Página 9 dos resultados de 45945 itens digitais encontrados em 0.036 segundos

‣ Recognition by toll-like receptor 2 induces antigen-presenting cell activation and Th1 programming during infection by Neospora caninum

MINEO, Tiago W. P.; OLIVEIRA, Carlo J. F.; GUTIERREZ, Fredy R. S.; SILVA, Joao S.
Fonte: NATURE PUBLISHING GROUP Publicador: NATURE PUBLISHING GROUP
Tipo: Artigo de Revista Científica
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Neospora caninum is an apicomplexan parasite responsible for major economic losses due to abortions in cattle. Toll-like receptors (TLRs) sense specific microbial products and direct downstream signaling pathways in immune cells, linking innate, and adaptive immunity. Here, we analyze the role of TLR2 on innate and adaptive immune responses during N. caninum infection. Inflammatory peritoneal macrophages and bone marrow-derived dendritic cells exposed to N. caninum-soluble antigens presented an upregulated expression of TLR2. Increased receptor expression was correlated to TLR2/MyD88-dependent antigen-presenting cell maturation and pro-inflammatory cytokine production after stimulation by antigens. Impaired innate responses observed after infection of mice genetically deficient for TLR2((-/-)) was followed by downregulation of adaptive T helper 1 (Th1) immunity, represented by diminished parasite-specific CD4(+) and CD8(+) T-cell proliferation, IFN-gamma:interleukin (IL)-10 ratio, and IgG subclass synthesis. In parallel, TLR2(-/-) mice presented higher parasite burden than wild-type (WT) mice at acute and chronic stages of infection. These results show that initial recognition of N. caninum by TLR2 participates in the generation of effector immune responses against N. caninum and imply that the receptor may be a target for future prophylactic strategies against neosporosis. Immunology and Cell Biology (2010) 88...

‣ c-Jun Induces Mammary Epithelial Cellular Invasion and Breast Cancer Stem Cell Expansion*

Jiao, Xuanmao; Katiyar, Sanjay; Willmarth, Nicole E.; Liu, Manran; Ma, Xiaojing; Flomenberg, Neal; Lisanti, Michael P.; Pestell, Richard G.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
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The molecular mechanisms governing breast tumor cellular self-renewal contribute to breast cancer progression and therapeutic resistance. The ErbB2 oncogene is overexpressed in ∼30% of human breast cancers. c-Jun, the first cellular proto-oncogene, is overexpressed in human breast cancer. However, the role of endogenous c-Jun in mammary tumor progression is unknown. Herein, transgenic mice expressing the mammary gland-targeted ErbB2 oncogene were crossed with c-junf/f transgenic mice to determine the role of endogenous c-Jun in mammary tumor invasion and stem cell function. The excision of c-jun by Cre recombinase reduced cellular migration, invasion, and mammosphere formation of ErbB2-induced mammary tumors. Proteomic analysis identified a subset of secreted proteins (stem cell factor (SCF) and CCL5) induced by ErbB2 expression that were dependent upon endogenous c-Jun expression. SCF and CCL5 were identified as transcriptionally induced by c-Jun. CCL5 rescued the c-Jun-deficient breast tumor cellular invasion phenotype. SCF rescued the c-Jun-deficient mammosphere production. Endogenous c-Jun thus contributes to ErbB2-induced mammary tumor cell invasion and self-renewal.

‣ Quantitative Proteomics Analysis of Cell Cycle-regulated Golgi Disassembly and Reassembly*

Chen, Xuequn; Simon, Eric S.; Xiang, Yi; Kachman, Maureen; Andrews, Philip C.; Wang, Yanzhuang
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
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During mitosis, the stacked structure of the Golgi undergoes a continuous fragmentation process. The generated mitotic fragments are evenly distributed into the daughter cells and reassembled into new Golgi stacks. This disassembly and reassembly process is critical for Golgi biogenesis during cell division, but the underlying molecular mechanism is poorly understood. In this study, we have recapitulated this process using an in vitro assay and analyzed the proteins associated with interphase and mitotic Golgi membranes using a proteomic approach. Incubation of purified rat liver Golgi membranes with mitotic HeLa cell cytosol led to fragmentation of the membranes; subsequent treatment of these membranes with interphase cytosol allowed the reassembly of the Golgi fragments into new Golgi stacks. These membranes were then used for quantitative proteomics analyses by combining the isobaric tags for relative and absolute quantification approach with OFFGEL isoelectric focusing separation and liquid chromatography-matrix assisted laser desorption ionization-tandem mass spectrometry. In three independent experiments, a total of 1,193 Golgi-associated proteins were identified and quantified. These included broad functional categories, such as Golgi structural proteins...

