Che-Hong Chen

The Coatomer Protein β′-COP, a Selective Binding Protein (RACK) for Protein Kinase Cε

Distinct subcellular localization of activated protein kinase C (PKC) isozymes is mediated by their binding to isozyme-specific RACKs (receptors for activatedC-kinase). Our laboratory has previously isolated one such protein, RACK1, and demonstrated that this protein displays specificity for PKCβ. We have recently shown that at least part of the PKCε RACK-binding site on PKCε lies within the unique V1 region of this isozyme (Johnson, J. A., Gray, M. O., Chen, C.-H., and Mochly-Rosen, D. (1996) J. Biol. Chem. 271, 24962–24966). Here, we have used the PKCε V1 region to clone a PKCε-selective RACK, which was identified as the COPI coatomer protein, β′-COP. Similar to RACK1, β′-COP contains seven repeats of the WD40 motif and fulfills the criteria previously established for RACKs. Activated PKCε colocalizes with β′-COP in cardiac myocytes and binds to Golgi membranes in a β′-COP-dependent manner. A role for PKC in control of secretion has been previously suggested, but this is the first report of direct protein/protein interaction of PKCε with a protein involved in vesicular trafficking.

Molecular transporters for peptides: delivery of a cardioprotective ϵPKC agonist peptide into cells and intact ischemic heart using a transport system, R7

BACKGROUND Recently, we reported a novel oligoguanidine transporter system, polyarginine (R(7)), which, when conjugated to spectroscopic probes (e.g., fluorescein) and drugs (e.g., cyclosporin A), results in highly water-soluble conjugates that rapidly enter cells and tissues. We report herein the preparation of the first R(7) peptide conjugates and a study of their cellular and organ uptake and functional activity. The octapeptide (psi)(epsilon)RACK was selected for this study as it is known to exhibit selective epsilon protein kinase C isozyme agonist activity and to reduce ischemia-induced damage in cardiomyocytes. However, (psi)(epsilon)RACK is not cell-permeable. RESULTS Here we show that an R(7)-(psi)(epsilon)RACK conjugate readily enters cardiomyocytes, significantly outperforming (psi)(epsilon)RACK conjugates of the transporters derived from HIV Tat and from Antennapedia. Moreover, R(7)-(psi)(epsilon)RACK conjugate reduced ischemic damage when delivered into intact hearts either prior to or after the ischemic insult. CONCLUSIONS Our data suggest that R(7) converts a peptide lead into a potential therapeutic agent for the ischemic heart.

Targeting Aldehyde Dehydrogenase 2: New Therapeutic Opportunities

A family of detoxifying enzymes called aldehyde dehydrogenases (ALDHs) has been a subject of recent interest, as its role in detoxifying aldehydes that accumulate through metabolism and to which we are exposed from the environment has been elucidated. Although the human genome has 19 ALDH genes, one ALDH emerges as a particularly important enzyme in a variety of human pathologies. This ALDH, ALDH2, is located in the mitochondrial matrix with much known about its role in ethanol metabolism. Less known is a new body of research to be discussed in this review, suggesting that ALDH2 dysfunction may contribute to a variety of human diseases including cardiovascular diseases, diabetes, neurodegenerative diseases, stroke, and cancer. Recent studies suggest that ALDH2 dysfunction is also associated with Fanconi anemia, pain, osteoporosis, and the process of aging. Furthermore, an ALDH2 inactivating mutation (termed ALDH2*2) is the most common single point mutation in humans, and epidemiological studies suggest a correlation between this inactivating mutation and increased propensity for common human pathologies. These data together with studies in animal models and the use of new pharmacological tools that activate ALDH2 depict a new picture related to ALDH2 as a critical health-promoting enzyme.

Reperfusion-Induced Translocation of δPKC to Cardiac Mitochondria Prevents Pyruvate Dehydrogenase Reactivation

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 &dgr;-isoform of protein kinase C (PKC) to the mitochondria occurred during reperfusion. Inhibition of this process resulted in full recovery of PDH activity. Infusion of the &dgr;PKC activator H2O2 during normoxic perfusion, to mimic one aspect of cardiac reperfusion, resulted in loss in PDH activity that was largely attributable to translocation of &dgr;PKC to the mitochondria. Evidence indicates that reperfusion-induced translocation of &dgr;PKC is associated with phosphorylation of the &agr;E1 subunit of PDH. A potential mechanism is provided by in vitro data demonstrating that &dgr;PKC specifically interacts with and phosphorylates pyruvate dehydrogenase kinase (PDK)2. Importantly, this results in activation of PDK2, an enzyme capable of phosphorylating and inhibiting PDH. Thus, translocation of &dgr;PKC to the mitochondria during reperfusion likely results in activation of PDK2 and phosphorylation-dependent inhibition of PDH.

