Diversifying interferon functions through combinatorial and structural biology

描述（由申请人提供）：在本 KO1 提案中，我们试图深入探讨分子识别和结构在受体-配体系统（I 型干扰素 (IFN)）中的作用，该系统对基本受体信号传导生物学以及人类健康具有根本性影响。我们打算阐明 IFN 诱导信号传导的结构-功能关系，并通过组合、计算和结构生物学增强 IFN 功能。培训和研究提案描述了在 K. Christopher Garcia 博士指导下的一项为期五年的指导计划，该计划旨在以 Mendoza 博士之前的培训为基础，以进一步提高组合、计算和结构生物学方面的专业知识。这将使门多萨博士成为一名独立的多学科科学家，拥有一套强大的工具，为他提供解决癌症生物学细胞因子工程问题的实验范围。我们将尝试了解干扰素细胞因子如何通过其对细胞生长、分化和增殖的影响来介导广泛的生理过程。 I 型干扰素具有抗增殖特性，对于恶性细胞的监测和控制非常重要。干扰素与其受体细胞外区域结合后，会差异性地激活细胞内区域 JAK/STAT 信号级联。 I 型 IFN 家族的成员尽管与一对共同的受体 IFN?R1 和 IFN?R2 结合，但仍引发不同的信号传导反应。从根本上讲，细胞因子信号传导的特异性很大程度上取决于信号传导复合物内受体链的组成。虽然我们对细胞因子激活的细胞内信号传导途径了解很多，但我们对配体结合诱导的细胞外结构变化如何转化为信号传导事件知之甚少。所提出的实验将使这个问题更加清晰。潜在的转化和癌症治疗应用基于十多年前发表的一项开创性研究，该研究表明通过体外进化实现 IFN 多样化可以产生具有增强功能特性的分子。现在，借助强大的新方法，我们希望重新审视这一概念，以创造具有多种功能特性的干扰素，同时为这些改变的活性提供结构和机制的基本原理，从而为未来设计干扰素用于癌症治疗的策略提供信息。我们将通过以下方式实现这一目标：1) IFNα1 的体外进化，2) 使用体外抗病毒和抗增殖测定鉴定具有改变功能的变体，3) 进化的 IFN 三元复合物的结构表征，以将结构与功能关联起来，4) 具有独特和新颖的信号传导和功能输出的嵌合细胞因子的计算设计和体外进化。通过了解细胞因子/受体相互作用与相关功能的分子基础，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，并扩展受体介导的信号传导、功能活性和治疗特性的能力和多样性。

DESCRIPTION (provided by applicant): 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.