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.
项目摘要/摘要
这项建议探讨了核O-岩藻糖作为弓形虫新的调节机制的作用。
弓形虫病,导致严重的神经和眼部疾病。John Samuelson实验室使用了抗岩藻糖阿列乌里亚
AAL表明弓形虫岩藻糖化蛋白形成紧密结合的点状集合体
核孔复合体。O-岩藻糖化蛋白的组装使人联想到Cajal、PML和
组蛋白在宿主核内,不被O-岩藻糖修饰。AAL的浓缩和质量
光谱分析显示O-岩藻糖结合在丝氨酸和苏氨酸上,在数十个
蛋白质,它们似乎参与转录、信使核糖核酸的处理和运输,以及信号传递。《克里斯》
West实验室鉴定了弓形虫O-转移酶(TgSpy),它含有11个N端四肽重复序列
(TPR)和一个C端的GT41糖基转移酶结构域。重组人TgSpy的细胞质制备
细菌本身可以降解GDP-岩藻糖和O-岩藻糖酸盐,形成PolySer/GST融合,并在裂解产物中产生蛋白质
SpyKO细胞。TgSpy不是必需的,但spyKO生长较慢,而过度表达的转染体
TgSpy成长得更快。与PolySer融合的绿色荧光蛋白被O-岩藻糖基化并聚集在AAL-
在亲本菌株中标记的组件,但在spyKO中被降解。
TgSpy是拟南芥中赤霉酸信号的负调控因子,是植物Spinly的同系物,是
与宿主O-GlcNAc转移酶(OGT)类似。在TG中没有的OGT本质上是修饰的
它是数百种蛋白质的无序区域,是抗逆性和营养信号的重要因素。
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中。此外,TgSpy的修改唯一导致
O-岩藻糖化蛋白定位在鼻咽癌附近,在那里它们受到保护,不会被降解。
萨缪尔森实验室和韦斯特实验室将共同研究O-岩藻糖在弓形虫中的作用。在目标1中,我们
将评估TPR和GT41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,它们是O-岩藻糖化的和
有助于TG健身,回应不同的Spy表情。我们将进行互动组研究,以解决
研究了O-岩藻糖化蛋白组装过程中的蛋白质包合机制和脉冲追逐标记、时间-时间标记等。
失光显微镜和光漂白以确定组件的稳定性。在目标3中,我们将比较
在培养、小鼠和猫的亲本菌株、间谍基因敲除和过度表达TgSpy的生物中生长。
此外,我们将对以速殖子或SILAC形式生长的三组原生生物进行RNA-SEQ和SILAC
在培养过程中转变为缓殖子。
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: