Página 28 dos resultados de 45945 itens digitais encontrados em 0.038 segundos

‣ Biophysical Properties of Cadherin Bonds Do Not Predict Cell Sorting*S⃞

Shi, Quanming; Chien, Yuan-Hung; Leckband, Deborah
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Publicado em 17/10/2008 Português
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Differential binding between cadherin subtypes is widely believed to mediate cell sorting during embryogenesis. However, a fundamental unanswered question is whether cell sorting is dictated by the biophysical properties of cadherin bonds, or by broader, cadherin-dependent differences in intercellular adhesion or membrane tension. This report describes atomic force microscope measurements of the strengths and dissociation rates of homophilic and heterophilic cadherin (CAD) bonds. Measurements conducted with chicken N-CAD, canine E-CAD, and Xenopus C-CAD demonstrated that all three cadherins cross-react and form multiple, intermolecular bonds. The mechanical and kinetic properties of the heterophilic bonds are similar to the homophilic interactions. The thus quantified bond parameters, together with previously reported adhesion energies were further compared with in vitro cell aggregation and sorting assays, which are thought to mimic in vivo cell sorting. Trends in quantified biophysical properties of the different cadherin bonds do not correlate with sorting outcomes. These results suggest that cell sorting in vivo and in vitro is not governed solely by biophysical differences between cadherin subtypes.

‣ Ephrin-B2-induced Cleavage of EphB2 Receptor Is Mediated by Matrix Metalloproteinases to Trigger Cell Repulsion*S⃞

Lin, Kai-Ti; Sloniowski, Slawomir; Ethell, Douglas W.; Ethell, Iryna M.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Publicado em 24/10/2008 Português
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EphB receptors provide crucial adhesive and repulsive signals during cell migration and axon guidance, but it is unclear how they switch between these opposing responses. Here we provide evidence of an important role for matrix metalloproteinases (MMPs) in repulsive EphB2 signaling. We found that EphB2 is cleaved by MMPs both in vitro and in vivo, and that this cleavage is induced by interaction with its ligand ephrin-B2. Our findings demonstrate that MMP-2/MMP-9-specific inhibition or cleavage-resistant mutations in the ectodomain of EphB2 can prevent EphB2-mediated cell-cell repulsion in HEK293 cells, and block ephrin-B1-induced growth cone withdrawal in cultured hippocampal neurons. Transient expression of wtEphB2, but not noncleavable EphB2–4/5 mutant, restored ephrin-B1-induced growth cone collapse and withdrawal in EphB-deficient neurons. The inhibition of EphB2 cleavage also had potent regulatory effects on EphB2 activity. This study provides the first evidence that MMP-mediated cleavage of EphB2 is induced by receptor-ligand interactions at the cell surface and that this event triggers cell-repulsive responses.

‣ Identification of Aurora-A as a Direct Target of E2F3 during G2/M Cell Cycle Progression*

He, Lili; Yang, Hua; Ma, Yihong; Pledger, W. Jack; Cress, W. Douglas; Cheng, Jin Q.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Publicado em 07/11/2008 Português
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Aurora-A is a centrosome kinase and plays a pivotal role in G2/M cell cycle progression. Expression of Aurora-A is cell cycle-dependent. Levels of Aurora-A mRNA and protein are low in G1/S, accumulate during G2/M, and decrease rapidly after mitosis. Previous studies have shown regulation of the Aurora-A protein level during the cell cycle through the ubiquitin-proteasome pathway. However, the mechanism of transcriptional regulation of Aurora-A remains largely unknown. Here, we demonstrated that E2F3 modulates Aurora-A mRNA expression during the cell cycle. Ectopic expression of E2F3 induces Aurora-A expression. Stable knockdown of E2F3 decreases mRNA and protein levels of Aurora-A and delays G2/M entry. Further, E2F3 directly binds to Aurora-A promoter and stimulates the promoter activity. Deletion and mutation analyses of the Aurora-A promoter revealed that a region located 96-bp upstream of the transcription initiation site is critical for the activation of the promoter by E2F3. In addition, expression of E2F3 positively correlates with the protein level of Aurora-A in human ovarian cancer examined. These results indicate for the first time that Aurora-A is transcriptionally regulated by E2F3 during the cell cycle and that E2F3 is a causal factor for up-regulation of Aurora-A in a subset of human ovarian cancer. Thus...

