A potential role for aldose reductase in steroid metabolism.
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20-Hydroxysteroid Dehydrogenases↗
Biomedical subjects
J M Petrash
Publications and source records attributed to J M Petrash.
Specifically targeted modification of human aldose reductase by physiological disulfides.
Aldose reductase is inactivated by physiological disulfides such as GSSG and cystine. To study the mechanism of disulfide-induced enzyme inactivation, we examined the rate and extent of enzyme inactivation using wild-type human aldose reductase and mutants containing cysteine-to-serine substitutions at positions 80 (C80S), 298 (C298S), and 303 (C303S). The wild-type, C80S, and C303S enzymes lost >80% activity following incubation with GSSG, whereas the C298S mutant was not affected. Loss of activity correlated with enzyme thiolation. The binary enzyme-NADP+ complex was less susceptible to enzyme thiolation than the apoenzyme. These results suggest that thiolation of human aldose reductase occurs predominantly at Cys-298. Energy minimization of a hypothetical enzyme complex modified by glutathione at Cys-298 revealed that the glycyl carboxylate of glutathione may participate in a charged interaction with His-110 in a manner strikingly similar to that involving the carboxylate group of the potent aldose reductase inhibitor Zopolrestat. In contrast to what was observed with GSSG and cystine, cystamine inactivated the wild-type enzyme as well as all three cysteine mutants. This suggests that cystamine-induced inactivation of aldose reductase does not involve modification of cysteines exclusively at position 80, 298, or 303.
Aldehyde Reductase↗
Cloning, expression, and chaperone-like activity of human alphaA-crystallin.
One of the major protein components of the ocular lens, alpha-crystallin, is composed of alphaA and alphaB chain subunits that have structural homology to the family of mammalian small heat shock proteins. Like other small heat shock proteins, alpha-crystallin subunits associate to form large oligomeric aggregates that express chaperone-like activity, as defined by the ability to suppress nonspecific aggregation of proteins destabilized by treatment with a variety of denaturants including heat, UV irradiation, and chemical modification. It has been proposed that age-related loss of sequences at the C terminus of the alphaA chain subunit may be a factor in the pathogenesis of cataract due to diminished capacity of the truncated crystallin to protect against nonspecific aggregation of lens proteins. To evaluate the functional consequences of alpha-crystallin modification, two mutant forms of alphaA subunits were prepared by site-directed mutagenesis. Like wild type (WT), aggregates of approximately 540 kDa were formed from a tryptophan-free alphaA mutant (W9F). When added in stoichiometric amounts, both WT and W9F subunits completely suppressed the heat-induced aggregation of aldose reductase. In contrast, subunits encoded by a truncation mutant in which the C-terminal 17 residues were deleted (R157STOP), despite having spectroscopic properties similar to WT, formed much larger aggregates with a marked reduction in chaperone-like activity. Similar results were observed when the chaperone-like activity was assessed through inhibition of gamma-crystallin aggregation induced by singlet oxygen. These results demonstrate that the structurally conservative substitution of Phe for Trp-9 has a negligible effect on the functional interaction of alphaA subunits, and that deletion of C-terminal sequences from the alphaA subunit results in substantial loss of chaperone-like activity, despite overall preservation of secondary structure.
Aldehyde Reductase↗
Kinetic and spectroscopic evidence for active site inhibition of human aldose reductase.
Aldose reductase is an NADPH-dependent oxidoreductase that catalyzes the reduction of a variety of aldehydes and carbonyls, including monosaccharides. Intense interest in the discovery and characterization of inhibitors has developed since the action of this enzyme has been linked to the pathogenesis of some diabetic complications. Since past studies indicated that most inhibitors act noncompetitively or uncompetitively versus substrate in the direction of aldehyde reduction, it was assumed that they bind at one or more sites distinct from the active site. However, the crystal structure of aldose reductase complexed with inhibitor [Wilson et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 9847-9851] unambiguously revealed the inhibitor bound at the active site. The present study was undertaken to address this apparent discrepancy. Using a fluorometric assay, it was determined that zopolrestat, an acetic acid-type inhibitor, bound to aldose reductase complexed with either NADPH or NADP+. In contrast, the spirohydantoin-type inhibitor sorbinil demonstrated preferential binding to the binary enzyme.NADPH complex. Prior incubation of the enzyme.NADPH complex with zopolrestat prevented subsequent sorbinil binding. These results, together with the published structure of the ternary enzyme.NADPH.zopolrestat complex, are consistent with the conclusion that both sorbinil and zopolrestat bind at the active site. We propose that mixed inhibition patterns previously observed with sorbinil are due to inhibitor binding to both structural isomers of the enzyme.NADPH complex. Similar patterns for inhibition by zopolrestat are due to tight binding of the inhibitor. Substrate inhibition in the direction of aldehyde reduction occurs as a result of substrate binding to the enzyme.NADP+ complex.
