COPPER ENTRY INTO HUMAN CELLS

铜进入人体细胞

• 批准号: 7690597
• 负责人: JACK H KAPLAN
• 金额: $ 34.13万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-15 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7690597
• 关键词: Acute; Affinity; Amino Acids; Animal Model; Antioxidants; Apical; Binding; Biotinylation; Blood; Blood Cells; Brain; Caco-2 Cells; Cell Count; Cell Fractionation; Cell membrane; Cell physiology; Cell surface; Cells; Collagen; Copper; Diet; Dietary Copper; Elements; Embryo; Endocytosis; Enterocytes; Epithelial Cells; Epithelium; Excretory function; Funding; Gel; Genetic; Growth; Health; Hepatolenticular Degeneration; Hereditary Disease; Homeostasis; Human; Image; Insecta; Intestines; Ions; Kidney; Kinetics; Laboratories; Light; Link; Liver; Mammalian Cell; Mammals; Measurement; Mediating; Membrane; Metabolic Biotransformation; Metals; Methods; Modification; Molecular Chaperones; Molecular Conformation; Mus; Mutagenesis; N-terminal; Nerve Degeneration; Nutrient; Pathway interactions; Play; Post-Translational Regulation; Process; Production; Proteins; Recycling; Regulation; Relative (related person); Reporting; Role; SLC11A2 gene; Series; Specificity; Structure; Superoxide Dismutase; Surface; Testing; Time; Tissues; Tracer; Work; absorption; apical membrane; cell growth regulation; extracellular; glycosylation; kidney cell; member; monolayer; mutant; neurotransmitter biosynthesis; orexin A receptor; orexin B receptor; overexpression; oxidation; programs; protein complex; research study; sensor; sugar; uptake

Copper is an essential nutrient required in many cellular processes. It is a co-factor in anti-oxidant defense by superoxide dismutase, in neurotransmitter biosynthesis, in collagen production and in many other important biotransformations. Adequate copper is essential for human health, but elevated levels of copper are toxic. Sophisticated mechanisms have evolved to regulate the absorbtion and excretion of dietary copper and provide the important homeostasis of cellular and organismal copper. The major protein responsible for the high affinity uptake of copper into human cells is hCTRl The protein consists of 190 aminoacid residues and the PI of this application has, during the previous funding period characterized the membrane topology of hCTRl and shown that it is composed of three transmembrane segments separating an extracellular amino-terminus and an intracellular carboxy-terminus. Structure-function studies utilizing copper transport measurements and mutagenesis in insect cells suggested a pore-like mechanism for transport that has found support in the structural studies from the lab of another member of this program (Dr V Linger), these studies will be extended in mammalian cells. The present application extends a series of findings made in the Pi's lab during the previous funding period, and utilizes methods developed to study hCTRl in mammalian cells and epithelia. The PI has demonstrated that hCTRl is post-translationally modified with Olinked (as well as the more familiar N-linked) sugars. The O-linked modification protects hCTRl against a specific cellular proteolytic cleavage. The mechanisms involved in this specific cleavage and their role in the cell physiology of hCTRl will be characterized. In addition, it has been demonstrated that endogenous hCTRl is present at the basolateral surface of intestinal and renal epithelial cells and intestinal tissue. Thus an hCTR1-independent process is involved in the first step of copper acquisition from the diet. The proposed studies will identify and characterize this pathway as well as investigate the regulation of copper uptake in a number of cells. The roles of a second copper transporter, hCTR2, will also be explored. The experiments will provide essential information about how cells acquire their necessary copper.

铜是许多细胞过程所需的必需营养素。它是抗氧化防御的辅助因子 通过超氧化物歧化酶，在神经递质生物合成、胶原蛋白生产和许多其他方面 重要的生物转化。充足的铜对人体健康至关重要，但铜含量升高 是有毒的。已经发展出复杂的机制来调节膳食铜的吸收和排泄 并提供细胞和有机体铜的重要稳态。主要负责的蛋白质 人体细胞对铜的高亲和力摄取是 hCTR1 该蛋白质由 190 个氨基酸残基组成 该应用程序的 PI 在之前的资助期间描述了膜拓扑结构 hCTR1 的结构，并表明它由三个跨膜片段组成，将细胞外 氨基末端和细胞内羧基末端。利用铜传输的结构功能研究 昆虫细胞的测量和诱变表明了一种类似孔的运输机制 在该计划的另一位成员（V Linger 博士）的实验室的结构研究中找到了支持，这些 研究将扩展到哺乳动物细胞。本申请扩展了一系列发现 Pi 的实验室在上一个资助期间，并利用开发的方法来研究 hCTRl 哺乳动物细胞和上皮细胞。 PI 已证明 hCTR1 经过 Olinked 翻译后修饰 （以及更熟悉的 N-连接）糖。 O-连接修饰保护 hCTR1 免受 特异性细胞蛋白水解裂解。参与这种特定裂解的机制及其在 将表征hCTR1的细胞生理学。此外，研究还表明，内源性 hCTR1存在于肠和肾上皮细胞以及肠组织的基底外侧表面。因此 从饮食中获取铜的第一步涉及一个独立于 hCTR1 的过程。拟议的 研究将识别和表征该途径，并研究铜吸收的调节 细胞数量。还将探讨第二种铜转运蛋白 hCTR2 的作用。实验 将提供有关细胞如何获取必需铜的重要信息。