‣ Transforming Growth Factor-β/Smad3 Signaling Regulates Insulin Gene Transcription and Pancreatic Islet β-Cell Function*S⃞

Lin, Huei-Min; Lee, Ji-Hyeon; Yadav, Hariom; Kamaraju, Anil K.; Liu, Eric; Zhigang, Duan; Vieira, Anthony; Kim, Seong-Jin; Collins, Heather; Matschinsky, Franz; Harlan, David M.; Roberts, Anita B.; Rane, Sushil G.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Publicado em 01/05/2009 Português
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Pancreatic islet β-cell dysfunction is a signature feature of Type 2 diabetes pathogenesis. Consequently, knowledge of signals that regulate β-cell function is of immense clinical relevance. Transforming growth factor (TGF)-β signaling plays a critical role in pancreatic development although the role of this pathway in the adult pancreas is obscure. Here, we define an important role of the TGF-β pathway in regulation of insulin gene transcription and β-cell function. We identify insulin as a TGF-β target gene and show that the TGF-β signaling effector Smad3 occupies the insulin gene promoter and represses insulin gene transcription. In contrast, Smad3 small interfering RNAs relieve insulin transcriptional repression and enhance insulin levels. Transduction of adenoviral Smad3 into primary human and non-human primate islets suppresses insulin content, whereas, dominant-negative Smad3 enhances insulin levels. Consistent with this, Smad3-deficient mice exhibit moderate hyperinsulinemia and mild hypoglycemia. Moreover, Smad3 deficiency results in improved glucose tolerance and enhanced glucose-stimulated insulin secretion in vivo. In ex vivo perifusion assays, Smad3-deficient islets exhibit improved glucose-stimulated insulin release. Interestingly...

‣ Sorting Nexin 33 Induces Mammalian Cell Micronucleated Phenotype and Actin Polymerization by Interacting with Wiskott-Aldrich Syndrome Protein*

Zhang, Juan; Zhang, Xiaofei; Guo, Yunqian; Xu, Liangliang; Pei, Duanqing
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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Sorting nexin 33 (SNX33) is a novel member of the sorting nexin superfamily with three predicted structural domains, SH3-PX-BAR. Very little is known about the cellular function of SNX33. In an effort to analyze its structure/function relationship, we attempted but failed to generate stable cell lines for short hairpin RNA or overexpression SNX33. Transient knockdown of SNX33 induces both HeLa and MCF7 cells to grow multiple long processes, delay the G1/M transition, and become more apoptotic, implying that SNX33 may control cell cycle process through influence the cytoskeleton. In vitro cell lineage analysis revealed that cells transfected with SNX33 failed to divide and became micronucleated, suggesting a specific defect in cytokinesis. Further analysis revealed that SNX33 induced the accumulation of actin at the perinuclear space, which might have disabled the cytokinetic machinery. However, SNX33 appears to mediate actin polymerization indirectly, as they do not interact with each other. SNX33 interacts with itself and SNX9. Interestingly, it also interacts with VCA domain of Wiskott-Aldrich syndrome protein (WASp), a protein known to be involved in actin polymerization. Indeed, cells overexpressing WASp failed to divide and form stable colonies as SNX33...

