Investigating the role of GRP170 in ENaC biogenesis

研究 GRP170 在 ENaC 生物发生中的作用

• 批准号: 9087782
• 负责人: Teresa M Buck
• 金额: $ 7.7万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-01 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9087782
• 关键词: ATPase Domain; Address; Amiloride; Anabolism; Apoptosis; Applications Grants; Atherosclerosis; Bartter Disease; Binding; Biochemical; Biogenesis; Biological Assay; Biological Models; Blood Pressure; C-terminal; Cell surface; Cells; Communities; Complement; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Cytosol; Data; Defect; Disease; Endocytosis; Endoplasmic Reticulum Degradation Pathway; Epithelial; Epithelium; Event; Foundations; Future; Goals; Guanine Nucleotide Exchange Factors; Homeostasis; Homologous Gene; Human Cell Line; Hypertension; Hypotension; In Vitro; Inbred F344 Rats; Individual; Ischemia; KCNJ1 gene; Kidney; Lead; Link; Liquid substance; Lung; Measurement; Measures; Membrane Proteins; Molecular Chaperones; Molecular Conformation; Molecular Weight; Monitor; Mutation; N-terminal; Nucleotides; Output; Parkinson Disease; Pathway interactions; Physiology; Play; Positioning Attribute; Process; Protein Family; Proteins; Proteolytic Processing; Pseudohypoaldosteronism; Quality Control; Rattus; Regulation; Research; Role; Series; Sodium; Sodium Channel; Sodium Chloride; Surface; Syndrome; System; Testing; Thyroid Gland; Translating; Ubiquitination; Vascular Endothelial Growth Factors; Water; Work; Xenopus oocyte; Yeasts; base; blood pressure regulation; epithelial Na+ channel; follow-up; gain of function mutation; glucose-regulated protein 170; in vitro Assay; insight; insulin secretion; interest; knock-down; loss of function mutation; member; monolayer; mouse model; mutant; novel; overexpression; prevent; protein misfolding; public health relevance; research study; response; targeted treatment; therapeutic development; trafficking; tumor; vasopressin resistant diabetes insipidus

 DESCRIPTION (provided by applicant): The major focus of the study proposed here is to characterize the role of the ER lumenal chaperone, GRP170, on the quality control and trafficking of the Epithelial Sodium Channel (ENaC). ENaC is a heterotrimeric protein composed of α, β, and γ subunits. ENaC is responsible for salt reabsorption across the epithelia of the kidney and lung, and plays a critical role in controlling blood pressure and airway fluid volume. Defects in ENaC degradation are associated with Liddle's Syndrome and pseudohypoaldosteronism type I that result in hyper- and hypotension, respectively. ENaC is monitored by the ER quality control machinery and targeted for ER associated degradation (ERAD). ER associated degradation (ERAD) is the process whereby proteins entering the secretory pathway are subject to degradation when they fail to attain a mature conformation. In fact, even a large percentage of WT ENaC is targeted for the ERAD machinery. Many other disease relevant proteins can also become ERAD substrates, including CFTR (cystic fibrosis), AQP2 (nephrogenic diabetes insipidus), and Pael-R (Parkinson's disease). The focus of this proposal on the role of GRP170 in ENaC quality control and trafficking arises from the following considerations. First, GRP170 is abundant and is a member of the high molecular weight Hsp70-like family of proteins. Second, GRP170 possesses two unique activities: GRP170 acts as a nucleotide exchange factor (NEF) for the ER lumenal Hsp70, and it acts as a "holdase" in vitro and binds to misfolded proteins within the secretory pathway. Third, GRP170 targets the α ENaC subunit for ERAD, but has no effect on the β and γENaC subunits. Fourth, in contrast to its effect on individual subunits, preliminary data suggest that GRP170 promotes the assembly and surface expression of the ENaC channel (αβγ) in Xenopus oocytes. A Fisher Rat Thyroid (FRT) cell system will be used to investigate the differential effect of GRP170 on the monomeric versus assembled ENaC channel. FRT cells form polarized monolayers in culture, allowing for measurement of ENaC subunit stability, surface expression, proteolytic processing, and ENaC function, as measured by amiloride-sensitive Na+ short circuit current. These outputs will be quantified in response to knockdown or overexpression of GRP170. To further characterize the mechanism of GRP170 action on ENaC a series of GRP170 constructs with domain deletions will also be assayed. In addition to its role in regulating ENaC and therefore blood pressure, GRP170 function has been tied to other relevant processes, including insulin secretion, secretion of vascular endothelial growth factor (VEGF), tumor survival, and programmed cell death. Moreover, GRP170 plays a cytoprotective role during ischemia and atherosclerosis. Together, understanding GRP170 mechanism of action will provide novel insights into ENaC function and associated disease states.

 描述（由申请方提供）：此处拟定研究的主要重点是表征ER内腔伴侣GRP 170对上皮钠通道（ENaC）的质量控制和运输的作用。ENaC是由α、β和γ亚基组成的异源三聚体蛋白。ENaC负责跨肾脏和肺的上皮细胞的盐重吸收，并且在控制血压和气道流体体积中起关键作用。ENaC降解缺陷与Liddle综合征和I型假性醛固酮减少症相关，分别导致高血压和低血压。ENaC由ER质量控制机构监测，并针对ER相关降解（ERAD）。ER相关降解（ERAD）是蛋白质进入分泌途径时，当它们不能达到成熟构象时发生降解的过程。事实上，即使是很大比例的WT ENaC也是ERAD机制的目标。许多其他疾病相关蛋白质也可以成为ERAD底物，包括CFTR（囊性纤维化），AQP 2（肾源性尿崩症）和Pael-R（帕金森病）。本提案的重点是GRP 170在ENaC质量控制和贩运中的作用，这是出于以下考虑。首先，GRP 170是丰富的，并且是高分子量Hsp 70样蛋白家族的成员。第二，GRP 170具有两种独特的活性：GRP 170作为ER内腔Hsp 70的核苷酸交换因子（NEF），并且其在体外作为“保持酶”并结合分泌途径内的错误折叠蛋白。第三，GRP 170靶向ERAD的α ENaC亚基，但对β和γENaC亚基没有影响。第四，与其对单个亚基的作用相反，初步数据表明GRP 170促进非洲爪蟾卵母细胞中ENaC通道（αβγ）的组装和表面表达。Fisher大鼠甲状腺（FRT）细胞系统将用于研究GRP 170对单体与组装ENaC通道的差异效应。FRT细胞在培养物中形成极化单层，允许测量ENaC亚基稳定性、表面表达、蛋白水解加工和ENaC功能，如通过阿米洛利敏感性Na+短路电流测量的。这些输出将响应于GRP 170的敲低或过表达而定量。为了进一步表征GRP 170对ENaC的作用机制，还将测定一系列具有结构域缺失的GRP 170构建体。除了其在调节ENaC和血压中的作用外，GRP 170功能还与其他相关过程有关，包括胰岛素分泌、血管内皮生长因子（VEGF）分泌、肿瘤存活和程序性细胞死亡。此外，GRP 170在缺血和动脉粥样硬化过程中发挥细胞保护作用。总之，了解GRP 170的作用机制将为ENaC功能和相关疾病状态提供新的见解。