JDP2、Phf2蛋白在非小细胞肺癌组织中的表达及其与病理特征的相关性

目的探讨二聚化蛋白(JDP2)、Phf2蛋白在非小细胞肺癌(NSCLC)组织中的表达水平,及其与病理特征相关性。 方法收集2018年1月至2019年3月我院切除并经病理证实的65例NSCLC标本,其中>肿瘤边缘5 cm的癌旁组织作为空白对照组,收集患者基线资料性别、年龄、BMI、肿瘤直径、分化程度、肿瘤转移、肿瘤分期、病理类型及三年生存情况,采用免疫组化染色法检测JDP2、Phf2蛋白表达量,分析JDP2、Phf2蛋白与NSCLC患者临床病理特征及预后生存期相关性。 结果癌旁组织中JDP2、Phf2阳性表达率高于癌组织中JDP2、Phf2阳性表达率(P<0.05)。采用Spearman值分析两者关联性,结果JDP2、Phf2在NSCLC组织中共同表达呈负相关关系,二者具有协同作用(P<0.05)。分期Ⅲ期、出现肿瘤转移的NSCLC患者JDP2、Phf2阳性表达率低于分期Ⅰ/Ⅱ期、无肿瘤转移NSCLC患者JDP2、Phf2阳性表达率,而分化程度高级的NSCLC患者JDP2、Phf2阳性表达率高于分化程度低级的NSCLC患者JDP2、Phf2阳性表达率(P<0.05)。3年随访期间存活患者42例(64.62%),死亡23例(35.38%)。肿瘤直径>3 cm、TNM分期Ⅲ期、出现肿瘤转移、JDP2阴性、Phf2阴性的NSCLC患者存活率低于肿瘤直径≤3 cm、TNM分期Ⅰ/Ⅱ期、无肿瘤转移、JDP2阳性、Phf2阳性的NSCLC患者(P<0.05)。 结论JDP2、Phf2蛋白在NSCLC组织中均呈低表达,其表达水平与患者肿瘤TNM分期、肿瘤转移及预后呈相关性。     

Structure of human Mad1 C-terminal domain reveals its involvement in kinetochore targeting

The spindle checkpoint prevents aneuploidy by delaying anaphase onset until all sister chromatids achieve proper microtubule attachment. The kinetochore-bound checkpoint protein complex Mad1-Mad2 promotes the conformational activation of Mad2 and serves as a catalytic engine of checkpoint signaling. How Mad1 is targeted to kinetochores is not understood. Here, we report the crystal structure of the conserved C-terminal domain (CTD) of human Mad1. Mad1 CTD forms a homodimer and, unexpectedly, has a fold similar to those of the kinetochore-binding domains of Spc25 and Csm1. Nonoverlapping Mad1 fragments retain detectable kinetochore targeting. Deletion of the CTD diminishes, does not abolish, Mad1 kinetochore localization. Mutagenesis studies further map the functional interface of Mad1 CTD in kinetochore targeting and implicate Bub1 as its receptor. Our results indicate that CTD is a part of an extensive kinetochore-binding interface of Mad1, and rationalize graded kinetochore targeting of Mad1 during checkpoint signaling.     

基于荧光共振能量转移技术的STAT3二聚化抑制剂筛选模型的建立

目的%20构建STAT3二聚化抑制剂筛选模型,为STAT3抑制剂筛选提供实验方法。方法%20分别构建pECFP-N1-STAT3和pEYFP-N1-STAT3的荧光报告载体,利用脂质体转染技术将二者共转入HEK-293T细胞,利用ECFP和EYFP两种荧光蛋白之间能量共振转移,检测磷酸化STAT3分子的二聚化水平,并检测Stattic对二聚化的影响。结果%20成功构建了pECFP-N1-STAT3和pEYFP-N1-STAT3的荧光报告载体。将两种荧光报告载体共转染HEK-293T细胞后,Western%20Blot%20检测结果显示STAT3以及p-STAT3的表达水平明显增加。以458nm波长激发ECFP,其发射波长可激发EYFP。加入STAT3二聚化抑制剂Stattic后,荧光强度降低,且呈现一定的剂量依赖性。结论%20基于荧光共振能量转移技术的STAT3二聚化抑制剂筛选模型构建成功。     

Role of capsid sequence and immature nucleocapsid proteins p9 and p15 in Human Immunodeficiency Virus type 1 genomic RNA dimerization

