Theresa M. Harter

Functional genomic studies of aldo-keto reductases

Aldose reductase (AR) is considered a potential mediator of diabetic complications and is a drug target for inhibitors of diabetic retinopathy and neuropathy in clinical trials. However, the physiological role of this enzyme still has not been established. Since effective inhibition of diabetic complications will require early intervention, it is important to delineate whether AR fulfills a physiological role that cannot be compensated by an alternate aldo-keto reductase. Functional genomics provides a variety of powerful new tools to probe the physiological roles of individual genes, especially those comprising gene families. Several eucaryotic genomes have been sequenced and annotated, including yeast, nematode and fly. To probe the function of AR, we have chosen to utilize the budding yeast Saccharomyces cerevisiae as a potential model system. Unlike Caenorhabditis elegans and D. melanogaster, yeast provides a more desirable system for our studies because its genome is manipulated more readily and is able to sustain multiple gene deletions in the presence of either drug or auxotrophic selectable markers. Using BLAST searches against the human AR gene sequence, we identified six genes in the complete S. cerevisiae genome with strong homology to AR. In all cases, amino acids thought to play important catalytic roles in human AR are conserved in the yeast AR-like genes. All six yeast AR-like open reading frames (ORFs) have been cloned into plasmid expression vectors. Substrate and AR inhibitor specificities have been surveyed on four of the enzyme forms to identify, which are the most functionally similar to human AR. Our data reveal that two of the enzymes (YDR368Wp and YHR104Wp) are notable for their similarity to human AR in terms of activity with aldoses and substituted aromatic aldehydes. Ongoing studies are aimed at characterizing the phenotypes of yeast strains containing single and multiple knockouts of the AR-like genes.

Hypotension Due to Kir6.1 Gain‐of‐Function in Vascular Smooth Muscle

Background KATP channels, assembled from pore‐forming (Kir6.1 or Kir6.2) and regulatory (SUR1 or SUR2) subunits, link metabolism to excitability. Loss of Kir6.2 results in hypoglycemia and hyperinsulinemia, whereas loss of Kir6.1 causes Prinzmetal angina–like symptoms in mice. Conversely, overactivity of Kir6.2 induces neonatal diabetes in mice and humans, but consequences of Kir6.1 overactivity are unknown. Methods and Results We generated transgenic mice expressing wild‐type (WT), ATP‐insensitive Kir6.1 [Gly343Asp] (GD), and ATP‐insensitive Kir6.1 [Gly343Asp,Gln53Arg] (GD‐QR) subunits, under Cre‐recombinase control. Expression was induced in smooth muscle cells by crossing with smooth muscle myosin heavy chain promoter–driven tamoxifen‐inducible Cre‐recombinase (SMMHC‐Cre‐ER) mice. Three weeks after tamoxifen induction, we assessed blood pressure in anesthetized and conscious animals, as well as contractility of mesenteric artery smooth muscle and KATP currents in isolated mesenteric artery myocytes. Both systolic and diastolic blood pressures were significantly reduced in GD and GD‐QR mice but normal in mice expressing the WT transgene and elevated in Kir6.1 knockout mice as well as in mice expressing dominant‐negative Kir6.1 [AAA] in smooth muscle. Contractile response of isolated GD‐QR mesenteric arteries was blunted relative to WT controls, but nitroprusside relaxation was unaffected. Basal KATP conductance and pinacidil‐activated conductance were elevated in GD but not in WT myocytes. Conclusions KATP overactivity in vascular muscle can lead directly to reduced vascular contractility and lower blood pressure. We predict that gain of vascular KATP function in humans would lead to a chronic vasodilatory phenotype, as indeed has recently been demonstrated in Cantu syndrome.

