Calcium-dependent conformational rearrangements and protein stability in chicken annexin A5.
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The conformational rearrangements that take place after calcium binding in chicken annexin A5 and a mutant lacking residues 3-10 were analyzed, in parallel with human annexin A5, by circular dichroism (CD), infrared spectroscopy (IR), and differential scanning calorimetry. Human and chicken annexins present a slightly different shape in the far-UV CD and IR spectra, but the main secondary-structure features are quite similar (70-80% alpha-helix). However, thermal stability of human annexin is significantly lower than its chicken counterpart (approximately 8 degrees C) and equivalent to the chicken N-terminally truncated form. The N-terminal extension contributes greatly to stabilize the overall annexin A5 structure. Infrared spectroscopy reveals the presence of two populations of alpha-helical structures, the canonical alpha-helices (approximately 1650 cm(-1)) and another, at a lower wavenumber (approximately 1634 cm(-1)), probably arising from helix-helix interactions or solvated alpha-helices. Saturation with calcium induces: alterations in the environment of the unique tryptophan residue of the recombinant proteins, as detected by near-UV CD spectroscopy; more compact tertiary structures that could account for the higher thermal stabilities (8 to 12 degrees C), this effect being higher for human annexin; and an increase in canonical alpha-helix percentage by a rearrangement of nonperiodical structure or 3(10) helices together with a variation in helix-helix interactions, as shown by amide I curve-fitting and 2D-IR. (+info)
Overview of the 7th European symposium on calcium-binding proteins in normal and transformed cells.
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The strong feature of the meeting was the continuing efforts described in many papers to resolve the multiple ways in which calcium ions are released into cells via messenger signals and then interact with receptors to cause differential internal cellular activation and cell/cell communication. An easy general way to relate these studies to cell components is to start analysis from the genetic structures lying behind all cell activities and then to explore the RNA production, the proteome, the small substrates and calcium levels themselves in turn while referring to the environment of a particular cell, organ or organism. There is then of course the overall physiology. I shall summarize the papers in this order of their main interests. (+info)
The penta-EF-hand domain of ALG-2 interacts with amino-terminal domains of both annexin VII and annexin XI in a Ca2+-dependent manner.
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The apoptosis-linked protein ALG-2 is a Ca(2+)-binding protein that belongs to the penta-EF-hand (PEF) protein family. ALG-2 forms a homodimer, a heterodimer with another PEF protein, peflin, and a complex with its interacting protein, named Alix or AIP1. We previously identified annexin XI as a novel ALG-2-binding partner. Both the N-terminal regulatory domain of annexin XI (Anx11N) and the ALG-2-binding domain of Alix/AIP1 are rich in Pro, Gly, Ala, Tyr and Gln. This PGAYQ-biased amino acid composition is also found in the N-terminal extension of annexin VII (Anx7N). Using recombinant ALG-2 proteins and the glutathione S-transferase (GST) fusion proteins of Anx7N and Anx11N, the direct Ca(2+)-dependent interaction was analyzed by a biotin-tagged ALG-2 overlay assay and by a real-time interaction analysis with a surface plasmon resonance (SPR) biosensor. Both GST-Anx7N and GST-Anx11N showed similar binding kinetics against ALG-2 as well as ALG-2-DeltaN23, which lacked the hydrophobic N-terminal region. Two binding sites were predicted in both Anx7N and Anx11N, and the dissociation constants (K(d)) were estimated to be approximately 40-60 nM for the high-affinity site and 500-700 nM for the low-affinity site. (+info)
Stress fibres-a Ca2+ -independent store for annexins?
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Annexins belong to a family of lipid-binding proteins that are implicated in membrane organization. Several members are capable of binding to actin and, in smooth muscle cells, annexin 6 is known to form a Ca(2+)-dependent, plasmalemmal complex with actin filaments. Annexins can also associate with F-actin containing stress fibres within cultured smooth muscle cells or fibroblasts in a Ca(2+)-independent manner. Depolymerization of stress-fibre systems with cytochalasin D leads to the translocation of actin-bound annexin 2 from the cytoplasm to the plasma membrane at high intracellular levels of Ca(2+). This type of Ca(2+)-dependent annexin mobility is observed only in cells of mesenchymal phenotype, which have a well-developed stress-fibre system; not in epithelial cells. (+info)
Maturation of epiphyseal growth plate chondrocytes plays an important role in endochondral bone formation. Previously, we demonstrated that retinoic acid (RA) treatment stimulated annexin-mediated Ca(2+) influx into growth plate chondrocytes leading to a significant increase in cytosolic Ca(2+), whereas K-201, a specific annexin Ca(2+) channel blocker, inhibited this increase markedly. The present study addressed the hypothesis that annexin-mediated Ca(2+) influx into growth plate chondrocytes is a major regulator of terminal differentiation, mineralization, and apoptosis of these cells. We found that K-201 significantly reduced up-regulation of expression of terminal differentiation marker genes, such as cbfa1, alkaline phosphatase (APase), osteocalcin, and type I collagen in RA-treated cultures. Furthermore, K-201 inhibited up-regulation of annexin II, V, and VI gene expression in these cells. RA-treated chondrocytes released mineralization-competent matrix vesicles, which contained significantly higher amounts of annexins II, V, and VI as well as APase activity than vesicles isolated from untreated or RA/K-201-treated cultures. Consistently, only RA-treated cultures showed significant mineralization. RA treatment stimulated the whole sequence of terminal differentiation events, including apoptosis as the final event. After a 6-day treatment gene expression of bcl-2, an anti-apoptotic protein, was down-regulated, whereas caspase-3 activity and the percentage of TUNEL-positive cells were significantly increased in RA-treated cultures compared with untreated cultures. Interestingly, the cytosolic calcium chelator BAPTA-AM and K-201 protected RA-treated chondrocytes from undergoing apoptotic changes, as indicated by higher bcl-2 gene expression, reduced caspase-3 activity, and the percentage of TUNEL-positive cells. In conclusion, annexin-mediated Ca(2+) influx into growth plate chondrocytes is a positive regulator of terminal differentiation, mineralization, and apoptosis events in growth plate chondrocytes. (+info)
Interaction of chondrocytes, extracellular matrix and growth factors: relevance for articular cartilage tissue engineering.
