Daria Mochly-rosen - Academia.edu (original) (raw)
Papers by Daria Mochly-rosen
Science Translational Medicine, 2011
Tolerance to nitroglycerin, a drug often used to treat chest pain, increases susceptibility to in... more Tolerance to nitroglycerin, a drug often used to treat chest pain, increases susceptibility to injury by myocardial infarction.
A family of detoxifying enzymes called aldehyde dehydrogenases (ALDHs) has been a subject of rece... more 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. I. INTRODUCTION 1 II. MOUSE MODELS WITH MODIFIED... 4 III.
Circulation, 2003
From the Departments of Molecular Pharmacology (K. Inagaki, LC, DM-R.), Cardiovascular Medicine (... more From the Departments of Molecular Pharmacology (K. Inagaki, LC, DM-R.), Cardiovascular Medicine (FI, FHL, MR, PGY), and Comparative Medicine (DMB), Stanford University School of Medicine, Stanford, Calif; and the Laboratory of Signal Transduction, National Institute of ...
In vivo measurement of aldehyde dehydrogenase-2 activity in rat liver ethanol model using dynamic MRSI of hyperpolarized [1- 13 C]pyruvate
NMR in Biomedicine, 2012
To date, measurements of the activity of aldehyde dehydrogenase-2 (ALDH2), a critical mitochondri... more To date, measurements of the activity of aldehyde dehydrogenase-2 (ALDH2), a critical mitochondrial enzyme for the elimination of certain cytotoxic aldehydes in the body and a promising target for drug development, have been largely limited to in vitro methods. Recent advancements in MRS of hyperpolarized (13) C-labeled substrates have provided a method to detect and image in vivo metabolic pathways with signal-to-noise ratio gains greater than 10 000-fold over conventional MRS techniques. However aldehydes, because of their toxicity and short T1 relaxation times, are generally poor targets for such (13) C-labeled studies. In this work, we show that dynamic MRSI of hyperpolarized [1-(13) C]pyruvate and its conversion to [1-(13) C]lactate can provide an indirect in vivo measurement of ALDH2 activity via the concentration of NADH (nicotinamide adenine dinucleotide, reduced form), a co-factor common to both the reduction of pyruvate to lactate and the oxidation of acetaldehyde to acetate. Results from a rat liver ethanol model (n = 9) show that changes in (13) C-lactate labeling following the bolus injection of hyperpolarized pyruvate are highly correlated with changes in ALDH2 activity (R(2) = 0.76).
Background—Protein kinase C (PKC) plays a major role in cardioprotection from ischemia/reperfusio... more Background—Protein kinase C (PKC) plays a major role in cardioprotection from ischemia/reperfusion injury. Using an HIV-1 Tat protein- derived peptide to mediate rapid and efficient transmembrane delivery of peptide regulators of PKC translocation and function, we examined the cardioprotective effect of selective -PKC inhibitor (V1-1) and -PKC activator (RACK) peptides for ischemia/reperfusion damage in isolated perfused rat hearts. Furthermore, we
The many hats of protein kinase Cδ: one enzyme with many functions
Biochemical Society transactions, 2014
A large number of protein substrates are phosphorylated by each protein kinase under physiologica... more A large number of protein substrates are phosphorylated by each protein kinase under physiological and pathological conditions. However, it remains a challenge to determine which of these phosphorylated substrates of a given kinase is critical for each cellular response. Genetics enabled the generation of separation-of-function mutations that selectively cause a loss of one molecular event without affecting others, thus providing some tools to assess the importance of that one event for the measured physiological response. However, the genetic approach is laborious and not adaptable to all systems. Furthermore, pharmacological tools of the catalytic site are not optimal due to their non-selective nature. In the present brief review, we discuss some of the challenges in drug development that will regulate the multifunctional protein kinase Cδ (PKCδ).
