Quantitative proteomic analysis by iTRAQ® for the identification of candidate biomarkers in ovarian cancer serum

<p>Abstract</p> <p>Background</p> <p>Ovarian cancer is the most lethal gynecologic malignancy, with the majority of cases diagnosed at an advanced stage when treatments are less successful. Novel serum protein markers are needed to detect ovarian cancer in its earliest stage; when detected early, survival rates are over 90%. The identification of new serum biomarkers is hindered by the presence of a small number of highly abundant proteins that comprise approximately 95% of serum total protein. In this study, we used pooled serum depleted of the most highly abundant proteins to reduce the dynamic range of proteins, and thereby enhance the identification of serum biomarkers using the quantitative proteomic method iTRAQ^®.</p> <p>Results</p> <p>Medium and low abundance proteins from 6 serum pools of 10 patients each from women with serous ovarian carcinoma, and 6 non-cancer control pools were labeled with isobaric tags using iTRAQ^®to determine the relative abundance of serum proteins identified by MS. A total of 220 unique proteins were identified and fourteen proteins were elevated in ovarian cancer compared to control serum pools, including several novel candidate ovarian cancer biomarkers: extracellular matrix protein-1, leucine-rich alpha-2 glycoprotein-1, lipopolysaccharide binding protein-1, and proteoglycan-4. Western immunoblotting validated the relative increases in serum protein levels for several of the proteins identified.</p> <p>Conclusions</p> <p>This study provides the first analysis of immunodepleted serum in combination with iTRAQ^®to measure relative protein expression in ovarian cancer patients for the pursuit of serum biomarkers. Several candidate biomarkers were identified which warrant further development.</p

Iron-sparing Response of Mycobacterium avium subsp. paratuberculosis is strain dependent

<p>Abstract</p> <p>Background</p> <p>Two genotypically and microbiologically distinct strains of <it>Mycobacterium avium </it>subsp. <it>paratuberculosis </it>(MAP) exist - S and C MAP strains that primarily infect sheep and cattle, respectively. Concentration of iron in the cultivation medium has been suggested as one contributing factor for the observed microbiologic differences. We recently demonstrated that S strains have defective iron storage systems, leading us to propose that these strains might experience iron toxicity when excess iron is provided in the medium. To test this hypothesis, we carried out transcriptional and proteomic profiling of these MAP strains under iron-replete or -deplete conditions.</p> <p>Results</p> <p>We first complemented <it>M. smegmatis</it>Δ<it>ideR </it>with IdeR of C MAP or that derived from S MAP and compared their transcription profiles using <it>M. smegmatis mc</it>^<it>2</it><it>155 </it>microarrays. In the presence of iron, sIdeR repressed expression of <it>bfrA </it>and MAP2073c, a ferritin domain containing protein suggesting that transcriptional control of iron storage may be defective in S strain. We next performed transcriptional and proteomic profiling of the two strain types of MAP under iron-deplete and -replete conditions. Under iron-replete conditions, C strain upregulated iron storage (BfrA), virulence associated (Esx-5 and antigen85 complex), and ribosomal proteins. In striking contrast, S strain downregulated these proteins under iron-replete conditions. iTRAQ (isobaric tag for relative and absolute quantitation) based protein quantitation resulted in the identification of four unannotated proteins. Two of these were upregulated by a C MAP strain in response to iron supplementation. The iron-sparing response to iron limitation was unique to the C strain as evidenced by repression of non-essential iron utilization enzymes (aconitase and succinate dehydrogenase) and upregulation of proteins of essential function (iron transport, [Fe-S] cluster biogenesis and cell division).</p> <p>Conclusions</p> <p>Taken together, our study revealed that C and S strains of MAP utilize divergent metabolic pathways to accommodate in vitro iron stress. The knowledge of the metabolic pathways these divergent responses play a role in are important to 1) advance our ability to culture the two different strains of MAP efficiently, 2) aid in diagnosis and control of Johne's disease, and 3) advance our understanding of MAP virulence.</p

Glucose Starvation Alters Heat Shock Response, Leading to Death of Wild Type Cells and Survival of MAP Kinase Signaling Mutant