Aldehyde Reductase↗
Conformational properties of substrate proteins bound to a molecular chaperone alpha-crystallin.
alpha-Crystallin, the major protein of the ocular lens, acts as a molecular chaperone by suppressing the nonspecific aggregation of damaged proteins. To investigate the mechanism of the interaction between alpha-crystallin and substrate proteins, we prepared a tryptophan-free mutant of human alpha A-crystallin and assessed the conformation of thermally destabilized proteins captured by this chaperone using fluorescence spectroscopy. The fluorescence emission characteristics of bound substrates (rhodanese and gamma-crystallin) and the results of fluorescence quenching experiments indicate that the proteins captured by alpha-crystallin are characterized by a very low degree of unfolding. In particular, the structure of rhodanese bound to alpha A-crystallin appears to be considerably more native-like compared to that of the enzyme bound to the chaperonin GroEL. We postulate that alpha-crystallin (and likely other small heat shock proteins) recognize preferentially the aggregation-prone conformers that occur very early on the denaturation pathway. With its ability to capture and stabilize these early non-native structures, alpha-crystallin appears to be uniquely well suited to chaperone the transparency properties of the ocular lens.
Base Sequence↗
1.7 A structure of FR-1, a fibroblast growth factor-induced member of the aldo-keto reductase family, complexed with coenzyme and inhibitor.
Murine FR-1 is a protein that is induced by fibroblast growth factor-1 and, therefore, may play a role in the regulation of the cell cycle. Sequence comparison indicates that it is a member of the NADPH-dependent aldo-keto reductase family. It bears 70% identity to human aldose reductase, an enzyme implicated in diabetic complications and a target for drug design. We have determined the 1.7 A resolution structure of the FR-1 in a ternary complex with NADPH and zopolrestat, a potent aldose reductase inhibitor. FR-1 folds into a (beta/alpha)8 barrel with an active site characterized by a preponderance of hydrophobic residues residing in a deep oblong cavity at the C-terminal end of the beta-barrel. The nicotinamide moiety of the coenzyme sits in the base of the cavity. Zopolrestat occupies the active site cavity and makes numerous contacts with several hydrophobic residues. The FR-1 ternary complex structure indicates that it uses the same general catalytic mechanism as aldose reductase and other members of the family whose structures have been determined. The protein exhibits reductase activity with DL-glyceraldehyde as a substrate and is strongly inhibited by zopolrestat. When compared with the structure of a similar ternary complex of aldose reductase, the binding site retains many of the interactions with the coenzyme and inhibitor from the conserved residues. Some differences in sequence, however, create a larger binding site that contains six more water molecules than in the aldose reductase ternary complex structure.(ABSTRACT TRUNCATED AT 250 WORDS)
Alcohol Oxidoreductases↗
Kinetic characteristics of ZENECA ZD5522, a potent inhibitor of human and bovine lens aldose reductase.
