Hasan Koc

Regulation of Succinate Dehydrogenase Activity by SIRT3 in Mammalian Mitochondria

A member of the sirtuin family of NAD(+)-dependent deacetylases, SIRT3, is identified as one of the major mitochondrial deacetylases located in mammalian mitochondria responsible for deacetylation of several metabolic enzymes and components of oxidative phosphorylation. Regulation of protein deacetylation by SIRT3 is important for mitochondrial metabolism, cell survival, and longevity. In this study, we identified one of the Complex II subunits, succinate dehydrogenase flavoprotein (SdhA) subunit, as a novel SIRT3 substrate in SIRT3 knockout mice. Several acetylated Lys residues were mapped by tandem mass spectrometry, and we determined the role of acetylation in Complex II activity in SIRT3 knockout mice. In agreement with SIRT3-dependent activation of Complex I, we observed that deacetylation of the SdhA subunit increased the Complex II activity in wild-type mice. In addition, we treated K562 cell lines with nicotinamide and kaempferol to inhibit deacetylase activity of SIRT3 and stimulate SIRT3 expression, respectively. Stimulation of SIRT3 expression decreased the level of acetylation of the SdhA subunit and increased Complex II activity in kaempherol-treated cells compared to control and nicotinamide-treated cells. Evaluation of acetylated residues in the SdhA crystal structure from porcine and chicken suggests that acetylation of the hydrophilic surface of SdhA may control the entry of the substrate into the active site of the protein and regulate the enzyme activity. Our findings constitute the first evidence of the regulation of Complex II activity by the reversible acetylation of the SdhA subunit as a novel substrate of the NAD(+)-dependent deacetylase, SIRT3.

NAD+-dependent Deacetylase SIRT3 Regulates Mitochondrial Protein Synthesis by Deacetylation of the Ribosomal Protein MRPL10

A member of the sirtuin family of NAD+-dependent deacetylases, SIRT3, is located in mammalian mitochondria and is important for regulation of mitochondrial metabolism, cell survival, and longevity. In this study, MRPL10 (mitochondrial ribosomal protein L10) was identified as the major acetylated protein in the mitochondrial ribosome. Ribosome-associated SIRT3 was found to be responsible for deacetylation of MRPL10 in an NAD+-dependent manner. We mapped the acetylated Lys residues by tandem mass spectrometry and determined the role of these residues in acetylation of MRPL10 by site-directed mutagenesis. Furthermore, we observed that the increased acetylation of MRPL10 led to an increase in translational activity of mitochondrial ribosomes in Sirt3−/− mice. In a similar manner, ectopic expression and knockdown of SIRT3 in C2C12 cells resulted in the suppression and enhancement of mitochondrial protein synthesis, respectively. Our findings constitute the first evidence for the regulation of mitochondrial protein synthesis by the reversible acetylation of the mitochondrial ribosome and characterize MRPL10 as a novel substrate of the NAD+-dependent deacetylase, SIRT3.

A new face on apoptosis: Death-associated protein 3 and PDCD9 are mitochondrial ribosomal proteins

Two proteins known to be involved in promoting apoptosis in mammalian cells have been identified as components of the mammalian mitochondrial ribosome. Proteolytic digestion of whole mitochondrial ribosomal subunits followed by analysis of the peptides present using liquid chromatography–tandem mass spectrometry revealed that the proapoptotic proteins, death‐associated protein 3 (DAP3) and the programmed cell death protein 9, are both components of the mitochondrial ribosome. DAP3 has motifs characteristic of guanine nucleotide binding proteins and is probably the protein that accounts for the nucleotide binding activity of mammalian mitochondrial ribosomes. The observations reported here implicate mitochondrial protein synthesis as a major component in cellular apoptotic signaling pathways.

Expression analysis of extracellular proteins from Phanerochaete chrysosporium grown on different liquid and solid substrates.

