RILP suppresses invasion of breast cancer cells by modulating the activity of RalA through interaction with RalGDS

RILP (Rab7-interacting lysosomal protein) is a key regulator for late endosomal/lysosomal trafficking, and probably a tumor suppressor in prostate cancer. However, the role of RILP in other cancers and the underlying mechanism for RILP in regulating the invasion of cancer cells remain to be investigated. In this study, we showed that overexpression of RILP in breast cancer cells inhibits the migration and invasion, whereas the depletion of RILP by RNAi-mediated knockdown promotes the migration and invasion. We identified RalGDS (Ral guanine nucleotide dissociation stimulator) as a novel interacting partner for RILP, and truncation analysis revealed the N-terminal region of RILP is responsible for interacting with the guanine nucleotide exchange factor (GEF) domain of RalGDS. Immunofluorescence microscopy revealed that RalGDS can be recruited to the late endosomal compartments by RILP. Further investigations indicated that the overexpression of RILP inhibits the activity of RalA, a downstream target of RalGDS. Our data suggest that RILP suppresses the invasion of breast cancer cells by interacting with RalGDS to inhibit its GEF activity for RalA.Diverse alternations of oncogenic factors can either activate or inactivate signaling pathways involved in cell proliferation, migration and apoptosis that are intimately associated with cancer development.^{1, 2, 3} Recent studies suggest that the derailed membrane trafficking is also closely related to cancer development. Activation or attenuation of signal transduction is usually linked to membrane trafficking. The recycling and degradation of surface receptors, such as EGFR, will influence downstream signaling pathways.^{4, 5} Therefore, the cross-talk between membrane trafficking and signaling pathway could be the novel mechanism associated with cancer development.Alternations of the membrane trafficking machineries are established as the causes for some cancers. For examples, Rab25 is overexpressed in breast and ovary caners,⁶ and recent investigations suggest that Rab25 is also related to other cancers.^{7, 8, 9} Arf6 is a vital regulator for the invasive activity of breast cancer cells.¹⁰ Disordered membrane trafficking is emerging as an important property during tumorigenesis, thus the membrane trafficking machineries are potential therapeutic targets for cancer treatment.Rab small GTPases are considered as the master regulators for membrane trafficking.¹¹ The interactions between Rab proteins and their downstream effectors are involved in various steps of vesicle trafficking such as tethering and fusion. Aberrant activities of Rab proteins are closely related to some cancers.^{12, 13, 14, 15} Some Rab proteins mediate the trafficking of cargos, especially membrane proteins on the plasma membrane, such as integrin and E-cadherin. Their aberrant trafficking is proposed to be the underlying mechanism for the functional regulation of Rab protein in cancer cells.^{16, 17}Rab7, together with its downstream effector RILP (Rab7-interacting lysosomal protein), are the key regulators for late endosomal/lysosomal trafficking. RILP interacts with activated GTP-bound Rab7 through its carboxylic terminal region, whereas interacting with dynein/dynactin complex is mediated through its amino region, driving late endosomal/lysosomal trafficking, especially lysosomal positioning.^{18, 19} Rab7 has been demonstrated to be an important factor for cell growth and survival.^{20, 21} Recently, Steffan et al.²² found that RILP suppresses the invasion of prostate cancer cells through inhibiting the anterograde trafficking of lysosomes.²³ Whether the potential role of Rab7-RILP in cell migration/invasion is also implicated in other cancers is of interest to investigate and the underlying molecular mechanism is yet to be defined.In this study, we found that RILP suppresses the proliferation, migration and invasion of breast cancer cells. We also identified (Ral guanine nucleotide dissociation stimulator (RalGDS) as a novel interacting partner for RILP. The interaction of RILP with RalGDS modulates the activity of RalA. Our results suggest that RILP suppresses the invasion of breast cancer cells by modulating the activity of RalA through interaction with RalGDS.     

Screening of binding proteins that interact with human Salvador 1 in a human fetal liver cDNA library by the yeast two-hybrid system

Salvador promotes both cell cycle exit and apoptosis through the modulation of both cyclin E and Drosophila inhibitor of apoptosis protein in Drosophila. However, the cellular function of human Salvador (hSav1) is rarely reported. To screen for novel binding proteins that interact with hSav1, the cDNA of hSav1 was cloned into a bait protein plasmid, and positive clones were screened from a human fetal liver cDNA library by the yeast two-hybrid system. hSav1 mRNA was expressed in yeast and there was no self-activation and toxicity in the yeast strain AH109. Twenty proteins were found to interact with hSav1, including HS1 (haematopoietic cell specific protein1)-associated protein X-1 (HAX-1); neural precursor cell expressed, developmentally down-regulated 9, pyruvate kinase, liver and RBC, cytochrome c oxidase subunit Vb, enoyl coenzyme A hydratase short chain 1, and NADH dehydrogenase (ubiquinone) 1 beta subcomplex, demonstrating that the yeast two-hybrid system is an efficient method for investigating protein interactions. Among the identified proteins, there were many mitochondrial proteins, indicating that hSav1 may play a role in mitochondrial function. We also confirmed the interaction of HAX-1 and hSav1 in mammalian cells. This investigation provides functional clues for further exploration of potential apoptosis-related proteins in disease biotherapy.     

