Different molecular arrangements of the tetrameric annexin 2 modulate the size and dynamics of membrane aggregation

Annexin 2, a member of the annexin family of Ca²⁺-dependent membrane binding proteins is found in monomeric and heterotetrameric forms and has been involved in different membrane related functions. The heterotetrameric annexin 2 is composed of a dimer of S100A10, a member of the S100 family of Ca²⁺ binding proteins and two annexin 2 molecules ((Anx2-S100A10)₂). Different molecular models including tetramers and octamers in which S100A10 is localized in the centre of the complex with the annexin 2 molecules positioned around S100A10 had been proposed. Herein, the organization of the (Anx2-S100A10)₂ complex in conditions in which membranes are able to bridge was studied. We performed Cryo-electron microscopy observations of the tetrameric annexin 2 on the membrane surface, and study the S100A10 accessibility to antibodies by flow “cytometry”. We also studied the kinetics and size evolution of vesicle aggregates by dynamic light scattering. The results show that the protein is able to organize in three different arrangements depending on the presence of Ca²⁺ and pH and that the aggregation is faster in the presence of Ca²⁺ compared with the aggregation in its absence. In one arrangement the S100A10 molecule is exposed to the solvent allowing its interaction with other proteins. The presented results will serve as a molecular basis to explain some of the functions of the tetrameric annexin 2.     

S100A6 - New facts and features

S100A6 (calcyclin) is a 10.5 kDa Ca²⁺-binding protein that belongs to the S100 protein family. S100A6 contains two EF-hand motifs responsible for binding of Ca²⁺. It also binds Zn²⁺ through not yet identified structures. Binding of Ca²⁺ induces a conformational change in the S100A6 molecule which in consequence increases its overall hydrophobicity and allows for interaction with target proteins. S100A6 was found in different mammalian and avian (chicken) tissues. A high level of S100A6 is observed in epithelial cells, fibroblasts and in different kinds of cancer cells. The function of S100A6 is not clear at present, but it has been suggested that it may be involved in cell proliferation, cytoskeletal dynamics and tumorigenesis. Additionally, S100A6 might have some extracellular activities. This review presents new facts and features concerning the S100A6 protein.     

S100A10‐Mediated Translocation of Annexin‐A2 to SNARE Proteins in Adrenergic Chromaffin Cells Undergoing Exocytosis

In neuroendocrine cells, annexin‐A2 is implicated as a promoter of monosialotetrahexosylganglioside (GM1)‐containing lipid microdomains that are required for calcium‐regulated exocytosis. As soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors (SNAREs) require a specific lipid environment to mediate granule docking and fusion, we investigated whether annexin‐A2‐induced lipid microdomains might be linked to the SNAREs present at the plasma membrane. Stimulation of adrenergic chromaffin cells induces the translocation of cytosolic annexin‐A2 to the plasma membrane, where it colocalizes with SNAP‐25 and S100A10. Cross‐linking experiments performed in stimulated chromaffin cells indicate that annexin‐A2 directly interacts with S100A10 to form a tetramer at the plasma membrane. Here, we demonstrate that S100A10 can interact with vesicle‐associated membrane protein 2 (VAMP2) and show that VAMP2 is present at the plasma membrane in resting adrenergic chromaffin cells. Tetanus toxin that cleaves VAMP2 solubilizes S100A10 from the plasma membrane and inhibits the translocation of annexin‐A2 to the plasma membrane. Immunogold labelling of plasma membrane sheets combined with spatial point pattern analysis confirmed that S100A10 is present in VAMP2 microdomains at the plasma membrane and that annexin‐A2 is observed close to S100A10 and to syntaxin in stimulated chromaffin cells. In addition, these results showed that the formation of phosphatidylinositol (4,5)‐bisphosphate (PIP₂) microdomains colocalized with S100A10 in the vicinity of docked granules, suggesting a functional interplay between annexin‐A2‐mediated lipid microdomains and SNAREs during exocytosis.     

