Regulation of the Unfolded Protein Response by eIF2 alpha phosphatases
eIF2 α 磷酸酶对未折叠蛋白反应的调节
基本信息
- 批准号:9377749
- 负责人:
- 金额:$ 30.52万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2017
- 资助国家:美国
- 起止时间:2017-09-01 至 2021-08-31
- 项目状态:已结题
- 来源:
- 关键词:AddressAlzheimer&aposs DiseaseAmino AcidsAmyotrophic Lateral SclerosisApoptosisAutophagocytosisBindingBiological ModelsCatalytic DomainCell DeathCellsCellular biologyCharcot-Marie-Tooth DiseaseComplexComplicationDataDiseaseDrug TargetingEmbryoEndoplasmic ReticulumEpidermisEquilibriumEtiologyEukaryotic CellFatty AcidsFatty LiverFutureGenesHepatocyteHumanIn Situ HybridizationInflammatoryLightLinkLiverLiver diseasesMalignant NeoplasmsMapsMessenger RNAMetabolic DiseasesModelingMolecularMolecular ChaperonesNeurodegenerative DisordersNeuronsNon-Insulin-Dependent Diabetes MellitusOrphan DrugsPathway interactionsPatternPharmaceutical PreparationsPharmacologyPhenotypePhosphoric Monoester HydrolasesProcessProtein BiosynthesisProteinsProteolysisPublic HealthPublicationsReagentRegulationReporterResearchReverse Transcriptase Polymerase Chain ReactionRoleSignal TransductionSpinal cord injuryStimulusStressStructureStructure of beta Cell of isletSymptomsTestingTimeTissuesTranslationsWestern BlottingWorkZebrafishclinical developmentclinically relevantendoplasmic reticulum stressexperimental studygenetic analysisgenetic approachgenome editinghuman diseasein vivoinhibitor/antagonistinsightinterestloss of functionmisfolded proteinmouse modelmutantneoplastic cellpreventprotective effectprotein foldingprotein transportresponsereverse geneticstargeted agent
项目摘要
Project Summary
Eukaryotic cells must be able to maintain balance between the amount of protein entering the secretory pathway
and the capacity to process, fold and traffic these proteins through the endoplasmic reticulum. Excessive protein
synthesis can cause proteins to unfold and aggregate in the ER causing ER stress. To survive the cells launch
a signal transduction cascade called the Unfolded Protein Response (UPR). The UPR leads to increases in
protein folding capacity by upregulating chaperones, also prevents new proteins from entering the ER by
lowering the rate of global translation, and the UPR also helps clear unfolded proteins from the ER by increase
proteolysis and autophagy. Numerous human disease are linked to ER-stress including, Alzheimer’s and other
neurodegenerative diseases, type II diabetes, fatty liver disease, spinal cord injury, among others. Multiple drugs
have been developed that modulate UPR signaling; among the most promising are inhibitors of eIF2 alpha
phosphatases (including salubrinal and sephin1). These drugs have shown great promise in delaying onset and
prolonging survival in mouse models of Amyotrophic lateral sclerosis (ALS), while sephin1 has been recently
approved as an orphan drug for treating Charcot-Marie-Tooth disease 1B (CMT). The eIF2 alpha phosphatase
complexes are comprised of a catalytic subunit PP1 and either of two regulatory subunits, GADD34 and CReP,
and are potent inhibitors of one branch of the UPR. This project uses the zebrafish model system to investigate
the molecular mechanisms of ER-stress induced apoptosis. In aim 1 we will take a genetic approach and seek
to understand how levels of GADD34 and CReP are regulated by ER stress. Our preliminary data indicates that
GADD34 mRNA rapidly increases and remains elevated after ER-stress, while CReP transiently increases
following ER stress and then declines to levels below baseline after prolonged stress. We will identify which of
the branches of UPR signaling are required for this dynamic expression pattern. In addition, we will utilize
CRIPSR-CAS9 genome editing for reverse-genetic analysis of the function of GADD34 and CReP. In aim 2 we
will then focus on two major stress-induced phenotypes in zebrafish, ER-stress induced apoptosis in the
developing caudal fin and ER-stress induced accumulation of fatty acids in the liver (steatosis). This will provide
us with a highly tractable model of ER-stress induced cell death and ER-stress induced metabolic disorders.
