Gene_locus Report for: human-AADAC

BACKGROUND: The prognosis of Borrmann type III advanced gastric cancer (AGC) is known to vary significantly among patients. This study aimed to determine which differentially expressed genes (DEGs) are directly related to the survival time of Borrmann type III AGC patients and to construct a prognostic model. METHODS: We selected 25 patients with Borrmann type III AGC who underwent radical gastrectomy. According to the difference in overall survival (OS), the patients were divided into group A (OS<1 year, n=11) and group B (OS>3 years, n=14). DEGs related to survival time in patients with Borrmann type III AGC were determined by mRNA sequencing. The prognosis and functional differences of DEGs in different populations were determined by The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) public databases. The expression of mRNA and protein in cell lines was detected by quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) and Western blot (WB). Immunohistochemical (IHC) staining was used to detect protein expression in the paraffin-embedded tissues of 152 patients with Borrmann type III AGC who underwent radical gastrectomy. After survival analysis, nomograms were constructed to predict the prognosis of patients with Borrmann type III AGC. RESULTS: Arylacetamide deacetylase (AADAC) is a survival-related DEG in patients with Borrmann type III AGC. The higher the expression level of its mRNA and protein is, the better the prognosis of patients. Bioinformatics analysis found that AADAC showed significant differences in prognosis and function in European and American populations and Asian populations. In addition, the mRNA and protein expression levels of AADAC were high in differentiated gastric cancer (GC) cells. We also found that AADAC was an independent prognostic factor for patients with Borrmann type III AGC, and its high expression was significantly correlated with better OS and disease-free survival (DFS). Nomogram models of AADAC expression level combined with clinicopathological features can be used to predict the OS and DFS of Borrmann type III AGC. CONCLUSION: AADAC can be used as a biomarker to predict the prognosis of Borrmann type III AGC and has the potential to become a new therapeutic target for GC.

Esterases catalyze the hydrolysis of therapeutic drugs containing esters or amides in their structures. Human carboxylesterase (CES) and arylacetamide deacetylase (AADAC) are the major enzymes that catalyze the hydrolysis of drugs in the liver. Characterization of the enzyme(s) responsible for drug metabolism is required in drug development and to realize optimal drug therapy. Because multiple enzymes may show a metabolic potency for a given compound, inhibition studies using chemical inhibitors are useful tools to determine the contribution of each enzyme in human tissue preparations. The purpose of this study was to find specific inhibitors for human CES1, CES2, and AADAC. We screened 542 chemicals for the inhibition potency toward hydrolase activities of p-nitrophenyl acetate by recombinant CES1, CES2, and AADAC. We found that digitonin and telmisartan specifically inhibited CES1 and CES2 enzyme activity, respectively. Vinblastine potently inhibited both AADAC and CES2, but no specific inhibitor of AADAC was found. The inhibitory potency and specificity of these compounds were also evaluated by monitoring the effects on hydrolase activity of probe compounds of each enzyme (CES1: lidocaine, CES2: CPT-11, AADAC: phenacetin) in human liver microsomes. Telmisartan and vinblastine strongly inhibited the hydrolysis of CPT-11 and/or phenacetin, but digitonin did not strongly inhibit the hydrolysis of lidocaine, indicating that the inhibitory potency of digitonin was different between recombinant CES1 and liver microsomes. Although we could not find a specific inhibitor of AADAC, the combined use of telmisartan and vinblastine could predict the responsibility of human AADAC to drug hydrolysis.

