Purification and cloning of aggrecanase-1: a member of the ADAMTS family of proteins.

We purified, cloned, and expressed aggrecanase, a protease that is thought to be responsible for the degradation of cartilage aggrecan in arthritic diseases. Aggrecanase-1 [a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4)] is a member of the ADAMTS protein family that cleaves aggrecan at the glutamic acid-373-alanine-374 bond. The identification of this protease provides a specific target for the development of therapeutics to prevent cartilage degradation in arthritis.

The MAPK cascade is required for mammalian associative learning.

Mitogen-activated protein kinase (MAPK) is an integral component of cellular signaling during mitogenesis and differentiation of mitotic cells. Recently MAPK activation in post-mitotic cells has been implicated in hippocampal long-term potentiation (LTP), a potential cellular mechanism of learning and memory. Here we investigate the involvement of MAPK in learning and memory in behaving animals. MAPK activation increased in the rat hippocampus after an associative learning task, contextual fear conditioning. Two other protein kinases known to be activated during hippocampal LTP, protein kinase C and alpha-calcium/calmodulin protein kinase II, also were activated in the hippocampus after learning. Inhibition of the specific upstream activator of MAPK, MAPK kinase (MEK), blocked fear conditioning. Thus, classical conditioning in mammals activates MAPK, which is necessary for consolidation of the resultant learning.

Dependence of Dbl and Dbs transformation on MEK and NF-kappaB activation.

Dbs was identified initially as a transforming protein and is a member of the Dbl family of proteins (>20 mammalian members). Here we show that Dbs, like its rat homolog Ost and the closely related Dbl, exhibited guanine nucleotide exchange activity for the Rho family members RhoA and Cdc42, but not Rac1, in vitro. Dbs transforming activity was blocked by specific inhibitors of RhoA and Cdc42 function, demonstrating the importance of these small GTPases in Dbs-mediated growth deregulation. Although Dbs transformation was dependent upon the structural integrity of its pleckstrin homology (PH) domain, replacement of the PH domain with a membrane localization signal restored transforming activity. Thus, the PH domain of Dbs (but not Dbl) may be important in modulating association with the plasma membrane, where its GTPase substrates reside. Both Dbs and Dbl activate multiple signaling pathways that include activation of the Elk-1, Jun, and NF-kappaB transcription factors and stimulation of transcription from the cyclin D1 promoter. We found that Elk-1 and NF-kappaB, but not Jun, activation was necessary for Dbl and Dbs transformation. Finally, we have observed that Dbl and Dbs regulated transcription from the cyclin D1 promoter in a NF-kappaB-dependent manner. Previous studies have dissociated actin cytoskeletal activity from the transforming potential of RhoA and Cdc42. These observations, when taken together with those of the present study, suggest that altered gene expression, and not actin reorganization, is the critical mediator of Dbl and Rho family protein transformation.

Oxylanosterols as modifiers of cholesterol biosynthesis.

In this review, the thinking and strategy that lead to the design of mechanism-based inhibitors of cholesterol biosynthesis have been recounted. This work began with a purely biochemical perspective on the mechanism of lanosterol demethylation. The final efforts focused on pharmacology and drug design thus bringing the basic science effort to a practical application. Most recently, a series of 15-oxalanosterols, which act as pure suppressors of HMG-CoA reductase lacking lanosterol demethylase inhibition properties has been identified. These molecules also lower serum cholesterol and show promise as potential agents for clinical evaluation. The utility of these compounds and validation of our hypothesis will have to await further testing.

Cytosolic modulators of activities of microsomal enzymes of cholesterol biosynthesis. Purification and characterization of a non-specific lipid-transfer protein.

