There is substantial interest in the development of drugs that limit the extent of ischemia-induced cardiac damage caused by myocardial infarction or by certain surgical procedures. Here an unbiased proteomic search identified mitochondrial aldehyde dehydrogenase 2 (ALDH2) as an enzyme whose activation correlates with reduced ischemic heart damage in rodent models. A high-throughput screen yielded a small-molecule activator of ALDH2 (Alda-1) that, when administered to rats prior to an ischemic event, reduced infarct size by 60%, most likely through its inhibitory effect on the formation of cytotoxic aldehydes. In vitro, Alda-1 was a particularly effective activator of ALDH2*2, an inactive mutant form of the enzyme that is found in 40% of East Asian populations. Thus, pharmacologic enhancement of ALDH2 activity may be useful for patients with wildtype or mutant ALDH2 subjected to cardiac ischemia, such as during coronary bypass surgery. (140/140 words)
Conflicting roles for protein kinase C (PKC) isozymes in cardiac disease have been reported. Here, ␦PKC-selective activator and inhibitor peptides were designed rationally, based on molecular modeling and structural homology analyses. Together with previously identified activator and inhibitor peptides of PKC, ␦PKC peptides were used to identify cardiac functions of these isozymes. In isolated cardiomyocytes, perfused hearts, and transgenic mice, ␦PKC and PKC had opposing actions on protection from ischemiainduced damage. Specifically, activation of PKC caused cardioprotection whereas activation of ␦PKC increased damage induced by ischemia in vitro and in vivo. In contrast, ␦PKC and PKC caused identical nonpathological cardiac hypertrophy; activation of either isozyme caused nonpathological hypertrophy of the heart. These results demonstrate that two related PKC isozymes have both parallel and opposing effects in the heart, indicating the danger in the use of therapeutics with nonselective isozyme inhibitors and activators. Moreover, reduction in cardiac damage caused by ischemia by perfusion of selective regulator peptides of PKC through the coronary arteries constitutes a major step toward developing a therapeutic agent for acute cardiac ischemia.
Protein kinase C (PKC) isozymes translocate to unique subcellular sites following activation. We previously suggested that translocation of activated isozymes is required for their function and that in addition to binding to lipids, translocation involves binding of the activated isozymes to specific anchoring proteins (receptors for activated protein kinase C. Using cultured cardiomyocytes we identified inhibitors, the V1 fragment of ⑀PKC (⑀V1), and an 8-amino acid peptide derived from it that selectively inhibited the translocation of ⑀PKC. Inhibition of ⑀PKC translocation but not inhibition of ␦ or PKC translocation specifically blocked phorbol ester-or norepinephrine-mediated regulation of contraction. These isozyme-selective translocation inhibitors provide novel tools to determine the function of individual PKC isozymes in intact cells. Activation of protein kinase C (PKC)1 isozymes is associated with translocation of the enzymes form the cell soluble to the cell particulate fraction (1). These isozymes are activated by binding to lipid-derived second messengers and negatively charged phospholipids present in the cell particulate fraction (2, 3). In addition to binding to lipids, specific anchoring proteins participate in binding the activated PKC isozymes to this fraction (4 -9). We collectively termed these proteins RACKs, for receptors for activated protein kinase C (7, 10).In cultured neonatal cardiomyocytes, immunofluorescence studies demonstrated isozyme-specific subcellular localization following activation with either 4- phorbol 12-myristate-13-acetate (PMA) or with norepinephrine (NE) via an ␣ 1 -adrenergic receptor (11,12). Similar isozyme-specific localization was found in other cells following PKC activation (e.g. (13)). This isozyme-specific localization suggests that unique sequences in each isozyme (14) contain at least part of the recognition site for the anchoring molecules, the isozyme-specific RACKs.Here, we focus on the ⑀PKC unique region, ⑀V1, which is the largest variable region in this isozyme. Some homology between ⑀V1 and the C2 region of the classical PKCs, ␣, , and ␥, was noted (15). Because C2 contains at least part of the RACKbinding site of classical PKCs (16,17), an ⑀PKC specific RACKbinding site may reside within ⑀V1. In that case, an ⑀V1 fragment should bind to the ⑀PKC-specific RACK when introduced into cells and thus inhibit PMA-or hormone-induced ⑀PKC translocation and binding to that RACK. Translocation of other PKC isozymes should not be affected by ⑀V1. The following study confirms these predictions and demonstrates the use of translocation inhibitors to determine the role of specific isozymes in regulating cardiac contraction. EXPERIMENTAL PROCEDURESPeptides and Reagents-Peptides ⑀V1-2 (EAVSLKPT; ⑀PKC (14 -21)), scrambled ⑀V1-2 (LSETKPAV), ⑀V1-3 (LAVFHDA; ⑀PKC (81-87)), V1-2 (EAVGLQPT; PKC (18 -25)), and C2-4 (SLNPEWNET; PKC (218 -226)) were synthesized at the Beckman Center Protein and Nucleic Acid Facility at Stanford. All the peptides used in this study were ...
