Showing papers by "Jennifer E. Van Eyk published in 2009"

c-Myc suppression of miR-23a/b enhances mitochondrial glutaminase expression and glutamine metabolism.

Abstract: Altered glucose metabolism in cancer cells is termed the Warburg effect, which describes the propensity of most cancer cells to take up glucose avidly and convert it primarily to lactate, despite available oxygen. Notwithstanding the renewed interest in the Warburg effect, cancer cells also depend on continued mitochondrial function for metabolism, specifically glutaminolysis that catabolizes glutamine to generate ATP and lactate. Glutamine, which is highly transported into proliferating cells, is a major source of energy and nitrogen for biosynthesis, and a carbon substrate for anabolic processes in cancer cells, but the regulation of glutamine metabolism is not well understood. Here we report that the c-Myc (hereafter referred to as Myc) oncogenic transcription factor, which is known to regulate microRNAs and stimulate cell proliferation, transcriptionally represses miR-23a and miR-23b, resulting in greater expression of their target protein, mitochondrial glutaminase, in human P-493 B lymphoma cells and PC3 prostate cancer cells. This leads to upregulation of glutamine catabolism. Glutaminase converts glutamine to glutamate, which is further catabolized through the tricarboxylic acid cycle for the production of ATP or serves as substrate for glutathione synthesis. The unique means by which Myc regulates glutaminase uncovers a previously unsuspected link between Myc regulation of miRNAs, glutamine metabolism, and energy and reactive oxygen species homeostasis.

Abstract #LB-186: c-Myc suppression of miR-23 enhances mitochondrial glutaminase and glutamine metabolism

Abstract: Notwithstanding the renewed interest in the Warburg effect, which describes the propensity for cancer cells to avidly metabolize glucose to lactate, cancer cells also depend on continued mitochondrial function for metabolism, specifically glutaminolysis that catabolizes glutamine to generate ATP and lactate. Glutamine is a major source for energy, carbon and nitrogen for anabolic processes in cancer cells, but the regulation of glutamine metabolism is not well understood. Here, we report that the c-Myc oncogenic transcription factor regulates glutamine metabolism by a previously unsuspected mechanism which involves c-Myc suppression of miR-23 microRNAs that target and inhibit mitochondrial glutaminase, or GLS, the first enzyme that catabolizes glutamine. c-Myc also transactivates expression of glutamine transporter genes. We studied the human P-493 B cells that bear a tetracycline-repressible c-Myc construct, such that tetracycline withdrawal induces c-Myc and mitochondrial biogenesis followed by cell proliferation. We found through analyzing the mitochondrial proteome that GLS was increased dramatically in response to c-Myc induction. siRNA targeting GLS1 diminishes cell proliferation and increased apoptosis, indicating that GLS1 is necessary for Myc induced cell proliferation. GLS converts glutamine to glutamate that is further catabolized through the TCA cycle for the production of ATP or serves as substrate for glutathione synthesis. In this regard, depletion of glutamine significantly diminished proliferation of P-493 cell and the human prostate PC3 cancer cell line. Although GLS protein levels are induced >10-fold by c-Myc in P-493 cells, GLS1 mRNA did not vary significantly, suggesting that regulation of GLS protein levels by c-Myc is post-transcriptional. We document that c-Myc transcriptionally represses miR-23, which can inhibit the expression of GLS protein through targeting the 3\#8217;UTR. In addition to the responses of wild-type and miR-23 seed sequence mutant luciferase-GLS1-3\#8217;UTR reporter constructs, antisense miR-23 LNA oligonucleotides were able to elevate GLS protein levels in low c-Myc expressing P493 and PC3 cells, indicating that GLS1 mRNA is a target of miR-23 that inhibits glutaminase translation. Since miR-23 expression is decreased in human prostate cancer, we immunoblotted and found a correlation between c-Myc and GLS protein levels in human prostate cancer samples as compared with lowered expression in the corresponding normal prostate tissues. The unique means by which Myc regulates GLS uncovers a previously unsuspected link between Myc regulation of miRNAs, glutamine metabolism, and energy and reactive oxygen species (ROS) homeostasis and provides a regulatory mechanism involving c-Myc and miRNAs for elevated expression of glutaminase and glutamine metabolism in human cancers. Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr LB-186.

Circulating transforming growth factor-beta in Marfan syndrome.

Abstract: Background— Marfan syndrome (MFS) is caused by mutations in the fibrillin-1 gene and dysregulation of transforming growth factor-β (TGF-β). Recent evidence suggests that losartan, an angiotensin II...

Preparation of Proteins and Peptides for Mass Spectrometry Analysis in a Bottom-Up Proteomics Workflow

Abstract: This unit outlines the steps required to prepare a sample for MS analysis following protein separation or enrichment by gel electrophoresis, liquid chromatography, and affinity capture within the context of a bottom-up proteomics workflow in which the protein is first broken up into peptides, either by chemical or enzymatic digestion, prior to MS analysis. Also included are protocols for enrichment at the peptide level, including phosphopeptide enrichment and reversed-phase chromatography for sample purification immediately prior to MS analysis. Finally, there is a discussion regarding the types of MS technologies commonly used to analyze proteomics samples, as well as important parameters that should be considered when analyzing the MS data to ensure stringent and robust protein identifications and characterization.

