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Medicine in Novel Technology and Devices 2024年第2期22卷 102-114页

作者： Hayder M Abdulhamza Muthanna S.Farhan Sara.S Hassan Hany Aqeel Al-Hussainy Amjad Ibrahim Oriabi Department of Pharmacy Kut University CollegeAlkutWasit52001Iraq Department of Pharmaceutical Chemistry College of PharmacyUniversity of BaghdadBaghdadIraq Al-Manara College for Medical Sciences Department of PharmacyIraq Department of Pharmacy Hilla University CollegeBabylonIraq Dr Hany Akeel Institute Iraqi Medical Research CenterBaghdad10001Iraq

Chikungunya virus(CHIKV),transmitted by arthropods,has gained global recognition for its impact on public health.It has expanded globally,including Africa,Asia,and the Indian subcontinent,and has a helicase protein in its genome that is crucial for its replication.Thus,the study targeted the helicase protein of CHIKV with 745 antiviral compounds using an ML-based QSAR model and molecular docking.Top binders(5279172,78161839,6474310,and 5330286)were selected for MD simulation based on the control(Silvestrol).All compounds had the highest binding scores,with 78161839 showing the most consistent RMSD and the least conformational variation,indicating high stability.It also showed the lowest binding free energy(ΔG¼31.31 kcal/mol),indicating energetically favourable binding.PCA and FEL also depicted the stable complex confirmation of the protein and 78161839 complex during the 100 ns simulation.Overall,this study aimed to identify helicase function antiviral binders that could be experimentally tested for treating CHIKV.

关键词： Chikungunya virus Machine learning based QSAR Molecular dynamic solution helicase

RecQ helicase-catalyzed DNA Unwinding Detected by Fluorescence Resonance Energy Transfer

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Acta Biochimica et Biophysica Sinica 2005年第9期37卷 593-600页

作者： Xing-Dong ZHANG Shuo-Xing DOU Ping XIE Peng-Ye WANG Xu Guang XI Laboratory of Soft Matter Physics,Beijing National Laboratory for Condensed Matter Physics,Institute of Physics,Chinese Academy of Sciences,Beijing 100080,China Laboratoire de Biotechnologies et Pharmacologie Génétique Appliquée CNRS UMR 8113.Ecole Normale Supérieure de Cachan,61 Avenue du Président Wilson,94235 Cachan cedex,France Laboratory of Soft Matter Physics Beijing National Laboratory for Condensed Matter Physics Institute of Physics Chinese Academy of SciencesBeijing 100080 China Laboratoire de Biotechnologies et Pharmacologie Génétique Appliquée CNRS UMR 8113 Ecole Normale Supérieure de Cachan61 Avenue du Président Wilson 94235 Cachan cedex France

A fluorometric assay was used to study the DNA unwinding kinetics induced by Escherichiacoli RecQ helicase.This assay was based on fluorescence resonance energy transfer and carried out onstopped-flow,in which DNA unwinding was monitored by fluorescence emission enhancement of fluoresceinresulting from helicase-catalyzed DNA unwinding.By this method,we determined the DNA unwinding rateof RecQ at different enzyme concentrations.We also studied the dependences of DNA unwinding magnitudeand rate on magnesium ion concentration.We showed that this method could be used to determine thepolarity of DNA unwinding.This assay should greatly facilitate further study of the mechanism for RecQ-catalyzed DNA unwinding.

关键词： helicase DNA unwinding kinetics stopped-flow fluorescence resonance energy transfer (FRET)

helicase activity and substrate specificity of RecQ5β

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Chinese Physics B 2017年第6期26卷 499-507页

作者：尤菁徐雅楠李辉吕袭明李伟王鹏业窦硕星奚绪光 Beijing National Laboratory for Condensed Matter Physics and CAS Key Laboratory of Soft Matter Physics Institute of Physics Chinese Academy of Sciences (CAS) Beijing 100190 China School of Physical Sciences University of Chinese Academy of Sciences Beijing 100049 China College of Life Science Inner Mongolia University for Nationalities Tongliao 028000 China College of Life Sciences Northwest Agriculture and Forestry University Yangling 712100 China LBPA IDA ENS Cachan CNRS Universite Paris-Saclay Cachan F-94235 France

RecQ5β is an essential DNA helicase in humans, playing important roles in DNA replication, repair, recombination and transcription. The unwinding activity and substrate specificity of RecQ5β is still elusive. Here, we used stopped-flow kinetic method to measure the unwinding and dissociation kinetics of RecQ5β with several kinds of DNA substrates, and found that RecQ5β could well unwind ss/ds DNA, forked DNA and Holiday junction, but was compromised in unwinding blunt DNA and G-quadruplex. Rec5β has the preferred unwinding specificity for certain DNA substrates containing the junction point, which may improve the binding affinity and unwinding activity of RecQ5β. Moreover, from a comparison with the truncated RecQ5β～（1-467）, we discovered that the C-terminal domain might strongly influence the unwinding activity and binding affinity of RecQ5β. These results may shed light on the physiological functions and working mechanisms of RecQ5β helicase.