Mitochondrial aldehyde dehydrogenase and cardiac diseases

Numerous conditions promote oxidative stress, leading to the build-up of reactive aldehydes that cause cell damage and contribute to cardiac diseases. Aldehyde dehydrogenases (ALDHs) are important enzymes that eliminate toxic aldehydes by catalysing their oxidation to non-reactive acids. The review will discuss evidence indicating a role for a specific ALDH enzyme, the mitochondrial ALDH2, in combating oxidative stress by reducing the cellular ‘aldehydic load’. Epidemiological studies in humans carrying an inactive ALDH2, genetic models in mice with altered ALDH2 levels, and small molecule activators of ALDH2 all highlight the role of ALDH2 in cardioprotection and suggest a promising new direction in cardiovascular research and the development of new treatments for cardiovascular diseases.

Activation of aldehyde dehydrogenase 2 (ALDH2) confers cardioprotection in protein kinase C epsilon (PKCɛ) knockout mice

Acute administration of ethanol can reduce cardiac ischemia/reperfusion injury. Previous studies demonstrated that the acute cytoprotective effect of ethanol on the myocardium is mediated by protein kinase C epsilon (PKCvarepsilon). We recently identified aldehyde dehydrogenase 2 (ALDH2) as a PKCvarepsilon substrate, whose activation is necessary and sufficient to confer cardioprotection in vivo. ALDH2 metabolizes cytotoxic reactive aldehydes, such as 4-hydroxy-2-nonenal (4-HNE), which accumulate during cardiac ischemia/reperfusion. Here, we used a combination of PKCvarepsilon knockout mice and a direct activator of ALDH2, Alda-44, to further investigate the interplay between PKCvarepsilon and ALDH2 in cardioprotection. We report that ethanol preconditioning requires PKCvarepsilon, whereas direct activation of ALDH2 reduces infarct size in both wild type and PKCvarepsilon knockout hearts. Our data suggest that ALDH2 is downstream of PKCvarepsilon in ethanol preconditioning and that direct activation of ALDH2 can circumvent the requirement of PKCvarepsilon to induce cytoprotection. We also report that in addition to ALDH2 activation, Alda-44 prevents 4-HNE induced inactivation of ALDH2 by reducing the formation of 4-HNE-ALDH2 protein adducts. Thus, Alda-44 promotes metabolism of cytotoxic reactive aldehydes that accumulate in ischemic myocardium. Taken together, our findings suggest that direct activation of ALDH2 may represent a method of harnessing the cardioprotective effect of ethanol without the side effects associated with alcohol consumption.

Mitochondrial reactive oxygen species at the heart of the matter: new therapeutic approaches for cardiovascular diseases.

Reactive oxygen species (ROS) have been implicated in a variety of age-related diseases, including multiple cardiovascular disorders. However, translation of ROS scavengers (antioxidants) into the clinic has not been successful. These antioxidants grossly reduce total levels of cellular ROS including ROS that participate in physiological signaling. In this review, we challenge the traditional antioxidant therapeutic approach that targets ROS directly with novel approaches that improve mitochondrial functions to more effectively treat cardiovascular diseases.

Aldehyde dehydrogenase 2 activation in heart failure restores mitochondrial function and improves ventricular function and remodelling

AIMS We previously demonstrated that pharmacological activation of mitochondrial aldehyde dehydrogenase 2 (ALDH2) protects the heart against acute ischaemia/reperfusion injury. Here, we determined the benefits of chronic activation of ALDH2 on the progression of heart failure (HF) using a post-myocardial infarction model. METHODS AND RESULTS We showed that a 6-week treatment of myocardial infarction-induced HF rats with a selective ALDH2 activator (Alda-1), starting 4 weeks after myocardial infarction at a time when ventricular remodelling and cardiac dysfunction were present, improved cardiomyocyte shortening, cardiac function, left ventricular compliance and diastolic function under basal conditions, and after isoproterenol stimulation. Importantly, sustained Alda-1 treatment showed no toxicity and promoted a cardiac anti-remodelling effect by suppressing myocardial hypertrophy and fibrosis. Moreover, accumulation of 4-hydroxynonenal (4-HNE)-protein adducts and protein carbonyls seen in HF was not observed in Alda-1-treated rats, suggesting that increasing the activity of ALDH2 contributes to the reduction of aldehydic load in failing hearts. ALDH2 activation was associated with improved mitochondrial function, including elevated mitochondrial respiratory control ratios and reduced H2O2 release. Importantly, selective ALDH2 activation decreased mitochondrial Ca(2+)-induced permeability transition and cytochrome c release in failing hearts. Further supporting a mitochondrial mechanism for ALDH2, Alda-1 treatment preserved mitochondrial function upon in vitro aldehydic load. CONCLUSIONS Selective activation of mitochondrial ALDH2 is sufficient to improve the HF outcome by reducing the toxic effects of aldehydic overload on mitochondrial bioenergetics and reactive oxygen species generation, suggesting that ALDH2 activators, such as Alda-1, have a potential therapeutic value for treating HF patients.

