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Chang Sik Park,
Shan Chen,
Hoyong Lee,
Hyeseon Cha,
Jae Gyun Oh,
Sunghee Hong,
Peidong Han,
Kenneth S Ginsburg,
Sora Jin,
Inju Park,
Vivek P Singh,
Hong-Sheng Wang,
Clara Franzini-Armstrong, Woo Jin Park,
Donald M Bers,
Evangelia G Kranias,
Chunghee Cho,
Do Han Kim
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ABSTRACT: The histidine-rich Ca(2+)-binding protein (HRC) is located in the lumen of the sarcoplasmic reticulum (SR) and exhibits high-capacity Ca(2+)-binding properties. Overexpression of HRC in the heart resulted in impaired SR Ca(2+) uptake and depressed relaxation through its interaction with SERCA2a. However, the functional significance of HRC in overall regulation of calcium cycling and contractility is not currently well defined. To further elucidate the role of HRC in vivo under physiological and pathophysiological conditions, we generated and characterized HRC-knockout (KO) mice. The KO mice were morphologically and histologically normal compared to wild-type (WT) mice. At the cellular level, ablation of HRC resulted in significantly enhanced contractility, Ca(2+) transients, and maximal SR Ca(2+) uptake rates in the heart. However, after-contractions were developed in 50 % of HRC-KO cardiomyocytes, compared to 11 % in WT mice under stress conditions of high-frequency stimulation (5 Hz) and isoproterenol application. A parallel examination of the electrical activity revealed significant increases in the occurrence of Ca(2+) spontaneous SR Ca(2+) release and delayed afterdepolarizations with ISO in HRC-KO, compared to WT cells. The frequency of Ca(2+) sparks was also significantly higher in HRC-KO cells with ISO, consistent with the elevated SR Ca(2+) load in the KO cells. Furthermore, HRC-KO cardiomyocytes showed significantly deteriorated cell contractility and Ca(2+)-cycling caused possibly by depressed SERCA2a expression after transverse-aortic constriction (TAC). Also HRC-null mice exhibited severe cardiac hypertrophy, fibrosis, pulmonary edema and decreased survival after TAC. Our results indicate that ablation of HRC is associated with poorly regulated SR Ca(2+)-cycling, and severe pathology under pressure-overload stress, suggesting an essential role of HRC in maintaining the integrity of cardiac function.
Archiv für Kreislaufforschung 05/2013; 108(3):344. · 7.35 Impact Factor
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ABSTRACT: Cardiac sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a) plays a crucial role in Ca(2+) handling in cardiomyocytes. Phospholamban (PLB) is an endogenous inhibitor of SERCA2a and its inhibitory activity is enhanced via dephosphorylation by protein phosphatase 1 (PP1). Therefore, inhibition of PP1-mediated dephosphorylation of PLB might be an efficient strategy for the restoration of reduced SERCA2a activity in failing hearts. We sought to develop decoy peptides that would mimic phosphorylated PLB and thus competitively inhibit the PP1-mediated dephosphorylation of endogenous PLB. The phosphorylation sites Ser16 and Thr17 are located within the flexible loop region (amino acids 14-22) of PLB. We therefore synthesized a 9-mer peptide derived from this region (ψPLB-wt) and two pseudo-phosphorylated peptides where Ser16 was replaced with Glu (ψPLB-SE) or Thr17 was replaced with Glu (ψPLB-TE). These peptides were coupled to the cell-permeable peptide TAT to facilitate cellular uptake. Treatment of adult rat cardiomyocytes with ψPLB-SE or ψPLB-TE, but not with ψPLB-wt, significantly elevated the phosphorylation levels of PLB at Ser16 and Thr17. This increased phosphorylation of PLB correlated with an increase in contractile parameters in vitro. Furthermore, perfusion of isolated rat hearts with ψPLB-SE or ψPLB-TE, but not with ψPLB-wt, significantly improved left ventricular developed pressure that had been previously impaired by ischemia. These data indicate that ψPLB-SE and ψPLB-TE efficiently prevented dephosphorylation of PLB by serving as decoys for PP1. Therefore, these peptides may provide an effective modality to regulate SERCA2a activity in failing hearts.
