Rational Design of Loss-of-Function Phospholamban Mutants for Gene Therapy

描述（由申请人提供）：本提案的总体目标是合理设计“功能丧失”磷蛋白（PLN）突变候选体，以通过重组腺相关病毒（rAAV）基因治疗改善心肌病的发病。PLN是sarco（endo）质网Ca2+- atp酶（SERCA）的内源性抑制剂，SERCA是一种完整的膜酶，负责70%的Ca2+穿梭进入SR，诱导人类心肌松弛。SERCA:PLN相互作用的功能障碍被认为在心脏病中起作用。由于S16E的成功，SERCA和PLN相互作用功能障碍的创新基因治疗途径最近成为该领域的一个新的兴趣，S16E是PLN的一种伪磷酸化形式，成功地减少了小鼠模型和大型动物心力衰竭的进展。研究发现，PLN的结构动态可以调整，从而对SERCA产生直接的功能影响，本研究旨在创新其他候选基因，如S16E，用于心力衰竭的基因治疗。本提案将重点关注PLN突变种面向结构域的理性设计的几个关键组成部分，并关注这些具体目标：目标1：定义结构域通信在PLN抑制SERCA能力中的作用。目的2：双伪磷酸化PLN （S16E/T17E）的结构-动态-功能表征。目的3：阐明PLN“超抑制剂”的意义。新的PLN突变种将使用定点诱变技术开发，在大肠杆菌中表达并使用既定方法纯化。为了在非磷酸化和磷酸化状态下表征这些新的PLN突变体，将使用十二烷基磷脂胆碱（DPC）胶束的溶液态核磁共振（NMR）光谱研究结构动力学。由此产生的结构动力学扰动将与SERCA存在下偶联酶测定中观察到的功能效应相关联。从这项工作的观点来看，本提案的目的是合理设计新的PLN突变候选基因治疗，并为成功设计治疗性PLN类似物建立新的原则。公共卫生相关性：通过作用于酶亚基的结构动力学来控制酶的功能，本研究的结果旨在从分子水平上理解结构动力学与控制生理的相互作用。

DESCRIPTION (provided by applicant): The overall goal of this proposal is to rationally design "loss of function" phospholamban (PLN) mutant candidates for the purpose of ameliorating the onset of cardiomyopathy by recombinant adeno-associated virus (rAAV) gene therapy. PLN is the endogenous inhibitor of the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), the integral membrane enzyme responsible for 70% of the Ca2+ shuttling into the SR, inducing cardiac muscle relaxation in humans. Dysfunctions in SERCA:PLN interactions have been implicated as having a role in cardiac disease. The pathway to innovating genetic treatments for dysfunctions of SERCA and PLN interactions has recently become a new interest in the field due to the success of S16E, a pseudo-phosphorylated form of PLN which successfully reduced the progression of the cardiac failure in murine models and large animals. It has been found that the structural dynamics of PLN can be tuned to result in a directed functional effect on SERCA , and this study aims to innovate other candidates like S16E for use in gene therapy in treatment of heart failure. This proposal will concentrate on several key components of the domain-oriented rational design of PLN mutant species, and focus on these specific aims: AIM 1: Define the role of domain communication in PLN's ability to inhibit SERCA. AIM 2: Structural-dynamic-functional characterization of dual pseudo-phosphorylated PLN (S16E/T17E). AIM 3: Elucidate the significance of "superinhibitors" of PLN. New PLN mutant species will be developed using site-directed mutagenesis and expressed in Escherichia coli and purified using established methods. To characterize these new PLN mutants in their unphosphorylated and phosphorylated states, the structural-dynamics will be studied using solution-state nuclear magnetic resonance (NMR) spectroscopy in dodecylphosphocholine (DPC) micelles. The resulting perturbations in the structural-dynamics will be then correlated to functional effects observed in coupled enzyme assays in the presence of SERCA. From the insight of this work, the aim of this proposal is to rationally design new PLN mutant candidates for gene therapy and establish new principles for the successful design of therapeutic PLN analogs. PUBLIC HEALTH RELEVANCE: By controlling the function of an enyzme by acting on the structural dynamic of its' subunits, the results of this study aim to move from understanding the interplay of structural dynamics of controlling physiology at a molecular level.