Rat liver cytosol contains proteins which in the presence of low-molecular-weight metabolites modulate activities of membrane-bound enzymes of cholesterol biosynthesis. In the preceding paper, we identified Z-protein as a mediator in fatty acyl-CoA modulation of microsomal cholesterol synthetic and metabolizing enzymes. In this communication, we describe a second cytosolic protein which displays cholesterol-exchange activity. Purification of the protein to over 10000-fold and homogeneity has been achieved by gel permeation HPLC on an analytical Spherogel TSK-2000 SW column. Elution of both a single peak of active protein and one SDS-polyacrylamide gel electrophoresis species upon HPLC-purification suggests that homogeneous protein aggregates, with loss of exchange activity. In addition to stimulating microsomal enzymes of sterol synthesis, incubations of microsomes with cholesterol-containing liposomes and the protein consistently yields a 2-3-fold stimulation of microsomal acyl CoA: cholesterol acyltransferase activity. Under similar incubation conditions the protein enhances only slightly the extent of inhibition of microsomal hydroxymethylglutaryl-CoA reductase by liposomal cholesterol. The protein also catalyzes net transfer of cholesterol between membranes of different cholesterol content. The lipid-transfer protein and another cytosolic protein, also implicated in the regulation of sterol synthetic enzymes, appear identical. Regulation of activities of several membrane-bound enzymes of cholesterol metabolism in which the lipid-transfer protein and cytosolic Z-protein modulate uptake of lower-molecular-weight water-insoluble and water-soluble effectors, respectively, is discussed.

Cytosolic modulators of activities of microsomal enzymes of cholesterol biosynthesis. Effects of Acyl-CoA inhibition and cytosolic Z-protein.

Physiological concentrations of long-chain fatty acyl-CoAs have now been shown to inhibit microsomal methyl sterol oxidase. Acyl-CoA inhibition of hydroxymethylglutaryl-CoA reductase as well as methyl sterol oxidase can be either prevented or reversed by the addition of purified Z-protein (fatty acid-binding protein). Concomitantly, Z-protein addition decreases the extent of binding of radioactively labeled oleoyl-CoA to microsomal membranes. Free heme also inhibits hydroxymethylglutaryl-CoA reductase, and Z-protein reverses the extent of observed inhibition by binding heme analogous to the effect observed with acyl-CoAs. Similarly, Z-protein reverses substrate inhibition of acyl-CoA:cholesterol acyltransferase at high concentrations of acyl-CoA substrate. All these observations are consistent with the suggestion that, by binding acyl-CoAs and other enzyme effectors such as free heme, Z-protein modulates the effects of fluctuations of concentrations of major cellular metabolites. Furthermore, because the concentration of Z-protein is very low in rapidly growing hepatomas, such tumors may be very poorly buffered against the effects of acyl-CoAs, free fatty acids, heme and other effectors that may vary markedly by either altered metabolism or release of metabolites from necrotic tumor tissue.

Cholesterol biosynthesis from lanosterol: regulation and purification of rat hepatic sterol 14-reductase.

We have previously characterized the membrane-bound sterol 14-reductase (14-reductase) that catalyzes anaerobically NADPH-dependent reduction of the 14-double bond of delta 8,14-diene or delta 7,14-diene sterols that are sterol intermediates in cholesterol biosynthesis in mammals (Paik et al. (1984) J. Biol. Chem. 259, 13413-13423). To elucidate the regulatory mechanism as well as molecular characteristics of the 14-reductase, we extended our investigation on the consequences of alteration of the enzymic activity under various physiological conditions. The enzymic activity of rat hepatic sterol 14-reductase was induced more than 11-fold by feeding 5% cholestyramine plus 0.1% lovastatin (the CL-diet) for 7 days but was severely suppressed by feeding 5% cholesterol or 0.01% AY-9944 (an inhibitor of 14-reductase) for the same period. The increase or decrease in the 14-reductase activity also parallels the same change in the cholesterol synthetic rate in hepatocytes from rats that had been fed either the CL-diet or 0.01% AY-9944. In vitro inhibition studies revealed that AY-9944 acts as a competitive inhibitor of the 14-reductase (Ki = 0.26 microM). A diurnal variation was observed for the 14-reductase with peak activity near the middle of the dark cycle (10 p.m.), which was abolished by administration of cycloheximide. With induced enzyme conditions 14-reductase has been further purified with chromatographic procedures to near homogeneity. Purified 14-reductase appears to be a M(r) = 70,000 protein that is composed of two equally-sized subunits having a M(r) = 38,000. All properties of the purified 14-reductase suggest that the solubilized enzyme is the principal 14-reductase of microsomes. Taken together, our results provide the first evidence in support of a previously unknown regulatory role for the 14-reductase in the overall cholesterol synthetic pathway.