关键词： helicase RecQ5β DNA substrate specificity unwinding kinetics

SARS-Coronavirus-2 Nsp13 Possesses NTPase and RNA helicase Activities That Can Be Inhibited by Bismuth Salts

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Virologica Sinica 2020年第3期35卷 321-329页

作者： Ting Shu Muhan Huang Di Wu Yujie Ren Xueyi Zhang Yang Han Jingfang Mu Ruibing Wang Yang Qiu Ding-Yu Zhang Xi Zhou Center for Translational Medicine Wuhan Jinyintan HospitalWuhan 430023China State Key Laboratory of Virology Wuhan Institute of VirologyCenter for Biosafety Mega-ScienceChinese Academy of SciencesWuhan 430071China Center for Precision Translational Medicine of Wuhan Institute of Virology and Guangzhou Women and Children’s Medical Center Guangzhou Women and Children’s Medical CenterGuangzhou 510120China State Key Laboratory of Quality Research in Chinese Medicine Institute of Chinese Medical SciencesUniversity of MacaoMacao SAR 999078China University of Chinese Academy of Sciences Beijing 100049China

The ongoing outbreak of Coronavirus Disease 2019(COVID-19)has become a global public health emergency.SARScoronavirus-2(SARS-CoV-2),the causative pathogen of COVID-19,is a positive-sense single-stranded RNA virus belonging to the family Coronaviridae.For RNA viruses,virus-encoded RNA helicases have long been recognized to play pivotal roles during viral life cycles by facilitating the correct folding and replication of viral RNAs.Here,our studies show that SARS-CoV-2-encoded nonstructural protein 13(nsp13)possesses the nucleoside triphosphate hydrolase(NTPase)and RNA helicase activities that can hydrolyze all types of NTPs and unwind RNA helices dependently of the presence of NTP,and further characterize the biochemical characteristics of these two enzymatic activities associated with SARS-CoV-2 nsp13.Moreover,we found that some bismuth salts could effectively inhibit both the NTPase and RNA helicase activities of SARS-CoV-2 nsp13 in a dose-dependent manner.Thus,our findings demonstrate the NTPase and helicase activities of SARS-CoV-2 nsp13,which may play an important role in SARS-CoV-2 replication and serve as a target for antivirals.

关键词： SARS-coronavirus-2(SARS-CoV-2) Nsp13 NTPase helicase Antiviral target

Structural basis of Zika virus helicase in recognizing its substrates

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Protein & Cell 2016年第8期7卷 562-570页

作者： Hongliang Tian Xiaoyun Ji Xiaoyun Yang Zhongxin Zhang zuoku Lu Kailin Yang Cheng Chen Qi Zhao Heng Chi Zhongyu Mu Wei Xie Zefang Wang Huiqiang Lou Haitao Yang Zihe Rao School of Life Sciences Tianjin University Tianjin 300072 China Tianjin Intemational Joint Academy of Biotechnology and Medicine Tianjin 300457 China The State Key Laboratory of Pharmaceutical Biotechnology School of Life Sciences Nanjing University Nanjing 210023 China State Key Laboratory of Agro-Biotechnology College of Biological Sciences China Agricultural University Beijing 100193 China College of Life Sciences Nankai University Tianjin 300071 China Cleveland Clinic Lemer College of Medicine of Case Western Reserve University Cleveland OH 44195 USA Department of Molecular Biophysics and Biochemistry Yale University New Haven CT 06520 USA

