ORF3蛋白促进猪流行性腹泻病毒在Vero细胞上的增殖

【背景】猪流行性腹泻(Porcine epidemic diarrhea,PED)是由猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)感染猪而引起的一种急性肠道传染病,常导致病猪水样腹泻、呕吐、脱水。自2010年起,其大规模的暴发给养猪业造成巨大的经济损失。由于对PEDV免疫机理及侵入机制知之甚少,至今仍缺乏有效的PED防治措施。【目的】研究orf3对PEDV体外增殖的影响。【方法】利用基于RNA同源重组的PEDV反向遗传学操作技术拯救一系列携带不同orf3基因及orf3基因缺失的重组PEDV;将获得的重组PEDV以MOI 0.1感染Vero细胞,分别于感染的第8、16、24、32、40、48 h测定其TCID_(50)并绘制病毒生长曲线;分别在感染25 h和36 h利用全自动细胞计数分析仪对6孔板内的细胞进行计数,并于感染后的第12、24、36、48 h用CCK-8试剂盒对其细胞活力进行测定。【结果】RT-PCR结果及细胞病变观察证明成功拯救到了携带不同orf3基因或orf3基因缺失的重组PEDV;进一步的免疫组化分析结果证实PEDV的ORF3蛋白可以在Vero细胞中合成。SPSS软件分析表明携带orf3基因的重组PEDV的滴度(TCID_(50))显著高于缺失orf3基因的重组PEDV的滴度;带有orf3基因的重组PEDV感染Vero细胞25 h和36 h时的活细胞数显著高于缺失orf3基因的重组病毒感染相同时间时的活细胞数;而且重组PEDV感染Vero细胞24 h后,带有orf3基因的重组PEDV的细胞活性显著高于缺失orf3基因的重组病毒。【结论】ORF3蛋白对于PEDV在Vero细胞中的增殖具有促进作用,该作用是通过延缓或减少感染细胞的死亡实现的。本研究为揭示PEDV orf3基因的功能和PEDV复制机制的研究提供理论基础。     

斜纹夜蛾核型多角体病毒ⅡORF146基因的克隆、表达与启动子活性分析

【目的】研究斜纹夜蛾核型多角体病毒II ORF146基因的结构与功能。【方法】根据SpltMNPV IIORF146基因序列设计引物,经PCR扩增克隆ORF146基因。在生物信息学分析基础上进行启动子活性分析和转录时相分析。构建ORF146片段的原核表达载体,表达并纯化融合蛋白后制备多克隆抗体。【结果】核苷酸序列分析表明,读码框含1383 bp,编码460个氨基酸的蛋白质,推定分子量为50.4 kDa。启动子活性分析和转录时相分析都表明该基因是个早、晚期都表达的基因,在病毒感染8 h和18 h有两个转录峰,24 h以后转录水平略有下降,但趋于稳定。pET-28a-ORF13原核表达的融合蛋白经纯化后制备的多克隆抗体特异性高,效价可达1∶3200以上。【结论】SpltMNPV II ORF146基因是一个早期和晚期都表达的病毒组成型结构蛋白基因。推测ORF146基因可能与SpltMNPV II病毒感染宿主细胞后病毒DNA复制有关。制备的多克隆抗体可用于深入研究该蛋白的生物学特性与功能。     

