Overexpression of CD38 Decreases Cellular NAD Levels and Alters the Expression of Proteins Involved in Energy Metabolism and Antioxidant Defense

Nicotinamide adenine dinucleotide (NAD) is a coenzyme found in all living cells and mediates multiple cellular signaling pathways. In the present study, a 35% decrease of cellular NAD level is achieved by stable expression of the N-terminal truncated CD38, a NAD hydrolase. CD38-expressing (CD38­(+)) cells have the lower growth rate and are more susceptive to oxidative stress than the wild type cells and empty vector-transfected (CD38(−)) cells. Quantitative proteomic analysis shows that 178 proteins are down-regulated in CD38­(+) cells, which involve in diverse cellular processes including glycolysis, RNA processing and protein synthesis, antioxidant, and DNA repair. Down regulation of six selected proteins is confirmed by Western blotting. However, down-regulation of mRNA expressions of genes associated with glycolysis, antioxidant, and DNA repair is less significant than the corresponding change in protein expression, suggesting the low NAD level impairs the protein translational machinery in CD38­(+) cells. Down-regulation of antioxidant protein and DNA-repair protein expression contributes to the susceptibility of CD38­(+) cells to oxidative stress. Taken together, these results demonstrate that CD38­(+) cells are a useful model to study effects of the cellular NAD levels on cellular processes and establish a new linker between cellular NAD levels and oxidative stress

Development of the Fc-III Tagged Protein Expression System for Protein Purification and Detection

<div><p>In the present work, we developed the Fc-III tagged protein expression system for protein purification and detection. The Fc-III sequence encodes for a 13 residue peptide and this peptide is cyclized by disulfide bond formation when the fusion protein is expressed. The Fc-III-fusion proteins selectively bind to immunoglobulin Fc domains (IgG-Fc) expressed from <em>E. coli</em>. We showed the efficient purification of Fc-III tagged proteins by immobilized non-native IgG-Fc and the detection of the cellular locations of fusion proteins by fluorescent-conjugated IgG-Fc. Our results prove that Fc-III tagged protein expression system is a simple and efficient tool for protein purification and detection and is a useful addition to the biochemistry and proteomics toolbox.</p> </div

Oxidative Stress Induces Monocyte Necrosis with Enrichment of Cell-Bound Albumin and Overexpression of Endoplasmic Reticulum and Mitochondrial Chaperones

<div><p>In the present study, monocytes were treated with 5-azacytidine (azacytidine), gossypol or hydrogen peroxide to induce cell death through oxidative stress. A shift from apoptotic to necrotic cell death occurred when monocytes were treated with 100 µM azacytidine for more than 12 hours. Necrotic monocytes exhibited characteristics, including enrichment of cell-bound albumin and up-regulation of endoplasmic reticulum (ER)- and mitochondrial-specific chaperones to protect mitochondrial integrity, which were not observed in other necrotic cells, including HUH-7, A2780, A549 and HOC1a. Our results show that the cell-bound albumin originates in the culture medium rather than from monocyte-derived hepatocytes, and that HSP60 is a potential binding partner of the cell-bound albumin. Proteomic analysis shows that HSP60 and protein disulfide isomerase are the most abundant up-regulated mitochondrial and ER-chaperones, and that both HSP60 and calreticulin are ubiquitinated in necrotic monocytes. In contrast, expression levels of the cytosolic chaperones HSP90 and HSP71 were down-regulated in the azacytidine-treated monocytes, concomitant with an increase in the levels of these chaperones in the cell culture medium. Collectively, our results demonstrates that chaperones from different organelles behave differently in necrotic monocytes, ER- and mitochondrial chaperones being retained and cytosolic and nuclear chaperones being released into the cell culture medium through the ruptured cell membrane. HSP60 may serve as a new target for development of <i>myeloid</i> leukemia treatment.</p> </div

Detection of ROS in untreated and azacytidine-treated THP-1 cells.

<p>Cells were labeled with carboxy-H₂DCFDA, which exhibits green fluorescence upon reaction with ROS, and nuclei were stained with blue-fluorescent Hoechst 33342. (a) Untreated THP-1 cells; and (b) azacytidine-treated THP-1 cells. (c) The 1D-SDS-PAGE gel image of proteins from untreated, gossypol-, azacytidine- or H₂O₂-treated THP-1 cells. Lane 1, proteins from untreated cells; Lane 2, proteins from 20 µM gossypol-treated cells; Lane 3, proteins from 100 µ M azacytidine-treated cells; Lane 4, proteins from 50 mM H₂O₂-treated cells. The bands with differentially expressed proteins are labeled A, B, C, and D.</p

Development of the Double Cyclic Peptide Ligand for Antibody Purification and Protein Detection

