Impaired methylation as a novel mechanism for proteasome suppression in liver cells

The proteasome is a multi-catalytic protein degradation enzyme that is regulated by ethanol-induced oxidative stress; such suppression is attributed to CYP2E1-generated metabolites. However, under certain conditions, it appears that in addition to oxidative stress, other mechanisms are also involved in proteasome regulation. This study investigated whether impaired protein methylation that occurs during exposure of liver cells to ethanol, may contribute to suppression of proteasome activity. We measured the chymotrypsin-like proteasome activity in Huh7CYP cells, hepatocytes, liver cytosols and nuclear extracts or purified 20S proteasome under conditions that maintain or prevent protein methylation. Reduction of proteasome activity of hepatoma cell and hepatocytes by ethanol or tubercidin was prevented by simultaneous treatment with S-adenosylmethionine (SAM). Moreover, the tubercidin-induced decline in proteasome activity occurred in both nuclear and cytosolic fractions. In vitro exposure of cell cytosolic fractions or highly purified 20S proteasome to low SAM:S-adenosylhomocysteine (SAH) ratios in the buffer also suppressed proteasome function, indicating that one or more methyltransferase(s) may be associated with proteasomal subunits. Immunoblotting a purified 20S rabbit red cell proteasome preparation using methyl lysine-specific antibodies revealed a 25 kDa proteasome subunit that showed positive reactivity with anti-methyl lysine. This reactivity was modified when 20S proteasome was exposed to differential SAM:SAH ratios. We conclude that impaired methylation of proteasome subunits suppressed proteasome activity in liver cells indicating an additional, yet novel mechanism of proteasome activity regulation by ethanol.     

Excess methionine suppresses the methylation cycle and inhibits neural tube closure in mouse embryos

Suppression of one-carbon metabolism or insufficient methionine intake are suggested to increase risk of neural tube defects (NTD). Here, exogenous methionine unexpectedly caused frequent NTD in cultured mouse embryos. NTD were associated with reduced cranial mesenchyme cell density, which may result from a preceding reduction in proliferation. The abundance ratio of S-adenosylmethionine to S-adenosylhomocysteine was also decreased in treated embryos, suggesting methylation reactions may be suppressed. Such an effect is potentially causative as NTD were also observed when DNA methylation was specifically inhibited. Thus, reduced cranial mesenchyme density and impairment of critical methylation reactions may contribute to development of methionine-induced NTD.     

Mechanisms of homocysteine neurotoxicity in neurodegenerative diseases with special reference to dementia

Mild to moderate hyperhomocysteinemia is a risk factor for neurodegenerative diseases. Human studies suggest that homocysteine (Hcy) plays a role in brain damage, cognitive and memory decline. Numerous studies in recent years investigated the role of Hcy as a cause of brain damage. Hcy itself or folate and vitamin B12 deficiency can cause disturbed methylation and/or redox potentials, thus promoting calcium influx, amyloid and tau protein accumulation, apoptosis, and neuronal death. The Hcy effect may also be mediated by activating the N-methyl-D-aspartate receptor subtype. Numerous neurotoxic effects of Hcy can be blocked by folate, glutamate receptor antagonists, or various antioxidants. This review describes the most important mechanisms of Hcy neurotoxicity and pharmacological agents known to reverse Hcy effects.     

Numerical analysis of hemodynamics in spastic middle cerebral arteries

Cerebral vasospasm (CVS) is the most common serious complication of subarachnoid hemorrhage. Among the many factors that are associated with the pathogenesis of CVS, hemodynamics plays an important role in the initiation and development of CVS. Numerical simulation was carried out to obtain the flow patterns and wall shear stress (WSS) distribution in spastic middle cerebral arteries. The blood was assumed to be incompressible, laminar, homogenous, Newtonian, and steady. Our simulations reveal that flow velocity and WSS level increase at the stenosis segment of the spastic vessels, but further downstream of stenosis, the WSS significantly decreases along the inner wall, and flow circulation and stagnation are observed. The hydrodynamic resistance increases with the increase of vessel spasm. Moreover, the change of flow field and hydrodynamic forces are not linearly proportional to the spasm level, and the rapid change of hemodynamic parameters is observed as the spasm is more than 50%. Accordingly, in the view of hemodynamic physiology, vessels with less than 30% stenosis are capable of self-restoration towards normal conditions. However, vessels with more than 50% stenosis may eventually lose their capacity to adapt to differing physiologic conditions due to the extreme non-physilogic hemodynamic environment, and the immediate expansion of the vessel lumen might be needed to minimize serious and non-reversible effects.     

Phosphatidylcholine biosynthesis and function in bacteria

Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes and is estimated to be present in about 15% of the domain Bacteria. Usually, PC can be synthesized in bacteria by either of two pathways, the phospholipid N-methylation (Pmt) pathway or the phosphatidylcholine synthase (Pcs) pathway. The three subsequent enzymatic methylations of phosphatidylethanolamine are performed by a single phospholipid N-methyltransferase in some bacteria whereas other bacteria possess multiple phospholipid N-methyltransferases each one performing one or several distinct methylation steps. Phosphatidylcholine synthase condenses choline directly with CDP-diacylglycerol to form CMP and PC. Like in eukaryotes, bacterial PC also functions as a biosynthetic intermediate during the formation of other biomolecules such as choline, diacylglycerol, or diacylglycerol-based phosphorus-free membrane lipids. Bacterial PC may serve as a specific recognition molecule but it affects the physicochemical properties of bacterial membranes as well. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.     

