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

基本信息

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
项目类别：
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

项目摘要

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博士是一位出色的分析化学家，有多个出版物，详细介绍了用于定量蛋白质的仪器的发展。现在，他希望从事寄生学研究时期，以实现自己的长期目标，即成为一名独立研究人员，将工具分析的工具应用于传染病问题。环境：西雅图Biomed的Stefan Kappe博士和系统生物学研究所的Robert Moritz博士将考虑Swearingen博士。 Kappe博士是疟原虫生物学专家，而Moritz博士是蛋白质质谱的专家。 Kappe博士和Moritz博士建立了一项既定的合作，该合作曾出版，描述了使用质谱法来表征疟原虫中蛋白质表达的情况。拟议的研究将在西雅图Biomed和系统生物学研究所平等进行，这是该奖项的赞助机构。系统生物学研究所与西雅图生物生物学研究所之间建立了机构间伙伴关系，其中包括联合任命和共享资源。来自两个组织的人员在两个组织之间自由移动（不到一个城市街区），共享核心设施和设备。 Swearingen博士将从合作研究环境和两家机构提供的最新设施中受益匪浅。研究：疾病疟疾是由疟原虫属寄生虫引起的。疟疾每年有数亿人患病，但治疗有限，没有执照的疫苗。疟原虫被引入蚊子唾液中的哺乳动物宿主中，于是寄生虫迁移到肝脏并入侵肝细胞。直到寄生虫以梅罗寄生的身份退出肝脏并侵入红细胞，疟疾的临床发作才会发生。一部分梅罗斯人区分了配子细胞。传播到蚊子后，配子细胞成熟到游戏中，性繁殖并产生更多的孢子虫，完成了生命周期。防止孢子岩感染宿主肝细胞会阻止疾病的发作。防止将梅罗斯人分化为配子细胞，有时游戏会阻止传播传播疾病的蚊子。磷酸化是调节细胞功能的蛋白质的可逆后翻译后修饰。用激酶抑制剂抑制磷酸化可以防止疟原虫侵袭和传播，但是已知参与该调节网络的蛋白质很少。我将确定对肝细胞的孢子岩感染和梅罗唑人的性分子分化所必需的磷酸化事件，然后我将识别出负责的激酶，从而揭示了新抗菌素的靶标。该提案的前两个目的是确定分别触发感染和传播的磷酸化事件。寄生虫中的蛋白质表达和磷酸化将通过质谱法进行定量。从蚊子中分离出非常低的感染的孢子岩将与未经处理的感染性唾液腺孢子菌相提并论，并用专辑治疗，以模仿血液中的spearive，并影响血液中的流动性或用肝素治疗，以模仿肝素和诱导肠胃。将未差异的梅罗龙与配子细胞和游戏进行比较。第三个目标将是对目标1和2中确定的重要磷酸化事件的验证。将使用模仿关键蛋白质的组成型或不存在磷酸化的氨基酸取代产生转基因寄生虫，并且将评估肝细胞或传播到摩squito的侵袭的影响。肝细胞侵袭将通过荧光辅助细胞分选和体内测量通过测量感染小鼠的血液期通畅的时间来测量肝细胞侵袭。将通过感染血液喂食蚊子来测量蚊子的传播并进行解剖以检查孢子虫的发展。最后，将确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暴露于细胞裂解物的珠子上。化学交联链将用于捕获激酶 - 基底络合物，并通过质谱鉴定激酶。