高表达蛋白酪氨酸磷酸酶-PEST促进HepG2肝癌细胞体外增殖

为深入探讨蛋白酪氨酸磷酸酶-PEST在肝癌中的作用, 构建了真核重组表达质粒pcDNA3.1/ PTP-PEST并转染HepG2肝癌细胞, 经荧光定量RT-PCR和Western blot对转染细胞进行筛选, 选出稳定高表达PTP-PEST基因的细胞株, 并采用MTT及Western blot对高表达PTP-PEST基因的细胞株的增殖速度和生长因子受体结合蛋白2表达量进行检测, 观察到高表达PTP-PEST的细胞株内Grb2蛋白表达上调, 细胞增殖速度加快. 本研究表明, 在HepG2肝癌细胞株内, PTP-PEST可能通过影响Grb2蛋白的表达来促进细胞增殖.     

乙肝相关性肝癌差异表达基因的获得及FOLR1分析

运用基因芯片技术获取以稳定转染HBx基因的肝癌细胞HepG2(HepG2-X)及非转染的肝癌细胞HepG2中差异表达的基因,并对其中一条基因进行的生物信息学分析。采用人肝癌G2细胞(HepG2)细胞系为对照组,以稳定转染HBx的HepG2细胞为实验组,抽取总RNA,经过反转录cDNA,对照组用Cy3实验组用Cy5荧光标记,获得cDNA探针;经杂交、洗涤后,通过ImaGene3.0软件进行分析统计。通过基因芯片筛选,获得643条与乙肝相关性肝细胞性癌相关的基因,其中FOLR1基因差异性表达最显著,HBx显著下调其表达,同源性比较分析结果表明,其碱基序列与已经报道的其他12种哺乳动物的相似率为67%-99%,且符合种属之间的进化关系。基因芯片筛选HBx诱导的HepG2差异表达基因具有样品用量少,高质量,高速度,高敏感等特性。FOLR1可能为HBV相关肝癌的发生、转移的诊断、靶基因治疗和预后评估提供一定的依据。     

RNA干扰CD151基因表达抑制肝癌细胞侵袭

目的研究RNA干扰(RNA interference RNAi)抑制CD151表达对人类肝癌细胞迁移侵袭的影响及分子机制。方法将CD151-siRNA在脂质体介导下瞬时转入人肝癌HepG2细胞,倒置荧光显微镜观察转染效率,用qPCR,western blot检测HepG2细胞CD151mRNA和蛋白表达,体外研究肿瘤细胞迁移和侵袭能力,并检测相关信号通路的变化。结果成功转染CD151-siRNA后,HepG2细胞CD151基因的表达与正常对照组和阴性对照组相比,mRNA和蛋白表达水平明显降低(P0.05),细胞迁移和侵袭能力明显下降(P0.05),同时,沉默CD151的表达,FAK,ERK的磷酸化受抑制。结论CD151-siRNA能有效抑制人肝癌细胞CD151基因mRNA和蛋白的表达,通过抑制FAK,ERK蛋白的磷酸化水平,降低细胞的迁移和侵袭力。     

CXCL1促进人肝癌细胞发生内质网应激

目的:探究趋化因子CXCL1过表达对人肝癌HepG2细胞内质网应激的影响。方法:将GFP-CXCL1质粒转染人肝癌HepG2细胞,荧光显微镜观察绿色荧光蛋白;将该质粒转染人肝癌HepG2细胞,24 h后收集细胞,提取细胞总mRNA后进行反转录,荧光定量PCR检测内质网应激相关蛋白IRE1、PERK、ATF6的表达。结果:CXCL1过表达后,通过荧光定量PCR检测出内质网应激相关蛋白IRE1、PERK、ATF6均升高。结论:过表达的CXCL1能够促进人肝癌HepG2细胞的内质网应激,为后续研究奠定了基础。     