Journal of Molecular and Cellular Cardiology 12/2012; · 5.17 Impact Factor
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ABSTRACT: The plant viral protease, NIa, has a strict substrate specificity for the consensus sequence of Val-Xaa-His-Gln, with a scissoring property after Gln. We recently reported that NIa efficiently cleaved the amyloid-β (Aβ) peptide, which contains the sequence Val-His-His-Gln in the vicinity of the cleavage site by α-secretase, and that the expression of NIa using a Lenti-viral system in the brain of AD mouse model reduced plaque deposition levels. In the present study, we investigated whether exogenous expression of NIa in the brain of AD mouse model is beneficial to the improvement of cognitive deficits. To address this question, Lenti-NIa was intracerebrally injected into the brain of Tg-APPswe/PS1dE9 (Tg-APP/PS1) mice at 7 months of age and behavioral tests were performed 15-30 days afterwards. The results of the water maze test indicated that Tg-APP/PS1micewhich had been injected with Lenti-GFP showed an increased latency in finding the hidden-platform and markedly enhanced navigation near the maze-wall, and that such behavioral deficits were significantly reversed in Tg-APP/PS1 mice injected with Lenti-NIa. In the passive avoidance test, Tg- APP/PS1 mice exhibited a severe deficit in their contextual memory retention, which was reversed by NIa expression. In the marble burying test, Tg-APP/PS1 mice buried marbles fewer than non-transgenic mice, which was also significantly improved by NIa. After behavioral tests, it was verified that the Tg-APP/PS1 mice with Lenti-NIa injection had reduced Aβ levels and plaque deposition when compared to Tg-APP/PS1 mice. These results showed that the plant viral protease, NIa, not only reduces Aβ pathology, but also improves behavioral deficits.
Experimental and Molecular Medicine 11/2012; · 2.48 Impact Factor
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ABSTRACT: MicroRNAs (miRNAs) are endogenous small noncoding RNA molecules that suppress gene expression via degradation or translational inhibition of their target genes. Many miRNAs are associated with cardiac hypertrophy and heart failure. In this study, we pursued to identify miRNAs that negatively regulate cardiac hypertrophy by utilizing a surgical model for regression of cardiac hypertrophy. Microarray analysis revealed that 15 miRNAs out of the 696 miRNAs tested were specifically up-regulated during the regression period. Among these regression-specific miRNAs, nine microRNAs, which have not been previously reported, were further tested for their effects on phenylephrine (PE)-treated neonatal cardiomyocytes. Consequently, five miRNAs (miR-101b, 142-3p, 181d, 24-2(∗), and 450a) completely abrogated PE-induced hypertrophy as determined by measurements of cell size and fetal gene expression. Conversely, antagomers of these miRNAs exacerbated the PE-induced hypertrophy. Collectively, these findings suggest that the five miRNAs newly identified by using our cardiac hypertrophy-regression surgical model negatively regulate cardiac hypertrophy and could be used as potential therapeutic targets for the treatment of heart diseases.
Biochemical and Biophysical Research Communications 10/2012; · 2.48 Impact Factor
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ABSTRACT: This study was aimed at examining nitrate (NO3) and phosphate (PO4) removal potentials of rosegold pussy willow (Salix gracilistyla), giant pussy willow (Salix chaenomeloides), Korean willow (Salix koreensis), and bald cypress (Taxodium distichum) from eutrophic aquatic environment. These plants were replanted in rubber pots 35-cm high and 30-cm diameter without holes
in the bottom. Water of different concentration levels in NO3 (5, 10, 20ppm) or PO4 (0.5, 1, 2ppm) was funneled into the pots, and the residence time of inflow was controlled ranging from 1 to 4h. Nitrate
abatement of 58.9% was observed in the giant pussy willow pots with 20ppm concentration and 4h residence. The rosegold pussy
willow pots showed the highest PO4 removal at 20.2% at 0.5ppm concentration and 4h residence. Removal potentials of NO3 and PO4 were also investigated on the supposition that the polluted water would reside in wetlands or treatment facilities for longer
than 5days. Except that the residence time of inflow ranged from 5 to 20days, the same experimental conditions were kept.
The percentage of NO3 removal in the rosegold pussy willow pots was higher than in those of the other two willow species, and bald cypress showed
the lowest NO3 abatement. Highest PO4 removal was observed in giant pussy willow pots and lowest in rosegold pussy willow pots.
