基于细胞热漂移测定(CETSA)技术鉴定活细胞内药物靶标的标准操作流程

【目的】细胞热漂移测定(cell thermal shift assay, CETSA)技术是一种检测细胞内药物(配体)和蛋白质(靶标)相互作用的技术,原理是当蛋白质结合药物后,其热稳定性会发生变化,通过测定这种变化去鉴定药物和蛋白之间的相互作用。本研究以治疗多发性骨髓瘤的靶向药帕比司他(panobinostat)为例,建立基于蛋白印迹杂交(Western blotting)和CETSA技术的药物靶蛋白鉴定的标准操作流程。【方法】首先用药物panobinostat处理培养的K562细胞,然后加热处理细胞、裂解细胞及提取可溶性蛋白,以及用抗靶蛋白的抗体经Western blotting定量可溶性蛋白。【结果】经Western blotting定量及曲线拟合,成功得到3个蛋白——组蛋白去乙酰化酶(histone deacetylase,HDAC1)、人突触蛋白(humansyntaxin-4,STX4)以及四三肽重复结构域(tetratricopeptiderepeat domain38,TTC38)随温度变化的热熔解曲线和恒定温度条件下的药物剂量反应曲线。【结论】HDAC1、STX4及T...     

Mechanisms and Effects on HBV Replication of the Interaction between HBV Core Protein and Cellular Filamin B

Hepatitis B virus (HBV) infection is one of the major problems that threatens global health. There have been many studies on HBV, but the relationship between HBV and host factors is largely unexplored and more studies are needed to clarify these interactions. Filamin B is an actin-binding protein that acts as a cytoskeleton protein, and it is involved in cell development and several signaling pathways. In this study, we showed that filamin B interacted with HBV core protein, and the interaction promoted HBV replication. The interaction between filamin B and core protein was observed in HEK 293T, Huh7 and HepG2 cell lines by co-immunoprecipitation and co-localization immnofluoresence. Overexpression of filamin B increased the levels of HBV total RNAs and pre-genome RNA (pgRNA), and improved the secretion level of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg). In contrast, filamin B knockdown inhibited HBV replication, decreased the level of HBV total RNAs and pgRNA, and reduced the secretion level of HBsAg and HBeAg. In addition, we found that filamin B and core protein may interact with each other via four blocks of argentine residues at the C-terminus of core protein. In conclusion, we identify filamin B as a novel host factor that can interact with core protein to promote HBV replication in hepatocytes. Our study provides new insights into the relationship between HBV and host factors and may provide new strategies for the treatment of HBV infection.     

Thymidylate Synthase Protein and p53 mRNA Form an In Vivo Ribonucleoprotein Complex

A thymidylate synthase (TS)-ribonucleoprotein (RNP) complex composed of TS protein and the mRNA of the tumor suppressor gene p53 was isolated from cultured human colon cancer cells. RNA gel shift assays confirmed a specific interaction between TS protein and the protein-coding region of p53 mRNA, and in vitro translation studies demonstrated that this interaction resulted in the specific repression of p53 mRNA translation. To demonstrate the potential biological role of the TS protein-p53 mRNA interaction, Western immunoblot analysis revealed nearly undetectable levels of p53 protein in TS-overexpressing human colon cancer H630-R10 and rat hepatoma H35(F/F) cell lines compared to the levels in their respective parent H630 and H35 cell lines. Polysome analysis revealed that the p53 mRNA was associated with higher-molecular-weight polysomes in H35 cells compared to H35(F/F) cells. While the level of p53 mRNA expression was identical in parent and TS-overexpressing cell lines, the level of p53 RNA bound to TS in the form of RNP complexes was significantly higher in TS-overexpressing cells. The effect of TS on p53 expression was also investigated with human colon cancer RKO cells by use of a tetracycline-inducible system. Treatment of RKO cells with a tetracycline derivative, doxycycline, resulted in 15-fold-induced expression of TS protein and nearly complete suppression of p53 protein expression. However, p53 mRNA levels were identical in transfected RKO cells in the absence and presence of doxycycline. Taken together, these findings suggest that TS regulates the expression of p53 at the translational level. This study identifies a novel pathway for regulating p53 gene expression and expands current understanding of the potential role of TS as a regulator of cellular gene expression.     