<div><p>A moderate heat shock induces <i>Neurospora crassa</i> to synthesize large quantities of heat shock proteins that are protective against higher, otherwise lethal temperatures. However, wild type cells do not survive when carbohydrate deprivation is added to heat shock. In contrast, a mutant strain defective in a stress-activated protein kinase does survive the combined stresses. In order to understand the basis for this difference in survival, we have determined the relative levels of detected proteins in the mutant and wild type strain during dual stress, and we have identified gene transcripts in both strains whose quantities change in response to heat shock or dual stress. These data and supportive experimental evidence point to reasons for survival of the mutant strain. By using alternative respiratory mechanisms, these cells experience less of the oxidative stress that proves damaging to wild type cells. Of central importance, mutant cells recycle limited resources during dual stress by undergoing autophagy, a process that we find utilized by both wild type and mutant cells during heat shock. Evidence points to inappropriate activation of TORC1, the central metabolic regulator, in wild type cells during dual stress, based upon behavior of an additional signaling mutant and inhibitor studies.</p></div

Vacuoles of germinating conidiospores of wt and os2 in transmission electron micrographs.

<p>Cells were incubated under 30°C control conditions (a + d) or subjected to heat shock (b + e) or dual stress (c + f) for 2 hr. The vacuoles (lysosomes) are light-colored and indicated by v. The horizontal bar in each micrograph indicates 1 micrometer.</p

Percentage of surviving colonies, representing viable conidiospores, in petri plate assays.

<p>The controls in each case consist of the particular strain incubated at room temperature. At least three plates were counted for each treatment, and experiments were performed at least two times with similar results. Typical examples of reproducible experiments are shown.</p

Multiomics Approach Reveals an Important Role of BNIP3 in Myocardial Remodeling and the Pathogenesis of Heart Failure with Reduced Ejection Fraction

Previous work showed a role of BNIP3 in myocardial remodeling and progression to HFrEF. We utilized a multiomics approach to unravel BNIP3-related molecular mechanisms in the pathogenesis of HFrEF. BNIP3 knockdown in HFrEF improved glycolysis, pyruvate metabolism, branched-chain amino acid catabolism, and oxidative phosphorylation, and restored endoplasmic reticulum (ER)–mitochondrial (mt) calcium and ion homeostasis. These effects of BNIP3 on cardiac metabolism were related to its interaction and downregulation, and/or phosphorylation, of specific mt-proteins involved in the aforementioned metabolic pathways, including the MICOS and SLC25A families of carrier proteins. BNIP3 affected ER–mt-calcium and ion homeostasis via its interaction-induced VDAC1 dimerization and modulation of VDAC1 phosphorylation at Ser104 and Ser241, and the downregulation of LETM1. At the ER level, BNIP3 interacted with the enzyme SERCA2a and the PKA signaling complex, leading to the downregulation of SERCA2a and PKA-mediated Ser16 phospholamban phosphorylation. Additionally, BNIP3 attenuated AMPK and PRKCE activity by modulating AMPK phosphorylation at Ser485/491 and Ser377 residues, and PRKCE phosphorylation at Thr521 and Thr710 residues. BNIP3 also interacted with sarcomeric, cytoskeletal, and cellular transcription and translation proteins, and affected their expression and/or phosphorylation. In conclusion, BNIP3 modulates multiple pathobiological processes and constitutes an attractive therapeutic target in HFrEF

Categories of transcripts whose levels increase or decrease during stress: Signaling.

<p>Numbers of up- or down-regulated transcripts discussed in this report, are indicated for wt (filled bars) and os2 (outlined bars) during heat shock (hs) or dual stress (ds). For details of transcript levels for individual proteins see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165980#sec049" target="_blank">Supporting Information</a>.</p

Categories of transcripts whose levels increase or decrease during stress: Metabolism.

<p>Numbers of up- or down-regulated transcripts discussed in this report, are indicated for wt (filled bars) and os2 (outlined bars) during heat shock (hs) or dual stress (ds). For details of transcript levels for individual proteins see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165980#sec049" target="_blank">Supporting Information</a>.</p

Differential activation of Os2 in Western blot of proteins from wt and stk10 cell extracts.

<p>The cells were subjected to DS for 10 min in the presence (+) or absence (-) of rapamycin. Proteins were separated by SDS-gel electrophoresis, and the blot was probed with antibody against mammalian (Thr180/Tyr182) phospho-p38, which detects phosphorylated Os2.</p