Activity of the Neuronal Cold Sensor TRPM8 Is Regulated by Phospholipase C via the Phospholipid Phosphoinositol 4,5-Bisphosphate

Cold temperatures robustly activate a small cohort of somatosensory nerves, yet during a prolonged cold stimulus their activity will decrease, or adapt, over time. This process allows for the discrimination of subtle changes in temperature. At the molecular level, cold is detected by transient receptor potential melastatin 8 (TRPM8), a nonselective cation channel expressed on a subset of peripheral afferent fibers. We and others have reported that TRPM8 channels also adapt in a calcium-dependent manner when activated by the cooling compound menthol. Additionally, TRPM8 activity is sensitive to the phospholipid phosphoinositol 4,5-bisphosphate (PIP₂), a substrate for the enzyme phospholipase C (PLC). These results suggest an adaptation model whereby TRPM8-mediated Ca²⁺ influx activates PLC, thereby decreasing PIP₂ levels and resulting in reduced TRPM8 activity. Here we tested this model using pharmacological activation of PLC and by manipulating PIP₂ levels independent of both PLC and Ca²⁺. PLC activation leads to adaptation-like reductions in cold- or menthol-evoked TRPM8 currents in both heterologous and native cells. Moreover, PLC-independent reductions in PIP₂ had a similar effect on cold- and menthol-evoked currents. Mechanistically, either form of adaptation does not alter temperature sensitivity of TRPM8 but does lead to a change in channel gating. Our results show that adaptation is a shift in voltage dependence toward more positive potentials, reversing the trend toward negative potentials caused by agonist. These data suggest that PLC activity not only mediates adaptation to thermal stimuli, but likely underlies a more general mechanism that establishes the temperature sensitivity of somatosensory neurons.The detection of temperature is a fundamental task of the nervous system. Temperature-sensing sensory afferent neurons reside in either the trigeminal (TG)² or dorsal root (DRG) sensory ganglia and project peripherally, terminating as free nerve endings that innervate areas of the head or trunk, respectively (1, 2). Subpopulations of these afferents respond to distinct sub-modalities of thermal stimuli, including noxious heat, innocuous cooling and warmth, and painfully cold temperatures. Each carries thermal information to the dorsal horn of the spinal cord, synapsing with neurons that project centrally (1, 3).The discovery of thermosensitive ion channels of the transient receptor potential (TRP) family demonstrated an underlying molecular mechanism for temperature detection (4). Cold temperature sensation is largely mediated by TRPM8, a nonselective cation channel expressed on a small subset of neurons (5, 6). TRPM8 is activated by cooling compounds, such as menthol, as well as cold temperatures below ∼28 °C, in vitro (7, 8). Recent reports on the behavioral phenotype of TRPM8-null mice suggest that this lone channel is required for the majority of cold sensing in vivo (5, 9–11). These and other data strongly implicate TRPM8 in not only the detection of both innocuous cool and some aspects of noxious cold but also injury-induced hypersensitivity to cold and, paradoxically, cooling-mediated analgesia (11, 12). Thus, understanding regulatory mechanisms that alter TRPM8 activity will provide keen insights into temperature sensation, nociception, and analgesia.One fundamental property of cold-sensitive neurons is an intrinsic ability to adapt to prolonged cold stimuli, a mechanism that is likely critical for discrimination of changing environmental conditions (13, 14). We and others have shown that cold-sensitive neurons adapt to cold and menthol over time in vitro (6, 15), a phenomenon also observed with recombinant TRPM8 channels activated by menthol (7). During sustained exposure to menthol, TRPM8 currents adapt in a manner that is dependent upon the presence of external calcium (7). Interestingly, cold- and menthol-evoked currents are highly sensitive to cellular manipulation. In heterologous cells, TRPM8 currents quickly decrease