Fyn kinase regulates translation in mammalian mitochondria.

BACKGROUND: Mitochondrial translation machinery solely exists for the synthesis of 13 mitochondrially-encoded subunits of the oxidative phosphorylation (OXPHOS) complexes in mammals. Therefore, it plays a critical role in mitochondrial energy production. However, regulation of the mitochondrial translation machinery is still poorly understood. In comprehensive proteomics studies with normal and diseased tissues and cell lines, we and others have found the majority of mitochondrial ribosomal proteins (MRPs) to be phosphorylated. Neither the kinases for these phosphorylation events nor their specific roles in mitochondrial translation are known. METHODS: Mitochondrial kinases are responsible for phosphorylation of MRPs enriched from bovine mitoplasts by strong cation-exchange chromatography and identified by mass spectrometry-based proteomics analyses of kinase rich fractions. Phosphorylation of recombinant MRPs and 55S ribosomes was assessed by in vitro phosphorylation assays using the kinase-rich fractions. The effect of identified kinase on OXPHOS and mitochondrial translation was assessed by various cell biological and immunoblotting approaches. RESULTS: Here, we provide the first evidence for the association of Fyn kinase, a Src family kinase, with mitochondrial translation components and its involvement in phosphorylation of 55S ribosomal proteins in vitro. Modulation of Fyn expression in human cell lines has provided a link between mitochondrial translation and energy metabolism, which was evident by the changes in 13 mitochondrially encoded subunits of OXPHOS complexes. CONCLUSIONS AND GENERAL SIGNIFICANCE: Our findings suggest that Fyn kinase is part of a complex mechanism that regulates protein synthesis and OXPHOS possibly by tyrosine phosphorylation of translation components in mammalian mitochondria.

Expanding the proteome of an RNA virus by phosphorylation of an intrinsically disordered viral protein.

The human proteome contains myriad intrinsically disordered proteins. Within intrinsically disordered proteins, polyproline-II motifs are often located near sites of phosphorylation. We have used an unconventional experimental paradigm to discover that phosphorylation by protein kinase A (PKA) occurs in the intrinsically disordered domain of hepatitis C virus non-structural protein 5A (NS5A) on Thr-2332 near one of its polyproline-II motifs. Phosphorylation shifts the conformational ensemble of the NS5A intrinsically disordered domain to a state that permits detection of the polyproline motif by using (15)N-, (13)C-based multidimensional NMR spectroscopy. PKA-dependent proline resonances were lost in the presence of the Src homology 3 domain of c-Src, consistent with formation of a complex. Changing Thr-2332 to alanine in hepatitis C virus genotype 1b reduced the steady-state level of RNA by 10-fold; this change was lethal for genotype 2a. The lethal phenotype could be rescued by changing Thr-2332 to glutamic acid, a phosphomimetic substitution. Immunofluorescence and transmission electron microscopy showed that the inability to produce Thr(P)-2332-NS5A caused loss of integrity of the virus-induced membranous web/replication organelle. An even more extreme phenotype was observed in the presence of small molecule inhibitors of PKA. We conclude that the PKA-phosphorylated form of NS5A exhibits unique structure and function relative to the unphosphorylated protein. We suggest that post-translational modification of viral proteins containing intrinsic disorder may be a general mechanism to expand the viral proteome without a corresponding expansion of the genome.

Post-translational modification and mitochondrial relocalization of histone H3 during apoptosis induced by staurosporine.

Post-translational modifications (PTMs) of histones such as phosphorylation, acetylation, and ubiquitination, collectively referred to as the "histone-code", have been known to regulate gene expression and chromatin condensation for over a decade. They are also implicated in processes such as DNA repair and apoptosis. However, the study of the phosphorylation of histones has been mainly focused on chromosome condensation and mitosis. Therefore, the phosphorylation of histones in apoptosis is not fully understood. It was recently demonstrated by Tang et al. that histones are released from nucleosome during apoptosis, an observation that is in agreement with our findings. In addition to the release of histones, the dephosphorylation of histone H3 at Thr-3 and Ser-10 was observed during apoptosis in some cancer cells. Our data suggest that the modification and release of histones could serve markers of apoptosis in human cancer cells. We also suggest that the released histones, especially H3, could be translocated to mitochondria during apoptosis.

