Modulation of methyl group metabolism by streptozotocin-induced diabetes and all-trans-retinoic acid

The hepatic enzyme glycine N-methyltransferase (GNMT) plays a major role in the control of methyl group and homocysteine metabolism. Because disruption of these vital pathways is associated with numerous pathologies, understanding GNMT control is important for evaluating methyl group regulation. Recently, gluconeogenic conditions have been shown to modulate homocysteine metabolism and treatment with glucocorticoids and/or all-trans-retinoic acid (RA)-induced active GNMT protein, thereby leading to methyl group loss. This study was conducted to determine the effect of diabetes, alone and in combination with RA, on GNMT regulation. Diabetes and RA increased GNMT activity 87 and 148%, respectively. Moreover, the induction of GNMT activity by diabetes and RA was reflected in its abundance. Cell culture studies demonstrated that pretreatment with insulin prevented GNMT induction by both RA and dexamethasone. There was a significant decline in homocysteine concentrations in diabetic rats, owing in part to a 38% increase in the abundance of the transsulfuration enzyme cystathionine beta-synthase; treatment of diabetic rats with RA prevented cystathionine beta-synthase induction. A diabetic state also increased the activity of the folate-independent homocysteine remethylation enzyme betaine-homocysteine S-methyltransferase, whereas the activity of the folate-dependent enzyme methionine synthase was diminished 52%. In contrast, RA treatment attenuated the streptozotocin-mediated increase in betaine-homocysteine S-methyltransferase, whereas methionine synthase activity remained diminished. These results indicate that both a diabetic condition and RA treatment have marked effects on the metabolism of methyl groups and homocysteine, a finding that may have significant implications for diabetics and their potential sensitivity to retinoids.     

Influence of acute cadmium exposure on the liver proteome of a teleost fish,ayu (<Emphasis Type="Italic">Plecoglossus altivelis</Emphasis>)

Cadmium (Cd) is a toxic heavy metal that causes the disruption of a variety of physiological processes. In this study, the effect of Cd on liver proteome of ayu, Plecoglossus altivelis, was investigated by two-dimensional gel electrophoresis (2-DE) and matrix assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF–MS/MS). Twenty-three altered protein spots were successfully identified. They were involved in oxidative stress response, metal metabolism, methylation, and so on. The mRNA expression of 60S acidic ribosomal protein P0, heat shock protein 70, apolipoprotein A-I, betaine-homocysteine S-methyltransferase, parahox cluster neighbor, and transferrin was subsequently determined by real-time PCR. The mRNA expression of these genes was consistent with proteomic results. These findings enrich our knowledge on the influence of Cd toxicity to teleost fish, and may be worthy of further investigation to develop biomarkers.     

Methionine and serine synergistically suppress hyperhomocysteinemia induced by choline deficiency, but not by guanidinoacetic acid, in rats fed a low casein diet

The effects of dietary supplementation with 0.5% methionine, 2.5% serine, or both on hyperhomocysteinemia induced by deprivation of dietary choline or by dietary addition of 0.5% guanidinoacetic acid (GAA) were investigated in rats fed a 10% casein diet. Hyperhomocysteinemia induced by choline deprivation was not suppressed by methionine alone and was only partially suppressed by serine alone, whereas it was completely suppressed by a combination of methionine and serine, suggesting a synergistic effect of methionine and serine. Fatty liver was also completely prevented by the combination of methionine and serine. Compared with methionine alone, the combination of methionine and serine decreased hepatic S-adenosylhomocysteine and homocysteine concentrations and increased hepatic betaine and serine concentrations and betaine-homocysteine S-methyltransferase activity. GAA-induced hyperhomocysteinemia was partially suppressed by methionine alone, but no interacting effect of methionine and serine was detected. In contrast, GAA-induced fatty liver was completely prevented by the combination of methionine and serine. These results indicate that a combination of methionine and serine is effective in suppressing both hyperhomocysteinemia and fatty liver induced by choline deprivation, and that methionine alone is effective in suppressing GAA-induced hyperhomocysteinemia partially.     

