hDaxx与细胞肿瘤抑制子p53在体内外的相互作用

与急性早幼粒细胞性白血病蛋白 (promyelocyticleukemiaprotein ,PML)在体内相互作用共定位于细胞核PML致癌结构域 (PMLoncogenicdomains,PODs)的人Daxx(humanDaxx ,hDaxx) ,能结合Fas死亡结构域诱导细胞凋亡。细胞肿瘤抑制子p5 3抑制细胞及病毒转录 ,提高细胞内Fas的表达并调节细胞凋亡。为了探索hDaxx与 p5 3在诱导细胞凋亡中有无相互作用及其作用效果 ,利用酵母双杂交体系测定发现p5 3通过C端与hDaxx结合 ,共免疫沉淀反应及Westernblot结果显示hDaxx与 p5 3能在体内外直接结合。hDaxx与 p5

hDaxx及其删除突变体在酵母菌中的表达及与p53的结合反应

目的：研究hDaxx与细胞肿瘤抑制子p53相互作用的结构部位，以便探索两种蛋白质相互作用后的生物学效应，方法：利用pGBDU-C及pGAD-C载体构建Daxx(human Daxx,hDaxx)及其删除突变体（ΔhDaxx)酵母菌表达质粒，利用Western blot检测它们在酵母菌中的表达产物，通过酵母双杂交试验测定hDaxx与p53结合并发生相互作用的结构部位，结果pGBDU-C或pGAD-C/hDaxx与ΔhDaxx能在PJ69－4酵母菌株表达目的蛋白，hDaxx及3种ΔhDaxx与p53结合并发生相互作用，2种ΔhDaxx不与p53结合发生相互作用。结论：hDaxx通过第396－499位氨基酸（aa)或C端241个aa残基与p53发生结合。

Protein Interaction Between p53 and △113p53 Is Required for the Anti-Apoptotic Function of △113p53

Zebrafish △113p53, an N-terminal truncated p53 isoform, is a p53-target gene that antagonises p53-mediated apoptotic activity. Interestingly, △113p53 does not act on p53 in a dominant-negative manner, but rather interferes with the p53 function by differentially modulating p53-target gene expression to protect cells from apoptosis. Previous studies showed that over-expressed △113p53 and p53 proteins formed a complex. However, it is not known whether endogenous p53 and △113p53 proteins also interact with each other, and if this interaction is required for △113p53 to inhibit the apoptotic activity of full-length p53. In this study, we used two available zebrafish p53 antibodies to address these questions. One, Zfp53-N, only recognises full-length p53, whereas the other, Zfp53-A7C10, detects both full-length p53 and △113p53. Using Zfp53-N for immunoprecipitation and Zfp53-A7C 10 for detection, we demonstrated that endogenous △113p53 and full-length p53 induced by a DNA-damaging drug formed a complex in vivo. Furthermore, of the six △113p53 mutants we generated with different point mutations in the oligomerisation domain, two failed to interact with p53 and lost the ability to modulate p53-target gene expression and inhibit p53-induced cell apoptosis. However, those △113p53 mutants that could interact with p53 retained the ability to antagonise the apoptotic activity of p53. Therefore, our data demonstrated that protein--protein interaction between △113p53 and p53 is essential for the anti-apoptotic function of △113p53. In addition, the two △113p53 mutants that failed to interact with p53 are also useful for the study of the mechanisms of other functions of △113p53.

Structural characteristics of the hydrophobic patch of azurin and its interaction with p53: a site-directed spin labeling study

Site-directed spin labeling (SDSL) is a powerful tool for monitoring protein structure, dynamics and conformational changes. In this study, the domain-specific properties of azurin and its interaction with p53 were studied using this technique. Mutations of six residues, that are located in the hydrophobic patch of azurin, were prepared and spin labeled. Spectra of the six azurin mutants in solution showed that spin labeled residues 45 and 63 are in a very restricted environment, residues 59 and 65 are in a spacious environment and have free movement, and residues 49 and 51 are located in a relatively closed pocket. Polarity experiments confirmed these results. The changes observed in the spectra of spin labeled azurin upon interaction with p53 indicate that the hydrophobic patch is involved in this interaction. Our results provide valuable insight into the topographic structure of the hydrophobic domain of azurin, as well as direct evidence of its interaction with p53 in solution via the hydrophobic patch. Cytotoxicity studies of azurin mutants showed that residues along the hydrophobic patch are important for its cytotoxicity.

Mapping the Binding Site of P53 on UBC9 by NMR Spectroscopy

Human UBC9 is a member of the E2 family of proteins. However, instead of conjugating to ubiquitin, it conjugates to a ubiquitin homologue SUMO-1 (also known as UBL1, GMP1, SMTP3, PICT-1 and sentrin). The SUMO-1 conjugation pathway is very similar to that of ubiquitin with regard to the primary sequences of the ubiquitin activating enzymes (E1), the three-dimensional structures of the ubiquitin conjugating enzymes (E2), and the chemistry of the overall conjugation pathway. The interaction of p53 and UBC9, the E2 of the SUMO-1 pathway, has been studied by nuclear magnetic resonance spectroscopy. A peptide corresponding to the nuclear localization domain of p53 specifically interacts with UBC9 and this interaction is likely to be important for conjugation of p53 with SUMO-1. The largest chemical shift changes on UBC9 occur at residues 94 and 129-135. This region is adjacent to the active site and has significant dynamic behavior on the μs-ms and ps-ns timescales. Correlation of chemical shift changes and mobility of these residues further suggest the importance of these residues in substrate recognition.

Design,synthesis and structure-activity relationship of 4,5-dihydropyrrolo[3,4-c]pyrazol-6(1H)-ones as potent p53-MDM2 inhibitors

In the past decade,the p53-MDM2 protein-protein interaction by small molecules has been confirmed as a successful strategy for cancer therapy.In our previous work,pyrrolo[3,4-c]pyrazol-6（1H）-ones were found to be potent p53-MDM2 inhibitors.Further optimization and structure-activity relationship studies were described in the present work.The result revealed that benzyl group on position N1 of imidazole and bromine on C4-phenyl of pyrrolidone showed higher inhibitory activities.In vitro antiproliferative assay demonstrated the potent p53-MDM2 inhibitor 5c with 4-fold selectivity for U2 OS and Saos-2 cells.These data indicated that 4,5-dihydropyrrolo[3,4-c]pyrazol-6（1H）-one moiety is a valuable scaffold for further development of p53-MDM2 inhibitors.

Oscillatory Activities in Regulatory Biological Networks and Hopf Bifurcation

Exploiting the nonlinear dynamics in the negative feedback loop, we propose a statistical signal-response model to describe the different oscillatory behaviour in a biological network motif. By choosing the delay as a bifurcation parameter, we discuss the existence of Hopf bifurcation and the stability of the periodic solutions of model equations with the centre manifold theorem and the normal form theory. It is shown that a periodic solution is born in a Hopf bifurcation beyond a critical time delay, and thus the bifurcation phenomenon may be important to elucidate the mechanism of oscillatory activities in regulatory biological networks.