Genetic variation in human carboxylesterase CES1 confers resistance to hepatic steatosis

Obesity often leads non-alcoholic fatty liver disease, insulin resistance and hyperlipidemia. Expression of carboxylesterase CES1 is positively correlated with increased lipid storage and plasma lipid concentration. Here we investigated structural and metabolic consequences of a single nucleotide polymorphism in CES1 gene that results in p.Gly143Glu amino acid substitution. We generated a humanized mouse model expressing CES1^WT (control), CES1^G143E and catalytically dead CES1^S221A (negative control) in the liver in the absence of endogenous expression of the mouse orthologous gene. We show that the CES1^G143E variant exhibits only 20% of the wild-type lipolytic activity. High-fat diet fed mice expressing CES1^G143E had reduced liver and plasma triacylglycerol levels. The mechanism by which decreased CES1 activity exerts this hypolipidemic phenotype was determined to include decreased very-low density lipoprotein secretion, decreased expression of hepatic lipogenic genes and increased fatty acid oxidation as determined by increased plasma ketone bodies and hepatic mitochondrial electron transport chain protein abundance. We conclude that attenuation of human CES1 activity provides a beneficial effect on hepatic lipid metabolism. These studies also suggest that CES1 is a potential therapeutic target for non-alcoholic fatty liver disease management.     

Examination of the carboxylesterase phenotype in human liver

Carboxylesterases (CES) metabolize esters. Two CES isoforms are expressed in human liver (CES1 and CES2) and liver extracts are used in reaction phenotyping studies to discern interindividual metabolic variation. We tested the hypothesis that an individual's CES phenotype can be characterized by reporter substrates/probes that interrogate native CES1 and CES2 activities in liver and immunoblotting methods. We obtained 25 livers and found that CES1 is the main hydrolytic enzyme. Moreover, although CES1 protein levels were similar, we observed large interindividual variation in bioresmethrin hydrolysis rates (17-fold), a pyrethroid metabolized by CES1 but not CES2. Bioresmethrin hydrolysis rates did not correlate with CES1 protein levels. In contrast, procaine hydrolysis rates, a drug metabolized by CES2 but not CES1, were much less variant (3-fold). Using activity-based fluorophosphonate probes (FP-biotin), which covalently reacts with active serine hydrolases, CES1 protein was the most active enzyme in the livers. Finally, using bioorthogonal probes and click chemistry methodology, the half-life of CES 1 and 2 in cultured HepG2 cells was estimated at 96 h. The cause of the differential CES1 activities is unknown, but the underlying factors will be important to understand because several carboxylic acid ester drugs and environmental toxicants are metabolized by this enzyme.     

Identification of regulatory variants of carboxylesterase 1 (CES1): A proof-of-concept study for the application of the Allele-Specific Protein Expression (ASPE) assay in identifying cis-acting regulatory genetic polymorphisms

It is challenging to study regulatory genetic variants as gene expression is affected by both genetic polymorphisms and non-genetic regulators. The mRNA allele-specific expression (ASE) assay has been increasingly used for the study of cis-acting regulatory variants because cis-acting variants affect gene expression in an allele-specific manner. However, poor correlations between mRNA and protein expressions were observed for many genes, highlighting the importance of studying gene expression regulation at the protein level. In the present study, we conducted a proof-of-concept study to utilize a recently developed allele-specific protein expression (ASPE) assay to identify the cis-acting regulatory variants of CES1 using a large set of human liver samples. The CES1 gene encodes for carboxylesterase 1 (CES1), the most abundant hepatic hydrolase in humans. Two cis-acting regulatory variants were found to be significantly associated with CES1 ASPE, CES1 protein expression, and its catalytic activity on enalapril hydrolysis in human livers. Compared to conventional gene expression-based approaches, ASPE demonstrated an improved statistical power to detect regulatory variants with small effect sizes since allelic protein expression ratios are less prone to the influence of non-genetic regulators (e.g., diseases and inducers). This study suggests that the ASPE approach is a powerful tool for identifying cis-regulatory variants.     

