MicroRNA Expression Influences Methylmercury-Induced Lipid Accumulation and Mitochondrial Toxicity in Caenorhabditis elegans.

Metabolic effects of methylmercury (MeHg) are gaining wider attention. We have previously shown that MeHg causes lipid dysregulation in Caenorhabditis elegans (C. elegans), leading to altered gene expression, increased triglyceride levels and lipid storage, and altered feeding behaviors. Transcriptional regulators, such as transcription factors and microRNAs (miRNAs), have been shown to regulate lipid storage, serum triglycerides, and adipogenic gene expression in human and rodent models of metabolic diseases. As we recently investigated adipogenic transcription factors induced by MeHg, we were, therefore, interested in whether MeHg may also regulate miRNA sequences to cause metabolic dysfunction. Lipid dysregulation, as measured by triglyceride levels, lipid storage sites, and feeding behaviors, was assessed in wild-type (N2) worms and in transgenic worms that either were sensitive to miRNA expression or were unable to process miRNAs. Worms that were sensitive to the miRNA expression were protected from MeHg-induced lipid dysregulation. In contrast, the mutant worms that were unable to process miRNAs had exacerbated MeHg-induced lipid dysregulation. Concurrent with differential lipid homeostasis, miRNA-expression mutants had altered MeHg-induced mitochondrial toxicity as compared to N2, with the miRNA-sensitive mutants showing mitochondrial protection and the miRNA-processing mutants showing increased mitotoxicity. Taken together, our data demonstrate that the expression of miRNAs is an important determinant in MeHg toxicity and MeHg-induced metabolic dysfunction in C. elegans.

Residues flanking scissile bonds in Factor VIII modulate rates of cleavage and proteolytic activation catalyzed by Factor Xa.

Factor Xa (FXa) proteolytically activates Factor VIII (FVIII) by cleaving P1 residues Arg(372), Arg(740), and Arg(1689). The Arg(372) site represents the rate-limiting step for procofactor activation, whereas cleavage at Arg(740) is a fast step. FXa also catalyzes inactivating cleavages that occur on a slower time scale than the activating ones. To assess the role of sequences flanking the Arg(372) and Arg(740) sites, recombinant FVIII variants in which P3-P3' sequences were swapped individually or in combination were prepared. Replacing the Arg(372) flanking sequence with that from the Arg(740) site increased the rate of cleavage at Arg(372), as judged by the ~5-fold increased rate in A1 subunit generation, and reduced the FVIIIa-dependent lag time for in situ FXa generation. The reciprocal swap yielded a nearly 2-fold increase in the rate of Arg(372) cleavage, while the combined double-swap variant showed a 10-fold rate increase at that site, consistent with the individual effects being additive. Although this cleavage represents the slow step for activation, the rate of this reaction appeared to be ~9-fold greater than the rate of the primary inactivating cleavage at Arg(336) in generating the A1(336) product. Interestingly, replacement of the Arg(372) flanking sequence with the Arg(740) sequence combined with an Arg(740)Gln mutation yielded both more rapid cleavage of the Arg(372) site and accelerated inactivating cleavages within the A1 subunit. These results indicate that flanking sequences in part modulate the reaction rates required for procofactor activation and influence the capacity of FXa as an initial activator of FVIII rather than an inactivator.

P3-P3' residues flanking scissile bonds in factor VIII modulate rates of substrate cleavage and procofactor activation by thrombin.

Thrombin-catalyzed activation of factor VIII (FVIII) occurs through proteolysis at three P1 Arg residues: Arg(372) and Arg(740) in the FVIII heavy chain and Arg(1689) in the FVIII light chain. Cleavage at the latter two sites is relatively fast compared with cleavage at Arg(372), which appears to be rate-limiting. Examination of the P3-P3' residues flanking each P1 site revealed that those sequences at Arg(740) and Arg(1689) are more optimal for thrombin cleavage than at Arg(372), suggesting these sequences may impact reaction rates. Recombinant FVIII variants were prepared with mutations swapping scissile bond flanking sequences in the heavy chain individually and in combination with a second swap or with a P1 point mutation. Rates of generation of A1 and A3-C1-C2 subunits were determined by Western blotting and correlated with rates of cleavage at Arg(372) and Arg(1689), respectively. Rates of thrombin cleavage at Arg(372) were increased ~10- and ~3-fold compared with that of wild-type FVIII when it was replaced with P3-P3' residues flanking Arg(740) and Arg(1689), respectively, and these values paralleled increased rates of A2 subunit generation and procofactor activation. Positioning of more optimal residues flanking Arg(372) abrogated the need for initial cleavage at Arg(740) to facilitate this step. These results show marked changes in cleavage rates correlate with the extent of cleavage-optimal residues flanking the scissile bond and modulate the mechanism for procofactor activation.

Notecarin D binds human factor V and factor Va with high affinity in the absence of membranes.

