Control of Mitochondrial Function by SPFH Proteins in Pathogenic Yeast
病原酵母中 SPFH 蛋白对线粒体功能的控制
基本信息
- 批准号:10332017
- 负责人:
- 金额:$ 11.91万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2021
- 资助国家:美国
- 起止时间:2021-09-16 至 2025-08-31
- 项目状态:未结题
- 来源:
- 关键词:AddressAntifungal AgentsAntifungal TherapyApoptosisApoptoticAreaBinding ProteinsBiological AssayBiologyBiomedical ResearchCRISPR/Cas technologyCandida albicansCarbonCell WallCell membraneCell physiologyCellsCellular AssayCessation of lifeChimeric ProteinsClustered Regularly Interspaced Short Palindromic RepeatsCo-ImmunoprecipitationsComplementComplexDisseminated candidiasisDrug usageEpitopesEukaryotaEukaryotic CellExposure toFamilyFamily memberFilamentFlow CytometryFluorescence MicroscopyFundingGenesGeneticGenetic TranscriptionGoalsGrowthHispanic-serving InstitutionHumanHyphaeInfectionInterventionInvertebratesInvestigationKnowledgeLeishmaniaLifeLife Cycle StagesMammalsMediatingMembraneMembrane PotentialsMembrane ProteinsMitochondriaMolecularMolecular BiologyMolecular GeneticsMorphogenesisNematodaOrganellesOxidative StressParasite ControlParasitesPathogenesisPathogenicityPathway interactionsPatientsPhenotypePhospholipidsPlanariansPlasmodiumProcessProductionProductivityPropertyProtein FamilyProtein Sequence AnalysisProteinsProteomicsPublishingReactive Oxygen SpeciesResearchRespirationRoleSavingsSignal TransductionSourceStressStudentsStudy modelsSystemTestingTimeTrainingVertebratesVirulenceYeastsbasecandidemiacollegecost effectivedrug efficacyflotillinfungusgene discoverygenome editinghuman pathogenin vivoinhibitor/antagonistinnovationlight microscopymembermicrobialmitochondrial membranemortalitymutantnovelpathogenpathogenic fungusprogramsprohibitinprotein complexprotein functionrespiratoryresponse
项目摘要
The mitochondrion is the major energy-producing organelle of the cell and coordinates key activities such
as signaling, apoptosis, and phospholipid synthesis. These processes are essential for survival and
pathogenesis of Candida albicans, the most common fungal pathogen in humans. C. albicans respiration
occurs via three distinct pathways compared to vertebrates and is associated with virulence properties such
as morphogenesis and cell wall synthesis. Our long-term research goal is to understand the cellular and
molecular mechanisms that govern C. albicans survival in response to environmental stress and antifungal
drugs. We previously discovered that gene classes involved mitochondrial functions are highly transcribed
in response to osmotic and cell wall stress, and the gene SLP3 (stomatin like protein 3) was significantly
upregulated. SLP3 is a member of the conserved SPFH (Stomatin, Prohibitin, Flotillin, HflK/HflC) protein
superfamily. In eukaryotes, SPFH proteins are required for essential mitochondrial processes such as
respiration, mitophagy, and apoptosis and mediate pathogenicity in several parasites. Our objective in this
proposal is to determine the role of the C. albicans SPFH protein family in mitochondrial function. We were
the first to demonstrate that Slp3p overproduction disrupted mitochondrial membrane potential and
triggered apoptotic-like death specifically following prolonged exposure to oxidative stress. However, the
molecular function Slp3p remains unknown. Thus, our central hypothesis is that C. albicans SPFH proteins
form membrane complexes to coordinate mitochondrial function. This hypothesis is based upon
observations with mammalian SPFH proteins and our published and preliminary findings. Human SPFH
complexes appear as punctate foci when viewed using fluorescence microscopy. We found that Slp3p form
puncta at the plasma membrane. Further, we identified a mitochondrial targeting signal in Slp2p, Phb1p and
Phb2p, and our Slp2p-GFP fusion protein formed mitochondrial puncta. We will utilize a high-throughput
molecular genetic and cellular approach that is cost-effective and time-saving to determine SPFH protein
localization, protein complex composition, cellular function, and role in C. albicans infection. We will create
SPFH-GFP fusion proteins and use fluorescence microscopy to determine cellular localization. We will
construct SPFH-epitope-tagged strains and perform Co-IP and LC-MS/MS analyses to identify putative
SPFH protein binding partners. We will create SPFH mutants via CRISPR-Cas9 genome editing and
phenotypically characterize mutant strains in growth and cellular assays. We will examine SPFH function in
C. albicans pathogenesis using a novel invertebrate infection system. Our findings will address a poorly
understood area in C. albicans biology and provide a model for studying SPFH proteins in pathogenic fungi.
