• Journal of Applied Biological Chemistry
• /
• v.50 no.2
• /
• pp.58-62
• /
• 2007

Identification of Dual-specificity protein phosphatase (DSP) substrates is essential in revealing physiological roles of DSPs. We isolated VHZ-interacting proteins from extracts of 293T cells overexpressing a VHZ (C95S, D65A) mutant known to be substrate- trapping mutant. Analysis of specific proteins bound to VHZ by 2D gel electrophoresis and mass spectroscopy revealed that these proteins contained Chaperonin containing TCP1, Type II phosphatidylinositol phosphate kinase ${\gamma}$, Intraflagellar transport 80 homolog, and Kinesin superfamily protein 1B. VHZ-interacting proteins showed that VHZ is involved in many important cellular signal pathways such as protein folding, molecular transportation, and tumor suppression.

• IMMUNE NETWORK
• /
• v.1 no.3
• /
• pp.221-229
• /
• 2001

Background: Inactivation of tumor suppressor genes is believed to be important in the development of many human malignancies. Recently, several lines of evidence have indicated that the wild type p53 gene located at 17p13.3, may function as a tumor suppressor gene and that a mutant p53 gene could promote transformation by inactivating normal p53 function in a dominant negative fashion. These broad spectrum of p53 mutation in human cancers provide that mutant p53 and their protein may be potential targets of tumor diagnostic and therapeutic interventions. Method: Colony formation was performed to investigate growth suppressional ability. p53 expression pattern was examined by western blot and p53-mediated transactivation ability was assessed by CAT activity. SNU C2A cells were observed in apoptotic aspects induced by etoposide and $H_2O_2$ treatment, detecting sensitivity on agent, DNA fragmentation through agarose gel, chromatin condensation by fluorescence microscope, and cell cycle distribution by FACS. Result: 1) p53 mutant his179arg ($histidine{\rightarrow}arginine$) detected in SNU C2A cells lost transcriptional activity and growth suppression ability, showing dominant negative effect on its wild type p53. 2) Etoposide-treated SNU C2A cells induced apoptosis, exhibiting dramatic reduction of cell growth, DNA fragmentation, nuclear condensation formation of apoptotic body and increment of sub-G1 cell fraction. 3) Etoposide and $H_2O_2$-treated SNU C2A cells have no high increase of p53 expression and overexpressed p53 protein changed localization, from cytoplasm to nucleus. Also, p53-mediated transcriptional activity was increased by agents-treatment. Conclusion: SNU C2A cells coexpress wild-type and mutant p53 protein induced apoptosis in the condition on DNA damage, through localizational shift from cytoplasm to nucleus of p53 protein rather than the induction of p53 protein. SNU C2A cells derived mutant p53 his179arg abrogated both the growth supression ability and transactivational activity, showing inhibition effect on transcriptional activity of wild type p53, but did not repress the activity of wild type p53 in SNU C2A cells owing to dominant activity of wild type. These cell condition may provide new gene therapeutic implications leading effective antiproliferation of cell when mutant and wild-type p53 protein were co-expressed in cell.

• Journal of Life Science
• /
• v.17 no.6 s.86
• /
• pp.783-790
• /
• 2007

G protein coupled receptors (GPCRs) transmit various extracellular signals into the cells. Upon binding of the ligands, conformational changes in the extracellular and/or transmembrane (TM) domains of CPCRs were propagated into the cytoplasmic (CP) domain of the molecule leading to the activation of their cognate heterotrimeric C proteins and kinases. Constitutively active GPCR mutants causing the activation of C Protein signaling even in the absence of ligand binding are of interest for the study of activation mechanism of GPCRs. Two classes of constitutively active mutations, categorized by their effects on the salt bridge between Ell3 and K296, were found in the TM domain of rhodopsin. Opsin mutants containing combinations of the mutations were constructed to study the conformational changes required for the activation of rhodopsin. Rhodopsin chromophore regenerated with 11-cis-retinal showed a thermal stability inversely correlated with its constitutive activity. In contrast, rhodopsin mutants exhibited a binding affinity to an agonist, all-trans-retinal, in a constitutive activity-dependent manner. In order to test whether the conformational changes responsible for the activation of trans-ducin (Gt) are the same as the conformation required for the recognition of rhodopsin kinase, analysis of the mutants were carried out with phosphorylation by rhodopsin kinase. Rhodopsin mutants containing combinations of different classes of the mutations showed a strong synergistic effect on the phosphorylation of the mutants in the dark as similar to that of Gt activation. The results suggest that at least two or three kinds of segmental and independent conformational changes are required for the activation of rhodopsin and the conformational changes responsible for activating rhodopsin kinase and Gt are similar to each other.

