Ronald P. Magnusson

Purification, Characterization, and Cloning of a Cytosolic Aspartyl Aminopeptidase

An aminopeptidase with a preference for N-terminal aspartyl and glutamyl residues but distinct from glutamyl aminopeptidase (EC 3.4.11.7) was purified to near homogeneity from rabbit brain cytosol. Its properties were similar to an enzyme described previously (Kelly, J. A., Neidle, E. L., and Neidle, A. (1983) J. Neurochem. 40, 1727–1734). Aspartyl aminopeptidase had barely detectable activity toward simple aminoacyl-naphthylamide substrates. Its activity was determined with the substrate Asp-Ala-Pro-naphthylamide in the presence of excess dipeptidyl-peptidase IV (EC 3.4.14.5). The native enzyme has a molecular mass of 440 kDa and migrates as a single band of 55 kDa after SDS-polyacrylamide gel electrophoresis. The sequences of three tryptic peptides were used to screen the GenBankTM data base of expressed sequence tags. Human and mouse clones described as “similar to a yeast vacuolar aminopeptidase” and containing full-length cDNAs were identified and sequenced. The human cDNA was expressed in Escherichia coli. The amino acid sequence has significant homology to yeast aminopeptidase I, placing it as the first identified mammalian member of the M18 family of metalloproteinases. Homologous sequences in Caenorhabditis elegans and in prokaryotes revealed three conserved histidines, three conserved glutamates and five conserved aspartates. Aspartyl aminopeptidase is found at relatively high levels in all mammalian tissues examined and is likely to play an important role in intracellular protein and peptide metabolism.

Distinct Characteristics of the Basal Activities of Adenylyl Cyclases 2 and 6

Regulation of basal activities of adenylyl cyclase (AC) 2 and 6, expressed in Sf9 cells by infection with recombinant baculovirus, was studied. An antipeptide antibody that recognizes AC2 and AC6 with equal sensitivity was used to establish that equivalent levels were expressed. Basal activities of AC2 and AC6 were compared at varying concentrations of Mg or Mn ions; AC2 had 15- and 10-fold greater activity than AC6, respectively. At 20 mM Mg, the K values for ATP were 88 and 39 μM for AC2 and AC6, respectively, whereas their Vmax values were 281 and 11 pmol/mg protein•min. With 100 μM forskolin and either Mg or Mn, the difference in activities between AC2 and AC6 was reduced to approximately 2-fold. Forskolin stimulated AC6 greater than 40-fold at 0.5-2 mM Mg, whereas AC2 was stimulated 4-6-fold. At 20 mM Mg, AC2 was stimulated 2-fold by forskolin, whereas AC6 was stimulated 18-fold. With Mg alone, activities of AC2 and AC6 were not saturable up to 20 mM and yielded curvilinear Hofstee transformations. With forskolin, activities of both AC2 and AC6 were saturable by 10 mM Mg and yielded linear Hofstee transformations. These data indicate that there are substantial differences in the basal enzymatic activities of adenylyl cyclase isoforms, due to differential regulation by Mg ions rather than intrinsic catalytic capabilities. Thus the presence and relative abundance of adenylyl cyclase subtypes could greatly affect the resting cellular cAMP levels with consequent effects on important biological functions, such as differentiation and proliferation.

Differential Regulation of Adenylyl Cyclases by Gαs

Regulation of adenylyl cyclases 1, 2, and 6 by Gαs was studied. All three mammalian adenylyl cyclases were expressed in insect (Sf9 or Hi-5) cells by baculovirus infection. Membranes containing the different adenylyl cyclases were stimulated by varying concentrations of mutant (Q227L) activated Gαs expressed in reticulocyte lysates. Gαsstimulation of AC1 involved a single site and had an apparentK act of 0.9 nm. Gαsstimulation of AC2 was best explained by a non-interactive two site model with a “high affinity” site at 0.9 nm and a “low affinity” site at 15 nm. Occupancy of the high affinity site appears to be sufficient for Gβγ stimulation of AC2. Gαs stimulation of AC6 was also best explained by a two-site model with a high affinity site at 0.6–0.8 nm and a low affinity site at 8–22 nm; however, in contrast to AC2, only a model that assumed interactions between the two sites best fit the AC6 data. With 100 μm forskolin, Gαs stimulation of all three adenylyl cyclases showed very similar profiles. Gαs stimulation in the presence of forskolin involved a single site with apparentK act of 0.1–0.4 nm. These observations indicate a conserved mechanism by which forskolin regulates Gαs coupling to the different adenylyl cyclases. However, there are fundamental differences in the mechanism of Gαs stimulation of the different adenylyl cyclases with AC2 and AC6 having multiple interconvertible sites. These mechanistic differences may provide an explanation for the varied responses by different cells and tissues to hormones that elevate cAMP levels.

