Structural and lipid-binding characterization of human annexin A13a reveals strong differences with its long A13b isoform

2017 ◽  
Vol 398 (3) ◽  
pp. 359-371 ◽  
Author(s):  
Sara Fernández-Lizarbe ◽  
Emilio Lecona ◽  
Angélica Santiago-Gómez ◽  
Nieves Olmo ◽  
María Antonia Lizarbe ◽  
...  
Keyword(s):  
Calcium Binding ◽  
Structural Characteristics ◽  
Colon Adenocarcinoma ◽  
Human Colon ◽  
Lipid Binding ◽  
Tryptophan Residue ◽  
Dependent Manner ◽  
Calcium Dependent ◽  
Protein Levels ◽  
Acidic Phospholipids

Abstract Annexin A13 is the founder member of the vertebrate family of annexins, which are comprised of a tetrad of unique conserved domains responsible for calcium-dependent binding to membranes. Its expression is restricted to epithelial intestinal and kidney cells. Alternative splicing in the N-terminal region generates two isoforms, A13a and A13b, differing in a deletion of 41 residues in the former. We have confirmed the expression of both isoforms in human colon adenocarcinoma cells at the mRNA and protein levels. We have cloned, expressed, and purified human annexin A13a for the first time to analyze its structural characteristics. Its secondary structure and thermal stability differs greatly from the A13b isoform. The only tryptophan residue (Trp186) is buried in the protein core in the absence of calcium but is exposed to the solvent after calcium binding even though circular dichroism spectra are quite similar. Non-myristoylated annexin A13a binds in a calcium-dependent manner to acidic phospholipids but not to neutral or raft-like liposomes. Calcium requirements for binding to phosphatidylserine are around 6-fold lower than those required by the A13b isoform. This fact could account for the different subcellular localization of both annexins as binding to basolateral membranes seems to be calcium-dependent and myristoylation-independent.

scholarly journals Functional assessment of perforin C2 domain mutations illustrates the critical role for calcium-dependent lipid binding in perforin cytotoxic function

Blood ◽  
2009 ◽  
Vol 113 (2) ◽  
pp. 338-346 ◽  
Author(s):  
Ramon Urrea Moreno ◽  
Juana Gil ◽  
Carmen Rodriguez-Sainz ◽  
Elena Cela ◽  
Victor LaFay ◽  
...  
Keyword(s):  
Calcium Binding ◽  
Critical Role ◽  
Lipid Binding ◽  
Inflammatory Disorder ◽  
C2 Domain ◽  
Biophysical Modeling ◽  
Binding Assays ◽  
Calcium Dependent ◽  
Pathogen Elimination ◽  
Cytotoxic Function

Abstract Perforin-mediated lymphocyte cytotoxicity is critical for pathogen elimination and immune homeostasis. Perforin disruption of target cell membranes is hypothesized to require binding of a calcium-dependent, lipid-inserting, C2 domain. In a family affected by hemophagocytic lymphohistiocytosis, a severe inflammatory disorder caused by perforin deficiency, we identified 2 amino acid substitutions in the perforin C2 domain: T435M, a previously identified mutant with disputed pathogenicity, and Y438C, a novel substitution. Using biophysical modeling, we predicted that the T435M substitution, but not Y438C, would interfere with calcium binding and thus cytotoxic function. The capacity for cytotoxic function was tested after expression of the variant perforins in rat basophilic leukemia cells and murine cytotoxic T lymphocytes. As predicted, cells transduced with perforin-T435M lacked cytotoxicity, but those expressing perforin-Y438C displayed intact cytotoxic function. Using novel antibody-capture and liposome-binding assays, we found that both mutant perforins were secreted; however, only nonmutated and Y438C-substituted perforins were capable of calcium-dependent lipid binding. In addition, we found that perforin-Y438C was capable of mediating cytotoxicity without apparent proteolytic maturation. This study clearly demonstrates the pathogenicity of the T435M mutation and illustrates, for the first time, the critical role of the human perforin C2 domain for calcium-dependent, cytotoxic function.

scholarly journals Safed Musli (Chlorophytum borivilianum L.) Callus-Mediated Biosynthesis of Silver Nanoparticles and Evaluation of their Antimicrobial Activity and Cytotoxicity against Human Colon Cancer Cells

2019 ◽  
Vol 2019 ◽  
pp. 1-8 ◽  
Author(s):  
Fengchang Huang ◽  
Yaxin Long ◽  
Qingqing Liang ◽  
Boregowda Purushotham ◽  
Mallappa Kumara Swamy ◽  
...  
Keyword(s):  
Biomedical Applications ◽  
Colon Adenocarcinoma ◽  
Human Colon ◽  
Dependent Manner ◽  
Face Centered Cubic ◽  
Human Colon Cancer ◽  
Chlorophytum Borivilianum ◽  
The Face ◽  
Average Size ◽  
Safed Musli

