Hes1调控 AKT-MDM2-p53-PTEN通路的一种物理机制

基于Hill动力学与Michaelis-Menten方程,建立理论模型研究发状分裂相关增强子1(hairy and enhancer of split 1,Hes1)调控蛋白激酶B (Protein Kinase B,AKT)-鼠双微体2 (Murine Double Minute2,MDM2)-抗癌基因p53(p53)-第10号染色体缺失的磷酸酶及张力蛋白同源的基因(Phosphatase and tensin homolog deleted on chromosome ten,PTEN)通路的一种物理机制.研究发现,Hes1通过与PTEN结合抑制PTEN表达,并调控AKT信号.表明了Hes1蛋白的合成,以及Hes1与PTEN相互作用调控AKTMDM2-p53-PTEN通路信号,将会有效地控制细胞结果 . Hes1作为AKT-MDM2-p53-PTEN信号通路中上游调节的重要因素,还可以在一定程度上通过影响p53蛋白功能,改变p53对肿瘤的抑制性.理论结果可用于预测Notch通路信号异常诱导的致癌性,并进一步揭示了Notch信号通路影响细胞AKT-MDM2-p53-PTEN通路的激活...     

新的光散射体系测定蛋白质的研究

以1,6-己二胺二吖啶为荧光探针,建立了一种新的蛋白质共振散射检测体系。实验考察了该吖啶荧光探针的共振散射特征光谱、散射反应稳定性,研究了缓冲介质pH、探针浓度等参数对测定蛋白质的影响。结果表明,当pH 8.9时,1,6-己二胺二吖啶染料的散射波长为470 nm;加入BSA,反应约9 min后,体系光散射强度达到最大并维持稳定,2 h内无明显变化。当1,6-己二胺二吖啶探针(浓度1.00×10-4mol·L-1)用量为3.00 mL时,蛋白质与1,6-己二胺二吖啶的光散射增强作用显著,共振散射强度随着BSA的增加而明显增强,光散射强度与蛋白质浓度成良好线性关系,线性浓度范围为1.00～5.00 μg·mL-1,检测限(3σ/K)为0.085 μg·mL-1。测定了血清样品,浓度为2.00～4.00 μg·mL-1,回收率为98.0%～101.7%,测定偏差小于1.7％。     

tBid蛋白引发磷脂膜透化过程的研究

Bid蛋白是仅有BH3结构域的Bcl-2家族蛋白,在溶酶体膜透化以及线粒体外膜透化引发的细胞凋亡过程中起着非常重要的调控作用,但是Bid蛋白与生物膜之间的相互作用导致脂膜透化的确切机制尚不十分清楚.本文利用激光扫描共聚焦显微成像技术及基于氧化石墨烯表面诱导荧光衰逝的单分子荧光技术,分别从单囊泡及单分子水平对tBid蛋白与磷脂膜之间的相互作用进行了系统的研究.结果表明,tBid蛋白在膜上聚集后可引起脂膜的透化,且脂膜透化的发生源于聚集体中一些tBid蛋白更深入地插入了脂膜中.     

The characteristics of molten globule states and folding pathways strongly depend on the sequence of a protein

ABSTRACT

The majority of proteins perform their cellular function after folding into a specific and stable native structure. Additionally, for many proteins less compact ‘molten globule’ states have been observed. Current experimental observations show that the molten globule state can show varying degrees of compactness and solvent accessibility; the underlying molecular cause for this variation is not well understood. While the specificity of protein folding can be studied using protein lattice models, current design procedures for these models tend to generate sequences without molten globule-like behaviour. Here we alter the design process so the distance between the molten globule ensemble and the native structure can be steered; this allows us to design protein sequences with a wide range of folding pathways, and sequences with well-defined heat-induced molten globules. Simulating these sequences we find that (1) molten globule states are compact, but have less specific configurations compared to the folded state, (2) the nature of the molten globule state is highly sequence dependent, (3) both two-state and multi-state folding proteins may show heat-induced molten globule states, as observed in heat capacity curves. The varying nature of the molten globules and typical heat capacity curves associated with the transitions closely resemble experimental observations.     

Using light to bioactivate surfaces: A new way of creating oriented, active immunobiosensors

Ultraviolet light can be used to immobilize biomolecules onto thiol reactive surfaces in order to, e.g., make biosensors. The mechanism involves light-induced formation of free, reactive thiol groups in disulphide containing molecules. This technology allows for the creation of arrays of biomolecules with a high degree of reproducibility, circumventing the need for often expensive nano/micro-dispensing technologies. The ultimate size of the immobilized spots is defined by the focal area of the UV beam. Light-induced immobilization has the added benefit that the immobilized molecules will be spatially oriented and covalently bound to the surface. In this paper, we demonstrate the utility of a sensor array created with the new sensor technology when integrated into a microfluidic system. Protein arrays made using light-induced immobilization showed successful antigen/antibody binding in a flow cell allowing the visualisation of real time binding and enzyme activity. This new technology is ideal for the creation of protein/DNA microarrays, can replace present micro-dispensing arraying technologies and is ideal as a molecular imprinting technology.     

