Structural basis of antagonism within the TGFbeta-superfamily

描述(申请人提供)：大约40个独特的分泌配体，分为三个亚类(转化生长因子？S、激活素和骨形态发生蛋白)，形成转化生长因子？超家族，其功能是协调许多细胞事件。转化生长因子家族配体已经成为治疗的靶点，通过阻断它们与受体的相互作用来缓解人类疾病，如癌症、纤维化和肌肉萎缩疾病。当然，配体是通过与其N末端前肽形成不活跃的共价复合体来调节的，该复合体在合成过程中被切割，或者被许多结构多样的细胞外拮抗剂之一中和。这些分子与配体的结合范围很广，其中多个配体被一个拮抗剂拮抗，或者非常具体，其中一个拮抗剂只识别一个不同的配体。尽管转化生长因子家族的配体在结构上是相似的，但结构多样化的拮抗剂如何选择性地中和配体亚群的分子基础仍不清楚。因此，我们这一应用的长期目标是阐明转化生长因子家族拮抗剂的特异性机制。这项建议将集中于三种激活素家族拮抗剂之间的不同配体特异性：卵泡抑素(FST)(宽配体特异性)、卵泡抑素样3(FSTL3)(中等特异性)和前肽(精细特异性)。我们的中心假设是，拮抗剂不同地与保守和非保守的配体表面相互作用，从而在激活素配体家族中赋予一系列特异性。在目标1中，我们将通过结合FST的X-射线结构分析：配体复合体和结合研究来确定FST拮抗剂是如何赋予广泛配体拮抗作用的。在目标2中，我们将确定FSTL3结构域的差异如何限制与少数配体的结合，在目标3中，我们将确定前肽如何与肌肉生长抑素配体形成非活性复合体。在这三个目标中，我们将结合X射线结构测定与竞争和表面等离子体共振结合实验以及基于细胞的分析来阐明每个拮抗剂的特异性机制。阐明中和转化生长因子家族配体的详细机制将有助于开发新的策略和优化目前旨在拮抗配体的治疗方法。与公共健康相关目前，转化生长因子家族的配体正在被中和治疗，以缓解人类疾病，如癌症、纤维化和肌肉萎缩。我们的研究意义重大，因为它有望提供对抗配体的结合分子的细节。这些信息将加强定制旨在中和转化生长因子家族配体的治疗方法的努力。

DESCRIPTION (provided by applicant): Approximately 40 unique secreted ligands, divided into three subgroups (TGF¿s, activins and BMPs) form the TGF¿-superfamily whose function is to coordinate numerous cellular events. TGF¿-family ligands are already being targeted therapeutically by blocking their interactions with receptors to alleviate human disorders such as cancer, fibrosis and muscle wasting disease. Naturally, ligands are regulated by either formation of an inactive covalent complex with their N-terminal propeptide that is cleaved during synthesis or by neutralization by one of a number of structurally-diverse extracellular antagonists. Binding of these molecules to ligands can range from very broad, where multiple ligands are antagonized by a single antagonist or very specific where one antagonist only recognizes a distinct ligand. While TGF¿-family ligands are structurally similar, the molecular basis of how structurally-diverse antagonists selectively neutralize subsets of ligands remains unclear. Therefore, our long-term goal of this application is to elucidate mechanisms of specificity among the TGF¿-family antagonists. This proposal will focus on the divergent ligand specificity among three activin-family antagonists: Follistatin (FST) (broad ligand specificity), Follistatin-like 3 (FSTL3) (moderate specificity), and propeptides (exquisite specificity). Our central hypothesis is that antagonists differentially interact with conserved and nonconserved ligand surfaces to confer a range of specificity within the activin-family of ligands. In Aim 1, we will determine how FST antagonists confer broad ligand antagonism through a combination of X-ray structure analysis of FST:ligand complexes and binding studies. In Aim 2, we will determine how FSTL3 domain differences restrict binding to a few ligands, and in Aim 3, we will establish how the propeptide forms an inactive complex with the myostatin ligand. In all three aims, we will combine X-ray structure determination with competition and surface plasmon resonance binding experiments along with cellular-based assays to elucidate mechanisms of specificity for each antagonist. Elucidating details of the mechanisms that neutralize TGF¿-family ligands will help in development of new strategies and optimize current therapies aimed at antagonizing ligands. PUBLIC HEALTH RELEVANCE Presently, TGF¿-family ligands are being neutralized therapeutically to alleviate human disorders such as cancer, fibrosis and muscle wasting. Our research is significant as it is expected to provide details of binding molecules that antagonize ligands. This information will strengthen efforts to customize therapies designed to neutralize TGF¿-family ligands.