应用微阵列技术筛选PS1TP1基因转染细胞差异表达基因

阐明乙型肝炎病毒(HBV)前S1蛋白反式激活蛋白1(PS1TP1)的表达对于肝细胞的基因表达谱的影响。应用基因芯片技术对于pcDNA3.1()和pcDNA3.1()PS1TP1分别转染的HepG2细胞的基因表达谱进行分析。以肝癌细胞系HepG2基因作为模板,应用聚合酶链反应(PCR)技术扩增PS1TP1基因片段,以常规的分子生物学技术构建表达载体pcDNA3.1()PS1TP1。以脂质体技术转染肝母细胞瘤细胞系HepG2,提取总RNA,逆转录为cDNA,与转染空白表达载体pcDNA3.1()的HepG2细胞进行DNA芯片分析并比较。在4096个基因表达谱的筛选中,发现有8个基因表达水平显著上调,14个基因表达水平显著下调。PS1TP1基因的表达对于肝细胞基因表达谱有显著影响。DNA芯片技术是分析反式调节靶基因的有效技术途径。     

盐酸普鲁卡因对人肝癌HepG2细胞Syk基因甲基化的影响

为探讨盐酸普鲁卡因(procaine,PCA)对人肝癌HepG2细胞Syk基因甲基化的影响,应用巢式双重甲基化特异性聚合酶链反应(Methylation-specific PCR,MSP)检测盐酸普鲁卡因处理前后HepG2细胞中Syk基因启动子的甲基化水平;采用蛋白印迹技术观察Syk蛋白表达情况。结果显示,MSP检测到人肝癌HepG2细胞中Syk基因发生甲基化,随盐酸普鲁卡因浓度升高和时间的延长Syk基因甲基化水平逐渐降低;蛋白印迹结果表明,Syk蛋白在HepG2细胞中低表达,经盐酸普鲁卡因处理可以上调Syk蛋白的表达。人肝癌HepG2细胞中Syk基因启动子区域发生甲基化可能是肝癌发病的机制之一;盐酸普鲁卡因能降低Syk基因启动子区域CpG岛甲基化,并使Syk蛋白表达上调。     

应用微阵列技术筛选PS1TP1基因转染细胞差异表达基因

阐明乙型肝炎病毒(HBV)前-S1蛋白反式激活蛋白1(PS1TP1)的表达对于肝细胞的基因表达谱的影响.应用基因芯片技术对于pcDNA3.1(-)和pcDNA3.1(-)-PS1TP1分别转染的HepG2细胞的基因表达谱进行分析.以肝癌细胞系HepG2基因作为模板,应用聚合酶链反应(PCR)技术扩增PS1TP1基因片段,以常规的分子生物学技术构建表达载体pcDNA3.1(-)-PS1TP1.以脂质体技术转染肝母细胞瘤细胞系HepG2,提取总RNA,逆转录为cDNA,与转染空白表达载体pcDNA3.1(-)的HepG2细胞进行DNA芯片分析并比较.在4096个基因表达谱的筛选中,发现有8个基因表达水平显著上调,14个基因表达水平显著下调.PS1TP1基因的表达对于肝细胞基因表达谱有显著影响.DNA芯片技术是分析反式调节靶基因的有效技术途径.     

利用pSOS系统筛选靶向HBV病毒X基因的siRNA

构建靶向乙肝病毒(HBV)X基因的真核表达载体pSOS-X-siRNA和pSOS-siRNA,转染肝癌细胞系HepG2和HepG2.2.15,筛选和验证高效siRNA。设计4个靶向X基因siRNA,将siRNA和HBx基因插入载体pSOS得重组质粒pSOS-X-siRNA;pSOS-X-siRNA经PacI酶切去除目的片段HBx后得pSOS-siRNA。将质粒pSOS-X-siRNA和pSOS-siRNA分别转HepG2和HepG2.2.15肝癌细胞株。荧光显微镜下观察HepG2细胞绿色荧光(GFP)减弱程度预估干扰效率。ELISA检测HepG2.2.15细胞上清HBsAg、HBeAg表达,Western blotting检测胞内蛋白HBsAg、HBcAg表达,Real time PCR检测胞内HBsmRNA、HBx mRNA的转录。转染4d后,siRNA4使HepG2细胞的GFP信号表达程度最低,为阴性对照的9%,预测其干扰效果最强。siRNA4使HepG2.2.15细胞转染后4d上清的HBsAg蛋白表达为对照的(13.92±1.14)%(P0.05)、HBeAg为(21.69±4.92)%(P0.05),胞内的HBsAg、HBcAg蛋白表达量灰度比值为0.175±0.025、0.0825±0.028,均为各处理组中最低(P0.01),HBs mRNA和HBx mRNA分别降为对照的0.237±0.028(P0.01)、0.110±0.022(P0.01),差异有统计学意义,证实siRNA4为高效干扰位点。利用pSOS成功筛选并验证构建靶向HBV X基因的siRNA靶点。     

