Biochemical and docking analysis of substrate interactions with polyisoprenylated methylated protein methyl esterase.

Polyisoprenylated proteins (PPs) methylation by polyisoprenylated protein methyl transferase (PPMTase) is counteracted by polyisoprenylated methylated protein methyl esterase (PMPMEase). This is the only reversible step of the polyisoprenylation pathway as the relative amounts of the acid and ester forms are determined by the two competing reactions. Since PMPMEase and PPMTase may influence both the structural/functional conformations of PPs, a thorough study of these enzymes is essential to our understanding of the structural/functional features of PPs. PMPMEase has been reported under such pseudonyms as human carboxylesterase 1 (hCE1) because of its apparent broad substrate spectrum. The current study aimed to show the complementarity between its active site and the polyisoprenylated substrates that it metabolizes. Kinetics analysis was conducted with N-, S- and O-substituted substrates using porcine liver PMPMEase and docking analysis using Arguslab. Consistent with the biochemical analysis, the S-ethyl analog yielded an AScore binding energy of -11.32 compared to -13.48, -14.88, -16.15, and -16.81 kcal/mol for S-prenyl (C-5), S-trans-geranyl (C-10), S-trans,trans-farnesyl (C-15) and S-all trans-geranylgeranyl (C-20), respectively. The all trans-geranylgeranyl moiety provides the optimal size for active site interactions. The data reveal that the trans,trans-farnesyl and all trans-geranylgeranyl groups, which are reminiscent of endogenous PPs modifications, have the highest affinity for PMPMEase. Since PPs such as monomeric G-proteins are oncogenic, PMPMEase may be viewed as a viable target for anticancer drug development. The analyses reveal the important structural elements for the design of specific PMPMEase inhibitors to serve in the modulation of oncogenic PPs activities. The results also show that hCE1's repertoire of substrates extends beyond xenobiotics to include PPs as its endogenous substrates.

Farnesyl-L-cysteine analogs block SAM-induced Parkinson's disease-like symptoms in rats.

Injection of the endogenous methyl donor, S-adenosyl methionine (SAM), into rat brain induces Parkinson's disease (PD)-like symptoms possibly by stimulating deleterious protein methylation. Gel-filtration chromatography of rat brain extracts treated with [3H-methyl]-SAM revealed the presence of radioactive peaks with apparent molecular weights of about 5 kDa. Treatment with guanidine HCl altered the elution volumes of the labeled peaks. Lyophilized peak fractions released volatile 3H-methanol on incubation with NaOH, indicating the presence of carboxyl methyl esters. Because prenylated proteins are avid methyl acceptors at the terminal carboxylic acid groups, 1 micromol S-farnesylcysteine (FC) analogs blocked the SAM-induced tremors in the experimental rats. FC analogs did not only reverse the associated rigidity, abnormal posture, and hypokinesia, but stimulated hyperactivity in the animals. This amphetamine-like effect was monitored for 20 min in an animal activity monitor and movement times between 400 +/- 100 and 560 +/- 125 s covering distances between 78 +/- 29 to 125 +/- 35 m were recorded for rats treated with FC analogs with or without SAM. Control animals moved only for 60 +/- 13 s covering about 6 +/- 1 m, indicating a 7-9-fold and 13-21-fold increase in duration of movement and distance covered, respectively. N-Acetyl-S-farnesylcysteine (AFC) potentiated amphetamine-induced ipsiversive rotation of 6-hydroxydopamine-lesioned rats from 390 +/- 130 to 830 +/- 110, with AFC alone having no significant effect on net rotation compared to controls. These data indicate that intracerebroventricular injection of SAM may induce PD symptoms by interfering with the methylation/demethylation homeostasis of prenylated proteins that function in the dopaminergic and other signaling pathways, and that the FC analogs may counteract the SAM effects by acting synergistically on events subsequent to neurotransmitter release.

Quantification of S-adenosylmethionine-induced tremors: a possible tremor model for Parkinson's disease.

Tremor is the most visible symptom of Parkinson's Disease (PD), and should be the appropriate parameter in models for its evaluation. Lack of reliable PD tremor models and methods to distinguish tremors from nontremor movements means that nontremor behavior such as rotation following basal ganglia damage are mostly used. Our laboratory has shown that S-adenosyl-methionine (SAM) injections into the brain of rats reliably produced tremors, rigidity, hypokinesia, and abnormal posture. Thus, SAM-induced tremors, when distinguished from nontremor activities, has the potential as a model for testing anti-PD agents. Tremor Monitor-recorded activity profiles of the rats injected with SAM showed low-amplitude signals interlaced with high-amplitude bursts of tremor episodes. Control activities were of low-medium amplitudes with no such patterns. The number of real and apparent episodes detected over 20 min were 92 +/- 12 and 84 +/- 14 lasting 470 +/- 50 and 210 +/- 50 s, indicating mean durations of 5.1, and 2.4 s, frequencies of 12 +/- 0.1 and 11 +/- 0.2 Hz, cycles (waves) per episode of 54 +/- 6 and 19 +/- 2 and amplitudes of 42.3 +/- 5 and 19.8 +/- 1 for the SAM-treated and control rats, respectively. The nontremor activities of rats injected with phosphate-buffered saline were distinguished and eliminated by raising the minimum amplitude and number of cycles to 20. This procedure is being enhanced for screening antitremor agents and for elucidating the possible mechanism for Parkinsonism.

The proteolytic maturation of prohormone convertase 2 (PC2) is a pH-driven process.

Recombinant proPC2 purified from the medium of CHO cells overexpressing both the prohormone convertase (PC) precursor proPC2 and the 21-kDa amino terminal portion of the neuroendocrine protein 7B2 can spontaneously convert to an active species. In the present report, we have characterized the proPC2 zymogen conversion process. Sequencing of the mature 66 kDa enzyme revealed a single site of cleavage at the paired basic site amino terminal to the GYRDI sequence. In contrast to mature PC2 activity, proPC2 conversion was inhibited neither by the eukaryotic subtilisin inhibitor pCMS nor by the specific PC2 inhibitor, 7B2 CT peptide, suggesting significant differences between the proPC2 conversion reaction and the hydrolysis of synthetic substrates by mature PC2. In support of this idea, proPC2 conversion was not calcium dependent and was unaffected by 5 mM EDTA. The rate of conversion of proPC2 remained similar with a 10-fold difference in zymogen concentration, implicating an intramolecular rather than intermolecular mechanism of activation. Interestingly, the rate of proPC2 conversion was extremely pH dependent, occurring most extensively between pHs 4.0 and 4.9. Taken together, our results suggest that cellular proPC2 maturation occurs via an autocatalytic, intramolecular process controlled not by 7B2 inhibition nor by calcium levels, but by the decreasing pH gradient along the secretory pathway.

Identification of inhibitors of prohormone convertases 1 and 2 using a peptide combinatorial library.

A positional scanning synthetic peptide combinatorial library containing approximately 52 million hexapeptides was used to identify potential inhibitory peptides for recombinant mouse prohormone convertase 1 (PC1) and PC2 and to provide information on the specificity of these enzymes. The library surveys revealed that a P6 Leu, a P4 Arg, a P2 Lys, and a P1 Arg were most inhibitory against PC1, and a P6 Ile and a P4 Arg were most inhibitory against PC2. Using information derived from the library surveys, hexapeptide sets were synthesized and screened for inhibition of PC1 and PC2. The data obtained revealed the preference of both enzymes for a P3 Val. At P5, many substitutions were well tolerated. PC1 and PC2 proved to differ mainly in the selectivity of their S6 subsites. In PC1, this subsite displayed a strong preference toward occupation by Leu; the Ki value for peptide Ac-Leu-Leu-Arg-Val-Lys-Arg-NH2 was 28 times lower than that for peptide Ac-Ile-Ile-Arg-Val-Lys-Arg-NH2. In contrast, PC2 discriminated little between Leu and Ile at P6, as evidenced by the small (1.5-fold) difference in Ki values for these two peptides. Several hexapeptides synthesized as a result of the screen were found to represent potent inhibitors of PC2 (with Ki values in the submicromolar range) and, particularly, of PC1 (with Ki values in the low nanomolar range). The most potent inhibitor, Ac-Leu-Leu-Arg-Val-Lys-Arg-NH2, proved to be the same peptide for both enzymes and inhibited PC1 and PC2 in a competitive, fast-binding manner with Ki values of 3.2 and 360 nM, respectively. The four most potent peptide inhibitors of PC1 and PC2 were also tested against soluble human furin and found to exhibit a different rank order of inhibition; for example, Ac-Leu-Leu-Arg-Val-Lys-Arg-NH2 was 440-fold less potent against furin than against PC1, with a Ki of 1400 nM.

