A Unique Role of Carboxylesterase 3 (Ces3) in ß-Adrenergic Signaling-Stimulated Thermogenesis.

Carboxylesterase 3 (Ces3) is a hydrolase with a wide range of activities in liver and adipose tissue. In this study, we identified Ces3 as a major lipid droplet surface-targeting protein in adipose tissue upon cold exposure by liquid chromatography-tandem mass spectrometry. To investigate the function of Ces3 in the ß-adrenergic signaling-activated adipocytes, we applied WWL229, a specific Ces3 inhibitor, or genetic inhibition by siRNA to Ces3 on isoproterenol (ISO)-treated 3T3-L1 and brown adipocyte cells. We found that blockage of Ces3 by WWL229 or siRNA dramatically attenuated the ISO-induced lipolytic effect in the cells. Furthermore, Ces3 inhibition led to impaired mitochondrial function measured by Seahorse. Interestingly, Ces3 inhibition attenuated an ISO-induced thermogenic program in adipocytes by downregulating Ucp1 and Pgc1α genes via peroxisome proliferator-activated receptor γ. We further confirmed the effects of Ces3 inhibition in vivo by showing that the thermogenesis in adipose tissues was significantly attenuated in WWL229-treated or adipose tissue-specific Ces3 heterozygous knockout (Adn-Cre-Ces3flx/wt) mice. As a result, the mice exhibited dramatically impaired ability to defend their body temperature in coldness. In conclusion, our study highlights a lipolytic signaling induced by Ces3 as a unique process to regulate thermogenesis in adipose tissue.

An epididymis-specific carboxyl esterase CES5A is required for sperm capacitation and male fertility in the rat.

Despite the fact that the phenomenon of capacitation was discovered over half century ago and much progress has been made in identifying sperm events involved in capacitation, few specific molecules of epididymal origin have been identified as being directly involved in this process in vivo . Previously, our group cloned and characterized a carboxyl esterase gene Ces5a in the rat epididymis. The CES5A protein is mainly expressed in the corpus and cauda epididymidis and secreted into the corresponding lumens. Here, we report the function of CES5A in sperm maturation. By local injection of Lentivirus -mediated siRNA in the CES5A -expressing region of the rat epididymis, Ces5a -knockdown animal models were created. These animals exhibited an inhibited sperm capacitation and a reduction in male fertility. These results suggest that CES5A plays an important role in sperm maturation and male fertility.

The structure of the deacetylase domain of Escherichia coli PgaB, an enzyme required for biofilm formation: a circularly permuted member of the carbohydrate esterase 4 family.

Bacterial biofilm formation is an extremely widespread phenomenon involving the secretion of a protective exopolysaccharide matrix which helps the bacteria to attach to surfaces and to overcome a variety of stresses in different environments. This matrix may also include proteins, lipids, DNA and metal ions. Its composition depends on the bacterial species and growth conditions, but one of the most widely found components is polymeric ß-1,6-N-acetyl-D-glucosamine (PGA). Several studies have suggested that PGA is an essential component of biofilm and it is produced by numerous bacteria, including Escherichia coli, Staphylococcus epidermis, Yersinia pestis, Bordetella spp. and Actinobacillus spp. In E. coli, PGA production and export are dependent on four genes that form a single operon, pgaABCD, which appears to have been transferred between various species. Biofilms themselves are recognized as environments in which such horizontal gene transfer may occur. The pga operon of E. coli, which is even found in innocuous laboratory strains, is highly homologous to that from the plague bacterium Yersinia pestis, and biofilm is believed to play an important role in the transmission of Yersinia. The crystal structure of the N-terminal domain of PgaB, which has deacetylase activity, is described and compared with models of other deacetylases.

Prolonged toxic effects after cocaine challenge in butyrylcholinesterase/plasma carboxylesterase double knockout mice: a model for butyrylcholinesterase-deficient humans.

Death and toxicity after cocaine use do not correlate with cocaine blood levels. One explanation for this observation is that cocaine abusers may posses one or more of the 58 possible known mutations in the butyrylcholinesterase gene (BCHE). Butyrylcholinesterase (BChE) serves as the primary cocaine hydrolase producing a nontoxic product ecgonine methyl ester. A reduction in endogenous levels of BChE may result in increased metabolism by hepatic carboxylesterase to produce norcocaine, a toxic product. Humans have carboxylesterase in tissues but not in plasma, whereas wild-type mice have significant amounts of carboxylesterase in tissues and plasma. Knockout mice with no plasma carboxylesterase were created to eliminate the contribution of plasma carboxylesterase in cocaine hydrolysis, thereby simulating human enzyme levels. This study tested the hypothesis that reductions in BChE such as those in humans with BChE mutations contribute to increased toxicity after cocaine use. Carboxylesterase and BChE double knockout mice, models for humans with BChE deficiency, were challenged with a nonlethal dose of 100 mg/kg (-)-cocaine. Carboxylesterase/BChE double knockout mice demonstrated toxic signs significantly longer than did wild-type and carboxylesterase knockout mice. The carboxylesterase/BChE-deficient mice took approximately 2.5 times as long to recover from cocaine toxicities, including the following: hypothermia, hyperactivity, stereotypical behavior, ocular effects, and dorsiflexion of the tail. The carboxylesterase/BChE double knockout mouse model demonstrates the importance of endogenous BChE for protection against cocaine toxicity and provides an in vivo system for studying drug sensitivity of humans who carry a BChE mutation.

