Natural and synthetic mechanisms of ligand formation

项目摘要/摘要 细胞严格调节分泌的信号蛋白，以便它们在正确的地点和时间发挥作用。MOST信令 蛋白质以不同的组合与其他信号形成复合体。此混合匹配策略部署在 后生动物进化的所有阶段，最终使细胞类型多样化和复杂的动物行为成为可能。但是什么？ 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 信令出错时的解决方案。该计划的第一个分支是研究转化生长因子-β 信号蛋白，Vg1和Nodal，它们必须以异源二聚体的形式组装，才能正确地诱导中胚层和 内胚层组织(如肌肉、骨骼、血液)。我们最近发现，有几个伴侣有助于强健的 Vg1-Nodal异源二聚体在动物胚胎中的选择性组装。这一发现开启了根本 关于伴侣介导的信号组装的机制问题：分子规则是什么(以及顺序 这些规则)，伴侣用来控制信号复合体的组成？我们将联合起来 胚胎学操作、体外生化重建和计算机建模以识别 控制信号蛋白异构体组装的蛋白质调节因子和结合基序。在第二个 作为该计划的一部分，我们的目标是确定内源肽的真正生理功能。例如，一个 单个多蛋白编码基因可以产生多达8个生物活性多肽。然而，细胞只使用了一小部分 用来处理数以千计分泌的前体蛋白和多肽的转化酶。在我们之前的工作中，我们 建立了一种新的处理分泌蛋白质的分子方法--合成加工(Synpro)系统。 Synpro系统由一系列分泌的、合成的、能够裂解同源序列的蛋白酶组成。 在任何分泌的蛋白质上。我们将进一步开发这些新的分泌型蛋白酶来裂解分泌型多蛋白。 以特定于序列的方式。使用Synpro系统，我们实验室将以两种方式指定多肽功能：(I) 将合成酶可切割的序列引入多蛋白或(Ii)合成酶的定向进化 处理多肽中每一种多肽的自然序列。使切割序列字母表多样化 将使我们能够指定肽的功能，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.