Protein Levels In Response Research Articles

Nestin is essential for mitogen-stimulated proliferation of neural progenitor cells

The intermediate filament (IF) protein nestin is a widely accepted molecular marker for neural progenitor cells (NPCs), but its function during neurogenesis remains largely unknown. We found that in embryonic cortical NPCs down-regulation of the expression of nestin, but not its co-polymer IF protein vimentin, resulted in a G1 cell-cycle arrest and a severe reduction in the generation of neurons. Furthermore, down-regulating nestin expression in cultured cortical NPCs markedly suppressed their colony-formation ability and blocked the elevation of the cyclin D1/E protein level in response to the treatment with bFGF. Interestingly, nestin down-regulation caused a marked suppression in the activation of the phosphoinositide 3-kinase (PI3K) pathway but not the mitogen-activated protein kinase (MAPK) pathway in these NPCs. Moreover, defects in the proliferation of cortical NPCs caused by nestin down-regulation could be prevented by up-regulating PI3K activity. Thus, nestin is essential for the proliferation of NPCs by promoting the activation of PI3K in response to mitogenic growth factors.

Correlation between quantitative HER2 protein level and pathologic complete response (pCR) in HER2-positive (+) breast cancer patients (pts) treated with neoadjuvant (NEO) dose-dense (dd) chemotherapy plus trastuzumab.

586 Background: We previously conducted a phase II study to evaluate a NEO regimen of docetaxel/carboplatin/trastuzumab (TCH) in 48 pts with clinical stage II-IIIB HER2 overexpressing breast cancer. The pCR rate was 42%. The current study sought to retrospectively investigate the correlation between total HER2 protein expression (H2T) and HER2 homodimers (H2D), by a novel quantitative HER2 assay, in pre-therapy tumor specimens from this trial and pCR and percent change in tumor size (ΔTS) following NEO ddTCH therapy. Methods: Pre-therapy formalin-fixed, paraffin-embedded (FFPE) tissue sections with ≥10 mm2 tumor area were available from 17 pts enrolled in the phase II NEO ddTCH study. H2T and H2D in these samples were quantified by the HERmark assay. Correlations between quantitative H2T and H2D measurements and pCR and ΔTS (as measured by longest tumor dimension) pre- versus post-therapy were assessed using the Mann-Whitney test and Spearman correlation, respectively. Results: All pts were previously assessed as HER2(+) by IHC 3+ or IHC 2+/FISH(+). 7/17 pts (41%) achieved pCR. pCR was associated with higher H2T (p = 0.033) and higher H2D (p = 0.066). Higher H2T and H2D were correlated with reduction of tumor size (ΔTS) following ddTCH (r2 = 0.461, p = 0.004, for H2T; and r2 = 0.604, p = 0.001, for H2D). H2T was higher in estrogen receptor (ER) (-) vs. ER (+) tumors (p = 0.043) and in progesterone (PR) (-) vs. PR (+) tumors (p = 0.021). Neither HER2 IHC nor tumor size at diagnosis correlated significantly with pCR or ΔTS. Conclusions: This is the first study to test the HERmark assay in the neoadjuvant setting. In this small group of breast cancer pts previously assessed as HER2 (+) by IHC and/or FISH, pts with higher H2T levels were more likely to achieve pCR following neoadjuvant treatment with a trastuzumab-containing regimen. These data are consistent with previous findings in the metastatic breast cancer setting and suggest that further investigation into the possible relationship between quantitative levels of HER2 expression and trastuzumab response, in larger patient cohorts, is warranted. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Monogram Biosciences

Engineering drug combinations

The level of an individual protein in cells treated with combinations of drugs is best explained by simple linear superposition of the protein levels in response to single drugs. This finding may facilitate rational design of higher order drug combinations.