The recent explosive outbreak of Zika virus （ZlKV） infection has been reported in South and Central America and the Caribbean. Neonatal microcephaly associated with ZIKV infection has already caused a public health emergency of international concern. No specific vaccines or drugs are currently available to treat ZIKV infection. The ZIKV helicase, which plays a pivotal role in viral RNA replication, is an attractive tar- get for therapy. We determined the crystal structures of ZIKV helicase-ATP-Mnz＋ and ZlKV helicase-RNA. This is the first structure of any flavivirus helicase bound to ATP. Comparisons with related flavivirus helicases have shown that although the critical P-loop in the active site has variable conformations among different species, it adopts an identical mode to recognize ATPIMn2＋. The structure of ZlKV helicase-RNA has revealed that upon RNA binding, rotations of the motor domains can cause significant conformational changes. Strikingly, although ZlKV and dengue virus （DENV） apo-helicases share conserved residues for RNA binding, their different manners of motor domain rotations result in distinct individual modes for RNA recognition. It suggests that flavivirus helicases could have evolved a conserved engine to convert chemical energy from nucleoside triphosphate to mechanical energy for RNA unwinding, but different motor domain rotations result in variable RNA recognition modes to adapt to individual viral replication.

关键词： Zika virus helicase ATP crystal structure,flavivirus

The interaction between the helicase DHX33 and IPS-1 as a novel pathway to sense double-stranded RNA and RNA viruses in myeloid dendritic cells

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Cellular & Molecular Immunology 2014年第1期11卷 49-57页

作者： Ying Liu Ning Lu Bin Yuan Leiyun Weng Feng Wang Yong-Jun Liu Zhiqiang Zhang Department of Infectious Diseases The First Hospital Jilin University Changchun China Department of Immunology Center for Cancer ImmunologyResearch University of Texas MD Anderson Cancer Center Houston TX USA Baylor Institute for Immunology Research Baylor Research InstituteDallas TX USA

In eukaryotes, there are at least 60 members of the DExD/H helicase family, many of which are able to sense viral nucleic acids. By screening all known family members, we identified the helicase DHX33 as a novel double-stranded RNA （dsRNA） sensor in myeloid dendritic cells （mDCs）. The knockdown of DHX33 using small heteroduplex RNA （shRNA） blocked the ability of mDCs to produce type I interferon （IFN） in response to poly hC and reovirus. The HELICc domain of DHX33 was shown to bind poly hC. The interaction between DHX33 and IPS-1 is mediated by the HELICc region of DHX33 and the C-terminal domain of IPS-1 （also referred to MAVS and VISA）. The inhibition of DHX33 expression by RNA interference blocked the poly hC-induced activation of MAP kinases, NF-KB and IRF3. The interaction between the helicase DHX33 and IPS-1 was independent of RIG-I/MDA5 and may be a novel pathway for sensing poly I-C and RNA viruses in mDCs.

关键词： DHX33 helicase innate immunity IPS-1 myeloid dendritic cell viral nucleic acid

Fusarivirus accessory helicases present an evolutionary link for viruses infecting plants and fungi

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Virologica Sinica 2022年第3期37卷 427-436页

作者： Assane Hamidou Abdoulaye Jichun Jia Aqleem Abbas Du Hai Jiasen Cheng Yanping Fu Yang Lin Daohong Jiang Jiatao Xie State Key Laboratory of Agricultural Microbiology Huazhong Agricultural UniversityWuhan430070China Hubei Key Laboratory of Plant Pathology College of Plant Science and TechnologyHuazhong Agricultural UniversityWuhan430070China Hubei Hongshan Laboratory Wuhan430070China

A significant number of mycoviruses have been identified that are related to plant viruses,but their evolutionary relationships are largely unexplored.A fusarivirus,Rhizoctonia solani fusarivirus 4(RsFV4),was identified in phytopathogenic fungus Rhizoctonia solani(R.solani)strain XY74 co-infected by an alphaendornavirus.RsFV4 had a genome of 10,833 nt(excluding the poly-A tail),and consisted of four non-overlapping open reading frames(ORFs).ORF1 encodes an 825 aa protein containing a conserved helicase domain(Hel1).ORF3 encodes 1550 aa protein with two conserved domains,namely an RNA-dependent RNA polymerase(RdRp)and another helicase(Hel2).The ORF2 and ORF4 likely encode two hypothetical proteins(520 and 542 aa)with unknown functions.The phylogenetic analysis based on Hel2 and RdRp suggest that RsFV4 was positioned within the fusarivirus group,but formed an independent branch with three previously reported fusariviruses of R.solani.Notably,the Hel1 and its relatives were phylogenetically closer to helicases of potyviruses and hypoviruses than fusariviruses,suggesting fusarivirus Hel1 formed an evolutionary link between these three virus groups.This finding provides evidence of the occurrence of a horizontal gene transfer or recombination event between mycoviruses and plant viruses or between mycoviruses.Our findings are likely to enhance the understanding of virus evolution and diversity.

