Defining a pathway for mitochondrial heme trafficking
定义线粒体血红素运输途径
基本信息
- 批准号:10733705
- 负责人:
- 金额:$ 54.42万
- 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:2023-09-01 至 2027-08-31
- 项目状态:未结题
- 来源:
- 关键词:ABCG2 geneAnabolismAnemiaAsthmaAutoimmunityAzolesBindingBiologicalBiological ProcessBiotinylationCYP2D6 geneCYP3A4 geneCardiovascular systemCell Culture TechniquesCell membraneCellsChargeChimeric ProteinsComplexCrystallizationCrystallographyCytochrome P450DioxygenasesDiseaseDrug ControlsElectron TransportEnzymesEukaryotaEventGoalsHealthHemeHemeproteinsHemoglobinHereditary DiseaseHumanImmune systemInfectionIronKineticsKnowledgeLungMammalian CellMammalsMedicalMissionMitochondriaModificationMolecularMolecular ChaperonesMonitorMutagenesisMyoglobinNADPH OxidaseNational Institute of General Medical SciencesNeurologicNitric Oxide SynthaseOrganismPathway interactionsPeroxidasesPharmaceutical PreparationsPhysiologyPlayProductionPropertyProteinsPublishingRegulationResearchRoleSignal TransductionSiteSoluble Guanylate CyclaseSourceSteroid biosynthesisStructureSurfaceSystemTestingTransport ProcessVasodilationWorkYeastscofactorcrosslinkexperimental studyheme aheme oxygenase-1heme oxygenase-2indoleaminemutantparticleprotein complexprotein functionprotein purificationreconstitutionsensortraffickingtumor
项目摘要
ABSTRACT
Hemeproteins play vital transport, enzymatic, and signaling roles that fundamentally impact our cardiovascular,
pulmonary, digestive, neurological, and immune systems. Cells must transport mitochondrial heme to proteins
that mature and function outside the mitochondria. Our goal is to understand how intracellular heme delivery
takes place and is regulated in mammals. Cytosolic heme delivery proteins have been postulated but their
identities are unclear. We found that GAPDH binds mitochondrially-generated heme and that its heme binding
is essential for delivery to eight different hemeprotein targets. We hypothesize that GAPDH-dependent heme
delivery is fundamental for hemeprotein function, and we propose to discern mechanisms, scope, and
regulation of GAPDH-dependent heme delivery. Our experiments utilize purified proteins and cell culture,
which provides a facile path to molecular-level discoveries and a robust means to validate their biological
relevance. We have ways to control cell heme production, monitor GAPDH-heme binding and transfer in live
cells and between proteins, assess heme delivery to targets, characterize GAPDH-target complexes, and
determine their relevance for heme delivery.
AIM 1. How does GAPDH participate in intracellular heme delivery? We hypothesize GAPDH may bind
directly to the target proteins to deliver heme and found it does so with at least four proteins (apo-sGCβ, IDO1,
TDO, and Mb) in cells and purified form. We will: (i) identify interface regions in each GAPDH-hemeprotein
complex using HDx-MS, MS-cleavable crosslinking, and NMR approaches; (ii) use mutagenesis to test the role
of GAPDH-target protein contacts in heme deliveries; (iii) structurally characterize the GAPDH-heme complex
and GAPDH-hemeprotein complexes by crystallography and single particle EM; (iv) utilize a GAPDH-biolD2-HA
fusion protein to biotinylate intermediate or middleman proteins that associate with GAPDH during heme
transfers; (v) deploy GAPDH surface charge mutants to independently probe GAPDH-target binding and role in
heme transfers; (vi) perform heme transfer experiments with purified GAPDH & target proteins; (vii) screen for
GAPDH-dependent heme deliveries to cell heme exporters (FLVCR1a & ABCG2), cytochrome P450’s (CYP2D6
& CYP3A4), two peroxidases (LPO & EPO), an NADPH oxidase (NOX5), and heme oxygenases 1 & 2.
AIM 2. What controls GAPDH heme acquisition & deliveries? Cell & molecular mechanisms that control
GAPDH heme acquisition and release are unknown. We will: (i) determine if mitochondrial heme exporter
FLVCR1b and/or ER heme exporter PGRMC2 is a heme source for GAPDH; (ii) test if GAPDH heme acquisition
involves direct interaction with the exporters; (iii) investigate if GAPDH-heme level in cells is regulated by cell
heme exporters FLVCR1a & ABCG2; (iv) examine if post-translational GAPDH modification, chaperone hsp90,
or azole drugs control GAPDH heme acquisition and delivery.
摘要
项目成果
期刊论文数量(0)
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科研奖励数量(0)
会议论文数量(0)
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{{ truncateString('DENNIS J STUEHR', 18)}}的其他基金
Coordinate control of hemeprotein maturation and function by cell chaperones, heme, and nitric oxide
细胞伴侣、血红素和一氧化氮协调控制血红素蛋白的成熟和功能
- 批准号:
10207671 - 财政年份:2019
- 资助金额:
$ 54.42万 - 项目类别:
Coordinate control of hemeprotein maturation and function by cell chaperones, heme, and nitric oxide
细胞伴侣、血红素和一氧化氮协调控制血红素蛋白的成熟和功能
- 批准号:
10428556 - 财政年份:2019
- 资助金额:
$ 54.42万 - 项目类别:
New mechanism and regulation of intracellular heme delivery in mammals
哺乳动物细胞内血红素输送的新机制和调控
- 批准号:
8241962 - 财政年份:2011
- 资助金额:
$ 54.42万 - 项目类别:
New mechanism and regulation of intracellular heme delivery in mammals
哺乳动物细胞内血红素输送的新机制和调控
- 批准号:
8636030 - 财政年份:2011
- 资助金额:
$ 54.42万 - 项目类别:
New mechanism and regulation of intracellular heme delivery in mammals
哺乳动物细胞内血红素输送的新机制和调控
- 批准号:
8449268 - 财政年份:2011
- 资助金额:
$ 54.42万 - 项目类别:
New mechanism and regulation of intracellular heme delivery in mammals
哺乳动物细胞内血红素输送的新机制和调控
- 批准号:
8082579 - 财政年份:2011
- 资助金额:
$ 54.42万 - 项目类别:
2009 Nitric Oxide Gordon Research Conference
2009 年一氧化氮戈登研究会议
- 批准号:
7600784 - 财政年份:2009
- 资助金额:
$ 54.42万 - 项目类别:
Nitric Oxide Synthases As Oxidative And Therapeutic Agents
作为氧化剂和治疗剂的一氧化氮合成酶
- 批准号:
7664775 - 财政年份:2008
- 资助金额:
$ 54.42万 - 项目类别:
Redor Pathways Regulating Nemeprotein Maturation in Asthma
Redor 通路调节哮喘中的 Neme 蛋白成熟
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9232190 - 财政年份:2006
- 资助金额:
$ 54.42万 - 项目类别:
Redor Pathways Regulating Nemeprotein Maturation in Asthma
Redor 通路调节哮喘中的 Neme 蛋白成熟
- 批准号:
9015473 - 财政年份:2006
- 资助金额:
$ 54.42万 - 项目类别:
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