BACKGROUND & AIMS: Very low density lipoproteins (VLDLs) are triacylglycerol (TG)-rich lipoproteins produced by the human liver. VLDLs derive the majority of their TG cargo from the lipolysis of TG stored in hepatocellular lipid droplets (LDs). Important roles for LDs and the VLDL secretory pathway in the cell culture production of infectious hepatitis C virus (HCV) have been established. We hypothesized that TG lipolysis and VLDL production are impaired during HCV infection so that these cellular processes can be diverted towards HCV production.
METHODS: We used an HCV permissive cell culture system (JFH-1/HuH7.5 cells) to examine the relationship between TG lipolysis, VLDL assembly, and the HCV lifecycle using standard biochemical approaches.
RESULTS: Lipolysis of cellular TG and VLDL production were impaired in HCV infected cells during the early peak of viral infection. This was partially explained by an apparent deficiency of a putative TG lipase, arylacetamide deacetylase (AADAC). The re-introduction of AADAC to infected cells restored cellular TG lipolysis, indicating a role for HCV-mediated downregulation of AADAC in this process. Defective lipolysis of cellular TG stores and VLDL production were also observed in HuH7.5 cells stably expressing a short hairpin RNA targeting AADAC expression, proving AADAC deficiency contributes to these defective pathways. Finally, impaired production of HCV was observed with AADAC knockdown cells, demonstrating a role for AADAC in the HCV lifecycle.
CONCLUSIONS: This insight into the biology of HCV infection and possibly pathogenesis identifies AADAC as a novel and translationally relevant therapeutic target.

BACKGROUND: The prognosis of Borrmann type III advanced gastric cancer (AGC) is known to vary significantly among patients. This study aimed to determine which differentially expressed genes (DEGs) are directly related to the survival time of Borrmann type III AGC patients and to construct a prognostic model. METHODS: We selected 25 patients with Borrmann type III AGC who underwent radical gastrectomy. According to the difference in overall survival (OS), the patients were divided into group A (OS<1 year, n=11) and group B (OS>3 years, n=14). DEGs related to survival time in patients with Borrmann type III AGC were determined by mRNA sequencing. The prognosis and functional differences of DEGs in different populations were determined by The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) public databases. The expression of mRNA and protein in cell lines was detected by quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) and Western blot (WB). Immunohistochemical (IHC) staining was used to detect protein expression in the paraffin-embedded tissues of 152 patients with Borrmann type III AGC who underwent radical gastrectomy. After survival analysis, nomograms were constructed to predict the prognosis of patients with Borrmann type III AGC. RESULTS: Arylacetamide deacetylase (AADAC) is a survival-related DEG in patients with Borrmann type III AGC. The higher the expression level of its mRNA and protein is, the better the prognosis of patients. Bioinformatics analysis found that AADAC showed significant differences in prognosis and function in European and American populations and Asian populations. In addition, the mRNA and protein expression levels of AADAC were high in differentiated gastric cancer (GC) cells. We also found that AADAC was an independent prognostic factor for patients with Borrmann type III AGC, and its high expression was significantly correlated with better OS and disease-free survival (DFS). Nomogram models of AADAC expression level combined with clinicopathological features can be used to predict the OS and DFS of Borrmann type III AGC. CONCLUSION: AADAC can be used as a biomarker to predict the prognosis of Borrmann type III AGC and has the potential to become a new therapeutic target for GC.

Human arylacetamide deacetylase (AADAC) plays a role in the detoxification or activation of drugs and is sometimes involved in the incidence of toxicity by catalyzing hydrolysis reactions. AADAC prefers compounds with relatively small acyl groups, such as acetyl groups. Eslicarbazepine acetate, an antiepileptic drug, is a prodrug rapidly hydrolyzed to eslicarbazepine. We sought to clarify whether AADAC might be responsible for the hydrolysis of eslicarbazepine acetate. Eslicarbazepine acetate was efficiently hydrolyzed by human intestinal and liver microsomes and recombinant human AADAC. The hydrolase activities in human intestinal and liver microsomes were inhibited by epigallocatechin gallate, a specific inhibitor of AADAC, by 82% and 88% of the control, respectively. The hydrolase activities in liver microsomes from 25 human livers were significantly correlated (r = 0.87, P < 0.001) with AADAC protein levels, suggesting that the enzyme AADAC is responsible for the hydrolysis of eslicarbazepine acetate. The effects of genetic polymorphisms of AADAC on eslicarbazepine acetate hydrolysis were examined by using the constructed recombinant AADAC variants with T74A, V172I, R248S, V281I, N366K, or X400Q. AADAC variants with R248S or X400Q showed lower activity than wild type (5% or 21%, respectively), whereas those with V172I showed higher activity than wild type (174%). Similar tendencies were observed in the other 4 substrates of AADAC; that is, p-nitrophenyl acetate, ketoconazole, phenacetin, and rifampicin. Collectively, we found that eslicarbazepine acetate is specifically and efficiently hydrolyzed by human AADAC, and several AADAC polymorphic alleles would be a factor affecting the enzyme activity and drug response. Significance Statement This is the first study to clarify that AADAC is responsible for the activation of eslicarbazepine acetate, an antiepileptic prodrug, to eslicarbazepine, an active form, in the human liver and intestines. In addition, we found that several AADAC polymorphic alleles would be a factor affecting the enzyme activity and drug response.

