NADPH oxidase is involved in angiotensin II-induced apoptosis in H9C2 cardiac muscle cells: effects of apocynin

Angiotensin II stimulates NADPH oxidase activity in vascular cells. However, it is not fully understood whether angiotensin II, which plays an important role in heart failure, stimulates NADPH oxidase activation and expression in cardiac myocytes. Previous studies have shown that angiotensin II induces myocyte apoptosis, but whether the change is mediated via NADPH oxidase remains to be elucidated. In this study we proposed to determine whether angiotensin II stimulated NADPH oxidase activation and NADPH oxidase subunit p47-phox expression in H9C2 cardiac muscle cells. If so, we would determine whether the NADPH oxidase inhibitor apocynin prevented angiotensin II-induced apoptosis. The results showed that angiotensin II increased NADPH oxidase activity, p47-phox protein and mRNA expression, intracellular reactive oxygen species, and apoptosis in H9C2 cells. Angiotensin II elevated p38 mitogen-activated protein kinase (MAPK) activity, decreased Bcl-2 protein, and increased Bax protein and caspase-3 activity. Apocynin treatment inhibited angiotensin II-induced NADPH oxidase activation and increases in p47-phox expression, intracellular reactive oxygen species, and apoptosis. The effect of apocynin on apoptosis was associated with reduced p38 MAPK activity, increased Bcl-2 protein, and decreased Bax protein and caspase-3 activity. These results suggest that angiotensin II-induced apoptosis is mediated via NADPH oxidase activation probably through p38 MAPK activation, a decrease in Bcl-2 protein, and caspase activation.     

Mechanism of mitochondrial stress-induced resistance to apoptosis in mitochondrial DNA-depleted C2C12 myocytes

In this study, we show that partial mitochondrial DNA (mtDNA) depletion (mitochondrial stress) induces resistance to staurosporine (STP)-mediated apoptosis in C2C12 myoblasts. MtDNA-depleted cells show a 3-4-fold increased proapoptotic proteins (Bax, BAD and Bid), markedly increased antiapoptotic Bcl-2, and reduced processing of p21 Bid to active tBid. The protein levels and also the ability to undergo STP-mediated apoptosis were restored in reverted cells containing near-normal mtDNA levels and restored mitochondrial transmembrane potential. Inhibition of apoptosis closely correlated with sequestration of Bax, Bid and BAD in the mitochondrial inner membrane, increased Bcl-2 and Bcl-X(L), and inability to process p21 Bid. These factors, together with the reduced activation of caspases 3, 9 and 8 are possible causes of mitochondrial stress-induced resistance to apoptosis. Our results suggest that a highly proliferative and invasive behavior of mtDNA-depleted C2C12 cells is related to their resistance to apoptosis.     

Hydrogen peroxide induces apoptosis of BJAB cells due to formation of hydroxyl radicals via intracellular iron-mediated Fenton chemistry in glucose oxidase-mediated oxidative stress

The aim of this study was to determine if hydrogen peroxide (H2O2) generated by glucose oxidase (GO) induces apoptosis or necrosis of BJAB cells and which radical is the direct mediator of cell death. We found that GO produced H2O2 continuously in low concentrations, similar to in vivo conditions, and decreased proliferation and cell viability in a dose-dependent manner. The GO-mediated cytotoxicity resulted from apoptosis, and was confirmed by monitoring the cells after H33342/Annexin V/propidium iodide staining. Decreases of mitochondrial membrane potential and intracellular glutathione level were found to be critical events in the H2O2-mediated apoptosis. Additional experiments revealed that H2O2 exerted its apoptotic action through the formation of hydroxyl radicals via the Fenton rather than the Haber-Weiss reaction. Moreover, intracellular redox-active iron, but not copper, participated in the H2O2-mediated apoptosis.     

