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.     

Agrobacterium-mediated transformation and regeneration of cotton plants

Cotton (Gossypium hirsutum L.) was transformed by the EHA101 strain of Agrobacterium tumefaciens harboring a binary vector pGA482GG plasmid carrying the marker genes for neomycin phosphotransferase II (nptII) determining resistance to kanamycin and β-glucuronidase (GUS). The cotyledons, hypocotyls, shoot meristem tissue, and its segments taken from in vitro growing seedlings were used as explants. Explants were cultured in a Murashige and Skoog (MS) medium containing various hormone combinations to induce shoot regeneration. The highest frequency of shoot formation was obtained from the shoot meristem. After selection in the MS medium containing kanamycin (50 mg/l), these tissues were tested by histochemical GUS assay. Shoots regenerated from excised shoot meristems or their halves were cultured for 4–6 weeks to obtain rooted plants, which then produced fully-developed plants and seeds in pots. Genomic integration of the kanamycin-resistance gene was detected by the PCR analysis. Seed germination percentage was 95% after the F1 seeds of transgenic cotton plants were cultured on half-strength MS medium supplemented with 50 mg/l kanamycin. Thus, a protocol for effective Agrobacterium-mediated genetic transformation of cotton was optimized. Published in Russian in Fiziologiya Rastenii, 2006, Vol. 53, No. 3, pp. 462–467. The text was submitted by the authors in English.     

Segregation of T-DNA Inserts in the Offspring of Arabidopsis Thaliana After Agrobacterium Transformation

Using various transformation methods, T-DNA constructions for insertional mutagenesis were introduced into Arabidopsis thaliana and the pattern of segregation of hygromycin resistance selectable marker was followed in succeeding generations in individual transgenic lines up to T₄ generation. Despite the low frequency of transformation, T-DNA was often inserted in two or more independent sites. Mendelian segregation ratios 3:1, 15:1, and irregular segregation ratios were observed. We have also shown continuous decrease of the expression of the resident hygromycin resistance transgenic trait in some lines. This revised version was published online in July 2006 with corrections to the Cover Date.     

Structure of the mammalian mitochondrial ribosome reveals an expanded functional role for its component proteins

The mitochondrial ribosome is responsible for the biosynthesis of protein components crucial to the generation of ATP in the eukaryotic cell. Because the protein:RNA ratio in the mitochondrial ribosome (approximately 69:approximately 31) is the inverse of that of its prokaryotic counterpart (approximately 33:approximately 67), it was thought that the additional and/or larger proteins of the mitochondrial ribosome must compensate for the shortened rRNAs. Here, we present a three-dimensional cryo-electron microscopic map of the mammalian mitochondrial 55S ribosome carrying a tRNA at its P site, and we find that instead, many of the proteins occupy new positions in the ribosome. Furthermore, unlike cytoplasmic ribosomes, the mitochondrial ribosome possesses intersubunit bridges composed largely of proteins; it has a gatelike structure at its mRNA entrance, perhaps involved in recruiting unique mitochondrial mRNAs; and it has a polypeptide exit tunnel that allows access to the solvent before the exit site, suggesting a unique nascent-polypeptide exit mechanism.     

A computer-based model for the regulation of mitogen activated protein kinase (MAPK) activation

Computer simulations and mathematical modeling of biological processes are becoming increasingly popular, and yet the complexity of the biochemical systems or the differences between experimental setups make it very difficult to establish a standard formula for these modeling projects. Before we can start using computer-based models for predictions or targeted experiment designs, it is very important to establish a reliable model on which those predictions can be based and experimentally tested. Here we attempt to present a computer model for the mitogen-activated protein kinase (MAPK) signaling cascade which is consistent with previously published experimental results. In this study we have focused our attention to a generic MAPK ERK (extracellular signal-regulated kinase) pathway activated by epidermal growth factor (EGF) in an attempt to understand how receptors may achieve different activation kinetics of the MAPK signaling. We successfully show that the level of receptor expression is one key determinant in this regulation, and that the binding affinity of the active receptor to adaptor proteins can have a small but albeit direct effect on the downstream activation.     

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.     

