,1,2Screening and Identification of Proteins Interacting with Bombyx mori IAP and Their Effects on BmNPV Proliferation
CHEN Peng1,2, BAO XiYan1, KANG TaoTao1, DONG ZhanQi1, ZHU Yan1, PAN MinHui1,2, LU Cheng,1,2通讯作者:
收稿日期:2018-08-31接受日期:2018-09-17网络出版日期:2019-02-13
| 基金资助: |
Received:2018-08-31Accepted:2018-09-17Online:2019-02-13
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陈鹏, 包希艳, 康涛涛, 董战旗, 朱艳, 潘敏慧, 鲁成. 家蚕IAP互作蛋白的筛选、鉴定及其对BmNPV增殖的影响[J]. 中国农业科学, 2019, 52(3): 558-567 doi:10.3864/j.issn.0578-1752.2019.03.016
CHEN Peng, BAO XiYan, KANG TaoTao, DONG ZhanQi, ZHU Yan, PAN MinHui, LU Cheng.
0 引言
【研究意义】杆状病毒是一类专门寄生于无脊椎动物的病毒,尤其是鳞翅目、双翅目和膜翅目昆虫。作为一种细胞内寄生物,杆状病毒与宿主细胞以及机体的相互作用关系决定了病毒感染、复制和致病的分子机制。细胞凋亡作为机体细胞内的免疫直接参与病毒感染的应答,启动宿主细胞的凋亡机制。家蚕核型多角体病毒（Bombyx mori nucleopolyhedrovirus,BmNPV）作为家蚕血液型脓病的病原,属于杆状病毒的一种,也是蚕业生产上危害最严重的病原微生物之一。对BmNPV致病机制的研究可为家蚕抗BmNPV病毒新品种的培育提供新的思路和重要理论支撑,同时,以细胞凋亡相关基因为研究对象对阐明其在杆状病毒感染过程中的作用具有重要的参考价值。【前人研究进展】杆状病毒是一类具有环状、超螺旋、双链DNA（80—180 kb）的病毒,其与宿主的相互作用机制一直是研究的热点[1]。当杆状病毒侵染宿主时,宿主细胞会通过调控自身的相关基因通路来应对病毒的入侵和增殖,如细胞凋亡[2,3]、DNA损伤应答[4]、热休克应答[5,6]和miRNA通路[7,8]等。研究表明,家蚕感染BmNPV后,在不同的时间点会引起一系列信号通路的变化,如能量代谢、细胞骨架、铁离子代谢和泛素蛋白酶体途径等[9]。同时,病毒在进化过程中,除了自身蛋白可以相互作用以促进其增殖复制[10],也获得了多种逃逸机体免疫反应的机制,如抑制宿主细胞凋亡[11,12,13]、挟持宿主细胞DNA损伤应答[14,15]、关闭宿主蛋白合成[16]、阻断宿主细胞周期[17,18]等。其中,细胞凋亡作为昆虫免疫应答的重要组成部分,在病毒与宿主的“斗争”过程中起着重要作用[19]。病毒的复制严格依赖于宿主细胞,过早的细胞凋亡会影响病毒的增殖和持续感染,因此,细胞凋亡被宿主作为为一种重要防御策略,可以有效抑制病毒复制增殖,降低病毒的扩散速度[20,21]。然而,在病毒与宿主相互作用的过程中,细胞凋亡相关基因的具体功能及作用机制仍需要深入的研究。笔者实验室前期研究表明,在家蚕中存在4个凋亡抑制基因（inhibitor of apoptosis,IAP）家族成员（Bmiap、Bmiap-2、BmSurvivin-1和BmSurvivin-2）[22],其中有研究表明Bmiap具有明确的抑制细胞凋亡功能[23,24]。然而,Bmiap在BmNPV侵染过程中的作用尚需进一步研究。【本研究切入点】以昆虫重要的先天免疫应答反应——细胞凋亡作为切入点,家蚕凋亡抑制基因Bmiap作为研究对象,鉴定在BmNPV侵染家蚕细胞过程中与其相互作用的蛋白,并对获得的蛋白功能进行验证。【拟解决的关键问题】鉴定在BmNPV侵染过程中与BmIAP相互作用的蛋白,并验证相应蛋白与BmIAP的调控关系及其对BmNPV增殖的影响,为抗BmNPV家蚕品种培育提供分子靶标,并为深入阐明杆状病毒和宿主相互作用机制提供理论依据。1 材料与方法
试验于2016—2018年在家蚕基因组生物学国家重点实验室（西南大学）完成。1.1 试验材料
家蚕卵巢细胞系BmN-SWU1[25]由家蚕基因组生物学国家重点实验室保存,用含10%胎牛血清的TC-100昆虫培养基在27℃条件下恒温培养。重组BmNPV病毒（v39Kprm-EGFP）[26]由家蚕基因组生物学国家重点实验室保存。1.2 质粒构建和转染
根据目的基因序列设计相应引物,并在上下游引物分别添加相应的酶切位点、保护碱基和标签序列（表1）,Bmiap在GenBank中的序列号为NM_001043559.1,基因克隆步骤参考张倩等[27]的方法。用于构建过表达和敲除质粒的载体分别为pIZ/V5-His载体和本研究小组构建的基于Cas9的转基因敲除载体[28]。家蚕BmN-SWU1细胞均匀铺到12孔板中,每孔按单转1 μg,共转各0.8 μg质粒计算;质粒和X-tremeGENE HP DNA Transfection Reagent（Roche）转染试剂按1﹕3,加入100 μL无抗培养基,轻轻呈滴状混匀,室温孵育30 min;将上述转染混合物逐滴加入细胞中,轻轻晃动混匀,置于27℃恒温箱培养,培养6—8 h换液,按试验需要进行后续处理。Table 1
表1
表1引物信息
Table 1
| 引物名称 Primer name | 引物序列 Primer sequence |
|---|---|
| pIZ/V5-Flag-Bmiap-F | 5′-CGCGGATCCATGGACTACAAAGACGATGACGACAAGGAGTTGACGAAAGTTGCTA-3′ |
| pIZ/V5-Flag-Bmiap-R | 5′-CCGCTCGAGTCACTTGTCGTCATCGTCTTTGTAGTCCGAGAAGTAGAGCCGCACCGCA-3′ |
| pIZ/V5-HA-Bmpp5-F | 5′-CGCGGATCCATGTACCCATACGATGTTCCAGATTACGCTTCTAATCACGAAGAAATCACTG-3′ |
| pIZ/V5-HA-Bmpp5-R | 5′-CCGCTCGAGTTAAGCGTAATCTGGAACATCGTATGGGTATTGGCAGAGGAAATTGAAG-3′ |
| Knockdown-Bmiap-F | 5′-AAGTGCCCGACATGCGTCGTGAAG-3′ |
| Knockdown-Bmiap-R | 5′-AAACCTTCACGACGCATGTCGGGC-3′ |
| Knockdown-Bmpp5-F | 5′-AAGTGGTCGAGTGCTTGTAATGCA-3′ |
| Knockdown-Bmpp5-R | 5′-AAACTGCATTACAAGCACTCGACC-3′ |
| Bmiap-qRT-PCR-F | 5′-CCTTAGTGACTCCTGCTTTACGAA-3′ |
| Bmiap-qRT-PCR-R | 5′-TAGAAACTTGCAAATGGCTTGTG-3′ |
| Bmpp5-qRT-PCR-F | 5′-GGTTTCCGTGGGGAGGTG-3′ |
| Bmpp5-qRT-PCR-R | 5′-AGGCGGCTGTTTGTTTCG-3′ |
| vp39-qRT-PCR-F | 5′-CTAATGCCCGTGGGTATGG-3′ |
| vp39-qRT-PCR-R | 5′-TTGATGAGGTGGCTGTTGC-3′ |
| sw22934-qRT-PCR-F | 5′-TTCGTACTGGCTCTTCTCGT-3′ |
| sw22934-qRT-PCR-R | 5′-CAAAGTTGATAGCAATTCCCT-3′ |
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1.3 总RNA提取和cDNA合成
家蚕BmN-SWU1细胞总RNA使用TRIzol Reagent（Invitrogen）提取,使用反转录试剂盒（Promega）合成cDNA,操作按说明书进行,保存于-20℃备用。按照1.2方法转染48 h,感染BmNPV 12、24、48、72 h后收集细胞。使用总RNA提取试剂盒（OMEGA）,加入500 μL的TRK Lysis Buffer充分裂解细胞,后收集到1.5 mL无RNA酶的EP管中;加入75%乙醇250 μL轻轻摇匀,将上述样品移至RNA吸附柱,室温静置5 min后,4℃,10 000×g离心1 min;用RNA Wash Buffer I、II,各清洗两次;用 60℃的35 μL DEPC水溶解RNA;采用紫外分光光度计检测洗脱下来RNA,合格后-80℃保存。按上述方法提取总RNA,使用逆转录试剂盒（TaKaRa）反转录cDNA。在4℃配制去除基因组DNA的混合液,将上述混合液加入总RNA中,42℃,2 min;4℃配制反转录混合液,将上述混合液加入已去除基因组的RNA中,37℃,1.5 min;85℃,5 s;12℃保存。将反转合成的cDNA稀释后用BmActin3的引物做PCR扩增,合格cDNA贮存于-20℃备用。
1.4 免疫荧光检测