White-rot fungi secret a large number of hydrolytic and oxidative enzymes for degradation of lignocellulosic material. The sequencing of the genome of the white-rot fungus Phanerochaete chrysosporium has facilitated the characterization of its complete extracellular proteome. P. chrysosporium was grown on liquid medium, containing glucose, cellulose or wood chips as the carbon source, and also in solid substrate fermentation bags. For liquid-grown cultures, the extracellular protein fraction was separated by 2D gel electrophoresis. Protein spots were analysed by in-gel digestion and liquid chromatography (LC)/MS/MS. A total of 18 additional protein spots from the 2D gels yielded hits from blast searches. From solid substrate cultures in which the fungus was grown in bags, the proteins were resolved by SDS-PAGE, subjected to in-gel digestion and then identified by LC/MS/MS. An additional 16 proteins yielded hits on blast searches. Enzymes involved in cellulose, hemicellulose, lignin and protein degradation were identified. Expression patterns were very similar between cellulose-grown cultures and wood-grown cultures. In addition to enzymes which act on lignocellulosic material, proteases were also found, indicating the need of fungi to scavenge for nitrogen in wood.

A Proteomics Approach to the Identification of Mammalian Mitochondrial Small Subunit Ribosomal Proteins

Mammalian mitochondrial small subunit ribosomal proteins were separated by two-dimensional polyacrylamide gel electrophoresis. The proteins in six individual spots were subjected to in-gel tryptic digestion. Peptides were separated by capillary liquid chromatography, and the sequences of selected peptides were obtained by electrospray tandem mass spectrometry. The peptide sequences obtained were used to screen human expressed sequence tag data bases, and complete consensus cDNAs were assembled. Mammalian mitochondrial small subunit ribosomal proteins from six different classes of ribosomal proteins were identified. Only two of these proteins have significant sequence similarities to ribosomal proteins from prokaryotes. These proteins correspond to Escherichia coliS10 and S14. Homologs of two human mitochondrial proteins not found in prokaryotes were observed in the genomes of Drosophila melanogaster and Caenorhabditis elegans. A homolog of one of these proteins was observed in D. melanogaster but not in C. elegans, while a homolog of the other was present in C. elegans but not in D. melanogaster. A homolog of one of the ribosomal proteins not found in prokaryotes was tentatively identified in the yeast genome. This latter protein is the first reported example of a ribosomal protein that is shared by mitochondrial ribosomes from lower and higher eukaryotes that does not have a homolog in prokaryotes.

Regulation of mammalian mitochondrial translation by post-translational modifications

Mitochondria are responsible for the production of over 90% of the energy in eukaryotes through oxidative phosphorylation performed by electron transfer and ATP synthase complexes. Mitochondrial translation machinery is responsible for the synthesis of 13 essential proteins of these complexes encoded by the mitochondrial genome. Emerging data suggest that acetyl-CoA, NAD(+), and ATP are involved in regulation of this machinery through post-translational modifications of its protein components. Recent high-throughput proteomics analyses and mapping studies have provided further evidence for phosphorylation and acetylation of ribosomal proteins and translation factors. Here, we will review our current knowledge related to these modifications and their possible role(s) in the regulation of mitochondrial protein synthesis using the homology between mitochondrial and bacterial translation machineries. However, we have yet to determine the effects of phosphorylation and acetylation of translation components in mammalian mitochondrial biogenesis. This article is part of a Special Issue entitled: Mitochondrial Gene Expression.

Using DNA and hemoglobin adducts to improve the risk assessment of butadiene.

The purpose of this paper is to review what we know about various biomarkers of butadiene in animal, human and in vitro studies, and to draw inferences from these data that impact on the accurate assessment of human risks for cancer. Studies comparing the DNA and hemoglobin adducts of butadiene with exposure, metabolism and genotoxicity have provided a great deal of insight that is applicable to biologically based risk assessment. First, the DNA and hemoglobin adduct data strongly support the conclusion that 3,4-epoxy-1,2-butanediol is the major electrophile available for binding to these macromolecules. Biomarker studies have also provided insight into the possibility of a sensitive population associated with the GSTT1 null genotype. While it is clear that lymphocytes from GSTT1 null individuals are more sensitive for the induction of sister chromatid exchanges (SCE) following in vitro exposure to 1,2,3,4-diepoxybutane, there was no such increase in SCE or other biomarkers of genotoxicity in workers exposed to 1-3 p.p.m. butadiene, regardless of GST genotype. The globin adduct data also demonstrate that there is roughly a tenfold range for interindividual differences in the metabolism of butadiene. This type of analysis represents an excellent means for providing scientific data for this critical determinant. Another useful application of hemoglobin adducts in risk assessment was demonstrated by regressing data for various endpoints for genotoxicity against that individuals biologically effective dose, thereby providing an independent mechanism for evaluation that excludes any possible confounding by inappropriate controls. Finally, biomarker studies have identified critical gaps in our knowledge that are needed for the accurate assessment of butadiene. Most notable of these is the lack of diepoxide-specific biomarkers in mice, rats and humans.