Activation of antioxidant defenses in response to freezing in freeze-tolerant painted turtle hatchlings

Background Hatchlings of the painted turtle, Chrysemys picta marginata can endure long-term freezing of their extracellular body fluids. We hypothesized that freezing survival would include adaptive up-regulation of antioxidant defenses to deal with ischemia–reperfusion injuries associated with the freeze–thaw cycle. A number of antioxidant enzymes are under the control of the NF-E2 related factor 2 (Nrf2) transcription factor including members of the glutathione S-transferase (GST) and aldo–keto reductase (AKR) families. Methods RT–PCR and Western immunoblotting were used to measure changes in transcript and protein levels in response to 5-h freezing exposure of hatchlings. Results Transcript levels of Nrf2 increased in turtle brain, liver, and muscle by 1.5- to 2-fold, and protein levels increased in the brain and muscle by 1.6- to 2.3-fold in response to freezing. GSTs responded strongly to freezing in turtle brain with amounts of GSTP1, M1, K1, and A3 elevated by 1.5- to 2.4-fold. GSTM3 and T1 rose by 1.8- to 2.3-fold in gut, whereas reduced levels of GSTP1, M1, M3, and K1 were found in livers of frozen animals. Levels of the AKR1B4 isozyme rose 2.1-fold during freezing in brain. Conclusions Freezing triggered tissue-specific changes in the antioxidant defenses in C. picta marginata organs. General significance These data indicate that activation of an antioxidant response is an important aspect of natural freeze tolerance in turtles.

Need-Based Up-Regulation of Protein Levels in Response to Deletion of Their Duplicate Genes

Author SummaryDespite sequence divergence over long evolutionary times, many genes that have undergone duplication can still compensate for the loss of their duplicates. This compensation depends, not only on functional overlap between the paralogous genes, but also on overlap in their expression patterns. It has been proposed that compensation might therefore involve active up-regulation of a gene in response to deletion of its paralog. To test for such paralog responsiveness in the yeast Saccharomyces cerevisiae, we systematically measured changes in single-cell protein levels for approximately 200 duplicate genes in the presence or absence of their paralogs. Only a small fraction (∼11%) of proteins increased in level in response to deletion of their paralog, but this set matched almost exclusively the subset of paralogs whose overlapping function is required for viability. Further, when we examined yeast grown in different media, we found that genes had either gained or lost paralog responsiveness exactly according to their importance for growth in the tested conditions. Responsiveness, therefore, is need-based: it appears only in conditions in which the function of one or both paralogs is required. We propose that such need-based responsiveness of duplicate genes could play an important adaptive role, not just in the artificial event of paralog deletion, but also in the maintenance of functions that are compromised by natural genetic, environmental, or stochastic perturbations.

CHARACTERIZATION AND EXPRESSION ANALYSIS OF THE Lhcf GENE FAMILY IN EMILIANIA HUXLEYI (HAPTOPHYTA) REVEALS DIFFERENTIAL RESPONSES TO LIGHT AND CO21

Emiliania huxleyi (Lohmann) W. W. Hay et H. Mohler is the most abundant marine unicellular coccolithophore in the ocean and belongs to the group of organisms that have chl c and fucoxanthins as pigments in the photosynthetic light-harvesting complexes (LHCs). In this study, we report on the isolation and characterization of the mRNAs encoding six light-harvesting complex fucoxanthin-binding proteins (LHCFs) from E. huxleyi. Phylogenetic analysis of these sequences has revealed that they form three distinct subgroups: haptophyte, diatom/haptophyte, and LI818-like. Expression analysis of the six Lhcf genes showed a clear down-regulation at the transcriptional level when the cultures were grown in high light (300 μmol · m-2 · s-1) when compared to equivalent samples in low light (30 μmol · m-2 · s-1). In contrast, little impact on transcript levels was observed between cultures grown in either low CO 2 (180 ppm) or high CO2 (750 ppm) at either light intensities. Using polyclonal antibodies to three of the LHCFs revealed a down-regulation in protein levels in response to increased light availability with a minor increase in two of the LHCFs in elevated CO2. This study has provided an insight into the diversity of LHCFs and how changes in the levels of these proteins, together with altered pigment composition, may contribute to the flexible response of E. huxleyi to changes in the light environment. © 2009 Phycological Society of America.