pcDNA3.1-Canstatin-3Flag真核表达载体的构建及转染HepG2细胞中的表达

目的:构建pcDNA3.1-Canstatin-3Flag载体并稳定转染肝癌HepG2细胞,检测canstatin在mRNA水平的表达。方法:胎盘中提取总RNA,RT-PCR法获得canstatinDNA,克隆至pcDNA3.1(-)载体中,并测序,重组质粒pcDNA3.1-Canstatin-3Flag转染肝癌HepG2细胞,G418筛选出稳定转染细胞,RT-PCR检测canstatin mRNA表达。结果:1.成功构建出pcDNA3.1-Canstatin-3Flag重组质粒;2.获得稳定转染pcDNA3.1-Canstatin-3Flag的肝癌HepG2细胞;3.发现转染后的肝癌HepG2细胞canstatin在mRNA水平比未转染细胞有明显的增强。结论:获得了稳定转染pcDNA3.1-Canstatin-3Flag的肝癌HepG2细胞,为后期canstatin在肝癌中的研究提供了支持。     

血红素加氧酶-1对肝癌细胞细胞周期调控因子的影响

目的观察血红素加氧酶-1（heme oxygenase 1,HO-1）对人肝癌细胞HepG2细胞周期调控因子的影响。方法构建含有野生型和突变型HO-1基因的重组载体pcDNA3．1（＋）-wtHO-1和pcDNA3．1（＋）-mHO-1G143H。利用脂质体介导的方法将构建好的重组载体转染肝癌细胞系HepG2,以空载体转染作为对照组。通过G418筛选建立稳定表达野生型和突变型HO-1的HepG2肝癌细胞系。经半定量RT—PCR、Western印迹检测转染细胞系中HO-1 mRNA和蛋白的表达水平。在HO-1表达改变的稳转细胞系中,利用Western印迹检测转染细胞系中P21、P27蛋白表达水平。结果成功实现了野生型和突变型HO-1在HepG2细胞中的过表达;野生型和突变型HO-1过表达均能诱导抑癌基因p21和p27的表达。结论HO．1过表达诱导抑癌基因p21和p27的表达与血红素分解产物无关。HO-1可能通过其它机制调节p21和p27的表达。     

CHCHD2 and CHCHD10 regulate mitochondrial dynamics and integrated stress response

Mitochondrial dysfunction is becoming one of the main pathology factors involved in the etiology of neurological disorders. Recently, mutations of the coiled-coil-helix-coiled-coil-helix domain containing 2 (CHCHD2) and 10 (CHCHD10) which encode two homologous proteins that belong to the mitochondrial CHCH domain protein family, are linked to Parkinson’s disease and amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD), respectively. However, the physiological and pathological roles of these twin proteins have not been well elaborated. Here, we show that, in physiological conditions, CHCHD2 and CHCHD10 interact with OMA1 and suppress its enzyme activity, which not only restrains the initiation of the mitochondrial integrated response stress (mtISR), but also suppresses the processing of OPA1 for mitochondrial fusion. Further, during mitochondria stress-induced by carbonyl cyanide m-chlorophenylhydrazone (CCCP) treatment, CHCHD2 and CHCHD10 translocate to the cytosol and interacte with eIF2a, which attenuates mtISR overactivation by suppressing eIF2a phosphorylation and its downstream response. As such, knockdown of CHCHD2 and CHCHD10 triggers mitochondrial ISR, and such cellular response is enhanced by CCCP treatment. Therefore, our findings demonstrate the first “mtISR suppressor” localized in mitochondria for regulating stress responses in mammalian cells, which has a profound pathological impact on the CHCH2/CHCH10-linked neurodegenerative disorder.Subject terms: Stress signalling, Mitochondria     

