Phosphatidic Acid Plays a Regulatory Role in Clathrin-mediated Endocytosis

Clathrin-mediated endocytosis (CME) is the main route of internalization of receptor-ligand complexes. Relatively little is known about the role of specific lipids in CME, in particular that of phosphatidic acid (PA). We examined the effect of altering cellular PA levels on CME by manipulating the activities and/or levels of either phospholipase D (PLD1 and PLD2) or diacylglycerol kinase (DGK), two enzyme classes involved in PA production. DGK inhibition resulted in a dramatic reduction of cellular PA, measured directly using an enzyme-coupled reaction, which resulted in a decreased rate of EGFR internalization measured biochemically. This corresponded to a decreased rate of clathrin-coated pit (CCP) initiation and increased lifetimes of productive CCPs, as determined by quantitative live-cell total internal reflection fluorescence microscopy. Unexpectedly, PLD inhibition caused an increase in cellular PA, suggesting that PLD activity negatively regulates PA synthesis by other more productive pathways. Consistent with opposite effects on cellular PA levels, PLD inhibition had opposite effects on EGFR internalization and CCP dynamics, compared with DGK inhibition. Importantly, the constitutive internalization of transferrin receptors was unaffected by either treatment. These findings demonstrate that PA plays a regulatory rather than obligatory role in CME and differentially regulates ligand-stimulated CME of EGFR.     

Characterization and Functional Aspects of Human Ninein Isoforms that Regulated by Centrosomal Targeting Signals and Evidence for Docking Sites to Direct Gamma-Tubulin

The functions of centrosomal protein ninein may be involved in microtubule minus end capping, centriole positioning, protein anchoring, and microtubule nucleation, but the true physiological function of various human hNinein isoforms remains to be determined. Here we describe the identification of four diverse CCII-termini of human hNinein isoforms, including a novel isoform 6, by differential expression in a tissue-specific manner. These hNinein isoforms exhibit centrosomal (concentrated) and noncentrosomal (aggregated) localization when GFP-tagged fusion proteins are expressed transiently in mammalian cells. In a kinase assay, we show that the CCII region of hNinein provides a differential phosphorylation site by GSK3β. In addition, our data indicate that either N-terminal or CCIIZ domain disruption may cause hNinein conformational change which recruits γ-tubulin to centrosomal or non-centrosomal hNinein-containing sites, implying that the γ-tubulin localization may be hNinein-dependent. Further, our RNA interference experiment against all hNinein isoforms caused a significant decrease in the γ-tubulin signal in the centrosome. In domain swapping, we clearly show that the CCIIX-CCIIY region provides docking sites for γ-tubulin. Moreover, our data also show that nucleation of microtubules from the centrosome is significantly affected by the presence of either the full-length hNinein or CCIIX-CCIIY region overexpression. Taken together, these results show that the centrosomal targeting signals of hNinein have a role not only in regulating hNinein conformation, resulting in localization change, but also provide docking sites to recruit γ-tubulin at centrosomal and non-centrosomal sites.     

Kinesin-5 Promotes Microtubule Nucleation and Assembly by Stabilizing a Lattice-Competent Conformation of Tubulin

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The Linker Region Plays a Regulatory Role in Assembly and Activity of the Vps4 AAA ATPase

The AAA-type ATPase Vps4 functions with components of the ESCRT (endosomal sorting complex required for transport) machinery in membrane fission events that are essential for endosomal maturation, cytokinesis, and the formation of retroviruses. A key step in these events is the assembly of monomeric Vps4 into the active ATPase complex, which is aided in part by binding of Vps4 via its N-terminal MIT (microtubule interacting and trafficking) domain to its substrate ESCRT-III. We found that the 40-amino acid linker region between the MIT and the ATPase domain of Vps4 is not required for proper function but plays a role in regulating Vps4 assembly and ATPase activity. Deletion of the linker is expected to bring the MIT domains into close proximity to the central pore of the Vps4 complex. We propose that this localization of the MIT domain in linker-deleted Vps4 mimics a repositioning of the MIT domain normally caused by binding of Vps4 to ESCRT-III. This structure would allow the Vps4 complex to engage ESCRT-III subunits with both the pore and the MIT domain simultaneously, which might be essential for the ATP-driven disassembly of ESCRT-III.     