Keywords
Salix gracilistyla
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Salix chaenomeloides
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Salix koreensis
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Taxodium distichum
-Residence time-Removal of nitrogen and phosphorus
Landscape and Ecological Engineering 05/2012; 6(2):211-217. · 0.64 Impact Factor
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Jae Gyun Oh,
Dongtak Jeong,
Hyeseon Cha,
Ji Myoung Kim,
Ekaterina Lifirsu,
Jihwa Kim,
Dong Kwon Yang,
Chang Sik Park,
Changwon Kho,
Soonyong Park,
Yung Joon Yoo,
Do Han Kim,
Jaetaek Kim,
Roger J Hajjar, Woo Jin Park
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ABSTRACT: Protein kinase C (PKC)-interacting cousin of thioredoxin (PICOT) has distinct anti-hypertrophic and inotropic functions. We have previously shown that PICOT exerts its anti-hypertrophic effect by inhibiting calcineurin-NFAT signaling through its C-terminal glutaredoxin domain. However, the mechanism underlying the inotropic effect of PICOT is unknown. The results of protein pull-down experiments showed that PICOT directly binds to the catalytic domain of PKCζ through its N-terminal thioredoxin-like domain. Purified PICOT protein inhibited the kinase activity of PKCζ in vitro, which indicated that PICOT is an endogenous inhibitor of PKCζ. The inhibition of PKCζ activity with a PKCζ-specific pseudosubstrate peptide inhibitor was sufficient to increase the cardiac contractility in vitro and ex vivo. Overexpression of PICOT or inhibition of PKCζ activity down-regulated PKCα activity, which led to the elevation of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) 2a activity, concomitant with the increased phosphorylation of phospholamban (PLB). Overexpression of PICOT or inhibition of PKCζ activity also down-regulated protein phosphatase (PP) 2A activity, which subsequently resulted in the increased phosphorylation of troponin (Tn) I and T, key myofilament proteins associated with the regulation of contractility. PICOT appeared to inhibit PP2A activity through the disruption of the functional PKCζ/PP2A complex. In contrast to the overexpression of PICOT or inhibition of PKCζ, reduced PICOT expression resulted in up-regulation of PKCα and PP2A activities, followed by decreased phosphorylation of PLB, and TnI and T, respectively, supporting the physiological relevance of these events. Transgene- or adeno-associated virus (AAV)-mediated overexpression of PICOT restored the impaired contractility and prevented further morphological and functional deterioration of the failing hearts. Taken together, the results of the present study suggest that PICOT exerts its inotropic effect by negatively regulating PKCα and PP2A activities through the inhibition of PKCζ activity. This finding provides a novel insight into the regulation of cardiac contractility.
Journal of Molecular and Cellular Cardiology 03/2012; 53(1):53-63. · 5.17 Impact Factor
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ABSTRACT: Histidine-rich calcium binding protein (HRC) is located in the lumen of sarcoplasmic reticulum (SR) that binds to both triadin (TRN) and SERCA affecting Ca(2+) cycling in the SR. Chronic overexpression of HRC that may disrupt intracellular Ca(2+) homeostasis is implicated in pathogenesis of cardiac hypertrophy. Ablation of HRC showed relatively normal phenotypes under basal condition, but exhibited a significantly increased susceptibility to isoproterenol-induced cardiac hypertrophy. In the present study, we characterized the functions of HRC related to Ca(2+) cycling and pathogenesis of cardiac hypertrophy using the in vitro siRNA- and the in vivo adeno-associated virus (AAV)-mediated HRC knock-down (KD) systems, respectively.
AAV-mediated HRC-KD system was used with or without C57BL/6 mouse model of transverse aortic constriction-induced failing heart (TAC-FH) to examine whether HRC-KD could enhance cardiac function in failing heart (FH). Initially we expected that HRC-KD could elicit cardiac functional recovery in failing heart (FH), since predesigned siRNA-mediated HRC-KD enhanced Ca(2+) cycling and increased activities of RyR2 and SERCA2 without change in SR Ca(2+) load in neonatal rat ventricular cells (NRVCs) and HL-1 cells. However, AAV9-mediated HRC-KD in TAC-FH was associated with decreased fractional shortening and increased cardiac fibrosis compared with control. We found that phospho-RyR2, phospho-CaMKII, phospho-p38 MAPK, and phospho-PLB were significantly upregulated by HRC-KD in TAC-FH. A significantly increased level of cleaved caspase-3, a cardiac cell death marker was also found, consistent with the result of TUNEL assay.
Increased Ca(2+) leak and cytosolic Ca(2+) concentration due to a partial KD of HRC could enhance activity of CaMKII and phosphorylation of p38 MAPK, causing the mitochondrial death pathway observed in TAC-FH. Our results present evidence that down-regulation of HRC could deteriorate cardiac function in TAC-FH through perturbed SR-mediated Ca(2+) cycling.