CHCM1/CHCHD6, novel mitochondrial protein linked to regulation of mitofilin and mitochondrial cristae morphology

The structural integrity of mitochondrial cristae is crucial for mitochondrial functions; however, the molecular events controlling the structural integrity and biogenesis of mitochondrial cristae remain to be fully elucidated. Here, we report the functional characterization of a novel mitochondrial protein named CHCM1 (coiled coil helix cristae morphology 1)/CHCHD6. CHCM1/CHCHD6 harbors a coiled coil helix-coiled coil helix domain at its C-terminal end and predominantly localizes to mitochondrial inner membrane. CHCM1/CHCHD6 knockdown causes severe defects in mitochondrial cristae morphology. The mitochondrial cristae in CHCM1/CHCHD6-deficient cells become hollow with loss of structural definitions and reduction in electron-dense matrix. CHCM1/CHCHD6 depletion also leads to reductions in cell growth, ATP production, and oxygen consumption. CHCM1/CHCHD6 through its C-terminal end strongly and directly interacts with the mitochondrial inner membrane protein mitofilin, which is known to also control mitochondrial cristae morphology. CHCM1/CHCHD6 also interacts with other mitofilin-associated proteins, including DISC1 and CHCHD3. Knockdown of CHCM1/CHCHD6 reduces mitofilin protein levels; conversely, mitofilin knockdown leads to reduction in CHCM1 levels, suggesting coordinate regulation between these proteins. Our results further indicate that genotoxic anticancer drugs that induce DNA damage down-regulate CHCM1/CHCHD6 expression in multiple human cancer cells, whereas mitochondrial respiratory chain inhibitors do not affect CHCM1/CHCHD6 levels. CHCM1/CHCHD6 knockdown in human cancer cells enhances chemosensitivity to genotoxic anticancer drugs, whereas its overexpression increases resistance. Collectively, our results indicate that CHCM1/CHCHD6 is linked to regulation of mitochondrial cristae morphology, cell growth, ATP production, and oxygen consumption and highlight its potential as a possible target for cancer therapeutics.     

The mitochondrial inner membrane protein mitofilin exists as a complex with SAM50, metaxins 1 and 2, coiled-coil-helix coiled-coil-helix domain-containing protein 3 and 6 and DnaJC11

A monoclonal antibody (mAb) has been produced which reacts with human mitofilin, a mitochondrial inner membrane protein. This mAb immunocaptures its target protein in association with six other proteins, metaxins 1 and 2, SAM50, CHCHD3, CHCHD6 and DnaJC11, respectively. The first three are outer membrane proteins, CHCHD3 has been assigned to the matrix space, and the other two proteins have not been described in mitochondria previously. The functional role of this new complex is uncertain. However, a role in protein import related to maintenance of mitochondrial structure is suggested as mitofilin helps regulate mitochondrial morphology and at least four of the associated proteins (metaxins 1 and 2, SAM50 and CHCHD3) have been implicated in protein import, while DnaJC11 is a chaperone-like protein that may have a similar role.     