• Journal of Photoscience
• /
• v.9 no.2
• /
• pp.466-469
• /
• 2002

An experiment has been conducted to measure the impact of UV radiation sensitivity on dermatophytes (Microsporum boullardii) by different UV radiation exposure time interval (1 min, 2 min 5 min, 10 min and 20 min) in degradation of keratin (Feather) in growth promoting substances of protein, cysteine, cystine and methionine from 7 to 28 days of incubation period. Mutant strain caused maximum weight loss with 1 minutes of UV radiation exposure at 21 day and mutant strain became immune in sensitivity at 14 days for decomposition of feathers. Maximum protein caused at 21st days with 20 minutes U.V radiation exposure and immune sensitivity had deducted with other UV radiation exposure time. On 28 days, mutant strains became immune with all exposure times, Whereas maximum methionine caused at 21st days with 20 minutes UV radiation exposure. Maximum cysteine caused at $14^{th}$ day with 5 minutes UV radiation exposure and mutant strain showed immune response at all time periods. Cystine production was also followed by cysteine at 21 day and also showed complete immune response with 1 and 2 minutes UV radiation exposure at7 and 14 days. Thus mutant strain of Microspornm boullardii can be used as a biotechnological tool for production of growth promoting substances.

• BMB Reports
• /
• v.35 no.4
• /
• pp.353-357
• /
• 2002

Singlet oxygen ($^1O_2$) is highly reactive form of molecular oxygen that may harm living systems by oxidizing critical cellular macromolecules. The oxyR gene product regulates the expression of the enzymes and proteins that are needed for cellular protection against oxidative stress. In this study, the role of oxyR in cellular defense against a singlet oxygen was investigated using Escherichia coli oxyR mutant strains. Upon exposure to methylene blue and visible light, which generates singlet oxygen, the oxyR overexpression mutant was much more resistant to singlet oxygen-mediated cellular damage when compared to the oxyR deletion mutant in regard to growth kinetics, viability and protein oxidation. Induction and inactivation of major antioxidant enzymes, such as superoxide desmutase and catalase, were observed after their exposure to a singlet oxygen generating system in both oxyR strains. However, the oxyR overexpression mutant maintained significantly higher activities of anticxidant enzymes than did the oxyR deletion mutant. These results suggest that the oxyR regulon plays an important protective role in singlet oxygen-mediated cellular damage, presumably through the protection of antioxidant enzymes.

• Molecules and Cells
• /
• v.21 no.3
• /
• pp.381-388
• /
• 2006

Heat shock proteins (Hsp) 70 are a ubiquitous family of molecular chaperones involved in many cellular processes. A yeast strain, ssa1/2, with two functionally redundant cytosolic Hsp70s (SSA1 and SSA2) deleted shows thermotolerance comparable to mildly heatshocked wild type yeast, as well as increased protein synthesis and ubiquitin-proteasome protein degradation. Since mRNA abundance does not always correlate well with protein expression levels it is essential to study proteins directly. We used a gel-based approach to identify stress-responsive proteins in the ssa1/2 mutant and identified 43 differentially expressed spots. These were trypsin-digested and analyzed by nano electrospray ionization liquid chromatography tandem mass spectrometry (nESI-LC-MS/MS). A total of 22 non-redundant proteins were identified, 11 of which were confirmed by N-terminal sequencing. Nine proteins, most of which were up-regulated (2-fold or more) in the ssa1/2 mutant, proved to be stress-inducible proteins such as molecular chaperones and anti-oxidant proteins, or proteins related to carbohydrate metabolism. Interestingly, a translational factor Hyp2p up-regulated in the mutant was also found to be highly phosphorylated. These results indicate that the cytosolic Hsp70s, Ssa1p and Ssa2p, regulate an abundance of proteins mainly involved in stress responses and protein synthesis.

• The Plant Pathology Journal
• /
• v.38 no.6
• /
• pp.593-602
• /
• 2022

Anthracnose, caused by the ascomycete fungus Colletotrichum scovillei, is a destructive disease in pepper. The fungus germinates and develops an infection structure called an appressorium on the plant surface. Several signaling cascades, including cAMP-mediated signaling and mitogen-activated protein kinase (MAPK) cascades, are involved in fungal development and pathogenicity in plant pathogenic fungi, but this has not been well studied in the fruit-infecting fungus C. scovillei. Ste50 is an adaptor protein interacting with multiple upstream components to activate the MAPK cascades. Here, we characterized the CsSTE50 gene of C. scovillei, a homolog of Magnaporthe oryzae MST50 that functions in MAPK cascades, by gene knockout. The knockout mutant ΔCsste50 had pleiotropic phenotypes in development and pathogenicity. Compared with the wild-type, the mutants grew faster and produced more conidia on regular agar but were more sensitive to osmotic stress. On artificial and plant surfaces, the conidia of the mutant showed significantly reduced germination and failed to form appressoria. The mutant was completely non-pathogenic on pepper fruits with or without wounds, indicating that pre-penetration and invasive growth were both defective in the mutant. Our results show that the adaptor protein CsSTE50 plays a role in vegetative growth, conidiation, germination, appressorium formation, and pathogenicity in C. scovillei.

