Mitochondria Of Mammalian Cells Research Articles

NOA36 Protein Contains a Highly Conserved Nucleolar Localization Signal Capable of Directing Functional Proteins to the Nucleolus, in Mammalian Cells

NOA36/ZNF330 is an evolutionarily well-preserved protein present in the nucleolus and mitochondria of mammalian cells. We have previously reported that the pro-apoptotic activity of this protein is mediated by a characteristic cysteine-rich domain. We now demonstrate that the nucleolar localization of NOA36 is due to a highly-conserved nucleolar localization signal (NoLS) present in residues 1–33. This NoLS is a sequence containing three clusters of two or three basic amino acids. We fused the amino terminal of NOA36 to eGFP in order to characterize this putative NoLS. We show that a cluster of three lysine residues at positions 3 to 5 within this sequence is critical for the nucleolar localization. We also demonstrate that the sequence as found in human is capable of directing eGFP to the nucleolus in several mammal, fish and insect cells. Moreover, this NoLS is capable of specifically directing the cytosolic yeast enzyme polyphosphatase to the target of the nucleolus of HeLa cells, wherein its enzymatic activity was detected. This NoLS could therefore serve as a very useful tool as a nucleolar marker and for directing particular proteins to the nucleolus in distant animal species.

The mode of action of isocyanide in three aquatic organisms, Balanus amphitrite, Bugula neritina and Danio rerio.

Isocyanide is a potential antifouling compound in marine environments. In this study, we investigated its mode of action in three aquatic organisms. Two of them, the bryozoan Bugula neritina and the barnacle Balanus amphitrite, are major marine fouling invertebrates, and the other organism is the non-target species zebrafish Danio rerio. In the swimming larvae of B. neritina, isocyanide did not affect the total attachment rate (≤50 µg ml−1), but it did change the attachment site by increasing the percentage of attachment on the bottom of the container rather than on the wall or air-water inter-surface. Isocyanide binds several proteins in B. neritina as identified via SDS-PAGE-LC-MS/MS: 1) a 30 kD protein band containing two proteins similar to voltage dependent anion channels (VDAC), which control the direct coupling of the mitochondrial matrix to the energy maintenance of the cytosol and the release of apoptogenic factors from mitochondria of mammalian cells; and 2) an unknown 39 kD protein. In B. amphitrite cyprids, the isocyanide binding protein were 1) a protein similar to NADH-ubiquinone oxidoreductase, which is the “entry enzyme” of oxidative phosphorylation in mitochondria; and 2) cytochrome P450. In Danio rerio embryos, isocyanide caused “wavy” notochords, hydrocephalus, pericardial edema, poor blood circulation, and defects in pigmentation and hematopoiesis, which phenocopied copper deficiency. This is the first report on isocyanide binding proteins in fouling organisms, as well as the first description of its phenotype and potential toxicology in zebrafish.

Turnover of nonessential fatty acids in cardiolipin from the rat heart

Cardiolipin (CL) is a unique phospholipid (PL) found in the mitochondria of mammalian cells. CL remodeling is accompanied by turnover of its fatty acid acyl groups. Abnormalities in CL remodeling have been found in Barth's syndrome, diabetes, and obesity. The objective of this study was to determine nonessential fatty acid turnover in CL and phosphatidylethanolamine (PE) in the rat heart in vivo. Sprague-Dawley rats were fed either a regular chow or a high-fat diet for 15 weeks, and consumed 6% deuterium-enriched drinking water as a tracer for 14 days. CL and PE were extracted from cardiac tissue and isolated by TLC. Fatty acids from CL, PE, and plasma were analyzed by GC/MS for deuterium incorporation. Results showed oleate and vaccenate turnover were the highest in CL whereas palmitate and stearate turnover were low. Among the nonessential fatty acids in PE, turnover of stearate and vaccenate were the highest. The high turnover rate in vaccenate was unexpected, because vaccenate previously had no known metabolic or physiologic function. In conclusion, the similarly high turnover rates of both oleate and vaccenate readily suggest that remodeling is an important functional aspect of PL metabolism in CL.

