Protein interactions and misfolding analyzed by AFM force spectroscopy

Protein misfolding is conformational transition dramatically facilitating the assembly of protein molecules into aggregates of various morphologies. Spontaneous formation of specific aggregates, mostly amyloid fibrils, was initially believed to be limited to proteins involved in the development of amyloidoses. However, recent studies show that, depending on conditions, the majority of proteins undergo structural transitions leading to the appearance of amyloidogenic intermediates followed by aggregate formation. Various techniques have been used to characterize the protein misfolding facilitating the aggregation process, but no direct evidence as to how such a conformational transition increases the intermolecular interactions has been obtained as of yet. We have applied atomic force microscopy (AFM) to follow the interaction between protein molecules as a function of pH. These studies were performed for three unrelated and structurally distinctive proteins, alpha-synuclein, amyloid beta-peptide (Abeta) and lysozyme. It was shown that the attractive force between homologous protein molecules is minimal at physiological pH and increases dramatically at acidic pH. Moreover, the dependence of the pulling forces is sharp, suggesting a pH-dependent conformational transition within the protein. Parallel circular dichroism (CD) measurements performed for alpha-synuclein and Abeta revealed that the decrease in pH is accompanied by a sharp conformational transition from a random coil at neutral pH to the more ordered, predominantly beta-sheet, structure at low pH. Importantly, the pH ranges for these conformational transitions coincide with those of pulling forces changes detected by AFM. In addition, protein self-assembly into filamentous aggregates studied by AFM imaging was shown to be facilitated at pH values corresponding to the maximum of pulling forces. Overall, these results indicate that proteins at acidic pH undergo structural transition into conformations responsible for the dramatic increase in interprotein interaction and promoting the formation of protein aggregates.     

p53 R175H hydrophobic patch and H‐bond reorganization observed by MD simulation

Molecular dynamics simulations probe the origins of aberrant functionality of R175H p53, which normally prevent tumorigenesis. This hotspot mutation exhibits loss of its essential zinc cofactor, aggregation, and activation of gain of function promoters, characteristics contributing to the loss of normal p53 activity. This study provided molecular level insight into the reorganization of the hydrogen bonding network and the formation of a hydrophobic patch on the surface of the protein. The hydrogen bonding network globally redistributes at the expense of the stability of the β‐sandwich structure, and surface residues reorganize to expose a 250 Ų hydrophobic patch of residues covering approximately 2% of the solvent accessible surface. These changes could both stabilize the protein in the conformation exposing the patch to solvent to mediate the reported aggregation, and cause a destabilization in the area associated with DNA binding residues to affect the specificity. The development of the patch prior to loss of zinc indicates that stabilizing the patch quickly may prevent zinc loss. Considerations for rational design of small molecule therapeutics in light of the structural insight has been discussed and it suggest the positive ring around the hydrophobic patch and conserved residues may constitute a druggable site. © 2015 Wiley Periodicals, Inc. Biopolymers 105: 176–185, 2016.     

Standards for Immunohistochemical Imaging: A Protein Reference Device for Biomarker Quantitation

We are developing a reference device to be used in the validation of immunohistochemical imaging of biomarkers by microscopy. The prototype device consists of p53 protein immobilized at various concentrations on a glass slide. The device is designed as a reference control to be used with assays that incorporate commercially available anti-p53 antibodies. p53 protein was characterized by mass spectrometry and covalently immobilized through amide linkage to the (3-aminopropyl)trietoxysilane-modified glass surface. This procedure is reproducible and provides a chemically stable product in high yield. The surface-bound protein was shown to be immunoreactive by its specific interaction with anti-p53 antibody (Ab) and detection by absorbance and fluorescence spectroscopy. Also, comparison was made with microscopic images of Ab-stained tissue samples, known to stain positive for p53. Further development will be required to establish accurate surface protein concentrations in the range required for specific clinical applications. (J Histochem Cytochem 58:1005–1014, 2010)     

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(promyelocytic leukemia protein,PML)在体内相互作用共定位于细胞核PML致癌结构城(PML oncogenic domains,PODs)的人Daxx(human Daxx,hDaxx),能结合Fas死亡结构域诱导细胞凋亡.细胞肿瘤抑制子p53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p53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p53通过C端与hDaxx结合,共免疫沉淀反应及Western blot结果显示hDaxx与p53能在体内外直接结合.hDaxx与p53结合并发生相互作用可能对细胞周期有一定的调节作用.     

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.     

