The MDM2 RING Domain and Central Acidic Domain Play Distinct Roles in MDM2 Protein Homodimerization and MDM2-MDMX Protein Heterodimerization

The oncoprotein murine double minute 2 (MDM2) is an E3 ligase that plays a prominent role in p53 suppression by promoting its polyubiquitination and proteasomal degradation. In its active form, MDM2 forms homodimers as well as heterodimers with the homologous protein murine double minute 4 (MDMX), both of which are thought to occur through their respective C-terminal RING (really interesting new gene) domains. In this study, using multiple MDM2 mutants, we show evidence suggesting that MDM2 homo- and heterodimerization occur through distinct mechanisms because MDM2 RING domain mutations that inhibit MDM2 interaction with MDMX do not affect MDM2 interaction with WT MDM2. Intriguingly, deletion of a portion of the MDM2 central acidic domain selectively inhibits interaction with MDM2 while leaving intact the ability of MDM2 to interact with MDMX and to ubiquitinate p53. Further analysis of an MDM2 C-terminal deletion mutant reveals that the C-terminal residues of MDM2 are required for both MDM2 and MDMX interaction. Collectively, our results suggest a model in which MDM2-MDMX heterodimerization requires the extreme C terminus and proper RING domain structure of MDM2, whereas MDM2 homodimerization requires the extreme C terminus and the central acidic domain of MDM2, suggesting that MDM2 homo- and heterodimers utilize distinct MDM2 domains. Our study is the first to report mutations capable of separating MDM2 homo- and heterodimerization.     

Ammonia-induced structural changes of the oxygen-evolving complex in photosystem II as revealed by light-induced FTIR difference spectroscopy

Light-induced Fourier transform infrared difference spectroscopy has been applied to studies of ammonia effects on the oxygen-evolving complex (OEC) of photosystem II (PSII). We found that NH(3) induced characteristic spectral changes in the region of the symmetric carboxylate stretching modes (1450-1300 cm(-1)) of the S(2)Q(A)(-)/S(1)Q(A) FTIR difference spectra of PSII. The S(2) state carboxylate mode at 1365 cm(-1) in the S(2)Q(A)(-)/S(1)Q(A) spectrum of the controlled samples was very likely upshifted to 1379 cm(-1) in that of NH(3)-treated samples; however, the frequency of the corresponding S(1) carboxylate mode at 1402 cm(-1) in the same spectrum was not significantly affected. These two carboxylate modes have been assigned to a Mn-ligating carboxylate whose coordination mode changes from bridging or chelating to unidentate ligation during the S(1) to S(2) transition [Noguchi, T., Ono, T., and Inoue, Y. (1995) Biochim. Biophys. Acta 1228, 189-200; Kimura, Y., and Ono, T.-A. (2001) Biochemistry 40, 14061-14068]. Therefore, our results show that NH(3) induced significant structural changes of the OEC in the S(2) state. In addition, our results also indicated that the NH(3)-induced spectral changes of the S(2)Q(A)(-)/S(1)Q(A) spectrum of PSII are dependent on the temperature of the FTIR measurement. Among the temperatures we measured, the strongest effect was seen at 250 K, a lesser effect was seen at 225 K, and little or no effect was seen at 200 K. Furthermore, our results also showed that the NH(3) effects on the S(2)Q(A)(-)/S(1)Q(A) spectrum of PSII are dependent on the concentrations of NH(4)Cl. The NH(3)-induced upshift of the 1365 cm(-1) mode is apparent at 5 mM NH(4)Cl and is completely saturated at 100 mM NH(4)Cl concentration. Finally, we found that CH(3)NH(2) has a small but clear effect on the spectral change of the S(2)Q(A)(-)/S(1)Q(A) FTIR difference spectrum of PSII. The effects of amines on the S(2)Q(A)(-)/S(1)Q(A) FTIR difference spectra (NH(3) > CH(3)NH(2) > AEPD and Tris) are inverse proportional to their size (Tris approximately AEPD > CH(3)NH(2) > NH(3)). Therefore, our results showed that the effects of amines on the S(2)Q(A)(-)/S(1)Q(A) spectrum of PSII are sterically selective for small amines. On the basis of the correlations between the conditions (dependences on the excitation temperature and NH(3) concentration and the steric requirement for the amine effects) that give rise to the NH(3)-induced upshift of the 1365 cm(-)(1) mode in the S(2)Q(A)(-)/S(1)Q(A) spectrum of PSII and the conditions that give rise to the altered S(2) state multiline EPR signal, we propose that the NH(3)-induced upshift of the 1365 cm(-1) mode is caused by the binding of NH(3) to the site on the Mn cluster that gives rise to the altered S(2) state multiline EPR signal. In addition, we found no significant NH(3)-induced change in the S(2)Q(A)(-)/S(1)Q(A) FTIR difference spectrum at 200 K. Under this condition, the OEC gives rise to the NH(3)-stabilized g = 4.1 EPR signal and a suppressed g = 2 multiline EPR signal. Our results suggest that the structural difference of the OEC between the normal g = 2 multiline form and the NH(3)-stabilized g = 4.1 form is small.     

