Department of Chemistry and Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA 30303, USA; Department of Pathophysiology, School of Medicine, Jinan University, Guangzhou 510632, Guangdong, China.
Department of Chemistry and Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA 30303, USA; Engineering Research Center of Cell and Therapeutic Antibody, Ministry of Education, and School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China.
Cell Chem Biol. 2021 Sep 16;28(9):1283-1297.e8. doi: 10.1016/j.chembiol.2021.02.005. Epub 2021 Mar 4.
Attachment of the ubiquitin (UB) peptide to proteins via the E1-E2-E3 enzymatic machinery regulates diverse biological pathways, yet identification of the substrates of E3 UB ligases remains a challenge. We overcame this challenge by constructing an "orthogonal UB transfer" (OUT) cascade with yeast E3 Rsp5 to enable the exclusive delivery of an engineered UB (xUB) to Rsp5 and its substrate proteins. The OUT screen uncovered new Rsp5 substrates in yeast, such as Pal1 and Pal2, which are partners of endocytic protein Ede1, and chaperones Hsp70-Ssb, Hsp82, and Hsp104 that counteract protein misfolding and control self-perpetuating amyloid aggregates (prions), resembling those involved in human amyloid diseases. We showed that prion formation and effect of Hsp104 on prion propagation are modulated by Rsp5. Overall, our work demonstrates the capacity of OUT to deconvolute the complex E3-substrate relationships in crucial biological processes such as endocytosis and protein assembly disorders through protein ubiquitination.
通过 E1-E2-E3 酶促机制将泛素（UB）肽附着到蛋白质上，调节多种生物途径，但鉴定 E3 UB 连接酶的底物仍然是一个挑战。我们通过构建带有酵母 E3 Rsp5 的“正交 UB 转移（OUT）级联”克服了这一挑战，使工程化 UB（xUB）能够专一地递送到 Rsp5 及其底物蛋白。OUT 筛选在酵母中发现了新的 Rsp5 底物，例如内吞蛋白 Ede1 的伴侣 Pal1 和 Pal2，以及对抗蛋白质错误折叠并控制自我持续淀粉样聚集（朊病毒）的伴侣 Hsp70-Ssb、Hsp82 和 Hsp104。我们表明，Rsp5 调节朊病毒的形成和 Hsp104 对朊病毒传播的影响。总的来说，我们的工作表明，OUT 有能力通过蛋白质泛素化来分解内吞作用和蛋白质组装障碍等重要生物学过程中复杂的 E3-底物关系。