Effect of overexpressing fibroblast growth factor 2 protein isoforms in osteoblastic ROS 17/2.8 cells

Fibroblast growth factor-2 (FGF-2) is made by osteoblasts and modulates their function. There are high molecular weight (HMW) protein isoforms of FGF-2 that have nuclear localization sequences and a low molecular weight (LMW) 18 kDa FGF-2 protein that is exported from cells. Since FGF-2 is a trophic factor and potent mitogen for osteoblasts, the goal of this study was to utilize targeted overexpression of FGF-2 as a novel means of assessing different FGF-2 isoforms on osteoblastic cell viability and proliferation. Either LMW or HMW human Fgf2 cDNAs were cloned downstream of 3.6 kb alpha1(I)-collagen 5' regulatory elements (Col 3.6). A set of expression vectors, called Col3.6-Fgf2 isoforms-IRES-GFPsaph, capable of concurrently overexpressing either LMW or HMW FGF-2 isoforms concomitant with GFPsaph from a single bicistronic mRNA were built. Viable cell number in ROS 17/2.8 cells stably transfected with Vector (Col3.6-IRES-GFPsaph) versus each of the Col3.6-Fgf2-IRES-GFPsaph constructs were compared. In the presence of 1 or 10% serum, DNA synthesis was increased in cells expressing any isoform of FGF-2 compared with vector. However, cells transfected with HMW isoform had augmented DNA synthesis in 1 or 10% serum compared with cells expressing either ALL or LMW FGF-2 isoforms. A neutralizing FGF-2 antibody significantly reduced the mitogenic response in cells harboring ALL or the LMW FGF-2 isoforms but did not block the mitogenic effect of cells harboring the HMW isoforms. In summary, overexpression of any isoform of FGF-2 protein increased viable cell number and OB proliferation in the presence of low or high concentrations of serum. However, the HMW/nuclear isoforms preferentially mediate augmented OB proliferation. We conclude that differential expression of FGF-2 proteins isoforms is important in modulating OB function.     

Inhibition of carboxylesterase activity of THP1 monocytes/macrophages and recombinant human carboxylesterase 1 by oxysterols and fatty acids

Two major isoforms of human carboxylesterases (CEs) are found in metabolically active tissues, CES1 and CES2. These hydrolytic enzymes are involved in xenobiotic and endobiotic metabolism. CES1 is abundantly expressed in human liver and monocytes/macrophages, including the THP1 cell line; CES2 is expressed in liver but not in monocytes/macrophages. The cholesteryl ester hydrolysis activity in human macrophages has been attributed to CES1. Here, we report the direct inhibitory effects of several endogenous oxysterols and fatty acids on the CE activity of THP1 monocytes/macrophages and recombinant human CES1 and CES2. Using THP1 whole-cell lysates we found: (1) 27-hydroxycholesterol (27-HC) is a potent inhibitor of carboxylesterase activity (IC₅₀ = 33 nM); (2) 24(S),25-epoxycholesterol had moderate inhibitory activity (IC₅₀ = 8.1 μM); and (3) cholesterol, 7-ketocholesterol, 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, and 25-hydroxycholesterol each had little inhibitory activity. 27-HC was a partially noncompetitive inhibitor of recombinant CES1 (K_iapp = 10 nM) and impaired intracellular CES1 activity following treatment of intact THP1 cells. In contrast, recombinant CES2 activity was not inhibited by 27-HC, suggesting isoform-selective inhibition by 27-HC. Furthermore, unsaturated fatty acids were better inhibitors of CES1 activity than saturated fatty acids, while CES2 activity was unaffected by any fatty acid. Arachidonic acid (AA) was the most potent fatty acid inhibitor of recombinant CES1 and acted by a noncompetitive mechanism (K_iapp = 1.7 μM); when not complexed to albumin, exogenous AA penetrated intact THP1 cells and inhibited CES1. Inhibition results are discussed in light of recent structural models for CES1 that describe ligand binding sites separate from the active site. In addition, oxysterol-mediated inhibition of CES1 activity was demonstrated by pretreatment of human liver homogenates or intact THP1 cells with exogenous 27-HC, which resulted in significantly reduced hydrolysis of the pyrethroid insecticide bioresmethrin, a CES1-specific xenobiotic substrate. Collectively, these findings suggest that CE activity of recombinant CES1, cell lysates, and intact cells can be impaired by naturally occurring lipids, which may compromise the ability of CES1 to both detoxify environmental pollutants and metabolize endogenous compounds in vivo.     