Involvement of carboxylesterase 1 and 2 in the hydrolysis of mycophenolate mofetil.

Mycophenolate mofetil (MMF) is the ester prodrug of the immunosuppressant agent mycophenolic acid (MPA) and is rapidly activated by esterases after oral administration. However, the role of isoenzymes in MMF hydrolysis remains unclear. Although human plasma, erythrocytes, and whole blood contain MMF hydrolytic activities, the mean half-lives of MMF in vitro were 15.1, 1.58, and 3.20 h, respectively. Thus, blood esterases seemed to contribute little to the rapid MMF disappearance in vivo. In vitro analyses showed that human intestinal microsomes exposed to 5 and 10 µM MMF exhibited hydrolytic activities of 2.38 and 4.62 nmol/(min · mg protein), respectively. Human liver microsomes exhibited hydrolytic activities of 14.0 and 26.1 nmol/(min · mg protein), respectively, approximately 6-fold higher than those observed for intestinal microsomes. MMF hydrolytic activities in human liver cytosols were 1.40 and 3.04 nmol/(min · mg protein), respectively. Because hepatic cytosols generally contain 5-fold more protein than microsomes, MMF hydrolysis in human liver cytosols corresponded to approximately 50% of that observed in microsomes. Fractions obtained by 9000g centrifugation of supernatants from COS-1 cells expressing human carboxylesterase (CES) 1 or 2 exhibited MMF hydrolytic activity, with CES1-containing fractions showing higher catalytic efficiency than CES2-containing fractions. The CES inhibitor bis-p-nitrophenylphosphate inhibited MMF hydrolysis in human liver microsomes and cytosols with IC(50) values of 0.51 and 0.36 µM, respectively. In conclusion, both intestinal and hepatic CESs and in particular CES1 may be involved in MMF hydrolysis and play important roles in MMF bioactivation. Hepatic CES1 activity levels may help explain the between-subject variability observed for MMF usage.

Nonclinical pharmacokinetics of oseltamivir and oseltamivir carboxylate in the central nervous system.

Oseltamivir, a potent and selective inhibitor of influenza A and B virus neuraminidases, is a prodrug that is systemically converted into the active metabolite oseltamivir carboxylate. In light of reported neuropsychiatric events in influenza patients, including some taking oseltamivir, and as part of a full assessment to determine whether oseltamivir could contribute to, or exacerbate, such events, we undertook a series of nonclinical studies. In particular, we investigated (i) the distribution of oseltamivir and oseltamivir carboxylate in the central nervous system of rats after single intravenous doses of oseltamivir and oseltamivir carboxylate and oral doses of oseltamivir, (ii) the active transport of oseltamivir and oseltamivir carboxylate in vitro by transporters located in the blood-brain barrier, and (iii) the extent of local conversion of oseltamivir to oseltamivir carboxylate in brain fractions. In all experiments, results showed that the extent of partitioning of oseltamivir and especially oseltamivir carboxylate to the central nervous system was low. Brain-to-plasma exposure ratios were approximately 0.2 for oseltamivir and 0.01 for oseltamivir carboxylate. Apart from oseltamivir being a good substrate for the P-glycoprotein transporter, no other active transport processes were observed. The conversion of the prodrug to the active metabolite was slow and limited in human and rat brain S9 fractions. Overall, these studies indicate that the potential for oseltamivir and oseltamivir carboxylate to reach the central nervous system in high quantities is low and, together with other analyses and studies, that their involvement in neuropsychiatric events in influenza patients is unlikely.

Arabidopsis GDSL lipase 2 plays a role in pathogen defense via negative regulation of auxin signaling.

