Regulating the Unfolded Protein Response in Yeast

调节酵母中未折叠蛋白的反应

• 批准号: 9750098
• 负责人: Madhusudan Dey
• 金额: $ 33.44万
• 依托单位: UNIVERSITY OF WISCONSIN MILWAUKEE
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-15 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9750098
• 关键词: 1-Phosphatidylinositol 3-Kinase; 3' Untranslated Regions; Alleles; Arthritis; Binding; C-terminal; Cell physiology; Cells; Consensus; Crystallization; Data; Defect; Diabetes Mellitus; Disease; Elements; Endoplasmic Reticulum; Enzymes; Gene Deletion; Genetic Screening; Genetic Translation; Guanosine Triphosphate Phosphohydrolases; Human; In Vitro; Investigation; LacZ Genes; Malignant Neoplasms; Mediating; Messenger RNA; Molecular; Monitor; Multiple Myeloma; Mutate; OmpR protein; Orthologous Gene; Pathogenesis; Pathway interactions; Phenotype; Phosphatidylinositols; Phosphorylation; Phosphorylation Site; Phosphotransferases; Play; Production; Protein-Folding Disease; Proteins; Publishing; RNA Splicing; Reporter; Response Elements; Ribonucleases; Role; Saccharomyces cerevisiae; Structure; Testing; Untranslated Regions; XBP1 gene; Yeasts; base; biological adaptation to stress; design; endoplasmic reticulum stress; genetic linkage; human disease; in vivo; insight; novel; novel strategies; overexpression; phosphoinositide-dependent kinase 1; prevent; protein aggregate; protein folding; proteostasis; response; rho; sensor; small molecule therapeutics; upstream kinase

The unfolded protein response (UPR) is a strategy to increase the protein folding capacity of cells and to maintain the proteostasis network (PN). If this strategy fails, unfolded proteins aggregate causing many human diseases, including diabetes, arthritis and certain cancers. Thus, molecular understanding of the UPR or PN is important to develop new approaches to prevent/reverse the protein folding diseases. Ire1, an endoplasmic reticulum (ER)-resident protein, activates the UPR from yeast to humans. Ire1 activates production of the protein Xbp1 in human cells or Hac1 in yeast cells by processing (cytoplasmic splicing)theirtranslationallyrepressedmRNAs.Hac1/Xbp1thenbindstoacis-regulatoryUPRelement (UPRE) and induces expression of folding enzymes and chaperons (e.g., yeast Kar2 or human BiP). Though Hac1 is thought to induce the Kar2 in yeast, it has been shown that the Kar2 level is significantly increased when Hac1 or Ire1 protein null strain is subjected to an ER stress. We found that the Kar2 level was decreased in the strain lacking a new UPR modulator Kin2 and remained unchanged even when the Hac1 protein level was reduced in a strain lacking the kinase Pkh1. These observations suggest that other sensors or components might activate the yeast UPR. To identify new components modulating the yeast UPR, we conducted a genetic screen with gene-deletion strains of the yeast Saccharomyces cerevisiae and found that the amount of Hac1 or UPRE-driven lacZ reporter was significantly reduced in a few strains each lacking a single protein. These proteins include Vps15 (an ortholog of human phosphoinositide-3- kinase (PI3K) regulatory subunit), Bmh2 (an ortholog of human 14-3-3 protein) and Yhr097C (a previously uncharacterized protein and hereafter referred to as Pdp2) and Cdc42 (a conserved Rho-like GTPase). These proteins (Kin2, Vps15, Bmh2, Cdc42 and Pdp2) have been shown to play important roles in distinct cellular processes, and our results reveal their novel role in the UPR. We also obtained data that provided insights on Kin2 activation, interactions among the new UPR regulators, and the involvement of Vps15 in the UPR. Here, we will test three hypotheses: (1) Cdc42 and Bmh2 bind to Kin2 releasing auto-inhibition and enabling an upstream kinase to phosphorylate/activate the Kin2 kinase domain (KD); (2) Pdp2 activates HAC1 mRNA translation by interacting with its 3’- untranslated region; and (3) Vps15 kinase augments the Hac1-mediated UPR, or alternatively promotes the UPR by phosphorylating a substrate. The insights on the fundamental mechanism of the UPR obtained from these studies in yeast will provide new avenues of investigation for the human UPR leading to the possible identification of new pathway components to target and treat protein-folding diseases.

未折叠蛋白反应（UPR）是一种增加细胞蛋白质折叠能力的策略， 维持蛋白质稳态网络（PN）。如果这种策略失败，未折叠的蛋白质就会聚集，导致许多 人类疾病，包括糖尿病、关节炎和某些癌症。因此，从分子角度理解 UPR 或 PN 对于开发预防/逆转蛋白质折叠疾病的新方法非常重要。 Ire1 是一种内质网 (ER) 驻留蛋白，可激活从酵母到人类的 UPR。伊雷1 通过加工（细胞质 Hac1/Xbp1 然后与 acis 调节 UPR 元件结合 (UPRE) 并诱导折叠酶和伴侣（例如酵母 Kar2 或人 BiP）的表达。 虽然 Hac1 被认为可以诱导酵母中的 Kar2，但已表明 Kar2 水平显着降低。 当 Hac1 或 Ire1 蛋白缺失菌株受到 ER 应激时增加。我们发现Kar2级别 在缺乏新的 UPR 调节剂 Kin2 的菌株中，该值降低，并且即使当 在缺乏激酶 Pkh1 的菌株中，Hac1 蛋白水平降低。这些观察表明其他 传感器或组件可能会激活酵母 UPR。鉴定调节酵母的新成分 UPR，我们对酿酒酵母的基因缺失菌株进行了遗传筛选 并发现少数菌株中 Hac1 或 UPRE 驱动的 lacZ 报告基因的数量显着减少 每个都缺乏一种蛋白质。这些蛋白质包括 Vps15（人磷酸肌醇 3- 的直系同源物） 激酶 (PI3K) 调节亚基）、Bmh2（人类 14-3-3 蛋白的直系同源物）和 Yhr097C（以前的 未表征的蛋白质，以下称为 Pdp2）和 Cdc42（一种保守的 Rho 样 GTP 酶）。 这些蛋白质（Kin2、Vps15、Bmh2、Cdc42 和 Pdp2）已被证明在不同的疾病中发挥重要作用。 细胞过程，我们的结果揭示了它们在 UPR 中的新作用。 我们还获得了有关 Kin2 激活、新 UPR 之间相互作用的见解的数据 监管机构以及 Vps15 参与 UPR 的情况。在这里，我们将测试三个假设：（1）Cdc42 和 Bmh2 与 Kin2 结合释放自动抑制并使上游激酶磷酸化/激活 Kin2 激酶结构域 (KD)； (2) Pdp2通过与其3'-相互作用激活HAC1 mRNA翻译 非翻译区； (3) Vps15 激酶增强 Hac1 介导的 UPR，或者促进 通过磷酸化底物来进行 UPR。对普遍定期审议基本机制的见解 从这些酵母研究中获得的结果将为人类 UPR 领先的研究提供新的途径 可能鉴定出新的途径成分来靶向和治疗蛋白质折叠疾病。