Evidence for the Involvement of Galectin-3 in Mesenchymal Stem Cell Suppression of Allogeneic T-Cell Proliferation

Human bone marrow-derived mesenchymal stem cells (MSC) are multipotent non-hematopoietic progenitors that have regulatory activity on immune cells. NOD- and Toll-like receptors (NLR, TLR) have several roles in immunity, including those relevant to pathogen recognition and shaping the course of immune responses by controlling gene expression. We have shown that these innate immune receptors are expressed by hematopoietic CD34+ progenitors and MSC. To uncover genes critical in MSC function, first we have used microarray to screen for potential transcripts whose levels are altered in response to NOD-1 and TLR-2 activation, and second we validated some candidate genes using real-time RT-PCR, Western blots and cellular assays. Amongst the altered genes, galectin-3 was upregulated at both mRNA and protein levels in response to TLR-2 activation. Interestingly, MSC secreted galectin-3, a protein known to modulate T-cell proliferation, gene expression, cell adhesion and migration. Knockdown of galectin-3 in MSC using small interfering RNA (siRNA) reduced the immunosuppressive effect of MSC on mixed lymphocyte cultures when compared to cells treated with an irrelevant siRNA (P < 0.05). Collectively, the data emphasize a new role of galectin-3 in the immunomodulatory function of MSC and indicate that NOD signalling pathway is also functional in these cells.

Induction of heme oxygenase-1 in normal and malignant B lymphocytes by 15-deoxy-Δ12,14-prostaglandin J2 requires Nrf2

Heme-oxygenase-1 (HO-1) is induced in response to oxidative stress and is believed to be a cytoprotective and anti-inflammatory enzyme. It is unknown whether normal or malignant human B-lineage cells express HO-1. 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) is an interesting electrophilic lipid mediator able to increase oxidative stress in B cells. Here, we tested normal and malignant human B-lineage cells for their ability to express HO-1 in response to 15d-PGJ(2), as well as the signaling pathways required for HO-1 expression. 15d-PGJ(2) potently induced HO-1 protein expression in normal and malignant B cells. Malignant B cells exhibited a greater induction of HO-1 protein compared to normal B lymphocytes. Using siRNA directed against the transcription factor Nrf2 and B cells isolated from Nrf2-deficient mice, we show that HO-1 induction by 15d-PGJ(2) is dependent on Nrf2. These results show that, compared to normal B lymphocytes, malignant B cells have a greater capacity to increase their HO-1 protein levels in response to 15d-PGJ(2). We speculate that the ability to highly express HO-1 by malignant B cells could confer a survival advantage.

Apoptosis and the DNA Damage Response: The Role of the RB Tumor Suppressor Pathway in Oxidative Stress Responses in the Hematopoietic System.

