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.

心脏肥大可导致心力衰竭，治疗选择很少。不可避免地会过度生长 需要蛋白质分布机制，因为研究表明mRNA的增加都参与其中 翻译和mRNA沿沿着运输。然而，膜蛋白的分布如何发生， 对肥大性生长的反应是未知的。虽然肌浆网（SR）钙处理是关键， 肥大，我们不知道SR蛋白的运输如何使用心肌细胞特异性机制发生， 也没有其对肥大的调节和反应，这妨碍了治疗的关键靶点的发现。到 探索这一机制，并确定网站的调控，我们已经集中在一个共同的部分，生物合成 粗面内质网（ER）组织和分类蛋白质以转运到 SR小管。利用种特异性表达和免疫分析研究SR的早期步骤 在成年心肌细胞的蛋白质运输中，我们发现新合成的蛋白质首先积累到 有组织的核周ER点与z线对齐，然后过渡到横向SR z小管， 有T型小管对于所有新制备的蛋白质，除了受磷蛋白， 这表明这些蛋白质分选位点在ER中具有共性。之后，马达可以驱动蛋白质的运输 径向和轴向通过微管到它们的稳态分布。这些数据引出了我们的假设 新合成的SR蛋白被分类在一组特定的核周ER亚结构域中， 通过与z线对齐并靠近核孔和translocon组织，包含关键ER 运输蛋白，并允许新产生的SR蛋白发展其最初的功能相互作用，以转运到 Sr.这样的细胞分选和运输步骤适应了生理需求，但不适应肥大细胞。 心力衰竭，这可能适合于开发调节膜蛋白运输的新疗法。到 解剖这些分选位点和运输步骤的生化特征，我们已经开发了关键的实验 系统，以确定三个小的单克隆抗体翻译后12 - 48小时的蛋白质分布 这些跨膜结构域蛋白质被指定到不同的亚细胞位置。包括受磷蛋白 (in核被膜和纵向SR中的Ca摄取），连接蛋白（连接SR中的Ca释放），和 磷膜蛋白（在肌膜和闰盘中调节Na，K泵）。目标1将确定：如果 单个核周ER亚结构域可以有效地积累和共定位新产生SR蛋白（连接蛋白 和受磷蛋白），但单独的磷脂，为他们的多样性和独特的稳态分布;如果 位点与核孔，translocons对齐;如果已知ER运输蛋白，在我们的研究中新发现， SERCA激活的SR蛋白质组将调节这些运输步骤。目标2将决定 肥大反应调节蛋白质在任何内质网分选位点的积累，它们共同的 在肥大的动物模型中，研究了心肌细胞生物合成途径，或心肌ER运输蛋白的新集合。