Regulating the Unfolded Protein Response in Yeast

未折叠蛋白质反应（UPR）是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，未折叠的蛋白质聚集， 人类疾病，包括糖尿病、关节炎和某些癌症。因此，分子的理解， UPR或PN对于开发预防/逆转蛋白质折叠疾病的新方法具有重要意义。 Ire 1是一种内质网（ER）驻留蛋白，激活从酵母到人类的UPR。IRE1 通过加工（细胞质）激活人细胞中蛋白Xbp 1或酵母细胞中Hac 1的产生 Hac 1/Xbp 1结合stoacis-regulating UPR元件 （UPRE）并诱导折叠酶和伴侣蛋白（例如，酵母Kar 2或人BiP）。 虽然Hac 1被认为在酵母中诱导Kar 2，但已经显示Kar 2水平显著降低。 当Hac 1或Ire 1蛋白缺失菌株经受ER应激时增加。我们发现Kar 2水平 在缺乏新的UPR调节剂Kin 2的菌株中， 在缺乏激酶Pkh 1的菌株中，Hac 1蛋白水平降低。这些观察表明，其他 传感器或组件可以激活酵母UPR。鉴定调节酵母的新成分 UPR，我们用酿酒酵母的基因缺失菌株进行了遗传筛选 并发现Hac 1或UPRE驱动的lacZ报告基因的量在少数菌株中显著减少 每一种都缺少一种蛋白质这些蛋白质包括Vps 15（人磷脂酰肌醇-3-磷酸的直系同源物， 激酶（PI 3 K）调节亚基）、Bmh 2（人14-3-3蛋白的直系同源物）和Yhr 097 C（先前的人14-3-3蛋白的直系同源物）。 未表征的蛋白质，下文称为Pdp 2）和Cdc 42（保守的Rho样GT3）。 这些蛋白质（Kin 2、Vps 15、Bmh 2、Cdc 42和Pdp 2）已被证明在不同的细胞中起重要作用。 细胞过程，我们的研究结果揭示了它们在UPR中的新作用。 我们还获得了一些数据，这些数据提供了关于Kin 2激活、新的UPR之间的相互作用、 监管机构，以及Vps 15参与普遍定期审议。在这里，我们将测试三个假设：（1）Cdc 42和 Bmh 2与Kin 2结合，释放自抑制，并使上游激酶磷酸化/激活 Kin 2激酶结构域（KD）;（2）Pdp 2通过与其3 '- （3）Vps 15激酶增强Hac 1介导的UPR，或者促进Hac 1介导的UPR。 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 从这些研究中获得的酵母将为人类UPR的研究提供新的途径， 可能鉴定新的途径组分以靶向和治疗蛋白质折叠疾病。

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.