Oleg A. Barski

Conditionally replicating adenoviruses expressing short hairpin RNAs silence the expression of a target gene in cancer cells.

RNA interference (RNAi) is a posttranscriptional silencing mechanism triggered by double-stranded RNA that was recently shown to function in mammalian cells. Expression of cancer-associated genes was knocked down by expressing short hairpin RNAs (shRNAs) in cancer cells. By virtue of its excellent target specificity, RNAi may be used as a new therapeutic modality for cancer. The success of this approach will largely depend on efficient delivery of shRNAs to tumor cells. Tumor-selective replication competent viruses are especially suited to efficiently deliver anticancer genes to tumors. In addition, their intrinsic capacity to kill cancer cells makes these viruses promising anticancer agents per se. In this study, conditionally replicating adenoviruses were constructed encoding shRNAs targeted against firefly luciferase. These replicating viruses were shown to specifically silence the expression of the target gene in human cancer cells down to 30% relative to control virus. This finding offers the promise of using RNAi in the context of cancer gene therapy with oncolytic viruses.

Dietary Carnosine Prevents Early Atherosclerotic Lesion Formation in ApoE-null Mice

Objective—Atherosclerotic lesions are associated with the accumulation of reactive aldehydes derived from oxidized lipids. Although inhibition of aldehyde metabolism has been shown to exacerbate atherosclerosis and enhance the accumulation of aldehyde-modified proteins in atherosclerotic plaques, no therapeutic interventions have been devised to prevent aldehyde accumulation in atherosclerotic lesions. Approach and Results—We examined the efficacy of carnosine, a naturally occurring &bgr;-alanyl-histidine dipeptide, in preventing aldehyde toxicity and atherogenesis in apolipoprotein E–null mice. In vitro, carnosine reacted rapidly with lipid peroxidation-derived unsaturated aldehydes. Gas chromatography mass-spectrometry analysis showed that carnosine inhibits the formation of free aldehydes 4-hydroxynonenal and malonaldialdehyde in Cu2+-oxidized low-density lipoprotein. Preloading bone marrow–derived macrophages with cell-permeable carnosine analogs reduced 4-hydroxynonenal–induced apoptosis. Oral supplementation with octyl-D-carnosine decreased atherosclerotic lesion formation in aortic valves of apolipoprotein E–null mice and attenuated the accumulation of protein-acrolein, protein-4-hydroxyhexenal, and protein-4-hydroxynonenal adducts in atherosclerotic lesions, whereas urinary excretion of aldehydes as carnosine conjugates was increased. Conclusions—The results of this study suggest that carnosine inhibits atherogenesis by facilitating aldehyde removal from atherosclerotic lesions. Endogenous levels of carnosine may be important determinants of atherosclerotic lesion formation, and treatment with carnosine or related peptides could be a useful therapy for the prevention or the treatment of atherosclerosis.

Catalytic Mechanism and Substrate Specificity of the β-Subunit of the Voltage-Gated Potassium Channel†

The beta-subunits of voltage-gated potassium (Kv) channels are members of the aldo-keto reductase (AKR) superfamily. These proteins regulate inactivation and membrane localization of Kv1 and Kv4 channels. The Kvbeta proteins bind to pyridine nucleotides with high affinity; however, their catalytic properties remain unclear. Here we report that recombinant rat Kvbeta2 catalyzes the reduction of a wide range of aldehydes and ketones. The rate of catalysis was slower (0.06-0.2 min(-1)) than those of most other AKRs but displayed the expected hyperbolic dependence on substrate concentration, with no evidence of allosteric cooperativity. Catalysis was prevented by site-directed substitution of Tyr-90 with phenylalanine, indicating that the acid-base catalytic residue, identified in other AKRs, has a conserved function in Kvbeta2. The protein catalyzed the reduction of a broad range of carbonyls, including aromatic carbonyls, electrophilic aldehydes and prostaglandins, phospholipids, and sugar aldehydes. Little or no activity was detected with carbonyl steroids. Initial velocity profiles were consistent with an ordered bi-bi rapid equilibrium mechanism in which NADPH binding precedes carbonyl binding. Significant primary kinetic isotope effects (2.0-3.1) were observed under single- and multiple-turnover conditions, indicating that the bond-breaking chemical step is rate-limiting. Structure-activity relationships with a series of para-substituted benzaldehydes indicated that the electronic interactions predominate during substrate binding and that no significant charge develops during the transition state. These data strengthen the view that Kvbeta proteins are catalytically active AKRs that impart redox sensitivity to Kv channels.

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.

