Quantitative analysis of the evolving genotype-to-phenotype map

描述（由申请人提供）：癌基因导向的癌症治疗和抗菌治疗经常受到快速获得耐药性的阻碍，这是持久缓解的主要障碍。一个看似遥远的领域--蛋白质折叠--正在出现有希望的见解。为了发挥功能，蛋白质必须采用复杂的，通常是亚稳态的构象。危险的是，许多疾病是由单一蛋白质的折叠或错误折叠引起的。我以前的研究集中在热休克蛋白90，分子伴侣，折叠亚稳态蛋白质的致癌转化和信号转导的关键。通过影响调节蛋白的精英群体的折叠和功能，这种分子伴侣有能力影响新性状的进化。在我剩余的指导培训和最初的独立研究生涯中，我将解决以下目标：（I）确定Hsp 90如何转化遗传变异。Hsp 90强烈影响Mec 1/ATR多态性的影响，Mec 1/ATR是癌症信号传导的核心参与者，能够以牺牲其他基因毒性应激为代价对某些基因毒性应激做出反应。生物化学和功能，我将研究如何影响DNA损伤反应，细胞周期和活力。此外，还将研究Hsp 90如何影响Pdr 8的转录网络，Pdr 8是一种能够对许多药物产生耐药性的转录因子。最后，我将研究如何热休克蛋白90抑制转换的影响，顺式调节元件的NDI 1的多态性，创造强大的抗氧化应激。(II)研究Hsp 90相关表型的同化作用。为了研究最终的突破性耐药性，我将在最初和同化后分离致病变异（当耐药性不依赖于Hsp 90时）。高通量遗传技术和下一代测序将提供对这一现象的机制性理解。(III)识别和表征允许高度突变细胞存活、增殖和进化新性状的其他因素。癌细胞必须承受大量的突变负荷，从而导致有毒的蛋白质组不稳定。我将筛选出能挽救高度突变菌株生长但不影响未突变亲本的蛋白质。(IV)识别和表征促进适应新环境的其他蛋白质机制。使用数字核糖体分析，酵母和细菌遗传学，以及生物化学，我将描述两种朊病毒，[PSI+]和[GAR+]，并确定它们如何影响适应不断变化的环境。这些研究的结果将提供详细的见解如何热休克蛋白90，更一般的蛋白质稳态，控制信号通路，使耐药性，以及这些机制如何有助于突破性耐药。他们也将暴露所有癌症的致命弱点--对维持大量突变负荷的因子上瘾。

DESCRIPTION (provided by applicant): Oncogene-directed cancer treatments and antimicrobial therapies are often thwarted by rapid acquisition of drug resistance, a chief barrier to lasting remission. Promising insight is emerging from a seemingly distant field - protein folding. To function, proteins must adopt complex, often metastable conformations. Perilously, many diseases arise from folding or misfolding of a single protein. My previous studies have focused on Hsp90, a molecular chaperone that folds metastable proteins critical for oncogenic transformation and signaling. By influencing the fold and function of an elite cohort of regulatory proteins, this chaperone has the power to influence the evolution of new traits. I will address the following aims during my remaining mentored training and initial independent research career: (I) Determine how Hsp90 transforms genetic variation. Hsp90 strongly impacts the effects of polymorphisms in Mec1/ATR, a central player in cancer signaling, enabling responses to certain genotoxic stresses at the expense of others. Biochemically and functionally, I will examine how this affects the DNA damage response, cell cycle, and viability. Additionally, is will investigate how Hsp90 impacts the transcriptional network of Pdr8, a transcription factor that enables resistance to many drugs. Finally, I will examine how Hsp90 inhibition transforms the effects of polymorphisms in cis-regulatory elements of NDI1, to create strong resistance to oxidative stresses. (II) Investigate assimilation of Hsp90-contingent phenotypes. To investigate eventual breakthrough drug resistance I will isolate causative variation initially and after assimilation (when resistance is Hsp90-independent). High-throughput genetic techniques and next-generation sequencing will provide a mechanistic understanding of this phenomenon. (III) Identify and characterize additional factors that allow highly mutated cells to survive, proliferate, and evolve new traits. Cancer cells must sustain massive mutation loads and consequently toxic proteome destabilization. I will screen for proteins that rescue growth of highly mutated strains but do not affect unmutated parents. (IV) Identify and characterize additional protein-based mechanisms that facilitate adaptation to new environments. Using digital ribosome profiling, yeast and bacterial genetics, and biochemistry I will characterize two prions, [PSI+] and [GAR+], and determine how they affect adaptation to changing environments. The results of these studies will offer detailed insight into how Hsp90, and protein homeostasis more generally, controls signaling pathways that enable drug resistance and how these mechanisms contribute to breakthrough resistance. They will also expose an Achilles' heel common to all cancers - addiction to factors that enable maintenance of massive mutation loads.