Natural and synthetic mechanisms of ligand formation

项目总结/摘要 细胞严格调节分泌的信号蛋白，使它们在正确的地点和时间发挥作用。大多数信号 蛋白质以各种组合与其它信号形成复合物。这种混搭策略部署在 后生动物进化的所有阶段，最终使细胞类型的多样性和复杂的动物行为。但 是控制信号配体形成的分子规则吗我研究的首要目标是 程序是描述信号组装和处理的基本机制，以及提供 解决方案时，信号出差错。该计划的第一个分支研究转化生长因子-β 信号蛋白，Vg 1和Nodal，必须组装成异二聚体才能正确诱导中胚层， 内胚层组织（例如，肌肉、骨骼、血液）。我们最近发现，几种伴侣有助于 和动物胚胎中Vg 1-Nodal异源二聚体的选择性组装。这一发现从根本上开启了 分子伴侣介导的信号组装的机制问题：分子规则是什么（和顺序） 这些规则），伴侣用来控制信号复合物的组成？我们将联合收割机 胚胎学操作，体外生化重建和计算机建模，以确定 蛋白调节剂和控制信号蛋白的异聚体组装的结合基序。在第二 该计划的手臂，我们的目标是分配内源性肽的真实生理功能。例如是 单个多聚蛋白编码基因可以产生多达八种生物活性肽。然而，细胞只使用少数 转化酶来处理数以千计的分泌前体蛋白和肽。在以前的工作中，我们 建立了一种新的分子方法来处理分泌的蛋白质，合成处理（Synpro）系统。 Synpro系统由一个分泌的合成蛋白酶家族组成，可以切割同源序列 任何分泌的蛋白质。我们将进一步开发这些新的分泌蛋白酶来切割分泌的多聚蛋白 以特定序列的方式。使用Synpro系统，我们的实验室将以两种方式分配肽功能：（i） 将Synpro-可切割序列引入多蛋白中，或（ii）Synpro蛋白酶的定向进化， 处理多聚蛋白中每个肽的天然序列。使裂解序列字母表多样化 的Synpro蛋白酶将使我们能够分配肽功能，解构复杂的行为，并破坏 由分泌的信号蛋白引起的疾病。

PROJECT SUMMARY/ABSTRACT Cells tightly regulate secreted signaling proteins so that they function at the right place and time. Most signaling proteins form complexes with other signals in various combinations. This mix-and-match strategy is deployed in all stages of metazoan evolution, ultimately enabling cell type diversity and complex animal behaviors. But what are the molecular rules that govern the formation of signaling ligands? The overarching goal of my research program is to describe the fundamental mechanisms of signal assembly and processing, as well as to provide solutions when signaling goes awry. The first arm of the program investigates transforming growth factor-beta signaling proteins, Vg1 and Nodal, that must assemble as heterodimers to properly induce the mesoderm and endoderm tissues (e.g., muscle, bone, blood). We recently discovered that several chaperones aid in the robust and selective assembly of Vg1-Nodal heterodimers in animal embryos. This finding has opened fundamental mechanistic questions on chaperone-mediated signal assembly: What are the molecular rules (and the order of these rules) that chaperones use to control the composition of signaling complexes? We will combine embryological manipulation, biochemical reconstitution in vitro, and computational modeling to identify the protein regulators and binding motifs that govern the heteromeric assembly of signaling proteins. In the second arm of the program, we aim to assign the true physiological function of endogenous peptides. For example, a single polyprotein-encoding gene can produce up to eight bioactive peptides. However, cells only use a handful of convertases to process the thousands of secreted precursor proteins and peptides. In our previous work, we established a new molecular approach to process secreted proteins, the Synthetic Processing (Synpro) system. The Synpro system is composed of a family of secreted, synthetic proteases that can cleave cognate sequences on any secreted protein. We will further develop these novel secreted proteases to cleave secreted polyproteins in a sequence-specific way. Using the Synpro system, our lab will assign peptide function in two ways: (i) introduction of Synpro-cleavable sequences into polyproteins or (ii) directed evolution of Synpro proteases to process the natural sequence of each peptide within a polyprotein. Diversifying the cleavage sequence alphabet of Synpro proteases will enable us to assign peptide function, deconstruct complex behaviors, and disrupt diseases that arise from secreted signaling proteins.