or run down upon membrane patch excision (16, 17). Moreover, in membrane patches excised from cold- and menthol-sensitive DRG neurons, cold thresholds for current activation exhibit a shift of ∼10 °C to colder temperatures in comparison with thresholds recorded in intact cells (18).Phosphatidylinositol 4,5-bisphosphate (PIP₂) is a membrane phospholipid that accounts for ∼1% of all lipids in the inner leaflet of the plasma membrane and is known to regulate a variety of ion channels, including TRPM8 (16, 17). When applied to the cytoplasmic face of excised membrane patches containing TRPM8 channels, PIP₂ can recover menthol-evoked currents to near pre-rundown levels (16, 17). PIP₂ is proposed to interact with channels either through electrostatic interactions or by binding to target proteins at specific phosphoinositide-binding sites (19, 20). Membrane PIP₂ levels are a product of enzymatic activity, such as phosphoinositide kinases that synthesize PIP₂ from membrane precursors and phospholipase C (PLC) that hydrolyzes it, creating membrane-bound diacylglycerol (DAG) and cytosolic inositol trisphosphate (IP₃), both of which function as second messengers. Of the three different PLC isotypes, PLCδ isoforms are modulated by increases in intracellular calcium (21).When taken in context with the sensitivity of TRPM8 currents to PIP₂ levels, a model has been proposed whereby adaption is a result of channel-mediated Ca²⁺ influx activating one or more PLCδ isoforms (16, 17). The subsequent reductions in PIP₂ levels then promote reduced or adapted TRPM8 currents. However, this hypothesis has not been conclusively shown in intact heterologous cells or in somatosensory neurons expressing TRPM8. Moreover, other alternative hypotheses for TRPM8 adaptation have been proposed, including Ca²⁺-dependent kinase activity mediated by protein kinase C (22, 23). Thus, the cellular and molecular mechanisms for Ca²⁺-mediated TRPM8 adaptation are unclear.Here we show, in both heterologous cells and native TRPM8-expressing neurons, that Ca²⁺-independent activation of PLC results in adapted TRPM8 currents. Moreover, PLC- and Ca²⁺-independent PIP₂ depletion in heterologous cells produces similar effects on TRPM8 activity, again reducing both cold- and menthol-evoked currents. Mechanistically, we find that all such manipulations do not alter the temperature sensitivity of the channel but do lead to a shift in the voltage dependence of TRPM8 channel gating.     

Tetrameric Orai1 Is a Teardrop-shaped Molecule with a Long, Tapered Cytoplasmic Domain

The Ca²⁺ release-activated Ca²⁺ channel is a principal regulator of intracellular Ca²⁺ rise, which conducts various biological functions, including immune responses. This channel, involved in store-operated Ca²⁺ influx, is believed to be composed of at least two major components. Orai1 has a putative channel pore and locates in the plasma membrane, and STIM1 is a sensor for luminal Ca²⁺ store depletion in the endoplasmic reticulum membrane. Here we have purified the FLAG-fused Orai1 protein, determined its tetrameric stoichiometry, and reconstructed its three-dimensional structure at 21-Å resolution from 3681 automatically selected particle images, taken with an electron microscope. This first structural depiction of a member of the Orai family shows an elongated teardrop-shape 150Å in height and 95Å in width. Antibody decoration and volume estimation from the amino acid sequence indicate that the widest transmembrane domain is located between the round extracellular domain and the tapered cytoplasmic domain. The cytoplasmic length of 100Å is sufficient for direct association with STIM1. Orifices close to the extracellular and intracellular membrane surfaces of Orai1 seem to connect outside the molecule to large internal cavities.Ca²⁺ is an intracellular second messenger that plays important roles in various physiological functions such as immune response, muscle contraction, neurotransmitter release, and cell proliferation. Intracellular Ca²⁺ is mainly stored in the endoplasmic reticulum (ER).