Synthetic Reconstruction of Human Chaperone Networks in Yeast Models of Neurodegeneration
神经退行性酵母模型中人类伴侣网络的综合重建
基本信息
- 批准号:10591799
- 负责人:
- 金额:$ 11.53万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2023
- 资助国家:美国
- 起止时间:2023-03-15 至 2025-02-28
- 项目状态:未结题
- 来源:
- 关键词:Advisory CommitteesAgeAgingAlzheimer&aposs disease related dementiaBiochemistryBiological AssayBrain DiseasesCRISPR-mediated transcriptional activationCell modelCellsCellular AssayCollaborationsComplementComplexCytoplasmEngineeringEnzymesEukaryotaFlow CytometryFrontotemporal DementiaFundingFutureGenesGeneticGenetic TechniquesGoalsHeat shock proteinsHumanIn VitroIndividualLearning SkillLibrariesMapsMeasuresMentorsMessenger RNAModelingMolecular ChaperonesMolecular GeneticsMutationNerve DegenerationNeurodegenerative DisordersNeuronsOrganismPathologicPathologyPennsylvaniaPhasePhenotypePopulationPositioning AttributePostdoctoral FellowPreparationPreventionProcessProteinsProteomeRNA SplicingReporter GenesResearchSpecificityStressSystemTDP-43 aggregationTestingToxic effectTrainingTranslatingTriplet Multiple BirthUnited States National Institutes of HealthUniversitiesWorkYeast Model SystemYeastscombatcombinatorialexperimental studygenetic approachgenetic technologygenetic variantheat-shock proteins 110in vitro Assayinsightmisfolded proteinmutantneuronal survivalnovel strategiesoverexpressionpost-doctoral trainingprogramsprotein TDP-43protein expressionprotein misfoldingprotein purificationproteostasisreconstructionscreeningskill acquisitionskillstargeted treatmenttau Proteinstau aggregationtherapeutic targettool
项目摘要
Project Summary
In aging neurons, the accumulation of key misfolded proteins into aggregates is a hallmark of many
neurodegenerative diseases. For example, pathological forms of TDP43 become mislocalized to the cytoplasm
and accumulate in aggregates in frontotemporal dementia (FTD) and other Alzheimer Disease-related
Dementias (ADRD). Highly intricate networks of enzymes called molecular chaperones combat these processes.
In ADRD, it is unknown how the chaperone networks fail against pathological forms of ADRD proteins such as
TDP43, FUS, and TAU. A major challenge to studying chaperone networks is the combinatorial complexity. The
canonical Hsp70 network consists of 54 Hsp40, 12 Hsp70, and 16 Hsp110 gene variants, creating a landscape
of 12,155 possible protein expression combinations. Unique combinations of the Hsp40-Hsp70-Hsp110 proteins
are hypothesized to confer specificity for different misfolded proteins in the complex human proteome. This
hypothesis is widely accepted but it has never been directly tested due to technical limitations. This NIH K99/R00
proposal outlines a plan to directly test this hypothesis by building the first exhaustive map of a chaperone
network against the ADRD-associated proteins TDP43, FUS, and TAU. To achieve this goal, aim 1 will leverage
a new genetic technique developed by Dr. Edward Barbieri to express and study all 12,155 possible
combinations of the human Hsp40-Hsp70-Hsp110 network in yeast models of ADRD. The chaperones identified
as having activity against TDP43 in yeast will be further studied in aim 2 using human cells and in vitro assays.
With human cell models, Dr. Barbieri will study the effect of the TDP43-active chaperones on cytoplasmic TDP43
aggregation and assess if the chaperones restore native TDP43 function in mRNA splicing in both HEK-293T
cells and neurons. Using in vitro biochemistry, he will measure chaperone activity for prevention and reversal of
TDP43 aggregation. During the R00 phase in aim 3 Dr. Barbieri will apply the chaperone network screen to study
the TAU aggregation and he will expand the chaperone networks studied in yeast by including Hsp40 pairs and
small HSPs. Lastly, Dr. Barbieri will combine the skills he learns during the K99 phase to develop a screen for
combinatorially overexpressing all 194 human chaperones directly in human cell models of ADRD to study
proteostasis networks in the native context. Together, the experiments outlined in this proposal will identify key
chaperones as therapeutic targets for ADRD. Dr. Barbieri will perform the K99 phase mentored in the Shorter
lab at the University of Pennsylvania, a world class biochemistry lab with expertise in the study of chaperones
and ADRD. This is an ideal training setting for Dr. Barbieri to acquire new skills in biochemistry. Furthermore,
Dr. Barbieri assembled an advisory committee to provide expertise in ADRD and formal training in the human
cell assays. The new skills will complement his current expertise in molecular genetics, and his outlined training
plan will provide the necessary preparation for Dr. Barbieri to progress to his goal of an independent position.
