Diversifying interferon functions through combinatorial and structural biology

描述(申请人提供)：在这项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，该系统对基础受体信号生物学以及人类健康具有基础意义。我们打算阐明干扰素诱导信号的结构-功能关系，并通过组合、计算和结构生物学来增强干扰素的功能。培训和研究提案描述了一项由K.Christopher Garcia博士指导的五年指导计划，旨在以门多萨博士之前的培训为基础，进一步发展组合、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，为他提供了解决癌症生物学细胞因子工程问题的实验范围。我们将试图了解干扰素细胞因子如何通过它们对细胞生长、分化和增殖的影响来调节广泛的生理过程。I型干扰素具有抗增殖的特性，对监测和控制恶性细胞很重要。当它们的受体胞外区结合时，IFN会不同地激活细胞内 JAK/STAT信令级联。I型干扰素家族的成员尽管与一对常见的受体--干扰素R1和干扰素R2结合，但仍会产生不同的信号反应。从根本上说，细胞因子信号转导的特异性在很大程度上取决于信号复合体中受体链的组成。虽然我们对细胞因子激活的细胞内信号通路了解很多，但我们对配体结合引起的细胞外结构变化如何转化为信号事件知之甚少。提出的实验将为这个问题提供更清晰的解释。潜在的翻译和癌症治疗应用是基于十多年前发表的一项开创性研究，该研究表明，通过体外进化实现干扰素多样化可以产生具有增强功能特性的分子。现在，有了强大的新方法，我们希望重新审视这一概念，以创建具有不同功能特性的IFN，同时也为这些改变的活动提供结构和机制上的理由，以便为未来设计用于癌症治疗的IFN提供策略。我们将通过1)干扰素的体外进化，2)使用体外抗病毒和抗增殖试验鉴定功能改变的变体，3)进化的IFN的三元复合体的结构特征来关联结构和功能，以及4)具有独特和新颖的信号和功能输出的嵌合细胞因子的计算设计和体外进化来实现这一点。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药物的未来发展，以及扩大受体介导的信号、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.