² This ER system is distributed through the cytoplasm from around the nucleus to the cell periphery close to the plasma membrane. In non-excitable cells, the ER releases Ca²⁺ through the inositol 1,4,5-trisphosphate (IP₃) receptor channel in response to various signals, and the Ca²⁺ store is depleted. Depletion of Ca²⁺ then induces Ca²⁺ influx from outside the cell to help in refilling the Ca²⁺ stores and to continue Ca²⁺ rise for several minutes in the cytoplasm (1, 2). This Ca²⁺ influx was first proposed by Putney (3) and was named store-operated Ca²⁺ influx. In the immune system, store-operated Ca²⁺ influx is mainly mediated by the Ca²⁺ release-activated Ca²⁺ (CRAC) current, which is a highly Ca²⁺-selective inwardly rectified current with low conductance (4, 5). Pathologically, the loss of CRAC current in T cells causes severe combined immunodeficiency (6) where many Ca²⁺ signal-dependent gene expressions, including cytokines, are interrupted (7). Therefore, CRAC current is necessary for T cell functions.Recently, Orai1 (also called CRACM1) and STIM1 have been physiologically characterized as essential components of the CRAC channel (8–12). They are separately located in the plasma membrane and in the ER membrane; co-expression of these proteins presents heterologous CRAC-like currents in various types of cells (10, 13–15). Both of them are shown to be expressed ubiquitously in various tissues (16–18). STIM1 senses Ca²⁺ depletion in the ER through its EF hand motif (19) and transmits a signal to Orai1 in the plasma membrane. Although Orai1 is proposed as a regulatory component for some transient receptor potential canonical channels (20, 21), it is believed from the mutation analyses to be the pore-forming subunit of the CRAC channel (8, 22–24). In the steady state, both Orai1 and STIM1 molecules are dispersed in each membrane. When store depletion occurs, STIM1 proteins gather into clusters to form puncta in the ER membrane near the plasma membrane (11, 19). These clusters then trigger the clustering of Orai1 in the plasma membrane sites opposite the puncta (25, 26), and CRAC channels are activated (27).Orai1 has two homologous genes, Orai2 and Orai3 (8). They form the Orai family and have in common the four transmembrane (TM) segments with relatively large N and C termini. These termini are demonstrated to be in the cytoplasm, because both N- and C-terminally introduced tags are immunologically detected only in the membrane-permeabilized cells (8, 9). The subunit stoichiometry of Orai1 is as yet controversial: it is believed to be an oligomer, presumably a dimer or tetramer even in the steady state (16, 28–30).Despite the accumulation of biochemical and electrophysiological data, structural information about Orai1 is limited due to difficulties in purification and crystallization. In this study, we have purified Orai1 in its tetrameric form and have reconstructed the three-dimensional structure from negatively stained electron microscopic (EM) images.     

Differential Roles of Blocking Ions in KirBac1.1 Tetramer Stability

Potassium channels are tetrameric proteins that mediate K⁺-selective transmembrane diffusion. For KcsA, tetramer stability depends on interactions between permeant ions and the channel pore. We have examined the role of pore blockers on the tetramer stability of KirBac1.1. In 150 mm KCl, purified KirBac1.1 protein migrates as a monomer (∼40 kDa) on SDS-PAGE. Addition of Ba²⁺ (K_1/2 ∼ 50 μm) prior to loading results in an additional tetramer band (∼160 kDa). Mutation A109C, at a residue located near the expected Ba²⁺-binding site, decreased tetramer stabilization by Ba²⁺ (K_1/2 ∼ 300 μm), whereas I131C, located nearby, stabilized tetramers in the absence of Ba²⁺. Neither mutation affected Ba²⁺ block of channel activity (using ⁸⁶Rb⁺ flux assay). In contrast to Ba²⁺, Mg²⁺ had no effect on tetramer stability (even though Mg²⁺ was a potent blocker). Many studies have shown Cd²⁺ block of K⁺ channels as a result of cysteine substitution of cavity-lining M2 (S6) residues, with the implicit interpretation that coordination of a single ion by cysteine side chains along the central axis effectively blocks the