Structural basis of Rho glucosylation by Clostridium difficile toxins
艰难梭菌毒素 Rho 糖基化的结构基础
基本信息
- 批准号:10308686
- 负责人:
- 金额:$ 23.55万
- 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:2020-12-01 至 2023-11-30
- 项目状态:已结题
- 来源:
- 关键词:ActinsAffinityAmino AcidsAnaerobic BacteriaAnimal ModelAntibioticsApoptoticBacteriaBindingBiological AssayC-terminalCaspaseCell DeathCell Surface ReceptorsCellsCenters for Disease Control and Prevention (U.S.)Cessation of lifeChimeric ProteinsClinicalClostridium difficileClostridium difficile tcdA proteinColitisComplexCrystallizationCytoskeletonCytosolDeveloped CountriesDevelopmentDiarrheaDiseaseDrug TargetingEndocytosisEndosomesEngineeringEpithelialExotoxinsFDA approvedFamilyGastroenteritisGlucosyltransferaseGoalsGuanosine Triphosphate PhosphohydrolasesHealth care facilityHospitalsHot SpotInfectionInfection preventionInflammatoryInnate Immune ResponseLeadLengthLong-Term CareMediatingMembraneMolecularMolecular ConformationMolecular WeightMonomeric GTP-Binding ProteinsMutagenesisN DomainN-terminalOligopeptidesPathogenesisPathogenicityPathologyPatient-Focused OutcomesPeptidesPhytic AcidPlant RootsPositioning AttributeProtease DomainProtein IsoformsProtein Sequence AnalysisProteinsReagentRecurrenceResolutionRoleSpecificityStructureSubstrate SpecificitySymptomsTNFRSF6B geneTargeted ToxinsTertiary Protein StructureTherapeutic InterventionTimeToxinValidationVirulenceVirulentX-Ray Crystallographyalpha Toxinantibiotic-associated diarrheaantitoxinbasecell injurydesigndrug developmentelectron densityenzyme substrategut bacteriagut microbiomegut microbiotahuman modelimprovedin vivoinhibitorinsightmembermolecular targeted therapiesmouse modelmutantneutralizing antibodynovelnovel therapeuticsolder patientprotein complexpublic health relevancereceptor bindingreceptor mediated endocytosisresistant strainrhorho GTP-Binding Proteinsstandard of caresuccesstargeted treatmentthree dimensional structure
项目摘要
Abstract
Clostridium difficile, a Gram-positive, anaerobic, sporogenic bacterium, is often seen in severely ill or
elderly patients in hospitals or in long-term care facilities. Clostridium difficile infection (CDI), which is the most
common cause of antibiotic-associated diarrhea in developed countries, is primarily caused by two
homologous exotoxins, TcdA and TcdB. These toxins target and disrupt the colonic epithelium, leading to
diarrhea and colitis through receptor mediated endocytosis. TcdA (~308 kDa) and TcdB (~270 kDa) contain
four functional domains: an N-terminal glucosyltransferase domain (GTD), a cysteine protease domain (CPD),
a central transmembrane delivery and receptor-binding domain (DRBD), and a C-terminal combined repetitive
oligopeptides (CROPs) domain. It is widely accepted that the toxins bind to cell surface receptors via the
DRBD and the CROPs, and enter the cells through endocytosis. Acidification in the endosome triggers
conformational changes in the toxins that prompt the DRBD to form a pore and deliver the GTD and the CPD
across the endosomal membrane. In the cytosol, the CPD is activated by eukaryotic-specific inositol
hexakisphosphate and subsequently undergoes autoproteolysis to release the GTD. The GTD then
glucosylates small GTPases of the Rho family, including Rho, Rac, and Cdc42. Glucosylation of Rho proteins
inhibits their functions, leading to alterations in the actin cytoskeleton, cell-rounding, and ultimately apoptotic
cell death. Therefore the GTD is an ideal molecular target for therapeutic interventions, which directly targets
the root cause of disease symptoms and cellular damage in CDI. While the relative roles of these two toxins in
the pathogenesis of CDI are not completely understood, TcdB is considered to be more virulent than TcdA and
more important for inducing the host inflammatory and innate immune responses. Therefore, we will focus on
TcdB in this project, and the goal of this proposal is to understand the molecular mechanism by which TcdB
covalently modifies its substrates, Rho family GTPases, by glucosylation. We propose two specific aims: (1) to
understand the structural basis for recognition of Rho GTPases by the GTD, and (2) to understand the affinity
and specificity requirements for the GTD–Rho recognition. We will use X-ray crystallography and structure-
based mutagenesis to examine interactions between the GTD and Rho proteins at the molecular level, as well
as to reveal the structural determinants of substrate specificity and vulnerabilities of the GTD. These findings
will provide new insights into the function of the GTD and the pathogenicity of TcdB, which could guide the
design of novel therapeutic reagents to treat CDI by inhibiting the activity of the GTD.