Annexin II tetramer: structure and function

The annexins are a family of proteins that bind acidic phospholipids in the presence of Ca²⁺. The interaction of these proteins with biological membranes has led to the suggestion that these proteins may play a role in membrane trafficking events such as exocytosis, endocytosis and cell-cell adhesion. One member of the annexin family, annexin II, has been shown to exist as a monomer, heterodimer or heterotetramer. The ability of annexin II tetramer to bridge secretory granules to plasma membrane has suggested that this protein may play a role in Ca²⁺-dependent exocytosis. Annexin II tetramer has also been demonstrated on the extracellular face of some metastatic cells where it mediates the binding of certain metastatic cells to normal cells. Annexin II tetramer is a major cellular substrate of protein kinase C and pp60^src. Phosphorylation of annexin II tetramer is a negative modulator of protein function.Supported by a grant from the Medical Research Council of Canada     

Lipid raft–dependent plasma membrane repair interferes with the activation of B lymphocytes

Cells rapidly repair plasma membrane (PM) damage by a process requiring Ca²⁺-dependent lysosome exocytosis. Acid sphingomyelinase (ASM) released from lysosomes induces endocytosis of injured membrane through caveolae, membrane invaginations from lipid rafts. How B lymphocytes, lacking any known form of caveolin, repair membrane injury is unknown. Here we show that B lymphocytes repair PM wounds in a Ca²⁺-dependent manner. Wounding induces lysosome exocytosis and endocytosis of dextran and the raft-binding cholera toxin subunit B (CTB). Resealing is reduced by ASM inhibitors and ASM deficiency and enhanced or restored by extracellular exposure to sphingomyelinase. B cell activation via B cell receptors (BCRs), a process requiring lipid rafts, interferes with PM repair. Conversely, wounding inhibits BCR signaling and internalization by disrupting BCR–lipid raft coclustering and by inducing the endocytosis of raft-bound CTB separately from BCR into tubular invaginations. Thus, PM repair and B cell activation interfere with one another because of competition for lipid rafts, revealing how frequent membrane injury and repair can impair B lymphocyte–mediated immune responses.     

STIM and Orai: Dynamic Intermembrane Coupling to Control Cellular Calcium Signals

Ca²⁺ signals controlling a vast array of cell functions involve both Ca²⁺ store release and external Ca²⁺ entry. These two events are coordinated through a dynamic intermembrane coupling between two distinct membrane proteins, STIM and Orai. STIM proteins are endoplasmic reticulum (ER) luminal Ca²⁺ sensors that undergo a profound redistribution into discrete junctional ER domains closely juxtaposed with the plasma membrane (PM). Orai proteins are PM Ca²⁺ channels that migrate and become tethered by STIM within the ER-PM junctions, where they mediate exceedingly selective Ca²⁺ entry. We describe a new understanding of the nature of the proteins and how they function to mediate this remarkable intermembrane signaling process controlling Ca²⁺ signals.     

Nutritional Immunity: S100 Proteins at the Host-Pathogen Interface

The S100 family of EF-hand calcium (Ca²⁺)-binding proteins is essential for a wide range of cellular functions. During infection, certain S100 proteins act as damage-associated molecular patterns (DAMPs) and interact with pattern recognition receptors to modulate inflammatory responses. In addition, these inflammatory S100 proteins have potent antimicrobial properties and are essential components of the immune response to invading pathogens. In this review, we focus on S100 proteins that exhibit antimicrobial properties through the process of metal limitation, termed nutritional immunity, and discuss several recent advances in our understanding of S100 protein-mediated metal sequestration at the site of infection.     

