Serine Hydrolase Activity Research Articles

Chemical strategies for the global analysis of protein function: Profiling hydrolytic enzymes in cancer

The molecular features specific to metastatic carcinomas that support their invasive behavior are complex and ill defined. Although one generally accepted notion assigns hydrolytic enzymes a central role in promoting the aggressive properties of metastatic tumors, the actual functions played by individual proteases and esterases in cancer remain elusive. To understand better how hydrolases and their endogenous inhibitors affect cancer, we are using a new chemical strategy, activity-based protein profiling. This method allows us to monitor simultaneously and directly the catalytic activities of numerous serine hydrolases from whole-cell, tissue and fluid samples. We have initiated a program to compare the serine hydrolase activity profiles of estrogen-receptor-positive and -negative human breast cancer cell lines. In breast carcinomas, a strong inverse correlation exists between estrogen receptor expression and several metastatic phenotypes, including cell invasiveness and motility. When this finding is coupled with the observation that serine protease inhibitors suppress estrogen-receptor-negative tumor cell migration and invasion, an intriguing model emerges in which serine hydrolases play a central role in mediating the aggressive behavior of these cells. Nonetheless, the identities of the participating enzymes remain unknown. Using activity-based protein profiling, we have identified several serine hydrolase activities that vary dramatically among these breast cancer lines. We anticipate that such studies will identify functional changes in key serine hydrolases involved in promoting or retarding tumorigenesis. These enzymes should in turn serve as both markers for cancer progression and targets for pharmaceutical efforts aimed at treating this disease.

Characterisation of a gene cluster involved in bacterial degradation of the cyanobacterial toxin microcystin LR

A novel pathway for degradation of the cyanobacterial heptapeptide hepatotoxin microcystin LR was identified in a newly isolated Sphingomonas sp. (Bourne et al. 1996 Appl. Environ. Microbiol. 62: 4086-4094). We now report the cloning and molecular characterisation of four genes from this Sphingomonas sp. that exist on a 5.8-kb genomic fragment and encode the three hydrolytic enzymes involved in this pathway together with a putative oligopeptide transporter. The heterologously expressed degradation pathway proteins are enzymatically active. Microcystinase (MlrA), the first enzyme in the degradative pathway, is a 336-residue endopeptidase, which displays only low sequence identity with a hypothetical protein from Methanobacterium thermoautotrophicum. Inhibition of microcystinase by EDTA and 1,10-phenanthroline suggests that it is a metalloenzyme. The most likely residues that could potentially chelate an active-site transition metal ion are in the sequence HXXHXE, which would be unique for a metalloproteinase. Situated immediately downstream of mlrA with the same direction of transcription is a gene mlrD, whose conceptual translation (MlrD, 442 residues) shows significant sequence identity and similar potential transmembrane spanning regions to the PTR2 family of oligopeptide transporters. A gene mlrB is situated downstream of the mlrA and mlrD genes, but transcribed in the opposite direction. The gene encodes the enzyme MlrB (402 residues) which cleaves linear microcystin LR to a tetrapeptide degradation product. This enzyme belongs to the "penicillin-binding enzyme" family of active site serine hydrolases. The final gene in the cluster mlrC, is located upstream of the mlrA gene and is transcribed in the opposite direction. It codes for MlrC (507 residues) which mediates further peptidolytic degradation of the tetrapeptide. This protein shows significant sequence identity to a hypothetical protein from Streptomyces coelicolor. It is suspected to be a metallopeptidase based on inhibition by metal chelators. It is postulated on the basis of comparison with other microorganisms that the genes in this cluster may all be involved in cell wall peptidoglycan cycling and subsequently act fortuitously in hydrolysis of microcystin LR.

Identification of a putative surfactant convertase in rat lung as a secreted serine carboxylesterase.