Cmc1p is a conserved mitochondrial twin CX9C protein involved in cytochrome c oxidase biogenesis

Copper is an essential cofactor of two mitochondrial enzymes: cytochrome c oxidase (COX) and Cu-Zn superoxide dismutase (Sod1p). Copper incorporation into these enzymes is facilitated by metallochaperone proteins which probably use copper from a mitochondrial matrix-localized pool. Here we describe a novel conserved mitochondrial metallochaperone-like protein, Cmc1p, whose function affects both COX and Sod1p. In Saccharomyces cerevisiae, Cmc1p localizes to the mitochondrial inner membrane facing the intermembrane space. Cmc1p is essential for full expression of COX and respiration, contains a twin CX9C domain conserved in other COX assembly copper chaperones, and has the ability to bind copper(I). Additionally, mutant cmc1 cells display increased mitochondrial Sod1p activity, while CMC1 overexpression results in decreased Sod1p activity. Our results suggest that Cmc1p could play a direct or indirect role in copper trafficking and distribution to COX and Sod1p.     

Presence of an inactive protein immunologically analogous to cytochrome c oxidase in the inner membrane of sweet potato root mitochondria

A protein, which was immunoreactive to antibody against cytochrome c oxidase, was found in the mitochondrial membrane fraction of sweet potato root tissue. The protein was associated relatively weakly with the mitochondrial inner membrane as compared with cytochrome c oxidase. It exerted no cytochrome c oxidase activity and contained no heme a. The protein was purified by phenyl-Sepharose column chromatography and polyacrylamide gel electrophoresis. The molecular weight of its polypeptide chain was 57,000. In addition, the protein decreased during aging of tissue slices. It is therefore not improbable that the protein is a precursor of cytochrome c oxidase composed of only the subunits of cytoplasmic origin, since aging of tissue slices has been shown to result in an increase in the enzyme activity which is inhibited by chloramphenicol but not by cycloheximide.     

Supplemental ascorbate or alpha-tocopherol induces cell death in Cu-deficient HL-60 cells

Cytochrome c oxidase (CCO) is the Cu-dependent, terminal respiratory complex of the mitochondrial electron transport chain. Inhibition of CCO can promote oxidative stress by increasing mitochondrial production of reactive oxygen species (ROS). Because mitochondria have an important role in apoptosis as both a target and source for ROS, enhanced ROS production resulting from inhibition of CCO by Cu deficiency may trigger apoptosis. The present study focuses on the mitochondrial effects of N,N'-bis(2-aminoethyl)-1,3-propanedi-amine (TET), which inhibits CCO by causing cellular Cu deficiency, and the antioxidants ascorbate and alpha-tocopherol in a human promyelocytic leukemia cell line (HL-60). The following effects were observed: (i) TET reduced both cell growth and viability only in the presence of ascorbate or alpha-tocopherol; (ii) TET reduced CCO activity and increased mitochondrial ROS production as indicated by increased expression of Mn super-oxide dismutase, but the induction of Mn superoxide dismutase was not affected by ascorbate or alpha-tocopherol; (iii) TET acted independently of ascorbate or alpha-tocopherol in disrupting mitochondrial membrane potential; (iv) TET did not increase caspase-8 activity in the absence of ascorbate or alpha-tocopherol; and (v) TET did not increase transfer of cytochrome c from mitochondria to the cytosol unless alpha-tocopherol was present. These findings indicate that reduction in CCO activity by TET-induced Cu deficiency increased oxidative stress in HL-60 cells sufficiently to disrupt the electrochemical gradient of the inner mitochondrial membrane but did not trigger cell death. Also, ascorbate and alpha-tocopherol did not alleviate oxidative stress but may have become pro-oxidants, adding to the oxidant burden sufficiently to trigger cell death in TET-treated cells.     

A role for Pet100p in the assembly of yeast cytochrome c oxidase: interaction with a subassembly that accumulates in a pet100 mutant