取待检测的细胞在室温用PBS洗2次;4%多聚甲醛室温固定15 min;用PBST在室温漂洗5次,每次6 min;0.1% Triton 100室温孵育10 min;用PBST在室温漂洗5次,每次6 min;封闭液（10%羊血清加3% BSA,用PBS配制）封闭1 h,37℃;37℃一抗（HA抗体或Flag抗体,1﹕500,Sigma）孵育1 h;用PBST在室温漂洗5次,每次6 min;在37℃用不同荧光标记的二抗（1﹕1 000,碧云天）孵育1 h;室温DAPI（碧云天）染细胞核10 min;用PBST在室温漂洗5次,每次6 min;将铺满细胞的爬片封到载玻片上,使用激光共聚焦显微镜进行观察拍照。1.5 免疫共沉淀与质谱鉴定
取转染目的质粒48 h后的细胞并倒掉培养基,PBS洗两遍;加入1 mL IP裂解液（IP裂解液﹕PMSF=100﹕1）冰浴轻摇30 min;将裂解下的细胞转移至1.5 mL离心管中,液氮反复冻融2—3次;10 000 ×g,4℃,离心30 min,取上清50 μL转移至1.5 mL离心管中,加入5×SDS-PAGE 蛋白上样缓冲液,煮10 min,作为总蛋白;重悬磁珠,取60 μL磁珠放到磁力架上吸去废液,以400 μL IP裂解液洗一遍;加400 μL IP裂解液和3 μL抗体,翻转摇床上常温孵育40—50 min;将上步中孵育了抗体的磁珠放至磁力架上,弃液;将剩余裂解液与上步中的磁珠混匀,翻转摇床上室温孵育2 h;将1.5 mL的离心管放至磁力架上,吸掉孵育液;用PBS洗3遍,每遍500 μL;加入60 μL的IP裂解液和15 μL的5×SDS-PAGE蛋白上样缓冲液,煮10 min;将离心管放到磁力架上,将样品吸出,进行SDS-PAGE分析;电泳结束后用硝酸银进行凝胶染色,对银染后呈现的差异条带进行切胶回收,通过LC-MS/MS分析,得到相应蛋白质信息,通过检索家蚕蛋白数据库（http://www.silkdb.org/silkdb/doc/download.html）和NCBI中的BmNPV蛋白数据库（http://www.ncbi.nlm.nih.gov/taxonomy/?term= BmNPV）对蛋白进行鉴定（此部分委托深圳华大完成）。1.6 Western blot和银染分析
制备12%的SDS-PAGE凝胶,上样并进行电泳;200 mA转膜50 min;把PVDF膜放入封闭液中,室温摇床孵育3 h;将PVDF膜从封闭液中取出放入一抗稀释液中,4℃孵育过夜;在1×TBST缓冲液中清洗5次（室温）,每次10 min;将PVDF膜放入二抗稀释液中,室温孵育2 h;将配好的ECL显色液均匀的滴在PVDF膜上,避光显色约5 min;在化学发光成像仪上曝光成像。1.7 荧光定量PCR（qRT-PCR）检测
qRT-PCR反应条件：95℃,4 min;95℃,15 s,60℃,31 s,40个循环;95℃,15 s;60℃,20 s;95℃,15 s。所用引物见表1,试剂为iTaqTM Universal SYBR? Green Supermix（Bio-Rad）,以家蚕真核翻译起始因子4A（探针号：sw22934）为内参,相对定量PCR的数据处理用2-ΔCT法。2 结果
2.1 BmNPV侵染过程中BmIAP相互作用蛋白筛查
为检测在BmNPV侵染家蚕细胞过程中与BmIAP相互作用的蛋白,通过免疫共沉淀技术获得了BmN- SWU1细胞中与BmIAP的免疫共沉淀复合物。将收集的免疫共沉淀蛋白复合物进行SDS-PAGE电泳,经硝酸银染色后分析发现,与对照组相比,BmIAP-Flag融合蛋白的免疫共沉淀结果中有1条明显的特异性条带,大小在40—50 kD（图1）。特异性差异条带经LC-MS/MS蛋白质谱分析,将多肽序列与SilkDB和BmNPV蛋白数据库比对,共鉴定到45个蛋白。根据蛋白分子量大小对鉴定到的蛋白进行筛选,获得7个可能与BmIAP相互作用的宿主蛋白,同时有1个BmNPV蛋白ORF76（表2）。进一步对获得的蛋白功能进行分析,发现BGIBMGA004807（编码丝氨酸/苏氨酸蛋白磷酸酶,Ser/Thr protein phosphatase 5,PP5）的同源基因具有凋亡相关的功能[29,30],因此在本研究中将其作为主要研究对象,并命名为BmPP5（Bombyx mori Ser/Thr protein phosphatase 5）。Table 2
表2
表2LC-MS/MS鉴定与BmIAP相互作用的蛋白
Table 2
| 蛋白编号 Protein ID | 蛋白分子量 Protein molecular weight (kD) | 蛋白描述 Protein description |
|---|---|---|
| BGIBMGA003212 | 43633.63 | 26S蛋白酶体非ATP酶调节亚基6 26S proteasome non-ATPase regulatory subunit 6 |
| BGIBMGA011237 | 46381.43 | 26S蛋白酶体非ATP酶调节亚基13 26S proteasome non-ATPase regulatory subunit 13 |
| BGIBMGA003587 | 48985.52 | 蛋白质二硫键异构酶类蛋白ERp57前体 Protein disulfide-isomerase like protein ERp57 precursor |
| BGIBMGA014181 | 53335.57 | 线粒体三功能酶β亚基 Trifunctional enzyme subunit beta, mitochondrial |
| BGIBMGA008046 | 55096.73 | 液泡蛋白分选蛋白33A Vacuolar protein sorting-associated protein 33A-like |
| BGIBMGA012549 | 55160.29 | H+转运ATP合成酶β亚基亚型 1 H+ transporting ATP synthase beta subunit isoform 1 |
| BGIBMGA004807 | 56617.87 | 丝氨酸/苏氨酸蛋白磷酸酶 Serine/threonine-protein phosphatase 5 |
| BmNPV ORF76 | 18385.21 | 家蚕核型多角体病毒ORF76 BmNPV ORF76 |
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图1
新窗口打开|下载原图ZIP|生成PPT图1与BmIAP相互作用蛋白质的免疫共沉淀检测
Marker：蛋白分子量预染Marker Protein molecular weight Marker;Input：BmNPV感染BmN-SWU1细胞总蛋白Input cell lysates;IgG：阴性血清IgG的免疫共沉淀结果IP with control mouse IgG;Flag：Flag抗体的免疫共沉淀结果;黑色箭头指示差异条带 IP with Flag antibody; Black arrow indicates the specific band
Fig. 1Co-immunoprecipitation detection of proteins interacting with BmIAP
2.2 Bmpp5克隆及序列特征分析
成功克隆了Bmpp5的CDS序列,其中开放阅读框（open reading frame,ORF）为1 473 bp,编码490个氨基酸,预测的蛋白分子量约为56 kD。利用SMART在线工具（http://smart.embl-heidelberg.de/）对BmPP5的结构域进行预测发现,该基因有3个Tetratricopeptide Repeat（TRP）结构域和1个PP2Ac结构域（图2-A）。分别利用家蚕、斜纹夜蛾（Spodoptera litura）、埃及伊蚊（Aedes aegypti）及果蝇（Drosophila serrata）的PP5同源蛋白进行多序列比对,结果表明该基因在昆虫中高度保守（图2-B）。图2
新窗口打开|下载原图ZIP|生成PPT图2家蚕BmPP5蛋白的结构域及多序列比对
A：SMART预测BmPP5的结构域 Prediction of BmPP5 domains by SMART,TPR：19—52 aa、53—86 aa、87—120 aa;PP2Ac：195—471 aa。B：BmPP5蛋白多序列比对 Multiple sequence alignment of BmPP5 protein。Bm：家蚕 Bombyx mori（XP_012553114.1）;Sl：斜纹夜蛾 Spodoptera litura（XP_022817467.1）;Aa：埃及伊蚊 Aedes aegypti（XP_001650298.2）;Ds：果蝇 Drosophila serrata（XP_020818529.1）
Fig. 2Domains and multiple sequence alignment of BmPP5 protein
2.3 BmIAP和BmPP5的相互作用鉴定
利用免疫荧光和免疫共沉淀进一步验证BmIAP蛋白与其钓取到的候选互作蛋白BmPP5是否存在相互作用。构建了Bmiap（融合Flag标签）和Bmpp5（融合HA标签）的真核过表达载体,转染家蚕BmN- SWU1细胞,48 h后通过免疫荧光观察其定位,结果发现带有Flag标签的BmIAP蛋白能够与带有HA标签的BmPP5蛋白共定位于细胞质中（图3-A）,暗示它们之间可能存在相互作用。为进一步确定BmIAP和BmPP5之间的关系,将以上两个过表达载体共转家蚕BmN-SWU1细胞48 h后,收集细胞,用Flag标签抗体为诱饵,小鼠IgG抗体为对照,通过免疫共沉淀对BmIAP和BmPP5的相互作用进行验证。结果显示,无论是用Flag抗体还是HA抗体进行Western blot检测,均能检测到相应目的蛋白的表达（图3-B）,表明BmIAP和BmPP5之间存在相互作用。图3
