线粒体电压依赖性阴离子通道1在阿尔茨海默病发生机制中的作用

线粒体电压依赖性阴离子通道(voltage-dependent anion channel,VDAC)是存在于线粒体外膜上的一系列孔道蛋白,其亚型之一VDAC1参与线粒体通透性转换(MPT)孔道的功能发挥,调控阳离子、阴离子、ATP、无机磷酸盐以及其他代谢产物进出线粒体。研究发现,随着阿尔茨海默病(AD)的发生发展,VDAC1蛋白表达水平提高,且其与β-淀粉样蛋白(Aβ)及磷酸化tau蛋白相互作用,干扰ATP/ADP、蛋白质等代谢产物的转运。现就VDAC1的结构、特性以及在AD发病中的机制作一综述。     

泛素特异性蛋白酶30在线粒体动力学和自噬中的作用研究进展

线粒体作为细胞的能量中心,在细胞内呈现高度的动态变化,其数量、质量及功能的稳定对维持细胞的正常活动至关重要。线粒体动力学与线粒体自噬之间可互相调控,共同构成线粒体质量控制的重要环节。泛素特异性蛋白酶30 (USP30)作为去泛素化酶,既可通过线粒体融合蛋白1/2 (Mfn1/2)、线粒体动力蛋白相关蛋白1 (Drp1)等融合与分裂蛋白参与调控线粒体动力学过程,还能通过E3泛素连接酶Parkin、泛素(Ub)及电压依赖性阴离子通道1 (VDAC1)等多种信号而调控PTEN诱导激酶1 (PINK1)/Parkin途径介导的线粒体自噬,但其详细机制尚未完全阐明。本文对USP30在调控线粒体动力学和线粒体自噬中的作用与其机制进行了综述。     

致凋亡的声动力疗法诱导巨噬细胞线粒体钙升高的机制研究

目的:研究致凋亡的声动力疗法诱导巨噬细胞线粒体钙升高的机制。方法:应用佛波酯(PMA)诱导THP-1单核细胞分化为巨噬细胞进行实验研究。选用5-氨基酮戊酸(ALA)作为声敏剂,进行声动力治疗(SDT)。应用流式细胞术证实SDT显著促进了细胞凋亡;应用Rhod 2/AM实时监测线粒体Ca~(2+)水平;通过蛋白质免疫印迹对全细胞蛋白中的Bax、Cleaved-caspase3、电压依赖性阴离子通道1(VDAC1)和三磷酸肌醇Ⅲ型受体(IP3R-Ⅲ)进行检测;应用VDAC1抗体进行免疫共沉淀,检测VDAC1和IP3R-Ⅲ之间的相互作用;实时监测线粒体Ca~(2+)水平,检测VDAC抑制剂DIDS和IP3Rs抑制剂2-ABP对SDT效果的影响。结果:与对照组相比,仅SDT组出现了显著的细胞凋亡(P0.001)。与对照组相比,ALA对线粒体Ca~(2+)水平无明显影响,超声诱导了线粒体Ca~(2+)水平的明显升高,SDT诱导了线粒体Ca~(2+)水平快速且大幅度的升高,且去除超声后仍维持在较高水平。与对照组相比,超声对Bax、Cleaved-caspase3、VDAC1和IP3R-Ⅲ的表达无明显影响,ALA诱导了VDAC1(P0.01)和IP3R-Ⅲ表达量的增加(P0.05),SDT诱导了Bax(P0.001)、Cleaved-caspase3(P0.001)、VDAC1(P0.01)和IP3R-Ⅲ(P0.05)表达量的增加,VDAC1和IP3R-Ⅲ的增加幅度与ALA组接近;ALA和SDT均诱导了VDAC1和IP3R-Ⅲ之间相互作用的显著增强(P0.05)。DIDS和2-ABP均明显抑制了SDT诱导的线粒体Ca~(2+)增加。结论:在致凋亡的SDT作用于THP-1巨噬细胞的过程中,ALA诱导了线粒体外膜Ca~(2+)转运通道VDAC1和内质网重要Ca~(2+)转运通道IP3R-Ⅲ的表达量增加与二者间相互作用的增强,在内质网和线粒体之间建立了大量的Ca~(2+)转运通道,超声的作用则在于触发这些Ca~(2+)转运通道的开放,进而引发线粒体钙的迅速增加。这是后续线粒体凋亡通路启动的重要机制之一。     