With the advancement of nanobiotechnology, eco-friendly approaches of plant-mediated silver nanomaterial (AgNP) biosynthesis have become more attractive for biomedical applications. The present study is a report of biosynthesizing AgNPs using Chlorophytum borivilianum L. (Safed musli) callus extract as a novel source of reducing agent. AgNO3 solution challenged with the methanolic callus extract displayed a change in color from yellow to brown owing to the bioreduction reaction. Further, AgNPs were characterized by using UV–visible spectrophotometry, X-ray Diffraction (XRD), Atomic Force Microscopy (AFM), and Fourier Transform Infrared Spectroscopy (FTIR). UV–vis spectrum revealed the surface plasmon resonance property of AgNPs at around 450 nm. XRD pattern with typical peaks indicated the face-centered cubic nature of silver. AFM analysis confirmed the existence of spherical-shaped and well-dispersed AgNPs having an average size of 52.0 nm. Further, FTIR analysis confirmed the involvement of different phytoconstituents of the callus extract role in the process of bioreduction to form nanoparticles. The AgNPs were more efficient in inhibiting the tested pathogenic microbes, namely, Pseudomonas aeruginosa, Bacillus subtilis, Methicillin-resistant Escherichia coli, Staphylococcus aureus, and Candida albicans compared to callus extract. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay confirmed the cytotoxic property of AgNPs against human colon adenocarcinoma cell line (HT-29) in a dose-dependent manner. At higher concentrations of 500 μg/mL AgNPs, the cell viability was observed to be only 7% after 24 hours with IC50 value of 254 μg/mL. Therefore, these AgNPs clearly endorse the manifold potential to be used in various biomedical applications in the near future.

scholarly journals Phosphofructokinase 2 and glycolysis in HT29 human colon adenocarcinoma cell line. Regulation by insulin and phorbol esters

1990 ◽  
Vol 268 (2) ◽  
pp. 465-470 ◽  
Author(s):  
C Denis-Pouxviel ◽  
T Gauthier ◽  
D Daviaud ◽  
J C Murat
Keyword(s):  
Phorbol Esters ◽  
Lactate Production ◽  
Colon Adenocarcinoma ◽  
Glucose Consumption ◽  
Human Colon ◽  
Kinetic Properties ◽  
Dependent Manner ◽  
Glycerol Phosphate ◽  
Ht29 Cells ◽  
Long Term Treatment

Kinetic properties of phosphofructokinase 2 (PFK2) and regulation of glycolysis by phorbol 12-myristate 13-acetate (PMA) and insulin were investigated in highly glycolytic HT29 colon cancer cells. PFK2 was found to be inhibited by citrate and, to a lesser extent, by phosphoenolpyruvate and ADP, but to be insensitive to inhibition by sn-glycerol phosphate. From these kinetic data, PFK2 from HT29 cells appears different from the liver form, but resembles somewhat the heart isoenzyme. Fructose 2,6-bisphosphate (Fru-2,6-P2) levels, glucose consumption and lactate production are increased in a dose-dependent manner in HT29 cells treated with PMA or insulin. The increase in Fru-2,6-P2 can be related to an increase in the Vmax. of PFK2, persisting after the enzyme has been precipitated with poly(ethylene glycol), without change in the Km for fructose 6-phosphate. The most striking effects of PMA and insulin on Fru-2,6-P2 production are observed after long-term treatment (24 h) and are abolished by actinomycin, cycloheximide and puromycin, suggesting that protein synthesis is involved. Furthermore, the effects of insulin and PMA on glucose consumption, lactate production, Fru-2,6-P2 levels and PFK2 activity are additive, and the effect of insulin on Fru-2,6-P2 production is not altered by pre-treatment of the cells with the phorbol ester. This suggests that these effects are exerted by separate mechanisms.

Expression of IGF-II and IGF binding proteins in differentiating human intestinal Caco-2 cells

1995 ◽  
Vol 269 (5) ◽  
pp. E804-E813 ◽  
Author(s):  
Y. Zhang ◽  
D. A. Wick ◽  
B. Seetharam ◽  
N. M. Dahms
Keyword(s):  
Conditioned Medium ◽  
Binding Proteins ◽  
Colon Adenocarcinoma ◽  
Human Colon ◽  
Metabolic Effects ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Proliferating Cells ◽  
Igf Binding Proteins ◽  
Igf Binding