Drug discovery today. Disease mechanisms, 2010
Heart failure (HF) in which the blood supply does not match the body's needs, affects 10% of ... more Heart failure (HF) in which the blood supply does not match the body's needs, affects 10% of the population over 65 years old. The protein kinase C (PKC) family of kinases has a key role in normal and disease states. Here we discuss the role of PKC in HF and focus on the use of specific PKC regulators to identify the mechanism leading to this Pathology and potential leads for therapeutics.
Cardioprotection from ischemia by a brief exposure to physiological levels of ethanol: role of epsilon protein kinase C
Proceedings of the National Academy of Sciences of the United States of America, Jan 26, 1999
Recent epidemiological studies indicate beneficial effects of moderate ethanol consumption in isc... more Recent epidemiological studies indicate beneficial effects of moderate ethanol consumption in ischemic heart disease. Most studies, however, focus on the effect of long-term consumption of ethanol. In this study, we determined whether brief exposure to ethanol immediately before ischemia also produces cardioprotection. In addition, because protein kinase C (PKC) has been shown to mediate protection of the heart from ischemia, we determined the role of specific PKC isozymes in ethanol-induced protection. We demonstrated that (i) brief exposure of isolated adult rat cardiac myocytes to 10-50 mM ethanol protected against damage induced by prolonged ischemia; (ii) an isozyme-selective epsilonPKC inhibitor developed in our laboratory inhibited the cardioprotective effect of acute ethanol exposure; (iii) protection of isolated intact adult rat heart also occurred after incubation with 10 mM ethanol 20 min before global ischemia; and (iv) ethanol-induced cardioprotection depended on PKC ac...
Isozyme-specific inhibitors and activators of protein kinase C
Methods in Enzymology, 2002
We describe here the methods we have used to generate selective peptide inhibitors and activators... more We describe here the methods we have used to generate selective peptide inhibitors and activators of PKC-mediated signaling. These approaches should be applicable to any signaling event that is dependent on protein-protein interaction. Furthermore, targeting downstream enzymes in signal transduction has been notoriously difficult as there are often families of related enzymes in each cell. The approaches we have used overcame this difficulty and may prove useful not only in basic research, but also in drug discovery.
Science translational medicine, Jan 27, 2014
Exogenous aldehydes can cause pain in animal models, suggesting that aldehyde dehydrogenase-2 (AL... more 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 (R(2) = 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 AL...
Experimental neurology, 2015
Many studies have shown that mitochondrial aldehyde dehydrogenase 2 (ALDH2) functions as a cellul... more Many studies have shown that mitochondrial aldehyde dehydrogenase 2 (ALDH2) functions as a cellular protector against oxidative stress by detoxification of cytotoxic aldehydes. Within dopaminergic neurons, dopamine is metabolized by monoamine oxidase to yield 3,4-dihydroxyphenylacetaldehyde (DOPAL) then converts to a less toxic acid product by ALDH. The highly toxic and reactive DOPAL has been hypothesized to contribute to the selective neurodegeneration in Parkinson's disease (PD). In this study, we investigated the neuroprotective mechanism and therapeutic effect of ALDH2 in rotenone models for parkinsonism. Overexpression of wild-type ALDH2 gene, but not the enzymatically deficient mutant ALDH2*2 (E504K), reduced rotenone-induced cell death. Application of a potent activator of ALDH2, Alda-1, was effective in protecting against rotenone-induced apoptotic cell death in both SH-SY5Y cells and primary cultured substantia nigra (SN) dopaminergic neurons. In addition, intraperiton...
The challenge in translating basic research discoveries to treatment of Huntington disease
Rare diseases (Austin, Tex.), 2014
Huntington disease is a rare neurodegenerative disease resulting from insertion and/or expansion ... more Huntington disease is a rare neurodegenerative disease resulting from insertion and/or expansion of a polyglutamine repeats close to the N-terminal of the huntingtin protein. Although unequivocal genetic tests have been available for about 20 years, current pharmacological treatments do not prevent or slow down disease progression. Recent basic research identified potential novel drug targets for the treatment of Huntington disease. However, there are clear challenges in translating these discoveries into treatment strategies for these patients. The following is a brief discussion of these challenges using our recent experience as an example.