氧化修饰在调控细胞凋亡信号转导中的作用

氧化修饰是细胞内的活性氧诱导生物大分子发生氧化反应引起的结构及构象改变,发挥调控信号转导和对应激作出反应的功能。氧化修饰发生在凋亡信号转导中的多个生物大分子,包括凋亡相关蛋白质的氧化,如caspase-9、线粒体通透性转变孔及电压依赖的阴离子通道(voltagedependent anion channel,VDAC),同时也包括膜磷脂的氧化修饰,如磷脂酰丝氨酸及线粒体特异的心磷脂。氧化修饰作用也涉及凋亡诱导因子、促凋亡的凋亡信号调控激酶1(apoptosis signalregulatin gkinasel,ASK1)信号转导途径及抗凋亡的转录因子NF—kB的激活和活性。所以氧化修饰可能是调控凋亡信号转导机制中除磷酸化、泛素化外的另一个新的分子机制。     

二甲双胍对人膀胱癌细胞能量代谢的调控作用分析

目的:检测二甲双胍对人膀胱肿瘤细胞能量代谢的作用及分子机制。方法:将膀胱肿瘤细胞分为4组,分别用终浓度为0、20、40、60 mmol/L的二甲双胍处理,比色法检测各组葡萄糖的消耗和乳酸的生成,用ATP检测试剂盒检测各组ATP水平,用JC-1膜电位检测试剂盒检测二甲双胍对膀胱肿瘤细胞膜电位的影响,通过real-time PCR检测己糖激酶2(HK2)和电压依赖性阴离子通道(VDAC)的mRNA表达水平,采用Western印迹检测每组中HK2、VDAC、磷酸化信号转导与转录激活因子3(p-STAT3)的蛋白表达水平变化。结果:在20 mmol/L二甲双胍下,膀胱肿瘤细胞葡萄糖消耗增加,乳酸产生受到抑制,ATP产生和细胞线粒体膜电位降低,HK2、VDAC和p-STAT3的表达降低。结论:二甲双胍可能通过抑制HK2、VDAC和p-STAT3的表达来阻断膀胱肿瘤的糖酵解和线粒体功能,这为研究二甲双胍对肿瘤的抑制作用机制奠定了理论和实验基础。     

LRRC8A细胞模型的构建及其生理特性研究

本文旨在构建容积调控性阴离子通道主要成分LRRC8A的细胞模型,并应用该模型研究LRRC8A的生理特性。构建LRRC8A和YFP-H148Q/I152L真核表达载体,应用脂质体转染、抗生素筛选和有限稀释,获取共表达LRRC8A和YFP-H148Q/I152L的Fisher大鼠甲状腺滤泡上皮(Fischer rat thyroid, FRT)细胞。倒置荧光显微镜观察目的基因表达情况,荧光淬灭动力学实验检测LRRC8A和YFP-H148Q/I152L的功能。获得用于研究LRRC8A容积调控性阴离子通道的细胞模型,并应用该细胞模型研究LRRC8A的生理特性,包括阴离子转运特性、渗透压对LRRC8A的开放、阴离子转运速度的影响以及氯离子通道抑制剂对LRRC8A的作用。结果显示:(1)成功获得共表达LRRC8A和YFP-H148Q/I152L的FRT细胞,该细胞模型可用于LRRC8A容积调控性氯离子通道生理特性的研究。(2)在低渗状态下,LRRC8A容积调控性阴离子通道激活,可转运阴离子,如:碘离子和氯离子等;YFP-H148Q/I152L可用于研究阴离子的转运速度;渗透压是LRRC8A容积调控性阴离子通道开放的调控因素,其开放与渗透压呈负相关;氯离子通道抑制剂对LRRC8A通道的转运功能具有抑制作用,并呈剂量依赖关系。上述结果提示,本研究成功构建LRRC8A细胞模型,且应用该模型研究显示LRRC8A具有经典的容积调控性阴离子通道的特性。     

miRNA调控线粒体功能的研究进展

MiRNA为小分子非编码RNA,通过与靶基因的相互作用调节靶基因的表达,参与调控细胞的多个生物学过程。本文综述了miRNA与线粒体生物合成、线粒体动力学、线粒体能量代谢、线粒体钙稳态、线粒体自噬间的关系及其调节机制,阐述了microRNA调节线粒体功能的研究进展。     

