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Large-scale Identification and Time-course Quantification of ubiquitylation Events During Maize Seedling De-etiolation

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Genomics, Proteomics & Bioinformatics 2019年第6期17卷 603-622页

作者： Yue-Feng Wang Qing Chao Zhe Li Tian-Cong Lu Hai-Yan Zheng Cai-Feng Zhao Zhuo Shen Xiao-Hui Li Bai-Chen Wang Photosynthesis Research Center CAS Key Laboratory of PhotobiologyInstitute of BotanyChinese Academy of SciencesBeijing 100093China Crop Germplasm Resources Institute Jilin Academy of Agricultural SciencesGongzhuling 136100China Precision Scientific(Beijing)Co. Beijing 100085China Advanced Biotechnology and Application Research Center School of Chemistry and Biological EngineeringUniversity of Science and Technology BeijingBeijing 100083China Center for Advanced Biotechnology and Medicine Biological Mass Spectrometry FacilityRutgers UniversityPiscatawayNJ 08855USA College of Life Sciences Graduate University of Chinese Academy of SciencesBeijing 100049China

The ubiquitin system is crucial for the development and fitness of higher ***-etiolation, during which green plants initiate photomorphogenesis and establish autotrophy, is a dramatic and complicated process that is tightly regulated by a massive number of ubiquitylation/de-ubiquitylation events. Here we present site-specific quantitative proteomic data for the ubiquitylomes of de-etiolating seedling leaves of Zea mays L.(exposed to light for 1, 6, or 12 h)achieved through immunoprecipitation-based high-resolution mass spectrometry(MS). Through the integrated analysis of multiple ubiquitylomes, we identified and quantified 1926 unique ubiquitylation sites corresponding to 1053 proteins. We analyzed these sites and found five potential ubiquitylation motifs, KA, AXK, KXG, AK, and TK. Time-course studies revealed that the ubiquitylation levels of 214 sites corresponding to 173 proteins were highly correlated across two replicate MS experiments, and significant alterations in the ubiquitylation levels of 78 sites(fold change >1.5) were detected after de-etiolation for 12 h. The majority of the ubiquitylated sites we identified corresponded to substrates involved in protein and DNA metabolism, such as ribosomes and ***, multiple ubiquitylation sites were detected in proteins whose functions reflect the major physiological changes that occur during plant de-etiolation, such as hormone synthesis/signaling proteins, key C4 photosynthetic enzymes, and light signaling proteins. This study on the ubiquitylome of the maize seedling leaf is the first attempt ever to study the ubiquitylome of a C4 plant and provides the proteomic basis for elucidating the role of ubiquitylation during plant de-etiolation.

关键词： Maize seedling leaf De-etiolation ubiquitylation C4 photosynthetic enzymes Ribosome

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基于机器学习的Pupylation和ubiquitylation蛋白及其修饰位点的预测研究

基于机器学习的Pupylation和Ubiquitylation蛋白及其修饰位点的预...

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作者：关孟月景德镇陶瓷大学

学位级别：硕士

蛋白质是各种细胞功能最重要的执行者,其功能正常与否决定着生命活动能否有序、高效的进行。其中翻译后修饰起着至关重要的作用,它可以使蛋白质具有更为复杂的结构和更为完善的功能,实现更为精细的调节。目前,已经检测出400多种蛋白质... 详细信息