23 Delta PKC in cerebral ischemic and reperfusion injury in vivo
Journal of Molecular and Cellular Cardiology, 2002
Dopamine and Ethanol Cause Translocation of PKC Associated with RACK: Cross-Talk between cAMP-Dependent Protein Kinase A and Protein Kinase C Signaling Pathways
Molecular Pharmacology, 2008
We found previously that neural responses to ethanol and the dopamine D2 receptor (D2) agonist 2,... more We found previously that neural responses to ethanol and the dopamine D2 receptor (D2) agonist 2,10,11-trihydroxy-N-propylnorapomorphine hydrobromide (NPA) involve both epsilon protein kinase C (epsilonPKC) and cAMP-dependent protein kinase A (PKA). However, little is known about the mechanism underlying ethanol- and D2-mediated activation of epsilonPKC and the relationship to PKA activation. In the present study, we used a new epsilonPKC antibody, 14E6, that selectively recognized active epsilonPKC when not bound to its anchoring protein epsilonRACK (receptor for activated C-kinase), and PKC isozyme-selective inhibitors and activators to measure PKC translocation and catalytic activity. We show here that ethanol and NPA activated epsilonPKC and induced translocation of both epsilonPKC and its anchoring protein, epsilonRACK to a new cytosolic site. The selective epsilonPKC agonist, pseudo-epsilonRACK, activated epsilonPKC but did not cause translocation of the epsilonPKC/epsilonRACK complex to the cytosol. These data suggest a step-wise activation and translocation of epsilonPKC after NPA or ethanol treatment, where epsilonPKC first translocates and binds to its RACK and subsequently the epsilonPKC/epsilonRACK complex translocates to a new subcellular site. Direct activation of PKA by adenosine-3',5'-cyclic monophosphorothioate, Sp-isomer (Sp-cAMPS), prostaglandin E1, or the adenosine A2A receptor is sufficient to cause epsilonPKC translocation to the cytosolic compartment in a process that is dependent on PLC activation and requires PKA activity. These data demonstrate a novel cross-talk mechanism between epsilonPKC and PKA signaling systems. PKA and PKC signaling have been implicated in alcohol rewarding properties in the mesolimbic dopamine system. Cross-talk between PKA and PKC may underlie some of the behaviors associated with alcoholism.
Biochemistry, 1999
The pleckstrin homology (PH) domain, identified in numerous signaling proteins including the -adr... more The pleckstrin homology (PH) domain, identified in numerous signaling proteins including the -adrenergic receptor kinase ( ARK), was found to bind to various phospholipids as well as the subunit of heterotrimeric G proteins (G ) [Touhara, K., et al. (1994) J. Biol. Chem. 269, 10217-10220]. Several PH domain-containing proteins are also substrates of protein kinase C (PKC). Because RACK1, an anchoring protein for activated PKC, is homologous to G (both contain seven repeats of the WD-40 motif), we determined (i) whether a direct interaction between various PH domains and RACK1 occurs and (ii) the effect of PKC on this interaction. We found that recombinant PH domains of several proteins exhibited differential binding to RACK1. Activated PKC and the PH domain of -spectrin or dynamin-1 concomitantly bound to RACK1. Although PH domains bind acidic phospholipids, the interaction between various PH domains and RACK1 was not dependent on the phospholipid activators of PKC, phosphatidylserine and 1,2-diacylglycerol. Binding of these PH domains to RACK1 was also not affected by either inositol 1,4,5-triphosphate (IP 3 ) or phosphatidylinositol 4,5-bisphosphate (PIP 2 ). Our in vitro data suggest that RACK1 binds selective PH domains, and that PKC regulates this interaction. We propose that, in vivo, RACK1 may colocalize the kinase with its PH domain-containing substrates.
PKC Isozymes in Chronic Cardiac Disease: Possible Therapeutic Targets?