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.

Phosphorylation of mammalian mitochondrial EF-Tu by Fyn and c-Src kinases.

Src Family Kinases (SFKs) are tyrosine kinases known to regulate glucose and fatty acid metabolism as well as oxidative phosphorylation (OXPHOS) in mammalian mitochondria. We and others discovered the association of the SFK kinases Fyn and c-Src with mitochondrial translation components. This translational system is responsible for the synthesis of 13 mitochondrial (mt)-encoded subunits of the OXPHOS complexes and is, thus, essential for energy generation. Mitochondrial ribosomal proteins and various translation elongation factors including Tu (EF-Tumt) have been identified as possible Fyn and c-Src kinase targets. However, the phosphorylation of specific residues in EF-Tumt by these kinases and their roles in the regulation of protein synthesis are yet to be explored. In this study, we report the association of EF-Tumt with cSrc kinase and mapping of phosphorylated Tyr (pTyr) residues by these kinases. We determined that a specific Tyr residue in EF-Tumt at position 266 (EF-Tumt-Y266), located in a highly conserved c-Src consensus motif is one of the major phosphorylation sites. The potential role of EF-Tumt-Y266 phosphorylation in regulation of mitochondrial translation investigated by site-directed mutagenesis. Its phosphomimetic to Glu residue (EF-Tumt-E266) inhibited ternary complex (EF-Tumt•GTP•aatRNA) formation and translation in vitro. Our findings along with data mining analysis of the c-Src knock out (KO) mice proteome suggest that the SFKs have possible roles for regulation of mitochondrial protein synthesis and oxidative energy metabolism in animals.

Contacts between mammalian mitochondrial translational initiation factor 3 and ribosomal proteins in the small subunit.

Mammalian mitochondrial translational initiation factor 3 (IF3(mt)) binds to the small subunit of the ribosome displacing the large subunit during the initiation of protein biosynthesis. About half of the proteins in mitochondrial ribosomes have homologs in bacteria while the remainder are unique to the mitochondrion. To obtain information on the ribosomal proteins located near the IF3(mt) binding site, cross-linking studies were carried out followed by identification of the cross-linked proteins by mass spectrometry. IF3(mt) cross-links to mammalian mitochondrial homologs of the bacterial ribosomal proteins S5, S9, S10, and S18-2 and to unique mitochondrial ribosomal proteins MRPS29, MRPS32, MRPS36 and PTCD3 (Pet309) which has now been identified as a small subunit ribosomal protein. IF3(mt) has extensions on both the N- and C-termini compared to the bacterial factors. Cross-linking of a truncated derivative lacking these extensions gives the same hits as the full length IF3(mt) except that no cross-links were observed to MRPS36. IF3 consists of two domains separated by a flexible linker. Cross-linking of the isolated N- and C-domains was observed to a range of ribosomal proteins particularly with the C-domain carrying the linker which showed significant cross-linking to several ribosomal proteins not found in prokaryotes.

Comparing the efficacy of nebulizer recombinant human DNase and hypertonic saline as monotherapy and combined treatment in the treatment of persistent atelectasis in mechanically ventilated newborns.