关键词： Fusarivirus Potyvirus Hypovirus helicase Horizontal gene transfer(HGT)

Computational biology approach to uncover hepatitis C virus helicase operation

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World Journal of Gastroenterology 2014年第13期20卷 3401-3409页

作者： Holger Flechsig Department of Physical Chemistry Fritz Haber Institute of the Max Planck Society14195 BerlinGermany

Hepatitis C virus(HCV)helicase is a molecular motor that splits nucleic acid duplex structures during viral replication,therefore representing a promising target for antiviral treatment.Hence,a detailed understanding of the mechanism by which it operates would facilitate the development of efficient drug-assisted therapies aiming to inhibit helicase activity.Despite extensive investigations performed in the past,a thorough understanding of the activity of this important protein was lacking since the underlying internal conformational motions could not be resolved.Here we review investigations that have been previously performed by us for HCV helicase.Using methods of structure-based computational modelling it became possible to follow entire operation cycles of this motor protein in structurally resolved simulations and uncover the mechanism by which it moves along the nucleic acid and accomplishes strand separation.We also discuss observations from that study in the light of recent experimental studies that confirm our findings.

关键词： Hepatitis C virus Viral replication helicase prote

Rig-I蛋白C端helicase结构域的原核表达、纯化及其多克隆抗体制备

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中国免疫学杂志 2010年第9期26卷 774-777,782页

作者：李金鞠任华王自强钱旻杜冰华东师范大学生命科学学院上海200062

目的:为Rig-I蛋白结构与模式识别功能的深入研究提供灵敏、高效的免疫学检测试剂。方法:PCR法克隆小鼠Rig-I基因helicase区编码序列(726～2240bp,编码241～746位氨基酸),构建带组氨酸标签的原核表达载体pET15b-mRig-I-H,阳性克隆经酶切... 详细信息

目的:为Rig-I蛋白结构与模式识别功能的深入研究提供灵敏、高效的免疫学检测试剂。方法:PCR法克隆小鼠Rig-I基因helicase区编码序列(726～2240bp,编码241～746位氨基酸),构建带组氨酸标签的原核表达载体pET15b-mRig-I-H,阳性克隆经酶切、测序鉴定后转化E.coliBL21进行诱导表达。目的蛋白电泳分离并割胶纯化后免疫家兔制备抗血清。抗血清用ELISA、Western blot、细胞免疫荧光方法进行鉴定。结果:目的蛋白在大肠杆菌中获得了高效表达,制备的抗Rig-I抗体效价达到1∶100000,Western blot、细胞免疫荧光结果显示该抗体能够有效地对细胞内Rig-I蛋白的表达水平进行检测。结论:成功地对mRig-I-H进行了原核表达、纯化,抗mRig-I-H的多克隆抗体具有较高的特异性,为进一步研究Rig-I尤其是helicase区的结构与功能奠定了坚实的基础。

关键词： Rig-I helicase 原核表达多克隆抗体

Characterization of ATPase Activity of Recombinant Human Pif1

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Acta Biochimica et Biophysica Sinica 2006年第5期38卷 335-341页

作者： Yu HUANG Deng-Hong ZHANG Jin-Qiu ZHOU Max-Planck Junior Research Group at the State Key Laboratory of Molecular Biology,Institute of Biochemistry and Cell Biology,Shanghai Institutes for Biological Sciences,Chinese Academy of Sciences,Shanghai 200031,China Max-Planck Junior Research Group at the State Key Laboratory of Molecular Biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of SciencesShanghai 200031 China

Saccharomyces cerevisiae Piflp helicase is the founding member of the Pifl subfamily that isconserved from yeast to human.The potential human homolog of the yeast PIFI gene has been cloned fromthe cDNA library of the Hek293 cell line.Here,we described a purification procedure of glutathione S-transferase（GST）-fused N terminal truncated human Pifl protein（hPif1ΔN）from yeast and characterizedthe enzymatic kinetics of its ATP hydrolysis activity.The ATPase activity of human Pif1 is dependent ondivalent cation,such as Mg2+,Ca2+and single-stranded DNA.Km for ATP for the ATPase activity isapproximately 200 μM.As the ATPase activity is essential for hPifl’s helicase activity,these results willfacilitate the further investigation on hPif1.

关键词： Pifl helicase telomere telomerase ATPase