Elucidating phosphorylation signaling networks in infectious Plasmodium parasites

阐明传染性疟原虫寄生虫中的磷酸化信号网络

• 批准号: 9281675
• 负责人: Kristian Edward Swearingen
• 金额: $ 13.64万
• 依托单位: INSTITUTE FOR SYSTEMS BIOLOGY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-06-15 至 2020-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9281675
• 关键词: Albumins; Amino Acid Substitution; Antimalarials; Appointment; Award; Binding; Biochemical; Biology; Blood; Blood Cells; Cell Separation; Cell physiology; Cells; Cessation of life; Chemicals; Cities; Clinical; Collaborations; Communicable Diseases; Complex; Core Facility; Culicidae; Cyclic AMP-Dependent Protein Kinases; Development; Disease; Environment; Enzymes; Equipment; Erythrocytes; Event; Exhibits; Exposure to; Fluorescence; Gametogenesis; Germ Cells; Goals; Heparin; Hepatocyte; Human Resources; Immobilization; In Vitro; Infection; Infection prevention; Institutes; Institution; Invaded; Joints; Life Cycle Stages; Liver; Malaria; Mass Spectrum Analysis; Measures; Mentors; Methods; Midgut; Mus; Mutation; Onset of illness; Oocysts; Parasites; Parasitology; Phosphorylation; Phosphotransferases; Plasmodium; Post-Translational Protein Processing; Protein Kinase; Proteins; Publications; Reproduction; Research; Research Personnel; Resource Sharing; Role; Saliva; Salivary Glands; Serine; Signal Transduction; Sporozoites; Stream; Surveys; Symptoms; Systems Biology; Threonine; Time; Transgenic Organisms; Tyrosine; Undifferentiated; Vaccines; Validation; asexual; cell motility; crosslink; experimental study; feeding; follow-up; in vivo; inhibitor/antagonist; inorganic phosphate; instrumentation; inter-institutional; kinase inhibitor; migration; new therapeutic target; parasite invasion; prevent; protein expression; public health relevance; tool; transmission process; vector mosquito

 DESCRIPTION (provided by applicant): Candidate: The candidate, Dr. Kristian Swearingen, is an accomplished analytical chemist with multiple publications detailing the development of instrumentation for the quantification of proteins. He now wishes to engage in a period of mentored research in parasitology in order to achieve his long-term goal of becoming an independent researcher applying the tools of instrumental analysis to the problem of infectious disease. Environment: Dr. Swearingen will be mentored by Dr. Stefan Kappe of Seattle BioMed and Dr. Robert Moritz of the Institute for Systems Biology. Dr. Kappe is an expert in Plasmodium biology, while Dr. Moritz is an expert in mass spectrometry of proteins. Dr. Kappe and Dr. Moritz have an established collaboration that has produced publications describing the use of mass spectrometry to characterize protein expression in Plasmodium. The proposed research will be carried out in equal part at Seattle BioMed and the Institute for Systems Biology, which is the sponsoring institution for this award. There is an established inter-institutional partnership between the Institute for Systems Biology and Seattle BioMed that includes joint appointments and shared resources. Personnel from both organizations move freely between the two organizations (separated by less than one city block), and core facilities and equipment are shared. Dr. Swearingen will benefit greatly from the collaborative research environments and the state-of-the-art facilities available at both institutes. Research: The disease malaria is caused by parasites of the genus Plasmodium. Malaria sickens hundreds of millions of people annually, yet there are limited treatments and no licensed vaccine. Plasmodium sporozoites are introduced into the mammalian host in the saliva of mosquitoes, whereupon the parasites migrate to the liver and invade hepatocytes. Clinical onset of malaria does not occur until the parasites exit the liver as merozoites and invade erythrocytes, reproducing asexually. A subset of merozoites differentiates into gametocytes. Upon transmission to mosquitoes, gametocytes mature into gametes, reproduce sexually, and produce more sporozoites, completing the life cycle. Preventing sporozoite infection of host hepatocytes would prevent onset of disease. Preventing differentiation of merozoites into gametocytes and eventually gametes would prevent transmission to the mosquito vectors that spread the disease. Phosphorylation is a reversible post-translational modification of proteins that regulates cell function. Inhibiting phosphorylation with kinase inhibitors can prevent Plasmodium invasion and transmission, but few of the proteins involved in this regulatory network are known. I will identify phosphorylation events essential to sporozoite infection of hepatocytes and sexual differentiation of merozoites, and then I will identify the kinases responsible, revealing targets for new antimalarials. The first two aims of this proposal are to identify phosphorylation events that trigger infectivity and transmission, respectively. Protein expression and phosphorylation in Plasmodium parasites will be quantified by mass spectrometry. Sporozoites isolated from the mosquito mid-gut, which have very low infectivity, will be compared with infectious salivary gland sporozoites that are untreated, treated with albumin to mimic arrival in the blood stream and induce gliding motility, or treated with heparin to mimic arrival in the liver and induce invasion. Undifferentiated merozoites will be compared with gametocytes and gametes. The third aim will be validation of important phosphorylation events identified in Aims 1 and 2. Transgenic parasites will be generated with amino acid substitutions that mimic the constitutive presence or absence of phosphorylation on key proteins, and the effect on invasion of hepatocytes or transmission to mosquitoes will be assessed. Hepatocyte invasion will be measured in vitro by fluorescence aided cell sorting and in vivo by measuring the time to blood stage patency in infected mice. Transmission to mosquitoes will be measured by feeding mosquitoes with infected blood and dissecting to check for development of sporozoites. Finally, the kinases responsible for the critical phosphorylation events identified above will be identified. Purified bait proteins will be immobilized on beads an exposed to cell lysate. Chemical cross-linkers will be used to capture the kinase-substrate complex, and the kinases will be identified by mass spectrometry.

