Functional analysis of functional membrane microdomains in the biosynthesis of menaquinone-7 / 生物工程学报

Functional membrane microdomains (FMMs) that are mainly composed of scaffold proteins and polyisoprenoids play important roles in diverse cellular physiological processes in bacteria. The aim of this study was to identify the correlation between MK-7 and FMMs and then regulate the MK-7 biosynthesis through FMMs. Firstly, the relationship between FMMs and MK-7 on the cell membrane was determined by fluorescent labeling. Secondly, we demonstrated that MK-7 is a key polyisoprenoid component of FMMs by analyzing the changes in the content of MK-7 on cell membrane and the changes in the membrane order before and after destroying the integrity of FMMs. Subsequently, the subcellular localization of some key enzymes in MK-7 synthesis was explored by visual analysis, and the intracellular free pathway enzymes Fni, IspA, HepT and YuxO were localized to FMMs through FloA to achieve the compartmentalization of MK-7 synthesis pathway. Finally, a high MK-7 production strain BS3AT was successfully obtained. The production of MK-7 reached 300.3 mg/L in shake flask and 464.2 mg/L in 3 L fermenter.

The relationship between expression of llotillin-1 and lymph node metastasis in early-stage cervical cancers / 中国医师杂志

Objective To investigate the expression of flotillin-1 in cervical cancer,and molecular mechanism and relationship between flotillin-1 and lymph node metastasis.Methods Real-time polymerase chain reaction (RT-PCR),Western blot and immunohistochemistry were used to detect the expression of flotillin-1 in cervical cancer cells and tissues,and detect the role of flotillin-1 in cervical cancer metastasis and the possible mechanism by over-expression and interference.Results The protein and mRNA expression of Flotillin-1 were significantly upregnlated in cervical cancer cell lines and cancer tissues.The elevated expression of flotillin-1 protein in early-stage cervical cancers was significantly associated with pelvic lymph node metastasis (P ＜0.01).Moreover,flotillin-1 up-and down-regulations remarkably affected the motility and invasion of cervical cancer cells through epithelial-mesenchymal transition (EMT) regulated by the Wnt//β-catenin and nuclear factor-κB (NF-κB) pathways.Conclusions Flotillin-1 regulates the EMT process through the catenin beta Wnt and NF-κB signaling pathway,and promotes the metastasis of cervical cancers.The flotillin-1 expression profile serves as novel independent predictor of lymph node metastasis in early-stage cervical cancers.

Lipid Raft Integrity Is Required for Survival of Triple Negative Breast Cancer Cells / 한국유방암학회지

PURPOSE: Lipid rafts are cholesterol enriched microdomains that colocalize signaling pathways involved in cell proliferation, metastasis, and angiogenesis. We examined the effect of methyl-β-cyclodextrin (MβCD)-mediated cholesterol extraction on the proliferation, adhesion, invasion, and angiogenesis of triple negative breast cancer (TNBC) cells. METHODS: We measured cholesterol and estimated cell toxicity. Detergent resistant membrane (DRM) and non-DRM fractions were separated using the OptiPrep gradient method. Cell cycles stages were analyzed by flow cytometry, apoptosis was assessed using the TdT-mediated dUTP nick end-labeling assay, and metastasis was determined using a Matrigel invasion assay. Neo-vessel pattern and levels of angiogenic modulators were determined using an in vitro angiogenesis assay and an angiogenesis array, respectively. RESULTS: The present study found that the cholesterol-depleting agent MβCD, efficiently depleted membrane cholesterol and caused concentration dependent (0.1–0.5 mM) cytotoxicity compared to nystatin and filipin III in TNBC cell lines, MDA-MB 231 and MDA-MB 468. A reduced proportion of caveolin-1 found in DRM fractions indicated a cholesterol extraction-induced disruption of lipid raft integrity. MβCD inhibited 52% of MDA-MB 231 cell adhesion on fibronectin and 56% of MDA-MB 468 cell adhesion on vitronectin, while invasiveness of these cells was decreased by 48% and 52% respectively, following MβCD treatment (48 hours). MβCD also caused cell cycle arrest at the G2M phase and apoptosis in MDA-MB 231 cells (25% and 58% cells, respectively) and in MDA-MB 468 cells (30% and 38% cells, respectively). We found that MβCD treated cells caused a 52% and 58% depletion of neovessel formation in both MDA-MB 231 and MDA-MB 468 cell lines, respectively. This study also demonstrated that MβCD treatment caused a respective 2.6- and 2.5-fold depletion of tyrosine protein kinase receptor (TEK) receptor tyrosine kinase levels in both TNBC cell lines. CONCLUSION: MβCD-induced cholesterol removal enhances alterations in lipid raft integrity, which reduces TNBC cell survival.