Specificity of prohormone convertase 2 on proenkephalin and proenkephalin-related substrates.

In the central and peripheral nervous systems, the neuropeptide precursor proenkephalin must be endoproteolytically cleaved by enzymes known as prohormone convertases 1 and 2 (PC1 and PC2) to generate opioid-active enkephalins. In this study, we have investigated the specificity of recombinant mouse PC2 for proenkephalin-related internally quenched (IQ) peptides, for methylcoumarin amide-based fluorogenic peptides, and for recombinant rat proenkephalin. IQ peptides exhibited specificity constants (kcat/Km) between 9.4 x 10(4) M-1 s-1 (Abz-Val-Pro-Arg-Met-Glu-Lys-Arg-Tyr-Gly-Gly-Phe-Met-Gln-EDDnp+ ++; where Abz is ortho-aminobenzoic acid and EDDnp is N-(2, 4-dinitrophenyl)ethylenediamine)) and 0.24 x 10(4) M-1 s-1 (Abz-Tyr-Gly-Gly-Phe-Met-Arg-Arg-Val-Gly-Arg-Pro-Glu-EDDnp), with the peptide B to Met-enk-Arg-Phe cleavage preferred (Met-enk is met-enkephalin). Fluorogenic substrates with P1, P2, and P4 basic amino acids were hydrolyzed with specificity constants ranging between 2.0 x 10(3) M-1 s-1 (Ac-Orn-Ser-Lys-Arg-MCA; where MCA is methylcoumarin amide) and 1.8 x 10(4) M-1 s-1 (

Hydrolysis of insect neuropeptides by an angiotensin-converting enzyme from the housefly, Musca domestica.

The presence in insect tissues of peptides with structural similarities to angiotensin I and to bradykinin, the two best known substrates of mammalian angiotensin-converting enzyme, has not been reported. As part of our study to identify potential substrates for insect angiotensin-converting enzyme, we have investigated the susceptibility of a number of known insect peptide hormones and neurotransmitters to hydrolysis by Musca domestica angiotensin-converting enzyme. Insect peptides belonging to the red pigment-concentrating hormone, leucokinin, locust tachykinin, and depolarizing peptide families were hydrolyzed by housefly angiotensin-converting enzyme, whereas proctolin and crustacean cardioactive peptide were not substrates. Cus-DP II, LK I, LK II, and Lom-TK I were all cleaved at the penultimate C-terminal peptide bond to release a dipeptide amide as a major fragment with Km values of 94 +/- 11, 634 +/- 8, and 296 +/- 35 microM for Cus-DP II, LK I, and Lom-TK I, respectively. The ability of insect angiotensin-converting enzyme to hydrolyze C-terminally amidated peptides in vitro might be of functional significance because the enzyme has been localized to neuropile regions of the insect brain and is present in the hemolymph of houseflies.

Involvement of a polyproline helix-like structure in the interaction of 7B2 with prohormone convertase 2.