Aldo–keto reductases as modulators of stress response

Human aldose reductase (AKR1B1) has been implicated as a factor in the pathogenesis of diabetic complications. However, little is known about the physiological role of this enzyme or of related aldo-keto reductases in human tissues. In mammalian systems, a gene knock out approach is often employed as an experimental strategy to probe for gene function. However, in the murine system, phenotypic characterization of an aldose reductase (AKR1B3) knock out is likely to be complicated due to functional compensation by redundant AKRs including AKRs 1A (aldehyde reductase), 1B7 (FR-1) and 1B8 (MVDP). As an alternate strategy, we are examining the budding yeast Saccharomyces cerevisiae as a model system for a functional genomics study of AKRs. A distinct advantage of this system centers on the ability to readily ablate multiple targeted genes in a single strain. In addition to providing insights into functional redundancy, this system allows us to use a genetic approach to study possible effector pathways associated with one or more individual genes. Yeast open reading frames (ORFs) encoding AKRs with functional similarity to human aldose reductase (AKR1B1) were identified by BLAST analysis and were functionally validated by studies of recombinant proteins. By ablating three of the yeast AKR genes most functionally similar to AKR1B1, we have created a unique strain of S. cerevisiae that shows enhanced sensitivity to stress. Ongoing studies with oligonucleotide arrays show that the triple null strain has an altered transcription profile consistent with an enhanced stress response in comparison with the parental strain. These data indicate that AKR-null strains may provide new insights into signaling mechanisms involving this family of proteins.

Aldo-Keto Reductases in the Eye

Aldose reductase (AKR1B1) is an NADPH-dependent aldo-keto reductase best known as the rate-limiting enzyme of the polyol pathway. Accelerated glucose metabolism through this pathway has been implicated in diabetic cataract and retinopathy. Some human tissues contain AKR1B1 as well as AKR1B10, a closely related member of the aldo-keto reductase gene superfamily. This opens the possibility that AKR1B10 may also contribute to diabetic complications. The goal of the current study was to characterize the expression profiles of AKR1B1 and AKR1B10 in the human eye. Using quantitative reverse transcriptase-PCR and immunohistochemical staining, we observed expression of both AKR genes in cornea, iris, ciliary body, lens, and retina. Expression of AKR1B1 was the highest in lens and retina, whereas AKR1B10 was the highest in cornea. Lenses from transgenic mice designed for overexpression of AKR1B10 were not significantly different from nontransgenic controls, although a significant number developed a focal defect in the anterior lens epithelium following 6 months of experimentally induced diabetes. However, lenses from AKR1B10 mice remained largely transparent following longterm diabetes. These results indicate that AKR1B1 and AKR1B10 may have different functional properties in the lens and suggest that AKR1B10 does not contribute to the pathogenesis of diabetic cataract in humans.

A Potential Role for Aldose Reductase in Steroid Metabolism

Aldose reductase (ALR2) is a monomeric NADPH-dependent reductase distinguished from other members of the aldo-keto reductase enzyme family in its ability to catalyze the reduction of a variety of hexoses and pentoses. The role of ALR2 in enhanced polyol synthesis in diabetic and galactosemic tissues is well documented (Kinoshita and Nishimura, 1988), as is the potential therapeutic use of ALR2 inhibitors to delay or prevent the onset and progression of metabolic complications leading to cataract and retinopathy (Sarges and Oates, 1993). New insights into the structure of ALR2, together with emerging data demonstrating that many individual aldo-keto reductases may participate in divergent metabolic pathways, lead us to question whether sugars represent the only physiologically-relevant substrate of this enzyme.

Kinetic Alteration of Human Aldose Reductase by Mutagenesis of Cysteine Residues

Aldose reductase (ALR21: alditol:NADPH oxidoreductase: E.C. 1.1.1.21) catalyzes the NADPH-linked reduction of aldoses to their corresponding alcohols or polyols, the first step of the polyol pathway. Enhanced flux of glucose through the polyol pathway and consequent biochemical imbalances are thought to be crucial to the onset and progression of many complications of diabetes mellitus including cataract, retinopathy, neuropathy and nephropathy (Kinoshita and Nishimura, 1988). In light of its rate-limiting position in the polyol pathway as well as its apparent metabolic dispensability (Yancey et al., 1990), strategies to control or prevent the onset of diabetic complications through inhibition of aldose reductase are being aggressively pursued. While a structurally-diverse array of aldose reductase inhibitors (ARI) have yielded impressive results in animal studies, their effectiveness in arresting or preventing diabetic neuropathy (Boulton et al., 1990) and retinopathy (Sorbinil Retinopathy Trial Research Group, 1990) in human trials has been less encouraging (Frank, 1990).