Mitochondrial calpain 10 is degraded by Lon protease after oxidant injury

Calpain 10 is ubiquitously expressed and is one of four mitochondrial matrix proteases. We determined that over-expression or knock-down of mitochondrial calpain 10 results in cell death, demonstrating that mitochondrial calpain 10 is required for viability. Thus, we studied calpain 10 degradation in isolated mitochondrial matrix, mitochondria and in renal proximal tubular cells (RPTC) under control and toxic conditions. Using isolated renal cortical mitochondria and mitochondrial matrix, calpain 10 underwent rapid degradation at 37°C that was blocked with Lon inhibitors but not by calpain or proteasome inhibitors. While exogenous Ca(2+) addition, Ca(2+) chelation or exogenous ATP addition had no effect on calpain 10 degradation, the oxidants tert-butyl hydroperoxide (TBHP) or H(2)O(2) increased the rate of degradation. Using RPTC, mitochondrial and cytosolic calpain 10 increased in the presence of MG132 (Lon/proteasome inhibitor) but only cytosolic calpain 10 increased in the presence of epoxomicin (proteasome inhibitor). Furthermore, TBHP and H(2)O(2) oxidized mitochondrial calpain 10, decreased mitochondrial, but not cytosolic calpain 10, and pretreatment with MG132 blocked TBHP-induced degradation of calpain 10. In summary, mitochondrial calpain 10 is selectively degraded by Lon protease under basal conditions and is enhanced under and oxidizing conditions, while cytosolic calpain 10 is degraded by the proteasome.     

Dynamic Regulation of Hepatic Lipid Droplet Properties by Diet

Cytoplasmic lipid droplets (CLD) are organelle-like structures that function in neutral lipid storage, transport and metabolism through the actions of specific surface-associated proteins. Although diet and metabolism influence hepatic CLD levels, how they affect CLD protein composition is largely unknown. We used non-biased, shotgun, proteomics in combination with metabolic analysis, quantitative immunoblotting, electron microscopy and confocal imaging to define the effects of low- and high-fat diets on CLD properties in fasted-refed mice. We found that the hepatic CLD proteome is distinct from that of CLD from other mammalian tissues, containing enzymes from multiple metabolic pathways. The hepatic CLD proteome is also differentially affected by dietary fat content and hepatic metabolic status. High fat feeding markedly increased the CLD surface density of perilipin-2, a critical regulator of hepatic neutral lipid storage, whereas it reduced CLD levels of betaine-homocysteine S-methyltransferase, an enzyme regulator of homocysteine levels linked to fatty liver disease and hepatocellular carcinoma. Collectively our data demonstrate that the hepatic CLD proteome is enriched in metabolic enzymes, and that it is qualitatively and quantitatively regulated by diet and metabolism. These findings implicate CLD in the regulation of hepatic metabolic processes, and suggest that their properties undergo reorganization in response to hepatic metabolic demands.     

Musical chairs during mitophagy

Mitophagy, or the autophagic degradation of mitochondria, is thought to be important in mitochondrial quality control, and hence in cellular physiology. Defects in mitophagy correlate with late onset pathologies and aging. Here, we discuss recent results that shed light on the interrelationship between mitophagy and mitochondrial dynamics, based on proteomic analyses of protein dynamics in wild-type and mutant cells. These studies show that different mitochondrial matrix proteins undergo mitophagy at different rates, and that the rate differences are affected by mitochondrial dynamics. These results are consistent with models in which phase separation within the mitochondrial matrix leads to unequal segregation of proteins during mitochondrial fission. Repeated fusion and fission cycles may thus lead to “distillation” of components that are destined for degradation.     