HIV-1 genomic RNA (gRNA) dimerization is important for viral infectivity and is regulated by proteolytic processing of the Gag precursor protein (Pr55gag) under the direction of the viral protease. The processing occurs in successive steps and, to date, the step associated with formation of a wild-type (WT) level of gRNA dimers has not been identified. The primary cleavage divides Pr55gag into two proteins. The C-terminal polypeptide is termed NCp15 (NCp7-p1-p6) because it contains the nucleocapsid protein (NC), a key determinant of gRNA dimerization and packaging. To examine the importance of precursor polypeptides NCp15 and NCp9 (NCp7-p1), we introduced mutations that prevented the proteolytic cleavages responsible for the appearance of NCp9 or NCp7. Using native Northern blot analysis, we show that gRNA dimerization was impaired when both the secondary (p1-p6) and tertiary (p7-p1) cleavage sites of NCp15 were abolished, but unaffected when only one or the other site was abolished. Though processing to NCp9 therefore suffices for a WT level of gRNA dimerization, we also show that preventing cleavage at the p7-p1 site abolished HIV-1 replication. To identify the minimum level of protease activity compatible with a WT level of gRNA dimers, we introduced mutations Thr26Ser and Ala28Ser in the viral protease to partially inactivate it, and we prepared composite HIV-1 resulting from the cotransfection of various ratios of WT and protease-inactive proviral DNAs. The results reveal that a 30% processing of Pr55gag into mature capsid proteins (CA/CA-p2) yielded a WT level of gRNA dimers, while a 10% Pr55gag processing hardly increased gRNA dimerization above the level seen in protease-inactive virions. We found that full gRNA dimerization required less than 50% WT NC in complementation asssays. Finally, we show that if we destroy alpha helix 1 of the capsid protein (CA), gRNA dimerization is impaired to the same extent as when the viral protease is inactivated. Cotransfection studies show that this CA mutation, in contrast to the NC-disabling mutations, has a dominant negative effect on HIV-1 RNA dimerization, viral core formation, and viral replication. This represents the first evidence that a capsid mutation can affect HIV-1 RNA dimerization.     

Three residues at the interface of factor XI (FXI) monomers augment covalent dimerization of FXI

Summary.  Background:  Human plasma factor XI is a homodimer, with each monomer comprising a catalytic domain and four homologous 'apple' domains. The monomers bind to each other through non-covalent bonds and through a disulfide bond between Cys321 residues in apple 4 domains. Objective:  To identify residues essential for dimerization in the FXI monomer interface. Methods:  Specificity-determining residues in apple 4 domains were sought by sequence alignment of FXI and prekallikrein apple domains in different species. Specific residues identified in apple 4 domains were mutagenized and expressed in baby hamster kidney (BHK) cells for evaluation of their effect on FXI dimerization, analyzed by non-reduced sodium dodecylsulfate polyacrylamide gel electrophoresis and size-exclusion chromatography. Results:  Among the 19 residues of the FXI monomer interface, Leu284, Ile290 and Tyr329 were defined as specificity-determining residues. Substitutions of these residues or pairs of residues did not affect FXI synthesis and secretion from transfected BHK cells, but did impair dimerization, despite the presence of cysteine at position 321. The double mutant 284A/290A yielded predominantly a monomer, whereas all other single or double mutants yielded monomers as well as disulfide-bonded dimers. Conclusions:  The data suggest that Leu284, Ile290 and Tyr329 in the interface of FXI monomers are essential for forming non-covalently bonded dimers that facilitate formation of a disulfide-bonded stable FXI dimer.     

The modulation by xanthines of the DNA-damaging effect of polycyclic aromatic agents. Part II. The stacking complexes of caffeine with doxorubicin and mitoxantrone

Recently accumulated statistical data indicate the protective effect of caffeine consumption against several types of cancer diseases. There are also reports about protective effect of caffeine and other xanthines against tumors induced by polycyclic aromatic hydrocarbons. One of the explanations of this phenomenon is based on biological activation of such carcinogens by cytochromes that are also known for metabolism of caffeine. In the accompanying paper [Kapuscinski et al., this issue] we provide evidence (flow cytometry and the cell cycle analysis) that the cytostatic effects of caffeine (CAF) on two DNA alkylating agents, which do not require the biological activation, depend on their ability to form stacking (pi-pi) complexes. In this study, we use physicochemical techniques (computer aided light absorption and microcalorimetry), and molecular modeling to examine previously published qualitative data. This is published both by our and other group's data, indicates that CAF is able to modify the cytotoxic and/or cytostatic action of the two well known antitumor drugs doxorubicin (DOX) and mitoxantrone (MIT). To obtain the quantitative results from the experimental data we used the statistical-thermodynamical model of mixed aggregation, to find the association constants K(AC) of the CAF-drug interaction (128+/-10 and 356+/-21M(-1) for DOX-CAF and MIT-CAF complex formation, respectively). In addition, the favorable enthalpy change of CAF-MIT (DeltaH=-11.3kcal/mol) was measured by microcalorimetry titration. The molecular modeling (semi-empirical and force field method) allowed us to obtain the geometry of these complexes, which indicated the favorable energy (DeltaE) of complex formation of the protonated drug's molecules in aqueous environment (-7.4 and -8.7kcal/mol for DOX-CAF.5H(2)O and MIT-CAF.8H(2)O complex, respectively). The molecular modeling calculation indicates the existence of CAF-drug complexes in which the MIT molecules are intercalated between two CAF molecules (DeltaE=-29.9kcal/mol). These results indicate that the attenuating effect of caffeine on cytotoxic or mutagenic effects of some polycyclic aromatic mutagens cannot be the result of metabolic activation in the cells, but simply is the physicochemical process of the sequestering of aromatic molecules (e.g. carcinogens or mutagens) by formation of the stacking complexes. The caffeine may then act as the "interceptor" of potential carcinogens (especially in the upper part of digesting track) where its concentration can reach the mM level). There is, however, no indication, both, in the literature or from our experiments, that the xanthines can reverse the damage to nucleic acids at the point when the damage to DNA has already occurred.     