Human arylacetamide deacetylase (AADAC) can hydrolyze clinical drugs such as flutamide, phenacetin, and rifamycins. AADAC is a glycoprotein, but the role of glycosylation remains unclear. In the present study, we investigated the effect of glycosylation on AADAC enzyme activity. Immunoblot analysis of mutant AADACs that contained an asparagine (N, Asn) to glutamine (Q, Gln) substitution at either residue 78 or 282 (N78Q or N282Q) showed a different migration compared with the wild-type protein. A mutant AADAC that contained N to Q substitutions at both residue 78 and 282 (N78Q/N282Q) showed a similar migration to AADAC in human liver microsomes (HLM) treated with endoglycosidase H (Endo H), which produces deglycosylated proteins. This result indicated that AADAC was glycosylated at both N78 and N282. Mutant types of AADAC with the N282Q and the N78Q/N282Q substitutions showed dramatically lower phenacetin hydrolase activity than did the wild-type protein. The treatment of wild-type AADAC-expressing HuH-7 cells with tunicamycin, which produces unglycosylated protein, decreased AADAC enzyme activity. However, the treatment of the HLM with Endo H caused no decrease of AADAC activity. Thus, the oligosaccharide chain, per se, was not important for AADAC activity in the mature form. The mutant types of AADAC containing the N282Q and the N78Q/N282Q substitutions were not detected by immunoblotting analysis after non-reducing SDS-PAGE, suggesting that the glycosylation of AADAC at N282 was important for proper protein folding. Overall, this study found that the translational, but not post-translational, N-glycosylation of AADAC plays a crucial role in regulating AADAC enzyme activity.

Esterases catalyze the hydrolysis of therapeutic drugs containing esters or amides in their structures. Human carboxylesterase (CES) and arylacetamide deacetylase (AADAC) are the major enzymes that catalyze the hydrolysis of drugs in the liver. Characterization of the enzyme(s) responsible for drug metabolism is required in drug development and to realize optimal drug therapy. Because multiple enzymes may show a metabolic potency for a given compound, inhibition studies using chemical inhibitors are useful tools to determine the contribution of each enzyme in human tissue preparations. The purpose of this study was to find specific inhibitors for human CES1, CES2, and AADAC. We screened 542 chemicals for the inhibition potency toward hydrolase activities of p-nitrophenyl acetate by recombinant CES1, CES2, and AADAC. We found that digitonin and telmisartan specifically inhibited CES1 and CES2 enzyme activity, respectively. Vinblastine potently inhibited both AADAC and CES2, but no specific inhibitor of AADAC was found. The inhibitory potency and specificity of these compounds were also evaluated by monitoring the effects on hydrolase activity of probe compounds of each enzyme (CES1: lidocaine, CES2: CPT-11, AADAC: phenacetin) in human liver microsomes. Telmisartan and vinblastine strongly inhibited the hydrolysis of CPT-11 and/or phenacetin, but digitonin did not strongly inhibit the hydrolysis of lidocaine, indicating that the inhibitory potency of digitonin was different between recombinant CES1 and liver microsomes. Although we could not find a specific inhibitor of AADAC, the combined use of telmisartan and vinblastine could predict the responsibility of human AADAC to drug hydrolysis.

