Mechanism, functions and conservation of SHRED, a novel pathway regulating protein quality control

蛋白质需要正确折叠才能完成它们的功能。各种应激条件会导致错误折叠蛋白的积累，对细胞的正常功能构成严重威胁。质量控制机制确保错误折叠的蛋白质被识别和消除。未能清除错误折叠的蛋白质会导致疾病，包括神经退行性疾病。我们最近在发芽酵母中发现了一种新的调控途径，它通过重新编程泛素连接酶Ubr1的底物特异性来促进有效的蛋白质质量控制。我们将这一途径称为应激诱导的稳态调节蛋白降解(Shred)。当压力增强ROQ1基因的转录时，Shred被激活。Roq1蛋白被蛋白酶Ynm3切割，导致在Roq1的新N末端暴露出一个带正电荷的精氨酸残基。通过这个精氨酸残基，切割的Roq1与Ubr1中的底物结合部位相互作用，Ubr1通常识别带有正电荷N-末端残基的底物作为N端规则途径的一部分。与Roq1结合的Ubr1可加速蛋白酶体对错误折叠蛋白质的降解，并增加细胞对蛋白质错误折叠的抵抗力。此外，Shred对Ubr1的重新编程促进了某些天然的、折叠良好的蛋白质的降解，这表明Shred在生理适应过程中的蛋白质组重构中具有额外的作用。这些发现提出了许多新的问题，包括Roq1是作为变构调节器还是底物适配器，Shred还影响了哪些其他细胞过程，以及Shred是否存在于高等真核生物中。我们提出的研究目的是全面了解Shred的机制、功能和进化保守。具体地说，我们的目标是(1)通过体外重组和结构研究阐明该途径的确切分子机制，(2)广泛评价Shred在发芽酵母中的功能，(3)确定哺乳动物中可能的Shred途径。这些研究有望为泛素连接酶的调控和质量控制提供新的总体见解。此外，它们最终可能使出于治疗目的的蛋白质质量控制的药理学操作成为可能。

Proteins need to fold properly in order to fulfill their functions. Various stress conditions result in an accumulation of misfolded proteins, which pose a serious threat to normal cell function. Quality control mechanisms ensure that misfolded proteins are recognized and eliminated. Failure to clear misfolded proteins leads to disease, including neurodegenerative disorders.We have recently discovered a novel regulatory pathway in budding yeast that promotes efficient protein quality control by reprograming the substrate specificity of the ubiquitin ligase Ubr1. We have termed this pathway stress-induced homeostatically regulated protein degradation (SHRED). SHRED is activated when stress enhances transcription of the ROQ1 gene. The Roq1 protein is cleaved by the protease Ynm3, leading to exposure of a positively charged arginine residue at the new N-terminus of Roq1. By means of this arginine residue, cleaved Roq1 interacts with a substrate binding site in Ubr1 that normally recognizes substrates with positively charged N-terminal residues as part of the N-end rule pathway. Roq1-bound Ubr1 accelerates degradation of misfolded proteins by the proteasome and increases cellular resistance to protein misfolding. Furthermore, Ubr1 reprogramming by SHRED promotes the degradation of certain native, well-folded proteins, suggesting that SHRED has additional roles in proteome remodelling during physiological adaptation. These findings raise many new questions, including whether Roq1 acts as an allosteric regulator or as a substrate adaptor, which other cellular processes are influenced by SHRED and whether SHRED exists in higher eukaryotes.The goal of our proposed research is a comprehensive understanding of the mechanism, functions and evolutionary conservation of SHRED. Specifically, we aim to (1) elucidate the precise molecular mechanism of the pathway through in vitro reconstitution and structural studies, (2) gain a broad appreciation of the functions of SHRED in budding yeast, and (3) identify a putative SHRED pathway in mammals. These investigations promise general new insights into the regulation of ubiquitin ligases and quality control. Furthermore, they may ultimately enable pharmacological manipulation of protein quality control for therapeutic purposes.