Shigella flexneri 3a Outer Membrane Protein C Epitope Is Recognized by Human Umbilical Cord Sera and Associated with Protective Activity

Shigella flexneri 3a is one of the five major strains of the Shigella genus responsible for dysentery, especially among children, in regions of high poverty and poor sanitation. The outer membrane proteins (OMP) of this bacterium elicit immunological responses and are considered a prime target for vaccine development. When injected into mice they elicit a protective immunological response against a lethal dose of the pathogen. The OMPs from S. flexneri 3a were isolated and resolved by two-dimension-SDS-PAGE. Two 38-kDa spots were of particular interest since in our earlier studies OMPs of such molecular mass were found to interact with umbilical cord sera. These two spots were identified as OmpC by ESI-MS/MS spectrometry. By DNA sequencing, the ompC gene from S. flexneri 3a was identical to ompC from S. flexneri 2a [Gene Bank: 24113600]. A 3D model of OmpC was built and used to predict B-cell type (discontinuous) antigenic epitopes. Six epitopes bearing the highest score were selected and the corresponding peptides were synthesized. Only the peptides representing loop V of OmpC reacted strongly with the umbilical cord serum immunoglobulins. To determine which amino acids are essential for the antigenic activity of the epitope, the loop V was scanned with a series of dodecapeptides. The peptide RYDERY was identified as a minimal sequence for the loop V epitope. Truncation at either the C- or N-terminus rendered this peptide inactive. Apart from C-terminal tyrosine, substitution of each of the remaining five amino acids with glycine, led to a precipitous loss of immunological activity. This peptide may serve as a ligand in affinity chromatography of OmpC-specific antibodies and as a component of a vaccine designed to boost human immune defenses against enterobacterial infections.     

The Regulatory Role of NAD in Human and Animal Cells

Nicotinamide adenine dinucleotide (NAD) and its phosphorylated form NADP are the major coenzymes in the redox reactions of various essential metabolic pathways. NAD⁺ also serves as a substrate for several families of regulatory proteins, such as protein deacetylases (sirtuins), ADP-ribosyltransferases, and poly(ADP-ribose) polymerases, that control vital cell processes including gene expression, DNA repair, apoptosis, mitochondrial biogenesis, unfolded protein response, and many others. NAD⁺ is also a precursor for calcium-mobilizing secondary messengers. Proper regulation of these NAD-dependent metabolic and signaling pathways depends on how efficiently cells can maintain their NAD levels. Generally, mammalian cells regulate their NAD supply through biosynthesis from the precursors delivered with the diet: nicotinamide and nicotinic acid (vitamin B3), as well as nicotinamide riboside and nicotinic acid riboside. Administration of NAD precursors has been demonstrated to restore NAD levels in tissues (i.e., to produce beneficial therapeutic effects) in preclinical models of various diseases, such as neurodegenerative disorders, obesity, diabetes, and metabolic syndrome.     

microRNA-489 Plays an Anti-Metastatic Role in Human Hepatocellular Carcinoma by Targeting Matrix Metalloproteinase-7

Dysregulation of microRNAs (miRNAs) is actively involved in the pathogenesis and tumorigenicity of hepatocellular carcinoma (HCC). miR-489 was found to play either oncogenic or tumor suppressive roles in human cancers. Recent study reported that the levels of miR-489 in late recurrent HCC patients were evidently higher than that in early recurrent cases, suggesting that miR-489 may function as a tumor suppressive miRNA in HCC. Yet, the clinical value and biological function of miR-489 remain rarely known in HCC. Here, we presented that miR-489 level in HCC tissues was notably reduced compared to matched non-cancerous specimens. Its decreased level was evidently correlated with adverse clinical parameters and poor prognosis of HCC patients. Accordingly, the levels of miR-489 were obviously down-regulated in HCC cells. Ectopic expression of miR-489 in HCCLM3 and MHCC97H cells prominently inhibits the migration and invasion of tumor cells and reduced lung metastases in vivo, while miR-489 knockdown increased these behaviors of HepG2 and MHCC97L cells. Mechanically, miR-489 negatively regulated matrix metalloproteinase-7 (MMP7) abundance in HCC cells. Herein, MMP7 was found to be a downstream molecule of miR-489 in HCC. An inversely correlation between miR-489 and MMP7 was confirmed in HCC specimens. MMP7 knockdown prohibited cell migration and invasion while MMP7 overexpression showed opposite effects on HCC cells. Furthermore, restoration of MMP7 expression could abrogate the anti-metastatic effects of miR-489 on HCCLM3 cells with enhanced cell migration and invasion. Altogether, miR-489 potentially acts as a prognostic predictor and a drug-target for HCC patients.     

