Identify 70 bp repeat-associated chromatin components by End-targeting Proteomics of Isolated Chromatin segments (PICh) and initiate their functional characterization
通过分离染色质片段 (PICh) 的末端靶向蛋白质组学鉴定 70 bp 重复相关染色质成分,并启动其功能表征
基本信息
- 批准号:10293165
- 负责人:
- 金额:$ 7.43万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2021
- 资助国家:美国
- 起止时间:2021-06-04 至 2023-05-31
- 项目状态:已结题
- 来源:
- 关键词:3&apos Untranslated RegionsAffectAfricaAfricanAfrican TrypanosomiasisAnimalsBindingBinding ProteinsCell surfaceChromatinComplexConserved SequenceCountryCoupledDNADNA Double Strand BreakDevelopmentDiseaseDrug resistanceEconomic BurdenEventFluorescent in Situ HybridizationFrequenciesFutureGene ConversionGenerationsGenesGenetic RecombinationHumanImmune responseImmunofluorescence ImmunologicImmunologic SurveillanceIn SituInfectionLinkMass Spectrum AnalysisMediatingMessenger RNAParasitesPathogenesisPathway interactionsPharmaceutical PreparationsPlayProteinsProteomicsProtocols documentationPseudogenesRNA InterferenceRNA ProbesRegulationRoleSaharaSequence HomologySiteSurface AntigensTimeTrypanosoma brucei bruceiWorkcell growthcell typederepressiongenetic informationimprovedmortalityside effecttelomere
项目摘要
Project Summary
Trypanosoma brucei causes human African trypanosomiasis, which is frequently fatal without treatment.
Few drugs are available for treating this disease, most of which have severe side-effects and are difficult to
administer, while drug-resistant T. brucei infection has been on the rise. In addition, T. brucei also causes
animal African trypanosomiasis, which has been a significant economic burden in sub-Sahara Africa. It is
therefore important to further study T. brucei pathogenesis and identify better targets for future development of
anti-parasite agents. T. brucei regularly switches its major surface antigen, VSG, to evade its mammalian host
immune response. VSG switching is a major pathogenesis mechanism that enables T. brucei to
establish and maintain a long-term infection. However, how VSG switching is initiated naturally in T.
brucei is still not clear. T. brucei has >2,500 VSG genes and pseudogenes, all of which are located at
subtelomeres. However, VSGs are expressed exclusively from subtelomeric polycistronic VSG expression
sites (ESs). VSG is the last gene in any ES and within 2 kb from the telomere repeats. T. brucei has multiple
ESs with very similar sequences, but only one ES is fully active at any time, resulting in a single type of VSG
being expressed on the cells surface. DNA recombination has been shown to be a major pathway for VSG
switching. Most VSG genes are flanked by two common sequences, which provide sequence homology for
recombination between the active and a silent VSG gene in DNA recombination-mediated VSG switching
events. First, all VSG 3’UTR has a common 14 nt sequences. Long telomere sequences are also found
downstream of ES-linked VSGs and VSGs at minichromosome subtelomeres. Second, upstream of most VSG
genes are 70 bp repeats. The 70 bp repeats in ESs can be several kb to several tens kb long. It has been
shown that introducing a DNA double strand break in the 70 bp repeats immediately upstream of the active
VSG gene increases the VSG switching rate for ~ 250 fold. In addition, DNA breaks are found in the 70 bp
repeats in WT cells. Therefore, 70 bp repeat integrity is expected to significantly affect VSG switching
frequency. However, it is unknown whether any proteins specifically associate with the 70 bp repeats,
even though their sequences are highly conserved, and their associated proteins are expected to help
maintain their integrity and to participate in the regulation of VSG switching. In this small project, we aim
to use an improved “end-targeting proteomics of isolated chromatin segments” (ePICh) approach to isolate the
70 bp repeat chromatin and identify proteins associated with the 70 bp repeats by mass spectrometry. We will
validate candidates identified in the 70 bp repeat ePICh and initiate their functional analysis. Our work will
reveal clearly whether any proteins specifically associate with the 70 bp repeats, which will allow us to
investigate how 70 bp repeats integrity is maintained. Our findings will open up new avenues for better
understanding of the T. brucei pathogenesis and contribute to eradicate this parasite eventually.