BACKGROUND & AIMS: Very low density lipoproteins (VLDLs) are triacylglycerol (TG)-rich lipoproteins produced by the human liver. VLDLs derive the majority of their TG cargo from the lipolysis of TG stored in hepatocellular lipid droplets (LDs). Important roles for LDs and the VLDL secretory pathway in the cell culture production of infectious hepatitis C virus (HCV) have been established. We hypothesized that TG lipolysis and VLDL production are impaired during HCV infection so that these cellular processes can be diverted towards HCV production.
METHODS: We used an HCV permissive cell culture system (JFH-1/HuH7.5 cells) to examine the relationship between TG lipolysis, VLDL assembly, and the HCV lifecycle using standard biochemical approaches.
RESULTS: Lipolysis of cellular TG and VLDL production were impaired in HCV infected cells during the early peak of viral infection. This was partially explained by an apparent deficiency of a putative TG lipase, arylacetamide deacetylase (AADAC). The re-introduction of AADAC to infected cells restored cellular TG lipolysis, indicating a role for HCV-mediated downregulation of AADAC in this process. Defective lipolysis of cellular TG stores and VLDL production were also observed in HuH7.5 cells stably expressing a short hairpin RNA targeting AADAC expression, proving AADAC deficiency contributes to these defective pathways. Finally, impaired production of HCV was observed with AADAC knockdown cells, demonstrating a role for AADAC in the HCV lifecycle.
CONCLUSIONS: This insight into the biology of HCV infection and possibly pathogenesis identifies AADAC as a novel and translationally relevant therapeutic target.

PURPOSE: To evaluate the pharmacokinetics and tolerability of once-daily eslicarbazepine acetate (ESL) and twice-daily oxcarbazepine (OXC) and their metabolites in cerebrospinal fluid (CSF) and plasma following repeated oral administration. METHODS: Single-center, open-label, randomized, parallel-group study in healthy volunteers. Volunteers in ESL group (n = 7) received 600 mg on days 1-3 and 1,200 mg on days 4-9, once daily. Volunteers in the OXC group (n = 7) received 300 mg on days 1-3 and 600 mg on days 4-9, twice daily. Plasma and CSF sampling was performed following the last dose. KEY FINDINGS: Eslicarbazepine was the major drug entity in plasma and CSF, accounting for, respectively, 93.84% and 91.96% of total exposure in the ESL group and 78.06% and 76.42% in the OXC group. The extent of exposure to drug entities R-licarbazepine and oxcarbazepine was approximately four-fold higher with OXC as compared with ESL. There was relatively little fluctuation from peak-to-trough (ratio) in the CSF for both eslicarbazepine (ESL = 1.5; OXC = 1.2) and R-licarbazepine (ESL = 1.2; OXC = 1.2). In contrast, oxcarbazepine showed larger differences between peak and trough (ESL = 3.1; OXC = 6.4). A total of 84 and 24 treatment-emergent adverse events (TEAEs) were reported with OXC and ESL, respectively. SIGNIFICANCE: In comparison to OXC, administration of ESL resulted in more eslicarbazepine, less R-licarbazepine, and less oxcarbazepine in plasma and CSF, which may correlate with the tolerability profile reported with ESL. The smaller peak-to-trough fluctuation of eslicarbazepine in CSF (a measure of sustained delivery to the brain) than in plasma supports once-daily dosing of ESL.