Structure-Function Studies of FR-1

The aldo-keto reductase (AKR) gene superfamily represents a collection of proteins expressed in a wide variety of plants, animals, yeast, and procaryotic organisms. Most AKRs were originally identified as enzymes capable of catalyzing the NADPH-dependent reduction of carbonyl groups contained in a broad range of substrates (Bachur, 1976). However, recent genetic studies mediated by genome and expression sequencing approaches have identified several new members of the AKR superfamily. Many of these new proteins are characterized by high sequence homology to AKR enzymes although little or no information is available about their potential catalytic activities. One such new protein, designated FR-1* was identified as the product of a gene upregulated in serum-starved mouse fibroblasts following treatment with fibroblast growth factor I (FGF-I) (Donohue et al., 1994). High amino acid sequence identity (~70%) was observed between FR-1 and aldose reductase as well as other AKRs. Many amino acid residues known to contribute to the catalytic mechanism in other AKR enzymes including aldose reductase (AKRlB I), aldehyde reductase (AKRIAI) and 3a.-hydroxysteroid dehydrogenase (AKR1C9) are conserved in FR-l. These residues include Tyr-48, His-ll 0, Lys-77 and Asp-43 (numbering is that of aldose reductase) (Barski et al. , 1995; Pawlowski & Penning, 1994; Schlegel et al., 1998; Tarle et al. , 1993). The present study was undertaken to evaluate whether FR -1 is a catalyst of carbonyl reduction and to measure the affinity of FR-1 for various ligands such as nucleotide cofactors, carbonyl substrates and aldose reductase inhibitors. Our studies show that FR-1 catalyzes the NADPH-dependent reduction of substrates representative of diverse structural classes of aliphatic and aromatic aldehydes. Both saturated and unsaturated aldehydes were excellent substrates. Unlike aldose reductase and aldehyde reductase, FR-1 catalyzed the reduction of simple ketones such as acetone and butanone; however virtually no catalytic activity could be detected using steroid and aldose substrates. FR-1 was inhibited by various aldose reductase inhibitors in a manner similar to human aldose reductase. Besides being an excellent substrate, 4-hydroxy-2-nonenal (HNE) inactivated the enzyme through a mechanism involving Michael addition to Cys-298.

Cardiovascular consequences of KATP overactivity in Cantu syndrome

Cantu syndrome (CS) is characterized by multiple vascular and cardiac abnormalities including vascular dilation and tortuosity, systemic hypotension, and cardiomegaly. The disorder is caused by gain-of-function (GOF) mutations in genes encoding pore-forming (Kir6.1, KCNJ8) and accessory (SUR2, ABCC9) ATP-sensitive potassium (KATP) channel subunits. However, there is little understanding of the link between molecular dysfunction and the complex pathophysiology observed, and there is no known treatment, in large part due to the lack of appropriate preclinical disease models in which to test therapies. Notably, expression of Kir6.1 and SUR2 does not fully overlap, and the relative contribution of KATP GOF in various cardiovascular tissues remains to be elucidated. To investigate pathophysiologic mechanisms in CS we have used CRISPR/Cas9 engineering to introduce CS-associated SUR2[A478V] and Kir6.1[V65M] mutations to the equivalent endogenous loci in mice. Mirroring human CS, both of these animals exhibit low systemic blood pressure and dilated, compliant blood vessels, as well dramatic cardiac enlargement, the effects being more severe in V65M animals than in A478V animals. In both animals, whole-cell patch-clamp recordings reveal enhanced basal KATP conductance in vascular smooth muscle, explaining vasodilation and lower blood pressure, and demonstrating a cardinal role for smooth muscle KATP dysfunction in CS etiology. Echocardiography confirms in situ cardiac enlargement and increased cardiac output in both animals. Patch-clamp recordings reveal reduced ATP sensitivity of ventricular myocyte KATP channels in A478V, but normal ATP sensitivity in V65M, suggesting that cardiac remodeling occurs secondary to KATP overactivity outside of the heart. These SUR2[A478V] and Kir6.1[V65M] animals thus reiterate the key cardiovascular features seen in human CS. They establish the molecular basis of the pathophysiological consequences of reduced smooth muscle excitability resulting from SUR2/Kir6.1-dependent KATP GOF, and provide a validated animal model in which to examine potential therapeutic approaches to treating CS.