Comprehensive Proteomic Analysis Reveals Intermediate Stage of Non-Lesional Psoriatic Skin and Points out the Importance of Proteins Outside this Trend.

To better understand the pathomechanism of psoriasis, a comparative proteomic analysis was performed with non-lesional and lesional skin from psoriasis patients and skin from healthy individuals. Strikingly, 79.9% of the proteins that were differentially expressed in lesional and healthy skin exhibited expression levels in non-lesional skin that were within twofold of the levels observed in healthy and lesional skin, suggesting that non-lesional skin represents an intermediate stage. Proteins outside this trend were categorized into three groups: I. proteins in non-lesional skin exhibiting expression similar to lesional skin, which might be predisposing factors (i.e., CSE1L, GART, MYO18A and UGDH); II. proteins that were differentially expressed in non-lesional and lesional skin but not in healthy and lesional skin, which might be non-lesional characteristic alteration (i.e., CHCHD6, CHMP5, FLOT2, ITGA7, LEMD2, NOP56, PLVAP and RRAS); and III. proteins with contrasting differential expression in non-lesional and lesional skin compared to healthy skin, which might contribute to maintaining the non-lesional state (i.e., ITGA7, ITGA8, PLVAP, PSAPL1, SMARCA5 and XP32). Finally, proteins differentially expressed in lesions may indicate increased sensitivity to stimuli, peripheral nervous system alterations, furthermore MYBBP1A and PRKDC were identified as potential regulators of key pathomechanisms, including stress and immune response, proliferation and differentiation.

Differential Proteomics Reveals Discrete Functions of Proteins Interacting with Hypo- versus Hyper-phosphorylated NS5A of the Hepatitis C Virus.

Protein phosphorylation is a reversible post-translational modification that regulates many biological processes in almost all living forms. In the case of the hepatitis C virus (HCV), the nonstructural protein 5A (NS5A) is believed to transit between hypo- and hyper-phosphorylated forms that interact with host proteins to execute different functions; however, little was known about the proteins that bind either form of NS5A. Here, we generated two high-quality antibodies specific to serine 235 nonphosphorylated hypo- vs serine 235 phosphorylated (pS235) hyper-phosphorylated form of NS5A and for the first time segregated these two forms of NS5A plus their interacting proteins for dimethyl-labeling based proteomics. We identified 629 proteins, of which 238 were quantified in three replicates. Bioinformatics showed 46 proteins that preferentially bind hypo-phosphorylated NS5A are involved in antiviral response and another 46 proteins that bind pS235 hyper-phosphorylated NS5A are involved in liver cancer progression. We further identified a DNA-dependent kinase (DNA-PK) that binds hypo-phosphorylated NS5A. Inhibition of DNA-PK with an inhibitor or via gene-specific knockdown significantly reduced S232 phosphorylation and NS5A hyper-phosphorylation. Because S232 phosphorylation initiates sequential S232/S235/S238 phosphorylation leading to NS5A hyper-phosphorylation, we identified a new protein kinase that regulates a delicate balance of NS5A between hypo- and hyper-phosphorylation states, respectively, involved in host antiviral responses and liver cancer progression.

Assessment of DNA-PKcs kinase activity by quantum dot-based microarray.

Therapeutic efficacy against cancer is often based on a variety of DNA lesions, including DNA double-strand breaks (DSBs) which are repaired by homologous recombination and non-homologous end joining (NHEJ) pathways. In the past decade, the functions of the DNA repair proteins have been described as a potential mechanism of resistance in tumor cells. Therefore, the DNA repair proteins have become targets to improve the efficacy of anticancer therapy. Given the central role of DNA-PKcs in NHEJ, the therapeutic efficacy of targeting DNA-PKcs is frequently described as a strategy to prevent repair of treatment-induced DNA damage in cancer cells. The screening of a new inhibitor acting as a sensitizer requires the development of a high-throughput tool in order to identify and assess the most effective molecule. Here, we describe the elaboration of an antibody microarray dedicated to the NHEJ pathway that we used to evaluate the DNA-PKcs kinase activity in response to DNA damage. By combining a protein microarray with Quantum-Dot detection, we show that it is possible to follow the modification of phosphoproteomic cellular profiles induced by inhibitors during the response to DNA damage. Finally, we discuss the promising tool for screening kinase inhibitors and targeting DSB repair to improve cancer treatment.

Apoptosis in human compressive myelopathy due to metastatic neoplasia.