新窗口打开|下载原图ZIP|生成PPT图3免疫荧光和免疫共沉淀分析BmIAP和BmPP5的相互作用
Fig. 3Analysis of the interaction between BmIAP and BmPP5 by immunofluorescence and co-immunoprecipitation
2.4 Bmiap和Bmpp5在BmNPV侵染过程中的调控分析
为了分析Bmiap和Bmpp5在BmNPV侵染过程中的调控关系,首先通过在BmN-SWU1细胞中过表达或敲除Bmiap,然后利用BmNPV侵染细胞,通过qRT-PCR检测Bmpp5的表达水平。结果显示,过表达Bmiap后,Bmpp5的表达量显著上调（图4-A）,其中24 h时Bmpp5上调不显著,推测可能是由于BmNPV不同的复制时期造成的;而敲除Bmiap后,Bmpp5的表达量显著下调（图4-B）,表明Bmiap对Bmpp5具有一定的促进作用。图4
新窗口打开|下载原图ZIP|生成PPT图4Bmpp5和Bmiap的相对表达量分析
A：过表达Bmiap对Bmpp5的影响 Effect of overexpression of Bmiap on Bmpp5;B：敲除Bmiap对Bmpp5的影响 Effect of knockout of Bmiap on Bmpp5;C：过表达Bmpp5对Bmiap的影响 Effect of overexpression of Bmpp5 on Bmiap;D：敲除Bmpp5对Bmiap的影响 Effect of knockout of Bmpp5 on Bmiap。*P<0.05;**P<0.01
Fig. 4Analysis of the relative expression of Bmpp5 and Bmiap
同样,通过分别在BmN-SWU1细胞中过表达或敲除Bmpp5,并检测Bmiap的表达情况。结果显示,过表达Bmpp5后,Bmiap显著上调表达（图4-C）,而敲除Bmpp5后,Bmiap的显著下调表达（图4-D）,表明Bmpp5同样能够促进Bmiap的表达。
2.5 Bmpp5对BmNPV增殖的影响
为了进一步验证Bmpp5在BmNPV增殖过程中的作用,分别在BmN-SWU1细胞中过表达或敲除Bmpp5后,利用BmNPV侵染细胞,并检测杆状病毒Vp39的表达水平。结果显示,过表达Bmpp5后在感染24 h时能够在一定程度上引起Vp39的下调表达,推测在BmNPV感染前期Bmpp5引起的宿主细胞免疫反应可能对BmNPV增殖有一定的抑制作用,而在感染后期能够引起Vp39的上调表达;敲除Bmpp5后,Vp39的表达下调（图5）,表明Bmpp5的表达利于BmNPV的增殖。图5
新窗口打开|下载原图ZIP|生成PPT图5Bmpp5对BmNPV增殖的影响
A：过表达Bmpp5 Overexpression of Bmpp5;B：敲除Bmpp5 Knockout of Bmpp5。**P<0.01
Fig. 5Effect of Bmpp5 on BmNPV proliferation
3 讨论
细胞凋亡作为昆虫免疫应答的重要组成部分,可以有效抵御病毒的复制增殖,而宿主和病毒中凋亡抑制基因（iap）的同时存在[31,32],暗示其可能在病毒与宿主相互作用过程中起着重要作用,同时也为研究其分子机制提供了极好的切入点。PP5属于蛋白磷酸酶家族成员,参与多种信号通路,如DNA损伤、细胞生长、细胞凋亡等[29-30,33-34],其能够使许多与细胞应激反应相关的蛋白去磷酸化,如凋亡信号调节激酶（ASK-1）[35]、DNA-PKcs[36]等。本研究证明了BmPP5能够与BmIAP蛋白发生相互作用,并且在BmNPV侵染过程中,Bmiap促进Bmpp5上调表达的同时,Bmpp5同样能够促进Bmiap的表达,进而使细胞凋亡受到抑制。由此,笔者推测BmIAP和BmPP5的相互作用能够促进二者的表达,但是如何相互作用并不清楚。PP5在DNA损伤修复以及下游细胞周期阻滞和细胞凋亡过程主要是通过对ATM（ataxia telangiectasia mutated）、53BP1等蛋白磷酸化修饰,参与非同源末端连接和同源重组修复。因此,BmPP5对BmIAP蛋白磷酸化修饰调控细胞凋亡通路的过程是未来研究的重点。此外,还发现Bmpp5能够促进BmNPV的增殖,表明在BmNPV侵染过程中,Bmiap和Bmpp5间可能存在正反馈调节作用,增强对宿主细胞的凋亡抑制作用,使BmNPV可以充分利用宿主细胞进行自身的复制增殖,这也进一步证明了细胞凋亡途径在杆状病毒与宿主的相互作用过程中起着重要作用。
本研究以家蚕Bmiap为研究对象,通过免疫共沉淀鉴定并筛选与其相互作用的基因,根据分子量大小共筛选得到7个家蚕基因,同时还获得1个BmNPV的基因ORF76。BmNPV ORF76几乎存在于所有的杆状病毒基因组中,暗示着其功能的重要性。该基因在AcMNPV中的同源基因为Ac93,研究表明敲除Ac93的突变体将不能产生感染性的BV,并且可能参与了核内微泡的形成[37]。同样,敲除ORF76后的BmNPV bacmid也不会产生BV[38],表明该基因的功能具有一定的保守性。BmNPV ORF76与BmIAP的相互作用机制及其功能的解析也将是后续研究的重点内容。
4 结论
鉴定了与BmIAP相互作用的蛋白BmPP5,该蛋白在昆虫中具有较高的保守性。证明了在家蚕核型多角体病毒（BmNPV）侵染过程中Bmiap和Bmpp5能够相互促进,同时Bmpp5能够促进BmNPV的复制增殖。参考文献 原文顺序
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被引期刊影响因子
[本文引用: 1]
DOI:10.1371/journal.pone.0036324URLPMID:22629315 [本文引用: 1]
The Heliothine insect complex contains some of the most destructive pests of agricultural crops worldwide, including the closely relatedHelicoverpa zeaandH. armigera. Biological control using baculoviruses is practiced at a moderate level worldwide. In order to enable more wide spread use, a better understanding of cell-virus interactions is required. While many baculoviruses have been sequenced, none of the Heliothine insect genomes have been available. In this study, we sequenced, assembled and functionally annotated 29,586 transcripts from culturedH. zeacells using Illumina 100 bps and paired-end transcriptome sequencing (RNA-seq). The transcript sequences had high assembly coverage (64.5 times). 23,401 sequences had putative protein functions, and over 13,000 sequences had high similarities to available sequences in other insect species. The sequence database was estimated to cover at least 85% of all H. zea genes. The sequences were used to construct a microarray, which was evaluated on the infection ofH. zeacells withH. Armigera single-capsid nucleopolyhedrovirus(HearNPV). The analysis revealed that up-regulation of apoptosis genes is the main cellular response in the early infection phase (18 hours post infection), while genes linked to four major immunological signalling pathways (Toll, IMD, Jak-STAT and JNK) were down-regulated. Only small changes (generally downwards) were observed for central carbon metabolism. The transcriptome and microarray platform developed in this study represent a greatly expanded resource base forH. zeainsect- HearNPV interaction studies, in which key cellular pathways such as those for metabolism, immune response, transcription and replication have been identified. This resource will be used to develop better cell culture-based virus production processes, and more generally to investigate the molecular basis of host range and susceptibility, virus infectivity and virulence, and the ecology and evolution of baculoviruses.