Human arylacetamide deacetylase (AADAC) is a major esterase responsible for the hydrolysis of clinical drugs such as flutamide, phenacetin, and rifampicin. Thus, AADAC is considered to be a relevant enzyme in preclinical drug development, but there is little information about species differences with AADAC. This study investigated the species differences in the tissue distribution and enzyme activities of AADAC. In human, AADAC mRNA was highly expressed in liver and the gastrointestinal tract, followed by bladder. In rat and mouse, AADAC mRNA was expressed in liver at the highest level, followed by the gastrointestinal tract and kidney. The expression levels in rat tissues were approximately 7- and 10-fold lower than those in human and mouse tissues, respectively. To compare the catalytic efficiency of AADAC among three species, each recombinant AADAC was constructed, and enzyme activities were evaluated by normalizing with the expression levels of AADAC. Flutamide and phenacetin hydrolase activities were detected by the recombinant AADAC of all species. In flutamide hydrolysis, liver microsomes of all species showed similar catalytic efficiencies, despite the lower AADAC mRNA expression in rat liver. In phenacetin hydrolysis, rat liver microsomes showed approximately 4- to 6.5-fold lower activity than human and mouse liver microsomes. High rifampicin hydrolase activity was detected only by recombinant human AADAC and human liver and jejunum microsomes. Taken together, the results of this study clarified the species differences in the tissue distribution and enzyme activities of AADAC and facilitate our understanding of species differences in drug hydrolysis.

Human arylacetamide deacetylase (AADAC) is responsible for the hydrolysis of clinically used drugs such as flutamide, phenacetin, and rifamycins. Our recent studies suggested that human AADAC is a relevant enzyme pharmacologically and toxicologically. To date, the genetic polymorphisms that affect enzyme activity in AADAC have been unknown. In this study, we found single-nucleotide polymorphisms in the human AADAC gene in a liver sample that showed remarkably low flutamide hydrolase activity. Among them, g.13651G > A (V281I) and g.14008T > C (X400Q) were nonsynonymous. The latter would be predicted to cause a C-terminal one-amino acid (glutamine) extension. The AADAC*2 allele (g.13651G > A) was found in all populations investigated in this study (European American, African American, Korean, and Japanese), at allelic frequencies of 52.6 to 63.5%, whereas the AADAC*3 allele (g.13651G > A/g.14008T > C) was found in European American (1.3%) and African American (2.0%) samples. COS7 cells expressing AADAC.1 (wild-type) exhibited flutamide, phenacetin, and rifampicin hydrolase activities with intrinsic clearance (CLint) values of 1.31 +/- 0.06, 1.00 +/- 0.02, and 0.39 +/- 0.02 mul x min(-1) x unit(-1), respectively. AADAC.2, which is a protein produced from the AADAC*2 allele, showed moderately lower or similar CLint values, compared with AADAC.1, but AADAC.3 showed substantially lower CLint values (flutamide hydrolase, 0.21 +/- 0.02 mul x min(-1) x unit(-1); phenacetin hydrolase, 0.12 +/- 0.00 mul x min(-1) x unit(-1); rifampicin hydrolase, 0.03 +/- 0.01 mul x min(-1) x unit(-1), respectively). Microsomes from a liver sample genotyped as AADAC*3/AADAC*3 showed decreased enzyme activities, compared with those genotyped as AADAC*1/AADAC*1, AADAC*1/AADAC*2, and AADAC*2/AADAC*2. In conclusion, we found an AADAC allele that yielded decreased enzyme activity. This study should provide useful information on interindividual variations in AADAC enzyme activity.