线粒体与细胞凋亡调控

细胞凋亡是一个受到一系列相关基因严格调控的细胞死亡过程。线粒体是细胞凋亡调控的活动中心。在凋亡因子的刺激下，线粒体释放出不同促凋亡因子如细胞色素C、Smac／Diablo等，激活细胞内凋亡蛋白酶Caspase。我们发现，活化后的Caspase可以反过来作用于线粒体，引发更大量线粒体细胞色素c的释放，构成细胞色素c释放的正反馈调节机制，从而导致电子传递链的中断、膜电势的丧失、胞内ROS的升高以及线粒体产生ATP功能的完全丧失。Bcl-2家族蛋白在细胞色素C释放和细胞凋亡调控中起关键作用。     

血管内皮细胞的Cl-通道--电特性和有关细胞功能

内皮细胞在心血管系统具有重要功能,除通过分泌内皮舒张因子－－一氧化氮（ＮＯ）及收缩性物质内皮素等控制血管平滑肌张力外,并能调节血管通透性。近年来发现内皮细胞上的Ｃ１＾－通道能调节细胞体积和细胞膜电位的稳定性。通过离子通道调控膜电位一机理,能较好理解血管内皮的功能,并可望由此开拓新型血管药物。本文综述了内皮细胞的Ｃ１＾－通道的电生理特性、类别,并探讨该通道调控细胞体积,ＮＯ的分泌及调控细胞膜电位的可     

肠道微生物与线粒体之间的互作

肠道微生物与肠道细胞线粒体功能之间的关系十分密切。一方面,肠道微生物可直接或通过短链脂肪酸、硫化氢和一氧化氮等代谢产物间接影响与线粒体相关的能量代谢过程,调节线粒体活性氧的产生,调控线粒体甚至整个机体的免疫反应。另一方面,肠道细胞线粒体功能紊乱和基因组的遗传变异也会影响肠道微生物的组成和功能。本文主要介绍了肠道微生物和线粒体之间的互作关系的最新研究进展,为靶向作用于肠道菌群和线粒体以调节肠道健康提供理论依据。     

The voltage-dependent anion channel

Recently, it has been recognized that there is a metabolic coupling between the cytosol and mitochondria, where the outer mitochondrial membrane (OMM), the boundary between these compartments, has important functions. In this crosstalk, mitochondrial Ca2+ homeostasis and ATP production and supply play a major role. The primary transporter of ions and metabolites across the OMM is the voltage-dependent anion channel (VDAC). The interaction of VDAC with Ca2+, ATP glutamate, NADH, and different proteins was demonstrated, and these interactions may regulate OMM permeability. This review includes information on VDAC purification methods, characterization of its channel activity (selectivity, voltage-dependence, conductance), and the regulation of VDAC channel by ligands, such as Ca2+, glutamate and ATP and touches on many aspects of the physiological relevance of VDAC to Ca2+ homeostasis and mitochondria-mediated apoptosis.     

The Voltage-dependent Anion Channel in Endoplasmic/Sarcoplasmic Reticulum: Characterization,Modulation and Possible Function

In recent years, it has been recognized that there is a metabolic coupling between the cytosol, ER/SR and mitochondria. In this cross-talk, mitochondrial Ca²⁺ homeostasis and ATP production and supply play a major role. The primary transporter of adenine nucleotides, Ca²⁺and other metabolites into and out of mitochondria is the voltage-dependent anion channel (VDAC) located at the outer mitochondrial membrane, at a crucial position in the cell. VDAC has been established as a key player in mitochondrial metabolite and ion signaling and it has also been proposed that VDAC is present in extramitochondrial membranes. Thus, regulation of VDAC, as the main interface between mitochondrial and cellular metabolism, by other molecules is of utmost importance. This article reviews localization and function of VDAC, and focuses on VDAC as a skeletal muscle sarcoplasmic reticulum channel. The regulation of VDAC activity by associated proteins and by inhibitors is also presented. Several aspects of the physiological relevance of VDAC to Ca²⁺ homeostasis and mitochondria-mediated apoptosis will be discussed.     