Antagonistic and agonistic effects of quinazoline tyrosine kinase inhibitors on mutant EGF receptor function

A mutated form of the EGF receptor (EGFRvIII), resulting from deletion of exons 2-7, is an oncogenic protein that is expressed in multiple human tumors. This mutation induces ligand-independent activation of the EGFR tyrosine kinase and thereby can initiate unregulated cell growth and tumorigenesis. Thus, inhibition of the kinase activity of EGFRvIII is a potential means of suppressing its oncogenic properties. Certain tyrosine kinase inhibitors (tyrphostins) specifically inhibit the wild-type EGFR and thereby inhibit tumor growth both in vitro and in vivo. We demonstrate that the quinazoline tyrphostins AG 1478 and AG 1517 can suppress morphologic transformation of cell lines by EGFRvIII. Quinazolines were found to inhibit receptor autophosphorylation and signaling through MAP kinase, but had minimal effects on association of EGFRvIII with Grb2/SOS. Low concentrations of quinazoline also increased receptor dimerization and phosphotyrosine content. This was associated with increases in colony formation in soft agar and increased invasion through matrigel for AG 1478. Thus, both AG 1478 and AG 1517 can inhibit multiple EGFRvIII signaling pathways, but at low concentrations AG 1478 can enhance colony formation, presumably related to augmented homodimerization of the receptor and activation of downstream signaling.     

Entropy and oligomerization in GPCRs

Evolutionary trace (ET) and entropy are two related methods for analyzing a multiple sequence alignment to determine functionally important residues in proteins. In this article, these methods have been enhanced with a view to reinvestigate the issue of GPCR dimerization and oligomerization. In particular, cluster analysis has replaced the subjective visual analysis element of the original ET method. Previous applications of the ET method predicted two dimerization interfaces on the external transmembrane lipid-facing region of GPCRs; these were discussed in terms of dimerization and linear oligomers. Removing the subjective element of the ET method gives rise to the prediction of functionally important residues on the external face of each transmembrane helix for a large number of class A GPCRs. These results are consistent with a growing body of experimental information that, taken over many receptor subtypes, has implicated each transmembrane helix in dimeric interactions. In this application, entropy gave superior results to those obtained from the ET method in that its use gives rise to higher z-scores and fewer instances of z-scores below 3.     

Cell membrane gp96 facilitates HER2 dimerization and serves as a novel target in breast cancer

HER2 receptor dimerization is a critical step in the HER2 activation process. Here, we demonstrated that heat shock protein gp96 on cell membrane interacts with HER2, facilitates HER2 dimerization and promotes cell proliferation. Cell membrane gp96 levels were observed to correlate with HER2 phosphorylation in primary breast tumors. Finally, we provide evidence that targeting gp96 with a specific monoclonal antibody led to decreased cell growth and increased apoptosis in vitro, and suppression of tumor growth in vivo. Our work represents a new therapeutic strategy for inhibiting HER2 signaling in cancer.     

On the regulation of fibroblast growth factor activity by heparin-like glycosaminoglycans

The activity of several angiogenic factors, like fibroblast growth factors (FGF), is modulated by heparin-like glycosaminoglycans (HLGAGs), which are acidic polysaccharides present in the extracellular matrix and at the cell surface. FGF binds to HLGAG in the matrix, where it is sequestered in a protected and inactive form, and at the cell surface, where it activates its cognate signaling receptor. Here we review recent progress in elucidating how HLGAG regulates FGF-induced signal transduction. Data from crystal structures of FGF complexed to active and inactive oligosaccharides is analyzed in the context of current models for HLGAG modulation of FGF activity. We propose that FGF can dimerize in several different modes, stabilized by HLGAGs. Individual dimer modes may represent active or inactive FGF and it is possible that different HLGAGs preferentially stabilize different FGF dimer modes. Understanding HLGAG–FGF interactions can provide leverage for new approaches to therapeutic control of angiogenesis.