Aldose reductase modulates cardiac glycogen synthase kinase-3ß phosphorylation during ischemia-reperfusion.

Earlier studies have demonstrated that aldose reductase (AR) plays a key role in mediating ischemia-reperfusion (I/R) injury. Our objective was to investigate if AR mediates I/R injury by influencing phosphorylation of glycogen synthase kinase-3ß (p-GSK3ß). To investigate this issue, we used three separate models to study the effects of stress injury on the heart. Hearts isolated from wild-type (WT), human expressing AR transgenic (ARTg), and AR knockout (ARKO) mice were perfused with/without GSK3ß inhibitors (SB-216763 and LiCl) and subjected to I/R. Ad-human AR (Ad-hAR)-expressing HL-1 cardiac cells were exposed to hypoxia (0.5% O(2)) and reoxygenation (20.9% O(2)) conditions. I/R in a murine model of transient occlusion and reperfusion of the left anterior descending coronary artery (LAD) was used to study if p-GSK3ß was affected through increased AR flux. Lactate dehydrogenase (LDH) release and left ventricular developed pressure (LVDP) were measured. LVDP was decreased in hearts from ARTg mice compared with WT and ARKO after I/R, whereas LDH release and apoptotic markers were increased (P < 0.05). p-GSK3ß was decreased in ARTg hearts compared with WT and ARKO (P < 0.05). In ARKO, p-GSK3ß and apoptotic markers were decreased compared with WT (P < 0.05). WT and ARTg hearts perfused with GSK3ß inhibitors improved p-GSK3ß expression and LVDP and exhibited decreased LDH release, apoptosis, and mitochondrial pore opening (P < 0.05). Ad-hAR-expressing HL-1 cardiac cells, exposed to hypoxia (0.5% O(2)) and reoxygenation (20.9% O(2)), had greater LDH release compared with control HL-1 cells (P < 0.05). p-GSK3ß was decreased and correlated with increased apoptotic markers in Ad-hAR HL-1 cells (P < 0.05). Treatment with phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) inhibitor increased injury demonstrated by increased LDH release in ARTg, WT, and ARKO hearts and in Ad-hAR-expressing HL-1 cells. Cells treated with protein kinase C (PKC) α/ß inhibitor displayed significant increases in p-Akt and p-GSK3ß expression, and resulted in decreased LDH release. In summary, AR mediates changes in p-GSK3ß, in part, via PKCα/ß and Akt during I/R.

Ascorbate synthesis pathway: dual role of ascorbate in bone homeostasis.

Using mouse gene knock-out models, we identify aldehyde reductase (EC 1.1.1.2, Akr1a4 (GR)) and aldose reductase (EC 1.1.1.21, Akr1b3 (AR)) as the enzymes responsible for conversion of D-glucuronate to L-gulonate, a key step in the ascorbate (ASC) synthesis pathway in mice. The gene knock-out (KO) mice show that the two enzymes, GR and AR, provide approximately 85 and approximately 15% of L-gulonate, respectively. GRKO/ARKO double knock-out mice are unable to synthesize ASC (>95% ASC deficit) and develop scurvy. The GRKO mice ( approximately 85% ASC deficit) develop and grow normally when fed regular mouse chow (ASC content = 0) but suffer severe osteopenia and spontaneous fractures with stresses that increase ASC requirements, such as pregnancy or castration. Castration greatly increases osteoclast numbers and activity in GRKO mice and promotes increased bone loss as compared with wild-type controls and additionally induces proliferation of immature dysplastic osteoblasts likely because of an ASC-sensitive block(s) in early differentiation. ASC and the antioxidants pycnogenol and resveratrol block osteoclast proliferation and bone loss, but only ASC feeding restores osteoblast differentiation and prevents their dysplastic proliferation. This is the first in vivo demonstration of two independent roles for ASC as an antioxidant suppressing osteoclast activity and number as well as a cofactor promoting osteoblast differentiation. Although humans have lost the ability to synthesize ASC, our mouse models suggest the mechanisms by which suboptimal ASC availability facilitates the development of osteoporosis, which has important implications for human osteoporosis.

Affinity chromatography of native SUMO proteins using His-tagged recombinant UBC9 bound to Co2+-charged talon resin.

Four small ubiquitin-related modifier (SUMO) genes have been identified in humans. The recently identified SUMO4 was detected in mRNA transcripts from HEK293 cells, and human kidney and spleen tissue and may be involved in regulation of NF-kappaB and susceptibility to autoimmune diseases. However, identification and characterization of a native SUMO4 protein has not yet been reported. Here, we analyzed for the presence of native SUMO proteins in HEK293 cells and human kidney tissue using an affinity purification procedure using a UBC9 matrix followed by mass spectroscopy analyses for SUMO-specific peptides. Identification by mass spectroscopy of peptides generated by Trypsin and Lys-C digestion did reveal peptides unique to SUMO1 and SUMO2/3, but not SUMO4. In control experiments, SUMO4 prepared by recombinant methods was isolated and even enriched by our UBC9 affinity purification. Thus, SUMO4 protein appears to be either in extremely low abundance in human kidney or HEK293 cells or it is not present at all. It remains possible that SUMO4 protein is more abundant in other cell types or can be induced by hormonal or environmental challenges and the procedures reported here should be extremely useful for detecting native SUMO4. Furthermore, using His-tagged recombinant proteins bound to Co(2+)-charged Talon resin has general applicability to isolate native proteins that have strong non-covalent interactions with the resin-bound His-tagged proteins.