Generation of regulatory oxysterols: 26-hydroxylation of cholesterol by ovarian mitochondria.

De novo synthesis of cholesterol and low-density lipoprotein (LDL) receptor levels are suppressed in the presence of cholesterol. Recent evidence suggests that a cholesterol metabolite (possibly a hydroxysterol), not cholesterol per se, is the effector that inhibits transcription of genes encoding enzymes involved in sterol synthesis and LDL receptors. We found that 26-hydroxycholesterol inhibits human ovarian cell sterol synthesis, and that luteinized human granulosa cells contain 26-hydroxylase messenger RNA (mRNA). We proceeded to characterize the enzyme generating 26-hydroxycholesterol in the rat ovary. Mitochondria derived from ovaries of PMSG-human CG (hCG) primed immature rats (day 3 post-hCG) metabolized [3H] cholesterol into [3H]26-hydroxycholesterol in the presence of nicotinamide adenine dinucleotide phosphate and aminoglutethimide (100 micrograms/ml), added to inhibit metabolism of sterols by the cholesterol side-chain cleavage system. The identity of the product was confirmed by chromatography in several systems; recrystallization to constant specific activity and mass spectrometry. Negligible 26-hydroxylase activity was detected in other ovarian subcellular fractions. 26-Hydroxycholesterol formation progressed at a linear rate for up to 40 min and was linearly related to mitochondrial protein added to the incubation mixture. 26-Hydroxylase was markedly stimulated (5-fold) by calcium (0.2 mM). Maximal rates of 26-hydroxycholesterol formation observed were 1 pmol/min.mg protein. This activity is substantially lower than cholesterol side-chain cleavage measured in the absence of aminoglutethimide. Ketoconazole (1-100 microM) inhibited 26-hydroxylase in a dose-dependent manner. Pregnenolone (1-1000 microM) and progesterone (1-100 microM) inhibited 26-hydroxylase in a dose-dependent manner, with appreciable inhibitory effects in the 1-10 microM range. We suggest that 26-hydroxycholesterol is an intracrine regulator that controls cellular sterol metabolism. Formation of 26-hydroxcholesterol in ovarian cells may be regulated by steroidogenic activity in such a way as to ensure availability of steroid hormone precursors. When steroidogenesis is active, 26-hydroxylase is inhibited by products of the side-chain cleavage system, allowing increased de novo sterol synthesis and LDL uptake. With reduced steroidogenic activity and less demand for cholesterol, 26-hydroxylase is not blocked, permitting formation of 26-hydroxycholesterol with attendant reduction in sterol synthesis and LDL receptor gene expression.

Antiinflammatory 4,5-diarylpyrroles. 2. Activity as a function of cyclooxygenase-2 inhibition.

The antiinflammatory activity of a series of 2-substituted- and 2,3-disubstituted-4-(4-fluorophenyl)-5-[4-(methylsulfonyl)phenyl]-1H- pyrroles was previously shown by quantitative structure-activity relationship (QSAR) studies to be correlated with the molar refractivity and inductive field effect of the 2-substituent and the lipophilicity of the 3-substituent. The present study demonstrates that much of the antiinflammatory activity of these pyrroles could be correlated with the inhibition of the inducible isoform of cyclooxygenase (COX2). Additional QSAR studies have been used to identify the molecular parameters necessary for maximizing COX2 inhibition while simultaneously minimizing the inhibition of constitutively expressed cyclooxygenase-1. Such an effort should facilitate the discovery and development of selective COX inhibitors that should lead to safer nonsteroidal antiinflammatory drugs.