Notecarin D (NotD) is a prothrombin (ProT) activator in the venom of the tiger snake, Notechis scutatus, and a factor Xa (FXa) homolog. NotD binds specifically to the FXa binding site expressed on factor V (FV) upon activation to factor Va (FVa) by thrombin. NotD active site-labeled with 5-fluorescein ([5F]FFR-NotD) binds FV and FVa with remarkably high affinity in the absence of phospholipids (K(D) 12 and ≤ 0.01 nm, respectively). In the presence of membranes, the affinity of [5F]FFR-NotD for FVa is similar, but increased ∼55-fold for FV. Binding of FXa active site-labeled with Oregon Green to FV and FVa in the presence of phospholipids is ∼5,000- and ∼80-fold weaker than [5F]FFR-NotD, respectively. NotD reports FVa and not FV binding by a 3-fold increase in tripeptide substrate hydrolysis, demonstrating allosteric regulation by FVa. The NotD·FVa·membrane complex activates ProT with K(m)((app)) similar to prothrombinase, and ∼85-fold weaker without membranes. Active site-blocked NotD exhibits potent anticoagulant activity in plasma thrombin generation assays, representing inhibition of productive prothrombinase assembly and possible disruption of FXa inhibition by the tissue factor pathway inhibitor. The results show that high affinity binding of NotD to FVa is membrane-independent, unlike the strict membrane dependence of FXa for high affinity FVa binding.

Methylmercury-Induced Metabolic Alterations in Caenorhabditis elegans Are Diet-Dependent.

Methylmercury (MeHg) is a well-known neurotoxicant; however, its role in metabolic diseases has been gaining wider attention. Chronic exposure to MeHg in human populations shows an association with diabetes mellitus and metabolic syndrome (MS). As the incidences of both obesity and MS are on the rise globally, it is important to understand the potential role of MeHg in the development of the disease. There is a dearth of information on dietary interactions between MeHg and lipids, which play an important role in developing MS. We have previously shown that MeHg increases food seeking behaviors, lipid levels, fat storage, and pro-adipogenic gene expression in C. elegans fed the standard OP50 Escherichia coli diet. However, we hypothesized that these metabolic changes could be prevented if the worms were fed a bacterial diet lower in lipid content. We tested whether C. elegans developed metabolic alterations in response to MeHg if they were fed two alternative E. coli strains (HT115 and HB101) that are known absorb significantly less lipids from their media. Additionally, to explore the effect of a high-lipid and high-cholesterol diet on MeHg-induced metabolic dysfunction, we supplemented the OP50 strain with twice the standard concentration of cholesterol in the nematode growth media. Wild-type worms fed either the HB101 or HT115 diet were more resistant to MeHg than the worms fed the OP50 diet, showing a significant right-hand shift in the dose-response survival curve. Worms fed the OP50 diet supplemented with cholesterol were more sensitive to MeHg, showing a significant left-hand shift in the dose-response survival curve. Changes in sensitivity to MeHg by differential diet were not due to altered MeHg intake in the worms as measured by inductively coupled mass spectrometry. Worms fed the low-fat diets showed protection from MeHg-induced metabolic changes, including decreased food consumption, lower triglyceride content, and lower fat storage than the worms fed either of the higher-fat diets. Oxidative stress is a common characteristic of both MeHg exposure and high-fat diets. Worms fed either OP50 or OP50 supplemented with cholesterol and treated with MeHg had significantly higher levels of reactive oxygen species, carbonylated proteins, and loss of glutathione than the worms fed the HT115 or HB101 low-lipid diets. Taken together, our data suggest a synergistic effect of MeHg and dietary lipid levels on MeHg toxicity and fat metabolism in C. elegans, which may affect the ability of MeHg to cause metabolic dysfunction.

Methylmercury Induces Metabolic Alterations in Caenorhabditis elegans: Role for C/EBP Transcription Factor.

Methylmercury (MeHg) is a well-known neurotoxicant; however, its role in metabolic diseases has been gaining wider attention. We have previously shown that MeHg causes metabolic alterations in Caenorhabditis elegans, leading to decreased nicotinamide adenine dinucleotide cofactor, mitochondrial dysfunction, and oxidative stress. We were, therefore, interested in whether MeHg also affects nutrient metabolism, particularly lipid homeostasis, which may contribute to the development of metabolic conditions such as obesity or metabolic syndrome (MS). RNA from wild-type worms exposed to MeHg was collected immediately after treatment and used for gene expression analysis by DNA microarray. MeHg differentially regulated 215 genes, 17 genes involved in lipid homeostasis, and 12 genes involved in carbohydrate homeostasis. Of particular interest was cebp-1, the worm ortholog to human C/EBP, a pro-adipogenic transcription factor implicated in MS. MeHg increased the expression of cebp-1 as well as pro-adipogenic transcription factors sbp-1 and nhr-49, triglyceride synthesis enzyme acl-6, and lipid transport proteins vit-2 and vit-6. Concurrent with the altered gene expression, MeHg increased triglyceride levels, lipid storage, and feeding behaviors. Worms expressing mutant cebp-1 were protected from MeHg-induced alterations in lipid content, feeding behaviors, and gene expression, highlighting the importance of this transcription factor in the worm's response to MeHg. Taken together, our data demonstrate that MeHg induces biochemical, metabolic, and behavioral changes in C. elegans that can lead to metabolic dysfunction.