The neuroendocrine protein 7B2 is known to be involved in the biosynthesis and activity of prohormone convertase 2 (PC2). Previous studies have demonstrated that while the carboxyl-terminal portion of 7B2 (residues 155-186) regulates the enzymatic activity of PC2, the amino terminus of the molecule (residues 1-151) is required for maturation of proPC2. In this study we employed four different experimental approaches (co-immunoprecipitation with proPC2, facilitation of pro-PC2 maturation, acquisition of enzymatic activity, and thermal protection assays) to identify structural elements of 7B2 important for bioactivity. Inspection of the sequence of 7B2 indicated potential involvement of a polyproline helix-like (PPII) structure, with similarities to those present within SH3 domain ligands, in the interaction of 7B2 with proPC2. Site-directed point mutagenesis of this proline-rich region confirmed the involvement of this area. Replacement of prolines in positions critical to helix formation (Pro90, Pro91, Pro93, and Pro95) either severely impaired or totally abolished 7B2 bioactivity, as gauged by the four assays described. In addition, constructs longer than residues 1-121 were still functional, whereas those shorter than residues 1-109 were not. Computer-assisted analysis predicts the presence of an alpha-helix structure between residues 107 and 123. We conclude that both the proline-rich region and the alpha-helix contribute to 7B2 activity. Polyproline-containing peptides have been shown to be involved in cytoplasmic protein-protein interactions; our results suggest that the polyproline helix motif may also be used to mediate protein-protein interactions within the secretory pathway.

Purification and enzymatic characterization of recombinant prohormone convertase 2: stabilization of activity by 21 kDa 7B2.

Although previous efforts to produce significant quantities of purified prohormone convertase 2 from either recombinant or natural sources have been unsuccessful, our recent finding that the neuroendocrine polypeptide 7B2 is necessary for the biosynthesis of enzymatically active prohormone convertase 2 (PC2) has enabled us to obtain active recombinant enzyme from the conditioned medium of PC2-producing CHO cells supertransfected with cDNA coding for 21 kDa 7B2. The recombinant enzyme was purified to apparent homogeneity, with a 40% recovery, in milligram quantities. Two protein bands of Mrs 71 and 75 kDa were observed after SDS-PAGE followed by either Coomassie staining or Western blotting with PC2 antiserum. Spontaneous conversion of the 71- and 75-kDa species to the 66-kDa form occurred during incubation at pH 5.0; the degree of conversion correlated with a dramatic increase in activity. Kms of 124 and 131 microM and Kcats of 0.49 and 0.81 s(-1) were obtained for the substrates Cbz-Arg-Ser-Lys-Arg-AMC and Pyr-Arg-Thr-Lys-Arg-AMC, respectively. The pH optimum was 5.0, and the enzyme was inhibited by h7B2(155-185') p-CMS, and EDTA but not by other inhibitors tested. Interestingly, 21 kDa 7B2 was observed to copurify with the enzyme in a molar ratio of about 1:100 (7B2:PC2). Prior addition of recombinant 21 kDa 7B2 to activated 66 kDa PC2 provided significant protection against thermal denaturation. When coassociated 7B2 was mostly removed from activated PC2 through gel filtration, subsequent addition of recombinant 7B2 exerted a significant stabilizing effect on enzyme activity. Millimolar Ca2+ and pHs between 5 and 6 were required to observe this effect. Since these conditions resemble those thought to occur within secretory granules, and since 21 kDa 7B2 represents a stored secretory granule protein, our data suggest a physiological role for 21 kDa 7B2 in the stabilization of PC2 activity.

Internal cleavage of the inhibitory 7B2 carboxyl-terminal peptide by PC2: a potential mechanism for its inactivation.

