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Colorimetric and electrochemical detection of ligase through ligation reaction-induced streptavidin assembly

Chinese Chemical Letters 2022年第6期33卷 3151-3155页

作者： Yaliang Huang Ting Sun Wendi Li Lin Liu Gang Liu Xinyao Yi Jianxiu Wang Hunan Provincial Key Laboratory of Micro&Nano Materials Interface Science College of Chemistry and Chemical EngineeringCentral South UniversityChangsha 410083China Henan Province of Key Laboratory of New Optoelectronic Functional Materials Anyang Normal UniversityAnyang 455000China Sanquan College of Xinxiang Medical University Xinxiang 453003China

We propose a concept for ligase detection by conversion of aggregation-based homogeneous analysis into surface-tethered electrochemical assay through streptavidin(SA)-biotin *** A(Srt A)served as the model analyte and two biotinylated peptides(bio-LPETGG and GGGK-bio)were used as the *** A-catalyzed ligation of the peptide substrates led to the generation of *** ligation product(bio-LPETGGGK-bio)induced the aggregation and color change of SA-modified gold nanoparticles(Au NPs)through the SA-biotin interactions,which could be assayed by the colorimetric ***,we found that the bio-LPETGGGK-bio could trigger the assembly of tetrameric SA proteins with the formation of the(SA-bio-LPETGGGK-bio)nassemblies through the same *** above results were further confirmed by atomic force microscopy and fluorescent *** insulated assemblies were in-situ fabricated at the SA-modified gold electrode,thus hindering the electron transfer of[Fe(CN)_(6)]^(3-/4-) and leading to an increase in the electron-transfer *** capability of the method for the detection of Srt A both in vitro and Staphylococcus aureus(***)has been *** A with a concentration down to 1 pmol/L has been determined by the electrochemical analysis,which is lower than that achieved by the colorimetric assay(50 pmol/L).By integrating the advantages of homogeneous reaction and heterogeneous detection,the strategy serves as an ideal means for the fabrication of various sensing platforms by adopting biotin-labeled and sequence-specific peptide or nucleic acid substrates.

关键词： ligase Electrochemical analysis Streptavidin Gold nanoparticles Colorimetric assay Electrochemical impedance spectroscopy

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A useful toolbox for the detection of SCF E3 ligase activity

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Molecular Plant 2022年第11期15卷 1654-1655页

作者： Federica Casagrande Qi Xie Giovanna Serino Dipartimento di Biologia e Biotecnologie“C.Darwin ”Sapienza Universitàdi RomaPiazzale Aldo Moro 500185 RomeItaly State Key Laboratory of Plant Genomics Institute of Genetics and Developmental BiologyThe Innovative Academy of Seed DesignChinese Academy of SciencesBeijing 100101China

Since its discovery about 50 years ago,the ubiquitin-proteasome system(UPS)has not stopped to surprise us for its level of sophistication and complexity in achieving substrate-specific protein degradation(Vierstra,2009).At the heart of this system lies the concerted activity of three major classes of enzymes:ubiquitin activators(E1),ubiquitin conjugators(E2),and ubiquitin ligases(or E3).E3s,together with selected E2s,are responsible for promoting the formation of a ubiquitin chain on a specific protein substrate.

关键词： stopped ligase ubiquitin

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The bacterial RNA ligase RtcB accelerates the repair process of fragmented rRNA upon releasing the antibiotic stress

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Science China(Life Sciences) 2020年第2期63卷 251-258页

作者： Muhammad Ramzan Manwar Changjun Shao Xing Shi Jian Wang Qiang Lin Yigang Tong Yu Kang Jun Yu CAS Key Laboratory of Genome Sciences and Information Beijing Institute of GenomicsChinese Academy of SciencesBeijing 100101China University of Chinese Academy of Sciences Beijing 100190China Beijing Institute of Microbiology and Epidemiology Beijing 100071China

