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

描述（由申请方提供）：本提案的总体目标是合理设计“功能丧失”受磷蛋白（PLN）突变体候选物，以通过重组腺相关病毒（rAAV）基因治疗改善心肌病的发作。PLN是肌浆网Ca 2 +-ATP酶（SERCA）的内源性抑制剂，SERCA是负责70%的Ca 2+穿梭进入SR的整合膜酶，诱导人类心肌松弛。SERCA：PLN相互作用中的功能障碍被认为在心脏病中起作用。SERCA和PLN相互作用功能障碍的创新遗传治疗途径最近成为该领域的新兴趣，这是由于S16 E的成功，S16 E是PLN的一种假磷酸化形式，成功地减少了小鼠模型和大型动物中心力衰竭的进展。已经发现，PLN的结构动力学可以被调节以导致对SERCA的定向功能效应，并且本研究旨在创新其他候选物如S16 E用于治疗心力衰竭的基因疗法。该提案将集中在几个关键组成部分的面向域的合理设计的PLN突变体物种，并专注于这些特定的目标：目的1：定义的作用域通信PLN的能力，抑制SERCA。目的2：双假磷酸化PLN（S16 E/T17 E）的结构-动力学-功能表征。目的3：阐明PLN“超级抑制剂”的意义。将使用定点诱变开发新的PLN突变种属，并在大肠杆菌中表达，并使用既定方法纯化。为了表征这些新的PLN突变体在其非磷酸化和磷酸化状态，结构动力学将使用溶液状态核磁共振（NMR）光谱在十二烷基磷酸胆碱（DPC）胶束进行研究。在结构动力学中产生的扰动，然后将与在SERCA的存在下，在偶联酶测定中观察到的功能效应相关。从这项工作的洞察力，这项建议的目的是合理地设计新的PLN突变体候选基因治疗，并建立新的原则，成功设计治疗PLN类似物。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理解在分子水平上控制生理学的结构动力学的相互作用。

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.