Our preliminary data indicates that inhibition of GADD34 and CReP protects fin epidermal cells from ER-stress
induced apoptosis, but worsens fatty liver symptoms in the liver. In aim 3 we will examine the assembly of the
active eIF2 alpha phosphatase by determining the amino acids required for in vivo function. In our preliminary
data experiments we mapped the minimal PP1-binding domain of CReP and GADD34 and generated a suite of
point mutants to map residues critical for function. Using zebrafish will allow us to combine reverse genetics with
in vivo cell biology, and will provide an ideal model to test the function of GADD34 and CReP and shed light onto
the mechanism of action of clinically relevant drugs that target these proteins.
项目摘要
真核细胞必须能够在进入分泌途径的蛋白质量之间保持平衡
以及通过内质网加工、折叠和运输这些蛋白质的能力。过量蛋白质
合成可引起蛋白质在ER中展开和聚集,引起ER应激。为了在细胞发射后存活下来
一种称为未折叠蛋白质反应(UPR)的信号转导级联。普遍定期审议导致
蛋白质折叠能力通过上调分子伴侣,也防止新的蛋白质进入ER,
降低整体翻译的速率,UPR也有助于通过增加蛋白质的表达来清除ER中的未折叠蛋白质。
蛋白水解和自噬。许多人类疾病都与ER应激有关,包括阿尔茨海默氏症和其他
神经变性疾病、II型糖尿病、脂肪肝疾病、脊髓损伤等。多种药物
已经开发了调节UPR信号传导的药物;其中最有前途的是eIF 2 α抑制剂
磷酸酶(包括salubrinal和sephin 1)。这些药物在延迟发病和
延长肌萎缩侧索硬化症(ALS)小鼠模型的存活时间,而sephin 1最近已被
被批准为治疗Charcot-Marie-Tooth病1B(CMT)的孤儿药。eIF 2 α磷酸酶
复合物由催化亚基PP 1和两个调节亚基GADD 34和CReP中的任一个组成,
并且是UPR的一个分支的有效抑制剂。本计画利用斑马鱼模式系统进行研究
内质网应激诱导细胞凋亡的分子机制。在目标1中,我们将采取遗传方法,
了解GADD 34和CReP水平如何受ER应激调节。我们的初步数据显示,
GADD 34 mRNA在ER应激后迅速增加并保持升高,而CReP瞬时增加,
在ER应激之后,然后在长时间应激之后下降到低于基线的水平。我们会找出
这种动态表达模式需要UPR信号传导的分支。此外,我们将利用
CRIPSR-CAS9基因组编辑用于GADD 34和CReP功能的反向遗传分析。在目标2中,
然后将集中在两个主要的压力诱导的表型在斑马鱼,雌激素受体应激诱导的细胞凋亡,
发育中的尾鳍和ER应激诱导脂肪酸在肝脏中的积累(脂肪变性)。这将提供
我们用ER应激诱导的细胞死亡和ER应激诱导的代谢紊乱的高度易处理的模型。
我们的初步数据表明,GADD 34和CReP的抑制保护鳍表皮细胞免受ER应激
诱导细胞凋亡,但在肝脏中抑制脂肪肝症状。在目标3中,我们将研究
活性eIF 2 α磷酸酶通过测定体内功能所需的氨基酸。在我们的初步调查中
数据实验,我们映射了CReP和GADD 34的最小PP 1结合结构域,并生成了一套
点突变体以定位对功能至关重要的残基。利用斑马鱼将使我们能够将反向遗传学与联合收割机
在体内细胞生物学,并将提供一个理想的模型来测试GADD 34和CReP的功能,并阐明
靶向这些蛋白质的临床相关药物的作用机制。
项目成果
期刊论文数量(3)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
The PPP1R15 Family of eIF2-alpha Phosphatase Targeting Subunits (GADD34 and CReP).
EIF2-Alpha磷酸酶靶向亚基的PPP1R15家族(GADD34和CREP)。
- DOI:10.3390/ijms242417321
- 发表时间:2023-12-10
- 期刊:
- 影响因子:5.6
- 作者:Hicks, Danielle;Giresh, Krithika;Wrischnik, Lisa A.;Weiser, Douglas C.
- 通讯作者:Weiser, Douglas C.
Alternative splicing of (ppp1r12a/mypt1) in zebrafish produces a novel myosin phosphatase targeting subunit.
- DOI:10.1016/j.gene.2018.06.092
- 发表时间:2018-10-30
- 期刊:
- 影响因子:3.5
- 作者:LaFlamme A;Young KE;Lang I;Weiser DC
- 通讯作者:Weiser DC
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