Aldose reductase (aldehyde reductase 2) catalyses the conversion of glucose to sorbitol, and methylglyoxal to acetol. Treatment with aldose reductase inhibitors (ARIs) is a potential approach to decrease the development of diabetic complications. The sulphonylnitromethanes are a recently discovered class of aldose reductase inhibitors, first exemplified by ICI215918. We now describe enzyme kinetic characterization of a second sulphonylnitromethane, 3',5'-dimethyl-4'-nitromethylsulphonyl-2-(2-tolyl)acetanilide (ZD5522), which is at least 10-fold more potent against bovine lens aldose reductase in vitro and which also has a greater efficacy for reduction of rat nerve sorbitol levels in vivo (ED95 = 2.8 mg kg-1 for ZD5522 and 20 mg kg-1 for ICI 215918). ZD5522 follows pure noncompetitive kinetics against bovine lens aldose reductase when either glucose or methylglyoxal is varied (K(is) = K(ii) = 7.2 and 4.3 nM, respectively). This contrasts with ICI 215918 which is an uncompetitive inhibitor (K(ii) = 100 nM) of bovine lens aldose reductase when glucose is varied. Against human recombinant aldose reductase, ZD5522 displays mixed noncompetitive kinetics with respect to both substrates (K(is) = 41 nM, K(ii) = 8 nM with glucose and K(is) = 52 nM, K(ii) = 3.8 nM with methylglyoxal). This is the first report of the effects of a sulphonylnitromethane on either human aldose reductase or utilization of methylglyoxal. These results are discussed with reference to a Di Iso Ordered Bi Bi mechanism for aldose reductase, where the inhibitors compete with binding of both the aldehyde substrate and alcohol product. This model may explain why aldose reductase inhibitors follow noncompetitive or uncompetitive kinetics with respect to aldehyde substrates, and X-ray crystallography paradoxically locates an ARI within the substrate binding site. Aldehyde reductase (aldehyde reductase 1) is closely related to aldose reductase. Inhibition of bovine kidney aldehyde reductase by ZD5522 follows uncompetitive kinetics with respect to glucuronate (K(ii) = 39 nM), indicating a selectivity greater than 5-fold for bovine aldose reductase relative to aldehyde reductase.
Acetanilides↗
Expression of recombinant bovine gamma B-, gamma C- and gamma D-crystallins and correlation with native proteins.
Despite the use of bovine gamma-crystallins in numerous biophysical and chemical studies, characterization of these proteins at the molecular level is incomplete. Bovine lenses have at least six gamma-crystallin protein fractions currently assigned as gamma s/I, gamma A/IVb, gamma B/II, gamma C/IIIb, gamma D/IIIa and gamma E/IVa. A lack of primary sequence data for corresponding gamma-crystallin genes and proteins, however, has made these assignments tenuous. To clarify these assignments, we have over-expressed recombinant bovine gamma-crystallin proteins in Escherichia coli using complementary DNAs corresponding to gamma B-, gamma C-, and gamma D-crystallin genes. The recombinant crystallins were characterized by chromatographic and spectroscopic comparisons with native bovine crystallin fractions gamma II, gamma IIIa and gamma IIIb. The elution of recombinant gamma B and native gamma II proteins was identical on cation-exchange chromatography as expected; however, recombinant gamma C coeluted with gamma IIIa and recombinant gamma D co-eluted with gamma IIIb. Sequential Edman degradation through the first 29 residues of the native gamma IIIa and gamma IIIb polypeptides confirmed the colinearity of their sequences with those predicted from the gamma C- and gamma D-crystallin genes, respectively. Absorption and UV circular dichroism (CD) spectra of the recombinant proteins were almost identical to those of their native counterparts, indicating that the secondary and tertiary structures of the recombinant proteins were characteristic for gamma-crystallins. Based on these data the bovine gamma-crystallins proteins and genes are correlated as follows: II/gamma B, IIIa/gamma C and IIIb/gamma D. The assignment of gamma IIIb (previously characterized as having a low critical temperature for phase separation) with gamma D rather than gamma C proves an exception to the hypothesis that the gamma ABC-crystallin group is more resistant to phase separation than the gamma DEF group. These corrected assignments should provide a more substantial base for investigations of residues responsible for phase separation and other biophysical characteristics. Additionally, expression of recombinant gamma-crystallins with structures similar to native proteins may prove to be useful in probing specific structure-function relationships of the gamma-crystallins.
Amino Acid Sequence↗
Aldose reductase catalysis and crystallography. Insights from recent advances in enzyme structure and function.
Enhanced metabolism of glucose via the polyol pathway may play an important role in the pathogenesis of diabetic retinopathy, neuropathy, and nephropathy. Aldose reductase catalyzes the NADPH-dependent conversion of glucose to sorbitol, the first step in the polyol pathway. Interruption of the polyol pathway by inhibition of aldose reductase holds considerable promise as a therapeutic measure to prevent or delay the onset and severity of these late complications of diabetes. Dramatic advances in our understanding of the molecular biology, enzymology, and three-dimensional structure of aldose reductase have occurred in recent years, providing new and challenging insights into the enzyme's catalytic mechanism. Recent developments in structure determination of aldose reductase and the implications for evaluation and development of aldose reductase inhibitors are summarized.
Aldehyde Reductase↗
Probing the active site of human aldose reductase. Site-directed mutagenesis of Asp-43, Tyr-48, Lys-77, and His-110.