The mitogenic and metabolic effects of insulin-like growth factor-II (IGF-II) can be modulated by six distinct IGF binding proteins (IGFBPs). As a first step toward understanding the role of IGFs and their binding proteins in intestinal epithelial cell differentiation, the expression of IGF-II and IGFBPs was characterized in the human colon adenocarcinoma Caco-2 cell line. Northern blot analysis revealed two IGF-II transcripts of 5.4 and 4.5 kb, and ribonuclease protection assays indicated that IGF-II mRNA levels are regulated during Caco-2 differentiation. A specific radioimmunoassay detected IGF-II in serum-free conditioned medium, the level of which was three- to fivefold higher in proliferating cells than in differentiated cells. Immunoprecipitation and ligand blot analyses of conditioned medium demonstrated that IGFBP-2, IGFBP-3, IGFBP-4, and IGFBP-6 are synthesized by Caco-2 cells, with IGFBP-2 and IGFBP-4 being the major IGFBPs secreted, and that the levels of IGFBP-2 and IGFBP-6 decreased as differentiation proceeded. These results indicate that the expression of IGF-II, IGFBP-2, and IGFBP-6 is regulated in a differentiation-dependent manner in Caco-2 cells.

scholarly journals The Interplay between Fe3O4 Superparamagnetic Nanoparticles, Sodium Butyrate, and Folic Acid for Intracellular Transport

2020 ◽  
Vol 21 (22) ◽  
pp. 8473
Author(s):  
Maria Teresa Cambria ◽  
Giusy Villaggio ◽  
Samuele Laudani ◽  
Luca Pulvirenti ◽  
Concetta Federico ◽  
...  
Keyword(s):  
Folic Acid ◽  
Sodium Butyrate ◽  
Intracellular Transport ◽  
Colon Adenocarcinoma ◽  
Human Colon ◽  
Superparamagnetic Nanoparticles ◽  
High Dose ◽  
Dependent Manner ◽  
The Difference ◽  
Surface Modified

Combined treatments which use nanoparticles and drugs could be a synergistic strategy for the treatment of a variety of cancers to overcome drug resistance, low efficacy, and high-dose-induced systemic toxicity. In this study, the effects on human colon adenocarcinoma cells of surface modified Fe3O4 magnetic nanoparticles (MNPs) in combination with sodium butyrate (NaBu), added as a free formulation, were examined demonstrating that the co-delivery produced a cytotoxic effect on malignant cells. Two different MNP coatings were investigated: a simple polyethylene glycol (PEG) layer and a mixed folic acid (FA) and PEG layer. Our results demonstrated that MNPs with FA (FA-PEG@MNPs) have a better cellular uptake than the ones without FA (PEG@MNPs), probably due to the presence of folate that acts as an activator of folate receptors (FRs) expression. However, in the presence of NaBu, the difference between the two types of MNPs was reduced. These similar behaviors for both MNPs likely occurred because of the differentiation induced by butyrate that increases the uptake of ferromagnetic nanoparticles. Moreover, we observed a strong decrease of cell viability in a NaBu dose-dependent manner. Taking into account these results, the cooperation of multifunctional MNPs with NaBu, taking into consideration the particular cancer-cell properties, can be a valuable tool for future cancer treatment.

scholarly journals Identification, localization, and functional implications of an abundant nematode annexin.

1996 ◽  
Vol 132 (6) ◽  
pp. 1079-1092 ◽  
Author(s):  
C E Creutz ◽  
S L Snyder ◽  
S N Daigle ◽  
J Redick
Keyword(s):  
Membrane Trafficking ◽  
Calcium Binding ◽  
Peptide Sequencing ◽  
Uterine Wall ◽  
Dependent Manner ◽  
Membrane Systems ◽  
Expression Library ◽  
Sequencing Project ◽  
Calcium Dependent ◽  
Chromosome Iii

Cultures of the nematode C. elegans were examined for the presence of calcium-dependent, phospholipid-binding proteins of the annexin class. A single protein of apparent mass on SDS-polyacrylamide gels of 32 kD was isolated from soluble extracts of nematode cultures on the basis of its ability to bind to phospholipids in a calcium-dependent manner. After verification of the protein as an annexin by peptide sequencing, an antiserum to the protein was prepared and used to isolate a corresponding cDNA from an expression library in phage lambda gt11. The encoded protein, herein referred to as the nex-1 annexin, has a mass of 35 kD and is 36-42% identical in sequence to 10 known mammalian annexins. Several unique modifications were found in the portions of the sequence corresponding to calcium-binding sites. Possible phosphorylation sites in the NH2-terminal domain of the nematode annexin correspond to those of mammalian annexins. The gene for this annexin (nex-1) was physically mapped to chromosome III in the vicinity of the dpy-17 genetic marker. Two other annexin genes (nex-2 and nex-3) were also identified in chromosome III sequences reported by the nematode genomic sequencing project (Sulston, J., Z. Du, K. Thomas, R. Wilson, L. Hillier, R. Staden, N. Halloran, P. Green, J. Thierry-Mieg, L. Qiu, et al. 1992. Nature (Lond.). 356:37-41). The nex-1 annexin was localized in the nematode by immunofluorescence and by electron microscopy using immunogold labeling. The protein is associated with membrane systems of the secretory gland cells of the pharynx, with sites of cuticle formation in the grinder in the pharynx, with yolk granules in oocytes, with the uterine wall and vulva, and with membrane systems in the spermathecal valve. The presence of the annexin in association with the membranes of the spermathecal valve suggests a novel function of the protein in the folding and unfolding of these membranes as eggs pass through the valve. The localizations also indicate roles for the annexin corresponding to those proposed in mammalian systems in membrane trafficking, collagen deposition, and extracellular matrix formation.