‣ Regulation of Cyclin B2 Expression and Cell Cycle G2/M Transition by Menin*

Wu, Ting; Zhang, Xiuli; Huang, Xiaohua; Yang, Yuqing; Hua, Xianxin
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
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Multiple endocrine neoplasia type 1 (MEN1) results from mutations in tumor suppressor gene Men1, which encodes nuclear protein menin. Menin up-regulates certain cyclin-dependent kinase inhibitors through increasing histone H3 lysine 4 (H3K4) methylation and inhibits G0/G1 to S phase transition. However, little is known as to whether menin controls G2/M-phase transition, another important cell cycle checkpoint. Here, we show that menin expression delays G2/M phase transition and reduces expression of Ccnb2 (encoding cyclin B2). Menin associates with the promoter of Ccnb2 and reduces histone H3 acetylation, a positive chromatin marker for gene transcription, at the Ccnb2 locus. Moreover, Men1 ablation leads to an increase in cyclin B2 expression, histone H3 acetylation at the Ccnb2 locus, and G2/M transition. In contrast, knockdown of cyclin B2 diminishes the number of cells at M phase and reduces cell proliferation. Furthermore, menin interferes with binding of certain positive transcriptional regulators, such as nuclear factor Y (NF-Y), E2 factors (E2Fs), and histone acetyltransferase CREB (cAMP-response element-binding protein)-binding protein (CBP) to the Ccnb2 locus. Notably, MEN1 disease-related mutations, A242V and L22R, abrogate the ability of menin to repress cyclin B2 expression and G2/M transition. Both of the mutants fail to reduce the acetylated level of the Ccnb2 locus. Together...

‣ Akt Activation Emulates Chk1 Inhibition and Bcl2 Overexpression and Abrogates G2 Cell Cycle Checkpoint by Inhibiting BRCA1 Foci*

Tonic, Ivana; Yu, Wan-Ni; Park, Youngku; Chen, Chia-Chen; Hay, Nissim
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
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Akt is perhaps the most frequently activated oncoprotein in human cancers. Overriding cell cycle checkpoint in combination with the inhibition of apoptosis are two principal requirements for predisposition to cancer. Here we show that the activation of Akt is sufficient to promote these two principal processes, by inhibiting Chk1 activation with concomitant inhibition of apoptosis. These activities of Akt cannot be recapitulated by the knockdown of Chk1 alone or by overexpression of Bcl2. Rather the combination of Chk1 knockdown and Bcl2 overexpression is required to recapitulate Akt activities. Akt was shown to directly phosphorylate Chk1. However, we found that Chk1 mutants in the Akt phosphorylation sites behave like wild-type Chk1 in mediating G2 arrest, suggesting that the phosphorylation of Chk1 by Akt is either dispensable for Chk1 activity or insufficient by itself to exert an effect on Chk1 activity. Here we report a new mechanism by which Akt affects G2 cell cycle arrest. We show that Akt inhibits BRCA1 function that induces G2 cell cycle arrest. Akt prevents the translocation of BRCA1 to DNA damage foci and, thereby, inhibiting the activation of Chk1 following DNA damage.

‣ Secreted Heat Shock Protein 90α Induces Colorectal Cancer Cell Invasion through CD91/LRP-1 and NF-κB-mediated Integrin αV Expression*

