Defining the mechanistic basis of a prion disaggregase
定义朊病毒解聚酶的机制基础
基本信息
- 批准号:9239262
- 负责人:
- 金额:$ 33.93万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2013
- 资助国家:美国
- 起止时间:2013-01-01 至 2020-11-30
- 项目状态:已结题
- 来源:
- 关键词:ATP HydrolysisATP phosphohydrolaseAmyloidAmyotrophic Lateral SclerosisBasic ScienceBiochemical GeneticsBiological AssayBiotechnologyClientCollaborationsConflict (Psychology)CouplesCryoelectron MicroscopyDataDeuteriumDirected Molecular EvolutionDiseaseDisease modelEngineeringEnzymesFrontotemporal DementiaGenetic studyGoalsHIVHumanHydrogenIndustryInvestigationLinkMass Spectrum AnalysisMedicineNerve DegenerationNervous system structureNeurodegenerative DisordersParkinson DiseasePharmacologic SubstancePrionsProtein FootprintingProteinsRattusResearchResolutionRoentgen RaysSecureStructureSubstantia nigra structureTechnologyTherapeuticTherapeutic AgentsVariantX-Ray CrystallographyYeastsalpha synucleinamyloidogenesisbasecomparativedesignnanomachineprotein TDP-43protein aggregationprotein foldingprotein misfoldingproteostasisreconstructiontranslocaseunfoldaseyeast protein
项目摘要
Project summary: Our research objective is to define the mechanistic basis of Hsp104, a protein
disaggregase and hexameric AAA+ (ATPases Associated with diverse Activities) protein from yeast, which
remains poorly understood. Hsp104 couples ATP hydrolysis to the dissolution and reactivation of diverse
proteins trapped in disordered aggregates, toxic preamyloid oligomers, amyloids, and prions. Hsp104 is the
only factor known to dissociate α-synuclein (α-syn) oligomers and amyloids connected with Parkinson's
disease (PD) and rescue α-syn-induced neurodegeneration in the substantia nigra of a rat PD model.
However, Hsp104 activity is limited against α-syn and very high Hsp104 concentrations are needed for optimal
effects. Thus, we engineered potentiated Hsp104 variants, which dissolve fibrils formed by neurodegenerative
disease proteins such as TDP-43, FUS, and -syn, and mitigate neurodegeneration in the metazoan nervous
system at concentrations where Hsp104 is inactive. Curiously, Hsp104 is absent from metazoa. Thus, Hsp104
and potentiated variants could represent a disruptive technology to enhance proteostasis to counter
neurodegenerative disease and enable purification of irksome, aggregation-prone proteins for valuable basic or
pharmaceutical purposes. However, these endeavors are frustrated by a limited mechanistic understanding of
Hsp104, which despite intense investigation remains stalled at a low level of resolution. Three critical barriers
impede our understanding of Hsp104. First, we do not understand how Hsp104 selects clients for
disaggregation, which limits our ability to tailor Hsp104 activity for specific substrates. This issue is pernicious
because potentiated Hsp104 variants can have damaging, off-target effects due to promiscuous activity, which
could restrict therapeutic or biotechnological applications. Second, Hsp104 sequence space remains largely
unexplored. It is unclear whether natural Hsp104 orthologues exist with divergent enhanced or selective
activity against neurodegenerative disease substrates. Third, there is no atomic structure of the Hsp104
hexamer and conflicting cryo-electron microscopy reconstructions have confused the field. Based on our
preliminary data, we hypothesize that: (1) potentiated Hsp104 variants can be engineered to be more
substrate specific to avoid damaging off-target effects; (2) natural Hsp104 orthologues exist with
enhanced activity against neurodegenerative disease substrates and minimal off-target effects; and (3)
large structural changes in Hsp104 hexamers upon ATP hydrolysis drive protein disaggregation. Thus,
we will meet three aims: (1) Define potentiated Hsp104 variants with enhanced substrate selectivity; (2) Define
conserved and divergent activities of natural Hsp104 orthologues; (3) Define high-resolution structural changes
in Hsp104 and potentiated variants that drive protein disaggregation. In this way, we will secure a high-
resolution mechanistic view of Hsp104, which will empower the engineering of new Hsp104 nanomachines
with selective potentiated activity for key applications in biotechnology and medicine.
