Effects of age and calorie restriction on tryptophan nitration,protein content,and activity of succinyl-CoA:3-ketoacid CoA transferase in rat kidney mitochondria

This study examined the protein targets of nitration and the consequent impact on protein function in rat kidney mitochondria at 4, 13, 19, and 24 months of age. Succinyl-CoA transferase (SCOT), a rate-limiting enzyme in the degradation of ketone bodies, was the most intensely reactive protein against anti-3-nitrotyrosine antibody in rat kidney mitochondria. However, subsequent mass spectrometric and amino acid analyses of purified SCOT indicated that tryptophan 372, rather than a tyrosine residue, was the actual site of simultaneous additions of nitro and hydroxy groups. This finding suggests that identification of nitrated tyrosine residues based solely on reactivity with anti-3-nitrotyrosine antibody can be potentially misleading. Between 4 and 24 months of age, the amounts of SCOT protein and catalytic activity, expressed per milligram of mitochondrial proteins, decreased by 55 and 45%, respectively. SCOT, and particularly its nitrated carboxy-terminal region, was relatively more susceptible to in vitro proteolysis than other randomly selected kidney mitochondrial proteins. The age-related decreases in SCOT protein amount and catalytic activity were prevented by a relatively long-term 40% reduction in the amount of food intake. Loss of SCOT protein in the aged rats may attenuate the capacity of kidney mitochondria to utilize ketone bodies for energy production.     

Identification of [1-14C]pantothenic-acid-mediated modified mitochondrial proteins

The in vivo administration of [1-14C]pantothenic acid, which is the precursor of coenzyme A, resulted in the radioactive labelling of several mitochondrial proteins in rat liver. The incorporated radioactivity could be released by glutathione or 2-mercaptoethanol. Two mitochondrial matrix proteins acetyl-CoA acetyltransferase (liver and heart), an enzyme involved in the biosynthesis or degradation of ketone bodies, and 3-oxoacyl-CoA thiolase (liver), a protein participating in fatty acid oxidation were identified as modified proteins. The radioactivity was localized exclusively in forms A1 and A2 indicating that these forms represent the modified states of the acetyl-CoA acetyltransferase protein. Kinetics of incorporation of radioactivity revealed an accumulation of the modified forms. The ratio of specific radioactivities of A2 compared to A1 was 2.41 +/- 0.15 (n = 10). After in vivo labelling with [14C]leucine, the specific radioactivity of acetyl-CoA acetyltransferase depended on the state of the enzyme protein. The unmodified enzyme exhibited a lower specific radioactivity than its modified forms suggesting different turnover rates of these proteins.     

Structure of the mammalian CoA transferase from pig heart

Ketoacidosis affects patients who are deficient in the enzyme activity of succinyl-CoA:3-ketoacid CoA transferase (SCOT), since SCOT catalyses the activation of acetoacetate in the metabolism of ketone bodies. Thus far, structure/function analysis of the mammalian enzyme has been predicted based on the three-dimensional structure of a CoA transferase determined from an anaerobic bacterium that utilizes its enzyme for glutamate fermentation. To better interpret clinical data, we have determined the structure of a mammalian CoA transferase from pig heart by X-ray crystallography to 2.5 A resolution. Instrumental to the structure determination were selenomethionine substitution and the use of argon during purification and crystallization. Although pig heart SCOT adopts an alpha/beta protein fold, resembling the overall fold of the bacterial CoA transferase, several loops near the active site of pig heart SCOT follow different paths than the corresponding loops in the bacterial enzyme, accounting for differences in substrate specificities. Two missense mutations found associated with SCOT of ketoacidosis patients were mapped to a location in the structure that might disrupt the stabilization of the amino-terminal strand and thereby interfere with the proper folding of the protein into a functional enzyme.     