Chen, Jinn-Shiun; Hsu, Yuan-Ming; Chen, Chia-Chi; Chen, Li-Li; Lee, Chun-Chung; Huang, Tze-Sing
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
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HCT-8 colon cancer cells secreted heat shock protein 90α (HSP90α) and had increased invasiveness upon serum starvation. The concentrated conditioned medium of serum-starved HCT-8 cells was able to stimulate the migration and invasion of non-serum-starved cells, which could be prevented by treatment with an anti-HSP90α antibody. Recombinant HSP90α (rHSP90α) also enhanced HCT-8 cell migration and invasion, suggesting a stimulatory role of secreted HSP90α in cancer malignancy. HSP90α binding to CD91α and Neu was evidenced by the proximity ligation assay, and rHSP90α-induced HCT-8 cell invasion could be suppressed by the addition of anti-CD91α or anti-Neu antibodies. Via CD91α and Neu, rHSP90α selectively induced integrin αV expression, and knockdown of integrin αV efficiently blocked rHSP90α-induced HCT-8 cell invasion. rHSP90α induced the activities of ERK, PI3K/Akt, and NF-κB p65, but only NF-κB activation was involved in HSP90α-induced integrin αV expression. Additionally, we investigated the serum levels of HSP90α and the expression status of tumor integrin αV mRNA in colorectal cancer patients. Serum HSP90α levels of colorectal cancer patients were significantly higher than those of normal volunteers (p < 0.001). Patients with higher serum HSP90α levels significantly exhibited elevated levels of integrin αV mRNA in tumor tissues as compared with adjacent non-tumor tissues (p < 0.001). Furthermore...

‣ Rad9 Is Required for B Cell Proliferation and Immunoglobulin Class Switch Recombination*

An, Lili; Wang, Yulan; Liu, Yuheng; Yang, Xiao; Liu, Chunchun; Hu, Zhishang; He, Wei; Song, Wenxia; Hang, Haiying
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
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B cell maturation and B cell-mediated antibody response require programmed DNA modifications such as the V(D)J recombination, the immunoglobulin (Ig) class switch recombination, and the somatic hypermutation to generate functional Igs. Many protein factors involved in DNA damage repair have been shown to be critical for the maturation and activation of B cells. Rad9 plays an important role in both DNA repair and cell cycle checkpoint control. However, its role in Ig generation has not been reported. In this study, we generated a conditional knock-out mouse line in which Rad9 is deleted specifically in B cells and investigated the function of Rad9 in B cells. The Rad9−/− B cells isolated from the conditional knock-out mice displayed impaired growth response and enhanced DNA lesions. Impaired Ig production in response to immunization in Rad9−/− mice was also detected. In addition, the Ig class switch recombination is deficient in Rad9−/− B cells. Taken together, Rad9 plays dual roles in generating functional antibodies and in maintaining the integrity of the whole genome in B cells.

‣ Hydrogen Peroxide Removes TRPM4 Current Desensitization Conferring Increased Vulnerability to Necrotic Cell Death*

Simon, Felipe; Leiva-Salcedo, Elías; Armisén, Ricardo; Riveros, Ana; Cerda, Oscar; Varela, Diego; Eguiguren, Ana Luisa; Olivero, Pablo; Stutzin, Andrés
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
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Necrosis is associated with an increase in plasma membrane permeability, cell swelling, and loss of membrane integrity with subsequent release of cytoplasmic constituents. Severe redox imbalance by overproduction of reactive oxygen species is one of the main causes of necrosis. Here we demonstrate that H2O2 induces a sustained activity of TRPM4, a Ca2+-activated, Ca2+-impermeant nonselective cation channel resulting in an increased vulnerability to cell death. In HEK 293 cells overexpressing TRPM4, H2O2 was found to eliminate in a dose-dependent manner TRPM4 desensitization. Site-directed mutagenesis experiments revealed that the Cys1093 residue is crucial for the H2O2-mediated loss of desensitization. In HeLa cells, which endogenously express TRPM4, H2O2 elicited necrosis as well as apoptosis. H2O2-mediated necrosis but not apoptosis was abolished by replacement of external Na+ ions with sucrose or the non-permeant cation N-methyl-d-glucamine and by knocking down TRPM4 with a shRNA directed against TRPM4. Conversely, transient overexpression of TRPM4 in HeLa cells in which TRPM4 was previously silenced re-established vulnerability to H2O2-induced necrotic cell death. In addition, HeLa cells exposed to H2O2 displayed an irreversible loss of membrane potential...