Annual Review of Pharmacology and Toxicology, 2008
Cardiovascular disease is the leading cause of death in the United States. Therefore, identifying... more Cardiovascular disease is the leading cause of death in the United States. Therefore, identifying therapeutic targets is a major focus of current research. Protein kinase C (PKC), a family of serine/threonine kinases, has been identified as playing a role in many of the pathologies of heart disease. However, the lack of specific PKC regulators and the ubiquitous expression and normal physiological functions of the 11 PKC isozymes has made drug development a challenge. Here we discuss the validity of therapeutically targeting PKC, an intracellular signaling enzyme. We describe PKC structure, function, and distribution in the healthy and diseased heart, as well as the development of rationally designed isozyme-selective regulators of PKC functions. The review focuses on the roles of specific PKC isozymes in atherosclerosis, fibrosis, and cardiac hypertrophy, and examines principles of pharmacology as they pertain to regulators of signaling cascades associated with these diseases.
Journal of Heart and Lung Transplantation, 2009
Purpose: The prevention of chronic allograft vasculopathy (CAV) after heart transplantation remai... more Purpose: The prevention of chronic allograft vasculopathy (CAV) after heart transplantation remains a major problem for long-term success. Epsilon protein kinase C (⑀PKC) is a PKC isoform that plays pivotal roles in myocardial infarction and in heart failure. Here we investigate whether PKC epsilon activation is also involved in the development of intimal hyperplasia. Methods and Materials: Rats underwent balloon denudation of the abdominal aorta and received either 3mM ⑀PKC activator (yeRACK), 3mM ⑀PKC inhibitor (⑀V1-2), the carrier control (TAT 47-57 ), or saline by osmotic pump at ϳ3mg/kg/day for 4 weeks (6 rats/group). The treatment began intraoperatively). Aortas were harvested for histologic evaluation, and luminal obliteration and intima/media ratios were analyzed using computer morphometry. Results: Histology of untreated animals revealed marked intimal hyperplasia with moderate luminal obliteration (19.9Ϯ9%). Neointima formation was significantly increased by the ⑀PKC activator (32Ϯ5.5%; pϭ0.017 vs. untreated) and significantly decreased by the ⑀PKC inhibitor (9.1Ϯ4.3%; pϭ0.016 vs. untreated). No difference was observed between the untreated control and the TAT carrier peptide contol groups (pϭ0.43). The intima/media ratio was significantly higher in the ⑀PKC activator group compared to the ⑀PKC inhibitor group (0.67Ϯ0.46 and 0.25Ϯ0.46, respectively; pϭ0.034). Treatment with either of the ePKC regulators was very well tolerated and the animals in the ⑀PKC activator as well as the ⑀PKC inhibitor groups gained weight during the 4 week treatment period (105Ϯ1.9% and 102Ϯ3.4%, respectively; pϭns). No differences in creatinine, BUN, cholesterol, triglycerides, ALT, and AST were observed between the four groups. Conclusions: These data suggest that ⑀PKC activity contributes to the non-immunological development of intimal hyperplasia and that an ⑀PKC-selective inhibitor, such as ⑀V1-2, could augment current therapeutic strategies to suppress the development of vascular stenosis.
Journal of Cardiac Failure, 2008
polymorphism and bucindolol's remodeling effects; 2) ischemic cardiomyopathy patients who carry t... more polymorphism and bucindolol's remodeling effects; 2) ischemic cardiomyopathy patients who carry the Gly b 1 389 allele have a lower LVEF response to bucindolol, and those who carry the b 1 389 Gly plus the a 2c Del allele have no detectable response; 3) the b 1 389 Gly + a 2c Del carrier genotype is also associated with a loss of clinical efficacy in Isc, adding further support to the idea that patients with this ''unfavorable'' genotype should not treated with bucindolol. Further studies will be necessary to confirm whether ischemic and non-ischemic cardiomyopathy patients have different pharmacogenetically predicted remodeling responses to bucindolol.
Aldehyde dehydrogenase 2 activation in heart failure restores mitochondrial function and improves ventricular function and remodelling
Cardiovascular Research, 2014
We previously demonstrated that pharmacological activation of mitochondrial aldehyde dehydrogenas... more 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. 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. 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.