Polymorphism and signalling of melatonin receptors.

Melatonin receptors belong to the superfamily of G protein-coupled receptors. Cloning of Mel1c receptors expressed in Xenopus skin revealed the existence of a polymorphism for these receptors. Heterologous expression of the two allelic isoforms, called Mel1c(alpha) and Mel1c(beta), indicated functional differences in their signalling properties. Both isoforms are coupled to the cAMP and cGMP pathways. However, the alpha isoform is preferentially coupled to the cAMP pathway, whereas the beta isoform couples preferentially to the cGMP pathway. Coupling differences may be explained by the fact that five of the six amino acid substitutions between the two isoforms are localized within intracellular receptor regions potentially involved in G protein coupling. Allelic isoforms were also observed for Mel1a receptors expressed in ovine pars tuberalis, suggesting that polymorphism is a general feature of the melatonin receptor family. We also evaluated the potential of the two human melatonin receptor subtypes, Mel1a and Mel1b, to modulate the cGMP pathway. Melatonin inhibited intracellular cGMP levels in a dose-dependent manner in HEK293 cells transfected with the human Mel1b receptor. This was not the case for HEK293 cells transfected with the human Mel1a receptor. In conclusion, our results indicate that the expression of receptor subtypes and isoforms may permit differential signalling between melatonin receptors.     

ERalpha gene expression in human primary osteoblasts: evidence for the expression of two receptor proteins.

The beneficial influence of E2 in the maintenance of healthy bone is well recognized. However, the way in which the actions of this hormone are mediated is less clearly understood. Western blot analysis of ERalpha in osteoblasts clearly demonstrated that the well characterized 66-kDa ERalpha was only one of the ERalpha isoforms present. Here we describe a 46-kDa isoform of ERalpha, expressed at a level similar to the 66-kDa isoform, that is also present in human primary osteoblasts. This shorter isoform is generated by alternative splicing of an ERalpha gene product, which results in exon 1 being skipped with a start codon in exon 2 used to initiate translation of the protein. Consequently, the transactivation domain AF-1 of this ERalpha isoform is absent. Functional analysis revealed that human (h)ERalpha46 is able to heterodimerize with the full-length ERalpha and also with ERbeta. Further, a DNA-binding complex that corresponds to hERalpha46 is detectable in human osteoblasts. We have shown that hERalpha46 is a strong inhibitor of hERalpha66 when they are coexpressed in the human osteosarcoma cell line SaOs. As a functional consequence, proliferation of the transfected cells is inhibited when increasing amounts of hERalpha46 are cotransfected with hERalpha66. In addition to human bone, the expression of the alternatively spliced ERalpha mRNA variant is also detectable in bone of ERalpha knockout mice. These data suggest that, in osteoblasts, E2 can act in part through an ERalpha isoform that is markedly different from the 66-kDa receptor. The expression of two ERalpha protein isoforms may account, in part, for the differential action that estrogens and estrogen analogs have in different tissues. In particular, the current models of the action of estrogens should be reevaluated to take account of the presence of at least two ERalpha protein isoforms in bone and perhaps in other tissues.     

Effects of alternate RNA splicing on glucokinase isoform activities in the pancreatic islet, liver, and pituitary