Abstract SCI-15Exposure to pro-oxidants and defects in repair of oxidative base damage is associated with disease and aging and also contributes to the development of anemia, bone marrow failure and hematopoietic malignancies. Our work examines the role of the RB tumor suppressor pathway in the response of the hematopoietic system to oxidative stress and DNA damage. Evidence from mouse models has identified a role for the Rb protein (pRb) in the regulation of hematopoiesis through cell intrinsic functions in blood cell types but also through effects on the bone marrow microenvironment (Spike et al, 2004; Walkley et al, 2007; Daria et al, 2008). Such models have also demonstrated that pRb is required under stress conditions but not under conditions of steady state hematopoiesis (Spike et al, 2004; Spike et al, 2007; Daria et al, 2008). In particular, pRb was required to modulate the response of the hematopoietic system to replicative stress and hypoxia (Spike et al, 2007; Daria et al, 2008). To explain the mechanisms underlying these unique properties of pRb in hematopoiesis, we hypothesized that pRb protein levels are regulated by oxidative stress, including hypoxia and ROS generated as a consequence of stem cell location in the bone marrow niche or in response to replicative stress induced by agents such as 5-fluorouracil. Notably, hypoxia within the bone marrow niche has been reported to promote stem cell expansion and we postulated that this may be due to reduced pRb protein levels in response to hypoxia. We present evidence that pRb protein levels are regulated in wild-type bone marrow in response to replicative stress and that this in turn modulates expansion of stem cells and myeloid progenitors and also impacts end-stage differentiation in the erythroid lineage. Acetylation of pRb stabilized the protein in an active conformation while de-acetylation de-stabilized the protein and promoted pRb protein turnover and increased progenitor cell proliferation. We will present on-going studies that examine how hypoxia and/or ROS affects hematopoietic stem cell proliferation, self-renewal and differentiation in vivo as a function of pRb protein levels using conditional mouse models. The significance of our findings for bone marrow failure in human patients will be discussed. References Spike, B.T. et al. The Rb tumor suppressor is required for stress erythropoiesis. The EMBO J. 2004: 23, 4319-29. Spike, B.T., Dibling, B.C. & Macleod, K.F. Hypoxic stress underlies defects in erythroblast island formation in the Rb null mouse. Blood 2007; 110, 2173-81. Walkley, C.R., Shea, J.M., Sims, N.A., Purton, L.E. & Orkin, S.H. Rb regulates interactions between hematopoietic stem cells and their bone marrow microenvironment. Cell 2007; 129, 1081-95. Daria, D. et al. The retinoblastoma tumor suppressor is a critical intrinsic regulator for hematopoietic stem and progenitor cells under stress. Blood 2008; 111, 1894-902. Funding: The author is grateful to the J.P. McCarthy Foundation, the Aplastic Anemia and MDS International Foundation and the National Heart Lung & Blood Institute (RO1 HL080262) for funding of work in her laboratory relating to oxidative stress, erythropoiesis and hematopoietic diseases. DisclosuresNo relevant conflicts of interest to declare.

CXCL4 Downregulates the Atheroprotective Hemoglobin Receptor CD163 in Human Macrophages

CXCL4 is a platelet-derived chemokine that promotes macrophage differentiation from monocytes. Deletion of the PF4 gene that encodes CXCL4 reduces atherosclerotic lesions in ApoE(-/-) mice. We sought to study effects of CXCL4 on macrophage differentiation with possible relevance for atherogenesis. Flow cytometry for expression of surface markers in macrophage colony-stimulating factor (M-CSF)- and CXCL4-induced macrophages demonstrated virtually complete absence of the hemoglobin scavenger receptor CD163 in CXCL4-induced macrophages. mRNA for CD163 was downregulated as early as 2 hours after CXCL4. CD163 protein reached a minimum after 3 days, which was not reversed by treatment of cells with M-CSF. The CXCL4 effect was entirely neutralized by heparin, which bound CXCL4 and prevented CXCL4 surface binding to monocytes. Pretreatment of cells with chlorate, which inhibits glycosaminoglycan synthesis, strongly inhibited CXCL4-dependent downregulation of CD163. Similar to recombinant CXCL4, releasate from human platelets also reduced CD163 expression. CXCL4-differentiated macrophages were unable to upregulate the atheroprotective enzyme heme oxygenase-1 at the RNA and protein level in response to hemoglobin-haptoglobin complexes. Immunofluorescence of human atherosclerotic plaques demonstrated presence of both CD68+CD163+ and CD68+CD163- macrophages. PF4 and CD163 gene expression within human atherosclerotic lesions were inversely correlated, supporting the in vivo relevance of CXCL4-induced downregulation of CD163. CXCL4 may promote atherogenesis by suppressing CD163 in macrophages, which are then unable to upregulate the atheroprotective enzyme heme oxygenase-1 in response to hemoglobin.