Science Translational Medicine, 2011
Tolerance to nitroglycerin, a drug often used to treat chest pain, increases susceptibility to in... more Tolerance to nitroglycerin, a drug often used to treat chest pain, increases susceptibility to injury by myocardial infarction.
A family of detoxifying enzymes called aldehyde dehydrogenases (ALDHs) has been a subject of rece... more 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. I. INTRODUCTION 1 II. MOUSE MODELS WITH MODIFIED... 4 III.
Circulation, 2003
From the Departments of Molecular Pharmacology (K. Inagaki, LC, DM-R.), Cardiovascular Medicine (... more From the Departments of Molecular Pharmacology (K. Inagaki, LC, DM-R.), Cardiovascular Medicine (FI, FHL, MR, PGY), and Comparative Medicine (DMB), Stanford University School of Medicine, Stanford, Calif; and the Laboratory of Signal Transduction, National Institute of ...
In vivo measurement of aldehyde dehydrogenase-2 activity in rat liver ethanol model using dynamic MRSI of hyperpolarized [1- 13 C]pyruvate
NMR in Biomedicine, 2012
To date, measurements of the activity of aldehyde dehydrogenase-2 (ALDH2), a critical mitochondri... more To date, measurements of the activity of aldehyde dehydrogenase-2 (ALDH2), a critical mitochondrial enzyme for the elimination of certain cytotoxic aldehydes in the body and a promising target for drug development, have been largely limited to in vitro methods. Recent advancements in MRS of hyperpolarized (13) C-labeled substrates have provided a method to detect and image in vivo metabolic pathways with signal-to-noise ratio gains greater than 10 000-fold over conventional MRS techniques. However aldehydes, because of their toxicity and short T1 relaxation times, are generally poor targets for such (13) C-labeled studies. In this work, we show that dynamic MRSI of hyperpolarized [1-(13) C]pyruvate and its conversion to [1-(13) C]lactate can provide an indirect in vivo measurement of ALDH2 activity via the concentration of NADH (nicotinamide adenine dinucleotide, reduced form), a co-factor common to both the reduction of pyruvate to lactate and the oxidation of acetaldehyde to acetate. Results from a rat liver ethanol model (n = 9) show that changes in (13) C-lactate labeling following the bolus injection of hyperpolarized pyruvate are highly correlated with changes in ALDH2 activity (R(2) = 0.76).
Background—Protein kinase C (PKC) plays a major role in cardioprotection from ischemia/reperfusio... more Background—Protein kinase C (PKC) plays a major role in cardioprotection from ischemia/reperfusion injury. Using an HIV-1 Tat protein- derived peptide to mediate rapid and efficient transmembrane delivery of peptide regulators of PKC translocation and function, we examined the cardioprotective effect of selective -PKC inhibitor (V1-1) and -PKC activator (RACK) peptides for ischemia/reperfusion damage in isolated perfused rat hearts. Furthermore, we
The many hats of protein kinase Cδ: one enzyme with many functions
Biochemical Society transactions, 2014
A large number of protein substrates are phosphorylated by each protein kinase under physiologica... more A large number of protein substrates are phosphorylated by each protein kinase under physiological and pathological conditions. However, it remains a challenge to determine which of these phosphorylated substrates of a given kinase is critical for each cellular response. Genetics enabled the generation of separation-of-function mutations that selectively cause a loss of one molecular event without affecting others, thus providing some tools to assess the importance of that one event for the measured physiological response. However, the genetic approach is laborious and not adaptable to all systems. Furthermore, pharmacological tools of the catalytic site are not optimal due to their non-selective nature. In the present brief review, we discuss some of the challenges in drug development that will regulate the multifunctional protein kinase Cδ (PKCδ).
Drug discovery today. Disease mechanisms, 2010
Heart failure (HF) in which the blood supply does not match the body's needs, affects 10% of ... more Heart failure (HF) in which the blood supply does not match the body's needs, affects 10% of the population over 65 years old. The protein kinase C (PKC) family of kinases has a key role in normal and disease states. Here we discuss the role of PKC in HF and focus on the use of specific PKC regulators to identify the mechanism leading to this Pathology and potential leads for therapeutics.