The biogenesis of multimeric protein complexes of the inner mitochondrial membrane in yeast requires a number of nuclear-coded ancillary proteins. One of these, Pet100p, is required for cytochrome c oxidase. Previous studies have shown that Pet100p is not required for the synthesis, processing, or targeting of cytochrome c oxidase subunits to the mitochondrion nor for heme A biosynthesis. Here, we report that Pet100p does not affect the localization of cytochrome c oxidase subunit polypeptides to the inner mitochondrial membrane but instead functions after they have arrived at the inner membrane. We have also localized Pet100p to the inner mitochondrial membrane in wild type cells, where it is present in a subassembly (Complex A) with cytochrome c oxidase subunits VII, VIIa, and VIII. Pet100p does not interact with the same subunits after they have been assembled into the holoenzyme. In addition, we have identified two subassemblies that are present in pet100 null mutant cells: one subassembly (Complex A') is composed of subunits VII, VIIa, and VIII but not Pet100p, and another subassembly (Complex B) is composed of subunits Va and VI. Because pet100 null mutant cells lack assembled cytochrome c oxidase but accumulate Complexes A' and B it appears likely that these subassemblies of cytochrome c oxidase subunits are intermediates along an assembly pathway for holocytochrome c oxidase and that Pet100p functions in this pathway to facilitate the interaction(s) between Complex A' and other cytochrome c oxidase subassemblies and subunits.     

Early events in Bcl-2-enhanced apoptosis

Transfection of PC12 pheochromocytoma cells with bcl-2 potentiates apoptosis induced by the antimitotic agent, neocarzinostatin (NCS). The mechanism of potentiation involves caspase 3-dependent cleavage of Bcl-2 to its pro-apoptotic counterpart, but the cellular events proximal to caspase 3 activation in this system are not known. Two min after initiation of NCS treatment, Bax begins to translocate from cytosol to the mitochondria; the mitochondrial localization of Bax persists for 30 min after NCS treatment. At the same time, cytochrome C is released from the mitochondria to cytosol. The mitochondrial membrane potential exhibits differential change in mock- and bcl-2-transfected PC12 cells. In mock-transfected PC12 cells, the mitochondrial membrane potential increases immediately, peaks at 15 min following initiation of NCS treatment, and drops thereafter. In contrast, in bcl-2-transfected PC12 cells, the membrane potential drops immediately following NCS treatment. Caspase 9 is activated and peaks at 10 min in both mock- and bcl-2 transfected PC12 cells, however, the peak activity of caspase 9 is higher and caspase 9 activation lasts longer (30 min) after the treatment in bcl-2 transfectants. Not until 30 min after initiation of a 1 h treatment with NCS is Bcl-2 protein cleaved in bcl-2-transfected cells. Thus, in bcl-2-transfected cells, the mitochondrial membrane potential drops and cytochrome C is released from the mitochondria despite the presence of large amounts of intact mitochondrial Bcl-2. This makes it unlikely that cleavage of Bcl-2 is the only factor involved in potentiation of NCS-induced apoptosis by Bcl-2.     

Mechanism for generation of hydrogen peroxide and change of mitochondrial membrane potential during rotenone-induced apoptosis

Rotenone, an inhibitor of NADH dehydrogenase complex, is a naturally occurring insecticide, which is capable of inducing apoptosis. Rotenone-induced apoptosis is considered to contribute to its anticancer effect and the etiology of Parkinson's disease (PD). We demonstrated that rotenone induced internucleosomal DNA fragmentation, DNA ladder formation, in human cultured cells, HL-60 (promyelocytic leukemia) and BJAB cells (B-cell lymphoma). Flow cytometry showed that rotenone induced H2O2 generation, followed by significant changes in the mitochondrial membrane potential (DeltaPsim). Caspase-3 activity increased in HL-60 cells in a time-dependent manner. These apoptotic events were delayed in HP100 cells, an H2O2-resistant clone of HL-60, confirming the involvement of H2O2 in apoptosis. Expression of anti-apoptotic protein, Bcl-2, in BJAB cells drastically inhibited DeltaPsim change and DNA ladder formation but not H2O2 generation, confirming the participation of mitochondrial dysfunction in apoptosis. NAD(P)H oxidase inhibitors prevented H2O2 generation and DNA ladder formation. These results suggest that rotenone induces O2(-)-derived H2O2 generation through inhibition of NADH dehydrogenase complex and/or activation of NAD(P)H oxidase, and H2O2 generation causes the disruption of mitochondrial membrane in rotenone-induced apoptosis.     

Interrelationships of copper, cytochrome oxidase, phospholipid synthesis and adenine nucleotide binding.

