Critical Sorting Steps and Pathways in the Trafficking of Cardiac Sarcoplasmic Reticulum Proteins

心脏肌浆网蛋白运输的关键分选步骤和途径

• 批准号: 10719667
• 负责人: Zhenhui Chen
• 金额: $ 66.51万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-15 至 2027-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10719667
• 关键词: Adrenergic Agents; Adult; Anabolism; Animal Model; Binding; Biochemical; Cardiac; Cardiac Myocytes; Carrier Proteins; Cell membrane; Cells; Chronic; Communication; Complex; Cytoplasm; Data; Dedications; Disease; Distal; Endoplasmic Reticulum; Failure; Functional disorder; Golgi Apparatus; Growth; Heart; Heart Diseases; Heart Hypertrophy; Heart failure; Hypertrophy; Immunologics; Immunology procedure; Intercalated disc; Knowledge; Lead; Location; Membrane; Membrane Proteins; Messenger RNA; Microtubules; Molecular; Morphology; Motor; Muscle Cells; Nuclear; Nuclear Envelope; Nuclear Pore; Organ failure; Pathway interactions; Peptide Signal Sequences; Physiological; Preparation; Process; Protein Biosynthesis; Protein Sortings; Proteins; Proteome; Proteomics; Pump; Radial; Regulation; Ribosomes; Rough endoplasmic reticulum; Ryanodine Receptor Calcium Release Channel; SERCA2a; Sarcolemma; Sarcomeres; Sarcoplasmic Reticulum; Site; Sorting; Subcellular structure; Symptoms; System; Tertiary Protein Structure; Testing; Therapeutic; Therapeutic Intervention; Translating; Translations; Transmembrane Domain; Tubular formation; design; guided inquiry; mRNA Translation; new therapeutic target; novel; novel therapeutics; phospholamban; phospholemman; protein distribution; protein transport; response; rough endoplasmic reticulum cisterna; sex; targeted treatment; therapeutic target; therapeutically effective; trafficking; uptake

Cardiac hypertrophy can lead to heart failure, with few treatment options. Hypertrophic growth inevitably requires mechanisms of protein distribution, as studies show the involvement of both increased mRNA translation and mRNA trafficking along microtubules. Yet, how the distribution of membrane proteins occurs and responds to hypertrophic growth is unknown. While sarcoplasmic reticulum (SR) Ca handling is key to hypertrophy, we do not know how trafficking of SR proteins occurs using cardiomyocyte specific mechanisms, nor its regulation and response to hypertrophy, which precludes discovery of key targets for therapeutics. To probe this mechanism and identify sites of regulation, we have focused on a common portion of the biosynthetic membrane pathway where the rough endoplasmic reticulum (ER) organizes and sorts proteins for transit into SR tubules. Using a species-specific expression and immunological analysis to study the early steps of SR protein trafficking in adult cardiomyocytes, we show that newly synthesized proteins first accumulate into organized perinuclear ER puncta aligned with z-lines, and then transit to transverse SR z-tubules that aligned with T-tubules. Such morphology is observed for all newly made proteins examined, except for phospholamban, suggesting commonality of these protein sorting sites in ER. Afterward, motors can power transport of proteins both radially and axially via microtubules to their steady state distributions. These data lead to our hypotheses that newly synthesized SR proteins are sorted in a specific set of perinuclear ER subdomains, which are organized by the alignment with z-lines and in proximity to nuclear pores and translocons, contain critical ER trafficking proteins, and allow newly made SR proteins to develop their initial functional interactions for transit to SR. Such cellular sorting and trafficking steps adapt to the physiological demands, but maladapt to hypertrophic heart failure, which are likely amenable to develop new therapeutics regulating membrane protein trafficking. To dissect biochemical features of these sorting sites and trafficking steps, we have developed key experimental systems to determine progressive protein distribution 12-48 h after translation of three small single transmembrane domain proteins that are destined to different subcellular locations. Included is phospholamban (in the nuclear envelope and in longitudinal SR for Ca uptake), junctin (in junctional SR for Ca release), and phospholemman (in sarcolemma and intercalated discs to regulate the Na, K-pump). Aim 1 will determine: if a single perinuclear ER subdomain can effectively accumulate and co-localize newly made SR proteins (junctin and phospholamban), but separate phospholemman, for their diverse and distinct steady state distributions; if sites are aligned with nuclear pores, translocons; and if known ER trafficking proteins, newly identified in our SERCA-activated SR proteome, will modulate these trafficking steps. Aim 2 will determine whether a hypertrophic response modulates the accumulations of protein into any of the ER sorting sites, their common biosynthesis pathways, or the novel sets of cardiac ER trafficking proteins, in animal models of hypertrophy.

心脏肥大会导致心力衰竭，几乎没有治疗选择。肥厚的生长不可避免地 需要蛋白质分布的机制，因为研究表明两个mRNA的参与 沿微管的翻译和mRNA运输。然而，膜蛋白的分布是如何发生的， 对肥厚性生长的反应尚不清楚。而肌质网（SR）CA处理是关键 肥大，我们不知道使用心肌细胞特定机制的SR蛋白的运输是如何发生的 它也不是其对肥大的调节和反应，这排除了治疗剂的关键靶标。到 探究这种机制并确定调节部位，我们专注于生物合成的共同部分 膜途径，粗糙的内质网（ER）组织和分类蛋白质以转运到 SR小管。使用特异性表达和免疫学分析来研究SR的早期步骤 蛋白质运输在成人心肌细胞中，我们表明新合成的蛋白质首先积聚 有组织的核周ER与Z-Lines对齐，然后转移到对齐的横向SR Z-小管中 与T管。除了磷团，所有新制成的蛋白质均观察到这种形态 表明这些蛋白质分类位点在ER中的共同点。之后，电动机可以为蛋白质的运输动力运输 径向和轴向通过微管到其稳态分布。这些数据导致我们的假设 新合成的SR蛋白是在一组特定的核周核子域中排序的 通过与Z线对齐以及靠近核孔和易位的临界的临界ER组织 运输蛋白质，并允许新制作的SR蛋白开发其初始功能相互作用以进行过渡到 Sr。这种细胞分类和贩运步骤适应生理需求，但不适用于肥厚 心力衰竭，可能会开发新的治疗剂来调节膜蛋白运输。到 这些分类站点和贩运步骤的剖析生化特征，我们开发了关键的实验 在翻译三个小单个单一单一单一后12-48小时确定进行性蛋白质分布的系统 跨膜结构域蛋白，注定为不同的亚细胞位置。其中包括磷团 （在核包膜中和纵向SR中，用于CA的吸收），Junctin（在CA释放的连接式SR中）和 PhosphoLemman（在肌体和插入式圆盘中调节Na，K-Pump）。 AIM 1将确定：是否 单核ER子域可以有效地积累并共定位新制成的SR蛋白（Junctin 和Phospholamban），但分开了磷光体，用于它们的多样化和独特的稳态分布；如果 位点与核孔，转运孔对齐；如果众所周知的运输蛋白，我们在我们的 SERCA激活的SR蛋白质组将调节这些贩运步骤。 AIM 2将确定是否 肥厚反应调节蛋白质的积累到任何ER分选位点，它们的共同 在肥大动物模型中，生物合成途径或心脏运输蛋白的新型心脏ER运输蛋白。