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

用于基因治疗的功能缺失磷酸兰班突变体的合理设计

• 批准号: 8035358
• 负责人: Kim N Ha
• 金额: $ 2.73万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-02-16 至 2012-02-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8035358
• 关键词: Affect; Affinity; Animals; Attenuated; Binding; Biochemical; Biological; Biological Assay; Ca(2+)-Transporting ATPase; Candidate Disease Gene; Cardiac; Cardiac Myocytes; Cardiomyopathies; Collaborations; Communication; Coupled; Cytoplasmic Tail; Data; Dilated Cardiomyopathy; Disease; Double Effect; Engineering; Enzymes; Escherichia coli; Functional disorder; Genetic; Goals; Health; Heart Diseases; Heart failure; Human; Learning; Membrane; Methods; Micelles; Modeling; Molecular; Mus; Muscle relaxation phase; Mutagenesis; Mutation; Myocardium; NMR Spectroscopy; Outcome; Paper; Pathway interactions; Phosphorylation; Physiology; Protein Biochemistry; Publications; Recombinant adeno-associated virus (rAAV); Regulation; Reticulum; Role; Site-Directed Mutagenesis; Solutions; Structure; Techniques; Testing; Therapeutic; Translating; Transmembrane Domain; Universities; Work; analog; base; design; dodecylphosphocholine; gene therapy; improved; inhibitor/antagonist; innovation; insight; interest; intermolecular interaction; loss of function; mutant; novel therapeutics; phospholamban; success

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.

描述(申请人提供)：这项建议的总体目标是合理设计“功能丧失”的磷蛋白(PLN)突变候选基因，以通过重组腺相关病毒(RAAV)基因治疗来改善心肌病的发病。PLN是肌浆网钙-ATPase(SERCA)的内源性抑制物，SERCA是一种完整的膜酶，负责70%的Ca~(2+)穿梭到SR中，引起人体心肌松弛。SERCA功能障碍：PLN相互作用被认为在心脏病中起作用。最近，由于S16E的成功，创新SERCA和PLN相互作用功能障碍的遗传治疗的途径成为该领域的新兴趣。S16E是PLN的一种伪磷酸化形式，成功地减缓了小鼠模型和大型动物心力衰竭的进展。已经发现PLN的结构动力学可以调节，从而对SERCA产生直接的功能作用，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。这项建议将集中在PLN突变物种面向区域的合理设计的几个关键组成部分，并集中于这些特定的目标：目标1：确定区域通信在PLN抑制SERCA能力中的作用。目的2：双伪磷酸化PLN(S16E/T17E)的结构-动力学-功能表征。目的3：阐明PLN“超级抑制物”的意义。将使用定点突变技术开发新的PLN突变物种，并在大肠杆菌中表达，并使用已建立的方法进行纯化。为了表征这些新的PLN突变体的非磷酸化和磷酸化状态，将使用十二烷基磷胆碱(DPC)胶束中的溶液状态核磁共振(NMR)光谱来研究结构动力学。由此产生的结构-动力学的扰动将与在SERCA存在的情况下观察到的偶联酶分析的功能效应相关联。从这项工作的观点来看，这项建议的目的是合理地设计新的PLN基因治疗突变候选，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控制生理学的结构动力学的相互作用。