In the alveolar lumen, pulmonary surfactant converts from the contents of secreted lamellar bodies to tubular myelin to apoprotein-depleted vesicles during respiration. Using an in vitro system, researchers have reported that the conversion of tubular myelin to vesicles is blocked by inhibitors of serine hydrolase activity and have tentatively ascribed "convertase" activity to a diisopropyl fluorophosphate (DFP)-binding protein in mouse bronchoalveolar lavage (BAL). We purified and sequenced the homologous enzyme from rat BAL fluid. Amino acid sequence from the amino terminus and an internal cyanogen bromide peptide of the purified rat DFP-binding protein perfectly match the sequence of the carboxylesterase ES-2. Although ES-2 was initially cloned from liver, we found a 1.8-kilobase mRNA for ES-2 in decreasing relative abundance in rat liver, kidney, and lung but not in heart or spleen. Although further studies are required to establish the identity between "convertase" and ES-2 or a homologous member of the carboxylesterase family, our results raise the possibility that a protein with esterase/lipase activity plays a role in extracellular surfactant metabolism.

Inhibition of Diabrotica Larval Growth by Patatin, the Lipid Acyl Hydrolase from Potato Tubers.

Patatin, the nonspecific lipid acyl hydrolase from potato (Solanum tuberosum L.) tubers, dose-dependently inhibits the growth of southern corn rootworm (SCR) and western corn rootworm when fed to them on artificial diet. The 50% growth reduction levels are somewhat cultivar dependent, ranging from 60 to 150 [mu]g/g diet for neonate SCR larvae. A single patatin isoform also inhibits larval growth. Neonate SCR continuously exposed to patatin are halted in larval development. Treatment with di-isopropylfluorophosphate essentially eliminates patatin's phospholipase, galactolipase, and acyl hydrolase activities. SCR growth inhibition is eliminated also, indicating that patatin's serine hydrolase activity is responsible for the observed activities. Patatin-mediated phospholipolysis is highly pH and cultivar dependent, with specific activities up to 300-fold less at pH 5.5 than at pH 8.5. Esterase or phospholipase activities do not correlate with insect growth inhibition. Galactolipase activity, being cultivar and pH independent, correlates significantly with SCR growth inhibition. Insect-growth inhibition of patatin is significantly reduced with increased dietary cholesterol levels. In conclusion, patatin represents a new class of insect-control proteins with a novel mode of action possibly involving lipid metabolism.

Inhibition of interleukin-2-stimulated enhancement of human natural killer (NK) cell activity by carbaryl, an anticholinesterase insecticide

The potency of the anticholinesterase (antiCHE) insecticides as serine hydrolase inhibitors, and evidence for serine hydrolase activity in interleukin-2 (IL2) signalling suggest that the natural killer (NK) cell may be a target for dysregulation by antiCHE insecticides. NK cells are large granular lymphocytes (LGL) that respond to IL2 by proliferating and increasing their cytolytic efficiency. In the present study, we assessed the effects of carbaryl (CA, an antiCHE insecticide) and α-naphthol (NA, the major metabolite of CA) on both target cell killing per se and IL2 enhancement of target cell killing by human NK cells. Human LGL, collected from the peripheral blood of normal donors, were cultured for 4 days with human recombinant IL2 (HRIL2), then assayed by a 51Chromium ( 51Cr) release assay for lytic activity against human K562 cells. When added at the beginning of the culture period, CA inhibited enhancement of cytolytic efficiency in a concentration-dependent manner; at concentrations (0.5 and 5.0 μM) compatible with no cholinergic toxicity. Reduction of the effector/target cell (E/T) ratio in the 51Cr release assay markedly enhanced the observed inhibition by CA. In one experiment, inhibition increased from 6% to 20%, 17% to 35%, and 53% to 73% at 0.5, 5.0, and 50 μM CA, respectively, when E/T was reduced from 10:1 to 2.5:1. This result is consistent with reduced cytolytic efficiency of individual NK cells exposed to CA. NA had no effect at 0.5 or 5.0 μM but caused some inhibition at 50 μM. Neither CA nor NA produced LGL death. When CA or NA was added directly to the 51Cr release assay, inhibition was not observed. The mechanism of inhibition of IL2-stimulated enhancement of target cell killing is not yet known, however, the results are consistent with impairment of IL2 signalling, by CA.

Reversible modulation of serine protease activity by phosphonate esters

Temporary modification of serine hydrolase activity with 4-nitrophenyl phenacyl and 4-nitrophenacyl methylphosphonates occurs with self-catalyzed intramolecular reactivation of chymotrypsin, trypsin, thrombin and plasmin