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理解生物过程，使我们能够 以可预测的方式操纵它们。这些知识为预防和治疗奠定了基础 这些疾病是由这些过程的功能障碍引起的。为了实现这一目标，我们必须发展 这些实验工具使我们能够精确地扰乱细胞过程。此外还要 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 我们将描绘出细胞过程的最小组成部分;我们将能够 以定量的方式预测该过程对扰动的响应;我们将制造出 操纵细胞途径的新工具-例如，设计代谢途径以产生 所需的产品，包括药物。在这个建议中，我们专注于蛋白质降解的关键过程。我们 提出了一个强大的和广泛适用的新策略，专门针对蛋白质降解酵母 （S. cerevisiae）。在酵母中工作为使用遗传筛选和选择提供了很大的范围， 实现我们的目标。此外，酵母可获得的蛋白质组学信息的丰富程度远远超过 任何其他有机体。我们利用我们在蛋白质工程和设计方面的专业知识， 降解途径我们将设计E3连接酶CHIP，通过以下方式改变其底物识别特异性： 将其三肽重复序列（TPR）结构域转换为我们设计的不同TPR结构域。芯片 不是内源性酵母蛋白，因此没有细胞过程依赖于其活性。因此， 我们可以在不影响正常细胞功能的情况下改变CHIP的数量和活性。等 能力将使我们能够测试CHIP介导的降解的定量模型， 干扰CHIP的细胞浓度和比活性。此外，我们的战略提供了 使用CHIP的靶向降解作为操纵代谢途径的手段， 定义生产的产品。我们所提出的方案的一个独特优势是，多个蛋白质可以 靶向降解，在同一个细胞中，每个由不同的特异性CHIP控制。我们将 通过工程化紫素途径，靶向该途径的两种酶（ 单独地或同时地）以限定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.