Digitalis and digitalislike compounds down-regulate gene expression of the intracellular signaling protein 14-3-3 in rat lens.

Na+,K+-ATPase activity in the epithelial layer is fundamental to the maintenance of ionic concentration gradients and transparency of the lens. Recently we have identified endogenous digitalislike compounds (DLC), 19-norbufalin and its peptide derivatives, in human cataractous lenses (Lichtstein et al. Eur J Biochem 216: 261-268, 1993). Lenses were treated with 10 nM ouabain, bufalin or 19-norbufalin derivative for 24 h and were compared to control lenses. Differential display analysis revealed that one of the down-regulated genes was 14-3-3 theta. Down-regulation was confirmed by Northern blot and by RT-PCR analysis. RT-PCR of additional 14-3-3 isoforms revealed that the eta and gamma isoforms of 14-3-3 are also down-regulated by ouabain, bufalin and 19-norbufalin derivative, whereas the zeta isoform is down-regulated only by bufalin. These results demonstrate that one of the consequences of Na+,K+-ATPase inhibition by exogenous or endogenous inhibitors is the down-regulation of mRNA transcripts encoding several isoforms of 14-3-3. Since the 14-3-3 proteins are multifunctional regulatory proteins, the reduction in the abundance of various isoforms will have profound effects on cell function. Furthermore, These results, together with the demonstration of digitalislike compounds in the normal lens, and their increased level in human cataractous lenses, strongly suggests their involvement in the molecular mechanisms responsible for cataract formation.

Na+, K+-ATPase inhibitors down-regulate gene expression of the intracellular signaling protein 14-3-3 in rat lens.

To identify genes that are differentially expressed by Na+, K+-ATPase inhibitors, we used the differential display technique to compare mRNA expression patterns in rat lens. Lenses were treated with 10 microM ouabain, bufalin, or 19-norbufalin derivative for 24 h and were compared with control lenses. Differential display analysis revealed that one of the down-regulated genes was 14-3-3 theta. Down-regulation was confirmed by Northern blot and by reverse transcription-polymerase chain reaction analysis. Reverse transcription-polymerase chain reaction of additional 14-3-3 isoforms revealed that the eta and gamma isoforms of 14-3-3 are also down-regulated by ouabain, bufalin, and 19-norbufalin derivative, whereas the zeta isoform is down-regulated only by bufalin. Down-regulation of the 14-3-3 isoforms occurred without a significant change in gamma-crystallin gene expression. These results demonstrate that one of the consequences of Na+, K+-ATPase inhibition by exogenous or endogenous inhibitors is the down-regulation of mRNA transcripts encoding several isoforms of 14-3-3. Because the 14-3-3 proteins are multifunctional regulatory proteins, the reduction in the abundance of various isoforms will have profound effects on cell function.

Probing the inhibitor-binding site of aldose reductase with site-directed mutagenesis.

Aldose reductase (AR) has been implicated in the etiology of the secondary complications of diabetes, and enzyme inhibitors have been proposed as therapeutic agents. While effectively preventing the development of diabetic complications in animals, results from clinical studies of AR inhibitors have been disappointing, possibly due to poor potency in man. To assist in the design of more potent and specific inhibitors, crystallographic studies have attempted to identify enzyme-inhibitor interactions. Resolution of crystal complexes has suggested that the inhibitors bind to the enzyme active site and are held in place through hydrogen bonding and van der Waals interactions formed within two hydrophobic pockets. To confirm and extend these findings we quantified inhibitor activity with single, site-directed, mutant, human AR enzymes in which the apolar active-site residues tryptophan 20, -79, -111 and phenylalanine 115 were replaced with alanine or tyrosine, decreasing the potential for van der Waals interactions. Consistent with molecular models, the inhibitory activity of Tolrestat, Sorbinil and Zopolrestat decreased 800-2000-fold when tested with the mutant enzyme in which Trp20 was replaced with alanine. Further, alanine substitution for Trp111 decreased Zopolrestat's activity 400-fold, while mutations to Trp79 and Phe115 had little effect on the activity of any of the inhibitors. The alanine mutation at Trp111 had no effect on Tolrestat's activity but decreased the activity of Sorbinil by about 1000-fold. These latter effects were unanticipated based on the number of non-bonded interactions between the inhibitors, Tolrestat and Sorbinil, and Trp20 and Trp111 that have been identified in the crystal structures. In spite of these unexpected findings, our results are consistent with the hypothesis that AR inhibitors occupy the enzyme active site and that hydrophobic interactions between the enzyme and inhibitor contribute to inhibitor binding stability.

Characterization of the mouse aldose reductase gene and promoter in a lens epithelial cell line.