Protein tyrosine nitration in the mitochondria from diabetic mouse heart. Implications to dysfunctional mitochondria in diabetes

Oxidative stress has been implicated in dysfunctional mitochondria in diabetes. Tyrosine nitration of mitochondrial proteins was observed under conditions of oxidative stress. We hypothesize that nitration of mitochondrial proteins is a common mechanism by which oxidative stress causes dysfunctional mitochondria. The putative mechanism of nitration in a diabetic model of oxidative stress and functional changes of nitrated proteins were studied in this work. As a source of mitochondria, alloxan-susceptible and alloxan-resistant mice were used. These inbred strains are distinguished by the differential ability to detoxify free radicals. A proteomic approach revealed significant similarity between patterns of tyrosine-nitrated proteins generated in the heart mitochondria under different in vitro and in vivo conditions of oxidative stress. This observation points to a common nitrating species, which may derive from different nitrating pathways in vivo and may be responsible for the majority of nitrotyrosine formed. Functional studies show that protein nitration has an adverse effect on protein function and that protection against nitration protects functional properties of proteins. Because proteins that undergo nitration are involved in major mitochondrial functions, such as energy production, antioxidant defense, and apoptosis, we concluded that tyrosine nitration of mitochondrial proteins may lead to dysfunctional mitochondria in diabetes.     

Obligate role for ketone body oxidation in neonatal metabolic homeostasis

To compensate for the energetic deficit elicited by reduced carbohydrate intake, mammals convert energy stored in ketone bodies to high energy phosphates. Ketone bodies provide fuel particularly to brain, heart, and skeletal muscle in states that include starvation, adherence to low carbohydrate diets, and the neonatal period. Here, we use novel Oxct1(-/-) mice, which lack the ketolytic enzyme succinyl-CoA:3-oxo-acid CoA-transferase (SCOT), to demonstrate that ketone body oxidation is required for postnatal survival in mice. Although Oxct1(-/-) mice exhibit normal prenatal development, all develop ketoacidosis, hypoglycemia, and reduced plasma lactate concentrations within the first 48 h of birth. In vivo oxidation of (13)C-labeled β-hydroxybutyrate in neonatal Oxct1(-/-) mice, measured using NMR, reveals intact oxidation to acetoacetate but no contribution of ketone bodies to the tricarboxylic acid cycle. Accumulation of acetoacetate yields a markedly reduced β-hydroxybutyrate:acetoacetate ratio of 1:3, compared with 3:1 in Oxct1(+) littermates. Frequent exogenous glucose administration to actively suckling Oxct1(-/-) mice delayed, but could not prevent, lethality. Brains of newborn SCOT-deficient mice demonstrate evidence of adaptive energy acquisition, with increased phosphorylation of AMP-activated protein kinase α, increased autophagy, and 2.4-fold increased in vivo oxidative metabolism of [(13)C]glucose. Furthermore, [(13)C]lactate oxidation is increased 1.7-fold in skeletal muscle of Oxct1(-/-) mice but not in brain. These results indicate the critical metabolic roles of ketone bodies in neonatal metabolism and suggest that distinct tissues exhibit specific metabolic responses to loss of ketone body oxidation.     

Oxidatively damaged proteins of heart mitochondrial electron transport complexes

Protein modifications, such as carbonylation, nitration and formation of lipid peroxidation adducts, e.g. 4-hydroxynonenal (HNE), are products of oxidative damage attributed to reactive oxygen species (ROS). The mitochondrial respiratory chain Complexes I and III have been shown to be a major source of ROS in vitro. Additionally, modifications of the respiratory chain Complexes (I-V) by nitration, carbonylation and HNE adduct decrease their enzymatic activity in vitro. However, modification of these respiratory chain complex proteins due to in vivo basal level ROS generation has not been investigated. In this study, we show a basal level of oxidative damage to specific proteins of adult bovine heart submitochondrial particle (SMP) complexes, and find that most of these proteins are localized in the mitochondrial matrix. We postulate that electron leakage from respiratory chain complexes and subsequent ROS formation may cause damage to specific complex subunits and contribute to long-term accumulation of mitochondrial dysfunction.     