抽象的
艰难梭菌是一种革兰氏阳性、厌氧、产孢子细菌,常见于重症或重症患者。
医院或长期护理机构中的老年患者中艰难梭菌感染(CDI)最多。
在发达国家,抗生素相关性腹泻的常见原因主要是由以下两种原因引起
同源外毒素 TcdA 和 TcdB 这些毒素靶向并破坏结肠上皮,导致结肠癌。
通过受体介导的内吞作用引起的腹泻和结肠炎含有 TcdA (~308 kDa) 和 TcdB (~270 kDa)。
四个功能域:N端葡萄糖基转移酶结构域(GTD)、半胱氨酸蛋白酶结构域(CPD)、
中央跨膜递送和受体结合结构域 (DRBD),以及 C 端组合重复结构域
人们普遍认为毒素通过寡肽(CROP)结构域与细胞表面受体结合。
DRBD 和 CROP,并通过内吞作用进入细胞内触发酸化。
毒素的构象变化促使 DRBD 形成孔并传递 GTD 和 CPD
在细胞质中,CPD 被真核特异性肌醇激活。
六磷酸,随后进行自蛋白水解以释放 GTD。
葡萄糖基化 Rho 家族的小 GTP 酶,包括 Rho、Rac 和 Rho 蛋白的 Cdc42。
抑制其功能,导致肌动蛋白细胞骨架的改变、细胞变圆,并最终导致细胞凋亡
因此,GTD 是治疗干预的理想分子靶标,它直接针对细胞死亡。
CDI 疾病症状和细胞损伤的根本原因,而这两种毒素在其中的相对作用。
CDI 的发病机制尚不完全清楚,TcdB 被认为比 TcdA 毒性更强,并且
对于诱导宿主炎症和先天免疫反应更为重要,因此,我们将重点关注。
本项目中的 TcdB,该提案的目标是了解 TcdB 的分子机制
通过糖基化共价修饰其底物 Rho 家族 GTPases,我们提出了两个具体目标:(1)
了解 GTD 识别 Rho GTPases 的结构基础,以及 (2) 了解亲和力
GTD-Rho 识别的特异性要求和特异性要求 我们将使用 X 射线晶体学和结构-
基于诱变,在分子水平上检查 GTD 和 Rho 蛋白之间的相互作用
揭示 GTD 底物特异性和脆弱性的结构决定因素。
将为 GTD 的功能和 TcdB 的致病性提供新的见解,这可以指导
设计新型治疗试剂通过抑制 GTD 活性来治疗 CDI。
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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Rongsheng Jin其他文献
Rongsheng Jin的其他文献
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{{ truncateString('Rongsheng Jin', 18)}}的其他基金
A versatile structure-based therapeutic platform for development of VHH-based antitoxin and antiviral agents
一个多功能的基于结构的治疗平台,用于开发基于 VHH 的抗毒素和抗病毒药物
- 批准号:
10560883 - 财政年份:2023
- 资助金额:
$ 23.55万 - 项目类别:
Structural basis for recognition of SV2 by type E botulinum neurotoxin
E型肉毒杆菌神经毒素识别SV2的结构基础
- 批准号:
10281936 - 财政年份:2021
- 资助金额:
$ 23.55万 - 项目类别:
Developing broad-spectrum therapeutics against C. difficile toxins
开发针对艰难梭菌毒素的广谱疗法
- 批准号:
10348784 - 财政年份:2021
- 资助金额:
$ 23.55万 - 项目类别:
Structural basis for recognition of SV2 by type E botulinum neurotoxin
E型肉毒杆菌神经毒素识别SV2的结构基础
- 批准号:
10448471 - 财政年份:2021
- 资助金额:
$ 23.55万 - 项目类别:
Developing broad-spectrum therapeutics against C. difficile toxins
开发针对艰难梭菌毒素的广谱疗法
- 批准号:
10548826 - 财政年份:2021
- 资助金额:
$ 23.55万 - 项目类别:
Developing broad-spectrum therapeutics against C. difficile toxins
开发针对艰难梭菌毒素的广谱疗法
- 批准号:
10181652 - 财政年份:2021
- 资助金额:
$ 23.55万 - 项目类别:
Molecular mechanisms of botulinum neurotoxin neutralization
肉毒杆菌神经毒素中和的分子机制
- 批准号:
9160875 - 财政年份:2016
- 资助金额:
$ 23.55万 - 项目类别:
Structural mechanism for recognition of host receptor by botulinum neurotoxin A
A型肉毒杆菌神经毒素识别宿主受体的结构机制
- 批准号:
9238660 - 财政年份:2016
- 资助金额:
$ 23.55万 - 项目类别:
Molecular mechanisms of botulinum neurotoxin neutralization
肉毒杆菌神经毒素中和的分子机制
- 批准号:
9271846 - 财政年份:2016
- 资助金额:
$ 23.55万 - 项目类别:
Molecular mechanisms of botulinum neurotoxin neutralization
肉毒杆菌神经毒素中和的分子机制
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9918242 - 财政年份:2016
- 资助金额:
$ 23.55万 - 项目类别:
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