Strack P R, Waxman L, Fagan J M
Department of Animal Sciences, Rutgers University, New Brunswick, New Jersey 08903-0231, USA.
Biochemistry. 1996 Jun 4;35(22):7142-9. doi: 10.1021/bi9518048.
It is well established that the functional properties of proteins can be compromised by oxidative damage and, in vivo, proteins modified by oxidants are rapidly degraded. It was hypothesized that oxidants may also affect the ability of proteases to hydrolyze peptides and proteins. We therefore examined the effect of oxidants on the endopeptidase activities of the 650 kDa 20S proteasome or multicatalytic endopeptidase (MCP), which is thought to play a central role in nonlysosomal protein breakdown. Treatment of the MCP with the oxidant system, FeSO4-EDTA-ascorbate, stimulated the peptidase activities of the MCP while H2O2 treatment showed little or no stimulation. However, treatment of the MCP with FeSO4-EDTA-ascorbate or H2O2 stimulated proteinase activity by 480% and 730%, respectively. An endogenous activator of the MCP, PA28, stimulated the acidic, basic, and hydrophobic peptidase activities of the MCP, but had no effect on proteolytic activity. Treatment of PA28 with oxidants in the presence of MCP or alone did not greatly affect PA28's ability to activate the peptidase activities of the MCP. Using nondenaturing polyacrylamide gel electrophoresis, structural alterations in the enzyme which may be responsible for the activation of peptidase and protease activities following exposure to oxidants were investigated. Treatment of the MCP with reagents that activate proteolysis, including H2O2, as well as the serine protease inhibitor 3,4-dichloroisocoumarin and the cysteine protease inhibitor p-(chloromercuri) benzenesulfonic acid, all caused dissociation of the 650 kDa MCP. However, exposure to FeSO4-EDTA-ascorbate resulted in little or no dissociation of the complex. The MCP complex dissociated by p-(chloromercuri) benzenesulfonic acid could be reassociated upon treatment with the reducing agent dithiothreitol, but dithiothreitol failed to completely reassociate 3,4-dichloroisocoumarin- or H2O2 treated MCP. Therefore, chemical modification of the MCP can cause activation with varying degrees of complex dissociation. These results suggest that metabolites, such as reactive oxygen species, in addition to endogenous proteins, such as PA28, are capable of modulating MCP activity.
众所周知，蛋白质的功能特性会因氧化损伤而受损，并且在体内，被氧化剂修饰的蛋白质会迅速降解。据推测，氧化剂可能还会影响蛋白酶水解肽和蛋白质的能力。因此，我们研究了氧化剂对650 kDa 20S蛋白酶体或多催化内肽酶（MCP）的内肽酶活性的影响，该酶被认为在非溶酶体蛋白质分解中起核心作用。用氧化剂系统FeSO4 - EDTA - 抗坏血酸处理MCP，可刺激MCP的肽酶活性，而用H2O2处理则几乎没有刺激作用。然而，用FeSO4 - EDTA - 抗坏血酸或H2O2处理MCP分别使蛋白酶活性提高了480%和730%。MCP的内源性激活剂PA28可刺激MCP的酸性、碱性和疏水性肽酶活性，但对蛋白水解活性没有影响。在有MCP存在的情况下或单独用氧化剂处理PA28，对PA28激活MCP肽酶活性的能力没有太大影响。使用非变性聚丙烯酰胺凝胶电泳，研究了在暴露于氧化剂后可能导致肽酶和蛋白酶活性激活的酶的结构改变。用包括H2O2在内的激活蛋白水解的试剂以及丝氨酸蛋白酶抑制剂3,4 - 二氯异香豆素和半胱氨酸蛋白酶抑制剂对 - （氯汞基）苯磺酸处理MCP，均导致650 kDa的MCP解离。然而，暴露于FeSO4 - EDTA - 抗坏血酸导致该复合物几乎没有解离或不解离。被对 - （氯汞基）苯磺酸解离的MCP复合物在用还原剂二硫苏糖醇处理后可以重新结合，但二硫苏糖醇未能使3,4 - 二氯异香豆素或H2O2处理的MCP完全重新结合。因此，MCP的化学修饰可导致不同程度的复合物解离并激活其活性。这些结果表明，除了内源性蛋白质如PA28外，代谢产物如活性氧也能够调节MCP的活性。