Different localization in rat brain of the novel cytosolic ketone body-utilizing enzyme, acetoacetyl-CoA synthetase, as compared to succinyl-CoA:3-oxoacid CoA-transferase

In lipogenic tissue cytosol, ketone bodies are known to be activated by acetoacetyl-CoA synthetase (AACS) and incorporated into cholesterol and fatty acids. In order to investigate the physiological role of AACS in the brain, we examined the localization of AACS mRNA in rat brain by in situ hybridization using a labeled probe. High labeling was observed in the midbrain, pons/medulla, cerebral cortex, hippocampus and cerebellum, and the localization profile of AACS mRNA was different from that of succinyl-CoA:3-oxoacid CoA-transferase (SCOT), a mitochondrial ketone body-activating enzyme. In addition, the expression of AACS mRNA in the cerebellum was restricted primarily to glial cells, while in the cerebral cortex, it was restricted to neuronal cells. Streptozotocin treatment caused remarkable decreases in AACS mRNA levels in all regions where expression was observed, but changes in SCOT mRNA levels were not observed. These results suggest that the physiological role of AACS is different from that of SCOT and varies depending upon its localization in the brain.     

Identification of proteins from non-model organisms using mass spectrometry: application to a hibernating mammal

A major challenge in the life sciences is the extraction of detailed molecular information from plants and animals that are not among the handful of exhaustively studied "model organisms." As a consequence, certain species with novel phenotypes are often ignored due to the lack of searchable databases, tractable genetics, stock centers, and more recently, a sequenced genome. Characterization of phenotype at the molecular level commonly relies on the identification of differentially expressed proteins by combining database searching with tandem mass spectrometry (MS) of peptides derived from protein fragmentation. However, the identification of short peptides from nonmodel organisms can be hampered by the lack of sufficient amino acid sequence homology with proteins in existing databases; therefore, a database search strategy that encompasses both identity and homology can provide stronger evidence than a single search alone. The use of multiple algorithms for database searches may also increase the probability of correct protein identification since it is unlikely that each program would produce false negative or positive hits for the same peptides. In this study, four software packages, Mascot, Pro ID, Sequest, and Pro BLAST, were compared in their ability to identify proteins from the thirteen-lined ground squirrel (Spermophilus tridecemlineatus), a hibernating mammal that lacks a completely sequenced genome. Our results show similarities as well as the degree of variability among different software packages when the identical protein database is searched. In the process of this study, we identified the up-regulation of succinyl CoA-transferase (SCOT) in the heart of hibernators. SCOT is the rate-limiting enzyme in the catabolism of ketone bodies, an important alternative fuel source during hibernation.     

Protein tyrosine nitration of mitochondrial carbamoyl phosphate synthetase 1 and its functional consequences

Mitochondria are the primary locus for the generation of reactive nitrogen species including peroxynitrite and subsequent protein tyrosine nitration. Protein tyrosine nitration may have important functional and biological consequences such as alteration of enzyme catalytic activity. In the present study, mouse liver mitochondria were incubated with peroxynitrite, and the mitochondrial proteins were separated by 1D and 2D gel electrophoresis. Nitrotyrosinylated proteins were detected with an anti-nitrotyrosine antibody. One of the major proteins nitrated by peroxynitrite was carbamoyl phosphate synthetase 1 (CPS1) as identified by LC-MS protein analysis and Western blotting. The band intensity of nitration normalized to CPS1 was increased in a peroxynitrite concentration-dependent manner. In addition, CPS1 activity was decreased by treatment with peroxynitrite in a peroxynitrite concentration- and time-dependent manner. The decreased CPS1 activity was not recovered by treatment with reduced glutathione, suggesting that the decrease of the CPS1 activity is due to tyrosine nitration rather than cysteine oxidation. LC-MS analysis of in-gel digested samples, and a Popitam-based modification search located 5 out of 36 tyrosine residues in CPS1 that were nitrated. Taken together with previous findings regarding CPS1 structure and function, homology modeling of mouse CPS1 suggested that nitration at Y1450 in an α-helix of allosteric domain prevents activation of CPS1 by its activator, N-acetyl-l-glutamate. In conclusion, this study demonstrated the tyrosine nitration of CPS1 by peroxynitrite and its functional consequence. Since CPS1 is responsible for ammonia removal in the urea cycle, nitration of CPS1 with attenuated function might be involved in some diseases and drug-induced toxicities associated with mitochondrial dysfunction.     