Toward genetic transformation of mitochondria in mammalian cells using a recoded drug-resistant selection marker

Due to technical difficulties, the genetic transformation of mitochondria in mammalian cells is still a challenge. In this report, we described our attempts to transform mammalian mitochondria with an engineered mitochondrial genome based on selection using a drug resistance gene. Because the standard drug-resistant neomycin phosphotransferase confers resistance to high concentrations of G418 when targeted to the mitochondria, we generated a recoded neomycin resistance gene that uses the mammalian mitochondrial genetic code to direct the synthesis of this protein in the mitochondria, but not in the nucleus (mitochondrial version). We also generated a universal version of the recoded neomycin resistance gene that allows synthesis of the drug-resistant proteins both in the mitochondria and nucleus. When we transfected these recoded neomycin resistance genes that were incorporated into the mouse mitochondrial genome clones into mouse tissue culture cells by electroporation, no DNA constructs were delivered into the mitochondria. We found that the universal version of the recoded neomycin resistance gene was expressed in the nucleus and thus conferred drug resistance to G418 selection, while the synthetic mitochondrial version of the gene produced no background drug-resistant cells from nuclear transformation. These recoded synthetic drug-resistant genes could be a useful tool for selecting mitochondrial genetic transformants as a precise technology for mitochondrial transformation is developed.

Alkylrhodamines as Cationic Protonophores

Mild uncoupling of mitochondria assumes that partial decrease in membrane potential is beneficial for cells especially under some pathological conditions, suggesting that uncouplers are good candidates for therapeutic uses. The majority of known protonophoric uncouplers are weak acids capable to permeate across membranes in neutral and anionic forms. In the present study, protonophoric activity of a series of derivatives of cationic rhodamine 19 (including dodecylrhodamine C12R1 and its conjugate with plastoquinone SkQR1) but not of similar derivatives of rhodamine B was revealed by using a variety of assays. In planar bilayer lipid membranes, diffusion potential of hydrogen ions was formed in the presence of C12R1 and SkQR1. These compounds induced pH equilibration in liposomes loaded with the pH probe pyranine similarly to the well-known uncoupler FCCP. C12R1 and SkQR1 also stimulated respiration of rat liver mitochondria and decreased their membrane potential as measured by DiS-C3-5. The protonophoric activity of alkylrhodamines strongly depended on the number of carbon atoms in their alkyl chain (n): it was maximal at n about 10-12 and substantially decreased at n = 16. We surmise that the proton transport involves translocation of neutral and protonated cationic forms of C12R1 across a membrane. Based on the fact that SkQR1 accumulates in mitochondria of mammalian cells and protects them from oxidative stress (Bakeeva et al. 2008, Biochemistry Moscow, 73:1288-1299), it can be proposed that the protonophore-induced uncoupling could be involved in the protective effect of SkQR1 along with the direct antioxidant effect of plastoquinone.

NOA1 is an essential GTPase required for mitochondrial protein synthesis

Nitric oxide associated-1 (NOA1) is an evolutionarily conserved guanosine triphosphate (GTP) binding protein that localizes predominantly to mitochondria in mammalian cells. On the basis of bioinformatic analysis, we predicted its possible involvement in ribosomal biogenesis, although this had not been supported by any experimental evidence. Here we determine NOA1 function through generation of knockout mice and in vitro assays. NOA1-deficient mice exhibit midgestation lethality associated with a severe developmental defect of the embryo and trophoblast. Primary embryonic fibroblasts isolated from NOA1 knockout embryos show deficient mitochondrial protein synthesis and a global defect of oxidative phosphorylation (OXPHOS). Additionally, Noa1⁻/⁻ cells are impaired in staurosporine-induced apoptosis. The analysis of mitochondrial ribosomal subunits from Noa1⁻/⁻ cells by sucrose gradient centrifugation and Western blotting showed anomalous sedimentation, consistent with a defect in mitochondrial ribosome assembly. Furthermore, in vitro experiments revealed that intrinsic NOA1 GTPase activity was stimulated by bacterial ribosomal constituents. Taken together, our data show that NOA1 is required for mitochondrial protein synthesis, likely due to its yet unidentified role in mitoribosomal biogenesis. Thus, NOA1 is required for such basal mitochondrial functions as adenosine triphosphate (ATP) synthesis and apoptosis.

Mitochondrial DNA: An Up‐and‐coming Actor in White Adipose Tissue Pathophysiology

Mitochondrial DNA: An Up‐and‐coming Actor in White Adipose Tissue Pathophysiology

Correction for Yang et al., Regulation of the protein disulfide proteome by mitochondria in mammalian cells

Correction for Yang et al., Regulation of the protein disulfide proteome by mitochondria in mammalian cells

ATP-Citrate Lyase Links Cellular Metabolism to Histone Acetylation

Histone acetylation in single-cell eukaryotes relies on acetyl coenzyme A (acetyl-CoA) synthetase enzymes that use acetate to produce acetyl-CoA. Metazoans, however, use glucose as their main carbon source and have exposure only to low concentrations of extracellular acetate. We have shown that histone acetylation in mammalian cells is dependent on adenosine triphosphate (ATP)-citrate lyase (ACL), the enzyme that converts glucose-derived citrate into acetyl-CoA. We found that ACL is required for increases in histone acetylation in response to growth factor stimulation and during differentiation, and that glucose availability can affect histone acetylation in an ACL-dependent manner. Together, these findings suggest that ACL activity is required to link growth factor-induced increases in nutrient metabolism to the regulation of histone acetylation and gene expression.