NADC30-like猪繁殖与呼吸综合征病毒样颗粒的制备与鉴定

【背景】猪繁殖和呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)可以造成怀孕母猪的繁殖障碍及仔猪的呼吸系统疾病,近年来,NADC30-like谱系PRRSV已成为国内的优势流行毒株。【目的】研制针对NADC30-like谱系PRRSV的病毒样颗粒(virus-like particle,VLP)疫苗。【方法】将PRRSV NADC30-like毒株编码GP5蛋白开放阅读框5(open reading frame 5,ORF5)、ORF6(编码M蛋白)分别连接至pFastBac^TMDual载体P₁₀和P_H启动子下游多克隆位点,获得穿梭质粒pFB-30-ORF5及pFB-30-ORF6,酶切鉴定后,将ORF6基因插入到穿梭质粒pFB-30-ORF5 P_H启动子下游,构建穿梭质粒pFB-30-ORF5-OPF6。将上述3种穿梭质粒分别转化大肠杆菌DH10Bac感受态细胞,通过蓝白斑筛选及PCR鉴定重组杆粒。再将获得的重组杆粒转染至SF9昆虫细胞,发现细胞病变后收获病毒液,继续盲传3代,在透射电镜下观察是否有病毒样颗粒。用第3代病毒液感染SF9细胞后,分别用GP5蛋白、His-tag、Flag-tag单克隆抗体作为一抗,通过免疫电镜、间接免疫荧光(indirect immunofluorescence assay,IFA)、Western blotting鉴定重组蛋白。【结果】成功构建了3种穿梭质粒pFB-30-ORF5、pFB-30-ORF6和pFB-30-ORF5-OPF6,酶切鉴定正确。通过蓝白斑筛选及PCR验证后获得重组杆粒,分别命名为Bacmind-30-ORF5、Bacmind-30-ORF6和Bacmind-30-ORF5-ORF6。重组杆粒感染SF9细胞120h时出现明显的细胞病变,收获病毒液后,在透射电子显微镜可观察到大小为50nm左右呈现球形结构的VLPs。免疫电镜可以观察到胶体金颗粒结合在VLPs周围;IFA结果显示实验组均出现了明显绿色的特异性荧光灶;Western blotting结果表明,3种VLPs均出现特异性条带,并与预期大小一致。【结论】制备了3种NADC30-like谱系PRRSV的病毒样颗粒,为针对PRRSV新谱系流行株疫苗的研发奠定了基础。     

猪流行性腹泻病毒ORF3蛋白40–91 aa是其细胞质定位的关键结构域

ORF3蛋白是猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)基因组编码的唯一的辅助蛋白,与病毒毒力相关。为确定PEDV ORF3细胞质定位信号,文中构建了系列PEDV DR13wt ORF3蛋白全长或截短肽重组表达载体,转染Vero细胞并利用激光共聚焦显微镜分析与EGFP融合表达的全长ORF3蛋白和其系列截短肽在细胞内的分布。结果表明,全长ORF3蛋白或所有包含2个跨膜域的40–91 aa基序的ORF3截短肽均只定位于细胞质中,而不包含40–91aa基序的ORF3截短肽分布于整个细胞中(细胞质和细胞核均有分布)。这表明40–91 aa是猪流行性腹泻病毒ORF3蛋白细胞质定位的关键结构域。PEDVORF3蛋白细胞质定位结构域的明确为进一步研究其细胞内转运和生物学功能提供了参考。     

hsc70-4基因促进家蚕核型多角体病毒复制增殖

【目的】热休克应答(heatshockresponse,HSR)是机体细胞应对环境压力的一种重要防御策略,鉴定热休克蛋白在杆状病毒侵染宿主过程中的功能,并揭示其作用的分子机制,为探明宿主与病毒相互作用的分子基础提供理论依据。【方法】通过分子克隆技术对Bmhsc70-4基因进行克隆,并利用BioEdit及GeneDoc对其进行多序列比对分析;分别通过真核表达和基于CRISPR/Cas9的基因编辑系统对Bmhsc70-4基因进行过表达和敲除;利用荧光定量PCR技术检测相应基因的表达量;通过对Caspase-9和Caspase-3/7活性的检测确定Bmhsc70-4基因对细胞凋亡的影响;通过免疫荧光验证BmHSC70-4和BmIAP的共定位情况,并进一步通过免疫共沉淀验证它们的相互作用。【结果】Bmhsc70-4基因开放阅读框为1950bp,编码649个氨基酸,在昆虫间具有较高的保守性;BmNPV能够诱导Bmhsc70-4基因上调表达,过表达Bmhsc70-4基因能够促进BmNPV的增殖,敲除Bmhsc70-4基因能够抑制BmNPV的增殖,表明Bmhsc70-4基因的表达利于BmNPV的增殖;Bmhsc70-4基因具有抑制家蚕细胞凋亡的功能;荧光共定位显示BmHSC70-4和BmIAP共定位于细胞质中,免疫共沉淀结果表明两者可以相互作用;BmNPV侵染过程中Bmhsc70-4基因能够促进Bmiap基因的表达。【结论】Bmhsc70-4基因具有抑制家蚕细胞凋亡的功能,在BmNPV侵染家蚕细胞过程中,能够与BmIAP相互作用,并促进BmNPV复制增殖。     