GLIP1 was isolated previously from Arabidopsis, as a salicylic acid-responsive secreted GDSL lipase that functions in resistance to Alternaria brassicicola [I.S. Oh, A.R. Park, M.S. Bae, S.J. Kwon, Y.S. Kim, J.E. Lee, N.Y. Kang, S. Lee, H. Cheong, O.K. Park, Secretome analysis reveals an Arabidopsis lipase involved in defense against Alternaria brassicicola. Plant Cell 17 (2005) 2832-2847.]. To extend our knowledge of the roles played by GLIPs in Arabidopsis, we conducted functional studies of another family member, GLIP2. GLIP2 transcripts were expressed in young seedlings, as well as in the root and stem tissues of mature plants. GLIP2 transcript levels were elevated by treatment with salicylic acid, jasmonic acid and ethylene. Recombinant GLIP2 proteins possessed lipase and anti-microbial activities, inhibiting germination of fungal spores. In comparison to wild type plants, T-DNA insertion glip2 mutants exhibited enhanced auxin responses, including increased lateral root formation and elevated AUX/IAA gene expression. When challenged with the necrotropic bacteria Erwinia carotovora, glip2 mutants exhibited more susceptible phenotypes than wild type plants. These results suggest that GLIP2 plays a role in resistance to Erwinia carotovora via negative regulation of auxin signaling.

[Hydrolysis by carboxylesterase and disposition of prodrug with ester moiety].

Prodrug is a useful approach for improving the bioavailability of therapeutic agents through increased passive transport. Carboxylesterases (CESs, EC.3.1.1.1.) that show ubiquitous expression profiles play an important role in the biotransformation of ester-containing prodrugs into their therapeutically active forms in the body. High levels of CESs are found in the liver, small intestine and lungs where prodrugs are firstly hydrolyzed before entering the systemic circulation. Rat intestine single-pass perfusion experiments have shown that CES is involved in the intestinal first-pass hydrolysis. Extensive pulmonary first-pass hydrolysis has been observed in accordance to the substrate specificity of CES1 isozyme. Hydrolysis in the human liver and lungs is mainly catalyzed by hCE1 (a human CES1 family isozyme), whereas that in the small intestine is predominantly mediated by hCE2 (a human CES2 family isozyme). hCE2 preferentially hydrolyzes substrates with a small acyl moiety such as CPT-11, due to conformational steric hindrance in its active site. In contrast, hCE1 is able to hydrolyze a variety of substrates due to spacious and flexible substrate binding region in its active site. In addition, hCE1 has been found to catalyze transesterification. Caco-2 cells mainly expresses CES1 isozyme but not CES2 isozyme. Because of the differences in substrate specificity between CES1 and CES2 enzymes, Caco-2 cell monolayer is not suitable for predicting intestinal absorption of prodrugs. These findings indicate that identification of substrate specificity of CES isozymes and development of an in vitro experimental method are essential to support rational design of prodrug.

Isolation and characterization of a microsomal acid retinyl ester hydrolase.

Previous work demonstrated both acid and neutral, bile salt-independent retinyl ester hydrolase activities in rat liver homogenates. Here we present the purification, identification, and characterization of an acid retinyl ester hydrolase activity from solubilized rat liver microsomes. Purification to homogeneity was achieved by sequential chromatography using SP-Sepharose cation exchange, phenyl-Sepharose hydrophobic interaction, concanavalin A-Sepharose affinity and Superose 12 gel filtration chromatography. The isolated protein had a monomer molecular mass of approximately 62 kDa, as measured by mass spectrometry. Gel filtration chromatography of the purified protein revealed a native molecular mass of approximately 176 kDa, indicating that the protein exists as a homotrimeric complex in solution. The purified protein was identified as carboxylesterase ES-10 (EC 3.1.1.1) by N-terminal Edman sequencing and extensive LC-MS/MS sequence analysis and cross-reaction with an anti-ES-10 antibody. Glycosylation analysis revealed that only one of two potential N-linked glycosylation sites is occupied by a high mannose-type carbohydrate structure. Using retinyl palmitate in a micellar assay system the enzyme was active over a broad pH range and displayed Michaelis-Menten kinetics with a K(m) of 86 microm. Substrate specificity studies showed that ES-10 is also able to catalyze hydrolysis of triolein. Cholesteryl oleate was not a substrate for ES-10 under these assay conditions. Real time reverse transcriptase-PCR and Western blot analysis revealed that ES-10 is highly expressed in liver and lung. Lower levels of ES-10 mRNA were also found in kidney, testis, and heart. A comparison of mRNA expression levels in liver demonstrated that ES-10, ES-4, and ES-3 were expressed at significantly higher levels than ES-2, an enzyme previously thought to play a major role in retinyl ester metabolism in liver. Taken together these data indicate that carboxylesterase ES-10 plays a major role in the hydrolysis of newly-endocytosed, chylomicron retinyl esters in both neutral and acidic membrane compartments of liver cells.