Diversifying interferon functions through combinatorial and structural biology

项目总结/摘要：在本KO 1提案中，我们试图深入探讨 受体-配体系统中的分子识别和结构，I型干扰素（IFN），具有基本的 对基本受体信号传导生物学以及人类健康的影响。我们打算解释它的结构- IFN诱导的信号传导的功能关系和通过组合增强IFN功能， 计算和结构生物学。培训和研究建议描述了一个为期五年的指导 在K博士的指导下。克里斯托弗·加西亚的设计是建立在门多萨博士以前的 培训，以进一步在组合，计算和结构生物学的专业知识。这将建立 博士门多萨是一位独立的多学科科学家，拥有一套强大的工具， 实验范围，以解决癌症生物学细胞因子工程的问题。 我们将试图了解干扰素细胞因子如何介导广泛的生理过程 通过它们对细胞生长、分化和增殖的影响。I型IFN具有抗增殖作用， 对于恶性细胞的监视和控制重要的性质。一旦它们的受体 在细胞外区域，IFN差异性地激活细胞内JAK/STAT信号级联。成员 I型IFN家族引起不同的信号传导反应，尽管与一对共同的受体IFN R1和 干扰素R2。从根本上说，细胞因子信号传导的特异性在很大程度上取决于细胞因子的组成。 信号复合物中的受体链。虽然对精氨酸激活的细胞内 信号通路，我们知道得少得多的细胞外结构的变化是如何诱导配体结合 转化为信号事件。所提出的实验将使这个问题更加清楚。的 潜在的转化和癌症治疗应用是基于一项开创性的研究， 十多年前，这表明IFN通过体外进化的多样化可以产生具有以下特征的分子： 增强的功能特性。现在，有了强大的新方法，我们希望重新审视这一概念 创造具有不同功能特性的IFN，但也为以下方面提供了结构和机制依据： 这些改变的活动在某种程度上可以为将来设计用于癌症治疗的IFN的策略提供信息。 我们将通过1）IFN？1的体外进化，2）鉴定功能改变的变体来实现这一目标 使用体外抗病毒和抗增殖测定，3）以下三元复合物的结构表征： 进化的IFN将结构与功能相关联，以及4）IFN的计算设计和体外进化， 嵌合细胞因子具有独特的和新的信号传导和功能输出。通过了解分子 基于细胞因子/受体相互作用与相关功能，这项工作的结果可能能够指导 细胞因子作为治疗剂的未来发展以及扩大受体的能力和多样性， 介导的信号传导、功能活性和治疗性质。

PROJECT SUMMARY/ABSTRACT: In this KO1 proposal, we attempt to deeply interrogate the role of molecular recognition and structure in a receptor-ligand system, type I Interferons (IFNs), that has fundamental implications for basic receptor signaling biology as well as human health. We intend to elucidate the structure- functional relationships of IFN induced signaling and to enhance IFN functions through combinatorial, computational, and structural biology. The training and research proposal describes a five year mentored program under the guidance of Dr. K. Christopher Garcia designed to build upon Dr. Mendoza's previous training in order to further expertise in combinatorial, computational, and structural biology. This will establish Dr. Mendoza as an independent multi-disciplinary scientist with a powerful set of tools to give him the experimental range to tackle problems in cytokine engineering for cancer biology. We will attempt to understand how IFN cytokines mediate a wide range of physiological processes through their effects on cell growth, differentiation, and proliferation. Type I IFNs have anti-proliferative properties important for the surveillance and control of malignant cells. Upon engagement of their receptor extracellular regions, IFNs differentially activate intracellular JAK/STAT signaling cascades. Members of the type I IFN family elicit different signaling responses despite binding to a common pair of receptors, IFN¿R1 and IFN¿R2. Fundamentally, the specificity of cytokine signaling is largely determined by the composition of receptor chains within the signaling complex. While much is known about cytokine-activated intracellular signaling pathways, we know much less about how extracellular structural changes induced by ligand binding translates into signaling events. The experiments proposed will provide more clarity to this question. The potential translational and cancer therapeutic applications are based on a pioneering study published more than ten years ago suggesting that IFN diversification through in vitro evolution could yield molecules with enhanced functional properties. Now, armed with powerful new methodologies, we wish to revisit this concept to create IFNs with diverse functional properties, but also to provide a structural and mechanistic rationale for these altered activities in a way that could inform strategies to engineer IFNs for cancer therapy in the future. We will achieve this by 1) in vitro evolution of IFN¿1, 2) identification of variants with altered function using in vitro anti-viral and anti-proliferative assays, 3) structural characterization of the ternary complexes of the evolved IFNs to correlate structure with function, and 4) the computational design and in vitro evolution of chimeric cytokines with distinct and novel signaling and functional outputs. By understanding the molecular basis of cytokine/receptor interactions with correlated function, the results from this work may be able to guide the future development of cytokines as therapeutics as well as expand the capabilities and diversity of receptor mediated signaling, functional activities, and therapeutic properties.