A proline-90 residue unique to SUMO-4 prevents maturation and sumoylation.

Four small ubiquitin-related modifier (SUMO) genes have been identified in humans. However, little is known about the basic biology of SUMO-4. Here, we report that SUMO-4 differs from SUMO-1, -2, and -3 in that the maturation process of SUMO-4 to active form containing C-terminal di-glycine residues is inhibited by a unique proline residue located at position 90 (Pro-90). Although, both the hydrolase and isopeptidase activities of SUMO peptidases are significantly diminished by Pro-90 as compared to Gln-90 (glutamine) in mutated SUMO genes, only the defective hydrolase activity appears to be biologically relevant. Native SUMO-4, thus, appears to be unable to form covalent isopeptide bonds with substrates. A biological role of SUMO-4, through non-covalent interactions is proposed.

The structure of Apo R268A human aldose reductase: hinges and latches that control the kinetic mechanism.

Aldose reductase (AR) catalyzes the NADPH-dependent reduction of glucose and other sugars to their respective sugar alcohols. The NADP+/NADPH exchange is the rate-limiting step for this enzyme and contributes in varying degrees to the catalytic rates of other aldo-keto reductase superfamily enzymes. The mutation of Arg268 to alanine in human recombinant AR removes one of the ligands of the C2-phosphate of NADP+ and markedly reduces the interaction of the apoenzyme with the nucleotide. The crystal structure of human R268A apo-aldose reductase determined to a resolution of 2.1 A is described. The R268A mutant enzyme has similar kinetic parameters to the wild-type enzyme for aldehyde substrates, yet has greatly reduced affinity for the nucleotide substrate which greatly facilitates its crystallization in the apoenzyme form. The apo-structure shows that a high temperature factor loop (between residues 214 and 226) is displaced by as much as 17 A in a rigid body fashion about Gly213 and Ser226 in the absence of the nucleotide cofactor as compared to the wild-type holoenzyme structure. Several factors act to stabilize the NADPH-holding loop in either the 'open' or 'closed' conformations: (1) the presence and interactions of the nucleotide cofactor, (2) the residues surrounding the Gly213 and Ser226 hinges which form unique hydrogen bonds in the 'open' or 'closed' structure, and (3) the Trp219 "latch" residue which interacts with an arginine residue, Arg293, in the 'open' conformation or with a cysteine residue, Cys298, in the 'closed' conformation. Several mutations in and around the high temperature factor loop are examined to elucidate the role of the loop in the mechanism by which aldose reductase binds and releases its nucleotide substrate.

Aldose reductase inhibition in the treatment of diabetic neuropathy: where are we in 2004?

Diabetic neuropathy is a major complication of poorly controlled diabetes mellitus. Aldose reductase, the first enzyme of the polyol pathway, is thought to play a role in initiating the metabolic damage to peripheral nerves during hyperglycemia. Aldose reductase inhibitors (ARIs) have been proposed to dampen the flux of glucose through the pathway during hyperglycemia; however, clinical trials in diabetic patients to demonstrate efficacy in the prevention or amelioration of diabetic neuropathy have failed thus far. Recent improved understanding of the pitfalls of past trials and some improved ARIs and clinical evaluation instruments show promise that success in the 20-plus year search for efficacious ARIs may soon be at hand.

Association of a CAG/CAA repeat sequence in the TBP gene with type I diabetes.

Type I diabetes is a complex disease in which multiple susceptibility loci have been implicated by whole genome scans. IDDM8, a susceptibility locus, is located on chromosome 6q27, however the specific susceptibility gene has yet to be identified. We have examined five potential candidate genes using 36 genetic markers, spanning 360kb located near the chromosome 6q27 terminus in 478 families for diabetes association. No associations with type I diabetes susceptibility were detected with the strength previously observed for IDDM1 or IDDM2. However, a novel CAG/CAA polymorphism was detected in exon 3 of the TATA box-binding protein gene, which shows preliminary evidence of association with diabetes susceptibility (p<0.05).

A 212-kb region on chromosome 6q25 containing the TAB2 gene is associated with susceptibility to type 1 diabetes.