S100 and S100 fused-type protein families in epidermal maturation with special focus on S100A3 in mammalian hair cuticles

Epithelial Ca²⁺-regulation, which governs cornified envelope formation in the skin epidermis and hair follicles, closely coincides with the expression of S100A3, filaggrin and trichohyalin, and the post-translational modification of these proteins by Ca²⁺-dependent peptidylarginine deiminases. This review summarizes the current nomenclature and evolutional aspects of S100 Ca²⁺-binding proteins and S100 fused-type proteins (SFTPs) classified as a separate protein family with special reference to the molecular structure and function of S100A3 dominantly expressed in hair cuticular cells. Both S100 and SFTP family members are identified by two distinct types of Ca²⁺-binding loops in an N-terminal pseudo EF-hand motif followed by a canonical EF-hand motif. Seventeen members of the S100 protein family including S100A3 are clustered with seven related genes encoding SFTPs on human chromosome 1q21, implicating their association with epidermal maturation and diseases. Human S100A3 is characterized by two disulphide bridges and a preformed Zn²⁺-pocket, and may transfer Ca²⁺ ions to peptidylarginine deiminases after its citrullination-mediated tetramerization. Phylogenetic analysis utilizing current genome databases suggests that divergence of the S100A3 gene coincided with the emergence of hair, a defining feature of mammals, and that the involvement of S100A3 in epithelial Ca²⁺-cycling occurred as a result of a skin adaptation in terrestrial mammals.     

Identification of a novel interaction between the Ca(2+)-binding protein S100A11 and the Ca(2+)- and phospholipid-binding protein annexin A6

S100A11 is a member of the S100 family of EF-hand Ca²⁺-binding proteins, which is expressed in smooth muscle and other tissues. Ca²⁺ binding to S100A11 induces a conformational change that exposes a hydrophobic surface for interaction with target proteins. Affinity chromatography with immobilized S100A11 was used to isolate a 70-kDa protein from smooth muscle that bound to S100A11 in a Ca²⁺-dependent manner and was identified by mass spectrometry as annexin A6. Direct Ca²⁺-dependent interaction between S100A11 and annexin A6 was confirmed by affinity chromatography of the purified bacterially expressed proteins, by gel overlay of annexin A6 with purified S100A11, by chemical cross-linking, and by coprecipitation of S100A11 with annexin A6 bound to liposomes. The expression of S100A11 and annexin A6 in the same cell type was verified by RT-PCR and immunocytochemistry of isolated vascular smooth muscle cells. The site of binding of S100A11 on annexin A6 was investigated by partial tryptic digestion and deletion mutagenesis. The unique NH₂ terminal head region of annexin A6 was not required for S100A11 binding, but binding sites were identified in both NH₂- and COOH-terminal halves of the molecule. We hypothesize that an agonist-induced increase in cytosolic free [Ca²⁺] leads to formation of a complex of S100A11 and annexin A6, which forms a physical connection between the plasma membrane and the cytoskeleton, or plays a role in the formation of signaling complexes at the level of the sarcolemma. smooth muscle; protein-protein interaction     

Plasma Membrane-associated Annexin A6 Reduces Ca2+ Entry by Stabilizing the Cortical Actin Cytoskeleton