基于线粒体Cytb基因全序列探讨两爪鳖和山瑞鳖的系统进化关系

采用PCR方法对两爪鳖(Carettochelys insculpta)和山瑞鳖(Pdea steindachneri)mtDNA的细胞色素b基因(Cytb)进行了扩增和测序,并结合GenBank中已公布的其它15种龟鳖类和两种鳄类的同源序列,进行了序列变异比较和系统发生分析.以凯门鳄和扬子鳄为外群,采用邻接(neighbor ioining,NJ)法、最大简约(maximum likelihood,ML)法和最大似然(maximum parsimony,MP)法重建分子系统树.结果均支持将龟鳖目分为3支:侧颈龟科(Pelomedusidae)(非洲侧颈龟Pelomedusa subrufa),两爪鳖科(Carettochelyidae)(两爪鳖Carettochelys insculpta)和一支由其它15种现生龟鳘类组成的进化支.在这里两爪鳘科被认为可能代表一新的亚目;而山瑞鳖聚于鳖科(Trionychidae)中,与中华鳌(Pelodiscus sinensis)最先聚成一支,再依次与马来鳖(Dogania subplana)、非洲鳖(Trionyx triunguis)、印度箱鳖(Lissemys punctata)聚成一组.     

Optimization of medium composition for the production of exopolysaccharides from Phellinus baumii Pilát in submerged culture and the immuno-stimulating activity of exopolysaccharides

Optimization of medium composition for the production of exopolysaccharides (EPS) from Phellinus baumii Pilát in submerged culture and the immuno-stimulating activity of EPS were carried out. Firstly, the medium components having significant effect on EPS production were screened out to be glucose, yeast extract and diammonium oxalate monohydrate by using a 2⁽⁷⁻³⁾ fractional factorial design. Secondly, the concentrations of the three factors were optimized using central composite design in response surface methodology. As results, a quadratic model was found to fit for EPS production, and the optimal medium composition was determined as following (g/l): 34.12 glucose, 4 peptone, 5.01 yeast extract, 0.88 diammonium oxalate monohydrate, 0.75 MgSO₄ and 1 KH₂PO₄ and 0.0075 thiamine (VB₁). A yield of 2.363 ± 0.04 g/l for EPS was observed in verification experiment. Finally, EPS from P. baumii Pilát was found to have direct immuno-stimulating activity in vitro on splenocyte proliferative response and acid phosphatase activity in peritoneal macrophages in a dose-dependent manner.     

Tumor necrosis factor receptor-associated factor family protein 2 is a key mediator of the epidermal growth factor-induced ribosomal S6 kinase 2/cAMP-responsive element-binding protein/Fos protein signaling pathway

TRAF2 has an important function in mediating the TNF-R signaling pathway toward activation of NF-κB and JNKs. Here we reveal a novel function of TRAF2 in the epidermal growth factor (EGF) signaling pathway. Knockdown of TRAF2 blocked EGF-induced AP-1 activity and anchorage- independent cell transformation. Notably, we showed that EGF induces ribosomal S6 kinase 2 (RSK2) ubiquitination, and knocking down TRAF2 suppresses ubiquitination of RSK2 induced by EGF. We also found that TRAF2 affects RSK2 activity through RSK2 ubiquitination. RSK2 plays a critical role in AP-1 activity mediated through CREB and c-Fos, which regulates anchorage-independent cell transformation. In addition, TRAF2 is overexpressed in colon cancer and required for colon cancer development, suggesting that TRAF2 might be a potential molecular target for cancer prevention and treatment.