The neuroendocrine protein 7B2 contains two domains, a 21-kDa protein required for prohormone convertase 2 (PC2) maturation and a carboxyl-terminal (CT) peptide that inhibits PC2 at nanomolar concentrations. To determine how the inhibition of PC2 is terminated, we studied the metabolic fate of the 7B2 CT peptide in RinPE-7B2, AtT-20/PC2-7B2, and alphaTC1-6 cells. Extracts obtained from cells labeled for 6 h with [3H]valine were subjected to immunoprecipitation using an antibody raised against the extreme carboxyl terminus of r7B2, and immunoprecipitated peptides were separated by gel filtration. All three cell lines yielded two distinct peaks at about 3.5 kDa and 1.5 kDa, corresponding to the CT peptide and a smaller fragment consistent with cleavage at an interior Lys-Lys site. These results were corroborated using a newly developed RIA against the carboxyl terminus of the CT peptide which showed that the intact CT peptide represented only about half of the stored CT peptide immunoreactivity, with the remainder present as the 1.5-kDa peptide. Both peptides could be released upon phorbol 12-myristate 13-acetate stimulation. We investigated the possibility that PC2 itself could be responsible for this cleavage by performing in vitro experiments. When 125I-labeled CT peptide was incubated with purified recombinant PC2, a smaller peptide was generated. Analysis of CT peptide derivatives for their inhibitory potency revealed that CT peptide 1-18 (containing Lys-Lys at the carboxyl terminus) represented a potent inhibitor, but that peptide 1-16 was inactive. Inclusion of carboxypeptidase E (CPE) in the reaction greatly diminished the inhibitory potency of the CT peptide against PC2, in line with the notion that the CT peptide cleavage product is not inhibitory after the removal of terminal lysines by CPE. In summary, our data support the idea that PC2 cleaves the 7B2 CT peptide at its internal Lys-Lys site within secretory granules; deactivation of the cleavage product is then accomplished by CPE, thus providing an efficient mechanism for intracellular inactivation of the CT peptide.

The endopeptidase activity and the activation by Cl- of angiotensin-converting enzyme is evolutionarily conserved: purification and properties of an an angiotensin-converting enzyme from the housefly, Musca domestica.

A soluble 67 kDa angiotensin-converting enzyme (ACE) has been purified by lisinopril-Sepharose affinity column chromatography from adult houseflies, Musca domestica. The dipeptidyl carboxypeptidase activity towards benzoyl-Gly-His-Leu was inhibited by captopril (IC50 50 nM) and fosinoprilat (IC50 251 nM), two inhibitors of mammalian ACE, and was activated by Cl- (optimal Cl- concentration 600 mM). Musca ACE removed C-terminal dipeptides from angiotensin I, bradykinin [Leu5]enkephalin and [Met5]enkephalin and also functioned as an endopeptidase by hydrolysing dipeptideamides from [Leu5]enkephalinamide and [Met5]enkephalinamide, and a dipeptideamide and a tripeptideamide from substance P. Musca ACE was also able to cleave a tripeptide from both the N-terminus and C-terminus of luteinizing hormone-releasing hormone, with C-terminal hydrolysis predominating. Maximal N-terminal tripeptidase activity occurred at 150 mM NaCl, whereas the C-terminal tripeptidase activity continued to rise with increasing concentration of Cl- (0-0.5 M). Musca ACE displays properties of both the N- and C-domains of human ACE, indicating a high degree of conservation during evolution of the substrate specificity of ACE and its response to Cl-.

Cloning and expression of an evolutionary conserved single-domain angiotensin converting enzyme from Drosophila melanogaster.

Mammalian somatic angiotensin converting enzyme (EC 3.4.15.1, ACE) consists of two highly homologous (N- and C-) domains encoded by a duplicated gene. We have identified an apparent single-domain (67 kDa) insect angiotensin converting enzyme (AnCE) in embryos of Drosophila melanogaster which converts angiotensin I to angiotensin II (Km, 365 microM), removes Phe-Arg from the C terminus of bradykinin (Km, 22 microM), and is inhibited by ACE inhibitors, captopril (IC50 = 1.1 x 10(-9) M) and trandolaprilat (IC50 = 1.6 x 10(-8) M). We also report the cloning and expression of a Drosophila AnCE cDNA which codes for a single-domain 615-amino acid protein with a predicted 17-amino acid signal peptide and regions with high levels of homology to both the N- and C-domains of mammalian somatic ACE, especially around the active site consensus sequence. Northern analysis identified a single 2.1-kilobase mRNA in Drosophila embryos, and Southern analysis of Drosophila genomic DNA indicates that the insect gene is not duplicated. When expressed in COS-7 cells, the AnCE protein is a secreted enzyme, which converts angiotensin I to angiotensin II and is inhibited by captopril (IC50 = 5.6 x 10(-9) M) and trandolaprilat (IC50 = 2 x 10(-8) M). The evolutionary significance of these results is discussed.