Mecanismo de acción de la toxina alfa hemolisina de Escherichia coli / Action mechanism of Escherichia coli alpha-hemolysin toxin / Mecanismo de ação da toxina alfa-hemolisina de Escherichia coli

Escherichia coli es una de las bacterias anaerobias facultativas más predominantes en el intestino, siendo, en la mayoría de los casos, inocua para el huésped. Existen cepas que traslocan al torrente sanguíneo causando enfermedades extraintestinales como infecciones urinarias, septicemia y meningitis. Dentro de éstas se encuentran las cepas uropatogénicas (Uropathogenic Escherichia coli: UPEC), que secretan varios factores de virulencia. Estos últimos incluyen: toxinas, sistemas de adquisición de hierro, adhesinas y antígenos capsulares. Las principales toxinas secretadas son: alfa-hemolisina (HlyA) y el factor necrotizante citotóxico 1 (CNF-1). En esta revisión se presenta una descripción exhaustiva de HlyA, incluyendo su síntesis, maduración y exportación desde la bacteria. La acilación de la proteína en dos residuos internos de lisina la convierte en una toxina muy virulenta al exponer regiones intrínsecamente desordenadas que son esenciales en diferentes pasos del mecanismo de acción de la misma. Específicamente, la exposición de estas regiones está involucrada en interacciones proteína-proteína dentro del proceso de oligomerización. La formación del oligómero es responsable de la permeabilidad inducida en las células blanco. Finalmente, basado en los conocimientos acerca de las características estructurales y funcionales de HlyA, se presentan potenciales usos de HlyA en terapias basadas en toxinas.

Escherichia coli is one of the predominant species of facultative anaerobes in the human gut, and in the majority of the cases it is harmless to the host. Some strains of this species can translocate to blood and cause infection such as urinary infection, septicemia and meningitis. These are the uropathogenic E. coli strains (UPEC) that secrete a number of virulence factors. The latter include a number of secreted toxins, iron-acquisition systems, adhesins, and capsular antigens. Secreted toxins include HlyA, the cytotoxic necrotizing factor-1 (CNF-1). In this review an exhaustive description of the toxin has been delineated, including its synthesis, maturation, and export from the bacteria. The acylation of the protein at two internal lysine residues gives the toxin its virulence, by exposing intrinsic disordered regions that are essential in different steps of the toxin's mechanism of action. The further exposure of regions involved in the protein-protein interaction within the oligomerization process is responsG-ible for the permeability induced in all the target cells. Based on the already known structural and functional characteristics of HlyA, the potential use in toxin-based therapy is presented.

Escherichia coli é uma das bactérias anaérobias facultativas mais predominantes no intestino, sendo na maioria dos casos inócua para o hóspede. Há cepas que passam ao torrente sanguíneo causando doenças extraintestinais como infecção urinária, septicemia e meningite. Dentro destas se encontram as cepas uropatogênicas (Uropathogenic Escherichia coli: UPEC) que secretam varios fatores de virulência. Estos últimos incluem: toxinas, sistemas de aquisição de ferro, adesinas e antígenos capsulares. As principais toxinas secretadas são: alfa hemolisina (HlyA) e o fator necrotizante citotóxico 1 (CNF-1). Nesta revisão apresenta-se uma descrição exaustiva de HlyA incluindo sua sintese, seu amadurecimento e exportação a partir da bactéria. A acilação da proteína em dois residuos internos de lisina a transforma numa toxina muito virulenta ao expor regiões intrinsecamente desordenadas que são essenciais em diferentes passos do mecanismo de ação da mesma. Especificamente, a exposição destas regiões esta envolvida em interações proteína-proteína dentro do processo de oligomerização. A formação do oligômero é responsável pela permeabilidade induzida nas células alvo. Finalmente, com base nos conhecimentos acerca das características estruturais e funcionais de HlyA, apresentam-se potenciais usos de HlyA em terapias baseadas em toxinas.

