Caveolin-1 mutants P132L and Y14F are dominant negative regulators of invasion, migration and aggregation in H1299 lung cancer cells

Caveolin-1 is an essential protein constituent of caveolae. Accumulating evidence indicates that caveolin-1 may act as a positive regulator of cancer progression. In this study, we investigated the function of caveolin-1 in human lung cancer cells. Caveolin-1 knockdown inhibited cell proliferation and reduced focal adhesion kinase (Fak) phosphorylation. Matrix invasion and cell migration as well as expression and activity of matrix metalloproteases were attenuated following caveolin-1 RNAi-mediated knockdown or overexpression of Y14F and P132L mutants, demonstrating dominant-negative activity of these mutants. Time-lapse fluorescence microscopy revealed that caveolin-1 and its mutants P132L and Y14F are localized to the trailing edge of migrating cells during both random and directed cell movement, implying an active role of caveolin-1 in the migration process. Suppression of caveolin-1 function greatly elevated the percentage of H1299 cells exhibiting focal adhesions. In addition, cell aggregation was increased by wild type caveolin-1 and attenuated by both P132L and Y14F mutants. Overexpression of wild type caveolin-1 increased caveolae density, however, P132L and Y14F mutants did not affect caveolae formation, suggesting that in this respect that the mutants do not act in a dominant negative manner, and that effects of caveolin-1 on caveolae and cell invasion, migration, focal adhesion and aggregation, are separable. Our data provide novel mechanistic insights into the role of caveolin-1 in cell motility, invasiveness and aggregation, therefore, expanding our understanding of the tumor-promoting activities of caveolin-1 in advanced-stage cancer.     

Dependence of folding dynamics and structural stability on the location of a hydrophobic pair in beta-hairpins

We study the dependence of folding time, nucleation site, and stability of a model beta-hairpin on the location of a cross-strand hydrophobic pair, using a coarse-grained off-lattice model with the aid of Monte Carlo simulations. Our simulations have produced 6500 independent folding trajectories dynamically, forming the basis for extensive statistical analysis. Four folding pathways, zipping-out, middle-out, zipping-in, and reptation, have been closely monitored and discussed in all seven sequences studied. A hydrophobic pair placed near the beta-turn or in the middle section effectively speed up folding; a hydrophobic pair placed close to the terminal ends or next to the beta-turn encourages stability of the entire chain.     

Conversion of type I 4:6 to 3:5 beta-turn types in human acidic fibroblast growth factor: effects upon structure, stability, folding, and mitogenic function

Human acidic fibroblast growth factor (FGF-1) is a member of the beta-trefoil superfold, a protein architecture that exhibits a characteristic threefold axis of structural symmetry. FGF-1 contains 11 beta-turns, the majority being type I 3:5; however, a type I 4:6 turn is also found at three symmetry-related locations. The relative uniqueness of the type I 4:6 turn in the FGF-1 structure suggests it may play a key role in the stability, folding, or function of the protein. To test this hypothesis a series of deletion mutations were constructed, the aim of which was to convert existing type I 4:6 turns at two locations into type I 3:5 turns. The results show it is possible to successfully substitute the type I 4:6 turn by a type I 3:5 turn with minimal impact upon protein stability or folding. Thus, these different turn structures, even though they differ in length, exhibit similar energetic properties. Additional sequence swapping mutations within the introduced type I 3:5 turns suggests that the turn sequence primarily affects stability but not turn structure (which appears dictated primarily by the local environment). Although the results suggest that a stable, foldable beta-trefoil protein may be designed utilizing a single turn type (type I 3:5), a type I 4:6 turn at turn 1 of FGF-1 appears essential for efficient mitogenic function.     

Specific short hairpin RNA-mediated inhibition of viral DNA packaging of human cytomegalovirus

To clearly demonstrate the role of the human cytomegalovirus (HCMV) portal protein pUL104 RNA interference was applied. Expressing cell lines were constructed by transduction of shRNAs via the infection with retroviral vectors. After infection of these cells with HCMV AD169 the expression of pUL104 was reduced up to 80% for shRNA S1 and 54% for shRNA S2 at late times of infection compared to controls. In addition, the inhibitory effect was corresponding with a decrease in viral mRNA and plaque formations. Electron microscopic analysis demonstrated that infection of cells expressing pUL104-specific shRNAs resulted in the inhibition of formation of replicative particles.     

The origin of the tRNA molecule: Independent data favor a specific model of its evolution

The properties, historical and empirical observations of a model of the origin of the tRNA molecule are discussed. This model would predict that this molecule originated by means of the assembly of two hairpin-like structures of RNA. The conclusion is that the model possesses a relevant part of the truth on the origin of the tRNA molecule.     

Intrinsic flexibility of snRNA hairpin loops facilitates protein binding

Stem–loop II of U1 snRNA and Stem–loop IV of U2 snRNA typically have 10 or 11 nucleotides in their loops. The fluorescent nucleobase 2-aminopurine was used as a substitute for the adenines in each loop to probe the local and global structures and dynamics of these unusually long loops. Using steady-state and time-resolved fluorescence, we find that, while the bases in the loops are stacked, they are able to undergo significant local motion on the picosecond/nanosecond timescale. In addition, the loops have a global conformational change at low temperatures that occurs on the microsecond timescale, as determined using laser T-jump experiments. Nucleobase and loop motions are present at temperatures far below the melting temperature of the hairpin stem, which may facilitate the conformational change required for specific protein binding to these RNA loops.     

shRNA抑制宫颈癌Hela细胞株HPV18 E6、E7基因表达的研究

应用短发夹RNA(Short hairpin RNA,shRNA)表达载体抑制宫颈癌Hela细胞株HPV18 E6、E7基因的表达。应用已鉴定的shRNA表达载体pHPV1、pHPV2转染Hela细胞,G418筛选阳性细胞,建立稳定转染细胞株;倒置荧光显微镜检测转染情况;提取细胞内总RNA,RT-PCR方法检测HPV18 E6、E7 mRNA;WesternBlot检测HPV18 E6、E7蛋白表达的变化;采用灰度分析软件对PCR扩增条带与蛋白质条带进行灰度分析。pHPV1实验组细胞内HPV18 E6、E7 mRNA含量分别为阴性对照组的31%、38%,E6、E7蛋白分别为阴性对照组的37%、31%;pHPV2实验组细胞内HPV18 E6、E7 mRNA含量分别为阴性对照组的54%、77%,E6、E7蛋白分别为阴性对照组的52%、83%。pHPV1、pHPV2表达载体能抑制Hela细胞HPV18 E6、E7的表达,针对外显子区434-452的pHPV1抑制作用更强。