PLoS ONE 01/2012; 7(8):e43282. · 4.09 Impact Factor
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Seung Hee Lee,
Jooyeon Kim,
Joo Young Ryu,
Suho Lee,
Dong Kwon Yang,
Dongtak Jeong,
Jaetaek Kim,
Sang-Hee Lee,
Jin Man Kim,
Roger J Hajjar, Woo Jin Park
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ABSTRACT: Despite its significant clinical implications, physiological hypertrophy remains poorly understood. In this study, the transcription coactivator Eya2 was shown to be up-regulated during physiological hypertrophy. Transgene- or adenovirus-mediated overexpression of Eya2 led to up-regulation of mTOR, a critical mediator of physiological hypertrophy. Luciferase reporter and chromatin immunoprecipitation assays revealed that Eya2 directly binds to and activates mTOR expression. The phosphorylation of mTOR downstream molecules was significantly enhanced in Eya2 transgenic (TG) hearts, implying that the Eya2-mediated induction of mTOR expression leads to an elevated mTOR activity. The transcription factor Six1 was also up-regulated during physiological hypertrophy and formed a complex with Eya2. Luciferase reporter and electrophoretic mobility shift assays revealed that the Eya2-Six1 complex binds to and enhances the expression of mTOR in a synergistic manner. Under pressure overload, Eya2 transgenic hearts developed hypertrophy which exhibited important molecular signatures of physiological hypertrophy, as assessed by gene expression profiling and measurements of expression levels of physiological hypertrophy-related genes by quantitative (q) RT-PCR. Examination of heart sections under electron microscopy revealed that the mitochondrial integrity remained largely intact in Eya2 transgenic mice, but not in wild-type littermates, under pressure overload. This finding was confirmed by measurements of mitochondrial DNA contents and the expression levels of mitochondrial function-related genes by qRT-PCR. These data suggest that Eya2 in a physical complex with Six1 plays a critical role in physiological hypertrophy. The cardioprotective effect of Eya2 appears to be due, at least in part, to its preservation of mitochondrial integrity upon pressure overload.
Journal of Molecular and Cellular Cardiology 12/2011; 52(3):718-26. · 5.17 Impact Factor
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ABSTRACT: The calcium-transporting ATPase ATP2A2, also known as SERCA2a, is a critical ATPase responsible for Ca(2+) re-uptake during excitation-contraction coupling. Impaired Ca(2+) uptake resulting from decreased expression and reduced activity of SERCA2a is a hallmark of heart failure. Accordingly, restoration of SERCA2a expression by gene transfer has proved to be effective in improving cardiac function in heart-failure patients, as well as in animal models. The small ubiquitin-related modifier (SUMO) can be conjugated to lysine residues of target proteins, and is involved in many cellular processes. Here we show that SERCA2a is SUMOylated at lysines 480 and 585 and that this SUMOylation is essential for preserving SERCA2a ATPase activity and stability in mouse and human cells. The levels of SUMO1 and the SUMOylation of SERCA2a itself were greatly reduced in failing hearts. SUMO1 restitution by adeno-associated-virus-mediated gene delivery maintained the protein abundance of SERCA2a and markedly improved cardiac function in mice with heart failure. This effect was comparable to SERCA2A gene delivery. Moreover, SUMO1 overexpression in isolated cardiomyocytes augmented contractility and accelerated Ca(2+) decay. Transgene-mediated SUMO1 overexpression rescued cardiac dysfunction induced by pressure overload concomitantly with increased SERCA2a function. By contrast, downregulation of SUMO1 using small hairpin RNA (shRNA) accelerated pressure-overload-induced deterioration of cardiac function and was accompanied by decreased SERCA2a function. However, knockdown of SERCA2a resulted in severe contractile dysfunction both in vitro and in vivo, which was not rescued by overexpression of SUMO1. Taken together, our data show that SUMOylation is a critical post-translational modification that regulates SERCA2a function, and provide a platform for the design of novel therapeutic strategies for heart failure.