RtcB, a highly conserved RNA ligase, is found in all three domains of life, and demonstrated to be an essential tRNA splicing component in archaea and metazoans. However, the biological functions of RtcB in bacteria, where there is no splicing, remains to be clarified. We first performed bioinformatics analysis which revealed highly conserved structures and presumably conserved functions of RtcB in bacteria. However, its orthologs only occur in ~0.5% of bacterial species across diverse phyla with significant signals of frequent horizontal transfer, highlighting its non-essential role in bacteria. Next, by constructing an rtcBknockout strain, we find that the removal of antibiotic stress induces a significant impact on rtcB expression in wild-type strain,and furthermore the depletion of RtcB(?RtcB strain) delays the recovery process. Our transcriptomic analysis, comprising the3′-end labeling of RNAs, highlights a significant increase in unmapped reads and cleaved rRNAs in the ?RtcB strain, particularly during recovery. Our observations suggest that the conserved RNA ligase RtcB, repairs damaged r RNAs following stress,which potentially saves energy and accelerates recovery of its host. We propose that acquisition of RtcB by diverse bacterial taxa provides a competitive advantage under stressful conditions.

关键词： RtcB bacteria RNA ligase stress response ribosomal RNA RNA repair

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Molecular cloning and functional characterization of apple U-box E3 ubiquitin ligase gene MdPUB29 reveals its involvement in salt tolerance

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Journal of Integrative Agriculture 2019年第7期18卷 1604-1612页

作者： HAN Peng-liang DONG Yuan-hua JIANG Han HU Da-gang HAO Yu-jin State Key Laboratory of Crop Biology National Research Center for Apple Engineering and TechnologyShandong Agricultural University

An E3 ubiquitin ligase gene(Genbank accession no.:MD01 G1010900) was cloned from the Royal Gala apple genome(Malus×domestica Borkh.).Sequence analysis showed that the length of the MdPUB29 gene was 1 275 bp,encoding 424 amino *** tree analysis indicated that the apple E3 ubiquitin ligase exhibited the greatest sequence similarity to Pyrus×*** predicted protein structural domain of MdPUB29 showed that it contained a U-box ***-PCR analysis showed that Md PUB29 was expressed widely in different tissues of the Royal Gala apple species,and was highly expressed in the root,while the expression of MdPUB29 was significantly inhibited by exogenous *** assays revealed that MdPUB29 protein abundance in tissue cultures of the Royal Gala apple accumulated under NaC l stress ***-dimensional protein structure prediction indicated that MdPUB29 was highly homologous with *** growing potential of MdPUB29-expressing apple calli and Arabidopsis were much stronger than that of the control under salt stress conditions,suggesting that MdPUB29 may positively regulate salt tolerance.

关键词： apple E3 ubiquitin ligase MdPUB29 salt stress

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The ubiquitin ligase Siah2 is revealed as an accomplice of the androgen receptor in castration resistant prostate cancer

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Asian Journal of Andrology 2013年第4期15卷 447-448页

作者： Michael R Freeman Research Division of Cancer Biology and Therapeutics Departments of Surgery Medicine and BiomedicalSciences Cancer Biology Program Samuel OschinComprehensive Cancer Institute Los Angeles CA90048 USA