项目摘要:我们的研究目标是确定 Hsp104(一种蛋白质)的机制基础
来自酵母的解聚酶和六聚体 AAA+(与多种活性相关的 ATP 酶)蛋白,
Hsp104 将 ATP 水解与多种物质的溶解和重新激活结合起来仍知之甚少。
被困在无序聚集体、有毒的前淀粉样蛋白寡聚体、淀粉样蛋白和 Hsp104 中的蛋白质是
唯一已知可解离与帕金森病相关的 α-突触核蛋白 (α-syn) 寡聚体和淀粉样蛋白的因子
疾病(PD)并挽救大鼠 PD 模型黑质中 α-syn 诱导的神经变性。
然而,Hsp104 对 α-syn 的活性有限,需要非常高的 Hsp104 浓度才能达到最佳效果。
因此,我们设计了增强的 Hsp104 变体,可以溶解神经退行性疾病形成的原纤维。
疾病蛋白,如 TDP-43、FUS 和 α-syn,并减轻后生动物神经中的神经变性
奇怪的是,后生动物中不存在 Hsp104。
增强的变体可能代表一种破坏性技术,可以增强蛋白质稳态以对抗
神经退行性疾病,并能够纯化令人厌烦的、易于聚集的蛋白质,以获得有价值的基本或
然而,这些努力因对机制的有限理解而受挫。
Hsp104,尽管进行了深入的研究,但仍停留在较低的分辨率水平。
阻碍了我们对Hsp104的理解,首先,我们不了解Hsp104是如何选择客户的。
分解,这限制了我们针对特定底物定制 Hsp104 活性的能力,这个问题是有害的。
因为强化的 Hsp104 变体可能会由于混杂的活动而产生破坏性的脱靶效应,
其次,Hsp104 序列空间仍然很大。
目前尚不清楚天然 Hsp104 直系同源物是否存在不同的增强型或选择性。
第三,Hsp104 没有原子结构。
六聚体和相互矛盾的冷冻电子显微镜重建使该领域感到困惑。
根据初步数据,我们发现:(1) 增强的 Hsp104 变体可以被设计得更
底物特异性以避免破坏性脱靶效应;(2) 天然 Hsp104 直向同源物存在
增强针对神经退行性疾病底物的活性并最小化脱靶效应;以及 (3)
ATP 水解后 Hsp104 六聚体的巨大结构变化驱动蛋白质解聚。
我们将实现三个目标:(1) 定义具有增强底物选择性的增强型 Hsp104 变体;(2) 定义
(3) 定义高分辨率结构变化
在 Hsp104 和驱动蛋白质解聚的增强变体中,我们将确保高水平。
Hsp104 的分辨率机制视图,这将为新型 Hsp104 纳米机器的工程设计提供支持
具有针对生物技术和医学关键应用的选择性增强活性。
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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James Shorter其他文献
James Shorter的其他文献
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{{ truncateString('James Shorter', 18)}}的其他基金
Engineering therapeutic TRIM11 disaggregases for Alzheimer's Disease-Related Dementias (ADRDs)
工程治疗 TRIM11 解聚酶治疗阿尔茨海默病相关痴呆症 (ADRD)
- 批准号:
10539674 - 财政年份:2022
- 资助金额:
$ 33.93万 - 项目类别:
Isolating small-molecule enhancers of HtrA1, an alpha-synuclein disaggregase
分离 HtrA1(一种 α-突触核蛋白解聚酶)的小分子增强子
- 批准号:
9374303 - 财政年份:2017
- 资助金额:
$ 33.93万 - 项目类别:
Exploring and enhancing Karyopherin beta-2 disaggregate activity
探索和增强核传递蛋白 beta-2 解聚活性
- 批准号:
9182306 - 财政年份:2016
- 资助金额:
$ 33.93万 - 项目类别:
Defining the mechanistic basis of a prion disaggregase
定义朊病毒解聚酶的机制基础
- 批准号:
8438661 - 财政年份:2013
- 资助金额:
$ 33.93万 - 项目类别:
Defining the mechanistic basis of a prion disaggregase
定义朊病毒解聚酶的机制基础
- 批准号:
8774612 - 财政年份:2013
- 资助金额:
$ 33.93万 - 项目类别:
Defining the mechanistic basis of a prion disaggregase
定义朊病毒解聚酶的机制基础
- 批准号:
8974843 - 财政年份:2013
- 资助金额:
$ 33.93万 - 项目类别:
Generating SEVI disaggregases to prevent HIV infection
生成 SEVI 分解以预防 HIV 感染
- 批准号:
8410697 - 财政年份:2012
- 资助金额:
$ 33.93万 - 项目类别:
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