The Biochemistry and Cell Biology of the Spindly O-fucosyltransferase of Toxoplasma
弓形虫纺锤体O-岩藻糖基转移酶的生物化学和细胞生物学
基本信息
- 批准号:9897291
- 负责人:
- 金额:$ 53.25万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2020
- 资助国家:美国
- 起止时间:2020-01-01 至 2023-11-30
- 项目状态:已结题
- 来源:
- 关键词:AcanthamoebaAdaptor Signaling ProteinAddressAdultAffectAleuria aurantia lectinAmino AcidsAmino SugarsAntibodiesArabidopsisBacteriaBiochemistryBiologyBlindnessBrainBurkholderiaC-terminalCatalytic DomainCategory B pathogenCell NucleusCellsCellular biologyChemistryChimeric ProteinsComplementCryptosporidiumCystCytoplasmDefectDiarrheaDictyosteliumDifferentiation and GrowthDiseaseEnzymesFelis catusFetusFucoseFucosyltransferaseFutureGenesGenetic TranscriptionGreen Fluorescent ProteinsGrowthGuanosine Diphosphate FucoseGuanosine Triphosphate PhosphohydrolasesHistonesHomologous GeneHumanHydroxyprolineInfectionKnock-outKnowledgeLabelLectinLife Cycle StagesLung infectionsMass Spectrum AnalysisMembraneMessenger RNAMethodsMicroscopyModificationMusN-terminalNeurologicNuclearNuclear Pore ComplexNuclear Pore Complex ProteinsNutrientO-GlcNAc transferaseOrganismOxygenParasitesParentsPersonsPhase TransitionPhotobleachingPhysiologic pulsePlantsPoint MutationPolymerasePost-Translational Protein ProcessingProtein RegionProteinsRadiolabeledReactionRecombinant ProteinsRecombinantsReporterResearch PersonnelResistanceRoleSignal TransductionSignaling ProteinSiteStressSurveysSystemTestingTimeToxoplasmaToxoplasma gondiiTransferaseVirulenceWorkX-linked intellectual disabilitybaseexperimental studyfitnessgibberellic acidglycosylationglycosyltransferasehuman modelhuman pathogenimmunosuppressedinorganic phosphatemolecular phenotypemouse modelnoveloverexpressionp19(SKP1) Proteinparalogous geneproteostasispublic health relevanceresponsesugartranscription factortranscriptome sequencingubiquitin-protein ligase
项目摘要
Project summary/Abstract
This proposal explores the role of nuclear O-fucose as a novel regulatory mechanism in Toxoplasma
gondii, cause of severe neurological and ocular diseases. The John Samuelson lab used the anti-fucose Aleuria
aurantia lectin (AAL) to show that fucosylated proteins of T. gondii form punctate assemblies in close association
with the nuclear pore complex. The assemblies of O-fucosylated proteins are reminiscent of Cajal, PML, and
histone locus bodies in the host nucleus, which are not modified by O-fucose. AAL-enrichment and mass
spectrometry showed O-fucose is attached to Ser and Thr in intrinsically disordered regions of dozens of
proteins, which appear to be involved in transcription, mRNA processing and transport, and signaling. The Chris
West lab identified the T. gondii O-transferase (TgSpy), which contains 11 N-terminal tetratricopeptide repeats
(TPRs) and a C-terminal GT41 glycosyltransferase domain. Recombinant TgSpy made in the cytoplasm of
bacteria hydrolyses GDP-fucose and O-fucosylates itself, a polySer/GST fusion, and proteins in lysates from
spyKO cells. TgSpy is not essential, but the spyKO grows more slowly, while transfectants that overexpress
TgSpy grow faster. Green fluorescent protein fused with polySer is O-fucosylated and accumulates in the AAL-
labeled assemblies in the parent strain but is degraded in the spyKO.