Different glucokinase isoforms are produced by tissue-specific alternative RNA splicing in the liver and pancreatic islet, the only tissues in which glucokinase activity has been detected. To determine whether differences in protein structure brought about by alternative RNA splicing have an effect on glucose phosphorylating activity, we expressed cDNAs encoding four different hepatic and islet glucokinase isoforms and determined the Km and Vmax of each. When the glucokinase B1 and L1 isoforms were expressed in eukaryotic cells, both high Km glucose phosphorylating activity and immunoreactive protein were detected. However, when the glucokinase B2 and L2 isoforms were expressed, both of which differ by deletion of 17 amino acids in a region between the putative glucose and ATP-binding domains, no high Km glucose phosphorylating activity and much less immunoreactive protein were detected. When the glucokinase B1 and B2 isoforms were expressed in Escherichia coli as fusion proteins with glutathione S-transferase, affinity-purified B1 fusion protein was able to phosphorylate glucose whereas the B2 fusion protein was not, thus indicating that the lack of glucose phosphorylating activity from both the B2 and L2 isoforms is due to lack of intrinsic activity in addition to accumulation of less protein. The Km values of the B1 and L1 isoforms, which differ from each other by 15 amino acids at the NH2 terminus, were similar, but the Vmax of the B1 isoform was 2.8-fold higher than that of the L1 isoform. Mutagenesis of the first two potential initiation codons in the glucokinase B1 cDNA from ATG to GTC (methionine to valine) indicated that the first ATG was crucial for activity and is, therefore, the likely translation initiation codon. Messenger RNAs encoding both the B2 and L2 isoforms of glucokinase were detected in islet and liver by polymerase chain reaction amplification of total cDNA, indicating that mRNAs utilizing this weak alternate splice acceptor site in the fourth exon are normally present in both the liver and islet but as minor components. A regulatory role for weak alternate splice acceptor and donor sites in the glucokinase gene was suggested by examining the expression of the gene in the pituitary and in AtT-20 cells. Interestingly, although glucokinase mRNAs of appropriate sizes were detected in both the AtT-20 cells and rat pituitaries, neither exhibited any detectable high Km glucose phosphorylating activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Protein kinase C in the human heart: differential regulation of the isoforms in aortic stenosis or dilated cardiomyopathy

Objectives Protein kinase C (PKC) is a central enzyme in the regulation of growth and hypertrophy. Little was known on PKC isoform regulation in human heart. Goal of this study was to characterize the isoforms of protein kinase C in human heart, their changes during ontogenesis, and their regulation in myocardial hypertrophy and heart failure. Methods In left ventricular and atrial samples from adults with end-stage dilated cardiomyopathy (DCM), from adults with severe aortic stenosis (AS), from small infants undergoing repair of ventricular septal defects, and from healthy organ donors (CO), activity of protein kinase C and the expression of its isozymes were examined. Results In the adult human heart, the isoforms PKC-α, PCK-β, PKC-δ, PKC-ε, PKC-λ/-ι, and PKC-ζ were detected both on protein and on mRNA level. All isozymes are subjected to downregulation during ontogenesis. No evidence, however, exists for an isoform shift from infancy to adulthood. DCM leads to a pronounced upregulation of PKC-β. Severe left ventricular hypertrophy in AS, however, recruits a distinct isoform pattern, i.e., isoforms PKC-α, PKC-δ, PKC-ε, PKC-λ/-ι, and PKC-ζ are upregulated, whereas PKC-β is not changed under this condition. Conclusion This work gives evidence for a differential recruitment of human PKC isoforms in various forms of myocardial hypertrophy and heart failure. Gregor Simonis and Steffen K. Briem contributed equally to this work.     

Type 3 inositol 1,4,5-trisphosphate receptor: A calcium channel for all seasons

Inositol 1,4,5 trisphosphate receptors (ITPRs) are a family of endoplasmic reticulum Ca²⁺ channels essential for the control of intracellular Ca²⁺ levels in virtually every mammalian cell type. The three isoforms (ITPR1, ITPR2 and ITPR3) are highly homologous in amino acid sequence, but they differ considerably in terms of biophysical properties, subcellular localization, and tissue distribution. Such differences underscore the variety of cellular responses triggered by each isoform and suggest that the expression/activity of specific isoforms might be linked to particular pathophysiological states. Indeed, recent findings demonstrate that changes in expression of ITPR isoforms are associated with a number of human diseases ranging from fatty liver disease to cancer. ITPR3 is emerging as the isoform that is particularly important in the pathogenesis of various human diseases. Here we review the physiological and pathophysiological roles of ITPR3 in various tissues and the mechanisms by which the expression of this isoform is modulated in health and disease.     