Effects of hyperhomocysteinemia and betaine–homocysteine S-methyltransferase inhibition on hepatocyte metabolites and the proteome

Both cardiovascular disease and liver injury are major public health issues. Hyperhomocysteinemia has been linked to cardiovascular diseases, and defects in methyl group metabolism, often resulting in hyperhomocysteinemia, are among the key molecular events postulated to play a role in liver injury. We employed proteomics and metabolomics analyses of human hepatocytes in primary cell culture to explore the spectrum of proteins and associated metabolites affected by the disruption of methyl group metabolism. We treated the hepatocytes with homocysteine (Hcy, 0.1 mM and 2 mM) to follow the impact of hyperhomocysteinemia, and in parallel, we used a specific inhibitor of betaine–homocysteine S-methyltransferase (BHMT) to extend our understanding of the physiological functions of the enzyme. The major effect of BHMT inhibition was a 50% decrease in S-adenosylmethionine levels. The treatments with Hcy resulted in multiple changes in the metabolite levels depending on the treatment modality. The BHMT inhibition and 0.1 mM Hcy treatment induced only moderate changes in the hepatocyte proteome and secretome, while the changes induced by the 2 mM Hcy treatment were extensive. Phosphatidylethanolamine carboxykinase and ornithine aminotransferase were up-regulated about two fold indicating an intervention into metabolism. Cellular proliferation was suspended, secretome composition was changed and signs of apoptosis were discernible. We have detected fibrinogen gamma dimers, which might have a role as a potentially new biomarker of early liver injury. Finally, we have demonstrated the failed maturation of apolipoprotein A1, which might be a new mechanism of disruption of cholesterol efflux from tissues.     

Structural characterization of BVU_3255, a methyltransferase from human intestine antibiotic resistant pathogen Bacteroides vulgatus

Methylation is important for various cellular activities. To date, there is no report of any methyltransferase structure from the human intestine antibiotic resistant pathogen Bacteroides vulgatus. The protein BVU_3255 from B. vulgatus ATCC 8482 belongs to a SAM-dependent methyltransferase. Here, we report the crystal structure of apo BVU_3255, and its complexes with SAM and SAH, which revealed a typical class I Rossmann Fold Methyltransferase. Isothermal titration calorimetric studies showed that both SAM and SAH bind with equal affinity. The structural and sequence analysis suggested that BVU_3255 is a small molecule methyltransferase and involved in methylating the intermediates in ubiquinone biosynthesis pathway.     

Dopamine but not l-dopa stimulates neural glutathione metabolism. Potential implications for Parkinson’s and other dopamine deficiency states

Dopamine is produced first by hydroxylalation of l-tyrosine to l-dihydroxyphenylalanine (l-dopa) and subsequently by the decarboxylation of l-dopa to dopamine catalysed by the enzymes tyrosine hydroxylase and aromatic l-amino acid decarboxylase (AADC) respectively. Reduced glutathione (GSH) acts as a major cellular antioxidant. We have investigated the role of dopamine in the control of GSH homeostasis in brain cells. The SH-SY5Y human neuroblastoma cell line was found to increase intracellular GSH levels in response to 50 μM dopamine treatment. Similarly the 1321N1 human astrocytoma cell line was found to increase GSH release in response to 50 μM dopamine. The same concentration of l-dopa was also found to increase intracellular GSH in SH-SY5Y cells, however when AADC was inhibited this affect was abolished. Furthermore 1321N1 cells which were found to have almost undetectable levels of AADC activity did not increase GSH release in response to 50 μM l-dopa. These results suggest that at these concentrations dopamine has the potential to act as a signal for the upregulation of GSH synthesis within neuronal-like cells and for the increased trafficking of GSH from astrocytes to neurons. This effect could potentially relate to the activation of antioxidant response elements leading to the induction of phase II detoxifying enzymes including those involved in GSH synthesis and release. The inability of l-dopa to produce a similar effect when AADC was inhibited or when AADC activity was absent indicates that these effects are relatively specific to dopamine. Additionally dopamine but not l-dopa treatment led in an increase in complex I activity of the respiratory chain in SH-SY5Y cells which may be related to the effect of dopamine on GSH levels.     

Oligomerization and Auto-methylation of the Human Lysine Methyltransferase SETD6

Signaling via lysine methylation by protein lysine methyltransferases (PKMTs), has been linked to diverse biological and disease processes. The mono-methyltransferase SETD6 (SET-domain-containing protein 6) is a member of the PKMT family and was previously shown to regulate essential cellular processes such as the NF-κB, WNT and the oxidative stress pathways. However, on the biochemical level, little is known about the enzymatic mode of action of SETD6. Here we provide evidence that SETD6 forms high-molecular-weight structures. Specifically, we demonstrate that SETD6 monomeric, dimeric and trimeric forms are stabilized by the methyl donor, S-adenosyl-l-methionine. We then show that SETD6 has auto-methylation activity at K39 and K179, which serves as the major auto-methylation sites with a moderate auto-methylation activity toward K372. A point mutation at K179 but not at K39 and K372, located at the SET domain of SETD6, impaired SETD6 ability to form a trimer, strongly implying a link between the auto-methylation and the oligomerization state. Finally, by radioactive in vitro methylation experiments and biochemical kinetics analysis, we show that the auto-methylation at K39 and K179 increases the catalytic rate of SETD6. Collectively, our data support a model by which SETD6 auto-methylation and self-interaction positively regulate its enzymatic activity in vitro and may suggest that other PKMTs are regulated in the same manner.