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Novel protein associated with cell stress response

a cell stress and protein technology, applied in the field of recombinant expression of a stressrelated protein, can solve the problems that stress and stress responses can have many deleterious effects on an organism, and achieve the effect of stimulating cell turnover

Inactive Publication Date: 2007-06-07
CHIRON CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The invention is about a new protein that is associated with the endoplasmic reticulum and is hyperphosphorylated in conditions of cell stress. The invention also relates to a native human Nogo protein, variants of the protein, polynucleotides encoding the protein, antibodies that recognize the protein, methods of identifying and quantifying the expression of the gene, methods of detecting cell stress, and methods of modulating phosphorylation of the protein. The invention also includes methods of inactivating the protein and stimulating cell turnover.

Problems solved by technology

Stress and stress responses can have many deleterious effects on an organism.

Method used

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  • Novel protein associated with cell stress response
  • Novel protein associated with cell stress response
  • Novel protein associated with cell stress response

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example 1

General Methods

[0110] Antibodies and other reagents. Antibodies against human Cdc2 (SC-54) and GRP94 (SC-1794) were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.). H2O2, suramine, specific p38 inhibitors SB202190 and PD169316 were purchased from Calbiochem (San Diego, Calif.). The plasmid pLIB-EGFP and the antibody against GFP were purchased from Clontech (Palo Alto, Calif.). (±)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDE) was purchased from ChemSyn Laboratories (Lenexa, Kans.).

[0111] Cell culture and treatment. Normal human fibroblast IMR90 was purchased from American Type Culture Collection (Manassas, Va.). Cells were routinely cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum, 2 mM L-glutamine, 4.5 g / liter glucose, 100 units penicillin and 100 μg streptomycin at 37° C. under an atmosphere of 5% CO2. For UV irradiation, cells were washed twice with phosphate-buffered saline (PBS) and then irradiated ...

example 2

Identification of a Protein Hyperphosphorylated During Stress

[0119] During a study of the effect of ultraviolet irradiation on cell cycle regulation, two closely-migrated proteins with apparent molecular weight around 46 KD were identified (FIG. 7A). Upon irradiation with 20 J / m2 of UVC, this doublet shifted very rapidly to a slower mobility form on a SDS polyacrylamide gel (FIG. 7A). At 2 hours after UV irradiation, about half of the protein changed back to the fast mobility form (FIG. 7B), as did most of the protein 20 hours later.

[0120] Since phosphorylation of a protein can lead to mobility shift on SDS-PAGE, the mobility shift of p46 was investigated to determine if it was due to phosphorylation. Protein lysates were prepared from irradiated cells in the absence of sodium orthovanadate. Incubation with alkaline phosphatase led to disappearance of the slow mobility form (FIG. 7C), indicating that the slow mobility form represented the hyperphosphorylated p46. Only the fastest ...

example2

P38 MAPK Activation Led to the P46 Hyperphosphorylation

[0125] It has been shown that elevated cellular stress level can activate p38 MAPK. The role of activation of p38 MAPK in p46 hyperphosphorylation was investigated. As shown in FIG. 5, p46 hyperphosphorylation was almost completely abolished in the presence of 270 nM of the p38-specific inhibitor SB202190 (IC50=300 nM) or in the presence of 90 nM of another p38-specific inhibitor PD169316 (IC50=89 nM). Another p38-specific inhibitor, SB203580, also showed similar effects. The control compound, SB202474, had no effect. The p46 hyperphosphorylation was not affected by the presence of 50 nM of wortmannin (IC50 for PI-3 kinase: 5 nM) or 20 uM of PD98059 (IC50 for MEK: 2 uM). It was concluded that p46 hyperphosphorylation was a result of p38 activation.

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Abstract

A stress-phosphorylated endoplasmic reticulum protein, Nogo B, is provided. The protein is hyperphosphorylated as a result of exposure of cells to stress. Two transcripts of Nogo B are identified in human tissues, and the longer transcript is predominant in human brain tumor samples.

Description

TECHNICAL FIELD [0001] This invention relates to the identification and recombinant expression of a stress-related protein associated with the endoplasmic reticulum. BACKGROUND OF THE INVENTION [0002] The endoplasmic reticulum (ER) is the site of production of most transmembrane proteins and lipids for cell organelles. The ER captures proteins from the cytosol as they are synthesized, and these proteins are transmembrane proteins or soluble proteins. The soluble proteins are fully translocated across the membrane and released into the ER lumen. In contrast, transmembrane proteins are only partially translocated across the ER membrane. During the course of protein synthesis and processing, the proteins fold to form their tertiary structure. [0003] When cells are exposed to conditions that disrupt protein folding in the ER, the transcription of genes encoding ER proteins may be upregulated. An unfolded protein response (UPR) exists in cells; this response follows detection of unfolded...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N9/12
CPCC07K14/47C12Q1/6886C12Q2600/158
Inventor WEI, DONGHALENBECK, ROBERT F.WILLIAMS, LEWIS T.
Owner CHIRON CORP
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