The IDDM5 gene, which is identified by whole-genome searches, is located on chromosome 6q25. TAB2 (MAP3K7IP2 [mitogen-activating protein kinase kinase kinase 7 interacting protein 2]) is a potential candidate gene for type 1 diabetes because it is located on chromosome 6q25 and is involved in nuclear factor (NF)-kappaB regulation. We have conducted familial association studies using 478 families and demonstrate that a type 1 diabetes susceptibility gene resides within a 212-kb region containing the TAB2 gene (Tsp = 1.0 x 10(-2) to 4.0 x 10(-4)). No amino acid polymorphisms were detected in TAB2; however, multiple single nucleotide polymorphisms (SNPs) found within 5' untranslated, 3' untranslated, and intron regions were associated with type 1 diabetes susceptibility. Two additional genes, LOC340152, a predicted gene with currently unknown function, and SMT3, which has homology to SUMO (small ubiquitin-related modifier) were found within the 212-kb region and were associated with type 1 diabetes susceptibility. Functional studies of the three genes will be required to determine their biological relevance to type 1 diabetes. However, both TAB2 and SUMO are involved in NF-kappaB activation and may thus be involved in type 1 diabetes through apoptosis in pancreatic beta-cells.

A M55V polymorphism in a novel SUMO gene (SUMO-4) differentially activates heat shock transcription factors and is associated with susceptibility to type I diabetes mellitus.

Three SUMO (small ubiquitin-related modifier) genes have been identified in humans, which tag proteins to modulate subcellular localization and/or enhance protein stability and activity. We report the identification of a novel intronless SUMO gene, SUMO-4, that encodes a 95-amino acid protein having an 86% amino acid homology with SUMO-2. In contrast to SUMO-2, which is highly expressed in all of the tissues examined, SUMO-4 mRNA was detected mainly in the kidney. A single nucleotide polymorphism was detected in SUMO-4, substituting a highly conserved methionine with a valine residue (M55V). In HepG2 (liver carcinoma) cells transiently transfected with SUMO-4 expression vectors, Met-55 was associated with the elevated levels of activated heat shock factor transcription factors as compared with Val-55, whereas the levels of NF-kappaB were suppressed to an identical degree. The SUMO-4M (Met) variant is associated with type I diabetes mellitus susceptibility in families (p = 4.0 x 10(-4)), suggesting that it may be involved in the pathogenesis of type I diabetes.

Regulation of aldehyde reductase expression by STAF and CHOP.

Aldehyde reductase is involved in the reductive detoxification of reactive aldehydes that can modify cellular macromolecules. To analyze the mechanism of basal regulation of aldehyde reductase expression, we cloned the murine gene and adjacent regulatory region and compared it to the human gene. The mouse enzyme exhibits substrate specificity similar to that of the human enzyme, but with a 2-fold higher catalytic efficiency. In contrast to the mouse gene, the human aldehyde reductase gene has two alternatively spliced transcripts. A fragment of 57 bp is sufficient for 25% of human promoter activity and consists of two elements. The 3' element binds transcription factors of the Sp1 family. Gel-shift assays and chromatin immunoprecipitation as well as deletion/mutation analysis reveal that selenocysteine tRNA transcription activating factor (STAF) binds to the 5' element and drives constitutive expression of both mouse and human aldehyde reductase. Aldehyde reductase thus becomes the fourth protein-encoding gene regulated by STAF. The human, but not the mouse, promoter also binds C/EBP homologous protein (CHOP), which competes with STAF for the same binding site. Transfection of the human promoter into ethoxyquin-treated mouse 3T3 cells induces a 3.5-fold increase in promoter activity and a CHOP-C/EBP band appears on gel shifts performed with the 5' probe from the human aldehyde reductase promoter. Induction is attenuated in similar transfection studies of the mouse promoter. Mutation of the CHOP-binding site in the human promoter abolishes CHOP binding and significantly reduces ethoxyquin induction, suggesting that CHOP mediates stimulated expression in response to antioxidants in the human. This subtle difference in the human promoter suggests a further evolution of the promoter toward responsiveness to exogenous stress and/or toxins.

Cyclosporine A inhibits the adaptive responses to hypertonicity: a potential mechanism of nephrotoxicity.

Cell survival in the hypertonic environment of the renal medulla is dependent on the intracellular accumulation of protective organic solutes through the induction of genes whose transcriptional regulation is mediated in part by interaction between osmotic response elements and the transcription nuclear factor of activated T lymphocyte 5. It is shown that cyclosporine A (CsA) prevents the nuclear translocation of the transcription nuclear factor of activated T lymphocyte 5 and inhibits osmotic response element-mediated reporter gene expression. The expression of mRNA for hypertonicity-induced genes (aldose reductase, betaine/gamma-amino-n-butyric acid transporter 1, and heat shock protein 70) is also decreased in the medulla of CsA-treated rats. CsA inhibits the increase of betaine/gamma-amino-n-butyric acid transporter 1 and heat shock protein 70 mRNA in osmotically stressed MDCK cells, blocks cell proliferation under isotonic conditions, and augments hypertonicity-induced apoptosis. Histologic examination of the kidneys of CsA-treated rats shows a marked increase in apoptosis in the renal medulla where hypertonicity normally prevails. The data are consistent with calcineurin-mediated induction of hypertonic stress-response genes, and they suggest that CsA nephrotoxicity may in part result from inhibition of the adaptive responses to hypertonicity occurring during the urinary concentrating mechanism.