Cardioprotection from ischemia by a brief exposure to physiological levels of ethanol: role of epsilon protein kinase C
Proceedings of the National Academy of Sciences of the United States of America, Jan 26, 1999
Recent epidemiological studies indicate beneficial effects of moderate ethanol consumption in isc... more Recent epidemiological studies indicate beneficial effects of moderate ethanol consumption in ischemic heart disease. Most studies, however, focus on the effect of long-term consumption of ethanol. In this study, we determined whether brief exposure to ethanol immediately before ischemia also produces cardioprotection. In addition, because protein kinase C (PKC) has been shown to mediate protection of the heart from ischemia, we determined the role of specific PKC isozymes in ethanol-induced protection. We demonstrated that (i) brief exposure of isolated adult rat cardiac myocytes to 10-50 mM ethanol protected against damage induced by prolonged ischemia; (ii) an isozyme-selective epsilonPKC inhibitor developed in our laboratory inhibited the cardioprotective effect of acute ethanol exposure; (iii) protection of isolated intact adult rat heart also occurred after incubation with 10 mM ethanol 20 min before global ischemia; and (iv) ethanol-induced cardioprotection depended on PKC ac...
Isozyme-specific inhibitors and activators of protein kinase C
Methods in Enzymology, 2002
We describe here the methods we have used to generate selective peptide inhibitors and activators... more We describe here the methods we have used to generate selective peptide inhibitors and activators of PKC-mediated signaling. These approaches should be applicable to any signaling event that is dependent on protein-protein interaction. Furthermore, targeting downstream enzymes in signal transduction has been notoriously difficult as there are often families of related enzymes in each cell. The approaches we have used overcame this difficulty and may prove useful not only in basic research, but also in drug discovery.
Science translational medicine, Jan 27, 2014
Exogenous aldehydes can cause pain in animal models, suggesting that aldehyde dehydrogenase-2 (AL... more 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 (R(2) = 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 AL...
Experimental neurology, 2015
Many studies have shown that mitochondrial aldehyde dehydrogenase 2 (ALDH2) functions as a cellul... more Many studies have shown that mitochondrial aldehyde dehydrogenase 2 (ALDH2) functions as a cellular protector against oxidative stress by detoxification of cytotoxic aldehydes. Within dopaminergic neurons, dopamine is metabolized by monoamine oxidase to yield 3,4-dihydroxyphenylacetaldehyde (DOPAL) then converts to a less toxic acid product by ALDH. The highly toxic and reactive DOPAL has been hypothesized to contribute to the selective neurodegeneration in Parkinson's disease (PD). In this study, we investigated the neuroprotective mechanism and therapeutic effect of ALDH2 in rotenone models for parkinsonism. Overexpression of wild-type ALDH2 gene, but not the enzymatically deficient mutant ALDH2*2 (E504K), reduced rotenone-induced cell death. Application of a potent activator of ALDH2, Alda-1, was effective in protecting against rotenone-induced apoptotic cell death in both SH-SY5Y cells and primary cultured substantia nigra (SN) dopaminergic neurons. In addition, intraperiton...
The challenge in translating basic research discoveries to treatment of Huntington disease
Rare diseases (Austin, Tex.), 2014
Huntington disease is a rare neurodegenerative disease resulting from insertion and/or expansion ... more Huntington disease is a rare neurodegenerative disease resulting from insertion and/or expansion of a polyglutamine repeats close to the N-terminal of the huntingtin protein. Although unequivocal genetic tests have been available for about 20 years, current pharmacological treatments do not prevent or slow down disease progression. Recent basic research identified potential novel drug targets for the treatment of Huntington disease. However, there are clear challenges in translating these discoveries into treatment strategies for these patients. The following is a brief discussion of these challenges using our recent experience as an example.