Role of TWIST in the apoptosis of Hep-2 cells induced by paclitaxel

To explore the relationship between the expression of transcription factor TWIST and apoptosis of Hep-2 cells induced by paclitaxel. Morphological changes of Hep-2 cells were observed by reserved microscopy and acridine orange cytochemistry staining. Viability of Hep-2 cells treated with various concentrations of paclitaxel was detected by MTT assay. Apoptosis was examined by flow cytometry. The expressions of transcription factor TWIST at both mRNA and protein level in response to paclitaxel at 24 h, 48 h and 72 h were then examined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot, respectively. Typical morphological changes of apoptotic cells, i.e., cellular rounding-up, cytoplasmic contraction, chromatin condensation and, particularly, apoptotic body, the main morphological characteristic of apoptosis, were observed by reserved microscopy and acridine orange cytochemistry staining. The cell surviving rates significantly decreased in a concentration- and time-dependent manner as evidenced by MTT assay (P < 0.05). Percent of apoptosis after 24 h, 48 h or 72 h paclitaxel-treatment was (22.6 +/- 5.3)%, (38.7 +/- 7.9)% and (52.4 +/- 14.3)%, respectively, whereas the percent of control was (9.85 +/- 5.83)%. There existed a statistically significant difference between treatment and control (F = 12.621, P < 0.05). The expression of TWIST at both mRNA and protein levels for 24 h, 48 h or 72 h in the paclitaxel-induced apoptosis of Hep-2 cells were decreased by 16.7%, 46.8%, 76.9% (F = 10.407, P < 0.05) and 16.4%, 33.6%, 69.6% (F = 18.013, P < 0.05) respectively. TWIST, which is significantly decreased in expression in response to paclitaxel in Hep-2 cells, may play a pivotal role in paclitaxel-induced apoptosis of Hep-2 cells.

Induction of RIP-2 kinase by proinflammatory cytokines is mediated via NF-κB signaling pathways and involves a novel feed-forward regulatory mechanism

The transcription factor NF-kappaB (nuclear factor kappaB) is a central mediator of inflammatory and apoptotic signaling in the cell. The protein kinase RIP-2 is a member of the CARD protein family (caspase activation and recruitment domain, also known as CARD3, Ripk2, CARDIAK, RICK, and CCK), and has been shown to be an activator of NF-kappaB. In this study, it was demonstrated by transcriptional profiling and protein expression analysis that the inflammatory cytokines TNF-alpha, IL-1 beta, and IFN-gamma induced RIP-2 transcription and translation in endothelial cells. Two mechanistically distinct inhibitors of NF-kappaB signaling, sulfasalazine (NF-kappaB inhibitor) and WY-14643 [PPAR alpha (peroxisome proliferator-activated receptor alpha) agonist] that interfere with the transcription factor RELA (p65), suppressed TNF-alpha induced RIP-2 gene expression, which indicated that NF-kappaB signaling was involved in the cytokine-induced transcriptional activation of RIP-2 gene expression. Consistent with these observations, multiple NF-kappaB response elements were found in the upstream regions of the human and mouse RIP-2 genes. NF-kappaB-mediated regulation of RIP-2 gene and protein expression suggests an additional step in the regulation of NF-kappaB function as RIP-2 has been shown to positively modulate NF-kappaB by binding IKK gamma (I kappaB kinase gamma), a component of the IKK complex. These findings support a positive feed-forward mechanism of NF-kappaB regulation that involves NF-kappaB-dependent induction of RIP-2 transcription and a subsequent increase in RIP-2 protein levels in response to inflammatory cytokines. Elevated RIP-2 protein levels are then available to promote NF-kappaB function via interaction with IKK gamma. RIP-2 is the first reported NF-kappaB-dependent protein kinase that positively regulates NF-kappaB activity.

Mitochondrial Proteomics in Experimental Autoimmune Uveitis Oxidative Stress

Photoreceptor mitochondrial oxidative stress is the initial pathologic event in experimental autoimmune uveitis. In this study, the authors determined alterations in retinal mitochondrial protein levels in response to oxidative stress during the early phase of experimental autoimmune uveitis (EAU). Retinal mitochondrial fractions during early EAU were prepared and subjected to two-dimensional difference in gel electrophoresis (2D-DIGE). Protein spots showing differential expression were excised and subjected to matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) for peptide identification. Levels of these proteins were also confirmed by Western blot analysis. mRNA expression of these proteins was confirmed by real-time PCR. TUNEL staining was performed to detect apoptosis. 2D-DIGE analysis revealed differential expression of 13 proteins. Ten proteins were overexpressed, including manganese-SOD, alphaA crystallin, beta crystallin, and four proteins were downregulated, including adenosine triphosphate (ATP) synthase, malate dehydrogenase, and calretinin. Increased levels of alphaA crystallin, betaB2 crystallin, MnSOD, and aconitase and decreased levels of ATP synthase were confirmed by Western blot analysis. qPCR also confirmed the increased expression of alphaA crystallin, betaB2 crystallin, MnSOD, and Hsp70. Apoptosis was absent during this phase. The presence of mitochondrial-specific oxidative stress-related proteins in the early EAU retina along with the downregulation of ATP synthase provides early evidence of stress-related retinal damage. The presence of high levels of alphaA and betaB2 crystallin in the mitochondria may prevent cell death during early EAU.

