Functional Analysis of RAD23 Protein

描述(申请人提供)：DMA修复蛋白RAD23可以结合多泛素蛋白和蛋白酶体。生化研究表明，RAD23是一种穿梭因子，可以结合和运送泛素化底物到蛋白酶体，并提示它可能促进DNA损伤后的蛋白质降解。RAD23在核苷酸切除修复(NER)中扮演两个不同的角色。其中一个功能涉及与DNA修复蛋白Rad4的相互作用。值得注意的是，这种相互作用通过Ub/蛋白酶体途径阻止了Rad4的降解。第二个功能涉及RAD23与蛋白酶体的相互作用，但这一功能与Rad4稳定无关，尽管它是NER所必需的。有令人信服的证据表明，RAD23/蛋白酶体相互作用是其所有不同功能所必需的，包括(NER)。然而，以前的研究是基于缺失UBL域的突变体，UBL域与蛋白酶体结合。由于UBL域有其他的生理伙伴，这些研究没有表征突变蛋白的特定缺陷。鉴定了RAD23中与RAD4(R4B)结合并稳定的序列。正如预期的那样，该序列的移除阻止了与Rad4的相互作用，并导致了紫外线敏感性。令人惊讶的是，R4B的缺失也取消了RAD23与多泛素化蛋白的相互作用。有人建议通过产生定义明确的突变来表征UBL和R4B基序。这些研究对于进一步检验RAD23是穿梭因子的假说具有重要意义。尽管对NER蛋白进行了广泛的表征，但Rad4的功能尚未被描述。我们的研究表明，RAD4可能会影响RAD23的功能，并建议对这一观点进行研究。RAD23和蛋白酶体亚基RpnIO的缺失导致严重的生长和蛋白降解缺陷。STS1作为这些缺陷的基因抑制因子被分离出来。初步研究表明，Sts1可能促进蛋白酶体在亚细胞内的适当分布，并可能特异性地调节核质运输。本研究的一个重要目的是确定Sts1是如何抑制Rac/23rpnlO突变体的缺陷的。由于RAD23和蛋白酶体在DNA损伤后定位于细胞核，这些研究有助于我们更好地理解RAD23在蛋白质降解和DNA修复中的作用。多泛素链被认为足以将底物靶向蛋白酶体。然而，新出现的证据表明，泛素化蛋白向蛋白酶体的转位需要调节因子，如RAD23。我们发现RAD23与蛋白酶体结合，从酵母到人类，其他UBL-UBA蛋白也有类似的发现。我们的研究将促进我们对底物转运到蛋白酶体的机制以及RAD23在NER中的作用的理解。

DESCRIPTION (provided by applicant): The DMA repair protein Rad23 can bind multiubiquitinated proteins and the proteasome. Biochemical studies suggested that Rad23 functions as a shuttle-factor that can bind and deliver ubiquitinated substrates to the proteasome, and suggested that it might promote protein degradation following DNA damage. Rad23 performs two distinct roles in nucleotide excision-repair (NER). One function involves interaction with the DNA repair protein Rad4. Remarkably, this interaction prevents Rad4 degradation by the Ub/proteasome pathway. A second function involves Rad23 interaction with the proteasome, but this function is not related to Rad4-stabilization, although it is required for NER. There is compelling evidence that Rad23/proteasome interaction is required for all its diverse functions, including (NER). However, previous studies were based on a deletion mutant that lacked the UbL domain, which binds the proteasome. Since the UbL domain has other physiological partners, these studies did not characterize the specific defect of the mutant protein. A sequence in Rad23 that binds and stabilizes Rad4 (R4B) was identified. As expected, removal of this sequence prevented interaction with Rad4, and caused UV sensitivity. Surprisingly, loss of R4B also abolished Rad23 interaction with multiubiquitinated proteins. Studies are proposed to characterize the UbL and R4B motifs, by generating well-defined mutations. These studies will be important for further testing the hypothesis that Rad23 is a shuttle-factor. Despite extensive characterization of NER proteins, a function for Rad4 has not been described. Our studies suggest that Rad4 might influence Rad23 function, and studies are proposed to investigate this idea. The loss of Rad23 and the proteasome subunit RpnIO caused severe growth and proteolytic defects. STS1 was isolated as genetic suppressor of these defects. Preliminary studies indicate that Sts1 might facilitate the proper sub-cellular distribution of proteasomes, and might specifically regulate nucleocytoplasmic transport. An important objective of this study is to determine how Sts1 suppressed the defects of the rac/23 rpnlO mutant. Since Rad23 and proteasomes are localized to the nucleus in response to DNA damage, these studies can improve our understanding of the function of Rad23 in protein degradation and DNA repair. Multiubiquitin chains were believed to be sufficient for targeting substrates to the proteasome. However, emerging evidence suggest that the translocation of ubiquitinated proteins to the proteasome requires regulatory factors, such as Rad23. We discovered that Rad23 binds the proteasome, and similar findings have now been described for other UbL-UBA proteins, from yeast to human. Our studies will advance our understanding of the mechanism of substrate delivery to the proteasome, and Rad23 function in NER.