Demonstration of diet-induced decoupling of fatty acid and cholesterol synthesis by combining gene expression array and 2H2O quantification

The liver is a crossroad for metabolism of lipid and carbohydrates, with acetyl-CoA serving as an important metabolic intermediate and a precursor for fatty acid and cholesterol biosynthesis pathways. A better understanding of the regulation of these pathways requires an experimental approach that provides both quantitative metabolic flux measurements and mechanistic insight. Under conditions of high carbohydrate availability, excess carbon is converted into free fatty acids and triglyceride for storage, but it is not clear how excessive carbohydrate availability affects cholesterol biosynthesis. To address this, C57BL/6J mice were fed either a low-fat, high-carbohydrate diet or a high-fat, carbohydrate-free diet. At the end of the dietary intervention, the two groups received (2)H(2)O to trace de novo fatty acid and cholesterol synthesis, and livers were collected for gene expression analysis. Expression of lipid and glucose metabolism genes was determined using a custom-designed pathway focused PCR-based gene expression array. The expression analysis showed downregulation of cholesterol biosynthesis genes and upregulation of fatty acid synthesis genes in mice receiving the high-carbohydrate diet compared with the carbohydrate-free diet. In support of these findings, (2)H(2)O tracer data showed that fatty acid synthesis was increased 10-fold and cholesterol synthesis was reduced by 1.6-fold in mice fed the respective diets. In conclusion, by applying gene expression analysis and tracer methodology, we show that fatty acid and cholesterol synthesis are differentially regulated when the carbohydrate intake in mice is altered.     

Diallyl trisulfide induces apoptosis and inhibits proliferation of A549 cells in vitro and in vivo

Lung cancer is the leading cause of cancer-related mortality all over the world. In recent years, pulmonary adenocarcinoma has surpassed squamous cell carcinoma in frequency and is the predominant form of lung cancer in many countries. Epidemiological investigations have shown an inverse relationship between garlic (Allium sativum) consumption and death rate from many cancers. Diallyl trisulfide (DATS) is one of the garlic-derived compounds (also known as: organosulfer compounds, OSC). DATS can induce apoptosis and inhibit the growth of many cancer cell lines. Our study demonstrated that the apoptotic incidents induced by DATS were a mitochondria-dependent caspase cascade through a significant decrease of the anti-apoptotic Bcl-2 that resulted in up-regulation of the ratio of Bax/Bcl-2 and the activity of caspase-3, -8, and -9. Eventually, DATS induced the apoptosis and inhibited the proliferation in a concentration- and time-dependent manner. Furthermore, by establishing an animal model of female BALB/c nude mice with A549 xenografts, we found that oral gavage of DATS significantly retarded growth of A549 xenografts in nude mice without causing weight loss or any other side effects compared with the control group. All the evidence both in vitro and in vivo suggested that DATS could be an ideal anti-cancer drug.     

Isolation and characterization of a mesophilic Arthrospira maxima strain capable of producing docosahexaenoic acid

A strain of the cyanobacterium Arthrospira was isolated from Lake Chahannaoer in northern China and was characterized according to microscopic morphology, photosynthetic oxygen-evolving activity, growth rate, and nutritional profile. Compared with thermophilic Arthrospira species occurring naturally in tropical and subtropical lakes, this isolate is mesophilic and grows optimally at ~20 degreesC. The total protein, fatty acid, phycocyanin, carotenoid, and chlorophyll a contents were 67.6, 6.1, 4.32, 0.29, and 0.76 grams per 100 grams of dry weight, respectively. The strain is rich in polyunsaturated fatty acids (PUFAs). An essential omega-3 fatty acid, docosahexaenoic acid (DHA), was detected, and gamma-linolenic acid (GLA) and DHA accounted for 28.3% of the total fatty acid content. These features of this newly isolated strain make it potentially useful in commercial mass culture in local areas or as a biofuel feedstock. It is also an alternative resource for studying the metabolic PUFA pathways and mechanisms of cold stress tolerance in cyanobacteria.     

The Hippo–YAP pathway regulates the proliferation of alveolar epithelial progenitors after acute lung injury

Regeneration of pulmonary epithelial cells plays an important role in the recovery of acute lung injury (ALI), which is defined by pulmonary epithelial cell death. However, the mechanism of the regenerative capacity of alveolar epithelial cells is unknown. Using a lung injury mouse model induced by hemorrhagic shock and lipopolysaccharide, a protein mass spectrometry‐based high‐throughput screening and linage tracing technology to mark alveolar epithelial type 2 cells (AEC2s), we analyzed the mechanism of alveolar epithelial cells proliferation. We demonstrated that the expression of Hippo‐yes‐associated protein 1 (YAP1) key proteins were highly consistent with the regularity of the proliferation of alveolar epithelial type 2 cells after ALI. Furthermore, the results showed that YAP1+ cells in lung tissue after ALI were mainly Sftpc lineage‐labeled AEC2s. An in vitro proliferation assay of AEC2s demonstrated that AEC2 proliferation was significantly inhibited by both YAP1 small interfering RNA and Hippo inhibitor. These findings revealed that YAP functioned as a key regulator to promote AEC2s proliferation, with the Hippo signaling pathway playing a pivotal role in this process.     

菌紫质的Ser和Lys残基修饰对BR结构和功能的影响

用化学修饰研究了菌紫质（ＢＲ）的结构和功能的变化。用氮氧自由基分别对赖氨酸和丝氨酸进行修饰,研究结果表明在圆二色谱上（ＣＤ谱）,与天然紫膜样品比较,两种自由基分别修饰赖氨酸（Ｌｙｓ）和丝氨酸（Ｓｅｒ）残基２４小时后的ＣＤ谱中均只有负峰,分别在５９６ｎｍ和６０２ｎｍ,５３５ｎｍ的正峰已消失,７２小时后５３５ｎｍ的正峰部分地恢复,但１２０小时后均未见进一步恢复。与未修饰的紫膜相比,两种自由基修饰的紫膜在Ｒａｍａｎ光谱上观察到中间体Ｍ４１２的相对量要明显增加。本文对这二种化学修饰引起的ＢＲ结构和功能变化进行了初步讨论。