Flutamide, an antiandrogen drug, is widely used for the treatment of prostate cancer. The initial metabolic pathways of flutamide are hydroxylation and hydrolysis. It was recently reported that the hydrolyzed product, 4-nitro-3-(trifluoromethyl)phenylamine (FLU-1), is further metabolized to N-hydroxy FLU-1, an assumed hepatotoxicant. However, the esterase responsible for the flutamide hydrolysis has not been characterized. In the present study, we found that human arylacetamide deacetylase (AADAC) efficiently hydrolyzed flutamide using recombinant AADAC expressed in COS7 cells. In contrast, carboxylesterase1 (CES1) and CES2, which are responsible for the hydrolysis of many drugs, could not hydrolyze flutamide. AADAC is specifically expressed in the endoplasmic reticulum. Flutamide hydrolase activity was highly detected in human liver microsomes (K(m), 794 +/- 83 microM; V(max), 1.1 +/- 0.0 nmol/min/mg protein), whereas the activity was extremely low in human liver cytosol. The flutamide hydrolase activity in human liver microsomes was strongly inhibited by bis-(p-nitrophenyl)phosphate [corrected], diisopropylphosphorofluoride, and physostigmine sulfate (eserine) but moderately inhibited by sodium fluoride, phenylmethylsulfonyl fluoride, and disulfiram. The same inhibition pattern was obtained with the recombinant AADAC. Moreover, human liver and jejunum microsomes showing AADAC expression could hydrolyze flutamide, but human pulmonary and renal microsomes, which do not express AADAC, showed slight activity. In human liver microsomal samples (n = 50), the flutamide hydrolase activities were significantly correlated with the expression levels of AADAC protein (r = 0.66, p < 0.001). In conclusion, these results clearly showed that flutamide is exclusively hydrolyzed by AADAC. AADAC would be an important enzyme responsible for flutamide-induced hepatotoxicity.

Carboxylesterases (CEs) are traditionally regarded as xenobiotic metabolizing enzymes that hydrolyze esterified xenobiotics to alcohol and carboxylic acid products. However, there is a growing appreciation for the role of CEs in the processing of endobiotics, including cholesteryl esters and triacylglycerols. Human liver microsomes (HLMs) are often used in reaction phenotyping studies to discern interindividual variability in xenobiotic metabolism. The two major CE isoforms expressed in human liver are hCE1 and hCE2. These two isoforms are different gene products. We have begun studies to evaluate the CE phenotype'' of human liver samples, i.e. to determine both the levels of hCE1 and hCE2 protein and the hydrolytic activity of each. We have previously shown that there is little variation in hCE1 protein expression in HLM samples from 11 individuals [a 1.3-fold difference between the highest and lowest individuals; coefficient of variation (CV), 9%]. hCE2 protein expression in individual HLMs is only slightly more variable than hCE1 (2.3-fold difference between the highest and lowest individuals; CV, 36%). However, hCE1 protein is found in 46-fold higher amounts in HLMs than hCE2 protein (64.4 +/- 16.5 microg hCE1/mg microsomal protein compared to 1.4 +/- 0.2 microg hCE2/mg microsomal protein). The hydrolytic activity specifically attributable to hCE1 and hCE2 in individual HLMs was measured using bioresmethrin (a pyrethroid insecticide hydrolyzed specifically by hCE1, but not by hCE2) and procaine (an analgesic drug hydrolyzed by hCE2, but not by hCE1). The hydrolytic activity of individual HLMs toward bioresmethrin and procaine did not correlate with the protein content of hCE1 and hCE2. Thus, the mere abundance of CE proteins is not a good predictor of CE activity in HLMs. Identification of the factors that lead to altered CE activities in HLMs will be important to characterize since several pharmaceutical agents, environmental toxicants, and endobiotics are metabolized by these enzymes.

After the completion of a draft human genome sequence, the International Human Genome Sequencing Consortium has proceeded to finish and annotate each of the 24 chromosomes comprising the human genome. Here we describe the sequencing and analysis of human chromosome 3, one of the largest human chromosomes. Chromosome 3 comprises just four contigs, one of which currently represents the longest unbroken stretch of finished DNA sequence known so far. The chromosome is remarkable in having the lowest rate of segmental duplication in the genome. It also includes a chemokine receptor gene cluster as well as numerous loci involved in multiple human cancers such as the gene encoding FHIT, which contains the most common constitutive fragile site in the genome, FRA3B. Using genomic sequence from chimpanzee and rhesus macaque, we were able to characterize the breakpoints defining a large pericentric inversion that occurred some time after the split of Homininae from Ponginae, and propose an evolutionary history of the inversion.