pore. We examined blocking and tetramer-stabilizing effects of Cd²⁺ on KirBac1.1 with cysteine substitutions in M2. Cd²⁺ block potency followed an α-helical pattern consistent with the crystal structure. Significantly, Cd²⁺ strongly stabilized tetramers of I138C, located in the center of the inner cavity. This stabilization was additive with the effect of Ba²⁺, consistent with both ions simultaneously occupying the channel: Ba²⁺ at the selectivity filter entrance and Cd²⁺ coordinated by I138C side chains in the inner cavity.Potassium channels are expressed in many cell types and are key players in a wide range of physiological processes. One subset of potassium channels, the inward-rectifying potassium (Kir) channels, are functionally blocked by cytosolic cations such as Mg²⁺ and polyamines and contribute to the regulation of membrane excitability, cardiac rhythm, vascular tone, insulin release, and salt flow across epithelia (1–3). There are seven subfamilies of eukaryotic Kir channel genes. Among them, Kir1 encodes weak rectifiers, whereas Kir2 and Kir5 encode strong rectifiers; Kir3 encodes G-protein-regulated channels; and Kir6 encodes ATP-sensitive channels (4). Recently, a related bacterial family of genes (KirBac) has been identified (5, 6), and in 2003, the first member (KirBac1.1) was crystallized (7), providing a structural model for eukaryotic channels.The crystal structure of KirBac1.1 revealed a tetrameric pore structure similar to that seen in KcsA and a novel cytoplasmic domain (7, 8). The selectivity filter of both KirBac1.1 and KcsA consists of an extremely conserved pore loop followed by a central cavity, forming a transmembrane ion-selective permeation pore (7, 8). The linear arrangement of five oxygen rings (four from carbonyl oxygens and one from a Thr side chain) in the selectivity filter coordinates with ions, compensating for the energy barrier caused by K⁺ dehydration, thereby facilitating the rapid diffusion of K⁺ across the membrane (8–12). Two-thirds of the KirBac1.1 amino acid residues constitute the cytosolic domain that is highly conserved among the Kir subfamilies and form the cytosolic vestibule (13–16), which, together with the transmembrane pore, generates an 88-Å-long ion conduction pore (7).The prototypic potassium channel KcsA exists very stably as a tetramer, even in the harsh conditions of SDS-PAGE (17). In addition to protein-protein interaction between monomers, protein-lipid and protein-ion interactions play important roles in stabilizing the KcsA tetramer (17–20). The selectivity filter of KcsA, coordinated with K⁺ ions, can serve as a bridge between the four monomers to maintain the structure of the selectivity filter and the tetrameric architecture of the channel as a whole (11, 21). Blocking ions, such as Ba²⁺, also act as strong stabilizers (17). In the crystal structure of KcsA, Ba²⁺ occupies a site equivalent to the S4 K⁺-binding site within the selectivity filter (22). Other permeant ions (Rb⁺, Cs⁺, Tl⁺, and NH⁺₄) and strong blockers (Sr²⁺) can also contribute to the thermostability of the KcsA tetramer in SDS-PAGE (17). In contrast, impermeant ions such as Na⁺ and Li⁺ or weak blockers such as Mg²⁺ tend to destabilize the KcsA tetramer (17, 19).Like KcsA, KirBac1.1 purified using decylmaltoside or tridecylmaltoside is active and presumably stable as a tetramer in mild detergent solutions. However, in SDS-PAGE, KirBac1.1 migrates exclusively as a monomer (23). Because KcsA and KirBac1.1 are structurally similar in the transmembrane region of the pore, we hypothesized that permeant and blocking ions would also affect KirBac1.1 tetramer stability in SDS-PAGE. In the present work, the effects of blocking ions such as Ba²⁺ and Mg²⁺ on KirBac1.1 tetramer stability were examined to provide insight to the physical nature of their interaction with KirBac1.1, particularly in the selectivity filter and TM2 cavity. The data reveal important differences in the nature of the interaction of Mg²⁺ and Ba²⁺ with the channel as well as provide previously unavailable evidence for the nature of Cd²⁺ coordination within the channel.     