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The abundant extracellular matrix of articular cartilage has to be maintained by a limited number of chondrocytes. Vice versa, the extracellular matrix has an important role in the regulation of chondrocyte function. OBJECTIVE: In this review we discuss the role of the extracellular matrix in the regulation of chondrocyte function and the relevance for cartilage tissue engineering. To reach this goal the international literature on this subject has been searched with a major focus on the last 5 years. RESULTS: Structural matrix macromolecules (e.g. collagen, hyaluronate), but also growth factors (e.g. IGF-I, TGF beta) entrapped in the matrix and released under specific conditions affect chondrocyte behavior. These factors communicate with the chondrocyte via specific membrane receptors. In this way there is a close interaction between the extracellular and intracellular milieu. Articular cartilage has a limited capacity of intrinsic repair, which has resulted in the development of tissue engineering approaches to repair damaged cartilage. Successful application of scaffolds has to take into account the important role of both soluble and insoluble matrix-derived factors in cartilage homeostasis. CONCLUSION: Functional tissue engineering will only be realized when the scaffolds used will provide cartilage cells with the correct extracellular signals. (+info)
Dysregulation of the annexin family protein family is associated with prostate cancer progression.
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Hormone refractory prostate cancer (PCa) is invariably lethal despite aggressive clinical treatment strategies. Detection strategies are needed to identify aggressive PCa before it becomes widely disseminated. Recently, two studies identified annexin 1 and 7 as potential biomarkers in the development of PCa progression. The annexins are a group of calcium-binding structural proteins that may play a role in the regulation of membrane trafficking, cellular adhesion, and cell signaling. Therefore the goal of this study is to simultaneously characterize the multiple members of the annexin family of genes in advanced PCa. Prostate samples from men with advanced hormone refractory PCa were compared to samples of hormone-naive PCa and noncancerous prostate tissue. Samples from 15 patients with advanced hormone refractory PCa were used. To examine the annexin family, gene expression profiles from 21 noncancerous prostate tissues, 16 clinically localized PCas, and 20 hormone refractory PCa samples were used. By cDNA microarray analysis, annexins 1, 2, 4, 7, and 11 were significantly decreased in hormone refractory PCa when compared to localized hormone-naive PCa with 2.2-, 1.5-, 1.3-, 1.4-, and 1.8-fold decreases, respectively (all P values <0.05). Interstudy validation of annexin family transcript expression was performed by meta-analysis of three other published prostate profiling studies. High-density tissue microarrays were used to validate a subset of annexins at the protein level by immunohistochemistry. Tissue microarray analysis revealed a significant decrease in protein expression for annexins 1, 2, 4, 7, and 11 in hormone refractory PCa as compared to localized PCa with 1.68-, 2.46-, 2.52-, and 3.01-fold decreases, respectively (Kruskal Wallis test, all P values P < 0.05). However, no significant differences were detected between the clinically localized PCa and noncancerous prostate tissues. These findings suggest that down-regulation of several members of the annexin family may contribute to PCa tumorigenesis. Annexins 1, 2, 4, 7, and 11 may play a role in tumor progression through distinct mechanisms or, alternatively, they may have redundant tumor suppressor activities. This study also suggests that a meta-analysis of existing gene expression data is useful in confirming findings from individual studies. Finally, down-regulation of several annexin family members may play a role in the development of the lethal PCa phenotype. (+info)
Attenuation of glucocorticoid functions in an Anx-A1-/- cell line.
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The Ca(2+)- and phospholipid-binding protein Anx-A1 (annexin 1; lipocortin 1) has been described both as an inhibitor of phospholipase A(2) (PLA(2)) activity and as a mediator of glucocorticoid-regulated cell growth and eicosanoid generation. Here we show that, when compared with Anx-A1(+/+) cells, lung fibroblast cell lines derived from the Anx-A1(-/-) mouse exhibit an altered morphology characterized by a spindle-shaped appearance and an accumulation of intracellular organelles. Unlike their wild-type counterparts, Anx-A1(-/-) cells also overexpress cyclo-oxygenase 2 (COX 2), cytosolic PLA(2) and secretory PLA(2) and in response to fetal calf serum, exhibit an exaggerated release of eicosanoids, which is insensitive to dexamethasone (10(-8)- 10(-6) M) inhibition. Proliferation and serum-induced progression of Anx-A1(+/+) cells from G(0)/G(1) into S phase, and the associated expression of extracellular signal-regulated kinase 2 (ERK2), cyclin-dependent kinase 4 (cdk4) and COX 2, is strongly inhibited by dexamethasone, whereas Anx-A1(-/-) cells are refractory to the drug. Loss of the response to dexamethasone in Anx-A1(-/-) cells occurs against a background of no apparent change in glucocorticoid receptor expression or sensitivity to non-steroidal anti-inflammatory drugs. Taken together, these observations suggest strongly that Anx-A1 functions as an inhibitor of signal-transduction pathways that lead to cell proliferation and may help to explain how glucocorticoids regulate these processes. (+info)