Secretion of adenylate kinase 1 is required for extracellular ATP synthesis in C2C12 myotubes

Extracellular ATP (exATP) has been known to be a critical ligand regulating skeletal muscle differentiation and contractibility. ExATP synthesis was greatly increased with the high level of adenylate kinase 1 (AK1) and ATP synthase beta during C2C12 myogenesis. The exATP synthesis was abolished by the knock-down of AK1 but not by that of ATP synthase beta in C2C12 myotubes, suggesting that AK1 is required for exATP synthesis in myotubes. However, membrane-bound AK1beta was not involved in exATP synthesis because its expression level was decreased during myogenesis in spite of its localization in the lipid rafts that contain various kinds of receptors and mediate cell signal transduction, cell migration, and differentiation. Interestingly, cytoplasmic AK1 was secreted from C2C12 myotubes but not from C2C12 myoblasts. Taken together all these data, we can conclude that AK1 secretion is required for the exATP generation in myotubes.

Multi-transmembrane protein K15 of Kaposi's sarcoma-associated herpesvirus targets Lyn kinase in the membrane raft and induces NFAT/AP1 activities

Viral proteins of gamma-2 herpesviruses, such as LMP2A of Epstein Barr virus (EBV) and Tip of herpesvirus saimiri (HVS) dysregulate lymphocyte signaling by interacting with Src family kinases. K15 open reading frame of Kaposi's sarcoma associated herpesvirus (KSHV), located at the right end of the viral genome, encodes several splicing variants differing in numbers of transmembrane domains. Previously, we demonstrated that the cytoplasmic tail of the K15 protein interfered with B cell receptor signal transduction to cellular tyrosine phosphorylation and calcium mobilization. However, the detailed mechanism underlying this phenomenon was not understood. In the C-terminal cytoplasmic region of K15, putative binding domains for Src-SH2 and -SH3 were identified. In this study, we attempted to characterize these modular elements and cellular binding protein(s) by GST pull down and co-immunoprecipitation assays. These studies revealed that K15 interacted with the major B cell tyrosine kinase Lyn. In vitro kinase and transient co-expression assays showed that the expression of K15 protein resulted in activation of Lyn kinase activity. In addition, GST pull down assay suggested that the SH2 domain of Lyn alone was necessary for interaction with the C-terminal SH2B (YEEV) of K15, but the addition of Lyn SH3 to the SH2 domain increases the binding affinity to K15 protein. The data from luciferase assays indicate that K15 expression in BJAB cells induced NFAT and AP1 activities. The tyrosine residue in the C-terminal end of K15 required for the Lyn interaction appeared to be essential for NFAT/AP1 activation, highlighting the significance of the C-terminal SH2B of K15 as a modular element in interfering with B lymphocyte signaling through interaction with Lyn kinase.

Extracellular ATP is generated by ATP synthase complex in adipocyte lipid rafts

Mitochondrial biogenesis is known to accompany adipogenesis to complement ATP and acetyl-CoA required for lipogenesis. Here, we demonstrated that mitochondrial proteins such as ATP synthase alpha and beta, and cytochrome c were highly expressed during the 3T3-L1 differentiation into adipocytes. Fully-differentiated adipocytes showed a significant increase of mitochondria under electron microscopy. Analysis by immunofluorescence, cellular fractionation, and surface biotinylation demonstrated the elevated levels of ATP synthase complex found not only in the mitochondria but also on the cell surface (particularly lipid rafts) of adipocytes. High rate of ATP (more than 30 micrometer) synthesis from the added ADP and Pi in the adipocyte media suggests the involvement of the surface ATP synthase complex for the exracellular ATP synthesis. In addition, this ATP synthesis was significantly inhibited in the presence of oligomycin, an ATP synthase inhibitor, and carbonyl cyanide m-chlorophenylhydrazone (CCCP), an ATP synthase uncoupler. Decrease of extracellular ATP synthesis in acidic but not in basic media further indicates that the surface ATP synthase may also be regulated by proton gradient through the plasma membrane.

Lipid rafts are important for the association of RANK and TRAF6

Rafts, cholesterol- and sphingolipid-rich membrane microdomains, have been shown to play an important role in immune cell activation. More recently rafts were implicated in the signal transduction by members of the TNF receptor (TNFR) family. In this study, we provide evidences that the raft microdomain has a crucial role in RANK (receptor activator of NF-kappaB) signaling. We found that the majority of the ectopically expressed RANK and substantial portion of endogenous TRAF2 and TRAF6 were detected in the low-density raft fractions. In addition, TRAF6 association with rafts was increased by RANKL stimulation. The disruption of rafts blocked the TRAF6 translocation by RANK ligand and impeded the interaction between RANK and TRAF6. Our observations demonstrate that proper RANK signaling requires the function of raft membrane microdomains.