‣ Characterization of Nuclear Localization Signal in the N Terminus of CUL4B and Its Essential Role in Cyclin E Degradation and Cell Cycle Progression*

Zou, Yongxin; Mi, Jun; Cui, Jinpeng; Lu, Defen; Zhang, Xiyu; Guo, Chenhong; Gao, Guimin; Liu, Qiji; Chen, Bingxi; Shao, Changshun; Gong, Yaoqin
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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CUL4A and CUL4B, which are derived from the same ancestor, CUL4, encode scaffold proteins that organize cullin-RING ubiquitin ligase (E3) complexes. Recent genetic studies have shown that germ line mutation in CUL4B can cause mental retardation, short stature, and other abnormalities in humans. CUL4A was observed to be overexpressed in breast and hepatocellular cancers, although no germ line mutation in human CUL4A has been reported. Although CUL4A has been known to be involved in a number of cellular processes, including DNA repair and cell cycle regulation, little is known about whether CUL4B has similar functions. In this report, we tested the functional importance of CUL4B in cell proliferation and characterized the nuclear localization signal (NLS) that is essential for its function. We found that RNA interference silencing of CUL4B led to an inhibition of cell proliferation and a prolonged S phase, due to the overaccumulation of cyclin E, a substrate targeted by CUL4B for ubiquitination. We showed that, unlike CUL4A and other cullins that carry their NLS in their C termini, NLS in CUL4B is located in its N terminus, between amino acid 37 and 40, KKRK. This NLS could bind to importin α1, α3, and α5. NLS-deleted CUL4B was distributed in cytoplasm and failed to promote cell proliferation. Therefore...

‣ Pleiotrophin Regulates Lung Epithelial Cell Proliferation and Differentiation during Fetal Lung Development via β-Catenin and Dlk1*

Weng, Tingting; Gao, Li; Bhaskaran, Manoj; Guo, Yujie; Gou, Deming; Narayanaperumal, Jeyaparthasarathy; Chintagari, Narendranath Reddy; Zhang, Kexiong; Liu, Lin
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 role of pleiotrophin in fetal lung development was investigated. We found that pleiotrophin and its receptor, protein-tyrosine phosphatase receptor β/ζ, were highly expressed in mesenchymal and epithelial cells of the fetal lungs, respectively. Using isolated fetal alveolar epithelial type II cells, we demonstrated that pleiotrophin promoted fetal type II cell proliferation and arrested type II cell trans-differentiation into alveolar epithelial type I cells. Pleiotrophin also increased wound healing of injured type II cell monolayer. Knockdown of pleiotrophin influenced lung branching morphogenesis in a fetal lung organ culture model. Pleiotrophin increased the tyrosine phosphorylation of β-catenin, promoted β-catenin translocation into the nucleus, and activated T cell factor/lymphoid enhancer factor transcription factors. Dlk1, a membrane ligand that initiates the Notch signaling pathway, was identified as a downstream target of the pleiotrophin/β-catenin pathway by endogenous dlk1 expression, promoter assay, and chromatin immunoprecipitation. These results provide evidence that pleiotrophin regulates fetal type II cell proliferation and differentiation via integration of multiple signaling pathways including pleiotrophin...

‣ Ectodomain Shedding of Interleukin-2 Receptor β and Generation of an Intracellular Functional Fragment*

de Oca B., Pavel Montes; Malardé, Valerie; Proust, Richard; Dautry-Varsat, Alice; Gesbert, Franck
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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Interleukin-2 (IL-2) regulates different functions of various lymphoid cell subsets. These are mediated by its binding to the IL-2 receptor (IL-2R) composed of three subunits (IL2-Rα, -β, and -γc). IL-2Rβ is responsible for the activation of several signaling pathways. Ectodomain shedding of membrane receptors is thought to be an important mechanism for down-regulation of cell surface receptor abundance but is also emerging as a mechanism that cell membrane-associated molecules require for proper action in vivo. Here, we demonstrate that IL-2Rβ is cleaved in cell lines of different origin, including T cells, generating an intracellular 37-kDa fragment (37βic) that comprises the full intracellular C-terminal and transmembrane domains. Ectodomain shedding of IL-2Rβ decreases in a mutant deleted of the juxtamembrane region, where cleavage is predicted to occur, and is inhibited by tissue inhibitor of metalloproteases-3. 37βic is tyrosine-phosphorylated and associates with STAT-5, a canonic signal transducer of IL-2R. Finally, lymphoid cell transfection with a truncated form of IL-2Rβ mimicking 37βic increases their proliferation. These data indicate that IL-2Rβ is subject to ectodomain shedding generating an intracellular fragment biologically functional...