蛋白质是各种细胞功能最重要的执行者,其功能正常与否决定着生命活动能否有序、高效的进行。其中翻译后修饰起着至关重要的作用,它可以使蛋白质具有更为复杂的结构和更为完善的功能,实现更为精细的调节。目前,已经检测出400多种蛋白质翻译后修饰(PTMs),其中Pupylation和ubiquitylation蛋白是重要的PTMs,在微生物的细胞功能中起着关键作用。准确预测Pupylation和植物ubiquitylation蛋白及其修饰位点对于研究基础生物学过程和相关药物的开发具有重要意义,而且到目前为止还没有提出对Pupylation和植物ubiquitylation蛋白的预测研究。本文提出了四种模型来分别预测Pupylation蛋白、Pupylation位点、植物ubiquitylation蛋白、植物ubiquitylation位点,具体的研究工作在下面进行详细展述。在Pupylation蛋白和植物ubiquitylation蛋白及其修饰位点的预测问题上,对于一条给定的蛋白质序列,如果能首先预测它是否是Pupylation蛋白或者ubiquitylation蛋白,然后再在被预测为Pupylation蛋白或者ubiquitylation蛋白序列的基础上进一步预测其具体位置的修饰位点,这将大大节省实验成本、提高工作效率。在预测Pupylation蛋白及其修饰位点的问题上,本文首先构建了识别Pupylation蛋白的模型,为改进Pupylation蛋白的预测模型,本文采用了基于功能域GO注释的KNN评分矩阵模型和词嵌入(Word Embedding)模型来对蛋白质序列进行特征提取。因为该蛋白质的数据集是不平衡的,本文应用了随机欠采样(RUS)和合成少数过采样技术(SMOT)来平衡特征。最后,将平衡的数据集输入到极限梯度提升(XGBoost)分类器中,经过10折交叉验证,各项评价指标性能表现良好,证明该模型具有较好的泛化能力。针对Pupylation位点的预测模型,研究人员已经开发了几种大规模的蛋白质组学方法来预测Pupylation位点,但仍有许多的位点有待发现。基于序列的预测方法将有助于预测Pupylation位点,本文采用了TPC、AAI、One-Hot、Pse AAC、CKSAAP、Word Embedding等六个特征编码方案对蛋白质序列片段进行特征提取,并采用卡方检验进行特征选择。经过10折交叉验证显示了准确率明显高于其他现有方法。最后,为了方便相关研究人员对其进行实验验证,本文建立一个名为“PUP-PS-Fuse”的预测平台,可以在“https://***/PUP-PS-Fuse”进行访问。在预测植物ubiquitylation蛋白及其修饰位点的问题上,在与ubiquitylation位点相关的文献中,他们的研究是跨物种的,生命模式尚不清楚,加上预测方法是针对物种特异性的。所以,在这项研究中,本文只选择植物中的ubiquitylation位点并对其研究。在这项工作中,本文首先构建了识别植物ubiquitylation蛋白的模型。为了更好地反映蛋白质序列信息,本文使用了基于功能域GO注释的KNN评分矩阵模型以及词嵌入模型(CBOW和Skip-Gram)。最后,提取的特征向量输入到Light Gradient Boosting Machine(LGBM)分类器中。在独立数据集上进行10折交叉验证,该模型取得了良好的效果。然后,本文构建了预测植物ubiquitylation位点的模型。Skip-Gram、CBOW以及EAAC这三种特征提取编码被用来提取蛋白质序列片段特征。经过10折交叉验证,在独立数据集上与现有预测模型进行对比,本文的模型在预测植物ubiquitylation蛋白和位点方面表现出良好的鲁棒性,性能优于之前的预测工具。本文中使用的数据集和源代码可以在“https://***/gmywqk/Ub-PS-Fuse”上免费获取。本文基于机器学习首次提出了预测Pupylation和植物ubiquitylation蛋白,并基于Pupylation和植物ubiquitylation蛋白进一步预测了Pupylation和植物ubiquitylation位点。通过实验对比分析,证实了基于机器学习的计算方法来预测Pupylation和植物ubiquitylation蛋白及其修饰位点,可以达到较好的预测效果。

关键词： Pupylation ubiquitylation 蛋白质翻译后修饰词嵌入采样技术

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Celastrol targeting Nedd4 reduces Nrf2-mediated oxidative stress in astrocytes after ischemic stroke

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Journal of Pharmaceutical Analysis 2023年第2期13卷 156-169页