Nature 09/2011; 477(7366):601-5. · 36.28 Impact Factor
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Inju Park,
Cecil Han,
Sora Jin,
Boyeon Lee,
Heejin Choi,
Jun Tae Kwon,
Dongwook Kim,
Jihye Kim,
Ekaterina Lifirsu, Woo Jin Park,
Zee Yong Park,
Do Han Kim,
Chunghee Cho
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ABSTRACT: Myosin II is an actin-binding protein composed of MHC (myosin heavy chain) IIs, RLCs (regulatory light chains) and ELCs (essential light chains). Myosin II expressed in non-muscle tissues plays a central role in cell adhesion, migration and division. The regulation of myosin II activity is known to involve the phosphorylation of RLCs, which increases the Mg2+-ATPase activity of MHC IIs. However, less is known about the details of RLC-MHC II interaction or the loss-of-function phenotypes of non-muscle RLCs in mammalian cells. In the present paper, we investigate three highly conserved non-muscle RLCs of the mouse: MYL (myosin light chain) 12A (referred to as MYL12A), MYL12B and MYL9 (MYL12A/12B/9). Proteomic analysis showed that all three are associated with the MHCs MYH9 (NMHC IIA) and MYH10 (NMHC IIB), as well as the ELC MYL6, in NIH 3T3 fibroblasts. We found that knockdown of MYL12A/12B in NIH 3T3 cells results in striking changes in cell morphology and dynamics. Remarkably, the levels of MYH9, MYH10 and MYL6 were reduced significantly in knockdown fibroblasts. Comprehensive interaction analysis disclosed that MYL12A, MYL12B and MYL9 can all interact with a variety of MHC IIs in diverse cell and tissue types, but do so optimally with non-muscle types of MHC II. Taken together, our study provides direct evidence that normal levels of non-muscle RLCs are essential for maintaining the integrity of myosin II, and indicates that the RLCs are critical for cell structure and dynamics.
Biochemical Journal 01/2011; 434(1):171-80. · 4.90 Impact Factor
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ABSTRACT: Polyglutamine (polyQ)-induced protein aggregation is the hallmark of a group of neurodegenerative diseases, including Huntington's disease. We hypothesized that a protease that could cleave polyQ stretches would intervene in the initial events leading to pathogenesis in these diseases. To prove this concept, we aimed to generate a protease possessing substrate specificity for polyQ stretches.
Hepatitis A virus (HAV) 3C protease (3CP) was subjected to engineering using a yeast-based method known as the Genetic Assay for Site-specific Proteolysis (GASP). Analysis of the substrate specificity revealed that 3CP can cleave substrates containing glutamine at positions P5, P4, P3, P1, P2', or P3', but not substrates containing glutamine at the P2 or P1' positions. To accommodate glutamine at P2 and P1', key residues comprising the active sites of the S2 or S1' pockets were separately randomized and screened. The resulting sets of variants were combined by shuffling and further subjected to two rounds of randomization and screening using a substrate containing glutamines from positions P5 through P3'. One of the selected variants (Var26) reduced the expression level and aggregation of a huntingtin exon1-GFP fusion protein containing a pathogenic polyQ stretch (HttEx1(97Q)-GFP) in the neuroblastoma cell line SH-SY5Y. Var26 also prevented cell death and caspase 3 activation induced by HttEx1(97Q)-GFP. These protective effects of Var26 were proteolytic activity-dependent.
These data provide a proof-of-concept that proteolytic cleavage of polyQ stretches could be an effective modality for the treatment of polyQ diseases.
PLoS ONE 01/2011; 6(7):e22554. · 4.09 Impact Factor
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Pyoung Oh Yoon,
Min-Ah Lee,
Hyeseon Cha,
Moon Hee Jeong,
Jooyeon Kim,
Seung Pil Jang,
Bo Youn Choi,
Dongtak Jeong,
Dong Kwon Yang,
Roger J Hajjar, Woo Jin Park
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ABSTRACT: CCN family members are matricellular proteins with diverse roles in cell function. The differential expression of CCN2 and CCN5 during cardiac remodeling suggests that these two members of the CCN family play opposing roles during the development of cardiac hypertrophy and fibrosis. We aimed to evaluate the role of CCN2 and CCN5 in the development of cardiac hypertrophy and fibrosis. In isolated cardiomyocytes, overexpression of CCN2 induced hypertrophic growth, whereas the overexpression of CCN5 inhibited both phenylephrine (PE)- and CCN2-induced hypertrophic responses. Deletion of the C-terminal (CT) domain of CCN2 transformed CCN2 into a CCN5-like dominant negative molecule. Fusion of the CT domain to the Carboxy-terminus of CCN5 transformed CCN5 into a CCN2-like pro-hypertrophic molecule. CCN2 transgenic (TG) mice did not develop cardiac hypertrophy at baseline but showed significantly increased fibrosis in response to pressure overload. In contrast, hypertrophy and fibrosis were both significantly inhibited in CCN5 TG mice. CCN2 TG mice showed an accelerated deterioration of cardiac function in response to pressure overload, whereas CCN5 TG mice showed conserved cardiac function. TGF-beta-SMAD signaling was elevated in CCN2 TG mice, but was inhibited in CCN5 TG mice. CCN2 is pro-hypertrophic and -fibrotic, whereas CCN5 is anti-hypertrophic and -fibrotic. CCN5 lacking the CT domain acts as a dominant negative molecule. CCN5 may provide a novel therapeutic target for the treatment of cardiac hypertrophy and heart failure.