‣ Low Density Lipoprotein Receptor-related Protein-1 (LRP1) Regulates Thrombospondin-2 (TSP2) Enhancement of Notch3 Signaling*

Meng, He; Zhang, Xiaojie; Lee, Soo Jung; Strickland, Dudley K.; Lawrence, Daniel A.; Wang, Michael 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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Intracellular trafficking of Notch and Notch ligands modulates signaling, suggesting that choreography of ligand and receptor translocation is essential for optimal Notch activity. Indeed, a major model for Notch signaling posits that Notch trans-endocytosis into the ligand-expressing (signal sending) cell is a key driving force for Notch signal transduction. The extracellular protein thrombospondin-2 (TSP2) enhances Notch signaling and binds to both Jagged1 and Notch3 ectodomains, potentially bridging two essential extracellular components of Notch signaling. We investigated the role of low density lipoprotein receptor-related protein-1 (LRP1), a TSP2 receptor, in the regulation of Notch3 signaling. TSP2 potentiation of Notch is blocked by the receptor-associated protein (an inhibitor of low density lipoprotein receptor-related protein function) and requires LRP1 expression in the signal-sending cell. TSP2 stimulates Notch3 endocytosis into wild type fibroblasts but not LRP1-deficient fibroblasts. Finally, recombinant Notch3 and Jagged1 interact with the LRP1 85-kDa B-chain, a subunit that lacks known ligand binding function. Our data suggest that LRP1 and TSP2 stimulate Notch activity by driving trans-endocytosis of the Notch ectodomain into the signal-sending cell and demonstrate a novel...

‣ Target of Rapamycin (TOR)-like 1 Kinase Is Involved in the Control of Polyphosphate Levels and Acidocalcisome Maintenance in Trypanosoma brucei*

de Jesus, Teresa Cristina Leandro; Tonelli, Renata Rosito; Nardelli, Sheila C.; da Silva Augusto, Leonardo; Motta, Maria Cristina M.; Girard-Dias, Wendell; Miranda, Kildare; Ulrich, Paul; Jimenez, Veronica; Barquilla, Antonio; Navarro, Miguel; Docampo, Ro
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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Target of rapamycin (TOR) kinases are highly conserved protein kinases that integrate signals from nutrients and growth factors to coordinate cell growth and cell cycle progression. It has been previously described that two TOR kinases control cell growth in the protozoan parasite Trypanosoma brucei, the causative agent of African trypanosomiasis. Here we studied an unusual TOR-like protein named TbTOR-like 1 containing a PDZ domain and found exclusively in kinetoplastids. TbTOR-like 1 localizes to unique cytosolic granules. After hyperosmotic stress, the localization of the protein shifts to the cell periphery, different from other organelle markers. Ablation of TbTOR-like 1 causes a progressive inhibition of cell proliferation, producing parasites accumulating in the S/G2 phase of the cell cycle. TbTOR-like 1 knocked down cells have an increased area occupied by acidic vacuoles, known as acidocalcisomes, and are enriched in polyphosphate and pyrophosphate. These results suggest that TbTOR-like 1 might be involved in the control of acidocalcisome and polyphosphate metabolism in T. brucei.