Structural models of human aldose reductase complexed with NADPH have revealed the apposition of C4 of the nicotinamide ring with tyrosine 48 and histidine 110, suggesting that either of these residues could function as the proton donor in the reaction mechanism. Tyrosine 48 is also part of a hydrogen-bonding network that includes lysine 77 and aspartate 43. In order to study the potential catalytic roles of these 4 residues, we evaluated the kinetic properties of mutants containing structurally conservative replacements at these sites. Enzymatic activity was undetectable when Tyr-48 was mutated to phenylalanine (Y48F) although affinity for NADPH was unchanged. In contrast, a mutant containing asparagine substituted for His-110 (H110N) was characterized by an almost 80,000-fold increase in Km, but only about a 14-fold reduction in kcat measured with D-glyceraldehyde. Modest changes in catalytic properties were observed in the mutant containing aspartate 43 substituted with asparagine (D43N): Km for aldehyde substrates was elevated up to 17-fold, and kcat decreased less than 16-fold. However, the Kd(NADP) values for D43N were about 5 times higher than those for wild type. Mutant enzyme containing methionine substituted for lysine 77 (K77M) was up to 1,460-fold less active than the wild type. These results are consistent with Tyr-48 acting as the acid-base catalyst in human aldose reductase and confirm the importance of Asp-43, Lys-77, and His-110 to the structure and function of the active site.
Aldehyde Reductase↗
Refined 1.8 A structure of human aldose reductase complexed with the potent inhibitor zopolrestat.
As the action of aldose reductase (EC 1.1.1.21) is believed to be linked to the pathogenesis of diabetic complications affecting the nervous, renal, and visual systems, the development of therapeutic agents has attracted intense effort. We report the refined 1.8 A x-ray structure of the human holoenzyme complexed with zopolrestat, one of the most potent noncompetitive inhibitors. The zopolrestat fits snugly in the hydrophobic active site pocket and induces a hinge-flap motion of two peptide segments that closes the pocket. Excellent complementarity and affinity are achieved on inhibitor binding by the formation of 110 contacts (< or = 4 A) with 15 residues (10 hydrophobic), 13 with the NADPH coenzyme and 9 with four water molecules. The structure is key to understanding the mode of action of this class of inhibitors and for rational design of better therapeutics.
Aldehyde Reductase↗
Kinetic alteration of human aldose reductase by mutagenesis of cysteine residues.
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Aldehyde Reductase↗
Prevention of diabetic vascular dysfunction by guanidines. Inhibition of nitric oxide synthase versus advanced glycation end-product formation.
This study was undertaken to compare the ability of two guanidine compounds (aminoguanidine and methylguanidine), with different in vitro effects on NO synthase activity and AGE formation, to inhibit diabetic vascular dysfunction developing early after the onset of diabetes. In rats with STZ-induced diabetes of 5-wk duration, regional vascular [125I]albumin permeation was increased about two- to threefold in ocular tissues, sciatic nerve, and aorta; in general, both guanidine compounds normalized albumin permeation in diabetic rats without affecting it in controls. Methylguanidine was only approximately 7% as effective as aminoguanidine as an inhibitor of AGE formation from L-lysine and G6P; both compounds were poor inhibitors of AR. Methylguanidine was approximately 1-5% as potent as aminoguanidine and L-NMMA as an inhibitor of the cytokine- and endotoxin-inducible isoform of NO synthase. In contrast, the potency of methylguanidine as an inhibitor of the constitutive isoform of NO synthase was comparable to that of aminoguanidine, and both guanidine compounds were much less effective than L-NMMA. These observations suggest a role for a relative or absolute increase in NO production in the pathogenesis of early diabetic vascular dysfunction and raise the possibility that inhibition of diabetic vascular functional changes by aminoguanidine may reflect inhibition of NO synthase activity rather than, or in addition to, prevention of AGE formation.
Aldehyde Reductase↗
The crystal structure of the aldose reductase.NADPH binary complex.
Aldose reductase is an NADPH-dependent oxidoreductase that catalyzes the reduction of a broad range of aldehydes, including glucose. Since aldose reductase has been strongly implicated in the development of the chronic complications of diabetes mellitus, much effort has been devoted to understanding the structure and mechanism of this enzyme, and many aldose reductase inhibitors have been developed as potential drugs for the treatment of these complications. We describe here the 2.75 A crystal structure of recombinant human aldose reductase (Cys-298 to Ser mutant) complexed with NADPH. This mutant displays unusual kinetic behavior characterized by high Km/high Vmax substrate kinetics and reduced sensitivity to certain aldose reductase inhibitors. The crystal structure revealed that the enzyme is a beta/alpha-barrel with the coenzyme-binding domain located at the carboxyl-terminal end of the parallel strands of the barrel. The enzyme undergoes a large conformational change upon binding NADPH which involves the reorientation of loop 7 to a position which appears to lock the coenzyme into place. NADPH is bound to aldose reductase in an unusual manner, more similar to FAD- rather than NAD(P)-dependent oxidoreductases. No disulfide bridges were observed in the crystal structure.
