Functional analysis of substrate-aggregation inhibition and unfoldase activities in the 20S proteasome

20S蛋白酶体是一种多功能蛋白酶复合物，可作为分子伴侣。ATP-ADP交换反应需要分子伴侣与其底物结合。我们发现20S蛋白酶体中的C6和C8亚基在ATP-ADP交换反应中是不可或缺的。虽然观察到伴侣蛋白对热诱导聚集蛋白的活性呈剂量依赖性，但未检测到对变性聚集蛋白的展开酶活性和折叠活性。而20S蛋白酶体两侧附着19S Cap调控亚基的26S则具有聚集抑制和展开酶活性。我们还发现，19S Cap仅具有这两种活动。26S的蛋白水解活性依赖于ATP，因为ATP是运输底物到圆柱体内部所必需的。然而，我们发现20S和26S对热诱导蛋白聚集和展开酶活性的抑制作用与ADP和ATP无关。此外，我们发现底物似乎在蛋白酶K的作用下被结合到环或圆柱外以获得结构变化，蛋白酶K消化变性蛋白质。一般来说，蛋白酶体作为伴侣的功能被认为是不依赖于atp的聚集和展开酶的抑制，以及依赖于atp的底物-运输到圆柱体内。

The 20S proteasome, a complex of multifunctional proteases acts as a molecular chaperone. ATP-ADP exchange reactions require upon the binding of molecular chaperone to its substrate. We have found C6 and C8 subunits in 20S proteasome are indispensable for ATP-ADP exchange reaction. Although the chaperone activities against heat-inducible aggregation of proteins were observed in a dose dependent manner, neither unfoldase activities not folding activities against denatured aggregated proteins were not detected. On the contrary the 26S of which the 19S Cap regulatory subunits attached on the both sides of the 20S proteasome, had aggregation inhibitory and unfoldase activities. We have also found the 19S Cap solely has the both activities. Proteolytic activities of the 26S are dependent on ATP because ATP is required to transport the substrates to inside the cylinder. Nevertheless, we found the inhibitory effects on the heat-inducible aggregation of the proteins and unfoldase activities of the 20S and the 26S were independent on ADP and ATP. In addition, we found substrates seem to be bound to outside the ring or cylinder to get a structural change by the effect of protease K which digests the denatured proteins. In general, the proteasome function as a chaperone is thought to be ATP-independent inhibition of aggregation plus unfoldase, and ATP-dependent substrates-transport to inside the cylinder.