Endocytic Rab proteins are required for hepatitis C virus replication complex formation.

During infection, hepatitis C virus (HCV) NS4B protein remodels host membranes to form HCV replication complexes (RC) which appear as foci under fluorescence microscopy (FM). To understand the role of Rab proteins in forming NS4B foci, cells expressing the HCV replicon were examined biochemically and via FM. First, we show that an isolated NS4B-bound subcellular fraction is competent for HCV RNA synthesis. Further, this fraction is differentially enriched in Rab1, 2, 5, 6 and 7. However, when examined via FM, NS4B foci appear to be selectively associated with Rab5 and Rab7 proteins. Additionally, dominant negative (DN) Rab6 expression impairs Rab5 recruitment into NS4B foci. Further, silencing of Rab5 or Rab7 resulted in a significant decrease in HCV genome replication. Finally, expression of DN Rab5 or Rab7 led to a reticular NS4B subcellular distribution, suggesting that endocytic proteins Rab5 and Rab7, but not Rab11, may facilitate NS4B foci formation.

DNA adducts: Effects of low exposure to ethylene oxide, vinyl chloride and butadiene

Dose-response relationships of genotoxic agents differ greatly depending on the agent and the endpoint being evaluated. Simple conclusions that genotoxic effects are linear cannot be applied universally. The shape of the molecular dose of DNA adducts varies from linear, to supralinear, to sublinear depending on metabolic activation and detoxication, and repair of individual types of DNA adducts. For mutagenesis and other genotoxicity endpoints, the dose-response reflects the molecular dose of each type of DNA adduct, cell proliferation, as well as endogenous factors that lead to mutagenesis such as the formation and repair of endogenous DNA adducts. These same factors are important when interpreting the shape of dose-response data for carcinogenesis of genotoxic agents, however, tumor background variability adds additional complexity. Endogenously formed DNA adducts may be identical to those formed by chemicals, as in the case of vinyl chloride and ethylene oxide, or they may be those associated with oxidative stress. Data presented in this paper demonstrate that the exogenous number of adducts induced by 5 days of exposure to 10 ppm vinyl chloride is only 2. 2-fold greater than that present as a steady-state amount in unexposed control rats. Similar data are shown for ethylene oxide. Extremely sensitive methods have been developed for measuring the molecular dose of genotoxins. These methods can detect DNA adducts as low as 1 per 10(9) to 10(10). However, in view of the high number of endogenous DNA adducts that are present in all cells, it is unlikely that causal relationships can be attributed to very low numbers of such DNA adducts. Effects of both exogenous and endogenous DNA adducts need to be factored into the interpretation of chemical exposures.

Identification of four proteins from the small subunit of the mammalian mitochondrial ribosome using a proteomics approach.

Proteins in the small subunit of the mammalian mitochondrial ribosome were separated by two‐dimensional polyacrylamide gel electrophoresis. Four individual proteins were subjected to in‐gel Endoprotease Lys‐C digestion. The sequences of selected proteolytic peptides were obtained by electrospray tandem mass spectrometry. Peptide sequences obtained from in‐gel digestion of individual spots were used to screen human, mouse, and rat expressed sequence tag databases, and complete consensus cDNAs for these species were deduced in silico. The corresponding protein sequences were characterized by comparison to known ribosomal proteins in protein databases. Four different classes of mammalian mitochondrial small subunit ribosomal proteins were identified. Only two of these proteins have significant sequence similarities to ribosomal proteins from prokaryotes. These proteins are homologs to Escherichia coli S9 and S5 proteins. The presence of these newly identified mitochondrial ribosomal proteins are also investigated in the Drosophila melanogaster, Caenorhabditis elegans, and in the genomes of several fungi.