Crystal structure of human annexin I at 2.5 A resolution.

cDNA coding for N-terminally truncated human annexin I, a member of the family of Ca(2+)-dependent phospholipid binding proteins, has been cloned and expressed in Escherichia coli. The expressed protein is biologically active, and has been purified and crystallized in space group P2(1)2(1)2(1) with cell dimensions a = 139.36 A, b = 67.50 A, and c = 42.11 A. The crystal structure has been determined by molecular replacement at 3.0 A resolution using the annexin V core structure as the search model. The average backbone deviation between these two structures is 2.34 A. The structure has been refined to an R-factor of 17.7% at 2.5 A resolution. Six calcium sites have been identified in the annexin I structure. Each is located in the loop region of the helix-loop-helix motif. Two of the six calcium sites in annexin I are not occupied in the annexin V structure. The superpositions of the corresponding loop regions in the four domains show that the calcium binding loops in annexin I can be divided into two classes: type II and type III. Both classes are different from the well-known EF-hand motif (type I).     

Cotton annexin proteins participate in the establishment of fiber cell elongation scaffold

Cotton plant is one of the most important economic crops in the world which supplies natural fiber for textile industry. The crucial traits of cotton fiber quality are fiber length and strength, which are mostly determined by the fiber elongation stage. Annexins are assumed to be involved in regulating fiber elongation, but direct evidences remain elusive. Recently, we have investigated the activities of fiber-specific expressed annexins AnGb5/6 and their interacted proteins in cotton. AnGb5 and 6 can interact reciprocally to generate a protein macro-raft in cell membrane. This macro-raft is probably a stabilized scaffold for Actin1 organization. The actin assembling direction and density are correlated with AnGb6 gene expression and fiber expanding rate among three fiber length genotypes. These results suggest that annexins may act as the adaptor that linked fiber cell membrane to actin assembling. Due to the strong Ca²⁺ and lipid binding ability of annexins, these results also indicate that annexins complex may function as an intermediate to receive Ca²⁺ or lipid signals during fiber elongation.     

Protein interactions between surface annexin A2 and S100A10 mediate adhesion of breast cancer cells to microvascular endothelial cells

Annexin A2 (AnxA2) and S100A10 are known to form a molecular complex. Using fluorescence-based binding assays, we show that both proteins are localised on the cell surface, in a molecular form that allows mutual interaction. We hypothesized that binding between these proteins could facilitate cell–cell interactions. For cells that express surface S100A10 and surface annexin A2, cell–cell interactions can be blocked by competing with the interaction between these proteins. Thus an annexin A2-S100A10 molecular bridge participates in cell–cell interactions, revealing a hitherto unexplored function of this protein interaction.     

Apical phosphatidylserine externalization in auditory hair cells

In hair cells of the inner ear, phosphatidylserine (PS), detected with fluorescent annexin V labeling, was rapidly exposed on the external leaflet of apical plasma membranes upon dissection of the organ of Corti. PS externalization was unchanged by caspase inhibition, suggesting that externalization did not portend apoptosis or necrosis. Consistent with that conclusion, mitochondrial membrane potential and hair-cell nuclear structure remained normal during externalization. PS externalization was triggered by forskolin, which raises cAMP, and blocked by inhibitors of adenylyl cyclase. Blocking Na⁺ influx by inhibiting the mechanoelectrical transduction channels and P2X ATP channels also inhibited external PS externalization. Diminished PS externalization was also seen in cells exposed to LY 294002, which blocks membrane recycling in hair cells by inhibiting phosphatidylinositol 3-kinase. These results indicate that PS exposure on the external leaflet, presumably requiring vesicular transport, results from elevation of intracellular cAMP, which can be triggered by Na⁺ entry into hair cells.     

Role of calcium-binding sites in calcium-dependent membrane association of annexin A4

Annexin A4 (Anx4) is a cytosolic calcium-binding protein with four repeat domains, each containing one calcium-binding site (CBS). The protein interacts with the phospholipid membrane through the CBS-coordinated calcium ion, although the role of each CBS in the calcium-dependent association is unclear. To determine the role of each CBS, 15 CBS-abolished variants were produced in various combinations by substitution of a calcium-liganding residue on each CBS by Ala. Various mutant combinations produced different influences on calcium-dependent membrane-binding behavior and on the sodium-dependent dissociation of membrane-bound Anx4. Our data suggest the interaction of Anx4 with the lipid membrane consists of strong and weak interactions. CBSs I and IV mediate formation of strong interactions, while CBSs II and III are important for weak interactions. We also suggest Anx4 binds the lipid membrane through CBSs I and IV in the cytoplasmic fluids.     

Annexin-actin interactions

The actin cytoskeleton is a malleable framework of polymerised actin monomers that may be rapidly restructured to enable diverse cellular activities such as motility, endocytosis and cytokinesis. The regulation of actin dynamics involves the coordinated activity of numerous proteins, among which members of the annexin family of Ca2+- and phospholipid-binding proteins play an important role. Although the roles of annexins in actin dynamics are not understood at a mechanistic level, annexins have the requisite properties to integrate Ca2+-signaling with actin dynamics at membrane contact sites. In this review we discuss the current state of knowledge on this topic, and consider how and where annexins may fit into the complex molecular machinery that regulates the actin cytoskeleton.     

Will imaging of apoptosis play a role in clinical care? A tale of mice and men

Programmed cell death (apoptosis) plays a role in the pathophysiology of many diseases and in the outcome of treatment. Apoptosis is the likely mechanism behind the cytoreductive effects of standard chemotherapeutic and radiation treatments, rejection of organ transplants, cellular damage in collagen vascular disorders, and delayed cell death due to hypoxic-ischemic injury in myocardial infarction and neonatal hypoxic ischemic injury. Observations about the role of apoptosis have fueled the development of novel agents and treatment strategies specifically aimed at inducing or inhibiting apoptosis.Despite these research developments there are no clinical entities where specific measures of apoptosis are used in either diagnosis or patient management. Part of the difficulty in bridging the gap between the basic science understanding of apoptosis and the clinical application of this information is the lack of a sensitive marker to monitor programmed cell death in association with disease progression or regression. Technetium-99m labeled annexin V localizes at sites of apoptosis in-vivo, due to its nanomolar affinity for membrane bound phosphatidylserine. Radiolabeled annexin V imaging permits identification of the site and extent of apoptosis in experimental animals. Annexin V has been successfully used in animal models to image organ transplant rejection, characterize successful therapy of tumors, pinpoint acute myocardial infarction, and identify hypoxic ischemic brain injury of the newborn and adult. Early studies in human subjects suggest that ^99mTc annexin imaging will be also be useful to identify rejection in transplant recipients, localize acute myocardial infarction, and characterize the effectiveness of a single treatment in patients with tumors.This review describes the imaging approaches to detect and monitor apoptosis in-vivo that are presently in early clinical trials. The preliminary data are extrapolated to identify conditions where apoptosis imaging may be valuable in clinical decision making. These conditions include: transplant rejection; hypoxic/ischemic injury of heart and brain; and determining the efficacy of therapy in cancer, heart failure and osteoporosis.