作者： Zexuan Hong Jun Cao Dandan Liu Maozhu Liu Mengyuan Chen Fanning Zeng Zaisheng Qin Jigang Wang Tao Tao Department of Anesthesiology Nanfang HospitalSouthern Medical UniversityGuangzhou510515China Department of Anesthesiology Central People's Hospital of ZhanjiangZhanjiangGuangdong524045China Artemisinin Research Center Institute of Chinese Materia MedicaChina Academy of Chinese Medical SciencesBeijing100700China Department of Pharmacy West China HospitalSichuan UniversityChengdu610000China Department of Pharmacy Xi'an Daxing HospitalXi'an710000China

Stroke is the second leading cause of death worldwide,and oxidative stress plays a crucial *** exhibits strong antioxidant properties in several diseases;however,whether it can affect oxidation in cerebral ischemic-reperfusion injury(CIRI)remains *** study aimed to determine whether celastrol could reduce oxidative damage during CIRI and to elucidate the underlying ***,we found that celastrol attenuated oxidative injury in CIRI by upregulating nuclear factor E2-related factor 2(Nrf2).Using alkynyl-tagged celastrol and liquid chromatography-tandem mass spectrometry,we showed that celastrol directly bound to neuronally expressed developmentally downregulated 4(Nedd4)and then released Nrf2 from Nedd4 in ***4 promoted the degradation of Nrf2 through K48-linked ubiquitination and thus contributed to astrocytic reactive oxygen species production in CIRI,which was significantly blocked by ***,by inhibiting oxidative stress and astrocyte activation,celastrol effectively rescued neurons from axon damage and *** study uncovered Nedd4 as a direct target of celastrol,and that celastrol exerts an antioxidative effect on astrocytes by inhibiting the interaction between Nedd4 and Nrf2 and reducing Nrf2 degradation in CIRI.

关键词： Celastrol Cerebral ischemia Reperfusion injury Oxidative stress Nedd4 Nrf2 ubiquitylation

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Chemical tools for E3 ubiquitin ligase study

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Chinese Chemical Letters 2023年第4期34卷 67-74页

作者： Yangwode Jing Chong Zuo Yun-Xiang Du Junxiong Mao Ruichao Ding Jiachen Zhang Lu-Jun Liang Qian Qu Institute of Translational Medicine Shanghai Jiao Tong UniversityShanghai 200240China Tsinghua-Peking Center for Life Sciences Ministry of Education Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical BiologyCenter for Synthetic and Systems BiologyDepartment of ChemistryTsinghua UniversityBeijing 100084China

E3 ubiquitin ligases catalyze the final step of ubiquitylation,a crucial post-translational modification involved in almost every process in eukaryotic cells.E3 ubiquitin ligases are key regulators of cellular events,and the investigation into their functions and functioning mechanisms are research areas with great *** or semi-synthetic tools have greatly facilitated the research about the enzyme activity,distribution in different physiological events,and catalytic mechanism of E3 ubiquitin *** this review,we summarize the development of chemical tools for E3 ubiquitin ligases with an emphasis on the synthetic *** show the utility of these chemical tools by briefly discussing their applications in biological research.

关键词： Chemical tools Protein chemical synthesis ubiquitylation E3 ligase Enzymatic mechanism

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Targeting deubiquitinase OTUB1 protects vascular smooth muscle cells in atherosclerosis by modulating PDGFRβ

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Frontiers of Medicine 2024年第3期18卷 465-483页

作者： Fei Xu Han Chen Changyi Zhou Tongtong Zang Rui Wang Shutong Shen Chaofu Li Yue Yu Zhiqiang Pei Li Shen Juying Qian Junbo Ge Department of Cardiology and Laboratory of Heart Valve Disease West China HospitalSichuan UniversityChengdu610041China Department of Cardiology Zhongshan HospitalFudan UniversityResearch Unit of Cardiovascular Techniques and DevicesChinese Academy of Medical SciencesShanghai200032China Shanghai Institute of Cardiovascular Diseases Shanghai200032China National Clinical Research Center for Interventional Medicine&Shanghai Clinical Research Center for Interventional Medicine(19MC1910300) Shanghai200032China