猪流行性腹泻病毒核衣壳蛋白与感染细胞核磷蛋白的共定位分析

【目的】阐明猪流行性腹泻病毒(PEDV)核衣壳蛋白与病毒感染细胞核仁成分B23.1蛋白的共定位特征。【方法】分别参照GenBank中PEDV CV777株的N基因序列(AF353511)和编码人细胞核仁蛋白B23.1基因序列(BC050628.1),设计、合成扩增N基因和B23.1基因的引物,利用RT-PCR技术扩增了N基因和Vero E6细胞的B23.1基因的cDNA,分别克隆到真核表达载体pAcGFP1-C1和pDsRed2-N1,获得重组质粒pAcGFP1-C1/N和pDsRed2-N1/B23.1,共转染Vero E6细胞。【结果】Western blots分析表明这些融合蛋白在转染的Vero E6细胞中表达;共聚焦显微镜技术分析表明在共转染Vero E6细胞中猪流行性腹泻病毒N蛋白与Vero E6细胞核磷蛋白B23.1发生共定位。【结论】为进一步鉴定PEDV N蛋白中核仁定位信号和N蛋白核仁定位机制提供可靠依据。     

与猪流行性腹泻病毒M蛋白互作的宿主蛋白的鉴定

【背景】猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)膜蛋白(M)在病毒粒子的组装、膜融合和病毒复制等方面具有重要的作用,但M蛋白与宿主细胞的互作机制尚不清楚。【目的】利用免疫沉淀技术和液质联用技术筛选细胞内与PEDVM蛋白相互作用的蛋白,为揭示M蛋白在病毒增殖过程中发挥的功能提供研究基础。【方法】将MOI=0.1的PEDV DR13疫苗株接种于长成单层的Vero细胞,感染36 h后,收集细胞并进行裂解。利用抗M的单克隆抗体沉淀与M相互作用蛋白复合物,通过液相色谱串联质谱(LC-MS/MS)进行鉴定并利用细胞功能富集分析(Gene ontology,GO)对感染组鉴定到的细胞蛋白进行分析,确定两个细胞内源性蛋白为候选蛋白,进行免疫共沉淀(Co-IP)验证和共定位分析。【结果】基于鉴定蛋白的肽段数的方法分析显示,感染组与对照组相比,鉴定了218个与M蛋白相互作用的细胞内源性蛋白,分别与蛋白质合成、代谢、细胞信号通路转导等密切相关,选择细胞分裂周期蛋白42 (Cell division cycle 42,CDC42)、真核翻译起始因子3亚基L蛋白(eIF3L)为候选蛋白进行Co-IP(Co-immunoprecipitation)验证和共定位分析,结果证实CDC42、eIF3L蛋白分别与M蛋白在细胞内存在相互作用。【结论】鉴定出PEDV M蛋白能够与宿主细胞CDC42和eIF3L蛋白相互作用,并鉴定出其他可能与M蛋白发生相互作用的宿主蛋白60个,为开展PEDV与宿主细胞蛋白相互作用研究提供了重要理论依据。     

基于CRISPR/Cas9系统筛选猪流行性腹泻病毒复制相关基因及其验证

【背景】成簇规律间隔的短回文重复序列相关蛋白(clustered regularly interspaced short palindromic repeats/CRISPR-associated protein,CRISPR/Cas9)已被广泛证实是高效、强大的第三代基因编辑工具,在发现功能基因等领域取得了重要进展,但至今尚无利用该方法挖掘猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)宿主基因的报道。【目的】利用CRISPR/Cas9系统在全基因组范围内筛选PEDV复制相关基因,并进行候选基因的初步验证,为培育抗PEDV种猪提供科学参考。【方法】通过CRISPR/Cas9技术构建人肝癌细胞系(Huh-7)全基因组敲除文库,利用PEDV感染Huh-7文库细胞,随后经过高通量测序筛选影响PEDV复制的关键宿主因子,结合基因干扰和检测病毒效价等相关试验对影响PEDV复制的候选基因进行初步验证。【结果】构建了CRISPR/Cas9系统在全基因组范围内筛选PEDV复制相关基因的方法,将富集程度排名靠前的整合素α11(integrin α11,ITGA11)、哺乳动物复制蛋白A2(replication protein A2,RPA2)、驱动蛋白家族成员2A(kinesin family member 2A,KIF2A)、诱导髓系白血病细胞分化蛋白1(induced myeloid leukemia cell differentiation protein 1,MCL1)、多聚ADP核糖化酶1[poly(ADP-ribose)polymerase 1,PARP1]和囊泡单胺转运蛋白(vesicular monoamine transporter,SLC18A1)基因进行了验证;采用siRNA对上述基因分别进行干扰后,结果与对照组相比,干扰ITGA11可显著降低PEDV猪源靶向细胞IPEC-J2中PEDV-NmRNA、蛋白表达水平及子代病毒滴度。【结论】基于CRISPR/Cas9系统的全基因组敲除文库可作为挖掘PEDV复制相关功能基因的有效工具,ITGA11基因可作为一种制备抗PEDV猪种潜在的靶基因。     