The annexins are a family of Ca²⁺- and phospholipid-binding proteins, which interact with membranes upon increase of [Ca²⁺]_i or during cytoplasmic acidification. The transient nature of the membrane binding of annexins complicates the study of their influence on intracellular processes. To address the function of annexins at the plasma membrane (PM), we fused fluorescent protein-tagged annexins A6, A1, and A2 with H- and K-Ras membrane anchors. Stable PM localization of membrane-anchored annexin A6 significantly decreased the store-operated Ca²⁺ entry (SOCE), but did not influence the rates of Ca²⁺ extrusion. This attenuation was specific for annexin A6 because PM-anchored annexins A1 and A2 did not alter SOCE. Membrane association of annexin A6 was necessary for a measurable decrease of SOCE, because cytoplasmic annexin A6 had no effect on Ca²⁺ entry as long as [Ca²⁺]_i was below the threshold of annexin A6-membrane translocation. However, when [Ca²⁺]_i reached the levels necessary for the Ca²⁺-dependent PM association of ectopically expressed wild-type annexin A6, SOCE was also inhibited. Conversely, knockdown of the endogenous annexin A6 in HEK293 cells resulted in an elevated Ca²⁺ entry. Constitutive PM localization of annexin A6 caused a rearrangement and accumulation of F-actin at the PM, indicating a stabilized cortical cytoskeleton. Consistent with these findings, disruption of the actin cytoskeleton using latrunculin A abolished the inhibitory effect of PM-anchored annexin A6 on SOCE. In agreement with the inhibitory effect of annexin A6 on SOCE, constitutive PM localization of annexin A6 inhibited cell proliferation. Taken together, our results implicate annexin A6 in the actin-dependent regulation of Ca²⁺ entry, with consequences for the rates of cell proliferation.Calcium entry into cells either through voltage- or receptor-operated channels, or following the depletion of intracellular stores is a major factor in maintaining intracellular Ca²⁺ homeostasis. Resting [Ca²⁺]_i is low (∼100 nm compared with extracellular [Ca²⁺]_ex of 1.2 mm) and can be rapidly increased by inositol triphosphate-mediated release from the intracellular Ca²⁺ stores (mostly endoplasmic reticulum (ER)³), or by channel-mediated influx across the plasma membrane (PM). Store-operated calcium entry (SOCE) has been proposed as the main process controlling Ca²⁺ entry in non-excitable cells (1), and the recent discovery of Orai1 and STIM provided the missing link between the Ca²⁺-release activated current (I_CRAC) and the ER Ca²⁺ sensor (2–4). Translocation of STIM within the ER, accumulation in punctae at the sites of contact with PM and activation of Ca²⁺ channels have been proposed as a model of its regulation of Orai1 activity (5, 6). However, many details of the functional STIM-Orai1 protein complex and its regulation remain to be elucidated. The actin cytoskeleton plays a major role in the regulation of SOCE, possibly by influencing the function of ion channels or by interfering with the interaction between STIM and Orai1 (7–9). However, the proteins connecting the actin cytoskeleton and SOCE activity at the PM have yet to be identified.The annexins are a multigene family of Ca²⁺- and phospholipid-binding proteins, which have been implicated in many Ca²⁺-regulated processes. Their C-terminal core is evolutionarily conserved and contains Ca²⁺-binding sites, their N-terminal tails are unique and enable the protein to interact with distinct cytoplasmic partners. At low [Ca²⁺]_i, annexins are diffusely distributed throughout the cytosol, however, after stimulation resulting in the increase of [Ca²⁺]_i, annexins are targeted to distinct subcellular membrane locations, such as the PM, endosomes, or secretory vesicles (10). Annexins are involved in the processes of vesicle trafficking, cell division, apoptosis, calcium signaling, and growth regulation (11), and frequent changes in expression levels of annexins are observed in disease (12, 13). Previously, using biochemical methods and imaging of fluorescent protein-tagged annexins in live cells, we demonstrated that annexins A1, A2, A4, and A6 interacted with the PM as well as with internal membrane systems in a highly coordinated manner (10, 14). In addition, there is evidence of Ca²⁺-independent membrane association of several annexins, including annexin A6 (15–19); some of which point to the existence of pH-dependent binding mechanisms (20–22). Given the fact that several annexins are present within any one cell, it is likely that they form a [Ca²⁺] and pH sensing system, with a regulatory influence on other signaling pathways.The role of annexins as regulators of ion channel activity has been addressed previously (23–25). In particular, annexin A6 has been implicated in regulation of the sarcoplasmic reticulum ryanodine-sensitive Ca²⁺ channel (25), the neuronal K⁺ and Ca²⁺ channels (26), and the cardiac Na⁺/Ca²⁺ exchanger (27). Cardiac-specific overexpression of annexin A6 resulted in lower basal [Ca²⁺], a depression of [Ca²⁺]_i transients and impaired cardiomyocyte contractility (28). In contrast, the cardiomyocytes from the annexin A6 null-mutant mice showed increased contractility and accelerated Ca²⁺ clearance (29). Consistent with its role in mediating the intracellular Ca²⁺ signals, especially Ca²⁺ influx, ectopic overexpression of annexin A6 in A431 cells, which lack endogenous annexin A6, resulted in inhibition of EGF-dependent Ca²⁺ entry (30).The difficulty of investigating the influence of annexins on signaling events occurring at the PM lies in the transient and reversible nature of their Ca²⁺ and pH-dependent lipid binding. Although the intracellular Ca²⁺ increase following receptor activation or Ca²⁺ influx promotes the association of the Ca²⁺-sensitive annexins A2 and A6 with the PM, the proteins quickly resume their cytoplasmic localization upon restoration of the basal [Ca²⁺]_i (14). Therefore, to investigate the effects of membrane-associated annexins on Ca²⁺ homeostasis and the cell signaling machinery, we aimed to develop a model system allowing for a constitutive membrane association of annexins. Here we used the PM-anchoring sequences of the H- and K-Ras proteins to target annexins A6 and A1 to the PM independently of [Ca²⁺]. The Ras GTPases are resident at the inner leaflet of the PM and function as molecular switches (31). The C-terminal 9 amino acids of H- and N-Ras and the C-terminal 14 amino acids of K-Ras comprise the signal sequences for membrane anchoring of Ras isoforms (32). Although the palmitoylation and farnesylation of the C terminus of H-Ras (tH) serves as a targeting signal for predominantly cholesterol-rich membrane microdomains at the PM (lipid rafts/caveolae) (33), the polybasic group and the lipid anchor of K-Ras (tK) ensures the association of K-Ras with cholesterol-poor PM membrane domains. Importantly, these minimal C-terminal amino acid sequences are sufficient to target heterologous proteins, for example GFP, to different microdomains at the PM and influence their trafficking (34).In the present study we fused annexins A6, A2, and A1 with fluorescent proteins and introduced the PM-anchoring sequences of either H-Ras (annexin-tH) or K-Ras (annexin-tK) at the C termini of the fusion constructs. We demonstrate that the constitutive PM localization of annexin A6 results in down-regulation of store-operated Ca²⁺ entry. Expression of membrane-anchored annexin A6 causes an accumulation of the cortical F-actin, and cytoskeletal destabilization with latrunculin A abolishes the inhibitory effect of PM-anchored annexin A6 on SOCE. Taken together, our results implicate annexin A6 in the maintenance of intracellular Ca²⁺ homeostasis via actin-dependent regulation of Ca²⁺ entry.     