 描述（由申请人提供）： 候选人：候选人 Kristian Swearingen 博士是一位卓有成就的分析化学家，发表了多篇论文，详细介绍了蛋白质定量仪器的开发。他现在希望从事寄生虫学方面的指导研究，以实现成为一名独立研究人员，将仪器分析工具应用于传染病问题的长期目标。 环境：Swearingen 博士将接受西雅图 BioMed 的 Stefan Kappe 博士和系统生物学研究所的 Robert Moritz 博士的指导。卡佩博士是疟原虫生物学方面的专家，而莫里茨博士是蛋白质质谱方面的专家。 Kappe 博士和 Moritz 博士建立了良好的合作关系，出版了一些出版物，描述了如何使用质谱来表征疟原虫中的蛋白质表达。拟议的研究将在西雅图生物医学中心和系统生物学研究所（该奖项的赞助机构）同时进行。系统生物学研究所和西雅图生物医学中心之间建立了机构间合作伙伴关系，包括联合任命和共享资源。 双方人员在两个机构之间自由流动（相距小于一个街区），核心设施设备共享。斯威林根博士将从两个研究所的合作研究环境和最先进的设施中受益匪浅。 研究：疟疾是由疟原虫属寄生虫引起的。疟疾每年使数亿人患病，但治疗方法有限，也没有获得许可的疫苗。疟原虫子孢子通过蚊子的唾液被引入哺乳动物宿主，随后寄生虫迁移到肝脏并侵入肝细胞。直到寄生虫以裂殖子的形式离开肝脏并侵入红细胞并进行无性繁殖后，疟疾才会出现临床症状。裂殖子的一个子集分化为配子细胞。传播给蚊子后，配子母细胞成熟为配子，有性繁殖，并产生更多的子孢子，完成生命周期。防止子孢子感染宿主肝细胞可以预防疾病的发生。防止裂殖子分化为配子细胞并最终分化为配子，将防止传播到传播疾病的蚊子媒介。磷酸化是调节细胞功能的蛋白质的可逆翻译后修饰。用激酶抑制剂抑制磷酸化可以阻止疟原虫的入侵和传播，但参与该调节网络的蛋白质很少为人所知。我将确定对肝细胞的子孢子感染和裂殖子的性分化至关重要的磷酸化事件，然后我将确定负责的激酶，揭示新抗疟药的靶点。该提案的前两个目标是确定分别触发感染性和传播的磷酸化事件。疟原虫寄生虫中的蛋白质表达和磷酸化将通过质谱法进行定量。从蚊子中肠分离的子孢子具有非常低的传染性，将与未经处理的传染性唾液腺子孢子进行比较，用白蛋白处理以模拟到达血流并诱导滑行运动，或用肝素处理以模拟到达肝脏并诱导侵袭。未分化的裂殖子将与配子母体和配子进行比较。第三个目标是验证目标 1 和 2 中确定的重要磷酸化事件。将通过模拟关键蛋白质磷酸化的组成性存在或不存在的氨基酸取代来产生转基因寄生虫，并将评估对肝细胞入侵或传播给蚊子的影响。将通过荧光辅助细胞分选在体外测量肝细胞侵袭，并通过测量感染小鼠的血液阶段通畅时间在体内测量肝细胞侵袭。通过用受感染的血液喂养蚊子并解剖以检查子孢子的发育来测量向蚊子的传播。 最后，将鉴定负责上述关键磷酸化事件的激酶。 纯化的诱饵蛋白将被固定在珠子上并暴露于细胞裂解物。化学交联剂将用于捕获激酶-底物复合物，并且激酶将通过质谱法进行鉴定。