Secretory Pathway Calcium and Manganese Pumps

分泌途径钙和锰泵

• 批准号: 7473439
• 负责人: RAJINI RAO
• 金额: $ 0.33万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-06-01 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7473439
• 关键词: Affinity; Animal Model; Binding; Biochemical; Biological; Biological Assay; Brain; Ca(2+)-Transporting ATPase; Caco-2 Cells; Calcium; Cell membrane; Cell model; Cell physiology; Characteristics; Collection; DNA Damage; Deposition; Disease; Drug Metabolic Detoxication; EF Hand Motifs; Engineering; Enterocytes; Familial benign pemphigus; Family; Family member; Foundations; Genes; Golgi Apparatus; Growth; Helix (Snails); Hepatocyte; Hippocampus (Brain); Homeostasis; Homologous Gene; Human; Ion Pumps; Ion Transport; Ions; Knock-out; Length; Link; Liver; Mammalian Cell; Mammary gland; Manganese; Measures; Mediating; Membrane; Milk; Modeling; Molecular; Mutation; N-terminal; Names; Neurons; Oxidative Stress; Parkinsonian Disorders; Pathway interactions; Phenocopy; Phenotype; Physiological; Physiological Processes; Pichia; Play; Process; Production; Property; Protein Binding; Protein Glycosylation; Protein Isoforms; Proteins; Pump; Range; Rattus; Research Personnel; Resolution; Retrieval; Role; Saccharomyces; Skin; Sorting - Cell Movement; Testing; Thinking; Tooth structure; Toxic effect; Ulcer; Up-Regulation; Yeast Model System; Yeasts; base; basolateral membrane; biological adaptation to stress; bone; ear helix; genome-wide analysis; intestinal epithelium; mutant; novel; polarized cell; programs; response; trafficking; trans-Golgi Network

Secretory Pathway Calcium-ATPases (SPCA) are a newly defined family of ion pumps that transport calcium and manganese into the lumen of the Golgi apparatus where they are essential for sorting, processing and glycosylation of proteins. The first member of this family, named PMR1, was described in Saccharomyces cerevisae, and more recently, two mammalian homologues of PMR1, SPCA1 and SPCA2, have been identified. Mutations in hSPCAl cause Hailey Hailey disease, a debilitating disorder characterized by severe ulceration of the skin, thought to result from dysregulation of cellular calcium. Excess manganese is deposited in the brain and leads to Parkinsonism. The SPCA have been implicated in diverse physiological processes, ranging from manganese detoxification in the liver, calcium transport across intestinal epithelia, and milk production by the mammary glands, although there is little molecular evidence for their specific roles. This proposal combines three parallel approaches to investigate the SPCA: biochemical studies using purified proteins or Golgi membranes, cell biological studies in polarized cultured mammalian cells, and large scale phenomic analysis of ion homeostasis in a model organism. In previous studies, we have defined the transmembrane helices and residues critical for ion transport and have identified a role for helix packing in determining ion selectivity. In Aim 1 of this proposal, we will shift our focus to understanding the ion binding and modulatory role of EF motifs in the cytoplasmic N-terminal domain. We have new evidence for trafficking of the pumps between the Golgi stacks and a novel, vesicular compartment in polarized cell models of hepatocytes and enterocytes. In Aim 2, we will determine if trafficking is ion-dependent and related to transcellular transport, and whether specific retrieval or PDZ-binding motifs at the C-terminus are important for localization. Gene knockdown approaches will be used to evaluate the isoform-specific contributions of the two SPCA pumps in the enterocyte model. Finally, we propose to use the yeast model organism for high resolution phenomic analysis that will identify new genes and pathways associated with the cellular function of the SPCA pumps (Aim 3). Taken together, this proposal lays the foundation for understanding the role of this novel family of transporters at the molecular, cellular and physiological level.

分泌途径钙-ATPases（SPCA）是一个新定义的离子泵家族， 将钙和锰运输到高尔基体的腔内，它们是必不可少的 用于分类，加工和糖基化蛋白质。这个家庭的第一个成员，名为PMR1， 在酿酒酵母中描述了 已鉴定出PMR1，SPCA1和SPCA2。 HSPCAL的突变导致Hailey Hailey 疾病是一种以皮肤严重溃疡为特征的令人衰弱的疾病，被认为是由 细胞钙的失调。多余的锰被沉积在大脑中，并导致 帕金森主义。 SPCA与不同的生理过程有关 肝脏中的锰排毒，钙跨肠上皮的钙转运和牛奶 乳腺的生产，尽管几乎没有分子证据证明其特异性 角色。该提案结合了研究SPCA的三种平行方法：生化 使用纯化蛋白或高尔基膜的研究，极化培养的细胞生物学研究 哺乳动物细胞和模型生物体中离子稳态的大规模现象分析。在 先前的研究，我们定义了跨膜螺旋和离子转运至关重要的残基 并确定了螺旋填料在确定离子选择性中的作用。在本提案的目标1中， 我们将把重点转移到理解EF基序中的离子结合和调节作用 细胞质N末端结构域。我们有新的证据表明贩运泵 高尔基堆和肝细胞极化细胞模型中的一种新颖的囊泡室 肠球菌。在AIM 2中，我们将确定运输是否依赖于离子，并且与跨细胞相关 运输，以及在C端的特定检索还是PDZ结合基序对 本土化。基因敲低方法将用于评估同工型特异性 肠道细胞模型中两个SPCA泵的贡献。最后，我们建议使用酵母 高分辨率现象分析的模型生物，该分析将识别新基因和途径 与SPCA泵的细胞功能相关（AIM 3）。综上所述，该提议提出 理解这个新型转运蛋白家族在分子中的作用的基础， 细胞和生理水平。