Milk Fat Globule Protein Epidermal Growth Factor-8. A Pivotal Relay Element Within the Angiotensin II and Monocyte Chemoattractant Protein-1 Signaling Cascade Mediating Vascular Smooth Muscle Cells Invasion

Abstract: Advancing age induces aortic wall thickening that results from the concerted effects of numerous signaling proteins, many of which have yet to be identified. To search for novel proteins associated with aortic wall thickening, we have performed a comprehensive quantitative proteomic study to analyze aortic proteins from young (8 months) and old (30 months) rats and identified 50 proteins that significantly change in abundance with aging. One novel protein, the milk fat globule protein epidermal growth factor 8 (MFG-E8), increases 2.3-fold in abundance in old aorta. Transcription and translation analysis demonstrated that aortic MFG-E8 mRNA and protein levels increase with aging in several mammalian species including humans. Dual immunolabeling shows that MFG-E8 colocalizes with both angiotensin II and monocyte chemoattractant protein (MCP)-1 within vascular smooth muscle cells (VSMCs) of the thickened aged aortic wall. Exposure of early passage VSMCs from young aorta to angiotensin II markedly increases MFG-E8 and enhances invasive capacity to levels observed in VSMCs from old rats. Treatment of VSMCs with MFG-E8 increases MCP-1 expression and VSMCs invasion that are inhibited by the MCP-1 receptor blocker vCCI. Silencing MFG-E8 RNA substantially reduces MFG-E8 expression and VSMCs invasion capacity. The data indicate that arterial MFG-E8 significantly increases with aging and is a pivotal relay element within the angiotensin II/MCP-1/VSMC invasion signaling cascade. Thus, targeting of MFG-E8 within this signaling axis pathway is a potential novel therapy for the prevention and treatment of the age-associated vascular diseases such as atherosclerosis.

Assessment of albumin removal from an immunoaffinity spin column: critical implications for proteomic examination of the albuminome and albumin-depleted samples.

Abstract: High abundance proteins in serum and plasma (e.g., albumin) are routinely removed during proteomic sample processing as they can mask lower abundance proteins and peptides of biological/clinical interest. A common method of albumin depletion is based on immunoaffinity capture, and many immunoaffinity devices are designed for multiple uses. In this case, it is critical that the albumin captured on the affinity matrix is stripped from the column prior to regeneration of the matrix and processing of subsequent samples, to ensure no carryover and that maximal binding sites are available for subsequent samples. The current study examines the ability of a manufacturer's protocol to remove the proteins and peptides captured by an immunoaffinity spin column. The data presented in the current work illustrate the difficulty in completely removing albumin from the immunoaffinity device, and consequently, may explain the variability and decreased efficiency shown for this device in previous studies. In summary, the current data present important considerations for the implementation of multiple-use immunoaffinity devices for processing subsequent clinical samples in a proteomic workflow.

Post-translational modifications of ATP synthase in the heart: biology and function

Abstract: The ATP synthase complex is a critical enzyme in the energetic pathways of cells because it is the enzyme complex that produces the majority of cellular ATP. It has been shown to be involved in several cardiac phenotypes including heart failure and preconditioning, a cellular protective mechanism. Understanding the regulation of this enzyme is important in understanding the mechanisms behind these important phenomena. Recently there have been several post-translational modifications (PTM) reported for various subunits of this enzyme complex, opening up the possibility of differential regulation by these PTMs. Here we discuss the known PTMs in the heart and other mammalian tissues and their implication to function and regulation of the ATP synthase.

Redox signaling and protein phosphorylation in mitochondria: progress and prospects.

Abstract: As we learn more about the factors that govern cardiac mitochondrial bioenergetics, fission and fusion, as well as the triggers of apoptotic and necrotic cell death, there is growing appreciation that these dynamic processes are finely-tuned by equally dynamic post-translational modification of proteins in and around the mitochondrion. In this minireview, we discuss the evidence that S-nitrosylation, glutathionylation and phosphorylation of mitochondrial proteins have important bioenergetic consequences. A full accounting of these targets, and the functional impact of their modifications, will be necessary to determine the extent to which these processes underlie ischemia/reperfusion injury, cardioprotection by pre/post-conditioning, and the pathogenesis of heart failure.

Mechanisms of gap junction traffic in health and disease.