‣ Desmosome Assembly and Cell-Cell Adhesion Are Membrane Raft-dependent Processes

Resnik, Nataša; Sepčić, Kristina; Plemenitaš, Ana; Windoffer, Reinhard; Leube, Rudolf; Veranič, Peter
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
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The aim of our study was to investigate the association of desmosomal proteins with cholesterol-enriched membrane domains, commonly called membrane rafts, and the influence of cholesterol on desmosome assembly in epithelial Madin-Darby canine kidney cells (clone MDc-2). Biochemical analysis proved an association of desmosomal cadherin desmocollin 2 (Dsc2) in cholesterol-enriched fractions that contain membrane raft markers caveolin-1 and flotillin-1 and the novel raft marker ostreolysin. Cold detergent extraction of biotinylated plasma membranes revealed that ∼60% of Dsc2 associates with membrane rafts while the remainder is present in nonraft and cholesterol-poor membranes. The results of immunofluorescence microscopy confirmed colocalization of Dsc2 and ostreolysin. Partial depletion of cholesterol with methyl-β-cyclodextrin disturbs desmosome assembly, as revealed by sequential recordings of live cells. Moreover, cholesterol depletion significantly reduces the strength of cell-cell junctions and partially releases Dsc2 from membrane rafts. Our data indicate that a pool of Dsc2 is associated with membrane rafts, particularly with the ostreolysin type of membrane raft, and that intact membrane rafts are necessary for desmosome assembly. Taken together...

‣ Engineered Annexin A5 Variants Have Impaired Cell Entry for Molecular Imaging of Apoptosis Using Pretargeting Strategies*

Ungethüm, Lisette; Kenis, Heidi; Nicolaes, Gerry A.; Autin, Ludovic; Stoilova-McPhie, Svetla; Reutelingsperger, Chris P. M.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
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Phosphatidylserine (PS) on apoptotic cells is a target for diagnosis and therapy using annexin A5 (anxA5). Pretargeting is a strategy developed to improve signal to background ratio for molecular imaging and to minimize undesired side effects of pharmacological and radiotherapy. Pretargeting relies on accessibility of the target finder on the surface of the target cell. anxA5 binds PS and crystallizes in a two-dimensional network covering the PS-expressing cell surface. Two-dimensional crystallization is the driving force for anxA5 internalization by PS-expressing cells. Here, we report structure/function analysis of anxA5 internalization. Guided by structural bioinformatics including protein-protein docking, we revealed that the amino acids Arg63, Lys70, Lys101, Glu138, Asp139, and Asn160 engage in intermolecular salt bridges within the anxA5 trimer, which is the basic building block of the two-dimensional network. Disruption of the salt bridges by site-directed mutagenesis does not affect PS binding but inhibits trimer formation and cell entry of surface-bound anxA5. The anxA5 variants with impaired internalization are superior molecular imaging agents in pretargeting strategies as compared with wild-type anxA5.

‣ Cell Fate Determination Factor Dachshund Reprograms Breast Cancer Stem Cell Function*

Wu, Kongming; Jiao, Xuanmao; Li, Zhaoming; Katiyar, Sanjay; Casimiro, Mathew C.; Yang, Wancai; Zhang, Qiong; Willmarth, Nicole E.; Chepelev, Iouri; Crosariol, Marco; Wei, Zhang; Hu, Junbo; Zhao, Keji; Pestell, Richard G.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
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The cell fate determination factor Dachshund was cloned as a dominant inhibitor of the hyperactive epidermal growth factor receptor ellipse. The expression of Dachshund is lost in human breast cancer associated with poor prognosis. Breast tumor-initiating cells (TIC) may contribute to tumor progression and therapy resistance. Here, endogenous DACH1 was reduced in breast cancer cell lines with high expression of TIC markers and in patient samples of the basal breast cancer phenotype. Re-expression of DACH1 reduced new tumor formation in serial transplantations in vivo, reduced mammosphere formation, and reduced the proportion of CD44high/CD24low breast tumor cells. Conversely, lentiviral shRNA to DACH1 increased the breast (B)TIC population. Genome-wide expression studies of mammary tumors demonstrated DACH1 repressed a molecular signature associated with stem cells (SOX2, Nanog, and KLF4) and genome-wide ChIP-seq analysis identified DACH1 binding to the promoter of the Nanog, KLF4, and Lin28 genes. KLF4/c-Myc and Oct4/Sox2 antagonized DACH1 repression of BTIC. Mechanistic studies demonstrated DACH1 directly repressed the Nanog and Sox2 promoters via a conserved domain. Endogenous DACH1 regulates BTIC in vitro and in vivo.