Nest Sanitation Plays a Role in Egg Burial by Yellow Warblers

Some hosts of the brown-headed cowbird ( Molothrus ater ) possess defences that eliminate all or most parasitism costs. Yellow warblers ( Dendroica petechia ) bury cowbird eggs, possibly to clean nests rather than serving strictly as an anti-parasite defence, as non-egg-shaped objects have been ejected, buried, or deserted by other hosts. With two experiments, we tested the 'nest sanitation' hypothesis by recording warblers' responses to objects (1) similar in volume, mass, and colour to cowbird eggs, and (2) half the mass and volume (more easily ejected), placed into nests before and during incubation. We compared outcomes at control nests with responses to objects that were dissimilar (stars) and moderately similar (dumbbells) to eggs, and to real cowbird and warbler eggs. We tested whether rejection (1) declines from stars through dumbbells and real eggs, (2) is similar between stages, and (3) non-egg-shaped objects are ejected because this is the least costly rejection method. Large stars were rejected (most buried) significantly more frequently (43.8%) than cowbird eggs (16.3%) in pre-incubation, suggesting that warblers reject objects shaped unlike their own eggs to rid nests of debris. Objects spent less time in nests the more they diverged from eggs. Warblers rarely rejected large stars and dumbbells, and cowbird eggs during incubation, possibly because burial and desertion are too costly by this time. Responses to small stars and dumbbells, and to foreign yellow warbler eggs did not differ between stages; also warblers rejected stars, mostly by ejection and selective burial, more frequently (28.8%) than dumbbells (1.3%) and warbler eggs (0%). Rejection by yellow warblers, especially burial, may keep nests clean, but also functions in rejecting cowbird eggs.     

Cytoglobin Up-regulated by Hydrogen Peroxide Plays a Protective Role in Oxidative Stress

Cytoglobin (Cygb) is a recently discovered intracellular respiratory globin, which exists in all types of cells. It has been suggested that Cygb has a role in protecting cells against oxidative stress. In the present study we have tested this hypothesis. The N2a neuroblastoma cells were exposed to various kinds of insults, including hydrogen peroxide (H₂O₂), hypoxia, kainic acid, high extracellular CaCl₂, high osmolarity, UV irradiation and heat shock. Among them, only H₂O₂-treatment induced a significant up-regulation of cytoglobin mRNA level. We stably transfected N2a cells with Cygb-siRNA vectors and successfully knocked down Cygb. The Cygb-siRNA could exacerbate cell death upon H₂O₂-treatment, as demonstrated by MTT cell viability assay. Thus, Cygb in neuronal cells might be specifically induced under oxidative stress to protect them from death.     

Nicotinic Acid Adenine Dinucleotide Phosphate Plays a Critical Role in Naive and Effector Murine T Cells but Not Natural Regulatory T Cells

Nicotinic acid adenine dinucleotide phosphate (NAADP), the most potent Ca²⁺ mobilizing second messenger discovered to date, has been implicated in Ca²⁺ signaling in some lymphomas and T cell clones. In contrast, the role of NAADP in Ca²⁺ signaling or the identity of the Ca²⁺ stores targeted by NAADP in conventional naive T cells is less clear. In the current study, we demonstrate the importance of NAADP in the generation of Ca²⁺ signals in murine naive T cells. Combining live-cell imaging methods and a pharmacological approach using the NAADP antagonist Ned-19, we addressed the involvement of NAADP in the generation of Ca²⁺ signals evoked by TCR stimulation and the role of this signal in downstream physiological end points such as proliferation, cytokine production, and other responses to stimulation. We demonstrated that acidic compartments in addition to the endoplasmic reticulum were the Ca²⁺ stores that were sensitive to NAADP in naive T cells. NAADP was shown to evoke functionally relevant Ca²⁺ signals in both naive CD4 and naive CD8 T cells. Furthermore, we examined the role of this signal in the activation, proliferation, and secretion of effector cytokines by Th1, Th2, Th17, and CD8 effector T cells. Overall, NAADP exhibited a similar profile in mediating Ca²⁺ release in effector T cells as in their counterpart naive T cells and seemed to be equally important for the function of these different subsets of effector T cells. This profile was not observed for natural T regulatory cells.     

Shugoshin 1 Plays a Central Role in Kinetochore Assembly and is Required for Kinetochore Targeting of Plk1

Physical connection between the sister chromatids is mediated by the cohesin protein complex. During prophase, cohesin is removed from the chromosome arms while the centromeres remain united. Shugoshin1 (Sgo1) is required for maintenance of centromeric cohesion from prophase to the metaphase-anaphase transition. Furthermore, Sgo1 has been proposed to regulate kinetochore microtubule stability and sense interkinetochore tension, two tasks which are tightly coupled with the function of the Chromosomal Passenger Complex (CPC) and Polo-like kinase 1 (Plk1). Here we show that depletion or chemical inhibition of Aurora B kinase (AurB), the catalytic subunit of the CPC, disrupts accumulation of Sgo1 on the kinetochores in HeLa cells and causes Sgo1 to localize on the chromosome arms. RNAi assays show that depletion of Sgo1 did not affect AurB localization but diminished Plk1 kinetochore binding. Furthermore, we demonstrate that vertebrate Sgo1 is phosphorylated by both AurB and Plk1 in vitro. The data presented here includes an extensive analysis of kinetochore targeting interdependencies of mitotic proteins that propose a novel branch in kinetochore assembly where Sgo1 and Plk1 have central roles. Furthermore our studies implicate Sgo1 in the tension sensing mechanism of the spindle checkpoint by regulating Plk1 kinetochore affinity.     