Downstream regulatory element antagonistic modulator regulates islet prodynorphin expression

2006 ◽  
Vol 291 (3) ◽  
pp. E587-E595 ◽  
Author(s):  
David A. Jacobson ◽  
Julie Cho ◽  
Luis R. Landa ◽  
Natalia A. Tamarina ◽  
Michael W. Roe ◽  
...  
Keyword(s):  
Calcium Binding ◽  
Regulatory Element ◽  
Glucagon Secretion ◽  
Dependent Manner ◽  
Β Cells ◽  
Dynorphin A ◽  
Calcium Dependent ◽  
Low Glucose ◽  
Stimulation Of

Calcium-binding proteins regulate transcription and secretion of pancreatic islet hormones. Here, we demonstrate neuroendocrine expression of the calcium-binding downstream regulatory element antagonistic modulator (DREAM) and its role in glucose-dependent regulation of prodynorphin (PDN) expression. DREAM is distributed throughout β- and α-cells in both the nucleus and cytoplasm. As DREAM regulates neuronal dynorphin expression, we determined whether this pathway is affected in DREAM−/− islets. Under low glucose conditions, with intracellular calcium concentrations of <100 nM, DREAM−/− islets had an 80% increase in PDN message compared with controls. Accordingly, DREAM interacts with the PDN promoter downstream regulatory element (DRE) under low calcium (<100 nM) conditions, inhibiting PDN transcription in β-cells. Furthermore, β-cells treated with high glucose (20 mM) show increased cytoplasmic calcium (∼200 nM), which eliminates DREAM's interaction with the DRE, causing increased PDN promoter activity. As PDN is cleaved into dynorphin peptides, which stimulate κ-opioid receptors expressed predominantly in α-cells of the islet, we determined the role of dynorphin A-(1–17) in glucagon secretion from the α-cell. Stimulation with dynorphin A-(1–17) caused α-cell calcium fluctuations and a significant increase in glucagon release. DREAM−/− islets also show elevated glucagon secretion in low glucose compared with controls. These results demonstrate that PDN transcription is regulated by DREAM in a calcium-dependent manner and suggest a role for dynorphin regulation of α-cell glucagon secretion. The data provide a molecular basis for opiate stimulation of glucagon secretion first observed over 25 years ago.

scholarly journals Glutathione conjugation of chlorambucil: measurement and modulation by plant polyphenols

1997 ◽  
Vol 325 (2) ◽  
pp. 417-422 ◽  
Author(s):  
Kai ZHANG ◽  
Kim Ping WONG
Keyword(s):  
Colon Adenocarcinoma ◽  
Human Colon ◽  
Thymidine Uptake ◽  
Dependent Manner ◽  
Glutathione S Transferase ◽  
Plant Polyphenols ◽  
Simultaneous Exposure ◽  
Adenocarcinoma Cells ◽  
Combined Action ◽  
Dose Dependent

Chlorambucil (CMB), an anticancer drug, was cytotoxic at concentrations of 5–20 μM to human colon adenocarcinoma cells. It inhibited [14C]thymidine uptake in a dose-dependent manner. Both effects were potentiated by simultaneous exposure of the cells to 10 μM plant polyphenols. In an attempt to explain the possible mechanism of action of the polyphenols in relation to these observations, an HPLC-radiometric method was developed to measure the conjugation of CMB with glutathione in these cells and to monitor the export of monochloromonoglutathionyl CMB (MG-CMB), its main glutathione conjugate. At micromolar concentrations, five polyphenols, namely quercetin, butein, tannic acid, 2′-hydroxychalcone and morin, inhibited the efflux of CMB significantly; an inhibition of 40% was observed with 10 μM quercetin. The glutathione S-transferase (GST) activity of the cancer cells, measured with 1-chloro-2,4-dinitrobenzene, was also inhibited by the polyphenols. Their combined action on GST and on the efflux of MG-CMB conjugate could provide an enhanced positive modulation of sensitivity of the tumour cells to CMB.

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