‣ The Bcl-2 Homology Domain 3 Mimetic Gossypol Induces Both Beclin 1-dependent and Beclin 1-independent Cytoprotective Autophagy in Cancer Cells*

Gao, Ping; Bauvy, Chantal; Souquère, Sylvie; Tonelli, Giovanni; Liu, Lei; Zhu, Yushan; Qiao, Zhenzhen; Bakula, Daniela; Proikas-Cezanne, Tassula; Pierron, Gérard; Codogno, Patrice; Chen, Quan; Mehrpour, Maryam
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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Gossypol, a natural Bcl-2 homology domain 3 mimetic compound isolated from cottonseeds, is currently being evaluated in clinical trials. Here, we provide evidence that gossypol induces autophagy followed by apoptotic cell death in both the MCF-7 human breast adenocarcinoma and HeLa cell lines. We first show that knockdown of the Bcl-2 homology domain 3-only protein Beclin 1 reduces gossypol-induced autophagy in MCF-7 cells, but not in HeLa cells. Gossypol inhibits the interaction between Beclin 1 and Bcl-2 (B-cell leukemia/lymphoma 2), antagonizes the inhibition of autophagy by Bcl-2, and hence stimulates autophagy. We then show that knockdown of Vps34 reduces gossypol-induced autophagy in both cell lines, and consistent with this, the phosphatidylinositol 3-phosphate-binding protein WIPI-1 is recruited to autophagosomal membranes. Further, Atg5 knockdown also reduces gossypol-mediated autophagy. We conclude that gossypol induces autophagy in both a canonical and a noncanonical manner. Notably, we found that gossypol-mediated apoptotic cell death was potentiated by treatment with the autophagy inhibitor wortmannin or with small interfering RNA against essential autophagy genes (Vps34, Beclin 1, and Atg5). Our findings support the notion that gossypol-induced autophagy is cytoprotective and not part of the cell death process induced by this compound.

‣ d-Maurocalcine, a Pharmacologically Inert Efficient Cell-penetrating Peptide Analogue*

Poillot, Cathy; Dridi, Kaouthar; Bichraoui, Hicham; Pêcher, Julien; Alphonse, Sebastien; Douzi, Badreddine; Ronjat, Michel; Darbon, Hervé; De Waard, Michel
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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Maurocalcine has been the first demonstrated animal toxin acting as a cell-penetrating peptide. Although it possesses competitive advantages, its use as a cell-penetrating peptide (CPP) requires that analogues be developed that lack its characteristic pharmacological activity on ryanodine-sensitive calcium channels without affecting its cell-penetrating and vector efficiencies. Here, we present the synthesis, three-dimensional 1H NMR structure, and activity of d-maurocalcine. We demonstrate that it possesses all of the desired features for an excellent CPP: preserved structure, lack of pharmacological action, conserved vector properties, and absence of cell toxicity. This is the first report of a folded/oxidized animal toxin in its d-diastereomer conformation for use as a CPP. The protease resistance of this new peptide analogue, combined with its efficient cell penetration at concentrations devoid of cell toxicity, suggests that d-maurocalcine should be an excellent vector for in vivo applications.

‣ Activation of AMP-activated Protein Kinase by Temozolomide Contributes to Apoptosis in Glioblastoma Cells via p53 Activation and mTORC1 Inhibition*

Zhang, Wen-bin; Wang, Zhuo; Shu, Fei; Jin, Yong-hua; Liu, Hong-yi; Wang, Qiu-juan; Yang, Yong
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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Methylating drugs such as temozolomide (TMZ) are widely used in the treatment of brain tumors including malignant glioblastoma. The mechanism of TMZ-induced glioblastoma cell death and apoptosis, however, is not fully understood. Here, we tested the potential involvement of AMP-activated protein kinase (AMPK) in this process. We found that methylating agents TMZ and N-methyl-N′-nitro-N-nitrosoguanidine induce AMPK activation in primary cultured human glioblastoma and glioblastoma cell lines. TMZ-induced O6-methylguanine production is involved in AMPK activation. O6-benzylguanine, an O6-methylguanine-DNA methyltransferase inhibitor, enhances TMZ-induced O6-methylguanine production, leading to enhanced reactive oxygen species production, which serves as an upstream signal for AMPK activation. Activation of AMPK is involved in TMZ-induced glioblastoma cell death and apoptosis. AMPK inhibitor (Compound C) or AMPKα siRNA knockdown inhibits TMZ-induced glioblastoma cell death and apoptosis, whereas AMPK activator 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside enhances it. In further studies, we found that activation of AMPK is involved in TMZ-induced p53 activation and subsequent p21, Noxa, and Bax up-regulation. Activation of AMPK by TMZ also inhibits mTOR complex 1 (mTORC1) signaling and promotes anti-apoptosis protein Bcl-2 down-regulation...