Molecular and genetic characterization of the gene family encoding the voltage-dependent anion channel in Arabidopsis

The voltage-dependent anion channel (VDAC), a major outer mitochondrial membrane protein, is thought to play an important role in energy production and apoptotic cell death in mammalian systems. However, the function of VDACs in plants is largely unknown. In order to determine the individual function of plant VDACs, molecular and genetic analysis was performed on four VDAC genes, VDAC1-VDAC4, found in Arabidopsis thaliana. VDAC1 and VDAC3 possess the eukaryotic mitochondrial porin signature (MPS) in their C-termini, while VDAC2 and VDAC4 do not. Localization analysis of VDAC-green fluorescent protein (GFP) fusions and their chimeric or mutated derivatives revealed that the MPS sequence is important for mitochondrial localization. Through the functional analysis of vdac knockout mutants due to T-DNA insertion, VDAC2 and VDAC4 which are expressed in the whole plant body are important for various physiological functions such as leaf development, the steady state of the mitochondrial membrane potential, and pollen development. Moreover, it was demonstrated that VDAC1 is not only necessary for normal growth but also important for disease resistance through regulation of hydrogen peroxide generation.     

Reflections on VDAC as a voltage-gated channel and a mitochondrial regulator

There is excellent agreement between the electrophysiological properties and the structure of the mitochondrial outer membrane protein, VDAC, ex vivo. However, the inference that the well-defined canonical “open” state of the VDAC pore is the normal physiological state of the channel in vivo is being challenged by several lines of evidence. Knowing the atomic structure of the detergent solubilized protein, a long sought after goal, will not be sufficient to understand the functioning of this channel protein. In addition, detailed information about VDAC’s topology in the outer membrane of intact mitochondria, and the structural changes that it undergoes in response to different stimuli in the cell will be needed to define its physiological functions and regulation.     

The VDAC channel as the mitochondria function regulator

Regulation of mitochondria physiology, indispensable for proper cell activity, requires an efficient exchange of molecules between mitochondria and cytoplasm at the level of the mitochondrial outer membrane. The common pathway for the metabolite exchange between mitochondria and cytoplasm is the VDAC channel (voltage dependent anion channel), known also as mitochondrial porin. The channel was identified for the first time in 1976 and since that time has been extensively studied. It has been recognized that the VDAC channel plays a crucial role in the regulation of metabolic and energetic functions of mitochondria. In this article we review the VDAC channel relevance to ATP rationing, Ca2+ homeostasis, protection against oxidative stress and apoptosis execution.     

Plasmalemmal VDAC controversies and maxi-anion channel puzzle

The maxi-anion channel has been observed in many cell types from the very beginning of the patch-clamp era. The channel is highly conductive for chloride and thus can modulate the resting membrane potential and play a role in fluid secretion/absorption and cell volume regulation. A wide nanoscopic pore of the maxi-anion channel permits passage of excitatory amino acids and nucleotides. The channel-mediated release of these signaling molecules is associated with kidney tubuloglomerular feedback, cardiac ischemia/hypoxia, as well as brain ischemia/hypoxia and excitotoxic neurodegeneration. Despite the ubiquitous expression and physiological/pathophysiological significance, the molecular identity of the maxi-anion channel is still obscure. VDAC is primarily a mitochondrial protein; however several groups detected it on the cellular surface. VDAC in lipid bilayers reproduced the most important biophysical properties of the maxi-anion channel, such as a wide nano-sized pore, closure in response to moderately high voltages, ATP-block and ATP-permeability. However, these similarities turned out to be superficial, and the hypothesis of plasmalemmal VDAC as the maxi-anion channel did not withstand the test by genetic manipulations of VDAC protein expression. VDAC on the cellular surface could also function as a ferricyanide reductase or a receptor for plasminogen kringle 5 and for neuroactive steroids. These ideas, as well as the very presence of VDAC on plasmalemma, remain to be scrutinized by genetic manipulations of the VDAC protein expression. This article is part of a Special Issue entitled: VDAC structure, function, and regulation of mitochondrial metabolism.     