23 Delta PKC in cerebral ischemic and reperfusion injury in vivo
Journal of Molecular and Cellular Cardiology, 2002
Dopamine and Ethanol Cause Translocation of PKC Associated with RACK: Cross-Talk between cAMP-Dependent Protein Kinase A and Protein Kinase C Signaling Pathways
Molecular Pharmacology, 2008
We found previously that neural responses to ethanol and the dopamine D2 receptor (D2) agonist 2,... more We found previously that neural responses to ethanol and the dopamine D2 receptor (D2) agonist 2,10,11-trihydroxy-N-propylnorapomorphine hydrobromide (NPA) involve both epsilon protein kinase C (epsilonPKC) and cAMP-dependent protein kinase A (PKA). However, little is known about the mechanism underlying ethanol- and D2-mediated activation of epsilonPKC and the relationship to PKA activation. In the present study, we used a new epsilonPKC antibody, 14E6, that selectively recognized active epsilonPKC when not bound to its anchoring protein epsilonRACK (receptor for activated C-kinase), and PKC isozyme-selective inhibitors and activators to measure PKC translocation and catalytic activity. We show here that ethanol and NPA activated epsilonPKC and induced translocation of both epsilonPKC and its anchoring protein, epsilonRACK to a new cytosolic site. The selective epsilonPKC agonist, pseudo-epsilonRACK, activated epsilonPKC but did not cause translocation of the epsilonPKC/epsilonRACK complex to the cytosol. These data suggest a step-wise activation and translocation of epsilonPKC after NPA or ethanol treatment, where epsilonPKC first translocates and binds to its RACK and subsequently the epsilonPKC/epsilonRACK complex translocates to a new subcellular site. Direct activation of PKA by adenosine-3',5'-cyclic monophosphorothioate, Sp-isomer (Sp-cAMPS), prostaglandin E1, or the adenosine A2A receptor is sufficient to cause epsilonPKC translocation to the cytosolic compartment in a process that is dependent on PLC activation and requires PKA activity. These data demonstrate a novel cross-talk mechanism between epsilonPKC and PKA signaling systems. PKA and PKC signaling have been implicated in alcohol rewarding properties in the mesolimbic dopamine system. Cross-talk between PKA and PKC may underlie some of the behaviors associated with alcoholism.
Biochemistry, 1999
The pleckstrin homology (PH) domain, identified in numerous signaling proteins including the -adr... more The pleckstrin homology (PH) domain, identified in numerous signaling proteins including the -adrenergic receptor kinase ( ARK), was found to bind to various phospholipids as well as the subunit of heterotrimeric G proteins (G ) [Touhara, K., et al. (1994) J. Biol. Chem. 269, 10217-10220]. Several PH domain-containing proteins are also substrates of protein kinase C (PKC). Because RACK1, an anchoring protein for activated PKC, is homologous to G (both contain seven repeats of the WD-40 motif), we determined (i) whether a direct interaction between various PH domains and RACK1 occurs and (ii) the effect of PKC on this interaction. We found that recombinant PH domains of several proteins exhibited differential binding to RACK1. Activated PKC and the PH domain of -spectrin or dynamin-1 concomitantly bound to RACK1. Although PH domains bind acidic phospholipids, the interaction between various PH domains and RACK1 was not dependent on the phospholipid activators of PKC, phosphatidylserine and 1,2-diacylglycerol. Binding of these PH domains to RACK1 was also not affected by either inositol 1,4,5-triphosphate (IP 3 ) or phosphatidylinositol 4,5-bisphosphate (PIP 2 ). Our in vitro data suggest that RACK1 binds selective PH domains, and that PKC regulates this interaction. We propose that, in vivo, RACK1 may colocalize the kinase with its PH domain-containing substrates.
PKC Isozymes in Chronic Cardiac Disease: Possible Therapeutic Targets?
