposed loop region 56-69 (Boilysin, BLN) have been probed with respect to stability toward water-miscible organic solvents and detergents. The solvent concentrations where 50% of enzyme activity were irreversibly lost (C(50)) decreased in the order methanol > 2-propanol > dimethylsulfoxide > dioxane > acetonitrile > dimethylformamide > acetone. The C(50) values were remarkably higher for the thermostable variants than for the wild-type enzymes. Therefore, the stabilization of this loop region also protects the molecule from irreversible inactivation by solvents, and inactivation seems to follow principally the same mechanism as thermal inactivation. However, in contrast to thermal inactivation where the corresponding T(50) values of DM and BLN differed by 10 K, the differences of the C(50) values of DM and BLN were not significant. Detergents had great effects on proteolytic activities which were dependent on the individual detergent and its concentration, but mostly without significant differences between the enzyme variants. These effects were inactivating (SDS, sulfobetaine) or strongly activating (CTAB, CHAPS). Triton X-100 and Tween 20 were activating or inactivating at low and high concentrations, respectively. In all detergents, stabilities of the enzymes were strongly decreased. However, the more thermostable variants were affected by the detergents to the same extent as the wild-type enzymes suggesting that the mechanism of detergent inactivation is different from that of thermal inactivation.
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PMID:The stability of engineered thermostable neutral proteases from Bacillus stearothermophilus in organic solvents and detergents. 1716 9