Brief periods of cardiac ischemia trigger protection from subsequent prolonged ischemia (preconditioning). Protein kinase C (PKC) has been suggested to mediate preconditioning. Here, we describe an PKC-selective agonist octapeptide, receptor for activated C-kinase (RACK), derived from an PKC sequence homologous to its anchoring protein, RACK. Introduction of RACK into isolated cardiomyocytes, or its postnatal expression as a transgene in mouse hearts, increased PKC translocation and caused cardio-protection from ischemia without any deleterious effects. Our data demonstrate that PKC activation is required for protection from ischemic insult and suggest that small molecules that mimic this PKC agonist octapeptide provide a powerful therapeutic approach to protect hearts at risk for ischemia.preconditioning ͉ hypoxia ͉ transgenic pseudoreceptors for activated C-kinase ͉ ischemia
In approximately one billion people, a point mutation inactivates a key detoxifying enzyme, aldehyde dehydrogenase (ALDH2). This mitochondrial enzyme metabolizes toxic biogenic and environmental aldehydes, including the endogenously produced 4-hydroxynonenal (4HNE) and the environmental pollutant, acrolein. ALDH2 also bioactivates nitroglycerin, but it is best known for its role in ethanol metabolism. The accumulation of acetaldehyde following the consumption of even a single alcoholic beverage leads to the Asian Alcohol-induced Flushing Syndrome in ALDH2*2 homozygotes. The ALDH2*2 allele is semi-dominant and heterozygotic individuals exhibit a similar, but not as severe phenotype. We recently identified a small molecule, Alda-1, which activates wild-type ALDH2 and restores near wild-type activity to ALDH2*2. The structures of Alda-1 bound to ALDH2 and ALDH2*2 reveal how Alda-1 activates the wild-type enzyme and how it restores the activity of ALDH2*2 by acting as a structural chaperone.
The occurrence of more than 200 diseases, including cancer, can be attributed to alcohol drinking. The global cancer deaths attributed to alcohol-consumption rose from 243,000 in 1990 to 337,400 in 2010. In 2010, cancer deaths due to alcohol consumption accounted for 4.2% of all cancer deaths. Strong epidemiological evidence has established the causal role of alcohol in the development of various cancers, including esophageal cancer, head and neck cancer, liver cancer, breast cancer, and colorectal cancer. The evidence for the association between alcohol and other cancers is inconclusive. Because of the high prevalence of ALDH2*2 allele among East Asian populations, East Asians may be more susceptible to the carcinogenic effect of alcohol, with most evidence coming from studies of esophageal cancer and head and neck cancer, while data for other cancers are more limited. The high prevalence of ALDH2*2 allele in East Asian populations may have important public health implications and may be utilized to reduce the occurrence of alcohol-related cancers among East Asians, including: 1) Identification of individuals at high risk of developing alcohol-related cancers by screening for ALDH2 polymorphism; 2) Incorporation of ALDH2 polymorphism screening into behavioral intervention program for promoting alcohol abstinence or reducing alcohol consumption; 3) Using ALDH2 polymorphism as a prognostic indicator for alcohol-related cancers; 4) Targeting ALDH2 for chemoprevention; and 5) Setting guidelines for alcohol consumption among ALDH2 deficient individuals. Future studies should evaluate whether these strategies are effective for preventing the occurrence of alcohol-related cancers.
