Designed proteins to study and modulate cellular processes

生物医学的一个重大挑战是在一个水平上了解生物过程，使我们能够 以可预测的方式操纵它们。这些知识为防治奠定了基础 由于这些过程的故障而导致的疾病。为了实现这一目标，我们必须发展 实验工具，使我们能够以精致的精度扰乱细胞过程。此外，我们必须 开发定量的、可测试的、可预测的模型。完成这一重大挑战的后果 有很多方面：我们将描绘出细胞过程的最小组成部分；我们将能够 以定量的方式预测这一过程对扰动的反应；然后我们就可以 用于操纵细胞途径的新工具-例如，设计代谢途径以产生 想要的产品，包括药品。在这个提案中，我们将重点放在蛋白质降解的关键过程上。我们 提出了一种有效且广泛适用的新策略，可以在酵母中特异性地针对降解的蛋白质 (酿酒酵母)。在酵母中的工作为使用遗传筛选和选择提供了很大的空间 完成我们的目标。此外，可用于酵母的蛋白质组信息的丰富远远超过了 任何其他有机体的。我们利用我们在蛋白质工程和设计方面的专业知识来创造一个‘正交’。 降解途径。我们将改造E3连接酶芯片，通过以下方式改变其底物识别特异性 将其四肽重复序列(TPR)结构域切换为我们设计的不同TPR结构域。芯片 不是一种内源性酵母蛋白，所以细胞过程不依赖于它的活性。因此，有巨大的 我们可以在不影响正常细胞功能的情况下改变芯片的数量和活性。是这样的 能力将使我们能够测试芯片介导的降解的量化模型，显著地 扰乱芯片的细胞浓度和比活性。此外，我们的战略提供了 利用芯片的靶向降解作为操纵代谢途径的一种手段，因此具有很大的潜力 定义所生产的产品。我们建议的方案的一个独特优势是可以将多种蛋白质 靶向降解，在同一细胞中，每个由不同的特异性芯片控制。我们会 通过设计紫罗兰素途径，靶向途径的两种酶(或 单独或同时)以定义在细胞中产生的代谢产物。

A grand challenge of biomedicine is to understand biological processes at a level that allows us to manipulate them in a predictable fashion. Such knowledge lays the foundation for preventing and treating diseases that result from the malfunction of such processes. To accomplish this goal, we must develop experimental tools that permit us to perturb cellular processes with exquisite precision. In addition, we must develop quantitative, testable, predictive models. The ramifications of accomplishing this grand challenge are many fold: We will have delineated the minimal components of a cellular process; we will be able to predict the responses of that process to perturbations in a quantitative fashion; and we will have fabricated new tools for manipulating cellular pathways – for example, to engineer metabolic pathways to produce desired products, including drugs. In this proposal, we focus on the key process of protein degradation. We present a powerful and widely applicable new strategy to specifically target proteins for degradation in yeast (S. cerevisiae). Working in yeast provides great scope for the use of genetic screens and selections to accomplish our goals. Moreover, the wealth of proteomic information available for yeast far surpasses that of any other organism. We employ our expertise in protein engineering and design to create an `orthogonal' degradation pathway. We will engineer the E3 ligase, CHIP, changing its substrate recognition specificity by switching its tetratricopeptide repeat (TPR) domain for different TPR domains that we have designed. CHIP is not an endogenous yeast protein, so no cellular processes depend on its activity. Thus, there is huge scope for us to change the amount and activity of CHIP without effecting normal cellular function. Such capability will allow us to test quantitative models of the CHIP-mediated degradation, by significantly perturbing both the cellular concentration and specific activity of CHIP. Moreover, our strategy provides great scope to use targeted degradation by CHIP as a means to manipulate metabolic pathways and thus define the products produced. A unique advantage of our proposed scheme is that multiple proteins can be targeted for degradation, in the same cell, each controlled by a different specificity CHIP. We will demonstrate this facility by engineering the Violacein pathway, targeting two enzymes of the pathway (either individually or at the same time) to define the metabolic products that are produced in the cell.