BACKGROUND: The purpose of the present study was to compare the cost-effectiveness and efficacy of nebulizer recombinant human DNase (rhDNase) and hypertonic saline (HS) as monotherapy and combined treatment in neonatal atelectasis. METHODS: Eighty-seven newborns with persistent atelectasis who did not respond to traditional treatment were studied retrospectively. Group 1 did not receive nebulizer drugs; Group 2 received 7%HS; Group 3 received rhDNase; and Group 4 received both 7%HS and rhDNase. Subjects' chest X-ray scores, partial pressure of CO(2), respiratory rate, fraction of inspired oxygen (FiO(2)) peak inspiratory pressure, atelectasis healing rate, median duration of nebulizer treatment and costs were compared. RESULTS: Percentages of improvement in atelectasis on Day 3 of treatment in Group 1, Group 2, Group 3 and Group 4 were 27, 70, 81 and 95%, respectively, while median duration of treatment was 8.1, 3.3, 2.9 and 2.4 days, respectively. Comparison of chest X-ray scores, partial pressure of CO(2), respiratory rate, FiO(2) and peak inspiratory pressure values before and 48 h after treatment did not yield a significant difference for the control group (P > 0.05), while a marked improvement was observed in other groups for all parameters (P < 0.05). The most distinct improvement was in Group 4, followed by Group 3. CONCLUSIONS: Although both the combined treatment with HS and rhDNase and their monotherapies are effective in the treatment of persistent atelectasis in newborns receiving mechanical ventilation, their combined use produces higher efficacy. The efficacy of rhDNase is superior to monotherapy with HS. Use of these two treatments concomitantly reduces the cost. To the best of our knowledge, the present study is the first to use HS alone or in combination with rhDNase in newborn patients.

Role of mitochondrial translation in remodeling of energy metabolism in ER/PR(+) breast cancer.

Remodeling of mitochondrial energy metabolism is essential for the survival of tumor cells in limited nutrient availability and hypoxic conditions. Defects in oxidative phosphorylation (OXPHOS) and mitochondrial biogenesis also cause a switch in energy metabolism from oxidative to aerobic glycolysis contributing to the tumor heterogeneity in cancer. Specifically, the aberrant expressions of mitochondrial translation components such as ribosomal proteins (MRPs) and translation factors have been increasingly associated with many different cancers including breast cancer. The mitochondrial translation is responsible for the synthesis 13 of mitochondrial-encoded OXPHOS subunits of complexes. In this study, we investigated the contribution of mitochondrial translation in the remodeling of oxidative energy metabolism through altered expression of OXPHOS subunits in 26 ER/PR(+) breast tumors. We observed a significant correlation between the changes in the expression of mitochondrial translation-related proteins and OXPHOS subunits in the majority of the ER/PR(+) breast tumors and breast cancer cell lines. The reduced expression of OXPHOS and mitochondrial translation components also correlated well with the changes in epithelial-mesenchymal transition (EMT) markers, E-cadherin (CHD1), and vimentin (VIM) in the ER/PR(+) tumor biopsies. Data mining analysis of the Clinical Proteomic Tumor Analysis Consortium (CPTAC) breast cancer proteome further supported the correlation between the reduced OXPHOS subunit expression and increased EMT and metastatic marker expression in the majority of the ER/PR(+) tumors. Therefore, understanding the role of MRPs in the remodeling of energy metabolism will be essential in the characterization of heterogeneity at the molecular level and serve as diagnostic and prognostic markers in breast cancer.

Properties of the C-terminal tail of human mitochondrial inner membrane protein Oxa1L and its interactions with mammalian mitochondrial ribosomes.

In humans the mitochondrial inner membrane protein Oxa1L is involved in the biogenesis of membrane proteins and facilitates the insertion of both mitochondrial- and nuclear-encoded proteins from the mitochondrial matrix into the inner membrane. The C-terminal approximately 100-amino acid tail of Oxa1L (Oxa1L-CTT) binds to mitochondrial ribosomes and plays a role in the co-translational insertion of mitochondria-synthesized proteins into the inner membrane. Contrary to suggestions made for yeast Oxa1p, our results indicate that the C-terminal tail of human Oxa1L does not form a coiled-coil helical structure in solution. The Oxa1L-CTT exists primarily as a monomer in solution but forms dimers and tetramers at high salt concentrations. The binding of Oxa1L-CTT to mitochondrial ribosomes is an enthalpy-driven process with a K(d) of 0.3-0.8 microM and a stoichiometry of 2. Oxa1L-CTT cross-links to mammalian mitochondrial homologs of the bacterial ribosomal proteins L13, L20, and L28 and to mammalian mitochondrial specific ribosomal proteins MRPL48, MRPL49, and MRPL51. Oxa1L-CTT does not cross-link to proteins decorating the conventional exit tunnel of the bacterial large ribosomal subunit (L22, L23, L24, and L29).