Omega oxidation of 3-hydroxy fatty acids by the human CYP4F gene subfamily enzyme CYP4F11

Long-chain 3-hydroxydicarboxylic acids (3-OHDCAs) are thought to arise via β-oxidation of the corresponding dicarboxylic acids (DCAs), although long-chain DCAs are neither readily transported into nor β-oxidized in mitochondria. We thus examined whether ω-hydroxylation of 3-hydroxy fatty acids (3-OHFAs), formed via incomplete mitochondrial oxidation, is a more likely pathway for 3-OHDCA production. NADPH-fortified human liver microsomes converted 3-hydroxystearate and 3-hydroxypalmitate to their ω-hydroxylated metabolites, 3,18-dihydroxystearate and 3,16-dihydroxypalmitate, respectively, as identified by GC-MS. Rates of 3,18-dihydroxystearate and 3,16-dihydroxypalmitate formation were 1.23 ± 0.5 and 1.46 ± 0.30 nmol product formed/min/mg protein, respectively (mean ± SD; n = 13). Polyspecific CYP4F antibodies markedly inhibited microsomal ω-hydroxylation of 3-hydroxystearate (68%) and 3-hydroxypalmitate (99%), whereas CYP4A11 and CYP2E1 antibodies had little effect. Upon reconstitution, CYP4F11 and, to a lesser extent, CYP4F2 catalyzed ω-hydroxylation of 3-hydroxystearate, whereas CYP4F3b, CYP4F12, and CYP4A11 exhibited negligible activity. CYP4F11 was the lone CYP4F/A enzyme that effectively oxidized 3-hydroxypalmitate. Kinetic parameters of microsomal 3-hydroxystearate metabolism were Km = 55 μM and Vmax = 8.33 min−1, whereas those for 3-hydroxypalmitate were Km = 56.4 μM and Vmax = 14.2 min−1. CYP4F11 kinetic values resembled those of native microsomes, with Km = 53.5 μM and Vmax = 13.9 min−1 for 3-hydroxystearate and Km = 105.8 μM and Vmax = 70.6 min−1 for 3-hydroxypalmitate. Our data show that 3-hydroxystearate and 3-hydroxypalmitate are converted to ω-hydroxylated 3-OHDCA precursors in human liver and that CYP4F11 is the predominant catalyst of this reaction. CYP4F11-promoted ω-hydroxylation of 3-OHFAs may modulate the disposition of these compounds in pathological states in which enhanced fatty acid mobilization or impairment of mitochondrial fatty acid β-oxidation increases circulating 3-OHFA levels.

Identification of histidine residues important in the catalysis and structure of aspartyl aminopeptidase.

Aspartyl aminopeptidase (DAP), a widely distributed and abundant cytosolic enzyme, removes glutamyl or aspartyl residues from N-terminal acidic amino acid-containing peptides. DAP is a member of the M18 family of the MH clan of cocatalytic metallopeptidases. The human and mouse enzymes have been cloned. We have identified 8 highly homologous eukaryotic sequences that are probable aspartyl aminopeptidases. Eight histidine residues of human DAP were sequentially mutated to phenylalanine. Mutation of His94, His170, and His440 abolished enzymatic activity. His94 and His440 are postulated to be involved in binding cocatalytic zinc atoms by homology with other members of the MH clan. Mutation of His352 dramatically reduced enzyme activity. Gel-filtration analysis of the His352 mutant revealed destabilization of the quaternary structure and dissociation of the native 440-kDa enzyme. Mutation of His33 and of histidines residing in a cluster at residues 349, 359, and 363 all decreased k(cat). These studies reveal an important role for histidine residues both in catalysis and in the structural integrity of DAP.

Expression of CYP4F2 in human liver and kidney: Assessment using targeted peptide antibodies

P450 enzymes comprising the human CYP4F gene subfamily are catalysts of eicosanoid (e.g., 20-HETE and leukotriene B4) formation and degradation, although the role that individual CYP4F proteins play in these metabolic processes is not well defined. Thus, we developed antibodies to assess the tissue-specific expression and function of CYP4F2, one of four CYP4F P450s found in human liver and kidney. Peptide antibodies elicited in rabbits to CYP4F2 amino acid residues 61-74 (WGHQGMVNPTEEG) and 65-77 (GMVNPTEEGMRVL) recognized on immunoblots only CYP4F2 and not CYP4F3b, CYP4F11 or CYP4F12. Immunoquantitation with anti-CYP4F2 peptide IgG showed highly variable CYP4F2 expression in liver (16.4+/-18.6pmol/mg microsomal protein; n=29) and kidney cortex (3.9+/-3.8 pmol/mg; n=10), with two subjects lacking the hepatic or renal enzyme entirely. CYP4F2 content in liver microsomes was significantly correlated (r> or =0.63; p<0.05) with leukotriene B4 and arachidonate omega-hydroxylase activities, which are both CYP4F2-catalyzed. Our study provides the first example of a peptide antibody that recognizes a single CYP4F P450 expressed in human liver and kidney, namely CYP4F2. Immunoquantitation and correlation analyses performed with this antibody suggest that CYP4F2 functions as a predominant LTB4 and arachidonate omega-hydroxylase in human liver.

Modulators of the activation of the proteasome by PA28 (11S Reg)

The degradation of chromogenic substrates and oligopeptides by the 20S proteasome is markedly enhanced and the generation of antigens for presentation by the MHC class-I system is facilitated by combination with an activator protein known as PA28 or 11S reg. We have described the properties of a PA28-proteasome modulator, N-benzyloxycarbonyl-Ile-Glu(O-t-Bu)-Ala-leucinol which shifts the pathway of peptide hydrolysis by the activated proteasome to products terminating in an acidic amino acid at the expense of products terminating in a hydrophobic amino acid. We now report that piperazinyl phenothiazines and several other antipsychotic drugs modulate the PA28-20S activated proteasome in an opposite manner. Fluphenazine, trifluoperazine and prochlorperazine antagonize the peptidylglutamyl peptide bond hydrolyzing activity of the activated proteasome much more strongly than the chymotrypsinlike activity. The chicken ovalbumin immunodominant epitope SIINFEKL is degraded by the activated proteasome to SIINFE and SIINF in approximately equimolar amounts. Piperazinyl phenothiazines promote formation of SIINF whereas Psi-ol promotes formation of SIINFE. PA28- proteasome modulators by modifying the profile of peptides produced by the activated proteasome, may either enhance or suppress the immune response.