Mutation Screening of the <Emphasis Type="Italic">CHCHD10</Emphasis> Gene in Chinese Patients with Amyotrophic Lateral Sclerosis

Mutations in the coiled-coil-helix-coiled-coil-helix domain-containing protein 10 gene (CHCHD10), involved in mitochondrial function, have recently been reported as a causative gene of amyotrophic lateral sclerosis (ALS). The aim of this study was to obtain the mutation prevalence of CHCHD10 and the phenotypes with mutations in Chinese ALS patients. A cohort of 499 ALS patients including 487 sporadic ALS (SALS) and 12 familial ALS (FALS), from the Department of Neurology, West China Hospital of Sichuan University, were screened for mutations of all exons of the CHCHD10 gene by Sanger sequencing. Novel candidate mutations or variants were confirmed by polymerase chain reaction-restriction fragment length polymorphism in 466 healthy individuals. All patients identified with mutations of CHCHD10 gene were screened for mutations of the common ALS causative genes including C9orf72, SOD1, TARDBP, FUS, PFN1, and SQSTM1. Three heterozygous variants, including two missense mutations (c.275A?>?G (p.Y92C) and c.306G?>?C (p.Q102H)) and a synonymous change c.306G?>?A (p.Q102Q), were found in exon 3 of CHCHD10 in three alive SALS individuals (with the longest disease duration of 8.6 years), all of which were not detected in healthy controls. No mutation in CHCHD10 was identified in FALS patients. No mutation was found in the aforementioned common ALS causative genes in the patients who carried CHCHD10 mutations. The mutation frequency of CHCHD10 (0.4 %, 2/487) in a Chinese SALS population suggests CHCHD10 gene mutation appears to be an uncommon cause of ALS in Chinese populations. CHCHD10 mutations are associated with a slow progression and long disease duration.     

AIFM1 is a component of the mitochondrial disulfide relay that drives complex I assembly through efficient import of NDUFS5

The mitochondrial intermembrane space protein AIFM1 has been reported to mediate the import of MIA40/CHCHD4, which forms the import receptor in the mitochondrial disulfide relay. Here, we demonstrate that AIFM1 and MIA40/CHCHD4 cooperate beyond this MIA40/CHCHD4 import. We show that AIFM1 and MIA40/CHCHD4 form a stable long‐lived complex in vitro, in different cell lines, and in tissues. In HEK293 cells lacking AIFM1, levels of MIA40 are unchanged, but the protein is present in the monomeric form. Monomeric MIA40 neither efficiently interacts with nor mediates the import of specific substrates. The import defect is especially severe for NDUFS5, a subunit of complex I of the respiratory chain. As a consequence, NDUFS5 accumulates in the cytosol and undergoes rapid proteasomal degradation. Lack of mitochondrial NDUFS5 in turn results in stalling of complex I assembly. Collectively, we demonstrate that AIFM1 serves two overlapping functions: importing MIA40/CHCHD4 and constituting an integral part of the disulfide relay that ensures efficient interaction of MIA40/CHCHD4 with specific substrates.     

Dependence of PINK1 accumulation on mitochondrial redox system

Accumulation of PINK1 on the outer mitochondrial membrane (OMM) is necessary for PINK‐mediated mitophagy. The proton ionophores, like carbonyl cyanide m‐chlorophenylhydrazone (CCCP) and carbonyl cyanide‐4‐(trifluoromethoxy)phenylhydrazone (FCCP), inhibit PINK1 import into mitochondrial matrix and induce PINK1 OMM accumulation. Here, we show that the CHCHD4/GFER disulfide relay system in the mitochondrial intermembrane space (IMS) is required for PINK1 stabilization when mitochondrial membrane potential is lost. Activation of CHCHD4/GFER system by mitochondrial oxidative stress or inhibition of CHCHD4/GFER system with antioxidants can promote or suppress PINK1 accumulation, respectively. Thus data suggest a pivotal role of CHCHD4/GFER system in PINK1 accumulation. The amyotrophic lateral sclerosis‐related superoxide dismutase 1 mutants dysregulated redox state and CHCHD4/GFER system in the IMS, leading to inhibitions of PINK1 accumulation and mitophagy. Thus, the redox system in the IMS is involved in PINK1 accumulation and damaged mitochondrial clearance, which may play roles in mitochondrial dysfunction‐related neurodegenerative diseases.     