DOI:10.1128/JVI.00667-11URLPMID:3147993Magsci [本文引用: 1]
Abstract Apoptosis is an important antivirus defense by virtue of its impact on virus multiplication and pathogenesis. To define molecular mechanisms by which viruses are detected and the apoptotic response is initiated, we examined the antiviral role of host inhibitor-of-apoptosis (IAP) proteins in insect cells. We report here that the principal IAPs, DIAP1 and SfIAP, of the model insects Drosophila melanogaster and Spodoptera frugiperda, respectively, are rapidly depleted and thereby inactivated upon infection with the apoptosis-inducing baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). Virus-induced loss of these host IAPs triggered caspase activation and apoptotic death. Elevation of IAP levels by ectopic expression repressed caspase activation. Loss of host IAP in both species was triggered by AcMNPV DNA replication. By using selected inhibitors, we found that virus-induced IAP depletion was mediated in part by the proteasome but not by caspase cleavage. Consistent with this conclusion, mutagenic disruption of the SfIAP RING motif, which acts as an E3 ubiquitin ligase, stabilized SfIAP during infection. Importantly, SfIAP was also stabilized upon the removal of its 99-residue N-terminal leader, which serves as a critical determinant of IAP turnover. These data indicated that a host pathway initiated by virus DNA replication and acting through instability motifs embedded within IAP triggers IAP depletion and thereby causes apoptosis. Taken together, the results of our study suggest that host modulation of cellular IAP levels is a conserved mechanism by which insects mount an apoptotic antiviral response. Thus, host IAPs may function as critical sentinels of virus invasion in insects.
DOI:10.1128/JVI.02246-12URLPMID:23035220Magsci [本文引用: 1]
Abstract The baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) initiates apoptosis in diverse insects through events triggered by virus DNA (vDNA) replication. To define the proapoptotic pathway and its role in antivirus defense, we investigated the link between the host's DNA damage response (DDR) and apoptosis. We report here that AcMNPV elicits a DDR in the model insect Drosophila melanogaster. Replication of vDNA activated DDR kinases, as evidenced by ATM-driven phosphorylation of the Drosophila histone H2AX homolog (H2Av), a critical regulator of the DDR. Ablation or inhibition of ATM repressed H2Av phosphorylation and blocked virus-induced apoptosis. The DDR kinase inhibitors caffeine and KU55933 also prevented virus-induced apoptosis in cells derived from the permissive AcMNPV host, Spodoptera frugiperda. This block occurred at a step upstream of virus-mediated depletion of the cellular inhibitor-of-apoptosis protein, an event that initiates apoptosis in Spodoptera and Drosophila. Thus, the DDR is a conserved, proapoptotic response to baculovirus infection. DDR inhibition also repressed vDNA replication and reduced virus yields 100,000-fold, demonstrating that the DDR contributes to virus production, despite its recognized antivirus role. In contrast to virus-induced phosphorylation of Drosophila H2Av, AcMNPV blocked phosphorylation of the Spodoptera H2AX homolog (SfH2AX). Remarkably, AcMNPV also suppressed SfH2AX phosphorylation following pharmacologically induced DNA damage. These findings indicate that AcMNPV alters canonical DDR signaling in permissive cells. We conclude that AcMNPV triggers a proapoptotic DDR that is subsequently modified, presumably to stimulate vDNA replication. Thus, manipulation of the DDR to facilitate multiplication is an evolutionarily conserved strategy among DNA viruses of insects and mammals.