Studies are reported on the interrelationships in liver mitochondria of copper status, cytochrome oxidase activity, adenine nucleotide binding capacity and phospholipid synthesis. Direct exposure of mitochondria to cyanide or diethyldithiocarbamate depressed cytochrome oxidase activity; ADP-binding and phospholipid synthesis. Fractionation of mitochondria to increase the specific activity of cytochrome oxidase about 10-fold did not increase the affinity to bind ADP. Ageing of mitochondria or dialysis of mitochondria or mitochondrial membrane preparations against water or diethyldithiocarbamate at 0--2 degrees for 18 h did not decrease cytochrome oxidase activity or copper content of reisolated and resuspended mitochondria or mitochondrial membrane preparations, but considerably reduced the affinity to bind ADP. The respiratory inhibitors, fluoride and azide, at concentrations inhibitory to cytochrome oxidase did not reduce ADP-binding or phospholipid synthesis. Atractyloside did not inhibit cytochrome oxidase activity but did inhibit ADP-binding and phospholipid synthesis. Pre-incubation of mitochondrial membrane preparations with Cu++ increased the copper content and ADP-binding affinity. The results indicate that cytochrome oxidase is not the ADP-binding site of the mitochondrial membrane system and that reduced cytochrome oxidase activity per se does not depress binding affinity. Copper appears to be a component of the adenine nucleotide binding sites of mitochondrial membranes because the copper-complexing agents, cyanide and diethyldithiocarbamate, depressed ADP-binding, while increased mitochondrial membrane copper content increased ADP-binding.     

Parthenolide regulates oxidative stress-induced mitophagy and suppresses apoptosis through p53 signaling pathway in C2C12 myoblasts

Muscle redox disturbances and oxidative stress have emerged as a common pathogenetic mechanism and potential therapeutic intervention in some muscle diseases. Parthenolide (PTL), a sesquiterpene lactone found in large amounts in the leaves of feverfew, possesses anti-inflammatory, anti-migraine, and anticancer properties. Although PTL was reported to alleviate cancer cachexia and improve skeletal muscle characteristics in a cancer cachexia model, its actions on oxidative stress-induced damage in C2C12 myoblasts have not been reported and the regulatory mechanisms have not yet been defined. In our study, PTL attenuated H₂O₂-induced growth inhibition and morphological changes. Furthermore, PTL exhibited scavenging activity against reactive oxygen species and protected C2C12 cells from apoptosis in response to H₂O₂. Meanwhile, PTL suppressed collapse of the mitochondrial membrane potential, thereby contributing to normalizing H₂O₂-induced autophagy flux and mitophagy, correlating with inhibiting degradation of mitochondrial marker protein TIM23, the increase in LC3-II expression and the reduction of mitochondria DNA. Besides its protective effect on mitochondria, PTL also prevented H₂O₂-induced lysosomes damage in C2C12 cells. In addition, the phosphorylation of p53, cathepsin B, and Bax/Bcl-2 protein levels, and the translocation of Bax from the cytosol to mitochondria induced by H₂O₂ in C2C12 cells was significantly reduced by PTL. In conclusion, PTL modulates oxidative stress-induced mitophagy and protects C2C12 myoblasts against apoptosis, suggesting a potential protective effect against oxidative stress-associated skeletal muscle diseases.     

Unfolding-resistant translocase targeting: a novel mechanism for outer mitochondrial membrane localization exemplified by the Bbeta2 regulatory subunit of protein phosphatase 2A

