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 表观遗传学机制已被用作重要的治疗药物靶点， 成功的癌症治疗抑制剂。尽管疟原虫具有许多表观遗传特性， 监管机构，它仍然是一个巨大的挑战，以发现独特的不同，从那些在人类 主持人根据以往的研究和我们最近的初步数据，PfSET 7，十个SET域之一， 含有赖氨酸（K）甲基转移酶（KMTs）的恶性疟原虫，被发现是一种 疟原虫特异性KMT具有独特的结构和功能。PfSET 7不属于任何 在模式生物中已知SET家族，因为它在SET结构域中含有独特的基序II。 发现PfSET 7定位于裂殖子的顶端，并且PfSET 7的下调约35%。 使用我们新开发的CRISPR干扰（CRISPRi）， 出口和入侵，让人想起的发现，弓形虫顶端复合物KMT （TgAKMT）也位于根尖尖端，然后TgAKMT流出和破坏导致缺损 在寄生虫的外出和入侵中，由于运动受损。然而，TgAKMT的作用方式， 尤其是其底物，仍然是未知的。值得注意的是，PfSET 7也被发现定位在 配子体细胞质和细胞核，PfSET 7下调约26%也导致缺陷 在配子母细胞发育中，与PfSET 7在配子母细胞中具有峰值表达的发现一致， 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，PfSET 7含有四个疟原虫特异性区域，并且与PfSET 7仅约41%相同。 TgAKMT，表明PfSET 7具有疟疾特异性结构。基于这些发现，我们 假设PfSET 7是疟原虫特异性KMT，在调节中具有双重功能， 配子体基因表达和裂殖子运动在出口/入侵过程中通过靶向组蛋白 和非组蛋白蛋白。为了破译关键功能和底物特异性 在PfSET 7中，我们将应用先进的新技术，包括更强大的CRISPRi KD， 优化的gRNA和用于基因功能研究的条件性敲除，以及定量 用于底物鉴定的甲基化组。预计本研究将为今后的研究奠定坚实的基础。 了解PfSET 7的作用机制和新型顶复体的开发， 特定的表观遗传药物