[本文引用: 1]
DOI:10.1111/j.1365-2583.2010.00993.xURLPMID:20201979 [本文引用: 1]
The infection profiles of the Bombyx mori nucleopolyhedrovirus (BmNPV) in B. mori larvae revealed that the virus invaded the fat body and haemocyte of both KN and 306 strains, which are highly resistant and susceptible, respectively, to BmNPV infection. However, viral proliferation was notably slowed in the resistant B. mori strain. Using suppression subtractive hybridization, two fat body cDNA libraries were constructed to compare BmNPV responsive gene expression levels between the two silkworm lines. In total, 96 differentially expressed genes were obtained. Real-time quantitative PCR (qPCR) analysis confirmed that eight genes were significantly up-regulated in the fat body and haemocyte of the KN strain following BmNPV injection. Our results suggest that these genes may have potential roles in B. mori antiviral infection mechanisms.
[本文引用: 1]
DOI:10.1371/journal.pone.0002997URLPMID:2500172 [本文引用: 1]
MicroRNAs (miRNAs) play crucial roles in various physiological processes through post-transcriptional regulation of gene expressions and are involved in development, metabolism, and many other important molecular mechanisms and cellular processes. The Bombyx mori genome sequence provides opportunities for a thorough survey for miRNAs as well as comparative analyses with other sequenced insect species. We identified 114 non-redundant conserved miRNAs and 148 novel putative miRNAs from the B. mori genome with an elaborate computational protocol. We also sequenced 6,720 clones from 14 developmental stage-specific small RNA libraries in which we identified 35 unique miRNAs containing 21 conserved miRNAs (including 17 predicted miRNAs) and 14 novel miRNAs (including 11 predicted novel miRNAs). Among the 114 conserved miRNAs, we found six pairs of clusters evolutionarily conserved cross insect lineages. Our observations on length heterogeneity at 5 and/or 3 ends of nine miRNAs between cloned and predicted sequences, and three mature forms deriving from the same arm of putative pre-miRNAs suggest a mechanism by which miRNAs gain new functions. Analyzing development-related miRNAs expression at 14 developmental stages based on clone-sampling and stem-loop RT PCR, we discovered an unusual abundance of 33 sequences representing 12 different miRNAs and sharply fluctuated expression of miRNAs at larva-molting stage. The potential functions of several stage-biased miRNAs were also analyzed in combination with predicted target genes and silkworm's phenotypic traits; our results indicated that miRNAs may play key regulatory roles in specific developmental stages in the silkworm, such as ecdysis. Taking a combined approach, we identified 118 conserved miRNAs and 151 novel miRNA candidates from the B. mori genome sequence. Our expression analyses by sampling miRNAs and real-time PCR over multiple developmental stages allowed us to pinpoint molting stages as hotspots of miRNA expression both in sorts and quantities. Based on the analysis of target genes, we hypothesized that miRNAs regulate development through a particular emphasis on complex stages rather than general regulatory mechanisms.
DOI:10.1128/JVI.07217-12URLPMID:22532689 [本文引用: 1]
Although microarray and expressed sequence tag (EST)-based approaches have been used to profile gene expression during baculovirus infection, the response of host genes to baculovirus infection and the interaction between baculovirus and its host remain largely unknown. To determine the host response to Bombyx mori nucleopolyhedrovirus infection and the dynamic interaction between the virus and its host, eight digital gene expression libraries were examined in a Bm5 cell line before infection and at 1.5, 3, 6, 12, 24, 48, and 96 h postinfection. Gene set enrichment analysis of differentially expressed genes at each time point following infection showed that gene sets including cytoskeleton, transcription, translation, energy metabolism, iron ion metabolism, and the ubiquitin-proteasome pathway were altered after viral infection. In addition, a time course depicting protein-protein interaction networks between the baculovirus and the host were constructed and revealed that viral proteins interact with a multitude of cellular machineries, such as the proteasome, cytoskeleton, and spliceosome. Several viral proteins, including IE2, CG30, PE38, and PK-1/2, were predicted to play key roles in mediating virus-host interactions. Based on these results, we tested the role of the ubiquitin-proteasome pathway and iron ion metabolism in the viral infection cycle. Treatment with a proteasome inhibitor and deferoxamine mesylate in vitro and in vivo confirmed that these pathways regulate viral infection. Taken together, these findings provide new insights into the interaction between the baculovirus and its host and identify molecular mechanisms that can be used to block viral infection and improve baculovirus expression systems.
[本文引用: 1]
[本文引用: 1]
DOI:10.2174/138945007782151405URLPMID:17979666 [本文引用: 1]
Apoptosis is used by metazoan organisms to dispose of damaged or unnecessary cells during development, tissue homeostasis, and disease. One of the situations where apoptosis is important is in defense against microbial pathogens, especially viruses. The demonstration that apoptosis could be stimulated by baculovirus infection was one of the first examples of apoptosis associated with virus infection, and this system remains one of the most valuable for studying how apoptosis can be a defense against viruses. In addition, studying how baculoviruses regulate apoptosis has led to many important findings in the field of apoptosis research, such as the discovery of P35, a caspase inhibitor that is widely used in studies of apoptosis, and IAP (inhibitor of apoptosis) proteins, which have homologs in cellular genomes that play important roles in regulating apoptosis and cytokinesis. This review highlights the range of apoptotic responses observed between different baculoviruses and different lepidopteran insects, and the diverse baculovirus genes that have evolved to regulate apoptosis. <br/> <br/> <br/>
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DOI:10.3389/fmicb.2012.00391URLPMID:3494084 [本文引用: 1]
Baculoviruses are insect viruses extensively exploited as eukaryotic protein expression vectors. Molecular biology studies have provided exciting discoveries on virus–host interactions, but the application of omic high-throughput techniques on the baculovirus–insect cell system has been hampered by the lack of host genome sequencing. While a broader, systems-level analysis of biological responses to infection is urgently needed, recent advances on proteomic studies have yielded new insights on the impact of infection on the host cell. These works are reviewed and critically assessed in the light of current biological knowledge of the molecular biology of baculoviruses and insect cells.
