• Biomolecules & Therapeutics
• /
• v.26 no.3
• /
• pp.298-305
• /
• 2018

Rhomboid family member 2 gene (Rhbdf2) is an inactive homologue lacking essential catalytic residues of rhomboid intramembrane serine proteases. The protein is necessary for maturation of tumor necrosis factor-alpha ($TNF-{\alpha}$) converting enzyme, which is the molecule responsible for the release of $TNF-{\alpha}$. In this study, Rhbdf2 knockout (KO) mice were produced by CRISPR/CAS9. To see the effects of the failure of $TNF-{\alpha}$ release induced by Rhbdf2 gene KO, collagen-induced arthritis (CIA), which is the representative $TNF-{\alpha}$ related disease, was induced in the Rhbdf2 mutant mouse using chicken collagen type II. The severity of the CIA was measured by traditional clinical scores and histopathological analysis of hind limb joints. A rota-rod test and grip strength test were employed to evaluate the severity of CIA based on losses of physical functions. The results indicated that Rhbdf2 mutant mice showed clear alleviation of the clinical severity of CIA as demonstrated by the significantly lower severity indexes. Moreover, a grip strength test was shown to be useful for the evaluation of physical functional losses by CIA. Overall, the results showed that the Rhbdf2 gene has a significant effect on the induction of CIA, which is related to $TNF-{\alpha}$.

• Proceedings of the Korean Society of Applied Pharmacology
• /
• 2002.07a
• /
• pp.113-113
• /
• 2002

Phylogenetically conserved Bcl-2 family proteins play a pivotal role in the regulation of apoptosis from virus to human. Members of the Bcl-2 family consist of antiapoptotic proteins such as Bcl-2, Bcl-xL, and Bcl-w, and proapoptotic proteins such as BAD, Bax, BOD, and Bok. It has been proposed that anti- and proapoptotic Bcl-2 proteins regulate cell death by binding to each other and forming heterodimers. A delicate balance between anti- and proapoptotic Bcl-2 family members exists in each cell and the relative concentration of these two groups of proteins determines whether the cell survives or undergoes apoptosis. Mcl-1 (Myeloid cell :leukemia-1) is a member of the Bcl-2 family proteins and was originally cloned as a differentiation-induced early gene that was activated in the human myeloblastic leukemia cell line, ML-1 . Mcl-1 is expressed in a wide variety of tissues and cells including neoplastic ones. We recently identified a short splicing variant of Mcl-1 short (Mcl-IS) and designated the known Mcl-1 as Mcl-1 long (Mcl-lL). Mcl-lL protein exhibits antiapoptotic activity and possesses the BH (Bcl-2 homology) 1, BH2, BH3, and transmembrane (TM) domains found in related Bcl-2 proteins. In contrast, Mcl-1 S is a BH3 domain-only proapoptotic protein that heterodimerizes with Mcl-lL. Although both Mc1-lL and Mcl-lS proteins contain BH domains fecund in other Bcl-2 family proteins, they are distinguished by their unusually long N-terminal sequences containing PEST (proline, glutamic acid, serine, and threonine) motifs, four pairs of arginine residues, and alanine- and glycine-rich regions. In addition, the expression pattern of Mcl-1 protein is different from that of Bcl-2 suggesting a unique role (or Mcl-1 in apoptosis regulation. Tankyrasel (TRF1-interacting, ankyrin-related ADP-related polymerasel) was originally isolated based on its binding to TRF 1 (telomeric repeat binding factor-1) and contains the sterile alpha motif (SAM) module, 24 ankyrin (ANK) repeats, and the catalytic domain of poly(adenosine diphosphate-ribose) polymerase (PARP). Previous studies showed that tankyrasel promotes telomere elongation in human cells presumably by inhibiting TRFI though its poly(ADP-ribosyl)action by tankyrasel . In addition, tankyrasel poly(ADP-ribosyl)ates Insulin-responsive amino peptidase (IRAP), a resident protein of GLUT4 vesicles, and insulin stimulates the PARP activity of tankyrase1 through its phosphorylation by mitogen-activated protein kinase (MAPK). ADP-ribosylation is a posttranslational modification that usually results in a loss of protein activity presumably by enhancing protein turnover. However, little information is available regarding the physiological function(s) of tankyrase1 other than as a PARP enzyme. In the present study, we found tankyrasel as a specific-binding protein of Mcl-1 Overexpression of tankyrasel led to the inhibition of both the apoptotic activity of Mel-lS and the survival action of Mcl-lL in mammalian cells. Unlike other known tankyrasel-interacting proteins, tankyrasel did not poly(ADP-ribosyl)ate either of the Mcl-1 proteins despite its ability to decrease Mcl-1 proteins expression following coexpression. Therefore, this study provides a novel mechanism to regulate Mcl-1-modulated apoptosis in which tankyrasel downregulates the expression of Mcl-1 proteins without the involvement of its ADP-ribosylation activity.