Mitochondrial nitric oxide metabolism in rat muscle during endotoxemia

In this study, heart and diaphragm mitochondria produced 0.69 and 0.77 nmol nitric oxide (NO)/min mg protein, rates that account for 67 and 24% of maximal cellular NO production, respectively. Endotoxemia and septic shock occur with an exacerbated inflammatory response that damages tissue mitochondria. Skeletal muscle seems to be one of the main target organs in septic shock, showing an increased NO production and early oxidative stress. The kinetic properties of mitochondrial nitric oxide synthase (mtNOS) of heart and diaphragm were determined. For diaphragm, the KM values for O2 and L-Arg were 4.6 and 37 microM and for heart were 3.3 and 36 microM. The optimal pH for mtNOS activity was 6.5 for diaphragm and 7.0 for heart. A marked increase in mtNOS activity was observed in endotoxemic rats, 90% in diaphragm and 30% in heart. Diaphragm and heart mitochondrial O2*- and H2O2 production were 2- to 3-fold increased during endotoxemia and Mn-SOD activity showed a 2-fold increase in treated animals, whereas catalase activity was unchanged. One of the current hypotheses for the molecular mechanisms underlying the complex condition of septic shock is that the enhanced NO production by mtNOS leads to excessive peroxynitrite production and protein nitration in the mitochondrial matrix, causing mitochondrial dysfunction and contractile failure.     

琥珀酰辅酶A转移酶的表达纯化及抗体制备

目的:琥珀酰辅酶A转移酶(SCOT)是酮体代谢过程中的关键限速酶,此酶缺陷多由SCOT基因突变引起,患者多有酮症酸中毒表现。为了进一步研究SCOT的功能,采用原核表达系统表达并纯化重组SCOT,制备SCOT多克隆抗体。方法:选择蛋鸡、肉鸡模式生物为研究对象,通过生物信息学对其抗原性和属间同源性进行分析,通过RT-PCR从鸡的骨骼肌cDNA中扩增了SCOT基因N端半长片段,克隆到表达载体pET28b中,在大肠杆菌BL21(DE3)中诱导表达,并用镍离子螯合柱(Ni-NTA)纯化重组SCOT;用纯化的重组SCOT免疫小鼠后得到多克隆抗体。结果:Western印迹表明,制备的SCOT抗体具有较高的特异性,可特异性识别鸡的SCOT蛋白,同时可特异性识别小鼠和人的相应SCOT蛋白。结论:SCOT多克隆抗体的制备为后续在鸡、鼠和人中研究SCOT基因提供基础。     

A comparative study of the metabolic capacity of hearts from reptiles and mammals

The metabolic capacities of reptilian and mammalian hearts have been investigated using two methods: measurement of mitochondrial enzyme activity (cytochrome oxidase) and measurement of both mitochondrial volume density and membrane surface area. The heart tissues from the reptiles and mammals showed 2-fold "weight specific" and 3-fold total organ metabolic capacity differences. Heart mitochondria from reptiles and mammals showed 2-fold differences in the activity of their enzymes per mg of mitochondrial protein yet showed very similar mitochondrial surface areas per cm3 of mitochondria. Heart mitochondria differ from liver mitochondria which have the same enzyme activities per mg of protein and the same mitochondrial surface area per cm3 of mitochondria in both the reptiles and mammals. A wide variety of reptiles and mammals both showed relationships between total heart metabolic capacity and body weight. Mammals have larger hearts than similar sized reptiles and their hearts have a greater proportion of cellular volume occupied by mitochondria.     

Regulation of glutamate dehydrogenase by reversible ADP-ribosylation in mitochondria

Mitochondrial ADP-ribosylation leads to modification of two proteins of approximately 26 and 53 kDA: The nature of these proteins and, hence, the physiological consequences of their modification have remained unknown. Here, a 55 kDa protein, glutamate dehydrogenase (GDH), was established as a specific acceptor for enzymatic, cysteine-specific ADP-ribosylation in mitochondria. The modified protein was isolated from the mitochondrial preparation and identified as GDH by N-terminal sequencing and mass spectrometric analyses of tryptic digests. Incubation of human hepatoma cells with [14C]adenine demonstrated the occurrence of the modification in vivo. Purified GDH was ADP-ribosylated in a cysteine residue in the presence of the mitochondrial activity that transferred the ADP-ribose from NAD+ onto the acceptor site. ADP- ribosylation of GDH led to substantial inhibition of its catalytic activity. The stoichiometry between incorporated ADP-ribose and GDH subunits suggests that modification of one subunit per catalytically active homohexamer causes the inactivation of the enzyme. Isolated, ADP-ribosylated GDH was reactivated by an Mg2+-dependent mitochondrial ADP-ribosylcysteine hydrolase. GDH, a highly regulated enzyme, is the first mitochondrial protein identified whose activity may be modulated by ADP-ribosylation.     

