Raasi Shahri, Pickart Cecile M
Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland 21205, USA.
J Biol Chem. 2003 Mar 14;278(11):8951-9. doi: 10.1074/jbc.m212841200.
Most substrates of the 26 S proteasome are recognized only following conjugation to a Lys48-linked polyubiquitin chain. Rad23 is one member of a family of proteins that possesses an N-terminal ubiquitin-like domain (UbL) and a C-terminal ubiquitin-associated domain(s) (UBA). Recent studies have shown that UbLs interact with 26 S proteasomes, whereas UBAs bind polyubiquitin chains. These biochemical properties suggest that UbL-UBA proteins may shuttle polyubiquitinated substrates to proteasomes. Here we show that contrary to prediction from this model, the effect of human Rad23A on the degradation of polyubiquitinated substrates catalyzed by purified proteasomes is exclusively inhibitory. Strong inhibition is dependent on the presence of both UBAs, independent of the UbL, and can be explained by competition between the UBA domains and the proteasome for binding to substrate-linked polyubiquitin chains. The UBA domains bind Lys48-linked polyubiquitin chains in strong preference to Lys63 or Lys29-linked chains, leading to selective inhibition of the assembly and disassembly of Lys48-linked chains. These results place constraints on the mechanism(s) by which UbL-UBA proteins promote proteasome-catalyzed proteolysis and reveal new properties of UBA domains.
26S蛋白酶体的大多数底物只有在与赖氨酸48连接的多聚泛素链结合后才能被识别。Rad23是一类蛋白质家族的成员之一，该家族蛋白质具有一个N端类泛素结构域（UbL）和一个C端泛素相关结构域（UBA）。最近的研究表明，UbL与26S蛋白酶体相互作用，而UBA结合多聚泛素链。这些生化特性表明，UbL-UBA蛋白可能将多聚泛素化的底物转运到蛋白酶体。在这里，我们表明，与该模型的预测相反，人Rad23A对纯化的蛋白酶体催化的多聚泛素化底物降解的影响完全是抑制性的。强烈的抑制作用依赖于两个UBA的存在，与UbL无关，并且可以通过UBA结构域和蛋白酶体之间竞争结合底物连接的多聚泛素链来解释。UBA结构域强烈优先结合赖氨酸48连接的多聚泛素链，而不是赖氨酸63或赖氨酸29连接的链，导致对赖氨酸48连接链的组装和拆卸的选择性抑制。这些结果对UbL-UBA蛋白促进蛋白酶体催化的蛋白水解的机制施加了限制，并揭示了UBA结构域的新特性。