Atherosclerosis is a chronic artery disease that causes various types of cardiovascular *** smooth muscle cells(VSMCs),the main components of atherosclerotic plaque,switch from contractile to synthetic phenotypes during *** is crucial in regulating VSMC phenotypes in atherosclerosis,and it can be reversely regulated by ***,the specific effects of deubiquitinases on atherosclerosis have not been thoroughly *** this study,RNAi screening in human aortic smooth muscle cells was performed to explore the effects of OTU family deubiquitinases,which revealed that silencing OTUB1 inhibited PDGF-BB-stimulated VSMC phenotype *** in vivo studies using Apoe−/−mice revealed that knockdown of OTUB1 in VSMCs alleviated atherosclerosis plaque burden in the advanced stage and led to a stable plaque ***,VSMC proliferation and migration upon PDGF-BB stimulation could be inhibited by silencing OTUB1 in *** RNA-sequencing data indicated that knocking down OTUB1 influenced VSMC differentiation,adhesion,and *** spectrometry of ubiquitinated protein confirmed that proteins related to cell growth and migration were differentially ***,we found that OTUB1 recognized the K707 residue ubiquitylation of PDGFRβwith its catalytic triad,thereby reducing the K48-linked ubiquitylation of PDGFRβ.Inhibiting OTUB1 in VSMCs could promote PDGFRβdegradation via the ubiquitin–proteasome pathway,so it was beneficial in preventing VSMCs’phenotype *** findings revealed that knocking down OTUB1 ameliorated VSMCs’phenotype switch and atherosclerosis progression,indicating that OTUB1 could be a valuable translational therapeutic target in the future.

关键词： atherosclerosis vascular smooth muscle cell ubiquitylation deubiquitinase OTUB1 PDGFRβ

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First small-molecule PROTACs for G protein-coupled receptors:inducing α1A-adrenergic receptor degradation

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Acta Pharmaceutica Sinica B 2020年第9期10卷 1669-1679页

作者： Zhenzhen Li Yuxing Lin Hui Song Xiaojun Qin Zhongxia Yu Zheng Zhang Gaopan Dong Xiang Li Xiaodong Shi Lupei Du Wei Zhao Minyong Li Department of Medicinal Chemistry Key Laboratory of Chemical Biology(MOE)School of PharmacyShandong UniversityJinan 250012China Department of Immunology Key Laboratory of Infection and Immunity of Shandong ProvinceSchool of Basic Medical ScienceShandong UniversityJinan 250012China Department of Chemistry University of South FloridaTampaFL 33620USA State Key Laboratory of Microbial Technology Shandong UniversityJinan 250100China

Proteolysis targeting chimeras(PROTACs)are dual-functional hybrid molecules that can selectively recruit an E3 ubiquitin ligase to a target protein to direct the protein into the ubiquitinproteasome system(UPS),thereby selectively reducing the target protein level by the ubiquitinproteasome ***,small-molecule PROTACs are gaining popularity as tools to desrade pathogenic ***,we present the first small-molecule PROTACs that can induce the alA-adrenergic receptor(α1 A-AR)degradation,which is also the first small-molecule PROTACs for G proteincoupled receptors(GPCRs)to our *** degradation inducers were developed through conjugation of knownα1-adrenergic receptors(α1-ARs)inhibitor prazosin and cereblon(CRBN)ligand pomalidomide through the different *** representative compound 9 c is proved to inhibit the proliferation of PC-3 cells and result in tumor growth regression,which highlighted the potential of our study as a new therapeutic strategy for prostate cancer.

关键词： Small-molecule PROTACs α1A-Adrenergic receptor ubiquitylation Degradation Prostate cancer

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Gentiopicroside targets PAQR3 to activate the PI3K/AKT signaling pathway and ameliorate disordered glucose and lipid metabolism

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Acta Pharmaceutica Sinica B 2022年第6期12卷 2887-2904页

作者： Haiming Xiao Xiaohong Sun Zeyuan Lin Yan Yang Meng Zhang Zhanchi Xu Peiqing Liu Zhongqiu Liu Heqing Huang Laboratory of Pharmacology&Toxicology School of Pharmaceutical SciencesSun Yat-sen UniversityGuangzhou 510006China School of Pharmaceutical Sciences Guangzhou University of Chinese MedicineGuangzhou 510006China National and Local United Engineering Lab of Druggability and New Drugs Evaluation School of Pharmaceutical SciencesSun Yat-sen UniversityGuangzhou 510006China

The obstruction of post-insulin receptor signaling is the main mechanism of insulin-resistant *** and adipoQ receptor 3(PAQR3),a key regulator of inflammation and metabolism,can negatively regulate the PI3 K/AKT signaling ***,we report that gentiopicroside(GPS),the main bioactive secoiridoid glycoside of Gentiana manshurica Kitagawa,decreased lipid synthesis and increased glucose utilization in palmitic acid(PA) treated HepG2 ***,GPS improved glycolipid metabolism in streptozotocin(STZ) treated high-fat diet(HFD)-induced diabetic *** findings revealed that GPS promoted the activation of the PI3 K/AKT axis by facilitating DNA-binding protein 2(DDB2)-mediated PAQR3 ubiquitinated ***,results of surface plasmon resonance(SPR),microscale thermophoresis(MST) and thermal shift assay(TSA) indicated that GPS directly binds to *** of molecular docking and cellular thermal shift assay(CETSA) revealed that GPS directly bound to the amino acids of the PAQR3 NH2-terminus including Leu40,Asp42,Glu69,Tyr125 and Ser129,and spatially inhibited the interaction between PAQR3 and the PI3 K catalytic subunit(P110α) to restore the PI3 K/AKT signaling *** summary,our study identified GPS,which inhibits PAQR3 expression and directly targets PAQR3 to restore insulin signaling pathway,as a potential drug candidate for the treatment of diabetes.

关键词： Gentiopicroside Insulin resistance PAQR3 PI3K/AKT DDB2 ubiquitylation Glucose metabolism disorder Lipid metabolism disorder

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A non-metabolic function of hexokinase 2 in small cell lung cancer:promotes cancer cell stemness by increasing USP11-mediated CD133 stability

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Cancer Communications 2022年第10期42卷 1008-1027页

作者： Juhong Wang Fei Shao Yannan Yang Wei Wang Xueying Yang Renda Li Hong Cheng Sijin Sun Xiaoli Feng Yibo Gao Jie He Zhimin Lu Department of Thoracic Surgery National Cancer Center/National Clinical Research Center for Cancer/Cancer HospitalChinese Academy of Medical Sciences and Peking Union Medical CollegeBeijing 100021P.R.China State Key Laboratory of Molecular Oncology National Cancer Center/National Clinical Research Center for Cancer/Cancer HospitalChinese Academy of Medical Sciences and Peking Union Medical CollegeBeijing 100021P.R.China Laboratory of Translational Medicine National Cancer Center/National Clinical Research Center for Cancer/Cancer HospitalChinese Academy of Medical Sciences and Peking Union Medical CollegeBeijing 100021P.R.China Department of Medical Oncology National Cancer Center/National Clinical Research Center for Cancer/Cancer HospitalChinese Academy of Medical Sciences and Peking Union Medical CollegeBeijing 100021P.R.China Department of Pathology National Cancer Center/National Clinical Research Center for Cancer/Cancer HospitalChinese Academy of Medical Sciences and Peking Union Medical CollegeBeijing 100021P.R.China Central Laboratory National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital&Shenzhen HospitalChinese Academy of Medical Sciences and Peking Union Medical CollegeShenzhenGuangdong 518116P.R.China Zhejiang Provincial Key Laboratory of Pancreatic Disease The First Affiliated Hospital and Institute of Translational MedicineZhejiang University School of MedicineHangzhouZhejiang 310029P.R.China Cancer Center Zhejiang UniversityHangzhouZhejiang 310029P.R.China