Regulation of HSH2 Adaptor Protein Expression in B cells is Mediated by Posttranscriptional Mechanisms (34.18)

Abstract Previous studies have shown that the adaptor protein hematopoietic src homology 2 (HSH2) is expressed at low levels in resting B cells. HSH2 expression is up-regulated in response to various stimuli that promote splenic B cell survival and differentiation including α-CD40 mAb, LPS, CpG, and Blys. HSH2 expression was found to be dependent upon NF-¿B stimulation in that inhibitors of NF-¿B blocked up-regulation of HSH2 expression. HSH2 expression did not increase upon stimulation of B cells via the BCR unless cells were previously stimulated through CD40. Preliminary data reveal that HSH2 protein expression is decoupled from transcript abundance. Real-time PCR analyses show that upon α-CD40 stimulation of splenic B cells, HSH2 transcript levels remain relatively constant, whereas protein expression increases. This suggests that there are one or more posttranscriptional mechanisms in place that control HSH2 expression at the protein level. One possibility is that the untranslated regions of HSH2 mRNA contain regulatory regions responsible for translational control, which is currently under investigation. A second finding is that HSH2 turns over rapidly at the protein level in response to conditions in which the 26S proteosome is blocked with pharmacological antagonists. Thus, it is apparent that HSH2 is subject to proteolytic degradation and that this is under tight regulation. In summary, HSH2 expression is controlled by posttranscriptional mechanisms that maintain low basal expression in resting B cells, but promote increased expression in cells exposed to agonists that promote survival and differentiation.

CAMP‐Induced Expression of the Orphan Nuclear Receptor Nur77 in MA‐10 Leydig Cells Involves a CaMKI Pathway

The Nur77 (Nr4a1) gene, encoding the orphan nuclear receptor NUR77 (NR4A1), is an immediate early response gene whose expression is rapidly induced by a variety of physiologic stimuli. Nur77 is expressed in several organs, including the classic steroidogenic tissues: gonads and adrenal. In MA-10 Leydig cells, NUR77 has been shown to regulate expression of several genes involved in steroidogenesis and male sex differentiation. In Leydig cells, androgen biosynthesis is controlled primarily by the pituitary gonadotropin luteinizing hormone (LH) acting via its receptor (LH-R), which in turn activates the adenylate cyclase/cyclic adenosine monophosphate (cAMP) signaling pathway. Even though Nur77 expression is induced both at the mRNA and protein levels in response to LH/forskolin/cAMP in Leydig cells, the mechanisms involved remain largely unknown. Here, we report that cAMP-mediated induction of Nur77 expression at the protein, mRNA, and promoter levels in MA-10 cells involves different mechanisms. We found that increased NUR77 protein requires transcription and translation, whereas increased Nur77 mRNA does not require de novo protein synthesis, and would therefore rely on transcription factors already present in the cell. In addition, our detailed analysis of the Nur77 promoter in MA-10 cells revealed that distinct regulatory elements are involved in basal and cAMP-induced Nur77 transcription. Finally, we found that maximal cAMP-mediated increase in Nur77 promoter activity involves a Ca(2+)/calmodulin kinase (CaMK)-dependent pathway and that Ca(2+)/calmodulin kinase I regulates Nur77 promoter activity in Leydig cells. Thus, our findings demonstrate the involvement of various mechanisms in the regulation of Nur77 expression in MA-10 Leydig cells, including a previously uncharacterized CaMK pathway.