牛病毒性腹泻病毒Erns蛋白激活NOD样受体热蛋白结构域相关蛋白3炎症小体诱发细胞焦亡的研究

【目的】牛病毒性腹泻病毒(bovine viral diarrhea virus, BVDV)是引起牛病毒性腹泻-黏膜病的关键病毒。BVDV的结构蛋白Erns可在病毒感染的初期削弱宿主的免疫防御,引发牛群炎症反应。核苷酸寡聚化结构域样受体(nucleotide-binding oligomerization domain, NOD)热蛋白结构域相关蛋白3 (NLRP3)炎症小体是NOD样受体(NOD-like receptor, NLRs)家族重要成员,调控炎症性疾病的发生发展,同时激活的NLRP3炎症小体能够引起宿主细胞焦亡,进而诱发级联放大的炎症反应。但BVDV Erns蛋白在BVDV感染诱发炎症反应的分子机制尚不清楚。【方法】为进一步探索Erns蛋白对BVDV感染激活NLRP3炎症小体诱发细胞焦亡的影响,构建了BVDV Erns蛋白的真核表达质粒pCMV-HA-Erns,过表达BVDV Erns蛋白,检测BVDV感染细胞中NLRP3炎症小体组分[半胱氨酸蛋白酶(caspase-1)、凋亡相关斑点样蛋白(apoptosis-associated speck-like protein, ASC)和NLRP3]、IL-1β的mRNA转录水平和蛋白表达水平,以及细胞死亡调节蛋白(gasdermin D, GSDMD)的基因表达和蛋白剪切情况,并通过扫描电镜观察牛睾丸(bovine testis, BT)细胞膜成孔及BT细胞内容物释放情况,以分析Erns蛋白诱导BT细胞产生细胞焦亡。【结果】Erns蛋白能够显著引起NLRP3炎症小体活化进而激活caspase-1,活化的caspase-1一方面切割GSDMD,形成有活性的GSDMD-N端并在BT细胞膜形成孔洞,释放内容物,诱导BT细胞发生细胞焦亡;另一方面活化的caspase-1切割pro-IL-1β,形成有活性的IL-1β,并释放到BT细胞外,引起BT细胞上清中IL-1β水平上升。【结论】系统解析了BVDV Erns蛋白激活NLRP3炎症小体介导细胞焦亡的产生,对疫苗及治疗药物的研制具有重要指导意义。     

汉滩病毒感染诱导热休克蛋白70表达

为了解汉滩病毒感染后细胞的应激反应及HSP70的表达与病毒复制的关系,在汉滩病毒A9株感染Vero-E6细胞后,用免疫组织化学及核酸分子原位杂交法,对细胞HSP70基因的表达进行了检测.结果表明,汉滩病毒感染细胞4h后即可诱导Vero-E6细胞表达HSP70,表达可持续至感染后5d,且HSP70在细胞内的分布也有改变.提示汉滩病毒可直接诱导HSP70的高表达.     

Roles for the E4 orf6, orf3, and E1B 55-Kilodalton Proteins in Cell Cycle-Independent Adenovirus Replication

Adenoviruses bearing lesions in the E1B 55-kDa protein (E1B 55-kDa) gene are restricted by the cell cycle such that mutant virus growth is most impaired in cells infected during G(1) and least restricted in cells infected during S phase (F. D. Goodrum and D. A. Ornelles, J. Virol. 71:548-561, 1997). A similar defect is reported here for E4 orf6-mutant viruses. An E4 orf3-mutant virus was not restricted for growth by the cell cycle. However, orf3 was required for enhanced growth of an E4 orf6-mutant virus in cells infected during S phase. The cell cycle restriction may be linked to virus-mediated mRNA transport because both E1B 55-kDa- and E4 orf6-mutant viruses are defective at regulating mRNA transport at late times of infection. Accordingly, the cytoplasmic-to-nuclear ratio of late viral mRNA was reduced in G(1) cells infected with the mutant viruses compared to that in G(1) cells infected with the wild-type virus. By contrast, this ratio was equivalent among cells infected during S phase with the wild-type or mutant viruses. Furthermore, cells infected during S phase with the E1B 55-kDa- or E4 orf6-mutant viruses synthesized more late viral protein than did cells infected during G(1). However, the total amount of cytoplasmic late viral mRNA was greater in cells infected during G(1) than in cells infected during S phase with either the wild-type or mutant viruses, indicating that enhanced transport of viral mRNA in cells infected during S phase cannot account for the difference in yields in cells infected during S phase and in cells infected during G(1). Thus, additional factors affect the cell cycle restriction. These results indicate that the E4 orf6 and orf3 proteins, in addition to the E1B 55-kDa protein, may cooperate to promote cell cycle-independent adenovirus growth.     