The androgen receptor （AR） remains the primary molecular target forprostate cancer （PCa） treatment and for development of novel therapies. Profiling and other analyses of human prostate adenocarcinoma have shown that the AR is still functional during latestage disease in the absence of circulating hormone fol lowing castration therapy. The molecular mechanisms that operate during this ＇cas tration resistant＇ phase are still not well understood. Qi et al. have now implicated the ubiquitin ligase Siah2 as an important mediator of AR action in castration resis tant prostate cancer （CRPC）. Siah2 was found to target repressed AR chromatin complexes for degradation, resulting in activation of ARregulated genes involved in tumor cell proliferation, cell motility and lipid metabolism. The authors show a requirement for Siah2 activity for PCa cell growth under conditions of low androgen, and also that targeting Siah2 results in tumor growth suppression under castrate conditions. These findings identify a new mechanism of AR regulation in progressing disease as well as a novel enzymatic target for therapeutic intervention. The AR is a wellstudied hormone receptor that belongs to the large nuclear receptor gene superfamily. AR is activated by androgen binding （principally 5：~dihydrotestosterone, 5DHT）, which elicits changes in AR resident cytosolic complexes, translocation of the receptor to cell nuclei, formation of multiprotein transcriptional complexes onchromatin, and activation or repression of gene expression. Experiments with in vivo model systems have provided evidence that virtually all physiologic processes affected by androgens require the AR as a mediator of molecular effects at the gene level.1 AR is widely expressed beyond the reproductive systems and is believed to play important roles in several nonreproductive tissues, including muscle and brain. The fact that PCa growth is initially dependent on the pre sence of androgens in the circulation, and that prostat

关键词： ligase revealed accomplice

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Study the effect of kidney stones on serum xanthine oxidase, ecto-5'-nucleotidase activity and E3 SUMO-protein ligase NSE2(NSMCE2) in Malaysian individuals

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Asian Pacific Journal of Tropical Biomedicine 2015年第8期5卷 665-668页

作者： Faridah Yusof Atheer Awad Mehde Wesen Adel Mehdi Hamid Ghazali Azlina Abd Rahman Department of Biotechnology Engineering College of Engineering International Islamic University Malaysia Department of Medical analysis Health and Medical Technical College Department of Chemistry College of Sciences for Women University of Baghdad Department of Urology Hospital Tengku Ampuan Afzan Department of Pathology Hospital Tengku Ampuan Afzan

Objective: To verify possible relations between 5'-nucleotidase, xanthine oxidase to E3 small ubiquitin-like modifier-protein ligase non structural maintenance of chromosomes elements 2 in sera patients with kidney stones and to evaluate the possibility of a new biomarker for the evaluation of kidney damage. Methods: A sixty patients with known kidney stones who appeared the government health clinics in Kuantan–Pahang and fifty apparently healthy were taken as control group. The 5'-nucleotidase,xanthine oxidase and other biochemical parameters were measured by colorimetric tests. The serum NSMCE2 were measured by enzyme linked immunosorbent ***: The mean serum xanthine oxidase [(39.98±19.70) IU/L] and ecto-5'-nucleotidase activity(40.03±9.53 IU/L) were significantly higher than the controls' levels of(18.04 ±6.26) and(16.06 ±4.61) IU/L respectively. There were 85.00% and 83.33%, of patients with kidney stones who had abnormal ecto-5'-nucleotidase activity and uric acid respectively while xanthine oxidase activity was less sensitive 58.33%.Conclusions: The present study suggests that the increase in serum of xanthine oxidase,ecto-5'-nucleotidase activities E3 small ubiquitin-like modifier-protein ligase NSE2 concentration can be used as biomarkers for diagnosis of kidney damage in patients with kidney stone,also in developments of change DNA damage and inflammation disorders in these patients.

关键词： Kidney stones Xanthine oxidase Ecto-5’-nucleotidase E3 SUMO-protein ligase NSE2

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Ovarian cancer and DNA repair: DNA ligase IV as a potential key

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World Journal of Clinical Oncology 2013年第1期4卷 14-24页

作者： Joana Assis Deolinda Pereira Rui Medeiros Molecular Oncology GRP-CI Portuguese Institute of Oncology4200-072 PortoPortugal Research Department of Portuguese League against Cancer(NRNorte) 4200-072 PortoPortugal Oncology Department Portuguese Institute of Oncology4200-072 PortoPortugal ICBAS Abel Salazar Institute for the Biomedical SciencesUniversity of Porto4200-072 PortoPortugal CEBIMED Faculty of Health Sciences of Fernando Pessoa University4200-150 PortoPortugal