Abstract-Cardiac ischemia and reperfusion are associated with loss in the activity of the mitochondrial enzyme pyruvate dehydrogenase (PDH). Pharmacological stimulation of PDH activity improves recovery in contractile function during reperfusion. Signaling mechanisms that control inhibition and reactivation of PDH during reperfusion were therefore investigated. Using an isolated rat heart model, we observed ischemia-induced PDH inhibition with only partial recovery evident on reperfusion. Translocation of the redox-sensitive ␦-isoform of protein kinase C (PKC) to the mitochondria occurred during reperfusion. Key Words: pyruvate dehydrogenase Ⅲ ␦PKC Ⅲ pyruvate dehydrogenase kinase Ⅲ free radicals Ⅲ mitochondria Ⅲ ischemia/reperfusion P yruvate dehydrogenase (PDH) is responsible for the conversion of pyruvate derived from glycolysis to acetylCoA for Krebs cycle activity. Enzyme activity is regulated, in part, by phosphorylation-and dephosphorylation-dependent inhibition and activation, respectively. 1,2 Phosphorylation is catalyzed by 4 PDH-associated pyruvate dehydrogenase kinases (PDK1-4) that exhibit tissue-specific expression patterns and differences in specific activity toward 3 phosphorylation sites on the E1␣ subunit of PDH. The PDH complex also contains 2 pyruvate dehydrogenase phosphatases (PDP 1 and PDP 2) responsible for reactivation of PDH. 2-4 PDH therefore represents a highly regulated and critical site for the control of glycolytic flux and ATP production.Cardiac ischemia/reperfusion is associated with alterations in metabolism that, depending on the severity of the ischemic insult, can progress to irreparable myocardial damage. 5 Although PDH activity in myocardial tissue has been reported to decline during flow-induced ischemia, 6 this is not universally observed. [7][8][9] The effects of reperfusion also exhibit considerable variability, with the majority of studies demonstrating a decrease in PDH activity. [7][8][9] Despite the disparity in evidence regarding PDH activity, cardiac efficiency and recovery of contractile function in postischemic hearts can be improved by pharmacological stimulation of PDH 8,10 -16 or infusion of pyruvate. [17][18][19][20][21][22][23] Identification of factors that regulate PDH activity during ischemia/reperfusion may therefore enhance the potential for therapeutic intervention.Reperfusion of ischemic myocardium is associated with enhanced free radical generation. 5,24 Pro-oxidants have been shown to regulate protein function either directly or indirectly through the modulation of other regulatory molecules. [25][26][27] One such example is the novel ␦-isoform of PKC. Exposure of purified ␦PKC to the thiol-specific oxidant diamide and glutathione (GSH) at concentrations that induce inactivation of other PKC isozymes results in ␦PKC activation. 28 Additionally, treatment of various cell types with H 2 O 2 , glutathione depleting agents, or the general PKC activator PMA results in tyrosine phosphorylation and/or activation and translocation of ␦PKC to the mitochondri...
Background: ALDH enzymes metabolize aldehydes in many pathways, including the inactivation of cyclophosphamide. Results: Covalent inhibitors against ALDH were discovered, and their mechanism of action was determined. Conclusion: Covalent inhibitors against ALDH potentiate cell killing in cyclophosphamide-resistant cells. Significance: These inhibitors represent novel research tools and can serve as leads toward therapeutics where increased ALDH activity is associated with disease.
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