PURPOSE: To clone and characterize the mouse aldose reductase (AR) gene and evaluate the functional promoter under basal and hypertonic conditions in mouse lens epithelial cells. METHODS: The mouse AR gene structure was determined by DNA sequencing, and its chromosomal localization was determined by fluorescent in situ hybridization. A luciferase reporter gene was utilized to assess promoter activities of mouse, rat, and human AR deletion constructs as well as mouse site-directed mutants containing specific deletions of an aldose reductase enhancer element (AEE) or a tonicity response element (TonE). Electrophoretic mobility shift assays were performed to evaluate binding of trans-acting factors to mouse AEE and TonE. RESULTS: The mouse AR gene (14.2 Kb) is located on chromosome 6. The basal AR promoter activity was greatest for the rat followed by mouse and human. All 3 species demonstrated increased promoter activity under hypertonic conditions. Deletion of TonE decreased mouse AR basal activity 2.5-fold and substantially reduced the osmotic response. Deletion of AEE had only a slight effect on AR promoter activity. Nevertheless, AEE strongly bound multiple trans-acting factors under nonstressed and stressed conditions, while weaker binding was evident for TonE. CONCLUSIONS: Species-specific differences in AR promoter activities suggest the presence of unique regulatory cis-acting elements. The effects of AEE or TonE on AR transcription appear to involve complex transcriptional regulatory mechanisms.

Studies on the inhibitor-binding site of porcine aldehyde reductase: crystal structure of the holoenzyme-inhibitor ternary complex.

Aldehyde reductase is an enzyme capable of metabolizing a wide variety of aldehydes to their corresponding alcohols. The tertiary structures of aldehyde reductase and aldose reductase are similar and consist of an alpha/beta-barrel with the active site located at the carboxy terminus of the strands of the barrel. We have determined the X-ray crystal structure of porcine aldehyde reductase holoenzyme in complex with an aldose reductase inhibitor, tolrestat, at 2.4 A resolution to obtain a picture of the binding conformation of inhibitors to aldehyde reductase. Tolrestat binds in the active site pocket of aldehyde reductase and interacts through van der Waals contacts with Arg 312 and Asp 313. The carboxylate group of tolrestat is within hydrogen bonding distance with His 113 and Trp 114. Mutation of Arg 312 to alanine in porcine aldehyde reductase alters the potency of inhibition of the enzyme by aldose reductase inhibitors. Our results indicate that the structure of the inhibitor-binding site of aldehyde reductase differs from that of aldose reductase due to the participation of nonconserved residues in its formation. A major difference is the participation of Arg 312 and Asp 313 in lining the inhibitor-binding site in aldehyde reductase but not in aldose reductase.

Characterization of the rat carbonic anhydrase II gene structure: sequence analysis of the 5' flanking region and 3' UTR.

The rat carbonic anhydrase II gene was characterized and found to be approximately 15.5 kb in length and to contain 7 exons and 6 introns. All intron/exon junction and branch point sequences conform to consensus sequences, and the overall rat CA II genomic structure appears to be conserved upon comparison with mouse, human, and chicken CA II genes. The putative cis-acting elements within the analyzed 1014 bp 5' flanking region include: TATA box, 4 Sp1 binding sites, 2 AP2 sites and putative tissue-specific beta-globin-like repeat elements. A CpG island of approximately 800 bp was identified that begins about 600 bp upstream of exon 1 and extends about 200 bp into intron 1. In the 3' UTR, two polyadenylation signals (AATAAA) are present, the second of which is believed to be utilized. Northern blot analysis reveals that the 1.7 kb rat CA II mRNA is abundantly expressed in adult male brain and kidney, while negligible amounts are detected in heart and liver.

Nucleotide sequence of a cDNA encoding rat brain carbonic anhydrase II and its deduced amino acid sequence.

A carbonic anhydrase II (CAII)-encoding cDNA clone was isolated from a rat brain lambda gt11 library. The 1459-bp cDNA codes for 260 amino acids with sequence similarity to mouse and human CAII and hybridizes to a single 1.7-kb mRNA.

High-level synthesis of recombinant HIV-1 protease and the recovery of active enzyme from inclusion bodies.

A complete chemical synthesis and assembly of genes for the production of human immunodeficiency virus type-I protease (HIV-PR) and its precursors are described. The T7 expression system was used to produce high levels of active HIV-PR and its precursors in Escherichia coli inclusion bodies. The gene encoding the open reading frames of HIV-PR was expressed in E. coli as a 10-kDa protein, while the genes encoding HIV-PR precursors were expressed as larger proteins, which were partially processed in E. coli to the 10-kDa form. These processing events are autoproteolytic, since a single-base mutation, changing the active-site aspartic acid to glycine, completely abolished the conversion. HIV-PR can be released with 8 M urea from washed cellular inclusion bodies, resulting in a preparation with few bacterial host proteins. After refolding, this preparation contains no nonspecific protease or peptidase activities. The recombinant HIV-PR isolated from inclusion bodies cleaves HIV-PR substrates specifically with a specific activity comparable to column-purified HIV-PR.