Journal of Molecular and Cellular Cardiology 08/2010; 49(2):294-303. · 5.17 Impact Factor
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Hye-Eun Han,
Saravanan Sellamuthu,
Bae Hyun Shin,
Yong Jae Lee,
Sungmin Song,
Ji-Seon Seo,
In-Sun Baek,
Jeomil Bae,
Hannah Kim,
Yung Joon Yoo,
Yong-Keun Jung,
Woo Keun Song,
Pyung-Lim Han, Woo Jin Park
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ABSTRACT: The nuclear inclusion a (NIa) protease of turnip mosaic virus (TuMV) is responsible for the processing of the viral polyprotein into functional proteins. NIa was previously shown to possess a relatively strict substrate specificity with a preference for Val-Xaa-His-Gln↓, with the scissile bond located after Gln. The presence of the same consensus sequence, Val(12)-His-His-Gln(15), near the presumptive α-secretase cleavage site of the amyloid-β (Aβ) peptide led us to hypothesize that NIa could possess activity against Aβ.
Western blotting results showed that oligomeric as well as monomeric forms of Aβ can be degraded by NIa in vitro. The specific cleavage of Aβ was further confirmed by mass spectrometry analysis. NIa was shown to exist predominantly in the cytoplasm as observed by immunofluorescence microscopy. The overexpression of NIa in B103 neuroblastoma cells resulted in a significant reduction in cell death caused by both intracellularly generated and exogenously added Aβ. Moreover, lentiviral-mediated expression of NIa in APP(sw)/PS1 transgenic mice significantly reduced the levels of Aβ and plaques in the brain.
These results indicate that the degradation of Aβ in the cytoplasm could be a novel strategy to control the levels of Aβ, plaque formation, and the associated cell death.
PLoS ONE 01/2010; 5(12):e15645. · 4.09 Impact Factor
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ABSTRACT: Eyes absent 2 (Eya2) is a transcription factor involved in a number of cellular and developmental processes. We have previously shown that Eya2 is up-regulated during regression of cardiac hypertrophy and blocks phenylephrine-induced development of cardiomyocyte hypertrophy in vitro, suggesting that Eya2 is a negative regulator of cardiac hypertrophy. In this study, we generated transgenic mice with cardiac-specific overexpression of Eya2 to elucidate the in vivo function of Eya2 in cardiac remodeling. Mild cardiac hypertrophy developed in Eya2 transgenic mice under baseline conditions with no obvious structural or functional defects. Eya2 overexpression inhibited development of cardiac hypertrophy as judged by the abrogation of increases in heart weight and cross-sectional cell surface areas and re-activation of fetal genes under pressure overload (4 weeks). Eya2 overexpression also prevented wall thinning, ventricular dilation, and deterioration of cardiac function as well as fibrosis and inflammation in the heart under long-term pressure overload (12 weeks). Gene expression profiling using the parametric analysis of gene set enrichment (PAGE) method revealed that gene sets related to mitochondrial biogenesis and metabolism were elevated in the Eya2 transgenic mice. We also observed that the PI3K/Akt/mTOR signaling cascade was preserved in the Eya2 transgenic mice, while it was significantly reduced in the wild type littermates under pressure overload. These results demonstrate that Eya2 prevents adverse cardiac remodeling under pressure overload partly through altering metabolic gene expression and preserving PI3K/Akt/mTOR signaling pathway.