Abstract: Gap junctions (GJs) allow direct communication between cells. In the heart, GJs mediate the electrical coupling of cardiomyocytes and as such dictate the speed and direction of cardiac conduction. A prominent feature of acquired structural heart disease is remodeling of GJ protein expression and localization concomitant with increased susceptibility to lethal arrhythmias, leading many to hypothesize that the two are causally linked. Detailed understanding of the cellular mechanisms that regulate GJ localization and function within cardiomyocytes may therefore uncover potential therapeutic strategies for a significant clinical problem. This review will outline our current understanding of GJ cell biology with the intent of highlighting cellular mechanisms responsible for GJ remodeling associated with cardiac disease.

Assessment of ProteoExtract subcellular fractionation kit reveals limited and incomplete enrichment of nuclear subproteome from frozen liver and heart tissue.

Abstract: The nuclear fraction of the ProteoExtract subcellular fractionation kit was assessed using frozen rat liver and heart tissue. Fractionation was evaluated by Western blot using protein markers for various subcellular compartments and followed up with LC/MS/MS analysis of the nuclear fractions. Of the proteins identified, nuclear proteins were in the minority (less than 15%) and there was poor representation of the various nuclear substructures when compared with liver nuclear isolations using a classical density-based centrifugation protocol. The ProteoExtract kit demonstrated poor specificity for the nucleus and offers limited promise for proteomics investigations of the nuclear subproteome in frozen tissue samples.

Biomarkers for myocardial ischemia

Abstract: This invention relates, e.g., to a method for determining if a subject has myocardial ischemia, comprising (a) providing a blood sample obtained from a subject suspected of having myocardial ischemia; (b) determining in the sample the amount of one or more of the following proteins: (i) Lumican and/or (ii) Extracellular matrix protein 1 and/or (iii) Carboxypeptidase N; and (c) comparing the amount(s) of the protein(s) to a baseline value that is indicative of the amount of the protein in a subject that does not have myocardial ischemia, wherein a statistically significantly increased amount of the protein(s) compared to the baseline value is indicative of myocardial ischemia. Other proteins indicative of myocardial ischemia are also described, as are methods for treating a subject based on a diagnostic procedure of the invention, and kits for carrying out a method of the invention.

Abstract 2944: Activation of Heat Shock Proteins and Shifts in Cytoskeleton Composition in a Cardiac-specific SERCA2 KO Mouse

Abstract: BACKGROUND: Depressed contractility is a key feature of the failing heart and has been linked to reduced Ca2+ availability due to decreased activity of the sarcoplasmic reticulum Ca2+ ATPase 2 (SER...

Abstract 3849: HNO Induces a Disulfide Bond Between Cysteine Residues on Actin (Cys 257) and Tropomyosin (Cys 190) Increasing Cardiac Force Development: A Novel, Redox-based Mechanism for Contractile Regulation

Abstract: Background: Nitroxyl (HNO) donors increase cardiac inotropy via combined enhancement of SR Ca2+ cycling and myofilament sensitization to Ca2+. HNO reacts with thiols, but the critical -SH targets o...

Abstract 3786: Cardiac Resynchronization Therapy Augments Myofilament Ca2+ Responsiveness in Failing Myocardium via Altered Phosphorylation

Abstract: Cardiac resynchronization therapy (CRT) has become one of the major clinical therapies that improve long term survival in heart failure (HF) patients The beneficial effects of CRT have been suggested, including upregulation of sarcoplamic reticulum ATPase, β 1 -adrenergic receptor expression, and improved chamber mechanoenergetics Here, we reveal that CRT also acts as a calcium sensitizer and restores maximum force when applied in a dog model of dyssynchronous HF (DHF, 6 wks rapid atrial pacing with LBBB) Trabeculae (in mm: 035±002 wide, 022±001 thick, 174±013 long, n=27) from the ventricles of control, DHF, always synchronous HF (HF-synch, atrial rapid pacing for 6 wks), and CRT (BiV rapid pacing for latter 3 wks) were dissected, mounted between a force transducer and a motor arm, and chemically skinned in 1% Triton Force-pCa relations were obtained with varied [Ca 2+ ] The Table compares maximal Ca 2+ -activated force (F max ), the [Ca 2+ ] for 50% of F max (Ca 50 ), and the Hill coefficient in these groups In parallel, myofibrillar ATPase activity as a function of Ca 2+ activation were studied in these four groups (n=4, and 3 runs in each dog sample) DHF displayed a substantial downward shift with depression of minimum and maximal Ca 2+ -activated myofibrillar ATPase compared with control or CRT, and HF-synch myofibrils also yielded a response quite similar to that of DHF Finally, 2D-DiGE and mass spectrometry of the myofilament proteins showed increased phosphorylation of troponin I, myosin light chain 2, and α -tropomyosin (at a novel site, T237), while troponin T had less phosphorylation These results demonstrate that (1) myofilament Ca 2+ responsiveness is markedly depressed in HF and this myofilament abnormality occurs in HF per se (ie regardless of synchrony), (2) CRT significantly improves myofilament Ca 2+ responsiveness, and (3) post-translational modification of myofil-ament proteins may underlie the beneficial myofilament effects of CRT