‣ Post-transcriptional Control of Na,K-ATPase Activity and Cell Growth by a Splice Variant of FXYD2 Protein with Modified mRNA*

Sweadner, Kathleen J.; Pascoa, Jennifer L.; Salazar, Cynthia A.; Arystarkhova, Elena
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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In kidney, FXYD proteins regulate Na,K-ATPase in a nephron segment-specific way. FXYD2 is the most abundant renal FXYD but is not expressed in most renal cell lines unless induced by hypertonicity. Expression by transfection of FXYD2a or FXYD2b splice variants in NRK-52E cells reduces the apparent Na+ affinity of the Na,K-ATPase and slows the cell proliferation rate. Based on RT-PCR, mRNAs for both splice variants were expressed in wild type NRK-52E cells as low abundance species. DNA sequencing of the PCR products revealed a base alteration from C to T in FXYD2b but not FXYD2a from both untreated and hypertonicity-treated NRK-52E cells. The 172C→T sequence change exposed a cryptic KKXX endoplasmic reticulum retrieval signal via a premature stop codon. The truncation affected trafficking of FXYD2b and its association with Na,K-ATPase and blocked its effect on enzyme kinetics and cell growth. The data may be explained by altered splicing or selective RNA editing of FXYD2b, a supplementary process that would ensure that it was inactive even if transcribed and translated, in these cells that normally express only FXYD2a. 172C→T mutation was also identified after mutagenesis of FXYD2b by error-prone PCR coupled with a selection for cell proliferation. Furthermore...

‣ Modeling Reveals That Dynamic Regulation of c-FLIP Levels Determines Cell-to-Cell Distribution of CD95-mediated Apoptosis*

Toivonen, Hannu T.; Meinander, Annika; Asaoka, Tomoko; Westerlund, Mia; Pettersson, Frank; Mikhailov, Andrey; Eriksson, John E.; Saxén, Henrik
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 expression levels of caspase-8 inhibitory c-FLIP proteins play an important role in regulating death receptor-mediated apoptosis, as their concentration at the moment when the death-inducing signaling complex (DISC) is formed determines the outcome of the DISC signal. Experimental studies have shown that c-FLIP proteins are subject to dynamic turnover and that their stability and expression levels can be rapidly altered. Even though the influence of c-FLIP on the apoptotic behavior of a single cell has been captured in mathematical simulation studies, the effect of c-FLIP turnover and stability has not been investigated. In this study, a mathematical model of apoptosis was developed to analyze how the dynamic turnover and stability of the c-FLIP isoforms regulate apoptotic signaling for both individual cells and cell populations. Intercellular parameter and concentration distributions were used to describe the behavior of cell populations. Monte-Carlo simulations of cell populations showed that c-FLIP turnover is a key determinant of death receptor responses. The fact that the developed model simulates the state of whole cell populations makes it possible to validate it by comparison with empirical data. The proposed modeling approach can be used to further determine limiting factors in the DISC signaling process.