• Journal of Life Science
• /
• v.15 no.3 s.70
• /
• pp.415-422
• /
• 2005

library of the mutants was prepared by transposon mutagenesis of the Mycobacterium bovis BCG. We screened this library for the resistance to an anti-tuberculosis antibiotic, PA-824. Most of the mutants resistant to the PA-824 were not able to synthesize the coenzyme $F_{420}$ which is normally produced by the wild type M. bovis BCG strains. HPLC analysis of the cellular extract showed that one of those mutants which lost the ability to synthesize $F_{420}$ still produced F0. The insertion site of the transposon in this mutant was determined by an inverse PCR and the transposon was found to be inserted in the Rv2435c open reading frame (ORF). Rv2435c ORF is predicted to encode an 80.3 kDa protein. Rv2435c protein appears to be bound to the cytoplasmic membrane, its N-terminal present in the periplasm and C-terminal in the cytoplasm. The C-terminal portion of this protein is highly homologous with the adenylyl cyclases of both prokaryotes and eukaryotes. There are 15 ORFs which have homology with the class III AC proteins in the genome of the M. tuberculosis and M. bovis. Two of those, Rv1625c and Rv2435c, are highly homologous with the mammalian ACs. We cloned the cytoplasmic domain of the Rv2435c ORF and expressed it with six histidine residues attached on its C-terminal in Escherichia coli to find out if this protein is a genuine AC. Production of that protein in E. coli was proved by purifying the histidine-tagged protein by using the Ni-NTA resin. This protein, however, failed to complement the cya mutation in E. coli, indicating that this protein lacks the AC activity. All of the further attempts to convert this protein to a functional AC by a mutagenesis with UV or hydroxylamine, or construction of several different fusion proteins with Rv1625c failed. It is, therefore, possible that Rv2435c protein might affect the conversion of F0 to $F_{420}$ not by synthesizing cAMP but by some other way.

• Biomedical Science Letters
• /
• v.2 no.1
• /
• pp.21-30
• /
• 1996

MAP kinases are a family of serine/threonine specific protein kinases becoming activated in response to different proliferative stimuli by phosphorylation at both threonine and tyrosine residue. Present study shows that MAP kinase was purified from P388 murine leukema cells by SP sephadex C-50, phenyl superose and Mono Q column chromatography and identified with anti-ERKl antibody by western blotting. Immnublotting analysis to the crude extract of P388 cell lysate shows 44 kD and other minor bands but partial purified fraction eluted from phenyl supherose column have 44kD and 66 kD isoform. Subcloned GST-fusion protein from N-terminal of $p56^{kk}$ was tested as a substrate for MAP kinase phosphorylation. It was showed that the wild type and mutant forms(S42A) were fully phosporylated by purified MAP kinase fraction as com-pare with the other mutant form(S59A). This finding suggest that those GST-fusion proteins may be used as substrate for the in vitro test of MAP kinase.

• Animal cells and systems
• /
• v.14 no.1
• /
• pp.1-10
• /
• 2010

Previously we showed that CHIP, a co-chaperone of Hsp70 and E3 ubiquitin ligase, can promote the degradation of mutant SOD1 linked to familial amyotrophic lateral sclerosis (fALS) via a mechanism not involving SOD1 ubiquitylation. Here we present evidence that CHIP functions in the interaction of mutant SOD1 with 26S proteasomes. Bag-1, a coupling factor between molecular chaperones and the proteasomes, formed a complex with SOD1 in an hsp70-dependent manner but had no direct effect on the degradation of mutant SOD1. Instead, Bag-1 stimulated interaction between CHIP and the proteasome-associated protein VCP (p97), which do not associate normally. Over-expressed CHIP interfered with the association between mutant SOD1 and VCP. Conversely, the binding of CHIP to mutant SOD1 was inhibited by VCP, implying that the chaperone complex and proteolytic machinery are competing for the common substrates. Finally we observed that mutant SOD1 strongly associated with the 19S complex of proteasomes and CHIP over-expression specifically reduced the interaction between S6/S6' ATPase subunits and mutant SOD1. These results suggest that CHIP, together with ubiquitin-binding proteins such as Bag-1 and VCP, promotes the degradation of mutant SOD1 by facilitating its translocation from ATPase subunits of 19S complex to the 20S core particle.