This proposal is innovative as we will characterize the SPFH family in the context of a critical, yet poorly
understood area in C albicans biology: the molecular framework underlying mitochondrial function.
线粒体是细胞的主要能量产生细胞器,并协调关键活动,
如信号传导、凋亡和磷脂合成。这些过程对生存至关重要,
白色念珠菌是人类最常见的真菌病原体。C.白色念珠菌呼吸
与脊椎动物相比,它通过三种不同的途径发生,并与毒力特性相关,例如
如形态发生和细胞壁合成。我们的长期研究目标是了解细胞和
控制C的分子机制环境应激和抗真菌药物对白念珠菌存活的影响
毒品我们以前发现,涉及线粒体功能的基因类是高度转录的,
SLP 3(stomatinlikeprotein 3)基因表达量显著高于对照组(P < 0.05)。
上调。SLP 3是保守的SPFH(Stomatin,Prohibitin,Flotilin,HflK/HflC)蛋白的成员
超家族在真核生物中,SPFH蛋白是必需的线粒体过程,如
呼吸,线粒体自噬和细胞凋亡,并介导几种寄生虫的致病性。我们的目标是
建议是确定C.白色念珠菌SPFH蛋白家族在线粒体功能中的作用。我们
第一个证明Slp 3 p的过度产生破坏了线粒体膜电位,
特别是在长时间暴露于氧化应激后引发了类似癫痫的死亡。但
Slp 3 p的分子功能仍然未知。因此,我们的中心假设是C。白色念珠菌SPFH蛋白
形成膜复合物以协调线粒体功能。这个假设是基于
与哺乳动物SPFH蛋白的观察和我们发表的初步研究结果。人SPFH
当使用荧光显微镜观察时,复合物表现为点状病灶。我们发现Slp 3 p形式
点在质膜上。此外,我们在Slp 2 p、Phb 1 p和
Phb 2 p和我们的Slp 2 p-GFP融合蛋白形成线粒体斑点。我们将利用高通量
分子遗传学和细胞学方法,是成本效益和节省时间,以确定SPFH蛋白
定位,蛋白质复合物的组成,细胞功能和C.白色念珠菌感染我们将创建
SPFH-GFP融合蛋白,并使用荧光显微镜来确定细胞定位。我们将
构建SPFH-表位标记的菌株,并进行Co-IP和LC-MS/MS分析,以鉴定推定的
SPFH蛋白结合伴侣。我们将通过CRISPR-Cas9基因组编辑创建SPFH突变体,
在生长和细胞测定中表型地表征突变菌株。我们将检查SPFH功能,
C.使用一种新的无脊椎动物感染系统研究白色念珠菌的发病机制。我们的发现将解决一个糟糕的
了解C中的区域。为研究病原真菌中SPFH蛋白提供了模型。
这项建议是创新的,因为我们将在一个关键的背景下描述SPFH家族,但很差,
白色念珠菌生物学的理解领域:线粒体功能的分子框架。
项目成果
期刊论文数量(0)
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Jason Malcolm Rauceo其他文献
Jason Malcolm Rauceo的其他文献
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{{ truncateString('Jason Malcolm Rauceo', 18)}}的其他基金
Control of Mitochondrial Function by SPFH Proteins in Pathogenic Yeast
病原酵母中 SPFH 蛋白对线粒体功能的控制
- 批准号:
10674870 - 财政年份:2021
- 资助金额:
$ 11.91万 - 项目类别:
Analysis of the Candida albicans adhesin Als5p
白色念珠菌粘附素 Als5p 的分析
- 批准号:
6942241 - 财政年份:2003
- 资助金额:
$ 11.91万 - 项目类别:
Analysis of the Candida albicans adhesin Als5p
白色念珠菌粘附素 Als5p 的分析
- 批准号:
6741334 - 财政年份:2003
- 资助金额:
$ 11.91万 - 项目类别:
Analysis of the Candida albicans adhesin Als5p
白色念珠菌粘附素 Als5p 的分析
- 批准号:
6807048 - 财政年份:2003
- 资助金额:
$ 11.91万 - 项目类别:
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