‣ Regulation of Purinergic Signaling in Biliary Epithelial Cells by Exocytosis of SLC17A9-dependent ATP-enriched Vesicles*

Sathe, Meghana N.; Woo, Kangmee; Kresge, Charles; Bugde, Abhijit; Luby-Phelps, Kate; Lewis, Matthew A.; Feranchak, Andrew P.
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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ATP in bile is a potent secretogogue, stimulating biliary epithelial cell (BEC) secretion through binding apical purinergic receptors. In response to mechanosensitive stimuli, BECs release ATP into bile, although the cellular basis of ATP release is unknown. The aims of this study in human and mouse BECs were to determine whether ATP release occurs via exocytosis of ATP-enriched vesicles and to elucidate the potential role of the vesicular nucleotide transporter SLC17A9 in purinergic signaling. Dynamic, multiscale, live cell imaging (confocal and total internal reflection fluorescence microscopy and a luminescence detection system with a high sensitivity charge-coupled device camera) was utilized to detect vesicular ATP release from cell populations, single cells, and the submembrane space of a single cell. In response to increases in cell volume, BECs release ATP, which was dependent on intact microtubules and vesicular trafficking pathways. ATP release occurred as stochastic point source bursts of luminescence consistent with exocytic events. Parallel studies identified ATP-enriched vesicles ranging in size from 0.4 to 1 μm that underwent fusion and release in response to increases in cell volume in a protein kinase C-dependent manner. Present in all models...

‣ Ultraviolet Irradiation Can Induce Evasion of Colon Cancer Cells from Stimulation of Epidermal Growth Factor*

Adachi, Seiji; Yasuda, Ichiro; Nakashima, Masanori; Yamauchi, Takahiro; Kawaguchi, Junji; Shimizu, Masahito; Itani, Masahiko; Nakamura, Momoko; Nishii, Yumi; Yoshioka, Takashi; Hirose, Yoshinobu; Okano, Yukio; Moriwaki, Hisataka; Kozawa, Osamu
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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Receptor down-regulation is the most prominent regulatory system of EGF receptor (EGFR) signal attenuation and a critical target for therapy against colon cancer, which is highly dependent on the function of the EGFR. In this study, we investigated the effect of ultraviolet-C (UV-C) on down-regulation of EGFR in human colon cancer cells (SW480, HT29, and DLD-1). UV-C caused inhibition of cell survival and proliferation, concurrently inducing the decrease in cell surface EGFR and subsequently its degradation. UV-C, as well as EGFR kinase inhibitors, decreased the expression level of cyclin D1 and the phosphorylated level of retinoblastoma, indicating that EGFR down-regulation is correlated to cell cycle arrest. Although UV-C caused a marked phosphorylation of EGFR at Ser-1046/1047, UV-C also induced activation of p38 MAPK, a stress-inducible kinase believed to negatively regulate tumorigenesis, and the inhibition of p38 MAPK canceled EGFR phosphorylation at Ser-1046/1047, as well as subsequent internalization and degradation, suggesting that p38 MAPK mediates EGFR down-regulation by UV-C. In addition, phosphorylation of p38 MAPK induced by UV-C was mediated through transforming growth factor-β-activated kinase-1. Moreover, pretreatment of the cells with UV-C suppressed EGF-induced phosphorylation of EGFR at tyrosine residues in addition to cell survival signal...

‣ Dual Processing of FAT1 Cadherin Protein by Human Melanoma Cells Generates Distinct Protein Products*

Sadeqzadeh, Elham; de Bock, Charles E.; Zhang, Xu Dong; Shipman, Kristy L.; Scott, Naomi M.; Song, Chaojun; Yeadon, Trina; Oliveira, Camila S.; Jin, Boquan; Hersey, Peter; Boyd, Andrew W.; Burns, Gordon F.; Thorne, Rick F.
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 giant cadherin FAT1 is one of four vertebrate orthologues of the Drosophila tumor suppressor fat. It engages in several functions, including cell polarity and migration, and in Hippo signaling during development. Homozygous deletions in oral cancer suggest that FAT1 may play a tumor suppressor role, although overexpression of FAT1 has been reported in some other cancers. Here we show using Northern blotting that human melanoma cell lines variably but universally express FAT1 and less commonly FAT2, FAT3, and FAT4. Both normal melanocytes and keratinocytes also express comparable FAT1 mRNA relative to melanoma cells. Analysis of the protein processing of FAT1 in keratinocytes revealed that, like Drosophila FAT, human FAT1 is cleaved into a non-covalent heterodimer before achieving cell surface expression. The use of inhibitors also established that such cleavage requires the proprotein convertase furin. However, in melanoma cells, the non-cleaved proform of FAT1 is also expressed at the cell surface together with the furin-cleaved heterodimer. Moreover, furin-independent processing generates a potentially functional proteolytic product in melanoma cells, a persistent 65-kDa membrane-bound cytoplasmic fragment no longer in association with the extracellular fragment. In vitro localization studies of FAT1 showed that melanoma cells display high levels of cytosolic FAT1 protein...