Immunological study of the tissue expression of D-beta-hydroxybutyrate dehydrogenase, a ketone body-converting enzyme

In rats, as in most mammal, ketone bodies are mainly produced in liver while they are metabolized in extrahepatic tissues. The expression of mitochondrial membrane-bound D-beta-hydroxybutyrate dehydrogenase (BDH), a ketone body-converting enzyme, has been estimated by two immunological techniques: immunohistofluorescence and Western blotting. The in situ labeling with anti-BDH antibody shows that the enzyme is expressed differently among the organs. Furthermore, within a given organ there are strong differences according to the cell type. The quantification of the enzyme by immunoblotting reveals that liver mitochondria have the highest content (more than 3% in protein mass). This content is 3,5 and 10 times lower in kidney, heart and brain mitochondria, respectively. Parallel D-beta-hydroxybutyrate dehydrogenase activity measurements on isolated mitochondria show differences in molecular activity of this enzyme according to the tissue origin. Due to the phospholipid requirement of this enzyme these differences in molecular activity are related to specific membrane lipid composition.     

Intrinsic protein kinase activity in mitochondrial oxidative phosphorylation complexes

Mitochondrial protein phosphorylation is a well-recognized metabolic control mechanism, with the classical example of pyruvate dehydrogenase (PDH) regulation by specific kinases and phosphatases of bacterial origin. However, despite the growing number of reported mitochondrial phosphoproteins, the identity of the protein kinases mediating these phosphorylation events remains largely unknown. The detection of mitochondrial protein kinases is complicated by the low concentration of kinase relative to that of the target protein, the lack of specific antibodies, and contamination from associated, but nonmatrix, proteins. In this study, we use blue native gel electrophoresis (BN-PAGE) to isolate rat and porcine heart mitochondrial complexes for screening of protein kinase activity. To detect kinase activity, one-dimensional BN-PAGE gels were exposed to [γ-(32)P]ATP and then followed by sodium dodecyl sulfate gel electrophoresis. Dozens of mitochondrial proteins were labeled with (32)P in this setting, including all five complexes of oxidative phosphorylation and several citric acid cycle enzymes. The nearly ubiquitous (32)P protein labeling demonstrates protein kinase activity within each mitochondrial protein complex. The validity of this two-dimensional BN-PAGE method was demonstrated by detecting the known PDH kinases and phosphatases within the PDH complex band using Western blots and mass spectrometry. Surprisingly, these same approaches detected only a few additional conventional protein kinases, suggesting a major role for autophosphorylation in mitochondrial proteins. Studies on purified Complex V and creatine kinase confirmed that these proteins undergo autophosphorylation and, to a lesser degree, tenacious (32)P-metabolite association. In-gel Complex IV activity was shown to be inhibited by ATP, and partially reversed by phosphatase activity, consistent with an inhibitory role for protein phosphorylation in this complex. Collectively, this study proposes that many of the mitochondrial complexes contain an autophosphorylation mechanism, which may play a functional role in the regulation of these multiprotein units.     