Reductive metabolism increases the proinflammatory activity of aldehyde phospholipids

The generation of oxidized phospholipids in lipoproteins has been linked to vascular inflammation in atherosclerotic lesions. Products of phospholipid oxidation increase endothelial activation; however, their effects on macrophages are poorly understood, and it is unclear whether these effects are regulated by the biochemical pathways that metabolize oxidized phospholipids. We found that incubation of 1-palmitoyl-2-(5′-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC) with THP-1-derived macrophages upregulated the expression of cytokine genes, including granulocyte/macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor (TNF)-α, monocyte chemotactic protein 1 (MCP-1), interleukin (IL)-1β, IL-6, and IL-8. In these cells, reagent POVPC was either hydrolyzed to lyso-phosphatidylcholine (lyso-PC) or reduced to 1-palmitoyl-2-(5-hydroxy-valeroyl)-sn-glycero-3-phosphocholine (PHVPC). Treatment with the phospholipase A2 (PLA2) inhibitor, pefabloc, decreased POVPC hydrolysis and increased PHVPC accumulation. Pefabloc also increased the induction of cytokine genes in POVPC-treated cells. In contrast, PHVPC accumulation and cytokine production were decreased upon treatment with the aldose reductase (AR) inhibitor, tolrestat. In comparison with POVPC, lyso-PC led to 2- to 3-fold greater and PHVPC 10- to 100-fold greater induction of cytokine genes. POVPC-induced cytokine gene induction was prevented in bone-marrow derived macrophages from AR-null mice. These results indicate that although hydrolysis is the major pathway of metabolism, reduction further increases the proinflammatory responses to POVPC. Thus, vascular inflammation in atherosclerotic lesions is likely to be regulated by metabolism of phospholipid aldehydes in macrophages.

Characterization of a Novel Murine Aldo-Keto Reductase

The aldo-keto reductase superfamily (Bohren et al., 1989) consists of many reductases that differ in their primary structure, substrate specficities and catalytic properties. Many subfamilies have been recognized, including the aldose reductase, aldehyde reductase, 3α-hydroxysteroid dehydrogenase, androgen regulated protein from the mouse vas deferens, and many more. In the course of cloning murine liver aldo-keto reductases, we discovered yet another aldo-keto reductase with some unique properties. We hereby describe the cloning, sequencing, over-expression and characterization of this novel murine aldo-keto reductase which appears to be uniquely different from any hitherto described member of the superfamily.

Human aldehyde reductase promoter allows simultaneous expression of two genes in opposite directions

Dual-Direction Tool Coordinated expression of two transgenes, such as equal production of antibody heavy and light chains, has proved a tricky problem. One solution involves a bicistronic message w...

Pyridine Nucleotide Dependence of Kv Beta - Induced Kv Inactivation: Role of Kv Alpha C-Terminus

Binding of ancillary β-subunits (Kvβ) to the N-terminal T1 domain of Kv1 and Kv4 regulates channel function and localization. The β subunits of Kv channels belong to the aldo-keto reductase superfamily (AKR6). These proteins bind NAD(P)(H) with high affinity, but the mechanisms by which nucleotides regulate channel gating are unclear. Herein we report that when coexpressed with Kv1.5 in COS-7 cells, Kvβ3 shifts the half-activation potential and imparts inactivation to slowly inactivating Kv1.5 current. Addition of NAD(P)H to the patch pipette increased rate and extent of inactivation, whereas NAD(P)+ reduced inactivation. These results conform to a model assuming that NAD(P)(H) binding regulates rate and extent of inactivation synergistically by altering the number of Kvβ monomers involved in inactivation. Deletion of 56 C-terminal amino acids of Kv1.5 (KvΔC56) did not significantly affect Kv association with Kvβ or Kvβ-mediated inactivation. KvΔC56 did not, however, respond to changes in intracellular pyridine nucleotide concentration when co-expressed with Kvβ3 and neither NAPDH nor NADP+ altered rate or extent of inactivation. Glutathione-S-transferase (GST) fusion protein containing peptides from the last 38 (Ile565-Leu602) and 60 (Arg543-Leu602), but not 19 (Asp584-Leu602), amino acids of Kv1.5 C-terminus precipitated Kvβ2 and Kvβ3 in pull-down assays from lysates of transformed bacteria. The C-terminal peptide (GST-C60) also precipitated Kvβ1 and Kvβ2 from mouse brain extracts. The GST-C60 construct did not bind to apoKvβ2, and it displayed higher affinity for Kvβ2:NADPH than for the Kvβ2:NADP+ binary complex. These results suggest that nucleotide binding provides an efficient mechanism to adjust potassium flux in response to metabolic changes. The C-terminal domain of Kv1.5 from Arg543-Asp584 interacts with Kvβ and this interaction may be involved in sensing different conformational states of Kvβ bound to either reduced or oxidized pyridine nucleotides.