Ultracytochemistry as a tool for the study of the cellular and subcellular localization of membrane-bound guanylate cyclase (GC) activity. Applicability to both receptor-activated and receptor-independent GC activity

Membrane-bound guanylate cyclase activity was detected by ultracytochemistry at the electron microscope level in several mammalian tissues. The technique used in these studies allows the detection of active enzyme at the membrane site where it is located. In a few cases, such as normal and regenerating peripheral nerves and placenta, membrane-bound guanylate cyclase could be detected in the absence of stimulators of enzyme activity. However, in the majority of these studies membrane-bound guanylate cyclase was investigated following stimulation with natriuretic peptides, guanylin, or the Ca²⁺ sensor proteins, S100B and S100A1. In general, membrane-bound guanylate cyclase was localized to plasma membranes, in accordance with the functional role of this enzyme. Yet, in secretory cells the enzyme activity was localized on intracellular membranes, suggesting a role of membrane-bound guanylate cyclase in secretory processes. Finally, S100B and S100A1 were found to colocalize with membrane-bound guanylate cyclase on photoreceptor disc membranes and to stimulate enzyme activity at these sites in dark-adapted retinas in a Ca²⁺-dependent manner. The results of these analyses are discussed in relation to the proposed functional role(s) of this enzyme.     

Triggered Ca2+ influx is required for extended synaptotagmin 1-induced ER-plasma membrane tethering

The extended synaptotagmins (E-Syts) are ER proteins that act as Ca²⁺-regulated tethers between the ER and the plasma membrane (PM) and have a putative role in lipid transport between the two membranes. Ca²⁺ regulation of their tethering function, as well as the interplay of their different domains in such function, remains poorly understood. By exposing semi-intact cells to buffers of variable Ca²⁺ concentrations, we found that binding of E-Syt1 to the PI(4,5)P₂-rich PM critically requires its C2C and C2E domains and that the EC₅₀ of such binding is in the low micromolar Ca²⁺ range. Accordingly, E-Syt1 accumulation at ER-PM contact sites occurred only upon experimental manipulations known to achieve these levels of Ca²⁺ via its influx from the extracellular medium, such as store-operated Ca²⁺ entry in fibroblasts and membrane depolarization in β-cells. We also show that in spite of their very different physiological functions, membrane tethering by E-Syt1 (ER to PM) and by synaptotagmin (secretory vesicles to PM) undergo a similar regulation by plasma membrane lipids and cytosolic Ca²⁺.     

The Zn2+ and Ca2+‐binding S100B and S100A1 proteins: beyond the myths

The S100 genes encode a conserved group of 21 vertebrate‐specific EF‐hand calcium‐binding proteins. Since their discovery in 1965, S100 proteins have remained enigmatic in terms of their cellular functions. In this review, we summarize the calcium‐ and zinc‐binding properties of the dimeric S100B and S100A1 proteins and highlight data that shed new light on the extracellular and intracellular regulation and functions of S100B. We point out that S100B and S100A1 homodimers are not functionally interchangeable and that in a S100A1/S100B heterodimer, S100A1 acts as a negative regulator for the ability of S100B to bind Zn²⁺. The Ca²⁺ and Zn²⁺‐dependent interactions of S100B with a wide array of proteins form the basis of its activities and have led to the derivation of some initial rules for S100B recognition of protein targets. However, recent findings have strongly suggested that these rules need to be revisited. Here, we describe a new consensus S100B binding motif present in intracellular and extracellular vertebrate‐specific proteins and propose a new model for stable interactions of S100B dimers with full‐length target proteins. A chaperone‐associated function for intracellular S100B in adaptive cellular stress responses is also discussed. This review may help guide future studies on the functions of S100 proteins in general.     

Ca2+‐dependent activation of guard cell anion channels,triggered by hyperpolarization,is promoted by prolonged depolarization

Rapid stomatal closure is driven by the activation of S‐type anion channels in the plasma membrane of guard cells. This response has been linked to Ca²⁺ signalling, but the impact of transient Ca²⁺ signals on S‐type anion channel activity remains unknown. In this study, transient elevation of the cytosolic Ca²⁺ level was provoked by voltage steps in guard cells of intact Nicotiana tabacum plants. Changes in the activity of S‐type anion channels were monitored using intracellular triple‐barrelled micro‐electrodes. In cells kept at a holding potential of ?100 mV, voltage steps to ?180 mV triggered elevation of the cytosolic free Ca²⁺ concentration. The increase in the cytosolic Ca²⁺ level was accompanied by activation of S‐type anion channels. Guard cell anion channels were activated by Ca²⁺ with a half maximum concentration of 515 nm (SE = 235) and a mean saturation value of ?349 pA (SE = 107) at ?100 mV. Ca²⁺ signals could also be evoked by prolonged (100 sec) depolarization of the plasma membrane to 0 mV. Upon returning to ?100 mV, a transient increase in the cytosolic Ca²⁺ level was observed, activating S‐type channels without measurable delay. These data show that cytosolic Ca²⁺ elevation can activate S‐type anion channels in intact guard cells through a fast signalling pathway. Furthermore, prolonged depolarization to 0 mV alters the activity of Ca²⁺ transport proteins, resulting in an overshoot of the cytosolic Ca²⁺ level after returning the membrane potential to ?100 mV.     