Background:Maintenance of cancer stem-like cell(CSC)stemness supported by aberrantly regulated cancer cell metabolism is critical for CSC self-renewal and tumor *** a key glycolytic enzyme,hexokinase 2(HK2)plays an instrumental role in aerobic glycolysis and tumor ***,whether HK2 directly contribute to CSC stemness maintenance in small cell lung cancer(SCLC)is largely *** this study,we aimed to investgate whether HK2 independent of its glycolytic activity is directly involved in stemness maintenance of CSC in ***:Immunoblotting analyses were conducted to determine the expression of HK2 in SCLC CSCs and their differentiated ***-like properties and tumorigenesis of SCLC cells with or without HK2 depletion or overexpression were examined by sphere formation assay and xenograft mouse *** and mass spectrometry analyses were performed to identify the binding proteins of *** expression levels of CD133-associated and CSC-relevant proteins were evaluated by immunoblotting,immunoprecipitation,immunofluorescence,and immunohistochemistry *** expression levels of Nanog,POU5F1,Lin28,HK2,Prominin-1 were analyzed through quantitative reverse transcription *** of CD133 was examined by in vitro or in vivo ubiquitination ***133+cells were sorted by flow cytometry using an anti-CD133 ***:We demonstrated that HK2 expression was much higher in CSCs of SCLC than in their differentiated ***2 depletion inhibited CSC stemness and promoted CSC ***,nonmitochondrial HK2 directly interacted with CD133 and enhanced CD133 expression without affecting CD133 mRNA *** interaction of HK2 and CD133 promoted the binding of the deubiquitinase ubiquitin-specific protease 11(USP11)to CD133,thereby inhibiting CD133 polyubiquitylation and ***2-mediated upregulation of CD133 expression enhanced the expression of cell rene

关键词： HK2 metabolic enzyme non-metabolic function cancer stem-like cell CD133 USP11 ubiquitylation SCLC

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Requirement of Rbx1 Cullin-RING ligase E3 and Ube2m neddylation E2 in the maintenance of the fitness of Treg cells

Requirement of Rbx1 Cullin-RING ligase E3 and Ube2m neddylat...

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第十四届全国免疫学学术大会

作者： Di Wu Haomin Li Mingwei Liu Jun Qin Yi Sun Zhejiang University

Cullin-RING Ligases(CRLs), upon activation by neddylation, play the key roles in regulating many biological processes. However, how CRL-neddylation regulates the function of Treg cells remains elusive. Here we show that mice with Treg-specific deletion of Rbx1, a RING component of CRLs required for its activity, developed an early-onset fetal inflammatory disorders and death at day ～25 after birth with disrupted homeostasis and impaired suppressive functions of Treg cells. Specifically, Rbx1 is essential for maintenance of the effector subpopulations in Treg cells, and regulates several inflammatory pathways. Similar phenotypes were seen in mice with deletion of Ube2m, a neddylation E2, in Treg cells, but with much lesser severity. Interestingly, Treg-specific deletion of Rbx2/Sag or Ube2f, the family member of Rbx1 or Ube2m, respectively, had no obvious phenotype. Thus, the Ube2m-Rbx1 axis is required for the maintenance of homeostasis and functions of Treg cells;and Rbx1 has Ube2m-independent roles in the fitness of Treg cells, suggesting a layer of complexity in neddylation activation of CRLs.

关键词： Treg cells Rbx1 Ube2m ubiquitylation neddylation

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