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.

Identification and characterization of 2'-deoxyadenosine adducts formed by isoprene monoepoxides in vitro.

Isoprene, the 2-methyl analogue of 1,3-butadiene, is ubiquitous in the environment, with major contributions to total isoprene emissions stemming from natural processes despite the compound being a bulk industrial chemical. Additionally, isoprene is a combustion product and a major component in cigarette smoke. Isoprene has been classified as possibly carcinogenic to humans (group 2B) by IARC and as reasonably anticipated to be a human carcinogen by the National Toxicology Program. Isoprene, like butadiene, requires metabolic activation to reactive epoxides to exhibit its carcinogenic properties. The mode of action has been postulated to be that of a genotoxic carcinogen, with the formation of promutagenic DNA adducts being essential for mutagenesis and carcinogenesis. In rodents, isoprene-induced tumors show unique point mutations (A→T transversions) in the K-ras protooncogene at codon 61. Therefore, we investigated adducts formed after the reaction of 2'-deoxyadenosine (dAdo ) with the two monoepoxides of isoprene, 2-ethenyl-2-methyloxirane (IP-1,2-O) and propen-2-yloxirane (IP-3,4-O), under physiological conditions. The formation of N1-2'-deoxyinosine (N1-dIno) due to the deamination of N1-dAdo adducts was of particular interest, since N1-dIno adducts are suspected to have high mutagenic potential based on in vitro experiments. Major stable adducts were identified by HPLC, UV-spectroscopy, and LC-MS/MS and characterized by (1)H NMR and (1)H,(13)C HSQC and HMBC NMR experiments. Adducts of IP-1,2-O that were fully identified are R,S-C1-N(6)-dAdo, R-C2-N(6)-dAdo, and S-C2-N(6)-dAdo; adducts of IP-3,4-O are S-C3-N(6)-dAdo, R-C3-N(6)-dAdo, R,S-C4-N(6)-dAdo, S-C4-N1-dIno, R-C4-N1-dIno, R-C3-N1-dIno, S-C3-N1-dIno, and C3-N7-Ade. Both monoepoxides formed adducts on the terminal and internal oxirane carbons. This is the first study to describe adducts of isoprene monoepoxides with dAdo. Characterization of adducts formed by isoprene monoepoxides with deoxynucleosides and subsequently with DNA represent the first step toward evaluating their potential for being converted into a mutation or as biomarkers of isoprene metabolism and exposure.

Purification of human mitochondrial ribosomal L7/L12 stalk proteins and reconstitution of functional hybrid ribosomes in Escherichia coli.

Bacterial ribosomal L7/L12 stalk is formed by L10, L11, and multiple copies of L7/L12, which plays an essential role in recruiting initiation and elongation factors during translation. The homologs of these proteins, MRPL10, MRPL11, and MRPL12, are present in human mitochondrial ribosomes. To evaluate the role of MRPL10, MRPL11, and MRPL12 in translation, we over-expressed and purified components of the human mitochondrial L7/L12 stalk proteins in Escherichia coli. Here, we designed a construct to co-express MRPL10 and MRPL12 using a duet expression system to form a functional MRPL10-MRPL12 complex. The goal is to demonstrate the homology between the mitochondrial and bacterial L7/L12 stalk proteins and to reconstitute a hybrid ribosome to be used in structural and functional studies of the mitochondrial stalk.

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.

c-Src kinase impairs the expression of mitochondrial OXPHOS complexes in liver cancer.