Comparative studies on the methionine synthesis in sheep and rat tissues

The important features of the enzymes involved in methionine synthesis in sheep were found to be the low activity of betaine-homocysteine methyltransferase and the high activity of 5-methyltetrahydrofolate-homocysteine methyltransferase. The rate of the methionine synthesis in sheep liver was significantly lower than that in rats due to the low activity of hepatic betaine-homocysteine methyltransferase. The hepatic methionine recycling was stimulated by the addition of betaine in both species. These results indicate that in sheep 5-methyltetrahydrofolate-homocysteine methyltransferase plays a significant role in hepatic methionine synthesis along with betaine-homocysteine methyltransferase. In contrast, in the rat hepatic system methionine synthesis is virtually dependent on betaine-homocysteine methyltransferase.     

Musical chairs during mitophagy

Mitophagy, or the autophagic degradation of mitochondria, is thought to be important in mitochondrial quality control, and hence in cellular physiology. Defects in mitophagy correlate with late onset pathologies and aging. Here, we discuss recent results that shed light on the interrelationship between mitophagy and mitochondrial dynamics, based on proteomic analyses of protein dynamics in wild-type and mutant cells. These studies show that different mitochondrial matrix proteins undergo mitophagy at different rates, and that the rate differences are affected by mitochondrial dynamics. These results are consistent with models in which phase separation within the mitochondrial matrix leads to unequal segregation of proteins during mitochondrial fission. Repeated fusion and fission cycles may thus lead to “distillation” of components that are destined for degradation.     

II - Insulin processing in mitochondria

Our objective was to know how insulin is processing in mitochondria; if IDE is the only participant in mitochondrial insulin degradation and the role of insulin degradation on IDE accumulation in mitoplasts. Mitochondria and its fractions were isolated as described by Greenwalt. IDE was purified and detected in immunoblot with specific antibodies. High insulin degradation was obtained through addition to rat’s diet of 25 g/rat of apple and 10 g/rat of hard-boiled eggs, 3 days a week. Mitochondrial insulin degradation was assayed with 5 % TCA, insulin antibody or Sephadex G50 chromatography. Degradation was also assayed 60 min at 37 °C in mitochondrial fractions (IMS and Mx) with diet or not and without IDE. Degradation in fractions precipitated with ammonium sulfates (60–80 %) were studied after mitochondrial insulin incubation (1 ng. insulin during 15 min, at 30 °C) or with addition of 2.5 mM ATP. Supplementary diet increased insulin degradation. High insulin did not increase mitoplasts accumulation and did not decrease mitochondrial degradation. High insulin and inhibition of degradation evidence insulin competition for a putative transport system. Mitochondrial incubation with insulin increased IDE in matrix as observed in immunoblot. ATP decreased degradation in Mx and increased it in IMS. Chromatography of IMS demonstrated an ATP-dependent protease that degraded insulin, similar to described by Sitte et al. Mitochondria participate in insulin degradation and the diet increased it. High insulin did not accomplish mitochondrial decrease of degradation or its accumulation in mitoplasts. Mitochondrial incubation with insulin increased IDE in matrix. ATP suggested being a regulator of mitochondrial insulin degradation.     

Cooperation of lysosomes and inner mitochondrial membrane in the degradation of carbamoyl phosphate synthetase and other proteins

Carbamoyl phosphate synthetase (CPS) from rat liver is proteolitically inactivated at acid pH by broken lysosomes. Inactivation increases when lysosomes are previously incubated with inner mitochondrial membrane, although this mitochondrial fraction does not inactivate CPS 'per se'. The increased degradation is due to membrane factor(s), most probably mitochondrial proteinase(s), solubilized by lysosomal matrix proteinases, after incubation of the inner mitochondrial membrane fraction with broken lysosomes. This (these ) factor(s) degrade(s) CPS and other proteins in the absence of lysosomal proteinases or when these are inhibited by leupeptin, chymostatin and pepstatin. We have also tested the possible regulation of this degradation and found that ATP and, particularly, acetyl glutamate accelerate the degradation of CPS by the factor(s) liberated from the inner mitochondrial membrane.     

