Diversifying interferon functions through combinatorial and structural biology

项目摘要/摘要：在这份KO1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 对基本受体信号生物学和人类健康的影响。我们打算阐明其结构-- 干扰素诱导信号转导与增强干扰素功能的功能关系 计算生物学和结构生物学。培训和研究提案描述了一个五年的指导 在K.Christopher Garcia博士指导下的计划旨在门多萨博士之前 为进一步掌握组合、计算和结构生物学方面的专业知识而进行的培训。这将确立 门多萨博士是一位独立的多学科科学家，拥有一套强大的工具，使他 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调节广泛的生理过程。 通过它们对细胞生长、分化和增殖的影响。I型干扰素具有抗增殖作用 对监测和控制恶性细胞很重要的特性。当它们的受体接合时 胞外区，IFN以不同的方式激活细胞内JAK/STAT信号级联。委员会成员 I型干扰素家族尽管与一对常见的受体--干扰素-1和干扰素-1结合，但仍可引起不同的信号反应 干扰素？R2。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 信号复合体内的受体链。虽然对细胞因子激活的细胞内有很多了解 信号通路，我们对配体结合引起的细胞外结构变化知之甚少 转化为信号事件。提出的实验将为这个问题提供更清晰的解释。这个 潜在的翻译和癌症治疗应用是基于一项开创性的研究，发表了更多 表明通过体外进化使干扰素多样化可以产生与 增强的功能特性。现在，用强大的新方法武装起来，我们希望重新审视这一概念 创建具有不同功能属性的IFN，但也为 这些改变的活动可能会为未来设计用于癌症治疗的IFN的策略提供信息。 我们将通过1)干扰素的体外进化，2)鉴定功能改变的变体来实现这一点 利用体外抗病毒和抗增殖试验，3)三元配合物的结构表征 进化的IFN将结构与功能联系起来，以及4)计算设计和体外进化 具有独特和新颖信号和功能输出的嵌合细胞因子。通过了解分子 在细胞因子/受体相互作用具有相关功能的基础上，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的能力和多样性 介导的信号、功能活动和治疗特性。

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.