A 2.91-billion base pair (bp) consensus sequence of the euchromatic portion of the human genome was generated by the whole-genome shotgun sequencing method. The 14.8-billion bp DNA sequence was generated over 9 months from 27,271,853 high-quality sequence reads (5.11-fold coverage of the genome) from both ends of plasmid clones made from the DNA of five individuals. Two assembly strategies-a whole-genome assembly and a regional chromosome assembly-were used, each combining sequence data from Celera and the publicly funded genome effort. The public data were shredded into 550-bp segments to create a 2.9-fold coverage of those genome regions that had been sequenced, without including biases inherent in the cloning and assembly procedure used by the publicly funded group. This brought the effective coverage in the assemblies to eightfold, reducing the number and size of gaps in the final assembly over what would be obtained with 5.11-fold coverage. The two assembly strategies yielded very similar results that largely agree with independent mapping data. The assemblies effectively cover the euchromatic regions of the human chromosomes. More than 90% of the genome is in scaffold assemblies of 100,000 bp or more, and 25% of the genome is in scaffolds of 10 million bp or larger. Analysis of the genome sequence revealed 26,588 protein-encoding transcripts for which there was strong corroborating evidence and an additional approximately 12,000 computationally derived genes with mouse matches or other weak supporting evidence. Although gene-dense clusters are obvious, almost half the genes are dispersed in low G+C sequence separated by large tracts of apparently noncoding sequence. Only 1.1% of the genome is spanned by exons, whereas 24% is in introns, with 75% of the genome being intergenic DNA. Duplications of segmental blocks, ranging in size up to chromosomal lengths, are abundant throughout the genome and reveal a complex evolutionary history. Comparative genomic analysis indicates vertebrate expansions of genes associated with neuronal function, with tissue-specific developmental regulation, and with the hemostasis and immune systems. DNA sequence comparisons between the consensus sequence and publicly funded genome data provided locations of 2.1 million single-nucleotide polymorphisms (SNPs). A random pair of human haploid genomes differed at a rate of 1 bp per 1250 on average, but there was marked heterogeneity in the level of polymorphism across the genome. Less than 1% of all SNPs resulted in variation in proteins, but the task of determining which SNPs have functional consequences remains an open challenge.

Microsomal arylacetamide deacetylase (DAC) competes against the activity of cytosolic arylamine N-acetyltransferase, which catalyzes one of the initial biotransformation pathways for arylamine and heterocyclic amine carcinogens in many species and tissues. Activity determination and immunoblot analysis of DAC in human target tissues for arylamine carcinogens revealed that in extrahepatic tissues, additional enzymes are responsible for any deacetylation activity, whereas a single enzyme predominantly catalyzes this hydrolytic reaction in liver. We isolated and characterized a full-length cDNA from a human liver lambda gt11 library. This clone encodes an open reading frame of 400 amino acids with a deduced molecular mass of 45.7 kDa and contains two putative glycosylation sites. The 3'-untranslated region contains two putative polyadenylation signals. The cDNA was confirmed to be that for DAC in tryptic peptides from the purified human liver protein. Highest sequence similarity of DAC was found in a series of prokaryotic esterases encompassing the putative active site. Two extended regions of significant sequence homology with hormone-sensitive lipase and with lipase 2 from Moraxella TA144 were identified, whereas similarity to carboxyl esterases was restricted to the region encompassing the putative active site, indicating that DAC should be classified as esterase. This cDNA provides an important tool to study deacetylation and its effects on the metabolic activation of arylamine and heterocyclic amine carcinogens.

Arylacetamide deacetylation is an important enzyme activity in the metabolic activation of arylamine substrates to ultimate carcinogens, best described as a carboxylesterase/amidase type of reaction. A 7-fold variation in the Vmax of 2-acetylaminofluorene deacetylation in 24 human livers was observed. An acetylaminofluorene deacetylase was purified 90 fold from human liver microsomes by PEG-fractionation, anion exchange and hydrophobic interaction chromatography. The purified 45kD protein showed no amino acid sequence homology to other carboxylesterases, neither in its N-terminus nor in tryptic peptides. Antibodies raised against the deacetylase recognized the protein with high specificity. This report thus describes the first arylacetamide deacetylase in human liver.