Src family kinases (SFKs) play a crucial role in the regulation of multiple cellular pathways, including mitochondrial oxidative phosphorylation (OXPHOS). Aberrant activities of one of the most predominant SFKs, c-Src, was identified as a fundamental cause for dysfunctional cell signaling and implicated in cancer development and metastasis, especially in human hepatocellular carcinoma (HCC). Recent work in our laboratory revealed that c-Src is implicated in the regulation of mitochondrial energy metabolism in cancer. In this study, we investigated the effect of c-Src expression on mitochondrial energy metabolism by examining changes in the expression and activities of OXPHOS complexes in liver cancer biopsies and cell lines. An increased expression of c-Src was correlated with an impaired expression of nuclear- and mitochondrial-encoded subunits of OXPHOS complexes I and IV, respectively, in metastatic biopsies and cell lines. Additionally, we observed a similar association between high c-Src and reduced OXPHOS complex expression and activity in mouse embryonic fibroblast (MEF) cell lines. Interestingly, the inhibition of c-Src kinase activity with the SFK inhibitor PP2 and c-Src siRNA stimulated the expression of complex I and IV subunits and increased their enzymatic activities in both cancer and normal cells. Evidence provided in this study reveals that c-Src impairs the expression and function of mitochondrial OXPHOS complexes, resulting in a significant defect in mitochondrial energy metabolism, which can be a contributing factor to the development and progression of liver cancer. Furthermore, our findings strongly suggest that SFK inhibitors should be used in the treatment of HCC and other cancers with aberrant c-Src kinase activity to improve mitochondrial energy metabolism.

Phosphorylated proteins of the mammalian mitochondrial ribosome: implications in protein synthesis.

Mitochondria, the powerhouse of eukaryotic cells, have their own translation machinery that is solely responsible for synthesis of 13 mitochondrially encoded protein subunits of oxidative phosphorylation complexes. Phosphorylation is a well-known post-translational modification in regulation of many processes in mammalian mitochondria including oxidative phosphorylation. However, there is still very limited knowledge on phosphorylation of mitochondrial ribosomal proteins and their role(s) in ribosome function. In this study, we have identified the mitochondrial ribosomal proteins that are phosphorylated at serine, threonine or tyrosine residues. Twenty-four phosphorylated proteins were visualized by phosphorylation-specific techniques including in vitro radiolabeling, residue specific antibodies for phosphorylated residues, or ProQ phospho dye and identified by tandem mass spectrometry. Translation assays with isolated ribosomes that were phosphorylated in vitro by kinases PKA, PKCdelta, or Abl Tyr showed up to 30% inhibition due to phosphorylation. Findings from this study should serve as the framework for future studies addressing the regulation mechanisms of mitochondrial translation machinery by phosphorylation and other post-translational modifications.

Treatment of secondary hyperparathyroidism with paricalcitol in patients with end-stage renal disease undergoing hemodialysis in Turkey: an observational study.

OBJECTIVE: To evaluate monthly percentage changes of intact parathyroid hormone (iPTH) and other major bone marker levels in patients with secondary hyperparathyroidism (SHPT) undergoing hemodialysis (HD) and receiving paricalcitol. METHODS: A total of 493 (F/M 244/249) adult patients with SHPT who were undergoing HD in 22 HD units and receiving paricalcitol treatment, with iPTH > 300 mg/mL, adjusted serum levels of calcium (Ca) < 10.2 mg/dL, and serum levels of inorganic phosphorus (iP) < 6 mg/dL were included in this multi-center, national, prospective, observational study. Data regarding efficacy, safety, and adverse events of paricalcitol treatment were collected during a 12-month follow-up period through monthly visits along with serum iPTH, Ca, iP, alkaline phosphatase (ALP) and other required biochemistry tests as necessary. Mortality data until 6 months after the end of the study were also investigated. RESULTS: The mean age was 58.3 ± 15.8 years and the mean duration of HD was 6.2 ± 5.5 years, respectively. As of 12th month, mean iPTH values decreased from 646 ± 424 pg/mL to 473 ± 387 pg/mL (p < 0.001); no statistically significant changes were observed in Ca levels (p > 0.05). Serum ALP levels also significantly decreased (p = 0.001) and serum phosphorus levels significantly increased (p < 0.001) during the study observation period. Reasons for early terminations were being lost to follow-up (n = 119, 24.1%), hyperphosphatemia (iP > 6 mg/dL, n = 108, 21.9%), low iPTH levels (iPTH < 150 mg/dL, n = 97, 19.7%), and withdrawal of consent (n = 41, 8.3%). In total 32 patients (6.5%) were prematurely terminated the study with hypercalcemia (Ca > 10.2 mg/dL). 46.9% of those hypercalcemic patients had other anomalies with iP and iPTH levels along with hypercalcemia. CONCLUSION: Paricalcitol treatment, resulted in successful iPTH control. In approximately 6.5% of the patients paricalcitol treatment was discontinued since Ca levels reached > 10.2 mg/dL in those patients. No unfavorable effects on serum phosphorus and Ca-phosphorus (Ca × P) product were observed.