Annual Review of Pharmacology and Toxicology, 2008
Cardiovascular disease is the leading cause of death in the United States. Therefore, identifying... more Cardiovascular disease is the leading cause of death in the United States. Therefore, identifying therapeutic targets is a major focus of current research. Protein kinase C (PKC), a family of serine/threonine kinases, has been identified as playing a role in many of the pathologies of heart disease. However, the lack of specific PKC regulators and the ubiquitous expression and normal physiological functions of the 11 PKC isozymes has made drug development a challenge. Here we discuss the validity of therapeutically targeting PKC, an intracellular signaling enzyme. We describe PKC structure, function, and distribution in the healthy and diseased heart, as well as the development of rationally designed isozyme-selective regulators of PKC functions. The review focuses on the roles of specific PKC isozymes in atherosclerosis, fibrosis, and cardiac hypertrophy, and examines principles of pharmacology as they pertain to regulators of signaling cascades associated with these diseases.
Journal of Heart and Lung Transplantation, 2009
Purpose: The prevention of chronic allograft vasculopathy (CAV) after heart transplantation remai... more Purpose: The prevention of chronic allograft vasculopathy (CAV) after heart transplantation remains a major problem for long-term success. Epsilon protein kinase C (⑀PKC) is a PKC isoform that plays pivotal roles in myocardial infarction and in heart failure. Here we investigate whether PKC epsilon activation is also involved in the development of intimal hyperplasia. Methods and Materials: Rats underwent balloon denudation of the abdominal aorta and received either 3mM ⑀PKC activator (yeRACK), 3mM ⑀PKC inhibitor (⑀V1-2), the carrier control (TAT 47-57 ), or saline by osmotic pump at ϳ3mg/kg/day for 4 weeks (6 rats/group). The treatment began intraoperatively). Aortas were harvested for histologic evaluation, and luminal obliteration and intima/media ratios were analyzed using computer morphometry. Results: Histology of untreated animals revealed marked intimal hyperplasia with moderate luminal obliteration (19.9Ϯ9%). Neointima formation was significantly increased by the ⑀PKC activator (32Ϯ5.5%; pϭ0.017 vs. untreated) and significantly decreased by the ⑀PKC inhibitor (9.1Ϯ4.3%; pϭ0.016 vs. untreated). No difference was observed between the untreated control and the TAT carrier peptide contol groups (pϭ0.43). The intima/media ratio was significantly higher in the ⑀PKC activator group compared to the ⑀PKC inhibitor group (0.67Ϯ0.46 and 0.25Ϯ0.46, respectively; pϭ0.034). Treatment with either of the ePKC regulators was very well tolerated and the animals in the ⑀PKC activator as well as the ⑀PKC inhibitor groups gained weight during the 4 week treatment period (105Ϯ1.9% and 102Ϯ3.4%, respectively; pϭns). No differences in creatinine, BUN, cholesterol, triglycerides, ALT, and AST were observed between the four groups. Conclusions: These data suggest that ⑀PKC activity contributes to the non-immunological development of intimal hyperplasia and that an ⑀PKC-selective inhibitor, such as ⑀V1-2, could augment current therapeutic strategies to suppress the development of vascular stenosis.
Journal of Cardiac Failure, 2008
polymorphism and bucindolol's remodeling effects; 2) ischemic cardiomyopathy patients who carry t... more polymorphism and bucindolol's remodeling effects; 2) ischemic cardiomyopathy patients who carry the Gly b 1 389 allele have a lower LVEF response to bucindolol, and those who carry the b 1 389 Gly plus the a 2c Del allele have no detectable response; 3) the b 1 389 Gly + a 2c Del carrier genotype is also associated with a loss of clinical efficacy in Isc, adding further support to the idea that patients with this ''unfavorable'' genotype should not treated with bucindolol. Further studies will be necessary to confirm whether ischemic and non-ischemic cardiomyopathy patients have different pharmacogenetically predicted remodeling responses to bucindolol.
Aldehyde dehydrogenase 2 activation in heart failure restores mitochondrial function and improves ventricular function and remodelling
Cardiovascular Research, 2014
We previously demonstrated that pharmacological activation of mitochondrial aldehyde dehydrogenas... more 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. 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. 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.