• Applied Chemistry for Engineering
• /
• v.7 no.3
• /
• pp.543-553
• /
• 1996

Oxygen reduction in an alkaline fuel cell was studied by using perovskite type oxides as an oxygen electrode catalyst. The high surface area catalysts were prepared by malic acid method and had a formula of $La_{0.6}Sr_{0.4}Co_{1-x}Fe_xO_3$(x=0.00, 0.01, 0.10, 0.20, 0.35 and 0.50). From the result of XRD pattern and specific surface area due to the amount of Fe substitution and the consumption of ammonia-water, the complex formation of Fe ion with $NH_3$ was the main factor for both the phase stability of perovskite and the increase of specific surface area. Multi-step calcination was necessary to give a single phase of perovskite in catalyst precursor. The crystal structure of the catalysts was simple cubic perovskite, which was verified from the XRD patterns of the catalysts. The activity of oxygen reduction was monitored by the techniques of cyclic voltammetry, static voltage-current method, and current interruption method. The activity(current density) of oxygen reduction showed its minimum at x=0.01 and its maximum between 0.20 and 0.35 of x-value in $La_{0.6}Sr_{0.4}Co_{1-x}Fe_xO_3$. This tendency was independent of the change of surface area.

• Journal of Life Science
• /
• v.18 no.1
• /
• pp.114-119
• /
• 2008

The molecular machinery controlling cell cycle is centered around the regulation of the activity of maturation-promoting factor (MPF), a complex composed of a catalytic Cdc2 and the cyclinB regulatory subunit. Cdc2 kinase is inactivated by phosphorylation of inhibitory kinase, Wee1. It has been known that there are three different Wee1 kinases in the mammalian cell, Wee1A, Wee1B and Myt1. To investigate the regulatory mechanism of Wee1 kinases, the phosphorylation and degradation of Wee1A and Wee1B were checked in the Xenopus oocyte cell cycle. When Wee1 kinases were injected into frog oocyte, Wee1B was more stable than Wee1A. Wee1A and Wee1B kinase were phosphorylated by many kinases such as PKA and Akt. The roles of amino or carboxyl terminal in mouse Wee1A or Wee1B kinase were investigated using chimeric constructs. The degree of protein phosphorylation, degradation and cell cycle progression were different between chimeric constructs. The amino domain of Wee1A was implicated in the protein phosphorylation and degradation while amino domain of Wee1B and carboxyl domain of Wee1A were involved in the activity regulation. These results suggested that the domains of Wee1 kinase have different and significant roles in regulating the Wee1 kinases in the cell cycle progression.

• The Korea Journal of Herbology
• /
• v.30 no.6
• /
• pp.7-15
• /
• 2015

Objectives : Rosa laevigata Michx. has been used for the treatment of renal disease in traditional Korean medicine. In this study, we investigated cytoprotective effect of R. laevigata water extract (RLE) against oxidative stress induced by arachidonic acid (AA) + iron.Methods : To evaluate the protective effects of RLE against AA + iron-induced oxidative stress in HepG2 cell, cell viability and changes on apoptosis-related proteins were assessed by MTT and immunoblot analyses. The effects of RLE on reduced glutathione level, production of reactive oxygen species and mitochondrial membrane potential were also monitored. Furthermore, to verify underlying molecular mechanism, NF-E2-related factor 2 (Nrf2) was examined by immunoblot analysis. Additionally, Nrf2 transactivation and its downstream target genes expression were also determined by reporter gene and realtime RT-PCR analyses.Results : RLE pretreatment (30-300 μg/ml) prevented cells from AA + iron-mediated cell death in a concentration dependent manner. In addition, 100 μg/ml RLE inhibited AA + iron-induced glutathione depletion, reactive oxygen species production and mitochondrial dysfunction. RLE accumulated nuclear Nrf2 and also transactivated Nrf2, which was evidenced by antioxidant response element- and glutathione S-transferase A2-driven luciferase activities and mRNA level of glutamate-cysteine ligase catalytic subunit, NAD(P)H:quinone oxidoreductase 1 and sestrin 2. Moreover, protective effect of RLE against AA + iron was abolished in Nrf2 knockout cells.Conclusions : These results indicate that RLE has the ability to protect hepatocyte against oxidative stress through Nrf2 activation.

• Journal of Life Science
• /
• v.16 no.7 s.80
• /
• pp.1133-1140
• /
• 2006

Human HtrA1 (High temperature requirement protein A1) is a homologue of the E. coli periplasmic serine protease HtrA. A recent study has demonstrated that HtrA1 is a serine protease involved in processing of insulin like growth factor binding protein (ICFBP), indicating that it serves as an important regulator of IGF activity. Additionally, several lines of evidence suggest a striking correlation between proteolytic activity of HtrA1 serine protease and the pathogenesis of several diseases; however, physiological roles of HtrA1 remain to be elucidated. We used the pGEX bacterial expression system to develop a simple and rapid method for purifying HtrA1, and the recombinant HtrA1 protein was utilized to investigate the optimal conditions in executing its proteolytic activity. The proteolytically active HtrA1 was purified to approximately 85% purity, although the yield of the recombinant HtrA1 protein was slightly low $460{\mu}g$ for 1 liter E. coli culture). Using in vitro endoproteolytic cleavage assay, we identified that the HtrA1 serine protease activity was dependent on the enzyme concentration and the incubation time and that the best reaction temperature was $42^{\circ}C$ instead of $37^{\circ}C$. We arbitrary defined one unit of proteolytic activity of the HtrA1 serine protease as 200nM of HtrA1 that cleaves half of $5{\mu}M\;of\;{\beta}-casein$ during 3 hr incubation at $37^{\circ}C$. Our study provides a method for generating useful reagents to investigate the molecular mechanisms by which HtrA1 serine protease activity contributes in regulating its physiological function and to identify natural substrates of HtrA1.