Apoptosis Induction is Associated with VDAC Oligomerization

Mitochondria-mediated apoptosis involves efflux of a number of potential apoptotic regulators, such as cytochrome c, to the cytosol, triggering the caspase cascade and cell destruction. The precise mechanism regulating cytochrome c release remains unknown, and the molecular architecture of the cytochrome c-conducting channel has also to be determined. There is substantial evidence suggesting that the voltage-dependent anion channel-1 (VDAC1) is a critical player in apoptosis by regulating the release of apoptogenic proteins from mitochondria in mammalian cells and interacting with pro- and anti-apoptotic proteins.

Redox regulation in radiation-induced cytochrome c release from mitochondria of human lung carcinoma A549 cells

Mitochondria in mammalian cells are well-known to play an important role in the intrinsic pathway of genotoxic-agent-induced apoptosis by releasing cytochrome c into cytosol and to be a major source of reactive oxygen species (ROS). The aim of this study was to examine whether mitochondrial ROS are involved in radiation-induced apoptotic signaling in A549 cells. Post-irradiation treatment with N-acetyl- l-cystein (NAC) inhibited cytochrome c release from mitochondria but did not affect expression levels of Bcl-2, Bcl-X L and Bax, suggesting that late production of ROS triggered cytochrome c release. Experiments using DCFDA (a classical ROS fluorescence probe) and MitoAR (a novel mitochondrial ROS probe) demonstrated that intracellular and mitochondrial ROS were enhanced 6 h after X irradiation. Furthermore, the O 2 - production ability of mitochondria isolated from A549 cells was evaluated by ESR spectroscopy combined with a spin-trapping reagent (CYPMPO). When isolated mitochondria were incubated with NADH, succinate and CYPMPO, an ESR spectrum due to CYPMPO–OOH was detected. This NADH/succinate-dependent O 2 - production from mitochondria of irradiated cells was significantly increased in comparison with that of unirradiated cells. These results indicate that ionizing radiation enhances O 2 - production from mitochondria to trigger cytochrome c release in A549 cells.

Selection by drug resistance proteins located in the mitochondria of mammalian cells

Transformation of mitochondria in mammalian cells is now a technical challenge. In this report, we demonstrate that the standard drug resistant genes encoding neomycin and hygromycin phosphotransferases can potentially be used as selectable markers for mammalian mitochondrial transformation. We re-engineered the drug resistance genes to express proteins targeted to the mitochondrial matrix and confirmed the location of the proteins in the cells by fusing them with GFP and by Western blot and mitochondrial content mixing analyses. We found that the mitochondrially targeted-drug resistance proteins confer resistance to high levels of G418 and hygromycin without affecting the viability of cells.

Academic Life: The Whole Package

When I received the unexpected but welcome invitation to write a Reflections article, I wondered which aspects of my life in science are sufficiently distinctive to make the article worth reading. One aspect may be my survival as a department chair for nearly 25 years while struggling to contribute as well in teaching and research, in other words, embracing the “whole package” of academic life: teaching, research, and service. Another part may be my experience as coauthor of a biochemistry textbook. To deal with both, I will begin with my introduction to biochemistry.

OPA1 Processing Reconstituted in Yeast Depends on the Subunit Composition of them-AAA Protease in Mitochondria

The morphology of mitochondria in mammalian cells is regulated by proteolytic cleavage of OPA1, a dynamin-like GTPase of the mitochondrial inner membrane. The mitochondrial rhomboid protease PARL, and paraplegin, a subunit of the ATP-dependent m-AAA protease, were proposed to be involved in this process. Here, we characterized individual OPA1 isoforms by mass spectrometry, and we reconstituted their processing in yeast to identify proteases involved in OPA1 cleavage. The yeast homologue of OPA1, Mgm1, was processed both by PARL and its yeast homologue Pcp1. Neither of these rhomboid proteases cleaved OPA1. The formation of small OPA1 isoforms was impaired in yeast cells lacking the m-AAA protease subunits Yta10 and Yta12 and was restored upon expression of murine or human m-AAA proteases. OPA1 processing depended on the subunit composition of mammalian m-AAA proteases. Homo-oligomeric m-AAA protease complexes composed of murine Afg3l1, Afg3l2, or human AFG3L2 subunits cleaved OPA1 with higher efficiency than paraplegin-containing m-AAA proteases. OPA1 processing proceeded normally in murine cell lines lacking paraplegin or PARL. Our results provide evidence for different substrate specificities of m-AAA proteases composed of different subunits and reveal a striking evolutionary switch of proteases involved in the proteolytic processing of dynamin-like GTPases in mitochondria.

