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.

项目概要 表观遗传学机制已被用作重要的治疗药物靶标 尽管疟疾寄生虫具有许多表观遗传特征，但它是癌症治疗的成功抑制剂。 对于监管者来说，发现与人类不同的独特基因仍然是一个巨大的挑战 根据之前的研究和我们最近的初步数据，PfSET7，十个 SET 域之一。 恶性疟原虫中含有赖氨酸 (K) 甲基转移酶 (KMT)，被发现是一种 具有独特结构和功能的疟原虫特异性KMT不属于任何一种。 模式生物中已知的 SET 家族，因为它在 SET 结构域中包含独特的基序 II。 发现 PfSET7 位于裂殖子的顶端，PfSET7 下调约 35% 使用我们新开发的 CRISPR 干扰 (CRISPRi) 导致裂殖子出现缺陷 流出和入侵，让人想起弓形虫顶端复合体 KMT 的发现 (TgAKMT) 在 TgAKMT 退出和破坏导致缺陷之前也定位于顶端 然而，TgAKMT 的作用方式， 尤其是它的底物，仍然未知。值得注意的是，PfSET7 也被发现定位于。 配子细胞的细胞质和细胞核，PfSET7 下调约 26% 也会导致缺陷 在配子细胞发育过程中，与 PfSET7 在配子细胞发育中具有峰值表达的发现相一致 在配子体组蛋白中鉴定出配子体和许多不同的甲基化赖氨酸。 值得注意的是，PfSET7 包含四个疟原虫特异性区域，并且仅约 41% 与 TgAKMT，表明 PfSET7 具有疟疾特异性结构。基于这些发现，我们。 PfSET7 是疟原虫特异性 KMT，具有双重调节功能 通过靶向组蛋白来影响配子体基因表达和裂殖子运动过程中的出口/入侵 和非组蛋白，分别破译关键功能和底物特异性。 PfSET7，我们将应用先进的新技术，包括更强大的 CRISPRi KD 用于基因功能研究和定量研究的优化 gRNA 和条件敲除 预计这项研究将为底物鉴定奠定坚实的基础。 了解 PfSET7 的作用机制和新型 apicomplexan 的开发 特定的表观遗传药物。