Direct modulation of the outer mitochondrial membrane channel,voltage-dependent anion channel 1 (VDAC1) by cannabidiol: a novel mechanism for cannabinoid-induced cell death

Cannabidiol (CBD) is a non-psychoactive plant cannabinoid that inhibits cell proliferation and induces cell death of cancer cells and activated immune cells. It is not an agonist of the classical CB1/CB2 cannabinoid receptors and the mechanism by which it functions is unknown. Here, we studied the effects of CBD on various mitochondrial functions in BV-2 microglial cells. Our findings indicate that CBD treatment leads to a biphasic increase in intracellular calcium levels and to changes in mitochondrial function and morphology leading to cell death. Density gradient fractionation analysis by mass spectrometry and western blotting showed colocalization of CBD with protein markers of mitochondria. Single-channel recordings of the outer-mitochondrial membrane protein, the voltage-dependent anion channel 1 (VDAC1) functioning in cell energy, metabolic homeostasis and apoptosis revealed that CBD markedly decreases channel conductance. Finally, using microscale thermophoresis, we showed a direct interaction between purified fluorescently labeled VDAC1 and CBD. Thus, VDAC1 seems to serve as a novel mitochondrial target for CBD. The inhibition of VDAC1 by CBD may be responsible for the immunosuppressive and anticancer effects of CBD.     

TSPO interacts with VDAC1 and triggers a ROS-mediated inhibition of mitochondrial quality control

The 18-kDa TSPO (translocator protein) localizes on the outer mitochondrial membrane (OMM) and participates in cholesterol transport. Here, we report that TSPO inhibits mitochondrial autophagy downstream of the PINK1-PARK2 pathway, preventing essential ubiquitination of proteins. TSPO abolishes mitochondrial relocation of SQSTM1/p62 (sequestosome 1), and consequently that of the autophagic marker LC3 (microtubule-associated protein 1 light chain 3), thus leading to an accumulation of dysfunctional mitochondria, altering the appearance of the network. Independent of cholesterol regulation, the modulation of mitophagy by TSPO is instead dependent on VDAC1 (voltage-dependent anion channel 1), to which TSPO binds, reducing mitochondrial coupling and promoting an overproduction of reactive oxygen species (ROS) that counteracts PARK2-mediated ubiquitination of proteins. These data identify TSPO as a novel element in the regulation of mitochondrial quality control by autophagy, and demonstrate the importance for cell homeostasis of its expression ratio with VDAC1.     

The mitochondrial channel VDAC has a cation-selective open state

The mitochondrial channel VDAC is known to have two major classes of functional states, a large conductance "open" state that is anion selective, and lower conductance substates that are cation selective. The channel can reversibly switch between open and half-open states, with the latter predominant at increasing membrane voltages of either polarity. We report the presence of a new functional state of VDAC, a cation-selective state with conductance approximately equal to that of the canonical open state. This newly described state of VDAC can be reached from either the half-open cation-selective state or from the open anion-selective state. The latter transition implies that a mechanism exists for selectivity gating in VDAC that is separate from partial closure, which may be relevant to the physiological regulation of this channel and mitochondrial outer membrane permeability.     

TSPO interacts with VDAC1 and triggers a ROS-mediated inhibition of mitochondrial quality control

The 18-kDa TSPO (translocator protein) localizes on the outer mitochondrial membrane (OMM) and participates in cholesterol transport. Here, we report that TSPO inhibits mitochondrial autophagy downstream of the PINK1-PARK2 pathway, preventing essential ubiquitination of proteins. TSPO abolishes mitochondrial relocation of SQSTM1/p62 (sequestosome 1), and consequently that of the autophagic marker LC3 (microtubule-associated protein 1 light chain 3), thus leading to an accumulation of dysfunctional mitochondria, altering the appearance of the network. Independent of cholesterol regulation, the modulation of mitophagy by TSPO is instead dependent on VDAC1 (voltage-dependent anion channel 1), to which TSPO binds, reducing mitochondrial coupling and promoting an overproduction of reactive oxygen species (ROS) that counteracts PARK2-mediated ubiquitination of proteins. These data identify TSPO as a novel element in the regulation of mitochondrial quality control by autophagy, and demonstrate the importance for cell homeostasis of its expression ratio with VDAC1.