We applied a combined proteomic and metabolomic approach to obtain novel mechanistic insights in PKCε-mediated cardioprotection. Mitochondrial and cytosolic proteins from control and transgenic hearts with constitutively active or dominant negative PKCε were analyzed using difference in-gel electrophoresis (DIGE). Among the differentially expressed proteins were creatine kinase, pyruvate kinase, lactate dehydrogenase, and the cytosolic isoforms of aspartate amino transferase and malate dehydrogenase, the two enzymatic components of the malate aspartate shuttle, which is required for the import of reducing equivalents from glycolysis across the inner mitochondrial membrane. These enzymatic changes appeared to be dependent on PKCε activity, as they were not observed in mice expressing inactive PKCε. High-resolution proton nuclear magnetic resonance (1H-NMR) spectroscopy confirmed a pronounced effect of PKCε activity on cardiac glucose and energy metabolism: normoxic hearts with constitutively active PKCε had significantly lower concentrations of glucose, lactate, glutamine and creatine, but higher levels of choline, glutamate and total adenosine nucleotides. Moreover, the depletion of cardiac energy metabolites was slower during ischemia/reperfusion injury and glucose metabolism recovered faster upon reperfusion in transgenic hearts with active PKCε. Notably, inhibition of PKCε resulted in compensatory phosphorylation and mitochondrial translocation of PKCδ. Taken together, our findings are the first evidence that PKCε activity modulates cardiac glucose metabolism and provide a possible explanation for the synergistic effect of PKCδ and PKCε in cardioprotection.
BackgroundA large subfamily of serine recombinases contains long polypeptide segments appended to the C-terminal end of the conserved catalytic domain. Members of this subfamily often function as phage integrases but also mediate transposition and regulate terminal differentiation processes in eubacteria. Although a few members of this subfamily have been studied in purified in vitro systems, key mechanistic aspects of reactions promoted by these recombinases remain to be determined, particularly with respect to the functions of the large C-terminal domain.ResultsWe have developed and characterized a robust in vitro recombination reaction by the Listeria phage A118 integrase, a member of the subfamily of serine recombinases containing a large C-terminal domain. The reaction occurs in a simple buffered salt solution and exhibits a modest stimulation by divalent cations or spermidine and DNA supercoiling. Recombination with purified A118 integrase is unidirectional, being efficient only between attP and attB DNA sites to either join separate DNA molecules (intermolecular recombination) or to generate deletions or inversions depending on the relative orientation of att sites in cis (intramolecular recombination). The minimal attP site is 50 bp but requires only 44 bp of base sequence information, whereas the minimal attB site is 42 bp and requires 38 bp of base sequence information. DNA exchange occurs between the central 2 bp of attP and attB. Identity between these two base pairs is required for recombination, and they solely determine the orientation of recombination sites. The integrase dimer binds efficiently to full att sites, including the attL and attR integration products, but poorly and differentially to each half-site. The large C-terminal domain can be separated from the N-terminal catalytic by partial proteolysis and mediates non-cooperative DNA binding to att sites.ConclusionsThe basic properties of the phage A118 integrase reaction and its substrate requirements have been elucidated. A118 integrase thus joins the handful of biochemically characterized serine integrases that are serving as models for mechanistic studies on this important class of recombinases. Information reported here will also be useful in exploiting this recombinase for genetic engineering.
We have previously reported arginase expression in human breast cancer cells and demonstrated that the inhibition of arginase by Nω hydroxy L-arginine (NOHA) in MDA-MB-468 cells induces apoptosis. However, arginase expression and its possible molecular targets in human breast tumor samples and potential clinical implications have not been fully elucidated. Here, we demonstrate arginase expression in human breast tumor samples, and several established breast cancer cell lines, in which NOHA treatment selectively inhibits cell proliferation. The over-expression of Bcl2 in MDA-MB-468 cells abolished NOHA-induced apoptosis, suggesting that the mitochondria may be the main site of NOHA’s action. We, therefore, undertook a proteomics approach to identify key mitochondrial targets of arginase in MDA-MB-468 cells. We identified 54 non-mitochondrial and 13 mitochondrial proteins that were differentially expressed in control and NOHA treated groups. Mitochondrial serine hydroxymethyltransferase (mSHMT) was identified as one of the most promising targets of arginase. Both arginase II (Arg II) and mSHMT expressions were higher in human breast tumor tissues compared to the matched normal and there was a strong correlation between Arg II and mSHMT protein expression. MDA-MB-468 xenografts had significant upregulation of Arg II expression that preceded the induction of mSHMT expression. Small inhibitory RNA (siRNA)-mediated inhibition of Arg II in MDA-MB-468 and HCC-1806 cells led to significant inhibition of both the mSHMT gene and protein expression. As mSHMT is a key player in folate metabolism, our data provides a novel link between arginine and folate metabolism in human breast cancer, both of which are critical for tumor cell proliferation.