DOI:10.1128/JVI.00231-08URLPMID:18508888 [本文引用: 1]
http://jvi.asm.org/cgi/doi/10.1128/JVI.00231-08
DOI:10.1128/JVI.05766-11URLPMID:3209345Magsci [本文引用: 1]
Several mammalian viruses have been shown to induce a cellular DNA damage response during replication, and in some cases, this response is required for optimal virus replication. However, nothing is known about whether a DNA damage response is stimulated by DNA viruses in invertebrates. Cell cycle arrest and apoptosis are two of the downstream effects of the DNA damage response, and both are stimulated by baculovirus infection, suggesting a possible relationship between baculoviruses and the DNA damage response. In the study described in this report, we found that replication of the baculovirus Autographa californica M nucleopolyhedrovirus (AcMNPV) in the cell line Sf9, derived from the lepidopteran insect Spodoptera frugiperda, stimulated a DNA damage response, as indicated by an increased abundance of the S. frugiperda P53 protein (SfP53) and phosphorylation of the histone variant protein H2AX. Stimulation of the DNA damage response was dependent on viral DNA replication. Inhibition of the DNA damage response prevented both the increase in SfP53 accumulation and H2AX phosphorylation and also caused a 10- to 100-fold reduction in virus production, along with decreased viral DNA replication and late gene expression. However, silencing of Sfp53 expression by RNA interference did not significantly affect AcMNPV replication or induction of apoptosis by a mutant of AcMNPV lacking the antiapoptotic gene p35, indicating that these processes are not dependent on SfP53 in Sf9 cells.
DOI:10.1128/JVI.02501-13URLPMID:24027328 [本文引用: 1]
The DNA damage response (DDR) of a host organism represents an effective antiviral defense that is frequently manipulated and exploited by viruses to promote multiplication. We report here that the large DNA baculoviruses, which require host DDR activation for optimal replication, encode a conserved replication factor, LEF-7, that manipulates the DDR via a novel mechanism. LEF-7 suppresses DDR-induced accumulation of phosphorylated host histone variant H2AX (H2AX), a critical regulator of the DDR. LEF-7 was necessary and sufficient to block H2AX accumulation caused by baculovirus infection or DNA damage induced by means of pharmacological agents. Deletion of LEF-7 from the baculovirus genome allowed H2AX accumulation during virus DNA synthesis and impaired both very late viral gene expression and production of infectious progeny. Thus, LEF-7 is essential for efficient baculovirus replication. We determined that LEF-7 is a nuclear F-box protein that interacts with host S-phase kinase-associated protein 1 (SKP1), suggesting that LEF-7 acts as a substrate recognition component of SKP1/Cullin/F-box (SCF) complexes for targeted protein polyubiquitination. Site-directed mutagenesis demonstrated that LEF-7's N-terminal F-box is necessary forH2AX repression and Autographa californica multiple nucleopolyhedrovirus (AcMNPV) replication events. We concluded that LEF-7 expedites virus replication most likely by selective manipulation of one or more host factors regulating the DDR, including 纬-H2AX. Thus, our findings indicate that baculoviruses utilize a unique strategy among viruses for hijacking the host DDR by using a newly recognized F-box protein.
DOI:10.1016/j.virol.2009.11.049URLPMID:20034650 [本文引用: 1]
We previously demonstrated that Bombyx mori nucleopolyhedrovirus (BmNPV) multiplication is restricted in permissive BmN-4 cells upon coinfection with Hyphantria cunea NPV (HycuNPV). Here, we show that HycuNPV-encoded hycu-ep32 gene is responsible for the restricted BmNPV multiplication in HycuNPV-coinfected BmN-4 cells. The only homologue for hycu-ep32 is in Orgyia pseudotsugata NPV. hycu-ep32 could encode a polypeptide of 312 amino acids, and it contains no characteristic domains or motifs to suggest its possible functions. hycu-ep32 is an early gene, and Hycu-EP32 expression reaches a maximum by 6 h postinfection. hycu-ep32-defective HycuNPV , vHycu ep32, was generated, indicating that hycu-ep32 is nonessential in permissive SpIm cells. In BmN-4 cells, HycuNPV infection resulted in a severe global protein synthesis shutdown, while vHycu ep32 did not cause any specific protein synthesis shutdown. These results indicate that the restriction of BmNPV multiplication by HycuNPV is caused by a global protein synthesis shutdown induced by hycu-ep32 upon coinfection with HycuNPV.
DOI:10.1128/JVI.00280-11URLPMID:3147967Magsci [本文引用: 1]
Chronic infection with the hepatitis C virus (HCV) is associated with increased risk for hepatocellular carcinoma (HCC). Chronic immune-mediated inflammation is likely to be an important factor in the development of HCV-associated HCC, but direct effects of HCV infection on the host cell cycle may also play a role. Although overexpression studies have revealed multiple interactions between HCV-encoded proteins and host cell cycle regulators and tumor suppressor proteins, the relevance of these observations to HCV-associated liver disease is not clear. We determined the net effect of these interactions on regulation of the cell cycle in the context of virus infection. Flow cytometry of HCV-infected carboxyfluorescein succinimidyl ester-labeled hepatoma cells indicated a slowdown in proliferation that correlated with abundance of viral antigen. A decrease in the proportions of infected cells in G(1) and S phases with an accumulation of cells in G(2)/M phase was observed, compared to mock-infected controls. Dramatic decreases in markers of mitosis, such as phospho-histone H3, in infected cells suggested a block to mitotic entry. In common with findings described in the published literature, we observed caspase 3 activation, suggesting that cell cycle arrest is associated with apoptosis. Differences were observed in patterns of cell cycle disturbance and levels of apoptosis with different strains of HCV. However, the data suggest that cell cycle arrest at the interface of G(2) and mitosis is a common feature of HCV infection.
DOI:10.1016/j.virusres.2013.09.036URLPMID:24095767 [本文引用: 1]
p53 signaling pathway plays an important role in the regulation of cell cycle. Our previous studies have demonstrated that TGEV infection induces the activation of p53 signaling pathway. In this study we investigated the effects of TGEV infection on the cell cycle of host cells and the roles of p53 activation in this process. The results showed that TGEV infection induced cell cycle arrest at S and G2/M phases in both asynchronous and synchronized PK-15 and ST cells, while UV-inactivated TGEV lost the ability of induction of cell cycle arrest. TGEV infection promoted p21 accumulation, down-regulated cell cycle-regulatory proteins cyclins B1, cdc2, cdk2 and PCNA. Further studies showed that inhibition of p53 signaling could attenuate the TGEV-induced S- and G2/M-phase arrest by reversing the expression of p21 and corresponding cyclin/cdk. In addition, TGEV infection of the cells synchronized in various stages of cell cycle showed that viral genomic RNA and subgenomic RNA, and virus titer were higher in the cells released from S-phase- or G2/M phase-synchronized cells than that in the cells released from the G0/G1 phase-synchronized or asynchronous cells after 18h p.i. Taken together, our data suggested that TGEV infection induced S and G2/M phase arrest in host cells, which might provide a favorable condition for viral replication.
DOI:10.1016/S0966-842X(99)01487-0URLPMID:10217831 [本文引用: 1]
Viruses can induce apoptosis of infected cells either directly, to assist virus dissemination, or by inadvertently triggering cellular sensors that initiate cell death. Cellular checkpoints that can function as ‘alarm bells’ to transmit pro-apoptotic signals in response to virus infections include death receptors, protein kinase R, mitochondrial membrane potential, p53 and the endoplasmic reticulum.