Heterotrimeric serine/threonine protein phosphatase 2A (PP2A) consists of scaffolding (A), catalytic (C), and variable (B, B', and B') subunits. Variable subunits dictate subcellular localization and substrate specificity of the PP2A holoenzyme. The Bbeta regulatory subunit gene is mutated in spinocerebellar ataxia type 12, and one of its splice variants, Bbeta2, targets PP2A to mitochondria to promote apoptosis in PC12 cells (Dagda, R. K., Zaucha, J. A., Wadzinski, B. E., and Strack, S. (2003) J. Biol. Chem. 278, 24976-24985). Here, we report that Bbeta2 is localized to the outer mitochondrial membrane by a novel mechanism, combining a cryptic mitochondrial import signal with a structural arrest domain. Scanning mutagenesis demonstrates that basic and hydrophobic residues mediate mitochondrial association and the proapoptotic activity of Bbeta2. When fused to green fluorescent protein, the N terminus of Bbeta2 acts as a cleavable mitochondrial import signal. Surprisingly, full-length Bbeta2 is not detectably cleaved and is retained at the outer mitochondrial membrane, even though it interacts with the TOM22 import receptor, as shown by luciferase complementation in intact cells. Mutations that open the C-terminal beta-propeller of Bbeta2 facilitate mitochondrial import, indicating that this rigid fold acts as a stop-transfer domain by resisting the partial unfolding step prerequisite for matrix translocation. Because hybrids of prototypical import and beta-propeller domains recapitulate this behavior, we predict the existence of other similarly localized proteins and a selection against highly stable protein folds in the mitochondrial matrix. This unfolding-resistant targeting to the mitochondrial translocase is necessary but not sufficient for the proapoptotic activity of Bbeta2, which also requires association with the rest of the PP2A holoenzyme.     

Differential gene expression during apoptosis induced by a serum factor: Role of mitochondrial F<Subscript>0</Subscript>-F<Subscript>1</Subscript> ATP synthase complex

The number of genes that are up regulated or down regulated during apoptosis is large and still increasing. In an attempt to characterize differential gene expression during serum factor induced apoptosis in AK-5 cells (a rat histiocytoma), we found subunit 6 and subunit 8 of the transmembrane proton channel and subunit alpha of the catalytic core of the mitochondrial F₀-F₁ ATP synthase complex to be up regulated during apoptosis. The increase in the expression levels of these subunits was concomitant with a transient increase in the intracellular ATP levels, suggesting that the increase in cellular ATP content is a result of the increase in the expression of ATP synthase subunits' gene and de novo protein synthesis. Depleting the cellular ATP levels with oligomycin inhibited apoptosis significantly, pointing to the requirement of ATP during apoptosis. Caspase 1 and caspase 3 activity and the loss of mitochondrial membrane potential were also inhibited by oligomycin during apoptosis in these cells, suggesting that the oligomycin induced inhibition of apoptosis could be due to inhibition of caspase activity and inhibition of mitochondrial depolarization. However, cytochrome C release during apoptosis was found to be completely independent of intracellular ATP content. Besides the ATP synthase complex genes, other mitochondrial genes like cytochrome C oxidase subunit II and III also showed elevated levels of expression during apoptosis. This kind of a mitochondrial gene expression profile suggests that in AK-5 cells, these genes are upregulated in a time-linked manner to ensure sufficient intracellular ATP levels and an efficient functioning of the mitochondrial respiratory chain for successful completion of the apoptotic pathway.     

COX16 is required for assembly of cytochrome c oxidase in human cells and is involved in copper delivery to COX2

Cytochrome c oxidase (COX), complex IV of the mitochondrial respiratory chain, is comprised of 14 structural subunits, several prosthetic groups and metal cofactors, among which copper. Its biosynthesis involves a number of ancillary proteins, encoded by the COX-assembly genes that are required for the stabilization and membrane insertion of the nascent polypeptides, the synthesis of the prosthetic groups, and the delivery of the metal cofactors, in particular of copper. Recently, a modular model for COX assembly has been proposed, based on the sequential incorporation of different assembly modules formed by specific subunits.We have cloned and characterized the human homologue of yeast COX16. We show that human COX16 encodes a small mitochondrial transmembrane protein that faces the intermembrane space and is highly expressed in skeletal and cardiac muscle. Its knockdown in C. elegans produces COX deficiency, and its ablation in HEK293 cells impairs COX assembly. Interestingly, COX16 knockout cells retain significant COX activity, suggesting that the function of COX16 is partially redundant.Analysis of steady-state levels of COX subunits and of assembly intermediates by Blue-Native gels shows a pattern similar to that reported in cells lacking COX18, suggesting that COX16 is required for the formation of the COX2 subassembly module. Moreover, COX16 co-immunoprecipitates with COX2. Finally, we found that copper supplementation increases COX activity and restores normal steady state levels of COX subunits in COX16 knockout cells, indicating that, even in the absence of a canonical copper binding motif, COX16 could be involved in copper delivery to COX2.     