Discovery of macrocyclic hydroxamic acids containing biphenylmethyl derivatives at P1', a series of selective TNF-alpha converting enzyme inhibitors with potent cellular activity in the inhibition of TNF-alpha release.

SAR exploration at P1' using an anti-succinate-based macrocyclic hydroxamic acid as a template led to the identification of several bulky biphenylmethyl P1' derivatives which confer potent porcine TACE and anti-TNF-alpha cellular activities with high selectivity versus most of the MMPs screened. Our studies demonstrate for the first time that TACE has a larger S1' pocket in comparison to MMPs and that potent and selective TACE inhibitors can be achieved by incorporation of sterically bulky P1' residues.

Design, synthesis, and structure-activity relationships of macrocyclic hydroxamic acids that inhibit tumor necrosis factor alpha release in vitro and in vivo.

To search for TNF-alpha (tumor necrosis factor alpha) converting enzyme (TACE) inhibitors, we designed a new class of macrocyclic hydroxamic acids by linking the P1 and P2' residues of acyclic anti-succinate-based hydroxamic acids. A variety of residues including amide, carbamate, alkyl, sulfonamido, Boc-amino, and amino were found to be suitable P1-P2' linkers. With an N-methylamide at P3', the 13-16-membered macrocycles prepared exhibited low micromolar activities in the inhibition of TNF-alpha release from LPS-stimulated human whole blood. Further elaboration in the P3'-P4' area using the cyclophane and cyclic carbamate templates led to the identification of a number of potent analogues with IC(50) values of

Aggrecanase. A target for the design of inhibitors of cartilage degradation.

In arthritic diseases there is a gradual erosion of cartilage that leads to a loss of joint function. Aggrecan, which provides cartilage with its properties of compressibility and elasticity, is the first matrix component to undergo measurable loss in arthritis. This loss of aggrecan appears to be due to an increased rate of degradation, that can be attributed to proteolytic cleavage of the core protein within the interglobular domain (IGD). Two major sites of cleavage have been identified within the IGD. One, between the amino acids Asn341-Phe342, where the matrix metalloproteinases (MMPs) have been shown to clip; and the other, between Glu373-Ala374, which is attributed to a novel protease, "aggrecanase." We have generated aggrecanase in conditioned media from IL-1-stimulated bovine nasal cartilage and have used an enzymatic assay to evaluate this proteinase activity. In these studies we follow the generation of aggrecanase and MMPs in response to IL-1 in this system and examine the contribution of these enzymes in aggrecan degredation. Our data suggest that aggrecanase is a key enzyme in cartilage aggrecan degradation that represents a novel target for cartilage protection therapy in arthritis.

Multicompartmental distribution of ribonuclease II in mouse liver.

Thirty percent of RNase II (EC 3.1.27.5) is present in the cytosol of mouse liver where it exists in an inactive complex with a protein inhibitor. The remaining 70% of RNase II is active, soluble enzyme unassociated with inhibitor and is distributed in a ratio of 1.3 to 1 between the lumen of reticular elements and the interior of heavy particles. Although heavy particle RNase II resembles acid hydrolases in centrifugal behavior, in other tests including density shift experiments the resemblance is incomplete. In experiments employing lysis induced by L-amino acid methyl esters, RNase II activity is much more latent than the activity of the lysosomal marker, acid RNase. It is postulated that the heavy particle component of RNase II is contained in a secretory vesicle rather than in classic lysosomes.

Comparative effects of the azole-based fungicide flusilazole on yeast and mammalian lanosterol 14 alpha-methyl demethylase.

