Whole-genome sequencing identifies recurrent mutations in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most deadly cancers worldwide and has no effective treatment, yet the molecular basis of hepatocarcinogenesis remains largely unknown. Here we report findings from a whole-genome sequencing (WGS) study of 88 matched HCC tumor/normal pairs, 81 of which are Hepatitis B virus (HBV) positive, seeking to identify genetically altered genes and pathways implicated in HBV-associated HCC. We find beta-catenin to be the most frequently mutated oncogene (15.9%) and TP53 the most frequently mutated tumor suppressor (35.2%). The Wnt/beta-catenin and JAK/STAT pathways, altered in 62.5% and 45.5% of cases, respectively, are likely to act as two major oncogenic drivers in HCC. This study also identifies several prevalent and potentially actionable mutations, including activating mutations of Janus kinase 1 (JAK1), in 9.1% of patients and provides a path toward therapeutic intervention of the disease.

Development of a quantitative biochemical and cellular sphingomyelin synthase assay using mass spectrometry.

The last step in sphingolipid biosynthesis is the conversion of ceramide (Cer) to sphingomyelin (SM), which is catalyzed by sphingomyelin synthase (SMS). Two isoforms of SMS have been identified with differential subcellular localizations. It is not clear whether the two isoforms have any differences in biochemical or cellular SMS activities. This report describes a mass spectrometry (MS)-based method that was used to characterize biochemical and cellular SMS activities of the two isoforms of SMS, namely SMS1 and SMS2. Cellular extracts of SMS1 or SMS2 expressed in SF9 cells displayed significant SMS activity. When these activities were measured by MS, both SMS1 and SMS2 demonstrated similar time- and substrate-dependent SMS activity. A previously reported SMS inhibitor, D609, inhibited both SMS1 and SMS2 activity. In HEK293 cells, overexpression of either SMS1 or SMS2 significantly increased SMS activity. These studies using MS methods to measure SMS activity of SMS1 and SMS2 represent the first quantitative measurement of SMS activities. The establishment of quantitative biochemical and cellular SMS assays may help to facilitate the discovery of novel SMS1- or SMS2-specific inhibitors.

Characterization of the expression and activity of carboxylesterases 1 and 2 from the beagle dog, cynomolgus monkey, and human.

The carboxylesterases (CESs) are a family of serine hydrolases that hydrolyze compounds containing an ester, amide, or thioester. In humans, two dominant forms, CES1 and CES2, are highly expressed in organs of first-pass metabolism and play an important role in xenobiotic metabolism. The current study was conducted to better understand species-related differences in substrate selectivity and tissue expression of these enzymes. To elucidate potential similarities and differences among these enzymes, a series of 4-nitrophenyl esters and a series of gemcitabine prodrugs were evaluated using enzyme kinetics as substrates of expressed and purified CESs from beagle dog, cynomolgus monkey, and human genes. For the substrates examined, human and monkey CES2 more efficiently catalyzed hydrolysis compared with CES1, whereas CES1 was the more efficient enzyme in dog. Quantitative real-time polymerase chain reaction and Western blot analyses indicate that the pattern of CES tissue expression in monkey is similar to that of human, but the CES expression in dog is unique, with no detectable expression of CES in the intestine. Loperamide, a selective human CES2 inhibitor, was also found to be a CES2-selective inhibitor in both dog and monkey. This is the first study to examine substrate specificity among dog, human, and monkey CESs.

Development of a novel sandwich ELISA for measuring cell lysate ABCA1 protein levels.