TgSpy is a homolog of plant Spindly, a negative regulator of gibberellic acid signaling in Arabidopsis, and is
also a paralog the host O-GlcNAc transferase (OGT). The OGT, which is absent in Tg, modifies intrinsically
disordered regions of hundreds of proteins and is an important factor in stress resistance and nutrient signaling.
According to precedent provided by OGT, we hypothesize that TgSpy modifies proteins and affects their activity
under normal growth conditions and in response to stress. In addition, modification by TgSpy uniquely causes
O-fucosylated proteins to localize near the NPC, where they are protected from degradation.
The Samuelson and West labs will work together to dissect how O-fucose works in T. gondii. In Aim 1 we
will evaluate the importance of the TPRs and GT41 glycosyltransferase for O-fucosylation and parasite growth.
In Aim 2 we will determine the stability, localization, and function of five proteins, which are O-fucosylated and
contribute to Tg fitness, in response to varied Spy expression. We will perform interactome studies to address
the mechanism of protein inclusion in assemblies of O-fucosylated proteins and pulse-chase labeling, time-
lapse microscopy, and photo-bleaching to determine the stability of assemblies. In Aim 3 we will compare the
growth in culture, mice, and cats of the parental strain, the spy knockout, and organisms overexpressing TgSpy.
In addition, we will perform RNA-seq and SILAC on the three sets of protists growing as tachyzoites or
converting to bradyzoites in culture.
项目概要/摘要
该建议探讨了核O-岩藻糖在弓形虫中作为一种新的调节机制的作用
弓形虫,导致严重的神经和眼部疾病。约翰·萨缪尔森实验室用抗岩藻糖粉糊
橙黄凝集素(AAL),以显示岩藻糖基化的T.弓形虫形成紧密结合的点状集合体
与核孔复合体结合。O-岩藻糖基化蛋白的组装让人想起Cajal、PML和
宿主细胞核中的组蛋白基因座体,其不被O-岩藻糖修饰。AAL富集和质量
光谱分析表明,O-岩藻糖连接到丝氨酸和苏氨酸的内在无序区域的几十个
这些蛋白质似乎参与转录、mRNA加工和运输以及信号传导。克里斯
西部实验室确认了T弓形虫O-转移酶(TgSpy),其含有11个N-末端三肽重复
在一个实施方案中,所述糖基转移酶包含TPRs和C-末端GT 41糖基转移酶结构域。重组TgSpy在细胞质中制备,
细菌水解GDP-岩藻糖和O-岩藻糖基化物本身,一种聚丝氨酸/GST融合物,以及来自
SpyKO细胞。TgSpy不是必需的,但spyKO生长较慢,而过表达的转染子
TgSpy增长更快。与polySer融合的绿色荧光蛋白被O-岩藻糖基化并在AAL-中积累。
标记的组装体在亲本菌株中,但在spyKO中降解。
TgSpy是植物Spindly的同源物,Spindly是拟南芥中赤霉酸信号传导的负调节因子,
也是宿主O-GlcNAc转移酶(OGT)的一部分。Tg中不存在的OGT本质上进行了修饰
它是数百种蛋白质的无序区域,是抗应激和营养信号传导的重要因素。
根据OGT提供的先例,我们假设TgSpy修饰蛋白质并影响其活性
在正常生长条件下和对压力的反应。此外,TgSpy的修改独特地导致
0-岩藻糖基化蛋白定位在NPC附近,在那里它们被保护免于降解。
萨缪尔森和韦斯特实验室将共同研究O-岩藻糖在T。刚地。在目标1中,
将评估TPR和GT 41糖基转移酶对O-岩藻糖基化和寄生虫生长的重要性。