Development of a peptide-based affinity matrix and detection reagent is important for biomedical research and the biopharmaceutical industry. In the present work, we designed and synthesized an immunoglobin G (IgG)-binding peptide ligand, Fc-III-4C. Fc-III-4C is composed of 15 residues, and the 4 cysteine residues form 2 disulfide bonds to generate a double cyclic structure. The binding affinity of the Fc-III-4C peptide toward human IgG was determined to be 2.45 nM (<i>K</i>_d), which is higher than that of IgG with Protein A/G (Pro-A/G). Importantly, the Fc-III-4C peptide displayed high affinity to various IgGs from different species. Fc-III-4C immobilized agarose beads exhibited high stability and reusability when compared with that of the Pro-A/G-immobilized beads. The conjugate of Fc-III-4C with FITC was demonstrated to be suitable for immunofluorescence detection of proteins expressed in cells. These results demonstrate that the Fc-III-4C peptide is a useful affinity ligand for antibody purification and as a protein detection reagent

Glutaredoxin Deletion Shortens Chronological Life Span in <i>Saccharomyces cerevisiae</i> via ROS-Mediated Ras/PKA Activation

Glutaredoxins (GRXs), small redox proteins that use reduced glutathione as an electron donor, are key components of the cellular antioxidant system. In this study, we used <i>Saccharomyces cerevisiae</i> as a model system to investigate the effects of GRX deletion on yeast chronological life span (CLS). Deletion of either Grx1 or Grx2 shortened yeast CLS. Quantitative proteomics revealed that GRX deletion decreased the expression of stress-response proteins, leading to increased cellular reactive oxygen species accumulation and, subsequently, intracellular acidification. This activated the Ras/protein kinase A (PKA) signaling pathway. Genetic and biochemical analyses demonstrated that Ras/PKA activation decreased stress resistance and increased biosynthesis, requiring yeast cells to grow under unfavorable conditions and resulting in a shortened CLS. Our results provided new insights into mechanisms underlying exacerbation of the aging process by oxidative stress

Comparison of protein expression and activities between His-tagged- and Fc-III tagged CA.

<p>(A) Lane 1, proteins containing His-tagged CA from crude whole cell lysate; Lane 2, proteins containing His Tagged CA from clear supernatant; Lane 3, proteins containing His-tagged CA from the flow-through fraction; Lane 4, the eluates of His-tagged CA from Ni-FF beads; Lane 5, proteins containing Fc-III tagged CA from crude whole cell lysate; Lane 6, proteins containing Fc-III tagged CA from clear supernatant; Lane 7, proteins containing Fc-III tagged CA from the flow-through fraction; Lane 8, the eluate of Fc-III tagged CA from the IgG-Fc beads; Lane 9, molecule maker. (B) Enzymatic activities of His-tagged CA and Fc-III tagged CA. The absorbance measurement was carried out at 348 nm wavelength and at 25°C in triplicate. Concentrations of both proteins were 1.0 mg/ml, respectively.</p

Glutaredoxin Deletion Shortens Chronological Life Span in <i>Saccharomyces cerevisiae</i> via ROS-Mediated Ras/PKA Activation

Glutaredoxins (GRXs), small redox proteins that use reduced glutathione as an electron donor, are key components of the cellular antioxidant system. In this study, we used <i>Saccharomyces cerevisiae</i> as a model system to investigate the effects of GRX deletion on yeast chronological life span (CLS). Deletion of either Grx1 or Grx2 shortened yeast CLS. Quantitative proteomics revealed that GRX deletion decreased the expression of stress-response proteins, leading to increased cellular reactive oxygen species accumulation and, subsequently, intracellular acidification. This activated the Ras/protein kinase A (PKA) signaling pathway. Genetic and biochemical analyses demonstrated that Ras/PKA activation decreased stress resistance and increased biosynthesis, requiring yeast cells to grow under unfavorable conditions and resulting in a shortened CLS. Our results provided new insights into mechanisms underlying exacerbation of the aging process by oxidative stress

Identification of cell-bound BSA.

<p>(a) The MS/MS spectrum of a doubly charged ion at m/z 722.32 for MH₂²⁺ corresponding to the mass of the peptide YICDNQDTISSK from bovine serum albumin. By searching a human protein database, this MS/MS spectrum matched YICENQDSISSK, which has an identical mass. The highlighted peaks matched only to the sequence of BSA. (b) Underlined peptides that were identified by MS/MS searching from the band B digestion cover 79% of the BSA sequence. (c) Western blot analysis of BSA from cells treated with 100 µM azacytidine for different periods of time. Lane 1, before treatment; Lane 2, 4 h; Lane 3, 8 h; Lane 4, 12 h; and Lane 5, 24 h.</p

Changes of ER- and mitochondrial-chaperones upon azacytidine treatments.

<p>(a) Western blot analysis of HSP60 and PDI from cells treated with 100 µM azacytidine for different periods of time. Lane 1, before treatment; Lane 2, 4 h; Lane 3, 8 h; Lane 4, 12 h; and Lane 5, 24 h. (b) Relative abundance and fold of changes of ER- and mitochondrial chaperones from the untreated and azacytidine-treated monocytes. The number on the y axis represents spectra counts for each identified protein.</p