A Novel Aldehyde Dehydrogenase-3 Activator Leads to Adult Salivary Stem Cell Enrichment In Vivo

Purpose: To assess aldehyde dehydrogenase (ALDH) expression in adult human and murine submandibular gland (SMG) stem cells and to determine the effect of ALDH3 activation in SMG stem cell enrichment. Experimental Design: Adult human and murine SMG stem cells were selected by cell surface markers (CD34 for human and c-Kit for mouse) and characterized for various other stem cell surface markers by flow cytometry and ALDH isozymes expression by quantitative reverse transcriptase PCR. Sphere formation and bromodeoxyuridine (BrdUrd) incorporation assays were used on selected cells to confirm their renewal capacity and three-dimensional (3D) collagen matrix culture was applied to observe differentiation. To determine whether ALDH3 activation would increase stem cell yield, adult mice were infused with a novel ALDH3 activator (Alda-89) or with vehicle followed by quantification of c-Kit+/CD90+ SMG stem cells and BrdUrd+ salispheres. Results: More than 99% of CD34+ huSMG stem cells stained positive for c-Kit, CD90 and 70% colocalized with CD44, Nestin. Similarly, 73.8% c-Kit+ mSMG stem cells colocalized with Sca-1, whereas 80.7% with CD90. Functionally, these cells formed BrdUrd+ salispheres, which differentiated into acinar- and ductal-like structures when cultured in 3D collagen. Both adult human and murine SMG stem cells showed higher expression of ALDH3 than in their non–stem cells and 84% of these cells have measurable ALDH1 activity. Alda-89 infusion in adult mice significantly increased c-Kit+/CD90+ SMG population and BrdUrd+ sphere formation compared with control. Conclusion: This is the first study to characterize expression of different ALDH isozymes in SMG stem cells. In vivo activation of ALDH3 can increase SMG stem cell yield, thus providing a novel means for SMG stem cell enrichment for future stem cell therapy. Clin Cancer Res; 17(23); 7265–72. ©2011 AACR.

Aldehyde dehydrogenase-2 regulates nociception in rodent models of acute inflammatory pain

Pain sensitivity can be modulated by an enzyme that degrades aldehydes in rodents, pointing to a promising therapeutic target. New Path to Pain Control Pain is a seriously undertreated condition, and new drugs are sorely needed. Now, Zambelli et al. find that an inactivating mutation of the enzyme that degrades aldehydes—a known pain-causing molecule—markedly increases rodents’ susceptibility to a painful stimulus. Conversely, activating the enzyme with the drug Alda-1 reverses this effect. Indeed, the authors found a tight correlation between the local concentration of aldehydes at the injury site and the magnitude of the pain response. New drugs that modulate aldehyde levels could prove beneficial for patients, possibly without the addiction that plagues opiate pain killers. In addition, these results may explain the greater sensitivity to pain reported in East Asian people, many of whom carry a mutation in this same aldehyde-degrading enzyme. Exogenous aldehydes can cause pain in animal models, suggesting that aldehyde dehydrogenase-2 (ALDH2), which metabolizes many aldehydes, may regulate nociception. To test this hypothesis, we generated a knock-in mouse with an inactivating point mutation in ALDH2 (ALDH2*2), which is also present in human ALDH2 of ~540 million East Asians. The ALDH2*1/*2 heterozygotic mice exhibited a larger response to painful stimuli than their wild-type littermates, and this heightened nociception was inhibited by an ALDH2-selective activator (Alda-1). No effect on inflammation per se was observed. Using a rat model, we then showed that nociception tightly correlated with ALDH activity (R2 = 0.90) and that reduced nociception was associated with less early growth response protein 1 (EGR1) in the spinal cord and less reactive aldehyde accumulation at the insult site (including acetaldehyde and 4-hydroxynonenal). Further, acetaldehyde- and formalin-induced nociceptive behavior was greater in the ALDH2*1/*2 mice than in the wild-type mice. Finally, Alda-1 treatment was even beneficial when given after the inflammatory agent was administered. Our data in rodent models suggest that the mitochondrial enzyme ALDH2 regulates nociception and could serve as a molecular target for pain control, with ALDH2 activators, such as Alda-1, as potential non-narcotic, cardiac-safe analgesics. Furthermore, our results suggest a possible genetic basis for East Asians’ apparent lower pain tolerance.