Structure-Function Studies of the Proton-Gated Urea Channel from H. Pylori

幽门螺杆菌质子门控尿素通道的结构功能研究

• 批准号: 8579827
• 负责人: HARTMUT LUECKE
• 金额: $ 46.25万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-02-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8579827
• 关键词: Affect; Affinity; Amides; Amino Acids; Antibiotic Therapy; Antibiotics; Architecture; Asthma; Binding; Biological Assay; C-terminal; Carboxylic Acids; Cessation of life; Charge; Chronic; Clarithromycin; Complement; Complement Inactivators; Developed Countries; Development; Discrimination; Drug Targeting; Duodenal Ulcer; Environment; Failure; Funding; Gastric Adenocarcinoma; Gastric mucosa; Gastritis; Gastroesophageal reflux disease; Goals; Helicobacter Infections; Helicobacter pylori; Histidine; Homo; Hydrogen Bonding; Incidence; Individual; Infection; Intervention; Intestinal Diseases; Knock-out; Knowledge; Lead; Measurement; Measures; Meta-Analysis; Metronidazole; Metronidazole resistance; Molecular Conformation; Mutagenesis; Mutate; Mutation; Organism; Outcome; Oxygen; Pathway interactions; Patients; Peptic Ulcer; Pharmaceutical Preparations; Point Mutation; Population; Positioning Attribute; Preventive; Protomer; Proton Pump Inhibitors; Protons; Research; Resistance; Resolution; Role; Side; Site; Specificity; Stomach; Stomach Carcinoma; Stomach Diseases; Structure; Structure-Activity Relationship; Surface; System; Tandem Repeat Sequences; Testing; Thiourea; Thrombocytopenic Purpura; Urea; Urease; Work; Xenopus oocyte; acid stress; base; design; disorder prevention; field study; high throughput screening; improved; infectious disease treatment; inhibitor/antagonist; innovation; malignant stomach neoplasm; microbiome; mimetics; mucosa-associated lymphoid tissue lymphoma; mutant; novel; pathogen; periplasm; prevent; proteoliposomes; public health relevance; small molecule; success; three dimensional structure; urea transporter

DESCRIPTION (provided by applicant): Infection of the gastric mucosa by Helicobacter pylori remains a worldwide problem and contributes to peptic ulcer disease and gastric cancer. Without active intervention, at least 20% of the population of developed countries will continue to be infected by this gastric pathogen. Eradication of the organism would contribute to prevention of disease. Current eradication requires triple therapy, a proton-pump inhibitor and two antibiotics given twice a day for 10 to 14 days. Resistance to either clarithromycin or metronidazole is > 20% and rising. No monotherapy is effective. Gastric infection by H. pylori depends on the expression of a channel unique to this pathogen, HpUreI. HpUreI is a proton-gated urea channel necessary for rapid access of urea to intrabacterial urease, essential for maintaining the periplasm at pH 6.1 in the acidic environment of the stomach, as low as pH 2.5, thus allowing colonization of the stomach. Expression of the channel is increased in the stomach. The channel has 195 residues with six transmembrane segments and three periplasmic regions. Mutagenesis studies have shown that the proton gating of HpUreI is regulated by hydrogen bonding between histidines and carboxylic acid residues in these three periplasmic regions. In the first funding cycle we determined the 3-dimensional structure of this channel, and we now propose to exploit that knowledge to increase our understanding of its proton-gating mechanism and to identify binding modes of inhibitors, some of which have been discovered already. Inhibition of this channel would be expected to result in specific and effective monotherapy for eradication of the organism and usher in an era of test and treat, rather than only treating symptomatic patients. This would provide a preventive approach to serious upper gastro-intestinal diseases, particularly stomach cancer, which causes approximately 750,000 deaths annually. Our specific aims are 1. Generate higher-resolution crystals and analyze the open and closed forms of HpUreI using new N- or C-terminal cleavable 6His- tag constructs of HpUreI. 2. Confirm the urea pathway and selectivity filter suggested by our HpUreI crystal structure by determining the effects of site-directed mutations on transport and on substrate selectivity of HpUreI using Xenopus oocyte and proteoliposome assays for urea transport as well as mutants expressed in H. pylori ureI knock-out strains. 3. Analyze the role of the hexameric arrangement by mutating residues conserved at the protomer interface of the hexamer to disrupt protomer association, followed by activity measurements of the monomeric state using Xenopus oocytes and proteoliposomes. 4. Discover and improve small molecule HpUreI inhibitors based on the structure of channel in the open form; measure their ability to increase the Tm of HpUreI, to inhibit urease activity in intact H. pylori and to affect survival in a novel gastro-mimetic incubation system.

描述（由申请人提供）：幽门螺杆菌对胃粘膜的感染仍然是全球问题，并导致了消化性溃疡疾病和胃癌。没有主动干预，至少有20％的发达国家将继续被这种胃病原体感染。根除生物体将有助于预防疾病。当前的根除需要三重治疗，一种质子 - 泵抑制剂和两种抗生素，每天两次进行10至14天。对克拉霉素或甲硝唑的耐药性> 20％且上升。没有单一治疗是有效的。幽门螺杆菌的胃感染取决于该病原体HPUREI独有的通道的表达。 HPUREI是一种质子门控的尿素通道，用于快速尿素进入肠内尿素酶，对于在胃的酸性环境中保持pH 6.1至关重要，低至pH 2.5，从而使胃重殖。通道的表达在胃中增加。该通道有195个残基，有6个跨膜段和三个周围区域。诱变研究表明，HPUREI的质子门控受到这三个周质区域的组氨酸和羧酸残基之间的氢键调节。在第一个融资周期中，我们确定了该通道的三维结构，现在我们建议利用这些知识来增强我们对质子门控机制的理解，并识别抑制剂的结合模式，其中一些已经被发现。预计该通道的抑制作用会导致特定而有效的单一疗法消除生物体，并在测试和治疗时代使用，而不仅仅是治疗有症状的患者。这将为严重的胃肠内疾病，尤其是胃癌提供预防方法，每年造成约75万人死亡。我们的具体目的是1。生成高分辨率的晶体，并使用HPUREI的新的N-或C末端可裂解的6His-tag构建体分析HPUREI的开放和封闭形式。 2.通过确定位置指导突变对使用Xenopus卵母细胞和蛋白质体的尿素转运蛋白脂质体分析以及在H.幽门螺杆菌urei urei nock-Onnoct-Ountion uncot-Ountion uncot-Ountion of trains中表达的突变体，确认了由我们的HPUREI晶体结构提出的尿素途径和选择性过滤器。 3。通过将六聚体在六聚体界面上的残基突变以破坏原始缔合，然后使用Xenopus卵母细胞和蛋白脂质体对单体状态进行活性测量，从而分析六聚体排列的作用。 4。根据开放形式的通道结构发现并改善小分子HPUREI抑制剂；衡量它们增加HPUREI TM的能力，抑制幽门螺杆菌完整的尿素酶活性并影响新型胃模拟孵化系统中的生存。