Metabolic epistasis among apoptosis-inducing factor and the mitochondrial import factor CHCHD4

Hypomorphic mutation of apoptosis-inducing factor (AIF) in the whole body or organ-specific knockout of AIF compromises the activity of respiratory chain complexes I and IV, as it confers resistance to obesity and diabetes induced by high-fat diet. The mitochondrial defect induced by AIF deficiency can be explained by reduced AIF-dependent mitochondrial import of CHCHD4, which in turn is required for optimal import and assembly of respiratory chain complexes. Here we show that, as compared to wild type control littermates, mice with a heterozygous knockout of CHCHD4 exhibit reduced weight gain when fed with a Western style high-fat diet. This finding suggests widespread metabolic epistasis among AIF and CHCHD4. Targeting either of these proteins or their functional interaction might constitute a novel strategy to combat obesity.     

CHCHD2 inhibits apoptosis by interacting with Bcl-x L to regulate Bax activation

Mitochondrial outer membrane permeabilization (MOMP) is a critical control point during apoptosis that results in the release of pro-apoptotic mitochondrial contents such as cytochrome c. MOMP is largely controlled by Bcl-2 family proteins such as Bax, which under various apoptotic stresses becomes activated and oligomerizes on the outer mitochondrial membrane. Bax oligomerization helps promote the diffusion of the mitochondrial contents into the cytoplasm activating the caspase cascade. In turn, Bax is regulated primarily by anti-apoptotic Bcl-2 proteins including Bcl-xL, which was recently shown to prevent Bax from accumulating at the mitochondria. However, the exact mechanisms by which Bcl-xL regulates Bax and thereby MOMP remain partially understood. In this study, we show that the small CHCH-domain-containing protein CHCHD2 binds to Bcl-xL and inhibits the mitochondrial accumulation and oligomerization of Bax. Our data show that in response to apoptotic stimuli, mitochondrial CHCHD2 decreases prior to MOMP. Furthermore, when CHCHD2 is absent from the mitochondria, the ability of Bcl-xL to inhibit Bax activation and to prevent apoptosis is attenuated, which results in increases in Bax oligomerization, MOMP and apoptosis. Collectively, our findings establish CHCHD2, a previously uncharacterized small mitochondrial protein with no known homology to the Bcl-2 family, as one of the negative regulators of mitochondria-mediated apoptosis.Apoptosis is a tightly regulated form of programmed cell death that is critical for proper embryonic development, tissue homeostasis and immune response. Aberrant regulation of apoptosis contributes to a wide range of ailments including autoimmune disorders, neurodegenerative diseases and cancer. Unlike necrotic cell death, apoptosis is a genetic program that is characterized by distinct morphological features such as membrane blebbing, chromatin condensation, DNA fragmentation and cell shrinkage.¹ In vertebrates, apoptosis can occur through two pathways: extrinsic, or receptor-mediated apoptosis, and intrinsic, or mitochondria-mediated apoptosis. Intrinsic apoptosis is induced by cellular stressors such as DNA damage, which lead to mitochondrial outer membrane permeabilization (MOMP), cytochrome c release from the mitochondrial intermembrane space, activation of cysteine proteases (caspases) and induction of apoptosis. Once MOMP occurs, cell death is thought to be inevitable. Therefore, much research has been devoted to elucidating the mechanisms and signaling pathways that govern this critical regulatory point in apoptosis.MOMP is controlled largely by the B-cell lymphoma 2 (Bcl-2) family of proteins,² all of which contain at least one of four BH (Bcl-2 homology) domains designated BH1–4. During apoptosis, the pro-apoptotic Bcl-2 proteins Bax and/or Bak become activated and oligomerize on the mitochondrial outer membrane³ increasing mitochondrial membrane permeabilization through a mechanism that is not entirely clear. Bax and Bak are activated by BH3-only Bcl-2 family proteins such as Bim, t-Bid and Puma.^{4, 5, 6, 7, 8, 9, 10, 11, 12, 13} Conversely, Bax and Bak are inhibited by pro-survival Bcl-2 family proteins such as Bcl-2, Mcl-1 and Bcl-xL.