Water-soluble formulation of Coenzyme Q10 inhibits Bax-induced destabilization of mitochondria in mammalian cells

Oxidative stress leads to mitochondrial dysfunction, which triggers the opening of the permeability transition pores (PTP) and the release of pro-apoptotic factors causing apoptotic cell death. In a limited number of cell systems, anti-oxidants and free-radical scavengers have been shown to block this response. We have previously reported that coenzyme Q(10) (CoQ(10)), an electron carrier in the mitochondrial respiratory chain, is involved in the reactive oxygen species (ROS) removal and prevention of oxidative stress-induced apoptosis in neuronal cells. However, the mechanism of this protection has not been fully elucidated. In the present study we investigated the effects of CoQ(10) on the mitochondrial events characteristic to apoptosis, especially on the function of pro-apoptotic protein Bax. Our results demonstrated that following a brief exposure of two human cell lines (fibroblasts and HEK293 cells) to H(2)O(2) the intracellular levels of ROS and the association of Bax with the mitochondria significantly increased and the cells underwent apoptosis. Both of these events, as well as the release of cytochrome c from the mitochondria, were blocked by a 24 h pre-treatment with CoQ(10). It is therefore believed that CoQ(10) prevented the collapse of the mitochondrial membrane potential in response to the H(2)O(2) treatment. Recombinant Bax protein alone caused the ROS generation and release of cytochrome c from isolated mitochondria and, again, CoQ(10) inhibited these Bax-induced mitochondrial dysfunctions.

Estrogen receptors α and β (ERα and ERβ) and androgen receptor (AR) in human sperm: localization of ERβ and AR in mitochondria of the midpiece

The central role of estrogens and androgens in the male reproductive system has focused attention on the presence and distribution of their cognate receptors [estrogen receptor (ER) alpha, ERbeta and androgen receptor (AR)] in male reproductive tissues and cells. Since the presence of steroid hormone receptors in mitochondria of mammalian cells has been well documented, we investigated the possibility of mitochondrial localization of sex steroid hormone receptors in sperm. Applying immunofluorescence labelling and confocal laser scanning microscopy we show that the estrogen receptor beta and the AR of human sperm are specifically enriched in the midpiece, at the site of the mitochondria, which were visualized by labelling with the vital dye CMX. Nuclear and mitochondrial localization of AR was also detected in LnCap human prostate cancer cells. Differentially, most of the ERalpha immunostaining is in the form of a compact zone at a region corresponding to the equatorial segment of the upper post-acrosomal region of the sperm head. Immunoblotting experiments using sperm extracts revealed the presence of a 66 and a 45 kDa protein reacting with the ERalpha antibody, one 64 kDa protein reacting with the ERbeta antibody and a 110 and a 90 kDa protein reacting with the antibody against AR. Our findings suggest that the differential localization of AR and ER isoforms in human sperm reveals distinct roles of these receptors in the physiology of sperm cells and, perhaps, also in the process of fertilization.

DNA repair initiated by glycosylases in the nucleus and mitochondria of mammalian cells; how our cells respond to a flood of oxidative DNA damage

Oxidative DNA damage causes blocks in transcription and replication and introduces errors leading to cell death and genomic instability. Extensive molecular analysis of repair mechanisms for oxidative DNA damage has revealed the strategy of mammalian cells against the threat posed by reactive oxygen species to the genetic information. Most oxidative base damage is first recognized by various glycosylases, which remove the base and initiate base excision repair, a repair mechanism widely distributed in life. However, disruptions of glycosylase genes have shown either no effect or rather mild phenotypes in mice, suggesting that the frontline defence against oxidative DNA damage has back-up systems. Indeed, a number of novel DNA glycosylases with overlapping substrate specificity were recently identified in mammalian cells. Several glycosylases are transported into the mitochondria too, indicating the importance of glycosylases and base damage repair in the mitochondria genome. Thus, mammalian cells survive the flood of oxidative DNA damage by means of extensive repair of the damage. Besides repair of DNA damage, mechanisms for tolerating DNA damage at replication have recently been discovered. Cells may tolerate residual damage at replication but thereby risk generating mutations.

