DEGRADATION OF ACTIVE OXYGEN DAMAGED PROTEIN BY THE MULTICATALYTIC PROTEASE

多催化蛋白酶降解活性氧损伤的蛋白质

• 批准号: 3779493
• 负责人: B FRIGUET
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3779493
• 关键词: Escherichia coli; aging; endopeptidases; enzyme activity; glucose 6 phosphate dehydrogenase; glutamate ammonia ligase; lipid peroxides; oxidative stress; ozone; protein degradation; proteolysis

Studies from the Laboratory of Biochemistry (NHLBI) have shown that there is an accumulation of oxidized proteins with age and in some disease states. These damaged proteins are degraded by the multicatalytic protease (MCP), a multicatalytic proteolytic complex found in eukaryotic cell. In order to gain insight into the mechanism of damaged protein degradation, we have started to examine this process with two enzymes, E. coli glutamine synthetase (GS) and L. mesenteroides glucose-6- phosphate dehydrogenase (Glu-6-PDH), that have been modified by different ways: metal catalyzed oxidations, treatment by ozone and reaction with 4-hydroxy-2-nonenal (HNE), a lipid peroxidation product. These damages which inactivate the enzymes lead, in most cases, to an enhanced susceptibility of the modified protein for proteolysis by the MCP. However, treatment with HNE results only in a small increase of proteolytic susceptibility with GS and no increase with Glu-6-PDH. In the case of the latter enzyme, we have shown that, in contrast to the HNE treated enzyme, the increase of proteolytic susceptibility of the oxidized enzyme is related to a decrease of its structural stability: thermolability and binding of the hydrophobic probe 8-anilino-1- naphthalene sulfonic acid (ANSA). Modified forms of GS exhibit also an increased ability to bind ANSA which correlates with their increased susceptibility to proteolysis by MCP. However, it remains to be determined if the signal for degradation is simply due to structural destabilization and/or is due also to more specific recognition patterns. Therefore, we have looked for the ability of the MCP to degrade various forms of GS in the presence of inhibitors specific to different proteolytic activities. No striking differences have been found so far, although some of the modified forms can be efficiently degraded even in the presence of inhibitor. We are currently investigating new ways for inhibiting specifically given proteolytic activities of the MCP and other modifications of the enzymes.

生物化学实验室（NHLBI）的研究表明， 是氧化蛋白质随着年龄和某些疾病的积累 州。这些受损的蛋白质被多催化降解 蛋白酶 (MCP)，一种在真核生物中发现的多催化蛋白水解复合物 细胞。 为了深入了解蛋白质受损的机制 降解，我们开始用两种酶检查这个过程， 大肠杆菌谷氨酰胺合成酶 (GS) 和肠系膜乳杆菌葡萄糖-6- 磷酸脱氢酶（Glu-6-PDH），已被不同的修饰 方式：金属催化氧化、臭氧处理、与 4-羟基-2-壬烯醛 (HNE)，一种脂质过氧化产物。 这些损害 在大多数情况下，使酶失活会导致增强 修饰蛋白对 MCP 蛋白水解的敏感性。 然而，HNE 治疗仅导致小幅增加 GS 的蛋白水解敏感性增加，而 Glu-6-PDH 则不增加。 在 对于后一种酶的情况，我们已经证明，与 HNE 相比 经过处理的酶，蛋白水解敏感性增加 酶的氧化与其结构稳定性的降低有关： 疏水性探针 8-anilino-1- 的热稳定性和结合 萘磺酸（ANSA）。 GS 的修改形式还表现出 结合 ANSA 的能力增强，这与它们的增强相关 对 MCP 蛋白水解的敏感性。 然而，仍有待 确定退化信号是否仅仅是由于结构原因造成的 不稳定和/或也是由于更具体的识别模式。 因此，我们寻找MCP降解各种物质的能力。 存在针对不同类型的特异性抑制剂时的 GS 形式 蛋白水解活性。 到目前为止还没有发现明显的差异 尽管一些修饰形式即使在 抑制剂的存在。 我们目前正在研究新方法 抑制 MCP 和其他特定蛋白水解活性 酶的修饰。