DOI:10.1371/journal.ppat.1004020URLPMID:3961361 [本文引用: 1]
Viral infection induces innate immunity and apoptosis. Apoptosis is an effective means to sacrifice virus-infected host cells and therefore restrict the spread of pathogens. However, the underlying mechanisms of this process are still poorly understood. Here, we show that the mitochondrial antiviral signaling protein (MAVS/VISA/Cardif/IPS-1) is critical for SeV (Sendai virus)-induced apoptosis. MAVS specifically activates c-Jun N-terminal kinase 2 (JNK2) but not other MAP kinases. Jnk2???/??? cells, but not Jnk1???/??? cells, are unable to initiate virus-induced apoptosis and SeV further fails to trigger apoptosis in MAPK kinase 7 (MKK7) knockout (Mkk7???/???) cells. Mechanistically, MAVS recruits MKK7 onto mitochondria via its 3D domain, which subsequently phosphorylates JNK2 and thus activates the apoptosis pathway. Consistently, Jnk2???/??? mice, but not Jnk1???/??? mice, display marked inflammatory injury in lung and liver after viral challenge. Collectively, we have identified a novel signaling pathway, involving MAVS-MKK7-JNK2, which mediates virus-induced apoptosis and highlights the indispensable role of mitochondrial outer membrane in host defenses.
[本文引用: 1]
DOI:10.1186/1471-2164-11-611URLPMID:3091752 [本文引用: 1]
Apoptosis is regulated in an orderly fashion by a series of genes, and has a crucial role in important physiological processes such as growth development, immunological response and so on. Recently, substantial studies have been undertaken on apoptosis in model animals including humans, fruit flies, and the nematode. However, the lack of genomic data for silkworms limits their usefulness in apoptosis studies, despite the advantages of silkworm as a representative of Lepidoptera and an effective model system. Herein we have identified apoptosis-related genes in the silkwormBombyx moriand compared them to those from insects, mammals, and nematodes. From the newly assembled genome databases, a genome-wide analysis of apoptosis-related genes inBombyx moriwas performed using both nucleotide and protein Blast searches. Fifty-two apoptosis-related candidate genes were identified, including five caspase family members, two tumor necrosis factor (TNF) superfamily members, one Bcl-2 family member, four baculovirus IAP (inhibitor of apoptosis) repeat (BIR) domain family members and 1 RHG (Reaper, Hid, Grim, and Sickle;Drosophilacell death activators) family member. Moreover, we identified a new caspase family member,BmCaspase-New, two splice variants ofBmDronc, and Bm3585, a mammalian TNF superfamily member homolog. Twenty-three of these apoptosis-related genes were cloned and sequenced using cDNA templates isolated from BmE-SWU1 cells. Sequence analyses revealed that these genes could have key roles in apoptosis. Bombyx moripossesses potential apoptosis-related genes. We hypothesized that the classic intrinsic and extrinsic apoptotic pathways potentially are active inBombyx mori. These results lay the foundation for further apoptosis-related study inBombyx mori.
DOI:10.1016/S0167-4889(00)00105-1URLPMID:11341966 [本文引用: 1]
We cloned a novel inhibitor of apoptosis protein (IAP) family member, BmIAP, from Bombyx mori BmN cells. BmIAP contains two baculoviral IAP repeat (BIR) domains followed by a RING domain. BmIAP shares striking amino acid sequence similarity with lepidopteran IAPs, SfIAP and TnIAP, and with two baculoviral IAPs, CpIAP and OpIAP, suggesting evolutionary conservation. BmIAP blocks programmed cell death (apoptosis) in Spodoptera frugiperda Sf-21 cells induced by p35 deficient Autographa californica nucleopolyhedrovirus (AcMNPV). This anti-apoptotic function requires both the BIR domains and RING domain of BmIAP. In mammalian cells, BmIAP inhibits Bax induced but not Fas induced apoptosis. Further biochemical data suggest that BmIAP is a specific inhibitor of mammalian caspase-9, an initiator caspase in the mitochondria/cytochrome- c pathway, but not the downstream effector proteases, caspase-3 and caspase-7. These results suggest that suppression of apoptosis by lepidopteran IAPs in insect cells may involve inhibition of an upstream initiator caspase in the conserved mitochondria/cytochrome- c pathway for apoptosis.
DOI:10.1016/j.ibmb.2016.10.012URLPMID:27989836 [本文引用: 1]
61Bombyx moriIAP (cBm-IAP1) depletion by RNAi-mediated silencing induced apoptosis in BM-N cells.61cBm-IAP1 suppressed the apoptosis triggered by transient expression of the initiator caspase Bm-Dronc.61cBm-IAP1 interacted strongly with Bm-Dronc, but only weakly with the effector caspase Bm-caspase-1.61cBm-IAP1 protein defective in the BIR or RING finger region induced apoptosis in BM-N cells.
[本文引用: 1]
DOI:10.1016/j.antiviral.2014.01.017URLPMID:24486953 [本文引用: 1]
Bombyx mori nucleopolyhedrovirus (BmNPV) is a major silkworm pathogen, causing substantial economic losses to the sericulture industry annually. We demonstrate a novel anti-BmNPV system expressing mature artificial microRNAs (amiRNAs) targeting the viral lef-11 gene. The mature amiRNAs inhibited the lef-11 gene in silkworm BmN-SWU1 cells. Antiviral assays demonstrated that mature amiRNAs silenced the gene and inhibited BmNPV proliferation efficiently. As constitutive overexpression of mature amiRNAs may induce acute cellular toxicity, we further developed a novel virus-induced amiRNA expression system. The amiRNA cassette is regulated by a baculovirus-induced fusion promoter. This baculovirus-induced RNA interference system is strictly regulated by virus infection, which functions in a negative feedback loop to activate the expression of mature amiRNAs against lef-11 and subsequently control inhibition of BmNPV replication. Our study advances the use of a regulatable amiRNA cassette as a safe and effective tool for research of basic insect biology and antiviral application.
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DOI:10.1016/j.antiviral.2016.03.009URLPMID:26979473 [本文引用: 1]
61CRISPR/Cas9 system is capable of specifically disruptingie-1gene in BmNPV genome.61The CRISPR/Cas9 system effectively edits BmNPV genome and inhibits virus replication.61Virus-inducible CRISPR/Cas9 system is expected to reduce the probability of off-target effects in the insect cells.
DOI:10.1007/s00436-013-3336-0URLPMID:23417098 [本文引用: 2]
AbstractScreening the anticoccidial drug targets is very important for developing novel drugs and revealing the molecular basis of drug resistance in coccidia. Due to high effectivity and safety, diclazuril was used widely in the poultry industry. To assess the roles of the serine/threonine protein phosphatase type 5 of second-generation merozoites in
DOI:10.1002/bab.1308URLPMID:25322973 [本文引用: 2]
Abstract Protein phosphatase 5 (PP5) is a unique member of the protein phosphatases family that functions in multiple signaling pathways involved in DNA damage, cell cycle control, cell growth, and apoptosis. Recent evidence indicated that PP5 may play a role in cancer progression. In this study, we aimed to examine the biological effect of PP5 on cell growth and apoptosis in human colorectal cancer (CRC). We first knocked down PP5 expression in RKO cells via a short hairpin RNA containing lentivirus system. Then, methylthiazoletetrazolium assay, colony formation assay, and flow cytometry analysis were performed. The proliferation and colony formation ability of RKO cells were remarkably suppressed in PP5-silenced groups, as compared with control groups. Moreover, downregulation of PP5 resulted in a significant G0/G1 phase cell cycle arrest and an induction of apoptosis. In all, these results demonstrated the importance of PP5 in CRC cell growth, and it might be used as a potential therapeutic target for the treatment of CRC.
