Mammalian telomeres comprise noncoding TTAGGG repeats in double-stranded regions with a single-stranded TTAGGG repeat 3' overhang and are bound by a multiprotein complex with a telomeric repeat-containing RNA (TERRA) containing a UUAGGG repeat as a G-quadruplex noncoding RNA. TLS/FUS is a human telomere-binding protein that was first identified as an oncogenic fusion protein in human myxoid and round-cell liposarcoma. Here, we show that the Arg-Gly-Gly domain in the C-terminal region of TLS forms a ternary complex with human telomere G-quadruplex DNA and TERRA in vitro. Furthermore, TLS binds to G-quadruplex telomere DNA in double-stranded regions and to G-quadruplex TERRA, which regulates histone modifications of telomeres and telomere length in vivo. Our findings suggest that the G-quadruplex functions as a scaffold for the telomere-binding protein, TLS, to regulate telomere length by histone modifications.

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.

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ABSTRACT: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 express- , 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.

ABSTRACT:Flutamide, an antiandrogen drug, is widely used for the treatment of prostate cancer. The major metabolic pathways of flutamide are hydroxylation and hydrolysis. The hydrolyzed metabolite, 5-amino-2-nitrobenzotrifluoride (FLU-1), is further metabolized to N-hydroxy FLU-1, an assumed hepatotoxicant. Our previous study demonstrated that arylacetamide deacetylase (AADAC), one of the major serine esterases expressed in the human liver and gastrointestinal tract, catalyzes the flutamide hydrolysis. However, the enzyme kinetics in human tissue microsomes were not consistent with the kinetics by recombinant human AADAC. Thus, it seemed that AADAC is not the sole enzyme responsible for flutamide hydrolysis in human. In the present study, we found that recombinant carboxylesterase (CES) 2 could hydrolyze flutamide at low concentrations of flutamide. In the inhibition assay, the flutamide hydrolase activities at a flutamide concentration of 5 M in human liver and jejunum microsomes were strongly inhibited by a selective CES2 inhibitor, 10 M loperamide, with the residual activities of 22.9 ؎ 3.5 and 18.6 ؎ 0.7%, respectively. These results suggest that CES2 is also involved in the flutamide hydrolysis in human tissues. Using six individual human livers, the contributions of AADAC and CES2 to flutamide hydrolysis were estimated by using the relative activity factor. The relative contribution of CES2 was approximately 75 to 99% at the concentration of 5 M flutamide. In contrast, the relative contribution of AADAC increased in parallel with the concentration of flutamide. Thus, CES2, rather than AA-DAC, largely contributed to the flutamide hydrolysis in clinical therapeutics.