Flusilazole inhibits the 14 alpha-demethylation of [24,25-3H-2]dihydrolanosterol by yeast and rat liver cell-free preparations. The 50% inhibitory concentration of demethylation shows that the yeast system is 100 times more sensitive than the rat system. Purified rat liver P-45014DM is about 10 times more sensitive to flusilazole than crude rat liver microsomes. Binding constants in microsomes indicate that yeast preparations (Kd, 21 nM) bind flusilazole with a higher affinity than rat liver (Kd, 496 nM). Purified rat liver P-45014DM (Kd, 45 nM) binds flusilazole with an affinity similar to that of the yeast enzyme. The P-450-dependent cholesterol 7 alpha-hydroxylase in rat liver microsomes is more sensitive to flusilazole than P-45014DM. The results indicate that selectivity of azole antimycotics is not due to inherent sensitivity differences between fungal and mammalian 14 alpha-demethylase; rather, in crude enzyme systems, a protective effect is afforded by other susceptible isozymes present in mammalian systems.

Identification of lanosterol 14 alpha-methyl demethylase in human tissues.

Lanosterol 14 alpha-methyl demethylase was investigated in human tissues using a radio-HPLC assay to detect the 4,4-dimethyl-5 alpha-cholesta-8, 14-dien-3 beta-ol (diene) metabolite. The sequence of events leading to the demethylated product in human liver microsomes involves the conversion of the diol to the aldehyde followed by diene formation. Enzyme activity displayed a greater than 10 fold variation among the 9 liver samples studied. Kinetic parameters were determined and shown to differ between two separate liver samples. Addition of inhibitors of yeast lanosterol 14 alpha demethylase, ketoconazole and miconazole, resulted in extensive inhibition of formation of the demethylated metabolite. The enzyme, detected in microsomes isolated from human kidney and lymphocytes, also catalyzed the conversion of dihydrolanosterol to oxylanosterol intermediates and the diene. The presence of this enzyme in microsomes from various human tissues suggests that it may play a role in cellular regulation of cholesterol synthesis.

Affinity chromatography of microsomal enzymes on immobilized detergent-solubilized cytochrome b5.

Highly selective chromatography of microsomal enzymes has been carried out on columns of immobilized cytochrome b5 that was obtained by detergent solubilization (d-b5) of the complete amphipathic molecule. Several partially purified isozymes of cytochrome P-450 are resolved on d-b5 columns, and one high-affinity isozyme has been readily purified to homogeneity. Chromatographic selectivity and correlation of elution order of isozymes of cytochrome P-450 with direct spectral measurements of affinity constants suggests affinity chromatography on d-b5 columns. Substantial one-step enrichments of NADH-cytochrome-b5 reductase and an unstable cytochrome b5-dependent oxidase of cholesterol synthesis, 4-methyl sterol oxidase, have been obtained on d-b5 columns which further supports this conclusion. Comparison of chromatographic behavior on columns of immobilized cytochrome b5 that was obtained by trypsin solubilization (t-b5) with d-b5 columns shows marked differences which must be attributed to the absence of the hydrophobic domain of the t-b5 molecule. NADH-cytochrome-b5 reductase and the high affinity isozyme of cytochrome P-450 purified by d-b5 affinity chromatography are poorly retained on t-b5 columns. A different cytochrome P-450 isozyme with lower affinity for cytochrome b5 is only retained on d-b5 columns. Cytochrome-P-450 reductase is not retained on either column. Because affinity chromatography is suggested on d-b5 columns, the procedure may be generally applicable for predicting protein-protein interactions of microsomal electron transport components that either donate electrons to, or receive electrons from, cytochrome b5. In addition, the procedure should have considerable utilitarian application for enzyme enrichment.

Mechanistic studies of lanosterol C-32 demethylation. Conditions which promote oxysterol intermediate accumulation during the demethylation process.