Ovarian cancer(OC)is the sixth most common cancer and the seventh cause of death from cancer in *** etiology and the ovarian carcinogenesis still need clarification although ovulation may be determinant due to its carcinogenic role in ovarian surface *** link between ovarian carcinogenesis and DNA repair is well established and it became clear that alterations in DNA damage response may affect the risk to develop *** are variations in the DNA sequence that exist in normal individuals of a population and are capable to change,among other mechanisms,the balance between DNA damage and cellular ***,genetic variability of the host has a great role in the development,progression and consequent prognosis of the oncologic patient as well as in treatment *** treatment for OC patients is based on cytoreductive surgery,followed by chemotherapy with a platinum agent and a *** 80%of the patients respond to the first-line therapy,the development of resistance is common although the mechanisms underlying therapy failure remain mostly *** of their role in oncology,enzymes involved in the DNA repair pathways,like DNA ligase IV(LIG4),became attractive study *** has been reported that variations in LIG4 activity can lead to a hyper-sensitivity to DNA damage,deregulation of repair and apoptosis mechanisms,affecting the susceptibility to cancer development and therapy *** overcome resistance mechanisms,several investigations have been made and the strategy to target crucial molecular pathways,such as DNA repair,became one of the important areas in clinical *** review aims to elucidate the link between DNA repair and OC,namely which concerns the role of LIG4 enzyme,and how genetic polymorphisms in LIG4 gene can modulate the activity of the enzyme and affect the ovarian carcinogenesis and treatment ***,we try to understand how LIG4 inhibition can be a potential cont

关键词： Ovarian Cancer DNA Repair DNA ligase IV Polymorphisms Susceptibility Treatment response

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Interaction between Mnk2 and CBC^(VHL) ubiquitin ligase E3 complex

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Science China(Life Sciences) 2006年第3期49卷 265-273页

作者： WANG Pingzhang, WANG Xin, WANG Feng, CAI Tianjing & LUO Ying Chinese National Human Genome Center, Beijing 100176, China Institute of Basic Medical Sciences, Chinese Acadamy of Medical Sciences, Beijing 100005, China Shanghai Genomics Inc., Shanghai 201203, China Chinese National Human Genome Center Beijing 100176 China Institute of Basic Medical Sciences Chinese Acadamy of Medical Sciences Beijing 100005 China Chinese National Human Genome Center Beijing 100176 China Shanghai Genomics Inc. Shanghai 201203 China

MAP kinase-interacting kinase-2 (Mnk2) is one of the downstream kinases activated by MAP kinases. It phosphorylates the eukaryotic initiation factor 4E (eIF4E), although the role of eIF4E phosphorylation and the role of Mnk2 in the process of protein translation are not well understood. Except for eIF4E, other physiological substrates of Mnk2 are still unidentified. To look for these unidentified substrates and to reveal the physiological function of Mnk2, we performed a yeast two-hybrid screening with Mnk2 as the bait. The results demonstrated Mnk2 could interact with VHL (von Hippel-Lindau tumor suppressor), Rbx1 (ring-box 1) and Cul2 (Cullin2) proteins in yeast cells. Furthermore, we validated the interaction between Mnk2 and VHL proteins in mammalian cells by co-immunoprecipitation analysis. Because the three proteins VHL, Rbx1 and Cul2 are all components of the CBCVHL ubiquitin ligase E3 complex, it has been shown that Mnk2 can interact with CBCVHL complex, and is probably one of the new substrates of the CBCVHL complex. Furthermore, during the interaction of Mnk2 with von Hippel-Lindau (VHL) tumor suppressor- binding protein 1 (VBP1), it appears that Mnk2 also joins to modulate cell shape as VBP1 plays an important role in the process of the maturation of the cytoskeleton and in the process of morphogenesis.