^{2, 14, 15, 16} Of the pro-survival Bcl-2 family proteins, Bcl-2 is found at the outer mitochondrial membrane, whereas Bcl-xL and Mcl-1 localize to the outer mitochondrial membrane and the mitochondrial matrix.^{17, 18} Matrix-localized Bcl-xL and Mcl-1 have been shown to promote mitochondrial respiration,¹⁹ suggesting that crosstalk exists between apoptotic pathways and other mitochondria-based biological events. Based on this recent discovery, one might reason that other mitochondrial proteins previously characterized as structural proteins or metabolism-associated enzymes could play an additional intermediate role in the regulation of apoptosis by interacting with Bcl-2 family proteins.We identified CHCHD2 in a mass spectrometry-based screen for binding partners of p32, a mitochondrial protein previously shown by our lab to bind and mediate the apoptotic effects of the tumor suppressor p14ARF.²⁰ CHCHD2 was subsequently detected in independent screens for proteins that regulate cellular metabolism and migration;^{21, 22} however, the functions of CHCHD2 remain unknown. CHCHD2 is encoded by the chchd2 gene (coiled-coil helix coiled-coil helix domain-containing 2), which spans 4921 base pairs, contains 4 exons, and is located on human chromosome 7p11.2, a chromosomal region that is often amplified in glioblastomas.²³ The protein encoded by the chchd2 gene is ubiquitously expressed²⁴ and is relatively small, as it codes for only 151 amino acids. CHCHD2 is well-conserved among different species from humans to yeast, and mouse and human CHCHD2 share 87% amino acid sequence identity (Supplementary Figures S1A and S1B). CHCHD2 contains a C-terminal CHCH (coiled-coil helix coiled-coil helix) domain, which is characterized primarily by four cysteine residues spaced 10 amino acids apart from one another (CX(9)C motif).²⁵ The function of the CHCH domain is not well understood, and the few characterized proteins that harbor this domain have diverse functions. Many CHCH domain-containing proteins localize to the mitochondrial inner membrane or the intermembrane space, including Cox12, Cox17, Cox19, Cox23, Mia40 (yeast homolog of human CHCHD4), CHCHD3 and CHCHD6. Cox17 and Cox19 aid in the assembly of the COX complex,^{26, 27} whereas Mia40/Tim40 has been shown to transport proteins into the mitochondrial intermembrane space.^{28, 29} Furthermore, CHCHD3 and CHCHD6 are essential for maintaining the integrity of mitochondrial cristae and thus mitochondrial function.^{30, 31, 32} Interestingly, a recent report has shown that CHCHD6 is regulated by DNA damage stress, and alterations in CHCHD6 expression affect the viability of breast cancer cells in response to genotoxic anticancer drugs.³²Despite advances in our understanding of how MOMP and apoptosis are regulated by the Bcl-2 family of proteins, much remains unknown with respect to the mechanisms that lead to Bax activation and oligomerization particularly concerning the roles that mitochondria-associated proteins play in the process. In this study, we characterize the small, mitochondria-localized protein CHCHD2 as a novel regulator of Bax oligomerization and apoptosis. Furthermore, we show evidence that CHCHD2 binds to Bcl-xL at the mitochondria under unstressed conditions. In response to apoptotic stimuli, CHCHD2 decreases and loses its mitochondria localization, which is accompanied by decreased Bcl-xL–Bax interaction and increased Bax homo-oligomerization and Bax–Bak hetero-oligomerization. Collectively, our results suggest that CHCHD2 negatively regulates the apoptotic cascade upstream of Bax oligomerization.     

Synergistic apoptosis induction in leukemic cells by miR-15a/16-1 and arsenic trioxide

Despite the mitochondria ubiquitous nature many of their components display divergences in their expression profile across different tissues. Using the bioinformatics-approach of guilt by association (GBA) we exploited these variations to predict the function of two so far poorly annotated genes: Coiled-coil-helix-coiled-coil-helix domain containing 10 (CHCHD10) and glioblastoma amplified sequence (GBAS). We predicted both genes to be involved in oxidative phosphorylation. Through in vitro experiments using gene-knockdown we could indeed confirm this and furthermore we asserted CHCHD10 to play a role in complex IV activity.