Zinc induces apoptosis that can be suppressed by lanthanum in C6 rat glioma cells

Zinc ions have both essential and toxic effects on mammalian cells. Here we report the ability of zinc to act as an inducer of apoptosis in C6 rat glioma cells. Incubation with 150 to 300 microM ZnCl2 caused cell death that was characterized as apoptotic by internucleosomal DNA fragmentation, formation of apoptotic bodies, nuclear fragmentation and breakdown of the mitochondrial membrane potential. On the other hand, zinc deprivation by the membrane permeable chelator TPEN [N,N,N',N',-tetrakis (2-pyridyl-methyl)-ethylenediamine] also induced programmed death in this cell line, indicating the existence of intracellular zinc levels below and above which apoptosis is induced. Zinc-induced apoptosis in C6 cells was independent of major signaling pathways (protein kinase C, mitogen activated protein kinase and guanylate cyclase) and protein synthesis, but was increased by facilitating zinc uptake with the ionophore pyrithione. Lanthanum(III)chloride was also able to increase the net zinc uptake, but nevertheless apoptotic features and zinc toxicity were reduced. Remarkably, lanthanum suppressed the zinc-induced breakdown of the mitochondrial membrane potential. We conclude that in C6 cells lanthanum acts in two different ways, as a promoter of net zinc uptake and as a suppressor of zinc-induced apoptosis.     

Decrease of cytochrome c oxidase protein in heart mitochondria of copper-deficient rats

Copper deficiency has been reported to be associated withdecreased cytochrome c oxidase activity, whichin turn may be responsible for theobserved mitochondrial impairment and cardiac failure. We isolatedmito-chondriafrom hearts of copper-deficient rats: cytochrome c oxidase activity was found to be lowerthan incopper-adequate mitochondria. The residual activity paralleled coppercontent of mitochondria and also corresponded with the heme amount associated with cytochromeaa3. In fact, lower absorption in thea-band region of cytochrome aa3 was foundfor copper-deficient rat heart mitochondria. Gel electrophoresisof protein extractedfrom mitochondrial membranes allowed measurements of protein content of thecomplexes ofoxidative phosphorylation, revealing a lower content of complex IV protein incopper-deficientrat heart mitochondria. The alterations caused by copper deficiency appear to bespecific forcytochrome c oxidase. Changes were not observed for F 0 F 1 ATP synthase activity,for heme contents ofcytochrome c and b, and for protein contents of complexes I, III and V.The present study demonstrates that the alteration of cytochrome c oxidase activityobserved in copper deficiency is due to a diminishedcontent of assembled protein and that shortnessof copper impairs heme insertion into cytochrome c oxidase.     

Role of Bcl-2 family of proteins in mediating apoptotic death of PC12 cells exposed to oxygen and glucose deprivation

Apoptotic cell death has been observed in many in vivo and in vitro models of ischemia. However, the molecular pathways involved in ischemia-induced apoptosis remain unclear. We have examined the role of Bcl-2 family of proteins in mediating apoptosis of PC12 cells exposed to the conditions of oxygen and glucose deprivation (OGD) or OGD followed by restoration of oxygen and glucose (OGD-restoration, OGD-R). OGD decreased mitochondrial membrane potential and induced necrosis of PC12 cells, which were both prevented by the overexpression of Bcl-2 proteins. OGD-R caused apoptotic cell death, induced cytochrome C release from mitochondria and caspase-3 activation, decreased mitochondrial membrane potential, and increased levels of pro-apoptotic Bax translocated to the mitochondrial membrane, all of which were reversed by overexpression of Bcl-2. These results demonstrate that the cell death induced by OGD and OGD-R in PC12 cells is potentially mediated through the regulation of mitochondrial membrane potential by the Bcl-2 family of proteins. It also reveals the importance of developing therapeutic strategies for maintaining the mitochondrial membrane potential as a possible way of reducing necrotic and apoptotic cell death that occurs following an ischemic insult.     