Identification and properties of a peptidyl dipeptidase in the housefly, Musca domestica, that resembles mammalian angiotensin-converting enzyme.

[D-Ala2,Leu5]Enkephalin was readily metabolized by membranes (40,000 g pellet) prepared from heads of the housefly, Musca domestica, with Gly3-Phe4 being the major site of cleavage. This hydrolysis was only partially inhibited (40%) by 10 microM phosphoramidon, an inhibitor of endopeptidase-24.11, but was almost totally abolished in the presence of a mixture of 10 microM phosphoramidon and 10 microM captopril, a potent inhibitor of mammalian angiotensin-converting enzyme (ACE). An assay for ACE employing Bz-Gly-His-Leu as the substrate was used to confirm the presence of an ACE-like peptidyl dipeptidase activity in fly head membranes. The peptidase had a Km of 1.91 mM for Bz-Gly-His-Leu and a pH optimum of 8.2. The activity was inhibited by 100 microM EDTA and was greatly activated by ZnCl2 but not other bivalent metal ions. Captopril, lisinopril, fosinoprilat and enalaprilat, all selective inhibitors of mammalian ACE, were also good inhibitors of the insect enzyme with IC50 values of 400 nM, 130 nM, 16 nM and 290 nM respectively. An M(r) value of around 87,000 was obtained for this enzyme from gel-filtration chromatography, indicating that the insect enzyme is similar in size to mammalian testicular ACE (M(r) = 90,000-110,000) and not the larger form of the enzyme (M(r) = 150,000-180,000) found in mammalian somatic tissues. The fly peptidyl dipeptidase was released from membranes into a soluble fraction by incubating the head membranes at 37 degrees C but not at 0 degree C, suggesting that the insect ACE-like enzyme can be solubilized from cell surfaces through the activity of a membrane-bound enzyme activity. In conclusion, we have shown the existence of a peptidyl dipeptidase in membranes from the heads of M. domestica, which has similar properties to those of mammalian ACE.

Metabolism of insect neuropeptides: properties of a membrane-bound endopeptidase from heads of Musca domestica.

A phosphoramidon-sensitive endopeptidase activity has been identified in membranes prepared from heads of Musca domestica. The enzyme hydrolyses the Gly3-Phe4 bond of the enkephalin analogue [D-Ala2,Leu5]enkephalin and the Asn3-Phe4 bond of AKH I. Phosporamidon (10 microM), a selective inhibitor of mammalian endopeptidase 24:11, was able to fully protect AKH I from degradation by head membranes. The breakdown of [D-Ala2,Leu5]enkephalin was only partially inhibited by phosphoramidon (10 microM), suggesting the presence of other enkephalin-degrading enzymes in this preparation. The endopeptidase activity was inhibited by 1 mM EDTA and 1 mM 1,10-phenanthroline and could be partially re-activated in the presence of ZnCl2 but not other divalent metal ions. The enzyme had a neutral pH optimum and behaved like an integral membrane protein when subjected to phase-separation with Triton X-114. Although they have a number of similar properties, the insect and mammalian enzymes could be distinguished by their sensitivity to site-directed inhibitors of endopeptidase 24:11. The fly endopeptidase was much less sensitive to phosphoramidon (IC50, 0.25 microM), thiorphan (IC50, 2.5 microM), SQ 28603 (IC50, 1.0 microM), SCH 39370 (IC50, 2.5 microM) and SCH 32615 (IC50, 30 microM). The fly enzyme is indistinguishable from the endopeptidase activity that is enriched in locust synaptic membranes and that found in membranes from heads of Drosophila melanogaster. In summary, we have identified a rich source of an insect neutral metallo-endopeptidase which is similar to endopeptidase 24:11, an enzyme known to play a key role in the metabolism and inactivation of neuropeptides in mammals.