Folate status modulates the induction of hepatic glycine N-methyltransferase and homocysteine metabolism in diabetic rats

A diabetic state induces the activity and abundance of glycine N-methyltransferase (GNMT), a key protein in the regulation of folate, methyl group, and homocysteine metabolism. Because the folate-dependent one-carbon pool is a source of methyl groups and 5-methyltetrahydrofolate allosterically inhibits GNMT, the aim of this study was to determine whether folate status has an impact on the interaction between diabetes and methyl group metabolism. Rats were fed a diet containing deficient (0 ppm), adequate (2 ppm), or supplemental (8 ppm) folate for 30 days, after which diabetes was initiated in one-half of the rats by streptozotocin treatment. The activities of GNMT, phosphatidylethanolamine N-methyltransferase (PEMT), and betaine-homocysteine S-methyltransferase (BHMT) were increased about twofold in diabetic rat liver; folate deficiency resulted in the greatest elevation in GNMT activity. The abundance of GNMT protein and mRNA, as well as BHMT mRNA, was also elevated in diabetic rats. The marked hyperhomocysteinemia in folate-deficient rats was attenuated by streptozotocin, likely due in part to increased BHMT expression. These results indicate that a diabetic state profoundly modulates methyl group, choline, and homocysteine metabolism, and folate status may play a role in the extent of these alterations. Moreover, the upregulation of BHMT and PEMT may indicate an increased choline requirement in the diabetic rat.     

Proteome analysis of human hepatocellular carcinoma tissues by two-dimensional difference gel electrophoresis and mass spectrometry

Proteome analysis of human hepatocellular carcinoma tissues was conducted using two-dimensional difference gel electrophoresis coupled with mass spectrometry. Paired samples from the normal and tumor region of resected human liver were labeled with Cy3 and Cy5, respectively while the pooled standard sample was labeled with Cy2. After analysis by the DeCyder software, protein spots that exhibited at least a two-fold difference in intensity were excised for in-gel tryptic digestion and matrix-assisted laser desorption/ionization-time of flight mass spectrometry. A total of 6 and 42 proteins were successfully identified from the well- and poorly-differentiated samples, respectively. The majority of these proteins are related to detoxification/oxidative stress and metabolism. Three down-regulated metabolic enzymes, methionine adenosyltransferase, glycine N-methyltransferase, and betaine-homocysteine S-methyltransferase that are involved in the methylation cycle in the liver are of special interest. Their expression levels, especially, methionine adenosyltransferase, seemed to have a major influence on the level of S-adenosylmethionine (AdoMet), a vital intermediate metabolite required for the proper functioning of the liver. Recent work has shown that chronic deficiency in AdoMet in the liver results in spontaneous development of steatohepatitis and hepatocellular carcinoma, and hence the down-regulation of hepatic methionine adenosyltransferase in our hepatocellular carcinoma samples is in line with this observation. Moreover, when a comparison is made between the differentially expressed proteins from our human hepatocellular carcinoma samples and from the liver tissues of knockout mice deficient in methionine adenosyltransferase, there is a fairly good correlation between them.     

Identification of new amine acceptor protein substrate candidates of transglutaminase in rat liver extract: use of 5-(biotinamido) pentylamine as a probe

Transglutaminases (TGs) are a family of enzymes that catalyze Ca(2+)-dependent post-translational modification of proteins by introducing protein-protein crosslinks (between specific glutamine and lysine residues), amine incorporation, and site-specific deamidation. In this study, new amine acceptor protein substrates of TG were isolated from rat liver extract and identified using 5-(biotinamido) pentylamine, a biotinylated primary amine substrate, as a probe. TG protein substrate candidates labeled with biotin by endogenous TG activity were isolated and recovered by avidin column chromatography. Proteins with molecular masses of 40, 42, and 45 kDa were the main components of the labeled proteins. Determination of their partial amino acid sequences and immunoblotting analyses were done to identify them. The 45-kDa protein was identical with betaine-homocysteine S-methyltransferase (EC 2.2.2.5), which was identified in our previous study. The 40- and 42-kDa proteins were identified as arginase-I (EC 3.5.3.1) and fructose-1,6-bisphosphatase (EC 3.1.3.11) respectively. TG catalyzed incorporation of 5-(biotinamido) pentylamine into both arginase-I and fructose-1,6-bisphosphatase purified from rat liver was confirmed in vitro. These results suggest that these two enzymes are the new protein substrate candidates of TG and that they can be modified post-translationally by cellular TG.     