Journal of Molecular and Cellular Cardiology 05/2009; 46(4):596-605. · 5.17 Impact Factor
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Jong-Ok Pyo,
Jihoon Nah,
Hyo-Jin Kim,
Jae-Woong Chang,
Young-Wha Song,
Dong-Kwon Yang,
Dong-Gyu Jo,
Hyung-Ryong Kim,
Han-Jung Chae,
Soo-Wan Chae,
Seung-Yong Hwang,
Seung-Jun Kim,
Hyo-Joon Kim,
Chunghee Cho,
Chang-Gyu Oh, Woo Jin Park,
Yong-Keun Jung
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ABSTRACT: The ischemic death of cardiomyocytes is associated in heart disease and heart failure. However, the molecular mechanism underlying ischemic cell death is not well defined. To examine the function of apoptosis repressor with a caspase recruitment domain (ARC) in the ischemic/hypoxic damage of cardiomyocytes, we generated cardio-specific ARC transgenic mice using a mouse alpha-myosin heavy chain promoter. Compared with the control, the hearts of ARC transgenic mice showed a 3-fold overexpression of ARC. Langendoff preparation showed that the hearts isolated from ARC transgenic mice exhibited improved recovery of contractile performance during reperfusion. The cardiomyocytes cultured from neonatal ARC transgenic mice were significantly resistant to hypoxic cell death. Furthermore, the ARC C-terminal calcium-binding domain was as potent to protect cardiomyocytes from hypoxic cell death as ARC. Genome-wide RNA expression profiling uncovered a list of genes whose expression was changed (>2-fold) in ARC transgenic mice. Among them, expressional regulation of developmentally regulated RNA-binding protein 1 (Drbp1) or the dimethylglycine dehydrogenase precursor (pMe(2)GlyDH) affected hypoxic death of cardiomyocytes. These results suggest that ARC may protect cardiomyocytes from hypoxic cell death by regulating its downstream, Drbp1 and pMe(2)GlyDH, shedding new insights into the protection of heart from hypoxic damages.
Journal of Biological Chemistry 10/2008; 283(45):30707-14. · 4.77 Impact Factor
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Hyeseon Cha,
Ji Myoung Kim,
Jae Gyun Oh,
Moon Hee Jeong,
Chang Sik Park,
Jaeho Park,
Hyeon Joo Jeong,
Byung Keon Park,
Young-Hoon Lee,
Dongtak Jeong,
Dong Kwon Yang,
Oliver Y Bernecker,
Do Han Kim,
Roger J Hajjar, Woo Jin Park
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ABSTRACT: PICOT (PKC-interacting cousin of thioredoxin) was previously shown to inhibit the development of cardiac hypertrophy, concomitant with an increase in cardiomyocyte contractility. To explore the physiological function of PICOT in the hearts, we generated a PICOT-deficient mouse line by using a gene trap approach. PICOT(-/-) mice were embryonic lethal indicating that PICOT plays an essential role during embryogenesis, whereas PICOT(+/-) mice were viable with no apparent morphological defects. The PICOT protein levels were reduced by about 50% in the hearts of PICOT(+/-) mice. Significantly exacerbated cardiac hypertrophy was induced by pressure overload in PICOT(+/-) mice relative to that seen in wild type littermates. In line with this observation, calcineurin-NFAT signaling was greatly enhanced by pressure overload in the hearts of PICOT(+/-) mice. Cardiomyocytes from PICOT(+/-) mice exhibited significantly reduced contractility, which may be due in part to hypophosphorylation of phospholamban and reduced SERCA activity. These data indicate that the precise PICOT protein level significantly affects the process of cardiac hypertrophy and cardiomyocyte contractility. We suggest that PICOT plays as a critical negative regulator of cardiac hypertrophy and a positive inotropic regulator.
Journal of Molecular and Cellular Cardiology 10/2008; 45(6):796-803. · 5.17 Impact Factor
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ABSTRACT: By using an improved genetic screening system, variants of the HAV 3CP protease which exhibit altered P2 specificity were obtained. We randomly mutated the His145, Lys146, Lys147, and Leu155 residues that constitute the S2 pocket of 3CP and then isolated variants that preferred substrates with Gln over the original Thr at the P2 position using a yeast-based screening method. One of the isolated variants cleaved the Gln-containing peptide substrate more efficiently in vitro, proving the efficiency of our method in isolating engineered proteases with desired substrate selectivity.
Biochemical and Biophysical Research Communications 07/2008; 371(1):122-6. · 2.48 Impact Factor
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ABSTRACT: Exploring biological systems from highly complex datasets is an important task for systems biology. The present study examined co-expression dynamics of mouse heart transcriptome by spectral graph clustering (SGC) to identify a heart transcriptomic network. SGC of microarray data produced 17 classified biological conditions (called condition spectrum, CS) and co-expression patterns by generating bi-clusters. The results showed dynamic co-expression patterns with a modular structure enriched in heart-related CS (CS-1 and -13) containing abundant heart-related microarray data. Consequently, a mouse heart transcriptomic network was constructed by clique analysis from the gene clusters exclusively present in the heart-related CS; 31 cliques were used for constructing the network. The participating genes in the network were closely associated with important cardiac functions (e. g., development, lipid and glycogen metabolisms). Online Mendelian Inheritance in Man (OMIM) database indicates that mutations of the genes in the network induced serious heart diseases. Many of the tested genes in the network showed significantly altered gene expression in an animal model of hypertrophy. The results suggest that the present approach is critical for constructing a heart-related transcriptomic network and for deducing important genes involved in the pathogenesis of various heart diseases.