STUDY DESIGN: Immunohistochemical assessment of apoptotic markers in human cases of compressive myelopathy due to neoplastic compression. OBJECTIVE: To characterize the role of apoptosis in neoplastic compressive myelopathy in human postmortem tissue with extramedullary tumor involvement. SUMMARY OF BACKGROUND DATA: Neoplasms, whether primary or metastatic, may lead to compression of the spinal cord and development of a compressive myelopathy syndrome. Apoptotic processes of cell death are thought to contribute to cell death in chronic compressive myelopathy because of degenerative spondylosis, but this has not previously been described in neoplastic compression. METHODS: Six postmortem cases of human neoplastic compressive myelopathy were assessed for apoptosis using a panel of immunohistochemical markers including Fas, B-cell lymphoma 2 (Bcl-2), caspase-3 and 9, DNA-dependent protein kinase catalytic subunit (DNA-PKcs), poly (ADP-ribose) polymerase (PARP), apoptosis-inducing factor (AIF), and terminal deoxynucleotide transferase dUTP Nick End Labeling (TUNEL). RESULTS: Apoptosis was maximal at the site of tumor compression. Glial cells, predominantly oligodendrocytes, were immunopositive for DNA-PKcs, PARP, AIF, and TUNEL. Axons were immunopositive for caspase 3, DNA-PKcs, and AIF. Neurons were immunopositive for DNA-PKcs, PARP, AIF, and TUNEL. CONCLUSION: The current study demonstrates that apoptosis plays a role in human neoplastic compressive myelopathy. Necrosis dominates the severe end of the spectrum of compression. The prominent oligodendroglial involvement is suggestive that apoptosis may be important in the ongoing remodeling of white matter due to sustained compression. LEVEL OF EVIDENCE: 4.

Scanning fluorescence correlation spectroscopy techniques to quantify the kinetics of DNA double strand break repair proteins after γ-irradiation and bleomycin treatment.

A common feature of DNA repair proteins is their mobilization in response to DNA damage. The ability to visualizing and quantifying the kinetics of proteins localizing/dissociating from DNA double strand breaks (DSBs) via immunofluorescence or live cell fluorescence microscopy have been powerful tools in allowing insight into the DNA damage response, but these tools have some limitations. For example, a number of well-established DSB repair factors, in particular those required for non-homologous end joining (NHEJ), do not form discrete foci in response to DSBs induced by ionizing radiation (IR) or radiomimetic drugs, including bleomycin, in living cells. In this report, we show that time-dependent kinetics of the NHEJ factors Ku80 and DNA-dependent protein kinase catalytic subunits (DNA-PKcs) in response to IR and bleomycin can be quantified by Number and Brightness analysis and Raster-scan Image Correlation Spectroscopy. Fluorescent-tagged Ku80 and DNA-PKcs quickly mobilized in response to IR and bleomycin treatments consistent with prior reports using laser-generated DSBs. The response was linearly dependent on IR dose, and blocking NHEJ enhanced immobilization of both Ku80 and DNA-PKcs after DNA damage. These findings support the idea of using Number and Brightness and Raster-scan Image Correlation Spectroscopy as methods to monitor kinetics of DSB repair proteins in living cells under conditions mimicking radiation and chemotherapy treatments.

Abnormal DNA-PKcs and Ku 70/80 expression may promote malignant pathological processes in gastric carcinoma.

AIM: To determine the expression of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) and the Ku70/Ku80 heterodimer (Ku 70/80) in gastric carcinoma. METHODS: Gastric biopsies were obtained from 146 gastric carcinoma patients [Helicobacter pylori (H. pylori)-negative: 89 and H. pylori-positive: 57] and 34 from normal subjects (H. pylori-negative: 16 and H. pylori-positive: 18) via surgery and endoscopic detection from April 2011 to August 2012 at the First Affiliated Hospital of Nanchang University. Pathological diagnosis and classification were made according to the criteria of the World Health Organization and the updated Sydney system. An ''in-house'' rapid urease test and modified Giemsa staining were employed to detect H. pylori infection. The expression of DNA-PKcs and the Ku 70/80 protein was detected by immunohistochemistry. RESULTS: Overall, the positive rates of both DNA-PKcs and Ku 70/80 were significantly increased in gastric cancer (χ(2) = 133.04, P < 0.001 for DNA-PKcs and χ(2) = 13.06, P < 0.01 for Ku) compared with normal gastric mucosa. There was hardly any detectable expression of DNA-PKcs in normal gastric mucosa, and the positive rate of DNA-PKcs protein expression in patients with a normal gastric mucosa was 0% (0/34), whereas the rate in gastric cancer (GC) was 93.8% (137/146). The difference between the two groups was statistically significant. Additionally, the positive rate of Ku 70/80 was 79.4% (27/34) in normal gastric mucosa and 96.6% (141/146) in gastric cancer. The DNA-PKcs protein level was significantly increased in gastric cancer (Mann-Whitney U = 39.00, P < 0.001), compared with normal gastric mucosa. In addition, there was a significant difference in the expression of Ku 70/80 (Mann-Whitney U = 1117.00, P < 0.001) between gastric cancer and normal gastric mucosa. There was also a significant difference in Ku70/80 protein expression between GC patients with and without H. pylori infection (P < 0.05). Spearman analysis showed a negative correlation between tumor differentiation and DNA-PKcs expression (r = -0.447, P < 0.05). Moreover, Ku70/80 expression was negatively correlated with both clinical stage (r = -0.189, P < 0.05) and H. pylori colonization (r = -0.168, P < 0.05). CONCLUSION: Overall, this research demonstrated that enhanced DNA-PKcs and Ku 70/80 expression may be closely associated with gastric carcinoma.

Accumulation of DNA damage in complex normal tissues after protracted low-dose radiation.

The biological consequences of low levels of radiation exposure and their effects on human health are unclear. Ionizing radiation induces a variety of lesions of which DNA double-strand breaks (DSBs) are the most biologically significant, because unrepaired or misrepaired DSBs can lead to genomic instability and cell death. Using repair-proficient mice as an in vivo system we monitored the accumulation of DNA damage in normal tissues exposed to daily low-dose radiation of 100mGy or 10mGy. Radiation-induced foci in differentiated and tissue-specific stem cells were quantified by immunofluorescence microscopy after 2, 4, 6, 8, and 10 weeks of daily low-dose radiation and DNA lesions were characterized using transmission electron microscopy (TEM) combined with immunogold-labeling. In brain, long-living cortical neurons had a significant accumulation of foci with increasing cumulative doses. In intestine and skin, characterized by constant cell renewal of their epithelial lining, differentiated enterocytes and keratinocytes had either unchanged or only slightly increased foci levels during protracted low-dose radiation. Significantly, analysis of epidermal stem cells in skin revealed a constant increase of 53BP1 foci during the first weeks of low-dose radiation even with 10mGy, suggesting substantial accumulations of DSBs. However, TEM analysis suggests that these remaining 53BP1 foci, which are predominantly located in compact heterochromatin, do not co-localize with phosphorylated Ku70 or DNA-PKcs, core components of non-homologous end-joining. The biological relevance of these persistent 53BP1 foci, particularly their contribution to genomic instability by genetic and epigenetic alterations, has to be defined in future studies.

Regulatory roles of tankyrase 1 at telomeres and in DNA repair: suppression of T-SCE and stabilization of DNA-PKcs.

Intrigued by the dynamics of the seemingly contradictory yet integrated cellular responses to the requisites of preserving telomere integrity while also efficiently repairing damaged DNA, we investigated roles of the telomere associated poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) tankyrase 1 in both telomere function and the DNA damage response following exposure to ionizing radiation. Tankyrase 1 siRNA knockdown in human cells significantly elevated recombination specifically within telomeres, a phenotype with the potential of accelerating cellular senescence. Additionally, depletion of tankyrase 1 resulted in concomitant and rapid reduction of the nonhomologous end-joining protein DNA-PKcs, while Ku86 and ATM protein levels remained unchanged; DNA-PKcs mRNA levels were also unaffected. We found that the requirement of tankyrase 1 for DNA-PKcs protein stability reflects the necessity of its PARP enzymatic activity. We also demonstrated that depletion of tankyrase 1 resulted in proteasome-mediated DNA-PKcs degradation, explaining the associated defective damage response observed; i.e., increased sensitivity to ionizing radiation-induced cell killing, mutagenesis, chromosome aberration and telomere fusion. We provide the first evidence for regulation of DNA-PKcs by tankyrase 1 PARP activity and taken together, identify roles of tankyrase 1 with implications not only for DNA repair and telomere biology, but also for cancer and aging.

PKBalpha/Akt1 acts downstream of DNA-PK in the DNA double-strand break response and promotes survival.

Protein kinase B (PKB/Akt) is a well-established regulator of several essential cellular processes. Here, we report a route by which activated PKB promotes survival in response to DNA insults in vivo. PKB activation following DNA damage requires 3-phosphoinositide-dependent kinase 1 (PDK1) and DNA-dependent protein kinase (DNA-PK). Active PKB localizes in the nucleus of gamma-irradiated cells adjacent to DNA double-strand breaks, where it colocalizes and interacts with DNA-PK. Levels of active PKB inversely correlate with DNA damage-induced apoptosis. A significant portion of p53- and DNA damage-regulated genes are misregulated in cells lacking PKBalpha. PKBalpha knockout mice show impaired DNA damage-dependent induction of p21 and increased tissue apoptosis after single-dose whole-body irradiation. Our findings place PKB downstream of DNA-PK in the DNA damage response signaling cascade, where it provides a prosurvival signal, in particular by affecting transcriptional p21 regulation. Furthermore, this function is apparently restricted to the PKBalpha isoform.