PEDV ORF3 encodes an ion channel protein and regulates virus production

Several studies suggest that the open reading frame 3 (ORF3) gene of porcine epidemic diarrhea virus (PEDV) is related to viral infectivity and pathogenicity, but its function remains unknown. Here, we propose a structure model of the ORF3 protein consisting of four TM domains and forming a tetrameric assembly. ORF3 protein can be detected in PEDV-infected cells and it functions as an ion channel in both Xenopus laevis oocytes and yeast. Mutation analysis showed that Tyr170 in TM4 is important for potassium channel activity. Furthermore, viral production is reduced in infected Vero cells when ORF3 gene is silenced by siRNA. Interestingly, the ORF3 gene from an attenuated PEDV encodes a truncated protein with 49 nucleotide deletions, which lacks the ion channel activity.     

Ovarian follicular atresia is mediated by heterophagy, autophagy, and apoptosis in Prochilodus argenteus and Leporinus taeniatus (Teleostei: Characiformes)

We investigated apoptosis, cell proliferation antigen (PCNA), and heat shock protein (HSP70) during ovarian follicular atresia in two freshwater teleost species from the São Francisco River basin, Brazil: curimatã-pacu, Prochilodus argenteus and piau-jejo, Leporinus taeniatus. Fishes were maintained in captivity after the reproductive period and ovarian regression was assessed by gonadosomatic index for three stages: early, advanced, and late regression. Follicular atresia was analysed by light and transmission electron microscopy, as well as by TUNEL and immunohistochemistry for HSP70 and PCNA. During early regression, atretic follicles exhibited zona pellucida breakdown, yolk degeneration, and hypertrophied follicular cells (e.g. granulosa in mammals). Intense heterophagy to engulf the yolk, and autophagy were detected in the follicular cells during advanced and late atresia. The TUNEL assay detected DNA fragmentation, mainly in late follicular atresia. The apoptosis rate of the follicular cells increased up to 10% during follicular atresia in both species and was negatively correlated with follicular area. Immunohistochemistry reaction for HSP70 stained the follicular cells strongly during advanced atresia, when they are intensively involved in yolk engulfment, whereas the reaction for PCNA labelled theca cells. We inferred that heterophagy, autophagy, and apoptosis contributed to follicular atresia in teleost ovaries, thereby achieving a more efficient removal of the degenerating oocyte and dying follicular cells. Additionally, HSP70 may protect the follicular cells before apoptosis when they are involved in yolk engulfment, and cell proliferation in the theca contributed to ovarian remodelling.     

The age-dependent induction of apoptosis-inducing factor (AIF) in the human semitendinosus skeletal muscle

To assess the dependence on age of the expression of apoptosis regulatory proteins in the human semitendinosus muscle, we measured the expression levels of several apoptosis-related genes, including apoptosis-inducing factor (AIF), Bax, Bcl-2, caspase-3 and heat shock protein 70 (HSP70), using RT-PCR, immunohistochemistry and TUNEL assays. We found that the DNA fragmentation was proportional to the age of the tissues sample donors. The expression levels of AIF were significantly elevated (by 10 to 25%) in semitendinosus tissue samples from older individuals, but the Bax, Bcl-2, caspase-3 and HSP 70 levels remained almost constant. This data suggests that the morphological and functional changes observed in aged human semitendinosus muscle correlates with the apoptosis of muscle cells through the induction of AIF.     