‣ Transducer of Cdc42-dependent Actin Assembly Promotes Epidermal Growth Factor-induced Cell Motility and Invasiveness*

Hu, Jinghui; Mukhopadhyay, Alka; Craig, Andrew W. B.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
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Toca-1 (transducer of Cdc42-dependent actin assembly) interacts with the Cdc42·N-WASP and Abi1·Rac·WAVE F-actin branching pathways that function in lamellipodia formation and cell motility. However, the potential role of Toca-1 in these processes has not been reported. Here, we show that epidermal growth factor (EGF) induces Toca-1 localization to lamellipodia, where it co-localizes with F-actin and Arp2/3 complex in A431 epidermoid carcinoma cells. EGF also induces tyrosine phosphorylation of Toca-1 and interactions with N-WASP and Abi1. Stable knockdown of Toca-1 expression by RNA interference has no effect on cell growth, EGF receptor expression, or internalization. However, Toca-1 knockdown cells display defects in EGF-induced filopodia and lamellipodial protrusions compared with control cells. Further analyses reveal a role for Toca-1 in localization of Arp2/3 and Abi1 to lamellipodia. Toca-1 knockdown cells also display a significant defect in EGF-induced motility and invasiveness. Taken together, these results implicate Toca-1 in coordinating actin assembly within filopodia and lamellipodia to promote EGF-induced cell migration and invasion.

‣ Zymophagy, a Novel Selective Autophagy Pathway Mediated by VMP1-USP9x-p62, Prevents Pancreatic Cell Death*♦

Grasso, Daniel; Ropolo, Alejandro; Lo Ré, Andrea; Boggio, Verónica; Molejón, María I.; Iovanna, Juan L.; Gonzalez, Claudio D.; Urrutia, Raúl; Vaccaro, María I.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
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Autophagy has recently elicited significant attention as a mechanism that either protects or promotes cell death, although different autophagy pathways, and the cellular context in which they occur, remain to be elucidated. We report a thorough cellular and biochemical characterization of a novel selective autophagy that works as a protective cell response. This new selective autophagy is activated in pancreatic acinar cells during pancreatitis-induced vesicular transport alteration to sequester and degrade potentially deleterious activated zymogen granules. We have coined the term “zymophagy” to refer to this process. The autophagy-related protein VMP1, the ubiquitin-protease USP9x, and the ubiquitin-binding protein p62 mediate zymophagy. Moreover, VMP1 interacts with USP9x, indicating that there is a close cooperation between the autophagy pathway and the ubiquitin recognition machinery required for selective autophagosome formation. Zymophagy is activated by experimental pancreatitis in genetically engineered mice and cultured pancreatic acinar cells and by acute pancreatitis in humans. Furthermore, zymophagy has pathophysiological relevance by controlling pancreatitis-induced intracellular zymogen activation and helping to prevent cell death. Together...

‣ α2B-Adrenergic Receptor Interaction with Tubulin Controls Its Transport from the Endoplasmic Reticulum to the Cell Surface*

Duvernay, Matthew T.; Wang, Hong; Dong, Chunmin; Guidry, Jesse J.; Sackett, Dan L.; Wu, Guangyu
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
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It is well recognized that the C terminus (CT) plays a crucial role in modulating G protein-coupled receptor (GPCR) transport from the endoplasmic reticulum (ER) to the cell surface. However the molecular mechanisms that govern CT-dependent ER export remain elusive. To address this issue, we used α2B-adrenergic receptor (α2B-AR) as a model GPCR to search for proteins interacting with the CT. By using peptide-conjugated affinity matrix combined with proteomics and glutathione S-transferase fusion protein pull-down assays, we identified tubulin directly interacting with the α2B-AR CT. The interaction domains were mapped to the acidic CT of tubulin and the basic Arg residues in the α2B-AR CT, particularly Arg-437, Arg-441, and Arg-446. More importantly, mutation of these Arg residues to disrupt tubulin interaction markedly inhibited α2B-AR transport to the cell surface and strongly arrested the receptor in the ER. These data provide the first evidence indicating that the α2B-AR C-terminal Arg cluster mediates its association with tubulin to coordinate its ER-to-cell surface traffic and suggest a novel mechanism of GPCR export through physical contact with microtubules.