Aldehyde Reductase↗
Involvement of cysteine residues in catalysis and inhibition of human aldose reductase. Site-directed mutagenesis of Cys-80, -298, and -303.
In order to study the potential role of cysteinyl residues in catalysis and inhibition of human aldose reductase, mutants containing cysteine to serine substitution at positions 80 (ALR2:C80S), 298 (ALR2:C298S), and 303 (ALR2:C303S) were constructed. Mutation of Cys298 resulted in the most profound changes, as ALR2:C298S displayed 4- to 5-fold elevation in K'm(NADPH), K'm(DL-glyceraldehyde), and kcat(DL-glyceraldehyde) relative to wild type aldose reductase as well as a 10-fold higher Ki for the aldose reductase inhibitor sorbinil. Wild type and mutant reductases were equally sensitive to tolrestat, a structurally different reductase inhibitor. Carboxymethylation of the wild type enzyme or the C80S and C303S mutants led to a modest decrease in kcat as well as an increase in K'm(DL-glyceraldehyde) and Ki(sorbinil). These parameters were not significantly changed when ALR2:C298S was subjected to carboxymethylation. Lithium sulfate caused activation of ALR2:WT, C80S, and C303S but did not significantly affect the activity of ALR2:C298S. The differential sensitivity of wild type and mutant reductases to inhibition by sorbinil and tolrestat, before and after carboxymethylation, indicates that these inhibitors bind at different sites. These results suggest that Cys-298 is present near the active site and constitutes a regulatory group which controls the catalytic activity and inhibitor sensitivity of the enzyme.
Aldehyde Reductase↗
Effects of osmotic stress and hyperglycemia on aldose reductase gene expression in human renal proximal tubule cells.
Sorbitol levels in proximal tubule cells cultured for 96 h in the presence of 16.5 mM and 27.5 mM glucose were significantly elevated when compared to cells cultured in 5.5 mM glucose. No changes in the levels of aldose reductase activity, mRNA and immunoreactivity were observed in cells cultured for up to 168 h in media containing either 5.5 mM or 27.5 mM glucose. In contrast, cells cultured in the presence of hypertonic media (600 mosmol/kg) containing either 5.5 mM or 27.5 mM glucose contained markedly elevated aldose reductase activity, mRNA and immunoreactivity. These results demonstrate that exposure of human renal proximal tubule cells to elevated glucose for up to 168 h does not result in enhancement of aldose reductase gene expression at transcriptional, translational or post-translational levels. However, exposure to a hyperosmotic milieu causes dramatic induction of aldose reductase gene expression.
Aldehyde Reductase↗
Applications of molecular biological techniques to the understanding of visual system disorders.
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Base Sequence↗
Detection of herpes simplex virus type 1 latency-associated transcripts in corneal cells of inbred mice by in situ hybridization.
We have previously recovered herpes simplex virus type 1 (HSV) from the corneas of latently infected mice by cultivation in vitro. It could be argued, however, that these data do not definitively distinguish between persistent and latent corneal infection. We have now used RNA hybridization in situ to resolve this question by determining the expression of HSV genes in the corneas of BALB/c mice during latent infection. Two to four months after topical corneal inoculation with HSV, when no active ocular disease or infectious virus was present, corneas were removed and digested with collagenase. Dissociated cells pooled from two corneas were hybridized with 3H- or 35S-labeled 2.6-kb single-stranded RNA probes to detect sense and antisense ICP-0 transcripts. Twenty-five percent of the pools hybridized with the probe for antisense ICP-0 (latency-associated transcript, LAT), while only 3% hybridized with the probe for ICP-0 (p less than 0.03). Of the cells in positive pools, 0.6-7.0% showed a positive hybridization signal for LAT. No infectious virus was found by culture of supernatants from the probed pools or control latently infected corneas. These data provide further evidence that HSV can establish a true latent infection in the mouse cornea.
Animals↗