Mechanism by which SepN modulates function of the RyR calcium release channel

描述（由申请人提供）：本提案的重点是测试硒蛋白N （SepN）的分子作用机制的流行模型。在人类中，导致SepN完全缺失的突变和影响RyR1蛋白功能的突变（RyR1蛋白是骨骼肌Ryanodine受体细胞内钙释放通道（RyR-CRC）的核心蛋白成分）导致具有类似细胞缺陷谱的先天性肌病。我们最近发表的工作（Jurynec， M. J. et al., 2008）。硒蛋白N是人体和斑马鱼肌肉中ryanodine受体钙释放通道活性所必需的。PNAS 105: 12485-90)发现SepN是体内正常钙动员和体外正常RyR- CRC功能所必需的因子。从斑马鱼胚胎或缺乏SepN的人病变肌肉中分离的RyR-CRC在体外不再对溶液氧化还原环境的变化做出反应，这表明SepN是RyR-CRC氧化还原传感器的重要组成部分。与这些发现一致，SepN具有指示氧化还原酶的序列基序。由于我们发现SepN在体内与RyR-CRC存在物理关联，我们提出i) SepN直接与钙释放通道相互作用，ii) SepN作为底物特异性氧化还原酶，有助于调节CRC的活性，以及iii) CRC功能缺陷导致SepN相关的肌病。最近提出了另一种模型，其中SepN的主要功能是维持细胞的整体氧化还原状态，仅间接影响肌肉功能（Arbogast， S. et al.(2009)）。sepn1相关肌病的氧化应激：从病理生理学到治疗。神经科杂志65:677-686。我们的目标是了解SepN介导通道功能调节的机制，从而更广泛地了解RyR-CRC活性如何在正常发育过程中被调节，在疾病过程中被干扰，或在治疗中被增强。本R03“小额资助”提案的目标是测试我们工作假设的要素，作为长期研究的重要第一步，旨在确定i) SepN与RyR-CRC之间的特定相互作用以及ii) SepN活性修饰的特定RyR-CRC靶点。在这里，我们将确定：目标#1)是否需要SepN的氧化还原功能来实现正常的RyR-CRC功能，以及目标#2)RyR-CRC是否是SepN氧化还原反应的直接靶标。了解SepN的功能将确定正常肌肉发育和功能所需的途径或生理状态。更具体地说，如果我们的工作模型是正确的，那么了解SepN的功能将确定一个关键机制，通过该机制，钙动员可以在多种细胞信号传导和细胞生理环境中得到调节。

DESCRIPTION (provided by applicant): The focus of this proposal is to test prevailing models of the molecular mechanism of action of Selenoprotein N (SepN). In humans, mutations that cause complete loss of SepN and mutations that affect the function of the RyR1 protein, the core protein component of the skeletal muscle Ryanodine Receptor intracellular Calcium Release Channel (RyR-CRC), result in congenital myopathies with a similar spectrum of cellular defects. Our recently published work (Jurynec, M. J. et al. (2008). "Selenoprotein N is required for ryanodine receptor calcium release channel activity in human and zebrafish muscle." PNAS 105: 12485-90.) identified SepN as a factor necessary for normal calcium mobilization in vivo and necessary for normal RyR- CRC function measured in vitro. RyR-CRCs isolated from zebrafish embryos or human diseased muscle lacking SepN no longer responded in vitro to changes in the solution redox environment indicating SepN as an essential component of the RyR-CRC redox sensor. Consistent with these findings, SepN has sequence motifs indicative of an oxidoreductase. As we found SepN physically associated with the RyR-CRC in vivo, we proposed that i) SepN interacts directly with the Calcium Release Channel, ii) SepN functions as a substrate- specific oxidoreductase that helps regulate activity of the CRC, and iii) defects in CRC function account for SepN-related myopathies. An alternative model has been recently set forth in which the primary function of SepN is to maintain the overall redox state of the cell and only indirectly affects muscle function (Arbogast, S. et al. (2009). "Oxidative stress in SEPN1-related myopathy: From pathophysiology to treatment." Ann Neurol 65: 677-686.). Our goal is to understand the mechanism by which SepN mediates the regulation of channel function as an entrance toward a broader understanding of how RyR-CRC activity may be regulated during normal development, perturbed in disease processes, or potentiated by treatments. The goal of this R03 "small grant" proposal is to test elements of our working hypothesis as an essential first step toward a longer-term study aimed at identifying i) the specific interactions between SepN and the RyR-CRC and ii) the specific RyR-CRC target sites modified by SepN activity. Here we will determine: Aim #1) if the redox function of SepN is required for normal RyR-CRC function, and Aim #2) if the RyR-CRC is a direct target of the SepN redox reaction. Understanding the function of SepN will identify pathways or physiological states required for normal muscle development and function. More specifically, if our working model is correct, understanding SepN function will identify a key mechanism by which calcium mobilization can be regulated in multiple cell signaling and cell physiology contexts. PUBLIC HEALTH RELEVANCE: Mutations that completely block expression or function of Selenoprotein N (SepN) cause birth defects affecting muscle development and function. Mutations affecting the intracellular Calcium Release Channel cause similar birth defects. Our recent studies indicate that SepN that helps regulate Calcium Release Channel function, explaining why loss of either factor has a common effect. The focus of this proposal is to understand the mechanism by which SepN regulates channel function as an entrance toward a broader understanding of how Calcium Release Channel activity may be regulated during normal development, perturbed in disease processes, or potentiated by treatments.