在目标2中,我们将确定五种蛋白质的稳定性、定位和功能,这五种蛋白质是O-岩藻糖基化的,
有助于TG适应性,以响应不同的Spy表达。我们将进行相互作用组研究,
O-岩藻糖基化蛋白质组装体中蛋白质内含物的机制和脉冲追踪标记,时间-
lapse显微镜和光漂白来确定组装体的稳定性。在目标3中,我们将比较
亲本菌株、spy敲除菌株和过表达TgSpy的生物体在培养物、小鼠和猫中的生长。
此外,我们将对三组原生生物进行RNA-seq和SILAC,这些原生生物生长为速殖子,
在培养中转化为缓殖子。
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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John C. Samuelson其他文献
Novel antibodies detect nucleocytoplasmic O-fucose in protist pathogens, cellular slime molds, and plants
新型抗体检测原生生物病原体、细胞黏菌和植物中的核质 O-岩藻糖
- DOI:
10.1128/msphere.00945-24 - 发表时间:
2025-02-03 - 期刊:
- 影响因子:3.100
- 作者:
Megna Tiwari;Elisabet Gas-Pascual;Manish Goyal;Marla Popov;Kenjiroo Matsumoto;Marianne Grafe;Ralph Gräf;Robert S. Haltiwanger;Neil Olszewski;Ron Orlando;John C. Samuelson;Christopher M. West - 通讯作者:
Christopher M. West
John C. Samuelson的其他文献
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{{ truncateString('John C. Samuelson', 18)}}的其他基金
The Biochemistry and Cell Biology of the SpindlyO-fucosyltransferase of Toxoplasma
弓形虫 SpindlyO-岩藻糖基转移酶的生物化学和细胞生物学
- 批准号:
10541113 - 财政年份:2020
- 资助金额:
$ 53.25万 - 项目类别:
The Biochemistry and Cell Biology of the SpindlyO-fucosyltransferase of Toxoplasma
弓形虫 SpindlyO-岩藻糖基转移酶的生物化学和细胞生物学
- 批准号:
10300056 - 财政年份:2020
- 资助金额:
$ 53.25万 - 项目类别:
Genetic modification of cultured Cryptosporidium to test the autoinfection model
对培养的隐孢子虫进行基因改造以测试自身感染模型
- 批准号:
9305341 - 财政年份:2017
- 资助金额:
$ 53.25万 - 项目类别:
Structure and Development of Oocyst and Sporocyst Walls
卵囊和孢子囊壁的结构和发育
- 批准号:
9206440 - 财政年份:2015
- 资助金额:
$ 53.25万 - 项目类别:
CYST WALL ENDOPROTEASES AND GLYCANS OF PARASITES
寄生虫的囊壁内切蛋白酶和聚糖
- 批准号:
8365537 - 财政年份:2011
- 资助金额:
$ 53.25万 - 项目类别:
CYST WALL ENDOPROTEASES AND GLYCANS OF PARASITES
寄生虫的囊壁内切蛋白酶和聚糖
- 批准号:
8170905 - 财政年份:2010
- 资助金额:
$ 53.25万 - 项目类别:
CYST WALL ENDOPROTEASES OF ENTAMOEBA INVADENS AND ENTAMOEBA HISTOLYTICA
入侵内阿米巴和溶组织内阿米巴的囊壁内切蛋白酶
- 批准号:
7955937 - 财政年份:2009
- 资助金额:
$ 53.25万 - 项目类别:
CYST WALL ENDOPROTEASES OF ENTAMOEBA INVADENS AND ENTAMOEBA HISTOLYTICA
入侵内阿米巴和溶组织内阿米巴的囊壁内切蛋白酶
- 批准号:
7723041 - 财政年份:2008
- 资助金额:
$ 53.25万 - 项目类别:
CYST WALL ENDOPROTEASES OF ENTAMOEBA INVADENS AND ENTAMOEBA HISTOLYTICA
入侵内阿米巴和溶组织内阿米巴的囊壁内切蛋白酶
- 批准号:
7602035 - 财政年份:2007
- 资助金额:
$ 53.25万 - 项目类别:
CYST WALL ENDOPROTEASES OF ENTAMOEBA INVADENS AND ENTAMOEBA HISTOLYTICA
入侵内阿米巴和溶组织内阿米巴的囊壁内切蛋白酶
- 批准号:
7369317 - 财政年份:2006
- 资助金额:
$ 53.25万 - 项目类别: