Crystallization of eukaryotic facilitated glucose transporters

真核促进葡萄糖转运蛋白的结晶

• 批准号: 7815678
• 负责人: Lan Guan
• 金额: $ 1.6万
• 依托单位: TEXAS TECH UNIVERSITY HEALTH SCIS CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-01 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7815678
• 关键词: 3-Dimensional; Binding Proteins; Biochemical; Biology; Blood - brain barrier anatomy; Cell membrane; Cell physiology; Crystallization; Data; Detergents; Diffusion; Disease; Drug Design; Epidemic; Erythrocytes; Escherichia coli; Exhibits; Family; Foundations; Future; GLUT-1 protein; Generations; Glucose; Glucose Transporter; Goals; Guidelines; Health; Homologous Gene; Human; Inherited; Insulin; Mammalian Cell; Medicine; Membrane; Membrane Proteins; Membrane Transport Proteins; Methods; Molecular; Molecular Cloning; Mutagenesis; Mutation; Neurologic; Non-Insulin-Dependent Diabetes Mellitus; Phospholipids; Play; Proteins; Research; Resolution; Roentgen Rays; Role; SLC2A1 gene; Screening procedure; Structure; Surface; Syndrome; System; Testing; Therapeutic Intervention; Tissues; X-Ray Crystallography; cancer cell; glucose disposal; glucose transport; improved; insight; lactose permease; member; mutant; novel strategies; overexpression; protein expression; protein protein interaction; protein structure; structural biology; success

DESCRIPTION (provided by applicant): The long term goals of this project are to understand the structure and function of physiologically and clinically important membrane transporters through a combination of X-ray crystallography and biochemical-biophysical approaches. Membrane proteins play crucial roles in many aspects of cell function; some are targets for pharmacologically and toxicologically active substances. The human facilitated glucose transporters (Gluts), members of the Major Facilitator Superfamily (MFS), comprise a relatively large family of structurally related proteins (GLUT1-12). Glut1, which is abundant in the human red blood cells (RBCs) and the blood-brain barrier, is an extensively-studied representative of these facilitated transporters. Glut4 is responsible for insulin-regulated glucose disposal. Several diseases have been identified with the mutation and malfunction of Gluts, such as GLUT1 deficiency syndrome, a hereditary neurological syndrome, and type II diabetes, one of major threats to human health that is becoming alarmingly epidemic in scale. Therefore, it is important to obtain a high-resolution structure, as well as characterize the transport mechanism at a molecular level. So far, there is no 3-D crystal structure available for any glucose facilitator, although many attempts have been made, particularly with Glut1. This proposal will focus on the crystallization of eukaryotic facilitated Gluts, particularly Glut1 from human RBCs. Isolation and purification of Glut1 from human RBCs was achieved more than two decades ago. Recently, the PI of this proposal observed that phospholipids (PL) play an important role in the crystallization of lactose permease of Escherichia coli, a member of MFS. Manipulating PL has dramatically improved our success rate for crystallization related membrane proteins. Therefore, the use of PL in crystallization should yield a promising approach for obtaining crystal structures of eukaryotic members of the MFS. Glut1, a human protein of MFS, will be purified from RBC membranes; over-expression systems of eukaryotic Glut1-4 in different systems will be simultaneously tested. Manipulation of PL content will be introduced in addition to conventional crystallization methods, to crystallize human Glut1 and/or the eukaryotic Gluts, which will lay the foundation for solving X-ray crystal structures of these important eukaryotic transporters in the future. Successful crystallization of eukaryotic Gluts is imperative to obtaining X-ray crystal structures; the expected structures will substantially improve our understanding of facilitated glucose transport and provide important clues for therapeutic intervention, which will have significant impact in biology and medicine.

描述（由申请人提供）：该项目的长期目标是通过X射线晶体学和生物化学-生物物理方法的组合来了解生理和临床上重要的膜转运蛋白的结构和功能。膜蛋白在细胞功能的许多方面起着至关重要的作用;有些是毒性和毒理学活性物质的靶点。人葡萄糖易化转运蛋白（Gluts）是主要易化因子超家族（MFS）的成员，包括一个相对较大的结构相关蛋白家族（GLUT 1 -12）。Glut 1在人红细胞和血脑屏障中含量丰富，是这些易化转运蛋白的代表，已被广泛研究。Glut 4负责胰岛素调节的葡萄糖处置。已经确定了几种疾病与Gluts的突变和功能障碍有关，例如GLUT 1缺乏综合征（一种遗传性神经系统综合征）和II型糖尿病（对人类健康的主要威胁之一，其流行程度令人担忧）。因此，重要的是获得高分辨率的结构，以及在分子水平上表征传输机制。到目前为止，还没有任何葡萄糖促进剂的三维晶体结构，尽管已经进行了许多尝试，特别是Glut 1。该建议将集中于真核易化Gluts的结晶，特别是来自人红细胞的Glut 1。二十多年前，从人红细胞中分离和纯化Glut 1。最近，该提案的PI观察到，磷脂（PL）在MFS成员大肠杆菌的乳糖通透酶的结晶中起重要作用。操纵PL显著提高了我们对结晶相关膜蛋白的成功率。因此，在结晶中使用PL应该产生一个有前途的方法获得晶体结构的真核成员的MFS。将从RBC膜中纯化MFS的人蛋白Glut 1;将同时测试真核Glut 1 -4在不同系统中的过表达系统。除了常规的结晶方法外，还将引入PL含量的调控，使人Glut 1和/或真核Glut蛋白结晶，这将为将来解决这些重要的真核转运蛋白的X射线晶体结构奠定基础。真核Gluts的成功结晶是获得X射线晶体结构的必要条件;预期结构将大大提高我们对葡萄糖易化转运的理解，并为治疗干预提供重要线索，这将在生物学和医学上产生重大影响。