de novo creation of base J in Leishmania

在利什曼原虫中从头创建 J 碱基

• 批准号: 10370926
• 负责人: BRYAN C JENSEN
• 金额: $ 28.28万
• 依托单位: SEATTLE CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-22 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10370926
• 关键词: Affinity Chromatography; Amino Acid Sequence; Anabolism; Annual Reports; Antibodies; Arginine; Binding; Biological Process; Case Study; Cell Survival; Cessation of life; ChIP-seq; Chromatin; Chromatin Remodeling Factor; Chromosomes; Code; Complex; Cutaneous; DNA; DNA Binding; DNA Sequence; DNA biosynthesis; Data; Development; Dioxygenases; Gene Cluster; Genes; Genetic Transcription; Genomics; Glucose; Glucosyltransferase; Histones; Hydroxylation; Individual; Lead; Left; Leishmania; Leishmaniasis; Lesion; Location; Lysine; Macromolecular Complexes; Maintenance; Mass Spectrum Analysis; Messenger RNA; Modeling; Modification; Molecular; Monitor; Names; Pathogenicity; Peptides; Polyadenylation; Post-Translational Protein Processing; Process; Proteins; Public Health; RNA; RNA Polymerase II; Reporting; Role; Site; Specificity; Symptoms; Tertiary Protein Structure; Testing; Tetracyclines; Therapeutic Agents; Thymidine; Thymine; Time; Trans-Splicing; Transcript; Transcription Initiation Site; Transferase; Visceral Leishmaniasis; arginyllysine; base; chromatin protein; chromosomal location; histone modification; hydroxyl group; insight; macromolecular assembly; mutant; neglect; novel; novel therapeutics; pathogen; recruit; telomere; therapeutic target; transcription termination; transcriptome sequencing

ABSTRACT There are over 20 pathogenic species of the genus Leishmania that are the causative agent for leishmaniasis. Symptoms for leishmaniasis range from self-limiting cutaneous lesion to visceral leishmaniasis that is fatal if left untreated. Globally there are more than 1 million reported cases in the last 5 years with over 20,000 fatalities reported annually. Leishmania resides in one of the most deeply branched eukaryotic lineages and harbors a number of distinct and novel biological processes, two of which are important for this proposal. The first is the organization of the protein coding genes transcribed by RNA polymerase II into long clusters of unrelated genes. The cluster of genes are transcribed as a single primary RNA from a transcription start site. Transcription then proceeds through the polycistronic cluster of genes with the individual mRNAs processed by trans-splicing until it reaches a transcription termination site. The second process is the modification of ~1% of thymidines (T) in DNA with glucose to form β-D-glucosyl-hydroxymethyluracil (J). The modification of T to J is a two-step process with the hydroxylation of T by either JBP1 or JBP2. Glucose is subsequently added to the hydroxyl group by a specific glucosyltransferase. JBP1 and JBP2 have distinct roles in the formation of J. JBP1 is responsible for maintenance of J, in regions already containing J, following DNA synthesis on the newly formed unmodified strand. JBP2 is responsible for de novo insertion of J, mostly at sites missed by JBP1, and ultimately determines which bases are modified to J. Most of J (~99%) is localized to the telomeres, but J is also found at internal sites most notably at all but one of the transcription termination sites. The modification of T to J is essential in Leishmania, so a deeper understanding of how Leishmania determines which bases are modified will be crucial in the development of potential therapeutic targeting the creation of J. The protein sequence of JBP2 does not contain any motif that would allow it to bind DNA, but it does contain a protein interaction domain. We determined that JBP2 interacts in a complex with a protein we named J2TDP. The protein sequence of J2TDP contains a Tudor domain, a motif known to interact with protein that contain dimethylated lysines or arginines, which are principally found in histones. The identification of J2TDP led us to the hypothesize that J2TDP binds a specific histone modification and then recruits JBP2 to the chromosome to modify adjacent Ts for conversion into J. In this proposal we break this hypothesis down in the follow three sub-hypotheses and then test key predictions of the hypotheses. 1) J2TDP binds a histone modification. 2) J2TDP recruits JBP2 to the chromosome. 3) Localization of JBP2 to the chromosome is sufficient for localized modification to J.

抽象的 利什曼原虫属有 20 多种致病菌，是利什曼病的病原体。 利什曼病的症状范围从自限性皮肤病变到内脏利什曼病，如果 未经治疗。过去 5 年，全球报告病例数超过 100 万例，其中 20,000 多例 每年报告死亡人数。利什曼原虫属于分支最深的真核谱系之一， 拥有许多独特且新颖的生物过程，其中两个对于本提案很重要。这 首先是由 RNA 聚合酶 II 转录成长簇的蛋白质编码基因的组织。 不相关的基因。基因簇从转录起始位点转录为单个初级 RNA。 然后转录通过基因的多顺反子簇进行，单个 mRNA 被处理 反式拼接直至到达转录终止位点。第二个过程是修改~1% DNA 中的胸苷 (T) 与葡萄糖形成 β-D-葡萄糖基-羟甲基尿嘧啶 (J)。 T 到 J 的修改是 T 通过 JBP1 或 JBP2 进行羟基化的两步过程。随后将葡萄糖添加到 羟基通过特定的葡萄糖基转移酶。 JBP1和JBP2在J的形成中具有不同的作用。JBP1 负责在已经含有 J 的区域中维持 J，在新的 DNA 合成之后 形成未修饰的链。 JBP2 负责 J 的从头插入，主要是在 JBP1 错过的位点，并且 最终决定哪些碱基被修饰为 J。大部分 J (~99%) 位于端粒，但 J 是 在内部位点也发现了最显着的情况，但转录终止位点之一除外。的修改 T 到 J 对于利什曼原虫至关重要，因此更深入地了解利什曼原虫如何确定哪些碱基 修饰对于开发针对 J. 蛋白的潜在治疗方法至关重要 JBP2 的序列不包含任何允许其结合 DNA 的基序，但它确实包含蛋白质 交互域。我们确定 JBP2 在复合物中与我们命名为 J2TDP 的蛋白质相互作用。这 J2TDP 的蛋白质序列包含一个 Tudor 结构域，该基序已知与含有以下成分的蛋白质相互作用： 二甲基化赖氨酸或精氨酸，主要存在于组蛋白中。 J2TDP 的识别使我们发现 假设 J2TDP 结合特定的组蛋白修饰，然后将 JBP2 招募到染色体上 修改相邻的T以转换为J。在这个提案中，我们将这个假设分解为以下三个 子假设，然后检验假设的关键预测。 1) J2TDP 结合组蛋白修饰。 2） J2TDP 将 JBP2 招募到染色体上。 3) JBP2在染色体上的定位足以用于定位 对 J 的修改