Malaria parasite harbors a unique protein lysine methyltransferase targeting both chromatin and motility machinery

疟原虫具有独特的蛋白质赖氨酸甲基转移酶，针对染色质和运动机制

• 批准号: 10741300
• 负责人: Jun Miao
• 金额: $ 22.49万
• 依托单位: UNIVERSITY OF SOUTH FLORIDA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-24 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10741300
• 关键词: Apical; Artemisinins; Biological Assay; Biology; Biomedical Research; Blood; CRISPR interference; Catalysis; Cell Nucleus; Cessation of life; Chromatin; Combined Modality Therapy; Complement; Complex; Cytoplasm; Cytosol; Data; Defect; Development; Down-Regulation; Drug Targeting; Enzymes; Epigenetic Process; Essential Genes; Face; Falciparum Malaria; Family; Foundations; Future; Gene Expression; Goals; Guide RNA; Histone-Lysine N-Methyltransferase; Histones; Human; Immunoprecipitation; Impairment; In Vitro; Intervention; Invaded; Knock-out; Lysine; Malaria; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Methylation; Methyltransferase; Parasite resistance; Parasites; Pharmaceutical Preparations; Phenotype; Plasmodium; Plasmodium falciparum; Process; Proteins; Proteomics; Regulation; SET Domain; Set protein; Sexual Development; Site; Solid; Specificity; Structure; Substrate Specificity; System; Theileria; Therapeutic; Toxoplasma gondii; Virulent; asexual; cancer therapy; cell motility; conditional knockout; epigenetic drug; genetic manipulation; histone methylation; human disease; inhibitor; insight; knock-down; knockout gene; methylome; model organism; new technology; new therapeutic target; non-histone protein; novel; overexpression; therapeutic development; transcriptome sequencing; transcriptomics; transmission process

PROJECT SUMMARY Epigenetics machinery has been used as important therapeutic drug targets evident by the successful inhibitors for cancer treatment. Although the malaria parasite harbors many epigenetic regulators, it remains a great challenge to discover unique ones diverse from those in the human host. Based on previous studies and our recent preliminary data, PfSET7, one of ten SET-domain containing lysine (K) methyltransferases (KMTs) in Plasmodium falciparum, is discovered to be a Plasmodium-specific KMT with unique structures and functions. PfSET7 does not belong to any known SET family in model organisms because it contains a unique motif II in the SET domain. PfSET7 was found localized at the apical tip of merozoite and downregulation of PfSET7 by ~35% using our newly developed CRISPR interference (CRISPRi) resulted in defects in merozoite egress and invasion, reminiscent of the findings that Toxoplasma gondii apical complex KMT (TgAKMT) is also localized at the apical tip before egress and disruption of TgAKMT led to defects in parasite egress and invasion due to impaired motility. However, TgAKMT’s mode of action, especially its substrates, is still unknown. Remarkably, PfSET7 was found also localized in the gametocyte cytoplasm and nucleus, and downregulation of PfSET7 by ~26% also led to defects in gametocyte development, coincidentally with the findings that PfSET7 has peak expression in gametocytes and many distinct methylated lysines were identified in gametocyte histones. Notably, PfSET7 contains four Plasmodium-specific regions and is only ~41% identical to TgAKMT, indicating PfSET7 harbors malaria-specific structures. Based on these findings, we hypothesize that PfSET7 is a Plasmodium-specific KMT and has a dual function in the regulation of gametocyte gene expression and merozoite motility during egress/invasion by targeting histone and non-histone proteins, respectively. To decipher the critical function and substrate specificity of PfSET7, we will apply advanced new technologies including a stronger CRISPRi KD with optimized gRNAs and a conditional knockout for gene functional study, and quantitative methylome for substrate identification. It is anticipated that this study will lay a solid foundation for understanding PfSET7’s mechanism of action and the development of novel apicomplexan- specific epigenetic drugs.

项目摘要 表观遗传学机械已被用作重要的治疗药物靶向证据 成功的癌症治疗抑制剂。尽管疟疾寄生虫具有许多表观遗传 监管机构，从人类中发现独特的潜水者仍然是一个巨大的挑战 主持人。根据先前的研究和我们最近的初步数据PFSET7，这是十个固定域之一 含有恶性疟原虫的含赖氨酸（K）甲基转移酶（KMTS）是一种 具有独特的结构和功能的疟原虫特异性KMT。 pfset7不属于任何 模型生物中已知的集合家族，因为它在设定域中包含一个独特的基序II。 发现PFSET7位于Merozoite的顶端，PFSET7的下调约为35％ 使用我们新开发的CRISPR干扰（CRISPRI）导致Merozoite缺陷 出口和入侵，让人联想到弓形虫弓形虫顶端复合物KMT的发现 （tgakmt）在出口和tgakmt的破坏之前也位于顶端的尖端，导致缺陷 由于运动受损而导致的寄生虫出口和入侵。但是，TGAKMT的行动方式， 尤其是其底物，仍然未知。值得注意的是，发现Pfset7也位于 配子细胞细胞质和核，PFSET7的下调约为26％，也导致缺陷 在配子细胞开发中，与PFSET7在 配子细胞和许多不同的甲基化赖氨酸在配子蛋白中鉴定出来。 值得注意的是，PFSET7包含四个特异性区域，仅与41％相同 TGAKMT，指示PFSET7具有疟疾特异性结构。基于这些发现，我们 假设PFSET7是疟原虫特异性KMT，并且在调节中具有双重功能 通过靶向组蛋白，在出口/入侵期间的配子细胞基因表达和梅罗洛罗兹岩运动 和非历史蛋白质分别。破译关键功能和底物特异性 在PFSET7中，我们将应用高级新技术，包括更强大的Crispri KD 优化的GRNA和用于基因功能研究的条件敲除和定量 用于底物鉴定的甲基团。预计这项研究将为 了解PFSET7的作用机理和新型Apicomplexan-的发展 特定的表观遗传药物。