Conditions have been established which promote the accumulation of the dihydrolanosterol C-32 demethylation intermediates lanost-8-en-3 beta,32-diol and 3 beta-hydroxylanost-8-en-32-aldehyde with intact hepatic microsomes. Accumulation of dihydrolanosterol-derived oxysterols occurs with a variety of assay manipulations which include short incubation times, limiting enzyme amounts, high pH, and increasing substrate concentration. In addition, competitive inhibition of dihydrolanosterol demethylation by lanosterol, or the reciprocal inhibition of lanosterol demethylation by dihydrolanosterol, leads to oxysterol accumulation at the expense of demethylated end product. Similarly, the nonsteroidal demethylase inhibitors miconazole and ketoconazole promote oxysterol accumulation in a concentration-dependent manner. Finally, cholesterol loading of isolated microsomes results in changes in the measured kinetic constants, Km and Vmax, and results in enhanced oxysterol accumulation above that seen in control microsomal preparations. The major oxysterol intermediate accumulated under all the conditions described above is the C-32 aldehyde in an approximate 3:1 ratio to the C-32 alcohol. These data support the conclusion that a single enzyme species is responsible for all three oxidations of the C-32 demethylation sequence. In addition, intermediates which do not routinely accumulate during demethylation are freely diffusible from the enzyme when appropriate conditions are established to prevent their further metabolism.

Sterol-mediated suppression of HMG-CoA reductase mRNA levels in cultured cells requires protein synthesis.

The mRNA levels for HMG-CoA reductase were measured in Chinese hamster lung Dede cells or ovary CHO cells by Northern blot analysis. It was observed that 25-hydroxycholesterol decreased the level of reductase mRNA by 40 to 70% in a 6 hour treatment. Inclusion of cycloheximide in the culture prevented the decrease observed with 25-hydroxycholesterol alone. Pretreatment of cells with 25-hydroxycholesterol for 6 hours prior to cycloheximide addition reveals that the protein synthesis inhibitor can return the suppressed levels of reductase mRNA back to control levels. Thus, protein synthesis is required for 25-hydroxycholesterol dependent suppression of HMG-CoA reductase mRNA.

Microsomal enzymes of cholesterol biosynthesis from lanosterol. Purification and characterization of delta 7-sterol 5-desaturase of rat liver microsomes.

Microsomal delta 7-sterol 5-desaturase of cholesterol biosynthesis is a multienzyme system which catalyzes the introduction of the delta 5-bond into delta 7-cholestenol to form 7-dehydrocholesterol. The detergent-solubilized 5-desaturase has been purified more than 70-fold and resolved from electron carriers and other rat liver microsomal enzymes of sterol biosynthesis by chromatography on DEAE-Sephacel, CM-Sepharose, and immobilized cytochrome b5; the 5-desaturase had not been fully resolved from cytochrom b5 reductase in earlier work. A functional electron transport system for the 5-desaturase has been reconstituted by combining the purified 5-desaturase and electron carriers with egg phosphatidylcholine liposomes. Optimizations of conditions for reconstitution have been obtained; both cytochrome b5 and NADH-cytochrome b5 reductase serve as electron carriers. A pyridine nucleotide-dependent flavoprotein is required and the requirement can be satisfied with either purified cytochrome b5 reductase or cytochrome P-450 reductase. Cyanide and iron-chelators strikingly inhibit the 5-desaturase activity, thus suggesting that 5-desaturase is a metalloenzyme as are other well-characterized cytochrome b5-dependent oxidases. 5-Desaturase is resolved from 4-methyl sterol oxidase activity of cholesterol biosynthesis by chromatography on the immobilized cytochrome b5. This resolution of the two oxidases not only indicates that introduction of the delta 5-bond and oxidation of 4 alpha-methyl groups are catalyzed by different terminal oxidases, but resolution affords enzymes of sufficient purity to carry out reconstitution experiments. A novel assay based on substrate-dependent increments of oxidation of alpha-NADH has been developed for measurement of 5-desaturase activity. Measurement of stoichiometry of 5-desaturase demonstrates that for each equivalent of cis-desaturation of delta 7-cholestenol, 1 eq of NADH is consumed. Along with strict dependence upon oxygen, this observation confirms, as suggested by previous workers, that the 5-desaturation is catalyzed by a mixed function oxidase rather than a dehydrogenase.