Probing Proteins in Solution by Xe NMR Spectroscopy

The interaction of xenon with different proteins in aqueous solution is investigated by ¹²⁹Xe NMR spectroscopy. Chemical shifts are measured in horse metmyoglobin, hen egg white lysozyme, and horse cytochrome c solutions as a function of xenon concentration. In these systems, xenon is in fast exchange between all possible environments. The results suggest that nonspecific interactions exist between xenon and the protein exteriors and the data are analyzed in term of parameters which characterize the protein surfaces. The experimental data for horse metmyoglobin are interpreted using a model in which xenon forms a 1:1 complex with the protein and the chemical shift of the complexed xenon is reported (Locci et al., Keystone Symposia “Frontiers of NMR in Molecular Biology VI”, Jan. 9–15, 1999, Breckenridge, CO, Abstract E216, p. 53; Locci et al., XeMAT 2000 “Optical Polarization and Xenon NMR of Materials”, June 28–30, 2000, Sestri Levante, Italy, p. 46).     

Sensitivity-enhanced sim-CT HMQC PFG-HBHA(CO)NH and PFG-CBCA(CO)NH triple-resonance experiments

Short transverse relaxation times of Calpha and Cbeta single-quantum coherences reduce the sensitivity of triple-resonance experiments involving transfers of Calpha/Cbeta or Halpha/Hbeta coherences. Multiple-quantum line-narrowing techniques improve the relaxation properties of 13C coherences, thereby increasing the sensitivity of the experiment. In the present work, we describe PFG-CBCA(CO)NH and PFG-HBHA(CO)NH experiments that utilize heteronuclear multiple-quantum coherences in a simultaneous constant-time period to obtain completely decoupled spectra with improved sensitivity. Results indicate that approximately 30% of cross peaks show an average enhancement of approximately 15% in the CBCA(CO)NH experiment. In the related HBHA(CO)NH experiment, approximately 97% of the cross peaks show an average enhancement of approximately 40%.     

吲哚基二聚体菁类探针同步荧光测定蛋白质的研究

基于蛋白质对双嵌吲哚染料具有良好的荧光增强作用,以新型水溶性吲哚基同型二聚体探针I,建立了一种灵敏的蛋白质同步荧光分析体系。实验考察了吲哚探针的荧光特征、吲哚探针浓度、缓冲体系pH、盐浓度等参数对体系荧光的影响。在酸性条件下,蛋白质分子与探针I发生结合作用,同步荧光明显增强并向长波方向发生红移,且同步荧光强度与蛋白质浓度成良好的线性关系。在最优条件下,牛血清白蛋白BSA的线性响应范围5.00×10-7～2.50×10-5 g·mL-1,检测限(3σ/K)为3 ×10-8 g·mL-1;测定了血清蛋白BSA的合成样品,不同浓度BSA样品回收率为98.6%～103.0%,相对标准偏差1.1%～1.9%;与蛋白质紫外标准测定法比较,测定偏差为0.4%～3.9％。     

Electron holography of biological samples

In this paper, we summarise the development of off-axis electron holography on biological samples starting in 1986 with the first results on ferritin from the group of Tonomura. In the middle of the 1990s strong interest was evoked, but then stagnation took place because the results obtained at that stage did not reach the contrast and the resolution achieved by conventional electron microscopy.

To date, there exist only a few (12) publications on electron holography of biological objects, thus this topic is quite small and concise. The reason for this could be that holography is mostly established in materials science by physicists. Therefore, applications for off-axis holography were powerfully pushed forward in the area of imaging, e.g. electric or magnetic micro- and nanofields. Unstained biological systems investigated by means of off-axis electron holography up to now are ferritin, tobacco mosaic virus, a bacterial flagellum, T5 bacteriophage virus, hexagonal packed intermediate layer of bacteria and the Semliki Forest virus. New results of the authors on collagen fibres and surface layer of bacteria, the so-called S-layer 2D crystal lattice are presented in this review. For the sake of completeness, we will shortly discuss in-line holography of biological samples and off-axis holography of materials related to biological systems, such as biomaterial composites or magnetotactic bacteria.     

Profiling of generic anti-phosphopeptide antibodies and kinases with peptide microarrays using radioactive and fluorescence-based assays

Kinases represent one of the largest enzyme families and key regulatory proteins in the cell. Only a small subset of these enzymes has been characterised so far. We have prepared different types of phosphopeptide and peptide microarrays displaying peptides deduced from annotated human phosphorylation sites and cytoplasmic domains of all annotated human membrane proteins. This approach was enabled by fully-automated high throughput micro-scale synthesis of peptides by the SPOT technology combined with chemo-selective immobilisation on modified glass slides. The phosphopeptide microarrays displaying 2923 peptides in total have been used for the characterisation of commercially available generic anti-phosphopeptide antibodies. This enabled us to detect Abl kinase activity on a microarray with anti-phosphotyrosine antibodies yielding results comparable to those obtained from a radioactive assay. More than 13 000 peptides deposited on six glass slides were used to profile casein kinase 2 (CK2) using a radioactive assay, since no generic antibody for the reliable detection of serine or threonine phosphorylation could be identified. All previously identified substrates were detected in the microarray experiment. In order to confirm whether substrates on the microarray are substrates in solution phase assays, more than 700 peptides were synthesised and tested with CK2 in a solution phase assay. All substrates identified in the solution phase assay were also detected on the microarray.