Biotechnology Journal 06/2008; 3(5):648-58.
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ABSTRACT: PICOT (protein kinase C-interacting cousin of thioredoxin) was previously shown to inhibit pressure overload-induced cardiac hypertrophy, concomitant with an increase in ventricular function and cardiomyocyte contractility. The combined analyses of glutathione S-transferase pull-down experiments and mass spectrometry enabled us to determine that PICOT directly interacts with muscle LIM protein (MLP) via its carboxyl-terminal half (PICOT-C). It was also shown that PICOT colocalizes with MLP in the Z-disc. MLP is known to play a role in anchoring calcineurin to the Z-disc in the sarcomere, which is critical for calcineurin-NFAT (nuclear factor of activated T cells) signaling. We, therefore, suggested that PICOT may affect calcineurin-NFAT signaling through its interaction with MLP. Consistent with this hypothesis, PICOT, or more specifically PICOT-C, abrogated phenylephrine-induced increases in calcineurin phosphatase activity, NFAT dephosphorylation/nuclear translocation, and NFAT-dependent transcriptional activation in neonatal cardiomyocytes. In addition, pressure overload-induced upregulation of NFAT target genes was significantly diminished in the hearts of PICOT-overexpressing transgenic mice. PICOT interfered with MLP-calcineurin interactions in a dose-dependent manner. Moreover, calcineurin was displaced from the Z-disc, concomitant with an abrogated interaction between calcineurin and MLP, in the hearts of PICOT transgenic mice. Replenishment of MLP restored the hypertrophic responses and the increase in calcineurin phosphatase activity that was inhibited by PICOT in phenylephrine-treated cardiomyocytes. Finally, PICOT-C inhibited cardiac hypertrophy to an extent that was comparable to that of full-length PICOT. Taken together, these data suggest that PICOT inhibits cardiac hypertrophy largely by negatively regulating calcineurin-NFAT signaling via disruption of the MLP-calcineurin interaction.
Circulation Research 04/2008; 102(6):711-9. · 9.49 Impact Factor
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Susumu Sakata,
Lifan Liang,
Naoya Sakata,
Yuri Sakata,
Elie R Chemaly,
Djamel Lebeche,
Yoshiaki Takewa,
Jiqiu Chen, Woo Jin Park,
Yoshiaki Kawase,
Roger J Hajjar
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ABSTRACT: 1. The aim of the present study was to examine the acute and chronic effects of adenoviral gene transfer on cardiac function in terms of left ventricular (LV) mechanoenergetic function. Recombinant adenoviral vector carrying beta-galactosidase and green fluorescent protein genes (Ad.betagal-GFP) was used. Cardiac function was examined in cross-circulated rat heart preparations, where end-systolic/diastolic pressure-volume relationships (ESPVR/EDPVR), systolic pressure-volume area (PVA), LV relaxation rate, equivalent maximal elastance at mid-range LV volume (eE(max) at mLVV), coronary blood flow, coronary vascular resistance and myocardial oxygen consumption (VO(2)) were also measured. 2. To examine the ex vivo acute effects of the adenoviral vector, data were obtained before and 30-90 min after intracoronary infusion of Ad.betagal-GFP in the excised, cross-circulated hearts that underwent serotonin pretreatment. To examine the in vivo chronic effects of adenoviral gene transfer, normal rat hearts received Ad.betagal-GFP or saline by a catheter-based technique and data were obtained 3 days after the injection of Ad.betagal-GFP or saline. 3. The ESPVR, EDPVR, LV relaxation rate, eE(max) at mLVV, coronary blood flow and coronary vascular resistance remained unchanged in Ad.betagal-GFP-transfected hearts in both ex vivo acute and in vivo chronic experiments. Moreover, the ex vivo and in vivo transfection caused no change in the slope and VO(2) intercept of the VO(2)-PVA relationship, VO(2) for basal metabolism and for Ca(2+) handling in excitation-contraction coupling and O(2) costs of LV contractility. 4. These results indicate that adenoviral gene transfer has neither acute nor chronic toxic effects on LV mechanical and energetic function. A special combination of in vivo adenoviral gene transfer and a cross-circulation experimental system may provide a useful novel strategy to explore the functional and mechanoenergetic role of specifically targeted genes in the diseased heart.
Clinical and Experimental Pharmacology and Physiology 01/2008; 34(12):1300-6. · 1.85 Impact Factor