Approximately 25,000 ovarian cancers are diagnosed in the U.S. annually, and 75% are in the advanced stage and largely incurable. There is critical need for early detection tools and novel treatments. Proteasomal ubiquitin receptor ADRM1 is a protein that is encoded by the ADRM1 gene. Recently, we showed that among 20q13-amplified genes in ovarian cancer, ADRM1 overexpression was the most highly correlated with amplification and was significantly upregulated with respect to stage, recurrence, and metastasis. Its overexpression correlated significantly with shorter time to recurrence and overall survival. Array-CGH and microarray expression of ovarian cancer cell lines provided evidence consistent with primary tumor data that ADRM1 is a 20q13 amplification target. Herein, we confirm the ADRM1 amplicon in a second ovarian cancer cohort and define a minimally amplified region of 262 KB encompassing seven genes. Additionally, using RNAi knock-down of ADRM1 in naturally amplified cell line OAW42 and overexpression of ADRM1 via transfection in ES2, we show that (1) ADRM1 overexpression increases proliferation, migration, and growth in soft agar, and (2) knock-down of ADRM1 results in apoptosis. Proteomic analysis of cells with ADRM1 knock-down reveals dysregulation of proteins including CDK-activating kinase assembly factor MAT1. Taken together, the results indicate that amplified ADRM1 is involved in cell proliferation, migration and survival in ovarian cancer cells, supporting a role as an oncogene and novel therapeutic target for ovarian cancer.
Limb girdle muscular dystrophy 2H is caused by mutations in the gene encoding the E3 ubiquitin ligase, TRIM32. Previously, we generated and characterized a Trim32 knockout mouse (T32KO) that displays both neurogenic and myopathic features. The myopathy in these mice is attributable to impaired muscle growth, associated with satellite cell senescence and premature sarcopenia. This satellite cell senescence is due to accumulation of the SUMO ligase PIASy, a substrate of TRIM32. The goal of this investigation was to identify additional substrates of TRIM32 using 2D fluorescence difference gel electrophoresis (2D-DIGE) in order to further explore its role in skeletal muscle. Because TRIM32 is an E3 ubiquitin ligase, we reasoned that TRIM32's substrates would accumulate in its absence. 2D-DIGE identified 19 proteins that accumulate in muscles from the T32KO mouse. We focused on two of these proteins, NDRG2 and TRIM72, due to their putative roles in myoblast proliferation and myogenesis. Follow-up analysis confirmed that both proteins were ubiquitinated by TRIM32 in vitro; however, only NDRG2 accumulated in skeletal muscle and myoblasts in the absence of TRIM32. NDRG2 overexpression in myoblasts led to reduced cell proliferation and delayed cell cycle withdrawal during differentiation. Thus, we identified NDRG2 as a novel target for TRIM32; these findings further corroborate the hypothesis that TRIM32 is involved in control of myogenic cells proliferation and differentiation.
The Crithidia fasciculata KAP2 and KAP3 proteins are closely related kinetoplast-specific histone-like DNA-binding proteins. The KAP2 and KAP3 genes are 46% identical and are arranged in tandem on the chromosomal DNA. Disruption of both alleles of either gene alone shows no detectable phenotype. However, replacement of both copies of the sequence encoding the entire KAP2 and KAP3 locus increases maxicircle mRNA levels two-to fourfold. These double-knockout cells are viable but grow extremely slowly, have reduced respiration and very abnormal cell morphologies, and accumulate numerous large vacuoles. The extreme phenotype of these mutant cells suggests an important role for the KAP2 and KAP3 proteins in mitochondrial metabolism and cell growth.
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