Identification of phosphorylation sites in mammalian mitochondrial ribosomal protein DAP3.

Mammalian mitochondrial ribosomes synthesize 13 proteins that are essential for oxidative phosphorylation. In addition to their role in protein synthesis, some of the mitochondrial ribosomal proteins have acquired functions in other cellular processes such as apoptosis. Death-associated protein 3 (DAP3), also referred to as mitochondrial ribosomal protein S29 (MRP-S29), is a GTP-binding pro-apoptotic protein located in the small subunit of the ribosome. Previous studies have shown that phosphorylation is one of the most likely regulatory mechanisms for DAP3 function in apoptosis and may be in protein synthesis; however, no phosphorylation sites were identified. In this study, we have investigated the phosphorylation status of ribosomal DAP3 and mapped the phosphorylation sites by tandem mass spectrometry. Mitochondrial ribosomal DAP3 is phosphorylated at Ser215 or Thr216, Ser220, Ser251 or Ser252, and Ser280. In addition, phosphorylation of recombinant DAP3 by Protein kinase A and Protein kinase Cdelta at residues that are endogenously phosphorylated in ribosomal DAP3 suggests both of these kinases as potential candidates responsible for the in vivo phosphorylation of DAP3 in mammalian mitochondria. Interestingly, the majority of the phosphorylation sites detected in our study are clustered around the highly conserved GTP-binding motifs, speculating on the significance of these residues on protein conformation and activity. Site-directed mutagenesis studies on selected phosphorylation sites were performed to determine the effect of phosphorylation on cell proliferation and PARP cleavage as indication of caspase activation. Overall, our findings suggest DAP3, a mitochondrial ribosomal small subunit protein, is a novel phosphorylated target.

A comprehensive proteomic analysis of the accessory sex gland fluid from mature Holstein bulls.

The expression of proteins in accessory sex gland fluid (AGF) of proven, high use mature Holstein bulls was evaluated. Thirty-seven bulls with documented fertility based on their non-return rates were studied. AGF was obtained by artificial vagina after bulls were surgically equipped with cannulae in the vasa deferentia. Samples of AGF were evaluated by two-dimensional SDS-PAGE, gels stained with Coomassie blue and polypeptide maps analyzed by PDQuest software. A master gel generated by the software representing the best pattern of spots in the AGF polypeptide maps was used as a reference for protein identification. Proteins were identified by Western blots and capillary liquid chromatography-nanoelectrospray ionization tandem-mass spectrometry (CapLC-MS/MS). The product ion spectra were processed using Protein Lynx Global Server 2.1 prior to database search with both PLGS and MASCOT (Matrix Science) software. The entire NCBI database was considered for mass fingerprint matching. An average of 52+/-5 spots was detected in the AGF 2D gels, which corresponded to proteins potentially involved in capacitation (bovine seminal plasma protein-BSP-A1/A2 and A3, BSP 30 kDa, albumin); sperm membrane protection, prevention of oxidative stress, complement-mediated sperm destruction and anti-microbial activity (albumin, clusterin, acidic seminal fluid protein--aSFP, 5'-nucleotidase--5'-NT, phospholipase A2--PLA2); acrosome reaction and sperm-oocyte interaction (PLA2, osteopontin); interaction with the extracellular matrix (tissue inhibitor of metalloproteinase 2, clusterin) and sperm motility (aSFP, spermadhesin Z13, 5'-NT). The 20 spots distinguished in all gels were matched to proteins associated with these functions. Proteins identified by tandem mass spectrometry as ecto-ADP-ribosyltransferase 5 and nucleobindin, never described before in the accessory sex gland secretions, were also detected. In summary, we identified a diverse range of components in the accessory sex gland fluid of a select group of Holstein bulls with documented fertility. Known characteristics of these proteins suggest that they play important roles in sperm physiology after ejaculation.