Comparative Temporal Proteomics of a Response Regulator (SO2426)-Deficient Strain and Wild-Type Shewanella oneidensis MR-1 During Chromate Transformation

Predicted orphan response regulators encoded in the Shewanella oneidensis MR-1 genome are poorly understood from a cellular function perspective. Our previous transcriptomic and proteomic analyses demonstrated that an annotated DNA-binding response regulator, SO2426, was significantly up-regulated in wild-type S. oneidensis cells at both the mRNA and protein levels in response to acute chromate [Cr(VI)] challenge, suggesting a potential regulatory role for this protein in metal stress pathways. To investigate the impact of SO2426 activity on chromate stress response at a genome-wide scale, we describe here comparative and temporal proteome characterizations using multidimensional HPLC-MS/MS and statistical analysis to identify differentially expressed proteins in biological replicates of wild-type S. oneidensis MR-1 and a so2426 deletion (Deltaso2426) strain, which exhibited an impaired Cr(VI) transformation rate compared to that of the parental strain. Global protein profiles were examined at different time intervals (0, 1, 3, 4 h) following exogenous chromate challenge. Results indicated that deletion of the so2426 gene negatively affected expression of a small protein subset (27 proteins) including those with annotated functions in siderophore biosynthesis (SO3032), Fe uptake (SO4743), intracellular Fe storage (Bfr1), and other transport processes. Cr(VI) exposure and subsequent transformation dramatically increased the number of differentially expressed proteins detected, with up-regulated abundance patterns observed largely for proteins involved in general stress protection and detoxification strategies, cell motility, and protein fate. In addition, the proteome data sets were mined for amino acids with potential post-translational modifications (PTMs) indicative of a level of gene expression regulation extending beyond the transcriptional control imposed by SO2426.

The cross-talk between NF-κB and HIF-1: further evidence for a significant liaison

1. The name of the author S. Bonello was spelt incorrectly in the contents list and indexes in the print version of this paper. The online journal is not affected.2. Important information and a reference were omitted from this online-only commentary in the first sentence of the third paragraph. The information (LPS), reference and the relevant citation in the text appear here:A cross-talk between the NF-κB pathway and the HIF pathway has been documented extensively at the protein level in response to TNF-α, colchicine, LPS or HGF (hepatocyte growth factor) [13–16,16a].

Brain-derived Neurotrophic Factor-Tropomyosin-related Kinase B Signaling Contributes to Activity-dependent Changes in Synaptic Proteins

The ability of synapses to undergo changes in structure and function in response to alterations of neuronal activity is an essential property of neural circuits. One way that this is achieved is through global changes in the molecular composition of the synapse; however, it is not clear how these changes are coupled to the dynamics of neuronal activity. Here we found that, in cultured rat cortical neurons, bidirectional changes of neuronal activity led to corresponding alterations in the expression of brain-derived neurotrophic factor (BDNF) and phosphorylation of its receptor tropomyosin-related kinase B (TrkB), as well as in the level of synaptic proteins. Exogenous BDNF reversed changes in synaptic proteins induced by chronic activity blockade, while inhibiting Trk kinase activity or depleting endogenous BDNF abolished the concentration changes induced by chronic activity elevation. Both tetrodotoxin and bicuculline had significant, but opposite, effects on synaptic protein ubiquitination in a time-dependent manner. Furthermore, exogenous BDNF was sufficient to increase ubiquitination of synaptic proteins, whereas scavenging endogenous BDNF or inhibiting Trk kinase activity prevented the ubiquitination of synaptic proteins induced by chronic elevation of neuronal activity. Inhibiting the proteasome or blocking protein polyubiquitination mimicked the effect of tetrodotoxin on the levels of synaptic proteins and canceled the effects of BDNF. Our study indicates that BDNF-TrkB signaling acts upstream of the ubiquitin proteasome system, linking neuronal activity to protein turnover at the synapse.