Discrimination of distinct proteinases at the four structural levels of rat liver mitochondria.

Rat liver mitochondrial fractions corresponding to four morphological structures (matrix, inner membrane, intermembrane space and outer membrane) contain proteinases that cleave casein components at different rates. Proteinases of the intermembrane space preferentially cleave kappa-casein, whereas the proteinases of the outer membrane, inner membrane and matrix fractions degrade alpha S1-casein more rapidly. Electrophoretic separation of the degradation products of alpha S1-casein and kappa-casein in polyacrylamide gels shows that different polypeptides are produced when the substrate is degraded by the matrix, by both membranes and by the intermembrane-space fraction. Some of the degradation products resulting from incubation of the caseins with the mitochondrial fractions are probably the result of digestion by contaminating lysosomal proteinase(s). The matrix has a high peptidase activity, since glucagon, a small peptide, is very rapidly degraded by this fraction. These observations strongly suggest that distinct proteinases, with different specificities, are associated respectively with the intermembrane space and with both membrane fractions.     

The ClpB homolog Hsp78 is required for the efficient degradation of proteins in the mitochondrial matrix

Molecular chaperones perform vital functions in mitochondrial protein import and folding. In yeast mitochondria, two members of the Clp/Hsp100 chaperone family, Hsp78 and Mcx1, have been identified as homologs of the bacterial proteins ClpB and ClpX, respectively. In this report we employed a novel quantitative assay system to assess the role of Hsp78 and Mcx1 in protein degradation within the matrix. Mitochondria were preloaded with large amounts of two purified recombinant reporter proteins exhibiting different folding stabilities. Proteolysis of the imported substrate proteins depended on the mitochondrial level of ATP and was mediated by the matrix protease Pim1/LON. Degradation rates were found to be independent of the folding stability of the reporter proteins. Mitochondria from hsp78Delta cells exhibited a significant defect in the degradation efficiency of both substrates even at low temperature whereas mcx1Delta mitochondria showed wild-type activity. The proteolysis defect in hsp78Delta mitochondria was independent from the aggregation behavior of the substrate proteins. We conclude that Hsp78 is a genuine component of the mitochondrial proteolysis system required for the efficient degradation of substrate proteins in the matrix.     

In vitro modification of betaine-homocysteine S-methyltransferase by tissue-type transglutaminase

Transglutaminases catalyze the cross-linking and amine incorporation of proteins, and are implicated in various biological phenomena. To elucidate the physiological roles of transglutaminase at the molecular level, we need to identify its physiological protein substrates and clarify the relationship between transglutaminase modification of protein substrates and biological responses. Here we examined whether betaine-homocysteine S-methyltransferase (BHMT: EC 2.1.1.5) can be a substrate of tissue-type transglutaminase by in vitro experiments using porcine liver BHMT and guinea pig liver transglutarninase. Guinea pig liver transglutaminase incorporated 5-(biotinamido) pentylamine and [3H] histamine into BHMT in a time-dependent manner. Putrescine and spermidine also seemed to be incorporated into BHMT by transglutaminase. In the absence of the primary amines, BHMT subunits were cross-linked intra- and intermolecularly. BHMT activity was decreased significantly through the cross-linking by transglutaminase. Histamine incorporation slightly reduced the BHMT activity. Peptide fragments of BHMT containing the glutamine residues reactive for transglutaminase reaction were isolated through biotin labelling, proteinase digestion, biotin-avidin a affinity separation, and reverse phase HPLC. The results of amino acid sequence analyses of these peptides and sequence homology alignment with other mammalian liver BHMT subunits showed that these reactive glutamine residues were located in the region near the carboxyl terminal of porcine BHMT subunit. These results suggested that the liver BHMT can be modified by tissue-type transglutaminase and its activity is regulated repressively by the modification, especially by the cross-linking. This regulatory reaction might be involved in the regulation of homocysteine metabolism in the liver.     