ATP-binding cassette transporter A-1 (ABCA1) mediates the transfer of cellular cholesterol to lipid-poor apolipoproteins. Liver X receptors (LXRs) are regulators of cholesterol homeostasis that increase transcription of ABCA1. Synthetic LXR agonists developed to date have been shown to induce ABCA1 mRNA expression and increase reverse cholesterol transport. Unfortunately, there have been few options for quantitatively measuring ABCA1 protein levels, including a previously described competitive ELISA standardized to an ABCA1 peptide with a sensitivity of 80 ng/ml. To address this unmet need, we developed a novel sandwich ELISA standardized to full-length human recombinant ABCA1 protein with sensitivity of approximately 0.5 ng/ml. To determine if the sandwich ELISA had adequate sensitivity to detect LXR-induced increases in ABCA1, we utilized it to measure ABCA1 levels in untreated and LXR agonist-treated human (THP-1) macrophage cells and human peripheral blood mononuclear cells (PBMC). Data obtained from the ELISA demonstrated an approximately eightfold increase in ABCA1 levels in both macrophages as well as PBMC in response to LXR agonist treatment, and results were highly correlated with those obtained by immunoprecipitation and western blotting. Together, these results suggest that the sandwich ELISA may be a sensitive and effective method for quantitating ABCA1 protein levels.

Effect of buffer components and carrier solvents on in vitro activity of recombinant human carboxylesterases.

INTRODUCTION: The effects of buffer and substrate solvent conditions on in vitro activity of carboxylesterases (CE) have not been previously described. Therefore, it is unknown if the many different assay conditions used by various laboratories have a substantial impact on the activity of CE enzymes. METHODS: Three human CEs were expressed and purified, and the hydrolysis of 4-nitrophenyl butyrate was measured to assess enzyme activity. Four buffers (HEPES, potassium phosphate, sodium phosphate, and Tris) were evaluated for their effects on enzyme activity at concentrations ranging from 5 to 900 mM, as well as phosphate buffered saline. Five commonly used substrate-carrier solvents (acetone, acetonitrile, dimethyl sulfoxide, ethanol, and methanol) ranging from 0.25 to 6% were also assessed for their effect on enzyme activity. RESULTS: The clearances for the CEs in HEPES, potassium phosphate, sodium phosphate, and Tris up to 100 mM were similar to the CE clearances obtained with phosphate buffered saline. Higher buffer concentrations resulted in differential activity of the CEs. All three CEs tolerated the substrate solvents up to 2% as indicated by little effect of solvent on catalytic activity. At substrate solvent concentrations above 2% the CE activities were found to gradually decrease. In general, CES3 displayed substantially lower activity than CES1 and CES2. DISCUSSION: In conclusion, any of the buffers examined up to 100 mM resulted in clearance values similar to that of phosphate buffered saline for the hydrolysis of 4-nitrophenyl butyrate by the human CEs. With regard to the substrate solvents tested, acetone, acetonitrile, or dimethyl sulfoxide appear to be well tolerated by the CEs up to 2% of the total reaction volume.

Secreted PCSK9 downregulates low density lipoprotein receptor through receptor-mediated endocytosis.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a protease that regulates low density lipoprotein receptor (LDLR) protein levels. The mechanisms of this action, however, remain to be defined. We show here that recombinant human PCSK9 expressed in HEK293 cells was readily secreted into the medium, with the prosegment associated with the C-terminal domain. Secreted PCSK9 mediated cell surface LDLR degradation in a concentration- and time-dependent manner when added to HEK293 cells. Accordingly, cellular LDL uptake was significantly reduced as well. When infused directly into C57B6 mice, purified human PCSK9 substantially reduced hepatic LDLR protein levels and resulted in increased plasma LDL cholesterol. When added to culture medium, fluorescently labeled PCSK9 was endocytosed and displayed endosomal-lysosomal intracellular localization in HepG2 cells, as was demonstrated by colocalization with DiI-LDL. PCSK9 endocytosis was mediated by LDLR as LDLR deficiency (hepatocytes from LDLR null mice), or RNA interference-mediated knockdown of LDLR markedly reduced PCSK9 endocytosis. In addition, RNA interference knockdown of the autosomal recessive hypercholesterolemia (ARH) gene product also significantly reduced PCSK9 endocytosis. Biochemical analysis revealed that the LDLR extracellular domain interacted directly with secreted PCSK9; thus, overexpression of the LDLR extracellular domain was able to attenuate the reduction of cell surface LDLR levels by secreted PCSK9. Together, these results reveal that secreted PCSK9 retains biological activity, is able to bind directly to the LDLR extracellular domain, and undergoes LDLR-ARH-mediated endocytosis, leading to accelerated intracellular degradation of the LDLR.