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CRL2^(APPBP2)-mediated TSPYL2 degradation counteracts human mesenchymal stem cell senescence

Science China(Life Sciences) 2024年第3期67卷 460-474页

作者： Daoyuan Huang Qian Zhao Kuan Yang Jinghui Lei Ying Jing Hongyu Li Chen Zhang Shuai Ma Shuhui Sun Yusheng Cai Guibin Wang Jing Qu Weiqi Zhang Si Wang Guang-Hui Liu Advanced Innovation Centerfor Human Brain Protection National Clinical Research Centerfor Geriatric DisordersXuanwu Hospital Capital Medical UniversityBeijing 100053China State Key Laboratory of Stem Cell and Reproductive Biology Institute of ZoologyChinese Academy of SciencesBeijing 100101China Aging Translational MedicineCenter International Centerfor Aging and CancerBejing Municipal GeriatricMedical Research CenterXuanwu HospitalCapital Medical UniversityBeijing 100053China State Key Laboratory of Membrane Biology Institute of ZoologyChinese Academy of SciencesBeijing 100101China University of Chinese Academy of Sciences Beijing 100049China Beijing Institute for Stem Cell and Regenerative Medicine Beijing100101China CAS Key Laboratory of Genomic and Precision Medicine Bejing Institute of Genomics and China National Center for BioinformationChinese Academy of SciencesBeijing 100101China Institute for Stem Cell and Regeneration CASBeijing 100101China The Fifth People's Hospital of Chongqing Chongqing 400062China Sino-Danish Cllege University of Chinese Academy of SciencesBeijing101408China National Laboratory of Biomacromolecules CAS Center for Excelence in BiomacromoleculesInstitute of BiophysicsChinese Academy of SciencesBeijing100101China State Key Laboratory of Proteomics Beijing Proteome Research CenterNational Center for Protein Sciences(Beijing)Beijing Institute of LifeomicsBeijing 102206China

Cullin-RING E3 ubiquitin ligases(CRLs),the largest family of multi-subunit E3 ubiquitin ligases in eukaryotic cells,represent core cellular machinery for executing protein degradation and maintaining ***,we asked what roles Cullin proteins play in human mesenchymal stem cell(hMSC)homeostasis and *** this end,we conducted a comparative aging phenotype analysis by individually knocking down Cullin members in three senescence models:replicative senescent hMSCs,Hutchinson-Gilford Progeria Syndrome hMSCs,and Werner syndrome *** all family members,we found that CUL2 deficiency rendered hMSCs the most susceptible to *** investigate CUL2-specific underlying mechanisms,we then applied CRISPR/Cas9-mediated gene editing technology to generate CUL2-deficient human embryonic stem cells(hESCs).When we differentiated these into h MSCs,we found that CUL2 deletion markedly accelerates hMSC ***,we identified that CUL2 targets and promotes ubiquitin proteasome-mediated degradation of TSPYL2(a known negative regulator of proliferation)through the substrate receptor protein APPBP2,which in turn downregulates one of the canonical aging marker-P21^(waf1/cip1),and thereby delays *** work provides important insights into how CRL2^(APPBP2)-mediated TSPYL2 degradation counteracts hMSC senescence,providing a molecular basis for directing intervention strategies against aging and aging-related diseases.

关键词： cullins stem cell senescence aging proteostasis ubiquitination APPBP2 TSPYL2

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Neddylation-dependent protein degradation is a nexus between synaptic insulin resistance,neuroinflammation and Alzheimer’s disease

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Translational Neurodegeneration 2022年第1期11卷 931-948页

作者： Alessandro Dario Confettura Eleonora Cuboni Mohamed Rafeet Ammar Shaobo Jia Guilherme M.Gomes PingAn Yuanxiang Rajeev Raman Tingting Li Katarzyna M.Grochowska Robert Ahrends Anna Karpova Alexander Dityatev Michael R.Kreutz RG Neuroplasticity Leibniz-Institute for Neurobiology39118 Magde-burgGermany German Center for Neurodegenerative Diseases(DZNE) 39120 MagdeburgGermany Center for Behavioral Brain Sciences Otto Von Guericke University39120 MagdeburgGermany Leibniz-Institut Für Analytische Wissenschaften-ISAS-e.V. 44227 DortmundGermany Leibniz Group‘Dendritic Organelles and Synaptic Function’ Center for Molecular NeurobiologyZMNHUniversity Medical Center Hamburg-Eppendorf20251 Ham-burgGermany Department of Analytical Chemistry Faculty of ChemistryUniversity of Vienna1090 WienAustria Medical Faculty Otto-von-Guericke University39120 MagdeburgGermany.