项目摘要
布氏锥虫引起人类非洲锥虫病,如果不治疗,往往是致命的。
治疗该病的药物很少,大多数药物副作用严重,难以治愈。
而耐药T.布氏杆菌感染呈上升趋势。此外,T.布氏杆菌还引起
动物非洲锥虫病,这一直是撒哈拉以南非洲的重大经济负担。是
因此,进一步研究T.布氏病发病机制,并确定更好的目标,为今后的发展,
抗寄生虫剂。T.布氏杆菌有规律地转换其主要表面抗原VSG,以逃避哺乳动物宿主
免疫反应VSG转换是T.布鲁塞岛
建立并维持长期感染。然而,如何VSG开关自然启动T。
布鲁塞还不清楚。T.布氏杆菌有超过2,500个VSG基因和假基因,所有这些基因都位于
亚端粒然而,VSG仅从亚端粒多顺反子VSG表达中表达,
站点(ES)。VSG是任何ES中的最后一个基因,距离端粒重复序列2kb以内。T.布氏杆菌有多种
ES具有非常相似的序列,但在任何时候只有一个ES完全激活,从而导致单一类型的VSG
在细胞表面表达。DNA重组已被证明是VSG的主要途径
切换大多数VSG基因的侧翼是两个共同的序列,这为VSG基因提供了序列同源性。
在DNA重组介导的VSG转换中活性和沉默VSG基因之间的重组
事件首先,所有VSG 3 'UTR具有共同的14 nt序列。长端粒序列也被发现
ES连锁的VSG下游和微小染色体亚端粒处的VSG。其次,大多数VSG的上游
基因是70 bp重复序列。ES中的70 bp重复序列可以是几kb到几十kb长。已经
显示在活性蛋白的上游直接引入70 bp重复序列中的DNA双链断裂,
VSG基因使VSG转换率增加约250倍。此外,在70 bp的DNA片段中发现了DNA断裂,
在WT细胞中重复。因此,预期70 bp重复完整性显著影响VSG转换
频率.然而,尚不清楚是否有任何蛋白质与70 bp重复序列特异性相关,
尽管它们的序列是高度保守的,它们的相关蛋白质有望帮助
保持其完整性,并参与VSG切换的调节。在这个小项目中,我们的目标是
使用改进的“分离染色质片段的末端靶向蛋白质组学”(ePICh)方法分离
70 bp重复染色质,并通过质谱鉴定与70 bp重复相关的蛋白质。我们将
验证在70 bp重复ePICh中鉴定的候选物,并启动其功能分析。我们的工作将
清楚地揭示了是否有任何蛋白质与70 bp重复序列特异性相关,这将使我们能够
研究如何保持70 bp重复序列的完整性。我们的发现将为更好地
对T.布氏杆菌致病性,并有助于最终根除这种寄生虫。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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{{ truncateString('Bibo Li', 18)}}的其他基金
Mechanisms of how Trypanosoma brucei TRF maintains telomere integrity
布氏锥虫 TRF 维持端粒完整性的机制
- 批准号:
10622535 - 财政年份:2022
- 资助金额:
$ 7.43万 - 项目类别:
Telomere end processing and telomere stability maintenance in trypanosomes
锥虫的端粒末端加工和端粒稳定性维持
- 批准号:
10503111 - 财政年份:2022
- 资助金额:
$ 7.43万 - 项目类别:
Mechanisms of how Trypanosoma brucei TRF maintains telomere integrity
布氏锥虫 TRF 维持端粒完整性的机制
- 批准号:
10526882 - 财政年份:2022
- 资助金额:
$ 7.43万 - 项目类别:
Telomere end processing and telomere stability maintenance in trypanosomes
锥虫的端粒末端加工和端粒稳定性维持
- 批准号:
10677878 - 财政年份:2022
- 资助金额:
$ 7.43万 - 项目类别:
Identify 70 bp repeat-associated chromatin components by End-targeting Proteomics of Isolated Chromatin segments (PICh) and initiate their functional characterization
通过分离染色质片段 (PICh) 的末端靶向蛋白质组学鉴定 70 bp 重复相关染色质成分,并启动其功能表征
- 批准号:
10417263 - 财政年份:2021
- 资助金额:
$ 7.43万 - 项目类别:
Characterize functions of T. brucei RAP1 and TRF in antigenic variation and telom
表征 T. brucei RAP1 和 TRF 在抗原变异和端粒中的功能
- 批准号:
8603220 - 财政年份:2007
- 资助金额:
$ 7.43万 - 项目类别:
Characterize functions of T. brucei RAP1 and TRF in antigenic variation and telom
表征 T. brucei RAP1 和 TRF 在抗原变异和端粒中的功能
- 批准号:
8107285 - 财政年份:2007
- 资助金额:
$ 7.43万 - 项目类别:
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