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.

心肌肥厚可能导致心力衰竭，治疗方案很少。肥厚性生长不可避免 需要蛋白质分布的机制，因为研究表明，这两种增加的mRNA都参与了 翻译和信使核糖核酸沿微管运输。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。而肌浆网(SR)钙的处理是 肥大，我们不知道SR蛋白是如何通过心肌细胞特有的机制进行运输的， 也不是它对肥大的调节和反应，这排除了发现治疗的关键靶点。至 探索这一机制并确定调控位点，我们重点介绍了生物合成的一个共同部分 粗面内质网(ER)组织和分类蛋白质以转运进入的膜途径 高级小管。利用物种特异性表达和免疫学分析研究SR的早期步骤 在成年心肌细胞中的蛋白质运输，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，然后过渡到对齐的横向SR z-微管 带着T形小管。除了磷蛋白外，所有新合成的蛋白质都观察到了这种形态， 提示内质网中这些蛋白质分选位点具有共性。之后，马达可以为蛋白质的运输提供动力。 径向和轴向均通过微管向其稳态分布。这些数据引出了我们的假设 新合成的SR蛋白被分选在一组特定的核周ER亚域中，这些亚域是 通过与z线对齐并在核孔和转座子附近组织，含有关键的内质网 运输蛋白，并允许新制造的SR蛋白发展其最初的功能相互作用，以转移到 高级这样的细胞分选和运输步骤适应生理需求，但不适应肥大 心力衰竭，这可能有助于开发调节膜蛋白运输的新疗法。至 剖析了这些分拣部位的生化特征和运输步骤，我们开发了关键的实验 确定三个小分子单链翻译后12-48小时递进蛋白质分布的系统 去往不同亚细胞位置的跨膜结构域蛋白。包括的是磷脂蛋白 (核膜和纵向SR中的钙摄取)、连接素(在连接SR中的钙释放)，以及 磷脂酶(在肌膜和间盘中调节Na，K-泵)。目标1将确定：如果一个 单个核周ER亚域可以有效地积聚和共定位新产生的SR蛋白(连接素 和磷蛋白)，但分离的磷莱曼，因为他们多样化和独特的稳态分布；如果 位点与核孔、转运蛋白排列一致；如果已知内质网运输蛋白，则在我们的 SERCA激活的SR蛋白质组，将调节这些转运步骤。目标2将决定一个 肥大反应调节蛋白质的积聚到任何内质网分选位置，它们共同的 肥厚动物模型中的生物合成途径，或新的一组心脏ER转运蛋白。