‣ Human Corin Isoforms with Different Cytoplasmic Tails That Alter Cell Surface Targeting*

Qi, Xiaofei; Jiang, Jingjing; Zhu, Mingqing; Wu, Qingyu
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
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Corin is a cardiac serine protease that activates natriuretic peptides. It consists of an N-terminal cytoplasmic tail, a transmembrane domain, and an extracellular region with a C-terminal trypsin-like protease domain. The transmembrane domain anchors corin on the surface of cardiomyocytes. To date, the function of the corin cytoplasmic tail remains unknown. By examining the difference between human and mouse corin cytoplasmic tails, analyzing their gene sequences, and verifying mRNA expression in hearts, we show that both human and mouse corin genes have alternative exons encoding different cytoplasmic tails. Human corin isoforms E1 and E1a have 45 and 15 amino acids, respectively, in their cytoplasmic tails. In transfected HEK 293 cells and HL-1 cardiomyocytes, corin isoforms E1 and E1a were expressed at similar levels. Compared with isoform E1a, however, isoform E1 was more active in processing natriuretic peptides. By cell surface labeling, glycosidase digestion, Western blotting, and flow cytometry, we found that corin isoform E1 was activated more readily as a result of more efficient cell surface targeting. By mutagenesis, we identified a DDNN motif in the cytoplasmic tail of isoform E1 (which is absent in isoform E1a) that promotes corin surface targeting in both HEK 293 and HL-1 cells. Our data indicate that the sequence in the cytoplasmic tail plays an important role in corin cell surface targeting and zymogen activation.

‣ Cell-surface Receptor for Complement Component C1q (gC1qR) Is a Key Regulator for Lamellipodia Formation and Cancer Metastasis*♦

Kim, Ki-Bum; Yi, Jae-Sung; Nguyen, Nga; Lee, Joo-Hyung; Kwon, Young-Chan; Ahn, Byung-Yoon; Cho, Hana; Kim, Yoon Ki; Yoo, Hee-Jung; Lee, Jae-Seon; Ko, Young-Gyu
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
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We previously demonstrated that the receptor for the complement component C1q (gC1qR) is a lipid raft protein that is indispensable for adipogenesis and insulin signaling. Here, we provide the first report that gC1qR is an essential component of lamellipodia in human lung carcinoma A549 cells. Cell-surface gC1qR was concentrated in the lamellipodia along with CD44, monosialoganglioside, actin, and phosphorylated focal adhesion kinase in cells stimulated with insulin, IGF-1, EGF, or serum. The growth factor-induced lamellipodia formation and cell migration were significantly decreased in gC1qR-depleted cells, with a concomitant blunt activation of the focal adhesion kinase and the respective receptor tyrosine kinases. Moreover, the gC1qR-depleted cells exhibited a reduced proliferation rate in culture as well as diminished tumorigenic and metastatic activities in grafted mice. We therefore conclude that cell-surface gC1qR regulates lamellipodia formation and metastasis via receptor tyrosine kinase activation.

‣ Differential Effects of Murine and Human Factor X on Adenovirus Transduction via Cell-surface Heparan Sulfate*

Zaiss, Anne K.; Lawrence, Roger; Elashoff, David; Esko, Jeffrey D.; Herschman, Harvey R.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
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Serum coagulation factor X (FX) is proposed to play a major role in adenovirus tropism, promoting transduction by bridging the virus to cell-surface heparan sulfate proteoglycans (HSPGs). Both murine FX and human FX increased transduction by Ad.CMVfLuc, an adenovirus vector, in murine hepatocyte-like cells and human hepatocarcinoma cells. In contrast, only hFX increased transduction of several non-hepatic cancer cell lines and Chinese hamster ovary (CHO) cells. Not only was mFX unable to promote transduction in these cells, it competitively blocked hFX-enhanced transduction. Competition and HSPG digestion experiments suggested mFX- and hFX-enhanced transduction in hepatocyte-derived cells, and hFX-enhanced transduction in epithelial cancer cells were dependent on HSPGs. Ad·hFX-mediated transduction of CHO mutants unable to produce HSPGs was also curtailed. Hepatocyte-derived cells expressed substantially more HSPGs than the cancer cell lines. Dose-response curves and heparin-Sepharose binding suggested Ad·hFX has greater affinity for HSPGs than does Ad·mFX. In coagulation factor-depleted mice hFX also had enhanced ability, compared with mFX, to reconstitute hepatic adenovirus transduction. The results suggest that differences in Ad·hFX and Ad·mFX affinity to HSPGs may result in differences in their ability to enhance adenovirus transduction of many cells. These findings may have implications for murine models of adenovirus vector targeting.

