Although biochemical properties of 2-Cys peroxiredoxins (Prxs) have been extensively studied, their real physiological functions in higher eukaryotic cells remain obscure and certainly warrant further study. Here we demonstrated that human (h) PrxII, a cytosolic isotype of human 2-Cys Prx, has dual functions as a peroxidase and a molecular chaperone, and that these different functions are closely associated with its adoption of distinct protein structures. Upon exposure to oxidative stress, hPrxII assumes a high molecular weight complex structure that has a highly efficient chaperone function. However, the subsequent removal of stressors induces the dissociation of this protein structure into low molecular weight proteins and triggers a chaperone-toperoxidase functional switch. The formation of a high molecular weight hPrxII complex depends on the hyperoxidation of its N-terminal peroxidatic Cys residue as well as on its C-terminal domain, which contains a "YF motif" that is exclusively found in eukaryotic 2-Cys Prxs. A C-terminally truncated hPrxII exists as low and oligomeric protein species and does not respond to oxidative stress. Moreover, this C-terminal deletion of hPrxII converted it from an oxidation-sensitive to a hyperoxidation-resistant form of peroxidase. When functioning as a chaperone, hPrxII protects HeLa cells from H 2 O 2 -induced cell death, as measured by a terminal deoxynucleotidyltransferase-mediated dUTP nickend labeling assay and fluorescence-activated cell sorting analysis.
We have purified the human lymphocyte Fc receptor specific for IgE (Fcc receptor) and its soluble form by using the anti-Fcc receptor monoclonal antibody H107. Using an oligonucleotide probe corresponding to the partial amino acid sequence of the soluble Fce receptor related to IgE binding factor, we cloned, sequenced, and expressed a cDNA for the receptor. The Fcc receptor has 321 amino acid residues with no NH2-terminal signal sequence. The receptor was separated into two domains by a putative 24-amino acid residue transmembrane region located near the NH2-terminal end. The Fce receptor showed a marked homology with animal lectins including human and rat asialoglycoprotein receptors, chicken hepatic lectin, and rat mannose binding proteins. Tresyl-Sepharose (Pharmacia) at 5 mg/1 ml of beads (10). Soluble FCE receptor was purified from the conditioned medium of RPMI 8866 cells using H107-Sepharose after absorption with the human gamma globulinSepharose. The FcE receptor was purified as described (8). The cell lysate of RPMI 8866 cells in 0.5% Nonidet P-40 was absorbed with bovine serum albumin-, human gamma globulin-, and DNase I-conjugated Sepharose, and then incubated with the H107-Sepharose. After extensive washing with a high-salt buffer (10 mM sodium phosphate, pH 7.5/0.5 M NaCl/0.1% Nonidet P-40), soluble FCE receptor and FcE receptors were eluted from H107-Sepharose with an elution buffer (0.1 M acetic acid, pH 4.0/0.5 M NaCl/0.1% Nonidet P-40). Affinity-purified, soluble FCE receptor was further fractionated on Superose column with Fast Protein Liquid Chromatography (FPLC; Pharmacia), to elucidate its IgE binding activity. NaDodSO4/PAGE and Protein Amino Acid Sequence. The method of NaDodSO4/PAGE is described elsewhere (8). For immunoblots, nonreducing 13% polyacrylamide gels were used. After electrotransfer to a nitrocellulose filter, the membrane, blocked with 3% (wt/vol) bovine serum albumin, was incubated with or without H107 mAb (1 ,g/ml) and then with goat anti-mouse IgG [F(ab')2] conjugated to horseradish peroxidase (Tago). The antibody-bound bands were visualized with 3,3'-diaminobenzidine tetrahydrochloride and H202. For amino acid sequencing, both affinity-purified, soluble FcE receptor and FcE receptor were further purified by reverse-phase HPLC (using a Synchropak RP-P C18 column) (Beckman) with a linear gradient of 2-propanol with Abbreviations: FC£ receptor, Fc receptor for IgE; IgE-BF, IgE binding factor; mAb, monoclonal antibody. tTo whom reprint requests should be addressed.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Tropomyosin-related kinase A (TrkA) plays an important role in cell survival, differentiation, and apoptosis in various neuronal and nonneuronal cell types. Here we show that TrkA overexpression by the Tet-On system mimics NGF-mediated activation pathways in the absence of nerve growth factor (NGF) stimulation in U2OS cells. In addition, p53 upregulation upon DNA damage was inhibited by TrkA, and p21 was upregulated by TrkA in a p53-independent manner. TrkA overexpression caused cell death by interrupting cell cycle progression, and TrkA-induced cell death was diminished in the presence of its specific inhibitor GW441756. Interestingly, TrkA-mediated cell death was strongly related to γH2AX production and poly (ADP-ribose) polymerase cleavage in the absence of DNA damage inducer. In this study, we also reveal that γH2AX production by TrkA is blocked by TrkA kinase inhibitors K-252a and GW441756, and it is also significantly inhibited by JNK inhibitor SP600125. Moreover, reduction of cell viability by TrkA was strongly suppressed by SP600125 treatment, suggesting a critical role of JNK in TrkA-induced cell death. We also found that γH2AX and TrkA were colocalized in cytosol in the absence of DNA damage, and the nuclear localization of γH2AX induced by DNA damage was partly altered to cytosol by TrkA overexpression. Our results suggest that the abnormal cytosolic accumulation of γH2AX is implicated in TrkA-induced cell death in the absence of DNA damage.
Renal cell carcinoma (RCC) is one of the most malignant tumors in urology, and due to its insidious onset patients frequently have advanced disease at the time of clinical presentation. Thus, early detection is crucial in management of RCC. To identify tumor specific proteins of RCC, we employed proteomic analysis. We prepared proteins from conventional RCC and the corresponding normal kidney tissues from seven patients with conventional RCC. The expression of proteins was determined by silver stain after two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). The overall protein expression patterns in the RCC and the normal kidney tissues were quite similar except some areas. Of 66 differentially expressed protein spots (p<0.05 by Student t-test), 8 different proteins from 11 spots were identified by MALDI-TOF-MS. The expression of the following proteins was repressed (p<0.05); aminoacylase-1, enoyl-CoA hydratase, aldehyde reductase, tropomyosin α-4 chain, agmatinase and ketohexokinase. Two proteins, vimentin and α-1 antitrypsin precursor, were dominantly expressed in RCC (p<0.05).
Rheumatoid arthritis (RA) is a chronic inflammatiory disease that mainly destroys cartilages or bones at the joints. This inflammatory disorder is initiated by self-attack using own immune system, but the detail of pathological mechanism is unclear. Features of autoantigens leading to autoimmune disease are also under veil although several candidates including type II collagen have been suggested to play a role in pathogenesis. In this report, we tried to identify proteins responding to antibodies purified from RA patients and screen proteins up-regulated or down-regulated in RA using proteomic approach. Fibronectin, semaphorin 7A precursor, growth factor binding protein 7 (GRB7), and immunoglobulin µ chain were specifically associated with antibodies isolated from RA synovial fluids. In addition, some metabolic proteins such as adipocyte fatty acid binding protein, galectin-1 and apolipoprotein A1 precursor were overexpressed in RA synovium. Also, expression of peroxiredoxin 2 was up-regulated in RA. On the contrary, expression of vimentin was severely suppressed in RA synoviocytes. Such findings might give some insights into understanding of pathological mechanism in RA.
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