关键词： Mnk2, yeast two-hybrid, CBCVHL ubiquitin ligase E3 complex, VBP1.

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Characterization of a Novel RING-Type Ubiquitin E3 ligase GhRING2 Differentially Expressed in Cotton Fiber

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American Journal of Plant Sciences 2014年第21期5卷 3364-3379页

作者： Shiva Soma Chuan-Yu Hsu Sukumar Saha Johnie N. Jenkins Din-Pow Ma Department of Biochemistry Molecular Biology Entomology and Plant Pa-thology Mississippi State University Mississippi State USA Institute for Genomics Biocomputing & Biotechnology Mississippi State Uni-versity Mississippi State USA Crop Science Research Laboratory Agricultural Research Service United States Department of Agriculture Mississippi State USA Department of Plant and Soil Sciences Mississippi State University Mississippi State USA

The ubiquitin-proteasome proteolysis pathway is responsible for the degradation of abnormal and short-lived proteins to regulate many important biochemical activities in eukaryotes. By employing affymetrix microarray analysis, we have identified a novel ubiquitin ligase E3 gene GhRING2 that is differentially expressed between two Gossypium hirsutum lines-Texas Marker-1 (TM-1) and Chromosome Substitution Line CS-B25. The CS-B25 line has chromosome 25 from G. barbadense substituted into TM-1. The complete nucleotide sequences of GhRING2 along with its 5’-flanking region were obtained by genomic walking. GhRING2 was highly expressed in elongating fiber, and GUS expression directed by the GhRING2 promoter was found in hypocotyls and young stems of transgenic Arabidopsis plants. Using a yeast two-hybrid assay GhRING2 was found to interact with a PROTODERMAL FACTOR1 (GhPDF1) protein. GhPDF1 was expressed preferentially in immature ovules and fiber initials, and the GhPDF1 gene had been suggested to play a role in cell fate determination and fiber development. Pull down and plasmid swap assays further confirmed the interaction between GhRING2 and GhPDF1. The expression and protein interaction data indicate that GhRING2 may be involved in the turnover of GhPDF1 and participation in the transition from initiation to elongation stages during fiber development. Our data strongly suggest that the ubiquitin-proteasome pathway may regulate cotton fiber growth and development. The nucleotide sequence data of GhRING2 in this article have been submitted to the Gen Bank Nucleotide Sequence Data Bases under the accession number BankIt 1,742,008 SeqKM 108,000.

关键词： Cotton Fiber Gossypium hirsutum G. barbadense RING-Type Ubiquitin ligase Ubiquitination

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抑制ligase4或Xrcc6活性增强斑马鱼原始生殖细胞中DNA同源重组的效率(英文)

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水生生物学报 2015年第2期39卷 339-348页

作者：魏志强熊凤何牡丹王厚鹏朱作言孙永华中国科学院水生生物研究所淡水生态与生物技术国家重点实验室武汉430072 中国科学院大学北京100049 湖北医药学院附属太和医院生物医学研究所十堰442000

利用斑马鱼作为体内模型,研究旨在提高斑马鱼原始生殖细胞(Primordial germ cells,PGCs)中同源重组(Homologous recombination,HR)的效率。首先,将UAS:m RFP-nos1载体显微注射到Tg(kop:Kal TA4)转基因胚胎中标记转基因PGCs,结果表明筛选PGCs特异表达m RFP的胚胎能够相对提高转基因的生殖系传递效率。随后建立了PGCs中HR效率的评估体系,并且证明抑制DNA ligase IV(Lig4)和Xrcc6(曾用名Ku70)的活性不但在全胚胎水平,而且在PGCs水平都能够显著提高HR的效率。研究表明Tg(kop:Kal TA4)转基因品系是开展HR介导的基因打靶的一个有效平台。

关键词：斑马鱼原始生殖细胞基因打靶同源重组 ligase 4 Xrcc6

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