Cytochrome c: a catalyst and target of nitrite-hydrogen peroxide-dependent protein nitration

Nitration of protein tyrosine residues to 3-nitrotyrosine (NO2Tyr) serves as both a marker and mediator of pathogenic reactions of nitric oxide (*NO), with peroxynitrite (ONOO-) and leukocyte peroxidase-derived nitrogen dioxide (*NO2) being proximal mediators of nitration reactions in vivo. Cytochrome c is a respiratory and apoptotic signaling heme protein localized exofacially on the inner mitochondrial membrane. We report herein a novel function for cytochrome c as a catalyst for nitrite (NO2-) and hydrogen peroxide (H2O2)-mediated nitration reactions. Cytochrome c catalyzes both self- and adjacent-molecule (hydroxyphenylacetic acid, Mn-superoxide dismutase) nitration via heme-dependent mechanisms involving tyrosyl radical and *NO2 production, as for phagocyte peroxidases. Although low molecular weight phenolic nitration yields were similar for cytochrome c and the proteolytic fragment of cytochrome c microperoxidase-11 (MPx-11), greater extents of protein nitration occurred when MPx-11 served as catalyst. Partial proteolysis of cytochrome c increased both the peroxidase and nitrating activities of cytochrome c. Extensive tyrosine nitration of Mn-superoxide dismutase occurred when exposed to either cytochrome c or MPx-11 in the presence of H2O2 and NO2-, with no apparent decrease in catalytic activity. These results reveal a post-translational tyrosine modification mechanism that is mediated by an abundant hemoprotein present in both mitochondrial and cytosolic compartments. The data also infer that the distribution of specific proteins capable of serving as potent catalysts of nitration can lend both spatial and molecular specificity to biomolecule nitration reactions.     

Glucocorticoid receptor nitration leads to enhanced anti-inflammatory effects of novel steroid ligands

It has recently emerged that posttranslational modification of proteins via nitration of tyrosine residues can alter their function. In this study, we describe that specific nitration of the glucocorticoid receptor (GR) by NCX-1015, a novel NO-donating prednisolone derivative (prednisolone 21-[4'-(nitrooxymethyl)benzoate), results in an enhancement of GR-mediated events. Incubation of PBMC and U937 cells with 1-10 micro M NCX-1015 caused faster activation of GR as assessed by augmented 1) binding to [(3)H]dexamethasone, 2) dissociation from heat shock protein 90, and 3) nuclear translocation. PBMCs treated with NCX-1015 contained GR that had undergone tyrosine nitration. The chemistry facilitating the increase in steroid binding capacity observed with NCX-1015 is specific, because changing the position of the NO-donating group or ubiquitous nitration by addition of an NO donor was unable to mimic this event. In vivo treatment with NCX-1015 provoked GR nitration and faster heat shock protein 90 dissociation as assessed in peritoneal cells. Accordingly, NCX-1015, but not prednisolone or other derivatives, produced a rapid inhibition of the early neutrophil recruitment and mediator generation in a model of peritonitis. In conclusion, we report here for the first time that posttranslational modification of GR by this novel nitrosteroid is associated with its enhanced anti-inflammatory activity.     

Induction of apoptosis in T lymphoma cells by long-term treatment with thyroxine involves PKCζ nitration by nitric oxide synthase

Thyroid hormones are important regulators of cell physiology, inducing cell proliferation, differentiation or apoptosis, depending on the cell type. Thyroid hormones induce proliferation in short-term T lymphocyte cultures. In this study, we assessed the effect of long-term thyroxine (T4) treatment on the balance of proliferation and apoptosis and the intermediate participants in T lymphoma cells. Treatment with T4 affected this balance from the fifth day of culture, inhibiting proliferation in a time-dependent manner. This effect was associated with apoptosis induction, as characterized through nuclear morphological changes, DNA fragmentation, and Annexin V-FITC/Propidium Iodide co-staining. In addition, increased iNOS gene and protein levels, and enzyme activity were observed. The generation of reactive oxygen species, depolarization of the mitochondrial membrane, and a reduction in glutathione levels were also observed. The imbalance between oxidants and antioxidants species is typically associated with the nitration of proteins, including PKCζ, an isoenzyme essential for lymphoma cell division and survival. Consistently, evidence of PKCζ nitration via proteasome degradation was also observed in this study. Taken together, these results suggest that the long-term culture of T lymphoma cells with T4 induces apoptosis through the increased production of oxidative species resulting from both augmented iNOS activity and the loss of mitochondrial function. These species induce the nitration of proteins involved in cell viability, promoting proteasome degradation. Furthermore, we discuss the impact of these results on the modulation of T lymphoma growth and the thyroid status in vivo.