Cytoplasmic Thioredoxin Reductase Is Essential for Embryogenesis but Dispensable for Cardiac Development

Two distinct thioredoxin/thioredoxin reductase systems are present in the cytosol and the mitochondria of mammalian cells. Thioredoxins (Txn), the main substrates of thioredoxin reductases (Txnrd), are involved in numerous physiological processes, including cell-cell communication, redox metabolism, proliferation, and apoptosis. To investigate the individual contribution of mitochondrial (Txnrd2) and cytoplasmic (Txnrd1) thioredoxin reductases in vivo, we generated a mouse strain with a conditionally targeted deletion of Txnrd1. We show here that the ubiquitous Cre-mediated inactivation of Txnrd1 leads to early embryonic lethality. Homozygous mutant embryos display severe growth retardation and fail to turn. In accordance with the observed growth impairment in vivo, Txnrd1-deficient embryonic fibroblasts do not proliferate in vitro. In contrast, ex vivo-cultured embryonic Txnrd1-deficient cardiomyocytes are not affected, and mice with a heart-specific inactivation of Txnrd1 develop normally and appear healthy. Our results indicate that Txnrd1 plays an essential role during embryogenesis in most developing tissues except the heart.

Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand–Mediated Activation of Mitochondria-Associated Nuclear Factor-κB in Prostatic Carcinoma Cell Lines

AbstractIt has been suggested that some nuclear transcription factors may participate in the regulation of mitochondrial functions through transcriptional control of mitochondrial DNA. Very little is known about the response of transcription factors within mitochondria to the activation of death receptors. Recent publications indicate that nuclear factor-κB (NF-κB) is localized in mitochondria of mammalian cells. Because of the critical role of mitochondria in the execution of many apoptotic pathways, we suggest that NF-κB-dependent mechanisms operating at the level of mitochondria contribute to its role in regulating death receptor signaling. We have found NF-κB p65 and p50 subunits with DNA binding activity in the mitochondria of prostatic carcinoma cell lines. Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) affects DNA binding activity of mitochondria-associated NF-κB but does not change the amount of p65 in mitochondria, which suggests activation of mitochondrial NF-κB without additional translocation of NF-κB subunits to mitochondria. We have also shown that TRAIL decreases mitochondrial genome encoded mRNA levels and inhibition of NF-κB prevents this decrease. TRAIL effects on mitochondrial NF-κB-DNA binding and mitochondrial genome encoded mRNA levels also depend on Bcl-2 overexpression. In addition, transcription factor activator protein-1 with DNA binding activity is also found in mitochondria of prostatic carcinoma cells and TRAIL treatment affects this binding. In summary, NF-κB is found in mitochondria of prostatic carcinoma cells, where it is thought to regulate mitochondria genome encoded mRNA levels in response to TRAIL treatment.

TRANSFEKSI DNA KE DALAM MITOKONDRIA SEL BETA PANKREAS TIKUS DAN SEL PRIMER MYOBLAST MANUSIA: LANGKAH MENUJU REKAYASA DNA MITOKONDRIA DAN TERAPI GEN

The delivery of an exogenous or modified DNA into the mitochondria of mammalian cells has been proposed as an alternative way to prevent or modify mitochondrial genetic defect. In  this study, a lipofection method was developed to transfer exogenous DNA into mitochondria of BRIN BD11 cells, a rat beta pancreas cell line, and human myoblast primary cells. An artificial mini-mitochondrial genome carrying a reporter gene encodes for a mitochondrial green fluorescent protein (mtGFP) was usedto track the fate of the exogenous DNA following its transfection into cellular mitochondria. Epifluorescence microscopic observation was carried out to detect the green fluorescence resulted from the GFPexpression. Immunohistochemical assays were also performed to validate and confirm the GFP expression. Collectively, the results indicate that the exogenous DNA pmtGFP could be  delivered into the mitochondrial compartment and was functional withinthe mitochondria of BRIN BD11 and human myoblast cells. Such achievement is a significant progress as both the artificial DNA construct and the mitochondrial transfection method could serve as a model to study the dynamic of mutant mtDNA as well as to provide a basis for the development of gene therapy for human mitochondrial diseases. Key words: DQAsome, pmtGFP, beta pancreas cell line, human myoblast primary cells