Impact of protein kinase CK2 on inhibitor of apoptosis proteins in prostate cancer cells

We have previously demonstrated that protein kinase CK2 is a potent suppressor of apoptosis in cells subjected to diverse mediators of apoptosis. The process of apoptosis involves a complex series of molecules localized in various cellular compartments. Among the various proteins that modulate apoptotic activity are inhibitors of apoptosis proteins (IAPs) which are elevated in cancers and have been proposed to block caspase activity. We have examined the impact of CK2 signal on these proteins in prostate cancer cells. Cellular IAPs demonstrate distinct localization and responsiveness to altered CK2 expression or activity in the cytoplasmic and nuclear matrix fractions. Modulation of cellular CK2 by various approaches impacts on cellular IAPs such that inhibition or downregulation of CK2 results in reduction in these proteins. Further, IAPs are also reduced when cells are treated with sub-optimal concentrations of chemical inhibitors of CK2 combined with low or sub-optimal levels of apoptosis-inducing agents (such as etoposide) suggesting that downregulation of CK2 sensitizes cells to induction of apoptosis which may be related to attenuation of IAPs. Decreased IAP protein levels in response to apoptotic agents such as TNFalpha or TRAIL were potently blocked upon forced overexpression of CK2 in cells. Together, our results suggest that one of the modes of CK2-mediated modulation of apoptotic activity is via its impact on cellular IAPs.

Cover Picture: Proteomics 11/2008

AbstractIn this issue of Proteomics you will find the following highlighted articles:Pancreatic cancer signs autograph on micro antibody arrayPancreatic cancer has been one of the nastier members of the “Discovered‐too‐late‐to‐do‐anything‐about‐it” disease club. Its 5‐year survival rate is 3–5 % because of late diagnosis and no effective therapy for advanced disease cases. This paper by Ingvarsson et al. reports their encouraging findings on the use of recombinant antibody microarrays to survey serum for diagnostic and prognostic proteins. In these “proof‐of‐concept” experiments they found a signature of 19 unique scFv antibodies, specific for immunoregulatory proteins, that could distinguish pancreatic cancer from normal and from Helicobacter pylori (an indicator of inflammation, 3 out of 14 overlap). The test panel distinguished long and short survivors (with only one long survivor misclassified). Data was classified using a Support Vector Machine. The classifier was validated by multiple splits of the data and leave‐one‐out tests.Ingvarsson, J. et al., Proteomics 2008, 8, 2211–2219.Of cadmium and zinc: Brothers or not?Cadmium and zinc occupy the same column in the periodic table so you might expect some biological similarities. Not much luck – mercury is also in that column. Zinc, under tight control, is an essential mineral; cadmium is toxic and induces a variety of defensive responses. A highly zinc‐resistant cell line (HZR) has been derived from the human HeLa line. Rousselet et al. have compared the proteomes of HZR and HeLa cultured in Cd and Zn using a variety of proteomic and genomic tools. MALDI‐TOF MS after 2‐DE revealed examples of a co‐chaperone, a heat‐shock organizing protein (Hop), ubiquitin and a number of reactive oxygen species control proteins elevated in HZR. Of special interest was 4‐hydroxyphenyl‐pyruvate dioxygenase (HPPD), catalyst of one of the first breakdown steps of tyrosine. The complex relationships revealed will require a lot more than one paragraph for explanation.Rousselet, E. et al., Proteomics 2008, 8, 2244–2255.Grey box proteomics of salty speciesIn the classic black box experiment you know nothing about the contents of the box. I propose a grey box for experiments directed by homologous knowledge – like these. Pandhal et al. have developed a protocol for proteomic analysis of an unsequenced species by homology. The organism of interest is a halotolerant cyanobacterium, Euhalothece sp. which can grow in NaCl concentrations ranging from 0% to &gt;9% NaCl. The nearest sequenced relative is a Synechocystis sp. By metabolic labeling with 15N/­14N, the researchers were able to use MS to match proteins from the two species and also quantitate changes in levels of proteins in response to salt levels. Three labelling experiments ([% NaCl], 0% +3%, 3% +6%, and 3% +9%) yielded 229, 212, and 96 proteins, respectively, by MASCOT search of proteins with two peptides of each isotope. MS BLAST found 32, 30, and 7 more proteins, respectively.Pandhal, J. et al., Proteomics 2008, 8, 2266–2284.