DOI:10.3390/cells2010163URLPMID:3972657 [本文引用: 1]
Inhibitors of Apoptosis (IAPs) are a family of proteins with various biological functions including regulation of innate immunity and inflammation, cell proliferation, cell migration and apoptosis. They are characterized by the presence of at least one N-terminal baculoviral IAP repeat (BIR) domain involved in protein-protein interaction. Most of them also contain a C-terminal RING domain conferring an E3-ubiquitin ligase activity. In drosophila, IAPs are essential to ensure cell survival, preventing the uncontrolled activation of the apoptotic protease caspases. In mammals, IAPs can also regulate apoptosis through controlling caspase activity and caspase-activating platform formation. Mammalian IAPs, mainly X-linked IAP (XIAP) and cellular IAPs (cIAPs) appeared to be important determinants of the response of cells to endogenous or exogenous cellular injuries, able to convert the survival signal into a cell death-inducing signal. This review highlights the role of IAP in regulating apoptosis in Drosophila and Mammals.
DOI:10.1016/S0968-0004(98)01198-0URLPMID:9612077 [本文引用: 1]
Trends Biochem Sci. 1998 May;23(5):159-62. Research Support, Non-U.S. Gov't; Review
DOI:10.1074/jbc.M402855200URLPMID:15155720 [本文引用: 1]
Abstract Serine/threonine protein phosphatase-5 (PP5) affects many signaling networks that regulate cell growth and cellular responses to stress. Here we report the crystal structure of the PP5 catalytic domain (PP5c) at a resolution of 1.6 A. From this structure we propose a mechanism for PP5-mediated hydrolysis of phosphoprotein substrates, which requires the precise positioning of two metal ions within a conserved Asp271-M1:M2-W1-His427-His304-Asp274 catalytic motif (where M1 and M2 are metals and W1 is a water molecule). The structure of PP5c provides a structural basis for explaining the exceptional catalytic proficiency of protein phosphatases, which are among the most powerful known catalysts. Resolution of the entire C terminus revealed a novel subdomain, and the structure of the PP5c should also aid development of type-specific inhibitors.
DOI:10.1038/sj.emboj.7600496URL [本文引用: 1]
Protein phosphatase 5 (Ppp5) is a serine/threonine protein phosphatase comprising a regulatory tetratricopeptide repeat (TPR) domain N-terminal to its phosphatase domain. Ppp5 functions in signalling pathways that control cellular responses to stress, glucocorticoids and DNA damage. Its phosphatase activity is suppressed by
DOI:10.1093/emboj/20.21.6028URLPMID:11689443 [本文引用: 1]
Apoptosis signal-regulating kinase 1 (ASK1) is a MAP kinase kinase kinase (MAPKKK) that activates the JNK and p38 MAP kinase cascades and is activated in response to oxidative stress such as hydrogen peroxide (H2O2). A yeast two-hybrid screening identified a serine/threonine protein phosphatase 5 (PP5) as a binding partner of ASK1. PP5 directly dephosphorylated an essential phospho-threonine residue within the kinase domain of ASK1 and thereby inactivated ASK1 activity in vitro and in vivo. The interaction between PP5 and ASK1 was induced by H2O2 treatment and was followed by the decrease in ASK1 activity. PP5 inhibited not only H2O2-induced sustained activation of ASK1 but also ASK1-dependent apoptosis. Thus, PP5 appears to act as a physiological inhibitor of ASK1090009JNK/p38 pathways by negative feedback.
DOI:10.1073/pnas.0307765100URLPMID:14734805 [本文引用: 1]
Unrepaired DNA double-strand breaks can lead to apoptosis or tumorigenesis. In mammals double-strand breaks are repaired mainly by nonhomologous end-joining mediated by the DNA-PK complex. The core protein of this complex, DNA-PKcs, is a DNA-dependent serine/threonine kinase that phosphorylates protein targets as well as itself. Although the (auto)phosphorylation activity has been shown to be essential for repair of both random double-strand breaks and induced breaks at the immunoglobulin locus, the corresponding phosphatase has been elusive. In fact, to date, none of the putative phosphatases in DNA double-strand break repair has been identified. Here we show that protein phosphatase 5 interacts with DNA-PKcs and dephosphorylates with surprising specificity at least two functional sites. Cells with either hypo- or hyperphosphorylation of DNA-PKcs at these sites show increased radiation sensitivity.
DOI:10.1128/JVI.05275-11URLPMID:21880748 [本文引用: 1]
Abstract Autographa californica nucleopolyhedrovirus (AcMNPV) orf93 (ac93) is a highly conserved uncharacterized gene that is found in all of the sequenced baculovirus genomes except for Culex nigripalpus NPV. In this report, using bioinformatics analyses, ac93 and odv-e25 (ac94) were identified as baculovirus core genes and thus p33-ac93-odv-e25 represent a cluster of core genes. To investigate the role of ac93 in the baculovirus life cycle, an ac93 knockout AcMNPV bacmid was constructed via homologous recombination in Escherichia coli. Fluorescence and light microscopy showed that the AcMNPV ac93 knockout did not spread by infection, and titration assays confirmed a defect in budded virus (BV) production. However, deletion of ac93 did not affect viral DNA replication. Electron microscopy indicated that ac93 was required for the egress of nucleocapsids from the nucleus and the formation of intranuclear microvesicles, which are precursor structures of occlusion-derived virus (ODV) envelopes. Immunofluorescence analyses showed that Ac93 was concentrated toward the cytoplasmic membrane in the cytoplasm and in the nuclear ring zone in the nucleus. Western blot analyses showed that Ac93 was associated with both nucleocapsid and envelope fractions of BV, but only the nucleocapsid fraction of ODV. Our results suggest that ac93, although not previously recognized as a core gene, is one that plays an essential role in the formation of the ODV envelope and the egress of nucleocapsids from the nucleus.
DOI:10.1016/j.virusres.2012.02.016URLPMID:22421381Magsci [本文引用: 1]
We constructed a series of gene knockout BmNPVs (KOVs) for each of 141 genes (Gomi et al., 1999; Katsuma et al., 2011) using the BmNPV T3 bacmid system (Ono et al., 2007) and lambda red recombination system (Datsenko and Wanner, 2000). In a subsequent analysis of the properties needed for infection using a marker gene, egfp (enhanced green fluorescent protein gene), inserted into the polyhedrin locus, the knockout viruses (KOVs) were subdivided into four phenotypic types, A to D. Type-A (86 KOVs) showed the ability to expand infections equivalent to the control while type-B (8 KOVs) spread infections more slowly. Type-C (37 KOVs) expressed egfp in transfected-BmN cells but the production of infectious viruses was not observed. Type-D (10 KOVs) showed no ability to express egfp even in the transfection experiments. KOVs lacking genes (pkip (Bm15), gp41 (Bm66), bro-d (Bm131), Bm20, 48, 65, 91, 93, or 101) previously identified as being essential, were placed in the viable type-A and B categories.