An LC-MS/MS method for the determination of digitoxigenin in skin samples and its application to skin permeation and metabolic stability studies.

An LC-MS/MS method was developed for the determination of digitoxigenin in mice skin samples. Chromatographic separation was achieved on an Agilent Poroshell 120 EC-C18 column. Mass spectrometric detection was achieved by a triple-quadrupole mass spectrometer equipped with an ESI interface operating in a positive ionization mode. Quantification was performed using selective reaction monitoring of the precursor-product ion transitions of m/z 375.5→339 for digitoxigenin and m/z 391.5→337 for internal standard (IS). The calibration curves were linear over the concentration range of 1.00-500ng/mL. The intra- and inter-batch precision was no more than 10.6% of the coefficient of variation and the accuracy was within ±8.1% of the actual values. This validated method has been successfully applied to skin permeation and skin metabolic stability studies of digitoxigenin in mice. The steady-state flux and lag time of digitoxigenin permeated across the full-thickness mice skin were 1.86±0.45µg/cm2/h and 0.46±0.18h, respectively. The metabolism of digitoxigenin in the skin was not detected in our study.

Effects of ethylene oxide and ethylene inhalation on DNA adducts, apurinic/apyrimidinic sites and expression of base excision DNA repair genes in rat brain, spleen, and liver.

Ethylene oxide (EO) is an important industrial chemical that is classified as a known human carcinogen (IARC, Group 1). It is also a metabolite of ethylene (ET), a compound that is ubiquitous in the environment and is the most used petrochemical. ET has not produced evidence of cancer in laboratory animals and is "not classifiable as to its carcinogenicity to humans" (IARC, Group 3). The mechanism of carcinogenicity of EO is not well characterized, but is thought to involve the formation of DNA adducts. EO is mutagenic in a variety of in vitro and in vivo systems, whereas ET is not. Apurinic/apyrimidinic sites (AP) that result from chemical or glycosylase-mediated depurination of EO-induced DNA adducts could be an additional mechanism leading to mutations and chromosomal aberrations. This study tested the hypothesis that EO exposure results in the accumulation of AP sites and induces changes in expression of genes for base excision DNA repair (BER). Male Fisher 344 rats were exposed to EO (100 ppm) or ET (40 or 3000 ppm) by inhalation for 1, 3 or 20 days (6h/day, 5 days a week). Animals were sacrificed 2h after exposure for 1, 3 or 20 days as well as 6, 24 and 72 h after a single-day exposure. Experiments were performed with tissues from brain and spleen, target sites for EO-induced carcinogenesis, and liver, a non-target organ. Exposure to EO resulted in time-dependent increases in N7-(2-hydroxyethyl)guanine (7-HEG) in brain, spleen, and liver and N7-(2-hydroxyethyl)valine (7-HEVal) in globin. Ethylene exposure also induced 7-HEG and 7-HEVal, but the numbers of adducts were much lower. No increase in the number of aldehydic DNA lesions, an indicator of AP sites, was detected in any of the tissues between controls and EO-, or ET-exposed animals, regardless of the duration or strength of exposure. EO exposure led to a 3-7-fold decrease in expression of 3-methyladenine-DNA glycosylase (Mpg) in brain and spleen in rats exposed to EO for 1 day. Expression of 8-oxoguanine DNA glycosylase, Mpg, AP endonuclease (Ape), polymerase beta (Pol beta) and alkylguanine methyltransferase were increased by 20-100% in livers of rats exposed to EO for 20 days. The only effects of ET on BER gene expression were observed in brain, where Ape and Pol beta expression were increased by less than 20% after 20 days of exposure to 3000 ppm. These data suggest that DNA damage induced by exposure to EO is repaired without accumulation of AP sites and is associated with biologically insignificant changes in BER gene expression in target organs. We conclude that accumulation of AP sites is not a likely primary mechanism for mutagenicity and carcinogenicity of EO.