项目摘要
在老化的神经元中,关键的错误折叠蛋白聚集成聚集体是许多
神经退行性疾病。例如,TDP43的病理形式会错误定位于细胞质
在额颞叶痴呆(FTD)和其他阿尔茨海默病相关疾病中聚集
痴呆(ADRD)。被称为分子伴侣的高度复杂的酶网络对抗这些过程。
在ADRD中,尚不清楚伴侣网络如何对抗ADRD蛋白的病理形式,如
TDP43、FUS和TAU。研究伴侣网络的一个主要挑战是组合的复杂性。这个
规范的Hsp70网络由54个Hsp40、12个Hsp70和16个Hsp110基因变体组成,创造了一个景观
12,155种可能的蛋白质表达组合。Hsp40-Hsp70-Hsp110蛋白的独特组合
被认为是对复杂的人类蛋白质组中不同错误折叠的蛋白质赋予特异性。这
假说被广泛接受,但由于技术限制,它从未被直接测试过。这是NIH K99/R00
提案概述了一项计划,通过构建第一个详尽的伴侣地图来直接测试这一假说
针对ADRD相关蛋白TDP43、FUS和TAU的网络。为了实现这一目标,目标1将利用
爱德华·巴比耶里博士开发的一种新的基因技术,用于表达和研究所有12,155种可能的
人Hsp40-Hsp70-Hsp110网络在ADRD酵母模型中的组合。已确认的监护人
在目标2中,将使用人体细胞和体外试验进一步研究酵母中具有抗TDP43活性的AS。
Barbieri博士将利用人类细胞模型研究TDP43活性伴侣对细胞质TDP43的影响
在HEK-293T中聚集和评估伴侣是否恢复了天然的TDP43在mRNA剪接中的功能
细胞和神经元。利用体外生物化学,他将测量伴侣蛋白的活性,以预防和逆转
TDP43聚集。在AIM 3的R00阶段,Barbieri博士将应用伴侣网络筛查进行研究
TAU聚集和他将扩大在酵母中研究的伴侣网络,包括Hsp40和
小型HSP。最后,Barbieri博士将结合他在K99阶段学到的技能来开发一种筛查
直接在ADRD人细胞模型中联合过表达所有194个人伴侣蛋白的研究
原生环境中的蛋白质平衡网络。总之,这项提案中概述的实验将确定关键
伴侣作为ADRD的治疗靶点。Barbieri博士将在较短的时间内进行K99阶段的指导
宾夕法尼亚大学的实验室,这是一个世界级的生化实验室,拥有研究伴侣的专业知识
还有阿德勒。这是Barbieri博士获得生物化学新技能的理想培训环境。此外,
Barbieri博士组建了一个咨询委员会,以提供ADRD方面的专业知识和人类
细胞分析。这些新技能将补充他目前在分子遗传学方面的专业知识,以及他概述的培训
该计划将为Barbieri博士迈向独立职位的目标提供必要的准备。
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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Edward Matthew Barbieri其他文献
Edward Matthew Barbieri的其他文献
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{{ truncateString('Edward Matthew Barbieri', 18)}}的其他基金
Programming Human Chaperone Systems Against Neurodegenerative Disease
对人类伴侣系统进行编程以对抗神经退行性疾病
- 批准号:
10238101 - 财政年份:2019
- 资助金额:
$ 11.53万 - 项目类别:
Programming Human Chaperone Systems Against Neurodegenerative Disease
对人类伴侣系统进行编程以对抗神经退行性疾病
- 批准号:
10026294 - 财政年份:2019
- 资助金额:
$ 11.53万 - 项目类别:
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