Role of heme in the synthesis of cytochrome c oxidase in Neurospora crassa

The role of heme in the synthesis of cytochrome c oxidase has been investigated in the mold Neurospora crassa. Iron-deficient cultures of the mold have low levels of cytochrome oxidase and delta-aminolevulinate dehydratase, the latter being the rate-limiting enzyme of the heme-biosynthetic pathway in this organism. Addition of iron to the iron-deficient cultures results in an immediate increase in the levels of delta-aminolevulinate dehydratase followed by an increase in the rate of heme synthesis and cytochrome oxidase levels. The rate of precursor labeling of the mitochondrial subunits of cytochrome oxidase is decreased preferentially under conditions of iron deficiency and addition of iron corrects this picture. Exogenous hemin addition which prevents iron-mediated induction of delta-aminolevulinate dehydratase also inhibits the increase in the activity of cytochrome oxidase and the enhanced precursor labeling of the mitochondrial subunits of cytochrome oxidase. Protein synthesis on mitoribosomes measured in vivo and in vitro is decreased under conditions of heme deficiency. Hemin addition in vitro to mitochondrial lysates prepared from heme-deficient mycelia restores a near normal rate of protein synthesis. It is concluded that heme is required for the optimal rate of translation on mitoribosomes.     

Nuclear mutants of Neurospora crassa temperature-sensitive for the synthesis of cytochrome aa3. Mitochondrial protein synthesis and analysis of the polypeptide composition of cytochrome c oxidase.

Three previously isolated mutants of Neurospora crassa, temperature-sensitive for the production of cytochrome aa3, have been further analyzed. These mutants have a slightly reduced capacity for mitochondrial protein synthesis when grown at 41 degrees C, although this relative deficiency appeared to be no greater than the deficiency in other cytochrome-aa3-deficient mutants. Thermolability studies revealed that the cytochrome c oxidase purified from each of the mutants grown at 23 degrees C is no more sensitive to heat inactivation than the enzyme isolated from wild-type cells. Sodium dodecylsulfate gel electrophoresis of immunoprecipitates obtained from the mitochondria of each of the mutants grown at 23 degrees C, using antiserum directed against holocytochrome c oxidase, indicated that all the subunits of cytochrome c oxidase were present in relative amounts similar to those found in mitochondria from wild-type cultures. However, when the mitochondria from mutant cultures grown at 41 degrees C were examined in the above fashion, only subunits 5 and 6 of the oxidase were detected. Nonetheless, the mitochondrially synthesized subunit 1, 2 and 3 polypeptides could be immunoprecipitated from mitochondria isolated from mutant cells grown at 41 degrees C and labelled with [3H]leucine in medium containing cycloheximide. Although subunits 4 and 7 could not be detected, because a suitable antibody was not available, the fact that five of the seven subunits were present, but not associated with each other, suggested that the genetic defects in these mutants may affect the process of cytochrome c oxidase assembly.     

Sestrin2对热暴露肺上皮细胞凋亡的干预作用及其机制*

目的:探讨Sestrin2蛋白对热暴露肺上皮细胞凋亡的干预作用及其作用机制。方法:体外培养的Beas-2B细胞分为对照组(37℃)和热暴露组(39℃、40℃和41℃),在上述温度中暴露不同时间(0、3、6和12 h),胰酶消化后收集细胞,分别通过Western blot、荧光分光光度计、流式细胞仪等方法检测细胞中的Sestrin2、超氧化物歧化酶(SOD)、活性氧自由基(ROS)表达水平,细胞线粒体膜电位及细胞凋亡率。基因序列克隆入高表达质粒pcDNA 3.1⁺中,采用Lipfectamine 2000方法转染Beas-2B细胞,构建Sestrin2和SOD高表达细胞,观察细胞线粒体膜电位及细胞凋亡等指标的变化。结果:随着暴露温度的升高,与对照组相比,热暴露组细胞Sestrin2蛋白表达水平下降。在41℃热暴露Beas-2B细胞,不同时间点ROS水平显著上升,线粒体膜电位显著下降,细胞凋亡率增加。Sestrin2和SOD高表达细胞,在41℃暴露条件下,与对照组比较,ROS表达水平显著降低,线粒体膜电位下降幅度减小,热暴露导致细胞凋亡率降低。结论: Sestrin2能够通过线粒体膜电位和SOD缓解热暴露引起肺上皮细胞的凋亡,对Beas-2B细胞具有保护作用。