‣ Usp18 Regulates Epidermal Growth Factor (EGF) Receptor Expression and Cancer Cell Survival via MicroRNA-7*

Duex, Jason E.; Comeau, Laurey; Sorkin, Alexander; Purow, Benjamin; Kefas, Benjamin
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
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Epidermal growth factor receptor (EGFR) is involved in development and progression of many human cancers. We have previously demonstrated that the ubiquitin-specific peptidase Usp18 (Ubp43) is a potent regulator of EGFR protein expression. Here we report that the 3′-untranslated region (3′-UTR) of the EGFR message modulates RNA translation following cell treatment with Usp18 siRNA, suggesting microRNA as a possible mediator. Given earlier evidence of EGFR regulation by the microRNA miR-7, we assessed whether miR-7 mediates Usp18 siRNA effects. We found that Usp18 depletion elevates miR-7 levels in several cancer cell lines because of a transcriptional activation and/or mRNA stabilization of miR-7 host genes and that miR-7 acts downstream of Usp18 to regulate EGFR mRNA translation via the 3′-UTR. Also, depletion of Usp18 led to a decrease in protein levels of other known oncogenic targets of miR-7, reduced cell proliferation and soft agar colony formation, and increased apoptosis. Notably, all of these phenotypes were reversed by a specific inhibitor of miR-7. Thus, our findings support a model in which Usp18 inhibition promotes up-regulation of miR-7, which in turn inhibits EGFR expression and the tumorigenic activity of cancer cells.

‣ Histone Deacetylase Inhibitor Belinostat Represses Survivin Expression through Reactivation of Transforming Growth Factor β (TGFβ) Receptor II Leading to Cancer Cell Death*

Chowdhury, Sanjib; Howell, Gillian M.; Teggart, Carol A.; Chowdhury, Aparajita; Person, Jonathan J.; Bowers, Dawn M.; Brattain, Michael G.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
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Survivin is a cancer-associated gene that functions to promote cell survival, cell division, and angiogenesis and is a marker of poor prognosis. Histone deacetylase inhibitors induce apoptosis and re-expression of epigenetically silenced tumor suppressor genes in cancer cells. In association with increased expression of the tumor suppressor gene transforming growth factor β receptor II (TGFβRII) induced by the histone deacetylase inhibitor belinostat, we observed repressed survivin expression. We investigated the molecular mechanisms involved in survivin down-regulation by belinostat downstream of reactivation of TGFβ signaling. We identified two mechanisms. At early time points, survivin protein half-life was decreased with its proteasomal degradation. We observed that belinostat activated protein kinase A at early time points in a TGFβ signaling-dependent mechanism. After longer times (48 h), survivin mRNA was also decreased by belinostat. We made the novel observation that belinostat mediated cell death through the TGFβ/protein kinase A signaling pathway. Induction of TGFβRII with concomitant survivin repression may represent a significant mechanism in the anticancer effects of this drug. Therefore, patient populations exhibiting high survivin expression with epigenetically silenced TGFβRII might potentially benefit from the use of this histone deacetylase inhibitor.

‣ Homeodomain-interacting Protein Kinase-2 Stabilizes p27kip1 by Its Phosphorylation at Serine 10 and Contributes to Cell Motility*

Pierantoni, Giovanna Maria; Esposito, Francesco; Tornincasa, Mara; Rinaldo, Cinzia; Viglietto, Giuseppe; Soddu, Silvia; Fusco, Alfredo
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
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HIPK2 is a serine/threonine kinase that acts as a coregulator of an increasing number of factors involved in cell survival and proliferation during development and in response to different types of stress. Here we report on a novel target of HIPK2, the cyclin-dependent kinase inhibitor p27kip1. HIPK2 phosphorylates p27kip1 in vitro and in vivo at serine 10, an event that accounts for 80% of the total p27kip1 phosphorylation and plays a crucial role in the stability of the protein. Indeed, HIPK2 depletion by transient or stable RNA interference in tumor cells of different origin was consistently associated with strong reduction of p27kip1 phosphorylation at serine 10 and of p27kip1 stability. An initial evaluation of the functional relevance of this HIPK2-mediated regulation of p27kip1 revealed a contribution to cell motility, rather than to cell proliferation, but only in cells that do not express wild-type p53.