Background:The metabolic syndrome is a consequence of modern lifestyle that causes synaptic insulin resistance and cognitive deficits and that in interaction with a high amyloid load is an important risk factor for Alzheimer’s *** has been proposed that neuroinflammation might be an intervening variable,but the underlying mechanisms are currently ***:We utilized primary neurons to induce synaptic insulin resistance as well as a mouse model of high-risk aging that includes a high amyloid load,neuroinflammation,and diet-induced obesity to test hypotheses on underly-ing ***:We found that neddylation and subsequent activation of cullin-RING ligase complexes induced synaptic insulin resistance through ubiquitylation and degradation of the insulin-receptor substrate IRS1 that organizes synap-tic insulin ***,inhibition of neddylation preserved synaptic insulin signaling and rescued memory deficits in mice with a high amyloid load,which were fed with a’western diet’.Conclusions:Collectively,the data suggest that neddylation and degradation of the insulin-receptor substrate is a nodal point that links high amyloid load,neuroinflammation,and synaptic insulin resistance to cognitive decline and impaired synaptic plasticity in high-risk aging.

关键词： Metabolic syndrome Alzheimer’s disease Neddylation cullins MLN-4924 Insulin IRS1 Amyloid-β TNFα

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类泛素蛋白NEDD8赖氨酸功能研究和修饰底物鉴定

类泛素蛋白NEDD8赖氨酸功能研究和修饰底物鉴定

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作者：隋沂言苏州大学

学位级别：硕士

背景和目的:泛素样蛋白NEDD8(Neural-precursor-cell-Expressed Developmentally Down-regulated 8)是最重要的类泛素化蛋白之一,其与底物蛋白的结合过程称为neddylation。Neddylation修饰与肿瘤细胞的增殖、转录因子的调控等高度相关。泛素的7个赖氨酸可以形成不同的泛素链结构,在各种生化过程中发挥着重要的作用。然而NEDD8上不同赖氨酸的功能至今尚未被研究。本课题中,我们将NEDD8中的各个赖氨酸分别突变成精氨酸,从而获得9个NEDD8突变体,研究了NEDD8上赖氨酸的潜在功能。作为E3泛素连接酶的一个组成蛋白,cullins是最早被发现可被NEDD8修饰的底物蛋白,cullins的neddylation修饰可以激活cullin-RING E3(CRL E3)泛素连接酶的活性。相比泛素修饰的底物蛋白研究,NEDD8修饰底物蛋白的研究少之又少。本课题中,我们通过亲和层析方法来分离富集NEDD8修饰的底物蛋白,并用质谱进行鉴定。方法:构建将各个赖氨酸分别突变成精氨酸的9个NEDD8突变体,在HEK293T细胞中过表达NEDD8野生型和突变体,免疫印迹在全蛋白液中检测各个突变体对neddylation修饰的影响,找到关键赖氨酸;共转染NEDD8野生型、突变体与NEDD8激活酶NAE1/Uba3、结合酶UBE2M,免疫印迹检测关键赖氨酸的突变对NEDD8的激活、结合过程的影响;共转染NEDD8野生型、突变体与cullins家族蛋白,纯化cullins后免疫印迹检测关键赖氨酸的突变对底物蛋白neddylation修饰及泛素修饰的影响;共转染NEDD8野生型、突变体与cullins家族成员与CRL E3泛素连接酶底物,纯化CRL E3泛素连接酶底物后免疫印迹检测关键赖氨酸的突变对底物蛋白泛素化的影响;大量过表达带His6亲和标签的NEDD8,通过标签亲和纯化NEDD8修饰蛋白,胰蛋白酶酶解后获得多肽片段,用质谱分析获得其底物蛋白。结果:(1)K27R NEDD8突变体抑制了蛋白质neddylation修饰。(2)K27R NEDD8未影响NEDD8的激活过程。(3)K27R NEDD8突变体通过与NEDD8结合酶UBE2M形成稳定的共价键,影响了NEDD8的结合过程。(4)K27R NEDD8突变体影响了cullins的neddylation修饰。(5)K27R NEDD8突变体也影响了cullins的泛素化修饰。(6)K27R NEDD8突变体大大降低了cullin RING E3泛素连接酶底物I?B?的泛素化修饰。(7)质谱分析共鉴定了84种NEDD8修饰蛋白,其中67种是新鉴定到的潜在NEDD8修饰底物蛋白,如染色体浓缩调节蛋白RCC等蛋白。结论:我们在NEDD8中发现了一个影响底物蛋白neddylation修饰的赖氨酸。NEDD8第27位赖氨酸的突变可以抑制neddylation修饰。其作用机制为K27R NEDD8突变体与结合酶UBE2M形成了某种稳定的共价键,从而无法与UBE2M形成硫酯键,进而阻断neddylation的下游进程。K27R NEDD8突变体可以抑制cullins的neddylation修饰与泛素化修饰,从而影响了cullins相关的E3泛素连接酶的活性及其底物蛋白质的泛素化。K27R NEDD8突变体在研究蛋白质neddylation修饰的生化功能中可以作为重要的显性负性突变体;NEDD8潜在底物的鉴定预示NEDD8或许参与细胞周期等某些重要生理反应的调控。

关键词： NEDD8 neddylation cullins cullin-RING E3泛素连接酶泛素化

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