Repair of HZE-particle-induced DNA double-strand breaks in normal human fibroblasts.

DNA damage generated by high-energy and high-Z (HZE) particles is more skewed toward multiply damaged sites or clustered DNA damage than damage induced by low-linear energy transfer (LET) X and gamma rays. Clustered DNA damage includes abasic sites, base damages and single- (SSBs) and double-strand breaks (DSBs). This complex DNA damage is difficult to repair and may require coordinated recruitment of multiple DNA repair factors. As a consequence of the production of irreparable clustered lesions, a greater biological effectiveness is observed for HZE-particle radiation than for low-LET radiation. To understand how the inability of cells to rejoin DSBs contributes to the greater biological effectiveness of HZE particles, the kinetics of DSB rejoining and cell survival after exposure of normal human skin fibroblasts to a spectrum of HZE particles was examined. Using gamma-H2AX as a surrogate marker for DSB formation and rejoining, the ability of cells to rejoin DSBs was found to decrease with increasing Z; specifically, iron-ion-induced DSBs were repaired at a rate similar to those induced by silicon ions, oxygen ions and gamma radiation, but a larger fraction of iron-ion-induced damage was irreparable. Furthermore, both DNA-PKcs (DSB repair factor) and 53BP1 (DSB sensing protein) co-localized with gamma-H2AX along the track of dense ionization produced by iron and silicon ions and their focus dissolution kinetics was similar to that of gamma-H2AX. Spatial co-localization analysis showed that unlike gamma-H2AX and 53BP1, phosphorylated DNA-PKcs was localized only at very specific regions, presumably representing the sites of DSBs within the tracks. Examination of cell survival by clonogenic assay indicated that cell killing was greater for iron ions than for silicon and oxygen ions and gamma rays. Collectively, these data demonstrate that the inability of cells to rejoin DSBs within clustered DNA lesions likely contributes to the greater biological effectiveness of HZE particles.

mTOR.RICTOR is the Ser473 kinase for Akt/protein kinase B in 3T3-L1 adipocytes.

The insulin-signaling pathway leading to the activation of Akt/protein kinase B has been well characterized except for a single step, the phosphorylation of Akt at Ser-473. Double-stranded DNA-dependent protein kinase (DNA-PK), ataxia telangiectasia mutated (ATM) gene product, integrin-linked kinase (ILK), protein kinase Calpha (PKCalpha), and mammalian target of rapamycin (mTOR), when complexed to rapamycin-insensitive companion of mTOR (RICTOR), have all been identified as playing a critical role in Akt Ser-473 phosphorylation. However, the apparently disparate results reported in these studies are difficult to evaluate, given that different stimuli and cell types were examined and that all of the candidate proteins have never been systematically studied in a single system. Additionally, none of these studies were performed in a classical insulin-responsive cell type or tissue such as muscle or fat. We therefore examined each of these candidates in 3T3-L1 adipocytes. In vitro kinase assays, using different subcellular fractions of 3T3-L1 adipocytes, revealed that phosphatidylinositol 3,4,5-trisphosphate-stimulated Ser-473 phosphorylation correlated well with the amount of DNA-PK, mTOR, and RICTOR but did not correlate with levels of ATM, ILK, and PKCalpha. PKCalpha was completely absent from compartments with Ser-473 phosphorylation activity. Although purified DNA-PK could phosphorylate a peptide derived from Akt that contains amino acid Ser-473, it could not phosphorylate full-length Akt2. Vesicles immunoprecipitated from low density microsomes using antibodies directed against mTOR or RICTOR had phosphatidylinositol 3,4,5-trisphosphate-stimulated Ser-473 activity that was sensitive to wortmannin but not staurosporine. In contrast, immunopurified low density microsome vesicles containing ILK could not phosphorylate Akt on Ser-473 in vitro. Small interference RNA knockdown of RICTOR, but not DNA-PK, ATM, or ILK, suppressed insulin-activated Ser-473 phosphorylation and, to a lesser extent, Thr-308 phosphorylation in 3T3-L1 adipocytes. Based on our cell-free kinase and small interference RNA results, we conclude that mTOR complexed to RICTOR is the Ser-473 kinase in 3T3-L1 adipocytes.