AHNAK interaction with the annexin 2/S100A10 complex regulates cell membrane cytoarchitecture

Remodelling of the plasma membrane cytoarchitecture is crucial for the regulation of epithelial cell adhesion and permeability. In Madin-Darby canine kidney cells, the protein AHNAK relocates from the cytosol to the cytosolic surface of the plasma membrane during the formation of cell-cell contacts and the development of epithelial polarity. This targeting is reversible and regulated by Ca(2+)-dependent cell-cell adhesion. At the plasma membrane, AHNAK associates as a multimeric complex with actin and the annexin 2/S100A10 complex. The S100A10 subunit serves to mediate the interaction between annexin 2 and the COOH-terminal regulatory domain of AHNAK. Down-regulation of both annexin 2 and S100A10 using an annexin 2-specific small interfering RNA inhibits the association of AHNAK with plasma membrane. In Madin-Darby canine kidney cells, down-regulation of AHNAK using AHNAK-specific small interfering RNA prevents cortical actin cytoskeleton reorganization required to support cell height. We propose that the interaction of AHNAK with the annexin 2/S100A10 regulates cortical actin cytoskeleton organization and cell membrane cytoarchitecture.     

Crystal structure of Ca2+ -free S100A2 at 1.6-A resolution

S100A2 is an EF hand-containing Ca^2 +-binding protein of the family of S100 proteins. The protein is localized exclusively in the nucleus and is involved in cell cycle regulation. It attracted most interest by its function as a tumor suppressor via p53 interaction. We determined the crystal structure of homodimeric S100A2 in the Ca^2 +-free state at 1.6-Å resolution. The structure revealed structural differences between subunits A and B, especially in the conformation of a loop that connects the N- and C-terminal EF hands and represents a part of the target-binding site in S100 proteins. Analysis of the hydrogen bonding network and molecular dynamics calculations indicate that one of the two observed conformations is more stable. The structure revealed Na⁺ bound to each N-terminal EF hand of both subunits coordinated by oxygen atoms of the backbone carbonyl and water molecules. Comparison with the structures of Ca^2 +-free S100A3 and S100A6 suggests that Na⁺ might occupy the S100-specific EF hand in the Ca^2 +-free state.     

Ca2+/S100 Proteins Act as Upstream Regulators of the Chaperone-associated Ubiquitin Ligase CHIP (C Terminus of Hsc70-interacting Protein)

The U-box E3 ubiquitin ligase CHIP (C terminus of Hsc70-interacting protein) binds Hsp90 and/or Hsp70 via its tetratricopeptide repeat (TPR), facilitating ubiquitination of the chaperone-bound client proteins. Mechanisms that regulate the activity of CHIP are, at present, poorly understood. We previously reported that Ca²⁺/S100 proteins directly associate with the TPR proteins, such as Hsp70/Hsp90-organizing protein (Hop), kinesin light chain, Tom70, FKBP52, CyP40, and protein phosphatase 5 (PP5), leading to the dissociation of the interactions of the TPR proteins with their target proteins. Therefore, we have hypothesized that Ca²⁺/S100 proteins can interact with CHIP and regulate its function. GST pulldown assays indicated that Ca²⁺/S100A2 and S100P bind to the TPR domain and lead to interference with the interactions of CHIP with Hsp70, Hsp90, HSF1, and Smad1. In vitro ubiquitination assays indicated that Ca²⁺/S100A2 and S100P are efficient and specific inhibitors of CHIP-mediated ubiquitination of Hsp70, Hsp90, HSF1, and Smad1. Overexpression of S100A2 and S100P suppressed CHIP-chaperone complex-dependent mutant p53 ubiquitination and degradation in Hep3B cells. The association of the S100 proteins with CHIP provides a Ca²⁺-dependent regulatory mechanism for the ubiquitination and degradation of intracellular proteins by the CHIP-proteasome pathway.     