基于乳腺癌ChIP-seq数据的p53抑癌机制研究

采用生物信息学方法分析野生型p53乳腺癌MCF7细胞的Ch IP-seq（染色质免疫共沉淀-测序）数据,以揭示p53的抑癌分子机制。从NCBI下载的编号为GSE47041的Ch IP-seq数据来源于三组试验,分别为：未经处理的乳腺癌MCF7细胞对照（NS_input）,Nutlin-3a（一种MDM2拮抗剂）处理的MCF7细胞对照（S_input）和Nutlin-3a刺激MCF7细胞后加入p53抗体的实验组（S_p53）。Ch IP获得的DNA数据的测序平台为Illumina Hi Seq 2000。利用Bowtie参照人基因组hg19进行序列比对;利用MACS进行峰信号检测,并利用自定义软件筛选p53可能的靶基因;利用DAVID在线工具对靶基因进行通路富集分析;最后利用STRING构建蛋白互作网络。研究共得到50个p53的靶基因,其中8个靶基因（CDKN1A、BBC3、BAX、DDB2、MDM2、CCNG1、XPC和PCNA）分别富集到p53信号转导通路和核苷酸切除修复通路两个通路上。在得到的由19个靶基因构成的蛋白质相互作用网络中,连通度最高的前5个基因分别是PCNA、MDM2、REV3L、CDKN1A和BAX。研究中采用的分析Ch IP-seq数据的方法能有效揭示野生型p53乳腺癌MCF7细胞中Nutlin-3a激活的p53的抑癌分子机制。     

p53基因及其功能研究进展

p53基因是一种肿瘤抑制基因,野生型p53对细胞周期和细胞凋亡起重要作用。其编码的蛋白P53相对分子质量为53×103,可刺激Cipt基因产生相对分子质量为21×103的蛋白,该蛋白可以抑制促使细胞通过细胞周期进入有丝分裂的酶的活性,进而抑制细胞生长表达而调控细胞生长,对于预防和治疗胆管癌、肝癌、胃癌等疾病有重要作用。我们在此简要阐述国内外对p53基因及其编码产物的结构、作用机制、检测、功能等方面的研究进展。     

MDM2-P53蛋白质相互作用的小分子抑制物

p53作为重要的抑癌基因已经成为一个治疗癌症重点的突破目标之一。直接调节p53基因或调节P53和MDM2蛋白质相互作用是再激活p53基因的两种重要机制。对于表达野生型P53的癌症设计小分子阻断剂阻断MDM2与P53蛋白相互作用是一个很有前景的治疗癌症的方向。文章主要总结了作为治疗癌症的新方法-MDM2-P53蛋白相互作用小分子抑制物的最新研究进展,其中最新的是人工合成化合物Nutlin-3和MI-219。     

原子力显微术在生物研究中的应用

原子力显微术不仅能够提供样品表面纳米级别分辨率的三维图像数据,而且能够对pN级微小力进行测量,同时将两者结合发展出的TREC（topography and recognition）显微术还能够在进行高分辨成像的同时实现对特定分子的定位。原子力显微术的这些特点使之成为生物化学、细胞生物学等生物研究的有利工具。本文主要介绍了原子力显微镜高分辨成像和检测生物分子识别的原理,以及TREC显微术在生物学上的应用。     

PRIMA-1 cytotoxicity correlates with nucleolar localization and degradation of mutant p53 in breast cancer cells

PRIMA-1 has been identified as a compound that restores the transactivation function to mutant p53 and induces apoptosis in cells expressing mutant p53. Studies on subcellular distribution of the mutant p53 protein upon treatment with PRIMA-1^Met, a methylated form of PRIMA-1, have suggested that redistribution of mutant p53 to nucleoli may play a role in PRIMA-1 induced apoptosis. Here, we specifically investigated the influence of PRIMA-1 on cellular localization of mutated p53-R280K endogenously expressed in tumour cells. By using immunofluorescence staining, we found a strong nucleolar redistribution of mutant p53 following PRIMA-1 treatment. This subcellular localization was associated to p53 degradation via ubiquitylation. When cells were treated with adriamycin, neither nucleolar redistribution nor mutant p53 down modulation and degradation were observed. Interestingly, cells where p53-R280K was silenced were more sensitive to PRIMA-1 than the parental ones. These results indicate that in some cellular context, the cell sensitivity to PRIMA-1 could depend on the abolition of a gain-of-function activity of the mutated p53, through a protein degradation pathway specifically induced by this compound.