The protective role of erdosteine on testicular tissue after testicular torsion and detorsion

Testicular torsion and detorsion are important clinical problems for infertile man and oxidative stress may have a role in this clinical situation. The aim of this study was to investigate the protective role of erdosteine, an antioxidant, on unilateral testicular reperfusion injury in rats. The rats were divided into four groups including seven rats in each group: control, torsion, torsion/detorsion and torsion/detorsion+erdosteine. Rats, except the sham operation group, were subjected to left unilateral torsion (720^∘ rotation in the clockwise direction) without including the epididymis. The experiments were finished after sham operation time for control, 120 min torsion for torsion group and 120 min torsion and 240 min detorsion for torsion/detorsion groups. Bilateral orchiectomy was performed for all groups of rats. The ipsilateral and controlateral testis were divided into two pieces to analyse biochemical parameters and to investigate the light microscopic view. Malondialdehyde level of ipsilateral testis was increased in torsion and torsion/detorsion groups in comparison with the other groups (p < 0.05). Erdosteine treatment ameliorated lipid peroxidation after torsion/detorsion in ipsilateral testis (p < 0.05). Also, xanthine oxidase activity of ipsilateral testis was increased in torsion/detorsion group in comparison with the others (p < 0.05). Nitric oxide (NO) level of ipsilateral testis was higher in all experimental groups than sham operated control group (p < 0.05). Also, NO level of torsion group was increased in comparison with detorsion groups (p < 0.05). Erdosteine treatment caused increased glutathione peroxidase activity in comparison with torsion and torsion/detorsion groups and catalase activity in comparison with the other groups in ipsilateral testis (p < 0.05). Superoxide dismutase activity of ipsilateral testis was higher in torsion/detorsion and torsion/detorsion+erdosteine groups than control and torsion groups (p < 0.05). The biochemical parameters were not affected in controlateral testis in all groups. Torsion, torsion/detorsion and torsion/detorsion+erdosteine groups showed ipsilateral testicular damage in the histological examination, but the specimens from torsion/detorsion had a significantly greater histological injury than those from the other groups (p < 0.05). Control rats showed normal seminiferous tubule morphology. Rats in torsion group had slight-to-moderate disruption of the seminiferous epithelium. Rats in torsion/detorsion group displayed moderate-to-severe disruption of the seminiferous epithelium. In all animals from torsion/detorsion+erdosteine group, the testicular tissues were affected with slight-to-moderate degenerative changes of the seminiferous epithelium. Administration of erdosteine resulted in a significantly reduced histological damage associated with torsion of the spermatic cord compared with torsion/detorsion. In all groups, the contralateral testes were histologically normal. In conclusion, the results clearly displayed that erdosteine treatment may have a protective role on testicular torsion/detorsion injury. (Mol Cell Biochem xxx: 193–199, 2005)     

Gap, a mycobacterial specific integral membrane protein, is required for glycolipid transport to the cell surface

The cell envelope of mycobacteria is a complex multilaminar structure that protects the cell from stresses encountered in the environment, and plays an important role against the bactericidal activity of immune system cells. The outermost layer of the mycobacterial envelope typically contains species-specific glycolipids. Depending on the mycobacterial species, the major glycolipid localized at the surface can be either a phenolglycolipid or a peptidoglycolipid (GPL). Currently, the mechanism of how these glycolipids are addressed to the cell surface is not understood. In this study, by using a transposon library of Mycobacterium smegmatis and a simple dye assay, six genes involved in GPLs synthesis have been characterized. All of these genes are clustered in a single genomic region of approximately 60 kb. We show by biochemical analyses that two non-ribosomal peptide synthetases, a polyketide synthase, a methyltransferase and a member of the MmpL family are required for the biosynthesis of the GPLs backbone. Furthermore, we demonstrate that a small integral membrane protein of 272 amino acids named Gap (gap: GPL addressing protein) is specifically required for the transport of the GPLs to the cell surface. This protein is predicted to contain six transmembrane segments and possesses homologues across the mycobacterial genus, thus delineating a new protein family. This Gap family represents a new paradigm for the transport of small molecules across the mycobacterial envelope, a critical determinant of mycobacterial virulence.