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: 8884527
• 负责人: HARTMUT LUECKE
• 金额: $ 48.82万
• 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 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8884527
• 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; Health; 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; 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(申请人提供)：幽门螺杆菌感染胃粘膜仍然是一个世界性的问题，并导致消化性溃疡疾病和胃癌。如果没有积极的干预，发达国家至少20%的人口将继续感染这种胃部病原体。根除这种生物将有助于预防疾病。目前的根除需要三联疗法，一种质子泵抑制剂和两种抗生素，每天两次，持续10至14天。克拉霉素或甲硝唑的耐药率为20%，而且还在上升。没有一种单一疗法是有效的。幽门螺杆菌对胃的感染依赖于该病原体所特有的HpUreI通道的表达。HpUreI是一种质子门控尿素通道，是尿素快速进入细菌内尿素酶所必需的，对于在胃的酸性环境中将周质维持在pH 6.1至低至pH 2.5是必不可少的，从而允许胃的定植。该通道在胃中的表达增加。该通道有195个残基，有6个跨膜片段和3个周质区域。诱变研究表明，HpUreI的质子门控受这三个周质区域组氨酸和羧酸残基之间的氢键调控。在第一个资金周期中，我们确定了这个通道的三维结构，现在我们建议利用这一知识来增加我们对其质子门控机制的理解，并确定抑制剂的结合模式，其中一些已经被发现。对这一渠道的抑制有望导致特定和有效的单一疗法来根除这种微生物，并开启一个测试和治疗的时代，而不是只治疗有症状的患者。这将为严重的上消化道疾病，特别是每年造成约75万人死亡的胃癌提供一种预防方法。我们的具体目标是1.利用新的N-端或C-端可切割的HpUreI 6His-tag构建HpUreI，生成更高分辨率的晶体，并分析HpUreI的开放和闭合形式。2.利用非洲爪哇卵母细胞和蛋白脂体尿素转运实验以及HpUreI基因敲除菌株中表达的突变体，通过测定定点突变对HpUreI转运和底物选择性的影响，证实了我们的HpUreI晶体结构所提出的尿素途径和选择性过滤器。3.通过突变六聚体界面上保守的残基来破坏六聚体的结合，分析六聚体排列的作用，然后用非洲爪哇卵母细胞和蛋白脂质体测量单体状态的活性。4.发现和改进基于开放通道结构的小分子HpUreI抑制剂；测量它们增加HpUreI的TM、抑制完整Hp的尿素酶活性以及影响其在新的胃肠道模拟孵育系统中的存活的能力。