‣ Inhibitor of Cyclin-dependent Kinase (CDK) Interacting with Cyclin A1 (INCA1) Regulates Proliferation and Is Repressed by Oncogenic Signaling*

Bäumer, Nicole; Tickenbrock, Lara; Tschanter, Petra; Lohmeyer, Lisa; Diederichs, Sven; Bäumer, Sebastian; Skryabin, Boris V.; Zhang, Feng; Agrawal-Singh, Shuchi; Köhler, Gabriele; Berdel, Wolfgang E.; Serve, Hubert; Koschmieder, Steffen; Müller-Tidow,
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 cycle is driven by the kinase activity of cyclin·cyclin-dependent kinase (CDK) complexes, which is negatively regulated by CDK inhibitor proteins. Recently, we identified INCA1 as an interaction partner and a substrate of cyclin A1 in complex with CDK2. On a functional level, we identified a novel cyclin-binding site in the INCA1 protein. INCA1 inhibited CDK2 activity and cell proliferation. The inhibitory effects depended on the cyclin-interacting domain. Mitogenic and oncogenic signals suppressed INCA1 expression, whereas it was induced by cell cycle arrest. We established a deletional mouse model that showed increased CDK2 activity in spleen with altered spleen architecture in Inca1−/− mice. Inca1−/− embryonic fibroblasts showed an increase in the fraction of S-phase cells. Furthermore, blasts from acute lymphoid leukemia and acute myeloid leukemia patients expressed significantly reduced INCA1 levels highlighting its relevance for growth control in vivo. Taken together, this study identifies a novel CDK inhibitor with reduced expression in acute myeloid and lymphoid leukemia. The molecular events that control the cell cycle occur in a sequential process to ensure a tight regulation, which is important for the survival of a cell and includes the detection and repair of genetic damage and the prevention of uncontrolled cell division.

‣ Dynamic Protein Pathway Activation Mapping of Adipose-Derived Stem Cell Differentiation Implicates Novel Regulators of Adipocyte Differentiation

Wilson, Bridget; Liotta, Lance A.; PetricoinIII, Emanuel
Fonte: The American Society for Biochemistry and Molecular Biology Publicador: The American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
448.3889%
Next to embryonic stem cell research, adult stem cell research is providing a promising alternative for enhanced tissue regeneration and transplantation. The key biochemical networks controlling the differentiation processes regulating stem cell biology remain largely disputed and or undefined, contributing to a lack of knowledge of the principle phosphoregulatory events propagating signal transduction. To effectively monitor these events relative to adipocyte differentiation, this study utilized a high throughput reverse phase protein microarray platform and characterized adult adipose-derived stem cell (ASC) differentiation through the monitoring of ∼100 phosphospecific endpoints with 33 distinct time points examined across 14 days. This kinetic-based analysis showed time ordered signal transduction ultimately implicating pathways correlated with adipogenic differentiation. To further validate the causal significance of these network activations, pharmacological targeting was implemented to include the chemical inhibitors MAPK inhibitor PD169316, rapamycin, and HNMPA-(AM)3 yielding partial or complete disruption of adipocytic differentiation, as noted by a decrease or lack of lipid formation within the mature adipocytes. Based on this analysis...