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水解酶的复合体，起着分子伴侣的作用。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交换反应中是必不可少的。虽然分子伴侣对蛋白质热诱导聚集的抑制作用呈剂量依赖关系，但既没有检测到解折叠酶活性，也没有检测到针对变性聚集蛋白的折叠活性。相反，19S-Cap调节亚基位于20S蛋白酶体两侧的26S蛋白酶体具有聚集抑制和解折酶活性。我们还发现，19s Cap只有这两种活动。26S的蛋白分解活性依赖于ATP，因为需要ATP将底物运输到圆柱体内。然而，我们发现20S和26S对蛋白质热诱导聚集和去折叠酶活性的抑制作用不依赖于ADP和ATP。此外，我们发现底物似乎结合在环或圆柱体之外，通过消化变性蛋白质的蛋白酶K的作用而获得结构变化。一般来说，蛋白酶体作为伴侣的作用被认为是不依赖于ATP的聚集抑制加上去折叠酶，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.