凤眼莲入侵程度对金鱼藻和黑藻生长及种间关系的影响

湿地生态系统中凤眼莲（Eichhornia crassipes）入侵造成湿地植物群落结构退化及功能崩溃,直接影响沉水植物的生长繁殖及初级生产力。目前关于凤眼莲的入侵机制有一定的研究,而关于凤眼莲入侵程度对沉水植物金鱼藻（Ceratophyllum demersum）和黑藻（Hydrilla verticillate）生长及种间关系的影响相对缺乏。以外来入侵植物凤眼莲,沉水植物金鱼藻和黑藻为研究对象,设计凤眼莲入侵程度（无入侵,轻度入侵对应盖度25%,重度入侵对应盖度75%）交叉定植方式（黑藻单种模式、金鱼藻单种模式,金鱼藻和黑藻混种模式）的控制实验,探究凤眼莲入侵强度对沉水植物金鱼藻和黑藻生长及种间关系的影响。结果表明,凤眼莲入侵程度显著降低了金鱼藻的生物量、分枝数;黑藻的株高、分枝数和分节数。无凤眼莲入侵时,两种沉水植物生物量均最大,两者种间竞争关系较强;随凤眼莲入侵盖度增加,两种沉水植物的生物量先急剧降低后略微增加,种间关系经过微弱促进后又变为竞争作用,其中黑藻表现出明显的竞争优势。此外,凤眼莲入侵略微降低了水体中的总氮、总磷含量。结构方程模型分析结果表明凤眼莲入侵以及水体总氮、总磷等水体理化性质对沉水植物生长均有显著负向影响（P<0.05）,且水体理化性质对沉水植物生长的影响强于凤眼莲入侵。总之,凤眼莲入侵显著降低了金鱼藻和黑藻生长繁殖,随着凤眼莲入侵程度增加,两种沉水植物种间关系由竞争转变为促进再转变为竞争。研究结果为凤眼莲入侵有效控制及湿地沉水植被的恢复与重建提供了一定的理论依据和技术支撑。     

Deficiency of β Common Receptor Moderately Attenuates the Progression of Myeloproliferative Neoplasm in NrasG12D/+ Mice

Activating Ras signaling is a major driver in juvenile and the myeloproliferative variant of chronic myelomonocytic leukemia (JMML/MP-CMML). Numerous studies suggest that GM-CSF signaling plays a central role in establishing and maintaining JMML/MP-CMML phenotypes in human and mouse. However, it remains elusive how GM-CSF signaling impacts on JMML/MP-CMML initiation and progression. Here, we investigate this issue in a well characterized MP-CMML model induced by endogenous Nras^G12D/+ mutation. In this model, Nras^G12D/+ hematopoietic stem cells (HSCs) are required to initiate and maintain CMML phenotypes and serve as CMML-initiating cells. We show that the common β chain of the GM-CSF receptor (βc) is dispensable for Nras^G12D/+ HSC function; loss of βc does not affect the expansion, increased self-renewal, or myeloid differentiation bias in Nras^G12D/+ HSCs. Therefore, βc^−/− does not abrogate CMML in Nras^G12D/+ mice. However, βc deficiency indeed significantly reduces Nras^G12D/+-induced splenomegaly and spontaneous colony formation and prolongs the survival of CMML-bearing mice, suggesting that GM-CSF signaling plays an important role in promoting CMML progression. Together, our results suggest that inhibiting GM-CSF signaling in JMML/MP-CMML patients might alleviate disease symptoms but would not eradicate the disease.