Activity-based Protein Profiling Identifies a Host Enzyme,Carboxylesterase 1, Which Is Differentially Active during Hepatitis C Virus Replication

Hepatitis C virus (HCV) relies on many interactions with host cell proteins for propagation. Successful HCV infection also requires enzymatic activity of host cell enzymes for key post-translational modifications. To identify such enzymes, we have applied activity-based protein profiling to examine the activity of serine hydrolases during HCV replication. Profiling of hydrolases in Huh7 cells replicating HCV identified CES1 (carboxylesterase 1) as a differentially active enzyme. CES1 is an endogenous liver protein involved in processing of triglycerides and cholesterol. We observe that CES1 expression and activity were altered in the presence of HCV. The knockdown of CES1 with siRNA resulted in lower levels of HCV replication, and up-regulation of CES1 was observed to favor HCV propagation, implying an important role for this host cell protein. Experiments in HCV JFH1-infected cells suggest that CES1 facilitates HCV release because less intracellular HCV core protein was observed, whereas HCV titers remained high. CES1 activity was observed to increase the size and density of lipid droplets, which are necessary for the maturation of very low density lipoproteins, one of the likely vehicles for HCV release. In transgenic mice containing human-mouse chimeric livers, HCV infection also correlates with higher levels of endogenous CES1, providing further evidence that CES1 has an important role in HCV propagation.     

Isoforms,Expression, Glycosylation,and Tissue Distribution of CTL2/SLC44A2

Antibodies to the solute carrier protein, CTL2/SLC44A2, cause hearing loss in animals, are frequently found in autoimmune hearing loss patients, and are implicated in transfusion-related acute lung injury. We cloned a novel CTL2/SLC44A2 isoform (CTL2 P1) from inner ear and identified an alternate upstream promoter and exon 1a encoding a protein of 704 amino acids which differs in the first 10–12 amino acids from the known exon 1b isoform (CTL2 P2; 706 amino acids). The expression of these CTL2/SLC44A2 isoforms, their posttranslational modifications in tissues and their localization in HEK293 cells expressing rHuCTL2/SLC44A2 were assessed. P1 and P2 isoforms with differing glycosylation are variably expressed in cochlea, tongue, heart, colon, lung, kidney, liver and spleen suggesting tissue specific differences that may influence function in each tissue. Because antibodies to CTL2/SLC44A2 have serious pathologic consequences, it is important to understand its distribution and modifications. Heterologous expression in X. laevis oocytes shows that while human CTL2-P1 does not transport choline, human CTL2-P2 exhibits detectable choline transport activity.     

2'-5' Oligoadenylate synthetase plays a critical role in interferon-gamma inhibition of respiratory syncytial virus infection of human epithelial cells

Respiratory syncytial virus (RSV), associated with bronchiolitis and asthma, is resistant to the antiviral effects of type-I interferons (IFN), but not IFN-gamma. However, the antiviral mechanism of IFN-gamma action against RSV infection is unknown. The molecular mechanism of IFN-gamma-induced antiviral activity was examined in this study using human epithelial cell lines HEp-2 and A549. Exposure of these cells to 100-1000 units/ml of IFN-gamma, either before or after RSV infection, results in a significant decrease in RSV infection. After 1 h of exposure, IFN-gamma induces protein expression of IFN regulatory factor-1 (IRF-1) but not IRF-2, double-stranded RNA-activated protein kinase, and inducible nitric-oxide synthase in these cells. The mRNA for IRF-1, p40, and p69 isoforms of 2'-5' oligoadenylate synthetase (2-5 AS) are detectable, respectively, at 1 and 4 h of IFN-gamma exposure. Studies using cycloheximide and antisense oligonucleotides to IRF-1 indicate a direct role of IRF-1 in activating 2-5 AS. Cells transfected with 2-5 AS antisense oligonucleotides inhibit the antiviral effect of IFN-gamma. A stable cell line of HEp-2 overexpressing RNase L inhibitor, RLI-14, which exhibits an IFN-gamma-induced gene expression pattern similar to that of the parent cell line, shows a significant reduction in RNase L activity and IFN-gamma-mediated antiviral effect, compared with HEp-2 cells. These results provide direct evidence of the involvement of 2-5 AS in IFN-gamma-mediated antiviral activity in these cells.