Identification of Multiple Novel Protein Biomarkers Shed by Human Serous Ovarian Tumors into the Blood of Immunocompromised Mice and Verified in Patient Sera

The most cancer-specific biomarkers in blood are likely to be proteins shed directly by the tumor rather than less specific inflammatory or other host responses. The use of xenograft mouse models together with in-depth proteome analysis for identification of human proteins in the mouse blood is an under-utilized strategy that can clearly identify proteins shed by the tumor. In the current study, 268 human proteins shed into mouse blood from human OVCAR-3 serous tumors were identified based upon human vs. mouse species differences using a four-dimensional plasma proteome fractionation strategy. A multi-step prioritization and verification strategy was subsequently developed to efficiently select some of the most promising biomarkers from this large number of candidates. A key step was parallel analysis of human proteins detected in the tumor supernatant, because substantially greater sequence coverage for many of the human proteins initially detected in the xenograft mouse plasma confirmed assignments as tumor-derived human proteins. Verification of candidate biomarkers in patient sera was facilitated by in-depth, label-free quantitative comparisons of serum pools from patients with ovarian cancer and benign ovarian tumors. The only proteins that advanced to multiple reaction monitoring (MRM) assay development were those that exhibited increases in ovarian cancer patients compared with benign tumor controls. MRM assays were facilely developed for all 11 novel biomarker candidates selected by this process and analysis of larger pools of patient sera suggested that all 11 proteins are promising candidate biomarkers that should be further evaluated on individual patient blood samples.     

Mycobacterium marinum MgtC Plays a Role in Phagocytosis but is Dispensable for Intracellular Multiplication

MgtC is a virulence factor involved in intramacrophage growth that has been reported in several intracellular pathogens, including Mycobacterium tuberculosis and Salmonella enterica serovar Typhimurium. MgtC participates also in adaptation to Mg²⁺ deprivation. Herein, we have constructed a mgtC mutant in Mycobacterium marinum to further investigate the role of MgtC in mycobacteria. We show that the M. marinum mgtC gene (Mma mgtC) is strongly induced upon Mg²⁺ deprivation and is required for optimal growth in Mg²⁺-deprived medium. The behaviour of the Mma mgtC mutant has been investigated in the Danio rerio infection model using a transgenic reporter zebrafish line that specifically labels neutrophils. Although the mgtC mutant is not attenuated in the zebrafish embryo model based on survival curves, our results indicate that phagocytosis by neutrophils is enhanced with the mgtC mutant compared to the wild-type strain following subcutaneous injection. Increased phagocytosis of the mutant strain is also observed ex vivo with the murine J774 macrophage cell line. On the other hand, no difference was found between the mgtC mutant and the wild-type strain in bacterial adhesion to macrophages and in the internalization into epithelial cells. Unlike the role reported for MgtC in other intracellular pathogens, Mma MgtC does not contribute significantly to intramacrophage replication. Taken together, these results indicate an unanticipated function of Mma MgtC at early step of infection within phagocytic cells. Hence, our results indicate that although the MgtC function is conserved among pathogens regarding adaptation to Mg²⁺ deprivation, its role towards phagocytic cells can differ, possibly in relation with the specific pathogen''s lifestyles.     

Antibiotic Production by Myxobacteria Plays a Role in Predation

Myxobacteria are predatory and are prolific producers of secondary metabolites. Here, we tested a hypothesized role that secondary metabolite antibiotics function as weapons in predation. To test this, a Myxococcus xanthus Δta1 mutant, blocked in antibiotic TA (myxovirescin) production, was constructed. This TA⁻ mutant was defective in producing a zone of inhibition (ZOI) against Escherichia coli. This shows that TA is the major M. xanthus-diffusible antibacterial agent against E. coli. Correspondingly, the TA⁻ mutant was defective in E. coli killing. Separately, an engineered E. coli strain resistant to TA was shown to be resistant toward predation. Exogenous addition of spectinomycin, a bacteriostatic antibiotic, rescued the predation defect of the TA⁻ mutant. In contrast, against Micrococcus luteus the TA⁻ mutant exhibited no defect in ZOI or killing. Thus, TA plays a selective role on prey species. To extend these studies to other myxobacteria, the role of antibiotic corallopyronin production in predation was tested and also found to be required for Corallococcus coralloides killing on E. coli. Next, a role of TA production in myxobacterial fitness was assessed by measuring swarm expansion. Here, the TA⁻ mutant had a specific swarm rate reduction on prey lawns, and thus reduced fitness, compared to an isogenic TA⁺ strain. Based on these observations, we conclude that myxobacterial antibiotic production can function as a predatory weapon. To our knowledge, this is the first report to directly show a link between secondary metabolite production and predation.