Developmental changes in the activities of enzymes related to methyl group metabolism in sheep tissues

The activities of choline oxidase and betaine-homocysteine methyltransferase increased markedly in pre-ruminant lamb liver after birth and subsequently decreased when the lambs reached the ruminant state, while the developmental changes in hepatic 5-methyl-H4folate-homocysteine methyltransferase were negatively correlated with those of betaine-homocysteine methyltransferase. Hepatic phospholipid methyltransferase was elevated almost four-fold by the 10th postnatal day, but declined thereafter. Hepatic glycine methyltransferase in one-day-old lambs increased 55-fold, compared with that of fetuses, and thereafter decreased dramatically with age. Guanidoacetate methyltransferase, glycine methyltransferase and betaine-homocysteine methyltransferase in sheep pancreas increased markedly with age and were many times higher than the hepatic enzymes in adult sheep. Choline oxidase, betaine-homocysteine methyltransferase, cystathionine beta-synthase and glycine methyltransferase in adult sheep liver were much lower than those in rat. These results illustrate the conservative features of methyl group metabolism in postruminant sheep.     

Characteristics of the turnover of uncoupling protein 3 by the ubiquitin proteasome system in isolated mitochondria

Uncoupling protein 3 (UCP3) is implicated in mild uncoupling and the regulation of mitochondrial ROS production. We previously showed that UCP3 turns over rapidly in C2C12 myoblasts, with a half-life of 0.5-4h, and that turnover can be reconstituted in vitro. We show here that rapid degradation of UCP3 in vitro in isolated brown adipose tissue mitochondria required the 26S proteasome, ubiquitin, ATP, succinate to generate a high membrane potential, and a pH of 7.4 or less. Ubiquitin containing lysine-48 was both necessary and sufficient to support UCP3 degradation, implying a requirement for polyubiquitylation at this residue. The 20S proteasome did not support degradation. UCP3 degradation was prevented by simultaneously blocking matrix ATP generation and import, showing that ATP in the mitochondrial matrix was required. Degradation did not appear to require a transmembrane pH gradient, but was very sensitive to membrane potential: degradation was halved when membrane potential decreased 10-20mV from its resting value, and was not significant below about 120mV. We propose that matrix ATP and a high membrane potential are needed for UCP3 to be polyubiquitylated through lysine-48 of ubiquitin and exported to the cytosolic 26S proteasome, where it is de-ubiquitylated and degraded.     

Parkin mediates proteasome-dependent protein degradation and rupture of the outer mitochondrial membrane

Upon mitochondrial depolarization, Parkin, a Parkinson disease-related E3 ubiquitin ligase, translocates from the cytosol to mitochondria and promotes their degradation by mitophagy, a selective type of autophagy. Here, we report that in addition to mitophagy, Parkin mediates proteasome-dependent degradation of outer membrane proteins such as Tom20, Tom40, Tom70, and Omp25 of depolarized mitochondria. By contrast, degradation of the inner membrane and matrix proteins largely depends on mitophagy. Furthermore, Parkin induces rupture of the outer membrane of depolarized mitochondria, which also depends on proteasomal activity. Upon induction of mitochondrial depolarization, proteasomes are recruited to mitochondria in the perinuclear region. Neither proteasome-dependent degradation of outer membrane proteins nor outer membrane rupture is required for mitophagy. These results suggest that Parkin regulates degradation of outer and inner mitochondrial membrane proteins differently through proteasome- and mitophagy-dependent pathways.