Ovarian follicular atresia is mediated by heterophagy,autophagy, and apoptosis in Prochilodus argenteus and Leporinus taeniatus (Teleostei: Characiformes)

We investigated apoptosis, cell proliferation antigen (PCNA), and heat shock protein (HSP70) during ovarian follicular atresia in two freshwater teleost species from the São Francisco River basin, Brazil: curimatã-pacu, Prochilodus argenteus and piau-jejo, Leporinus taeniatus. Fishes were maintained in captivity after the reproductive period and ovarian regression was assessed by gonadosomatic index for three stages: early, advanced, and late regression. Follicular atresia was analysed by light and transmission electron microscopy, as well as by TUNEL and immunohistochemistry for HSP70 and PCNA. During early regression, atretic follicles exhibited zona pellucida breakdown, yolk degeneration, and hypertrophied follicular cells (e.g. granulosa in mammals). Intense heterophagy to engulf the yolk, and autophagy were detected in the follicular cells during advanced and late atresia. The TUNEL assay detected DNA fragmentation, mainly in late follicular atresia. The apoptosis rate of the follicular cells increased up to 10% during follicular atresia in both species and was negatively correlated with follicular area. Immunohistochemistry reaction for HSP70 stained the follicular cells strongly during advanced atresia, when they are intensively involved in yolk engulfment, whereas the reaction for PCNA labelled theca cells. We inferred that heterophagy, autophagy, and apoptosis contributed to follicular atresia in teleost ovaries, thereby achieving a more efficient removal of the degenerating oocyte and dying follicular cells. Additionally, HSP70 may protect the follicular cells before apoptosis when they are involved in yolk engulfment, and cell proliferation in the theca contributed to ovarian remodelling.     

Newcastle disease virus V protein is a determinant of host range restriction

It has been demonstrated that the V protein of Newcastle disease virus (NDV) functions as an alpha/beta interferon (IFN-alpha/beta) antagonist (M. S. Park, M. L. Shaw, J. Mu?oz-Jordan, J. F. Cros, T. Nakaya, N. Bouvier, P. Palese, A. García-Sastre, and C. F. Basler, J. Virol. 77:1501-1511, 2003). We now show that the NDV V protein plays an important role in host range restriction. In order to study V functions in vivo, recombinant NDV (rNDV) mutants, defective in the expression of the V protein, were generated. These rNDV mutants grow poorly in both embryonated chicken eggs and chicken embryo fibroblasts (CEFs) compared to the wild-type (wt) rNDV. However, insertion of the NS1 gene of influenza virus A/PR8/34 into the NDV V(-) genome [rNDV V(-)/NS1] restores impaired growth to wt levels in embryonated chicken eggs and CEFs. These data indicate that for viruses infecting avian cells, the NDV V protein and the influenza NS1 protein are functionally interchangeable, even though there are no sequence similarities between the two proteins. Interestingly, in human cells, the titer of wt rNDV is 10 times lower than that of rNDV V(-)/NS1. Correspondingly, the level of IFN secreted by human cells infected with wt rNDV is much higher than that secreted by cells infected with the NS1-expressing rNDV. This suggests that the IFN antagonist activity of the NDV V protein is species specific. Finally, the NDV V protein plays an important role in preventing apoptosis in a species-specific manner. The rNDV defective in V induces apoptotic cell death more rapidly in CEFs than does wt rNDV. Taken together, these data suggest that the host range of NDV is limited by the ability of its V protein to efficiently prevent innate host defenses, such as the IFN response and apoptosis.     

Identification of a conserved anti-apoptotic protein that modulates the mitochondrial apoptosis pathway

Here we identified an evolutionarily highly conserved and ubiquitously expressed protein (C9orf82) that shows structural similarities to the death effector domain of apoptosis-related proteins. RNAi knockdown of C9orf82 induced apoptosis in A-549 and MCF7/casp3-10b lung and breast carcinoma cells, respectively, but not in cells lacking caspase-3, caspase-10 or both. Apoptosis was associated with activated caspases-3, -8, -9 and -10, and inactivation of caspases 10 or 3 was sufficient to block apoptosis in this pathway. Apoptosis upon knockdown of C9orf82 was associated with increased caspase-10 expression and activation, which was required for the generation of an 11 kDa tBid fragment and activation of Caspase-9. These data suggest that C9orf82 functions as an anti-apoptotic protein that modulates a caspase-10 dependent mitochondrial caspase-3/9 feedback amplification loop. We designate this ubiquitously expressed and evolutionarily conserved anti-apoptotic protein Conserved Anti-Apoptotic Protein (CAAP). We also demonstrated that treatment of MCF7/casp3-10b cells with staurosporine and etoposides induced apoptosis and knockdown of CAAP expression. This implies that the CAAP protein could be a target for chemotherapeutic agents.