RNA interference targeting against S100A4 suppresses cell growth and motility and induces apoptosis in human pancreatic cancer cells

S100A4 protein belongs to the S100 subfamily, which has grown to be one of the large subfamilies of the EF-hand Ca²⁺-binding proteins, and overexpression of S100A4 is suggested to associate with cell proliferation, invasion, and metastasis. We observed frequent overexpression of S100A4 in pancreatic cancer cell lines and further analyzed RNAi-mediated knockdown to address the possibility of its use as a therapeutic target for pancreatic cancer. The specific knockdown of S100A4 strongly suppressed cell growth, induced G2 arrest and eventual apoptosis, and decreased cell migration. Furthermore, microarray analyses revealed that knockdown of S100A4 induced expression of the tumor suppressor genes PRDM2 and VASH1. Our present results suggest the possibility that the inhibition of S100A4 can be utilized in antitumor applications for patients with pancreatic cancer.     

Investigation of the Ca2+-dependent interaction of trifluoperazine with S100a: A19F NMR and circular dichroism study

S100a is a heterodimeric, acidic calcium-binding protein that interacts with calmodulin antagonists in a Ca²⁺-dependent manner. In order to study the behavior of the hydrophobic domain on S100a when bound to Ca²⁺, its interaction with trifluoperazine (TFP) was investigated using¹⁶F nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy. The dissociation constant (K _d) values of TFP, as estimated from the chemical shifts of¹⁹F NMR, were 191 and 29 m in the absence and presence of Ca²⁺, respectively, and were similar to those previously reported for S100b. However, the TFP linewidth in the presence of Ca²⁺-bound S100a was 65 Hz greater than in the presence of Ca²⁺-bound S100b. This suggests a slower TFP exchange rate for S100a than for S100b. Thus, the TFP linewidths observed for each isoform may reflect differences in structural and modulatory properties of the Ca²⁺-dependent hydrophobic domains on S100a and S100b. Additionally, the presence of magnesium had no effect on the observed Ca²⁺-induced TFP spectral changes in S100a solutions. Circular dichroism studies indicate that Ca²⁺ induces a small transition from -helix to random coil in S100a; in contrast, the opposite transition is reported for calmodulin (Hennesseyet al., 1987). However, TFP did not significantly alter the secondary structure of Ca²⁺-bound S100a; this observation is similar to the effect of TFP on Ca²⁺-bound calmodulin and troponin C (Shimizu and Hatano, 1984; Gariépy and Hodges, 1983). It is, therefore, proposed that TFP binds to a hydrophobic domain on S100a in a fashion similar to other calcium-modulated proteins.     

The S100B Protein Inhibits Phosphorylation of GFAP and Vimentin in a Cytoskeletal Fraction from Immature Rat Hippocampus

The S100B protein belongs to a family of small Ca²⁺-binding proteins involved in several functions including cytoskeletal reorganization. The effect of S100B on protein phosphorylation was investigated in a cytoskeletal fraction prepared from immature rat hippocampus. An inhibitory effect of 5 M S100B on total protein phosphorylation, ranging from 25% to 40%, was observed in the presence of Ca²⁺ alone, Ca²⁺ plus calmodulin or Ca²⁺ plus cAMP. Analysis by two dimensional electrophoresis revealed a Ca²⁺/calmodulin-dependent and a Ca²⁺/cAMP-dependent inhibitory effect of S100B, ranging from 62% to 67% of control, on the phosphorylation of the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin. The fact that S100B binds to the N-terminal domain of GFAP and that the two proteins are co-localized in astrocytes suggests a potential in vivo role for S100B in modulating the phosphorylation of intermediate filament proteins in glia.