Effects of Combined Phosphorylation at Ser-617 and Ser-1179 in Endothelial Nitric-oxide Synthase on EC50(Ca2+) Values for Calmodulin Binding and Enzyme Activation

We have investigated the possible biochemical basis for enhancements in NO production in endothelial cells that have been correlated with agonist- or shear stress-evoked phosphorylation at Ser-1179. We have found that a phosphomimetic substitution at Ser-1179 doubles maximal synthase activity, partially disinhibits cytochrome c reductase activity, and lowers the EC₅₀(Ca²⁺) values for calmodulin binding and enzyme activation from the control values of 182 ± 2 and 422 ± 22 nm to 116 ± 2 and 300 ± 10 nm. These are similar to the effects of a phosphomimetic substitution at Ser-617 (Tran, Q. K., Leonard, J., Black, D. J., and Persechini, A. (2008) Biochemistry 47, 7557–7566). Although combining substitutions at Ser-617 and Ser-1179 has no additional effect on maximal synthase activity, cooperativity between the two substitutions completely disinhibits reductase activity and further reduces the EC₅₀(Ca²⁺) values for calmodulin binding and enzyme activation to 77 ± 2 and 130 ± 5 nm. We have confirmed that specific Akt-catalyzed phosphorylation of Ser-617 and Ser-1179 and phosphomimetic substitutions at these positions have similar functional effects. Changes in the biochemical properties of eNOS produced by combined phosphorylation at Ser-617 and Ser-1179 are predicted to substantially increase synthase activity in cells at a typical basal free Ca²⁺ concentration of 50–100 nm.The nitric-oxide synthases catalyze formation of NO and l-citrulline from l-arginine and O₂, with NADPH as the electron donor (1). The role of NO generated by endothelial nitricoxide synthase (eNOS)² in the regulation of smooth muscle tone is well established and was the first of several physiological roles for this small molecule that have so far been identified (2). The nitric-oxide synthases are homodimers of 130–160-kDa subunits. Each subunit contains a reductase and oxygenase domain (1). A significant difference between the reductase domains in eNOS and nNOS and the homologous P450 reductases is the presence of inserts in these synthase isoforms that appear to maintain them in their inactive states (3, 4). A calmodulin (CaM)-binding domain is located in the linker that connects the reductase and oxygenase domains, and the endothelial and neuronal synthases both require Ca²⁺ and exogenous CaM for activity (5, 6). When CaM is bound, it somehow counteracts the effects of the autoinhibitory insert(s) in the reductase. The high resolution structure for the complex between (Ca²⁺)₄-CaM and the isolated CaM-binding domain from eNOS indicates that the C-ter and N-ter lobes of CaM, which each contain a pair of Ca²⁺-binding sites, enfold the domain, as has been observed in several other such CaM-peptide complexes (7). Consistent with this structure, investigations of CaM-dependent activation of the neuronal synthase suggest that both CaM lobes must participate (8, 9).Bovine eNOS can be phosphorylated in endothelial cells at Ser-116, Thr-497, Ser-617, Ser-635, and Ser-1179 (10–12). There are equivalent phosphorylation sites in the human enzyme (10–12). Phosphorylation of the bovine enzyme at Thr-497, which is located in the CaM-binding domain, blocks CaM binding and enzyme activation (7, 11, 13, 14). Ser-116 can be basally phosphorylated in cells (10, 11, 13, 15), and dephosphorylation of this site has been correlated with increased NO production (13, 15). However, it has also been reported that a phosphomimetic substitution at this position has no effect on enzyme activity measured in vitro (13). Ser-1179 is phosphorylated in response to a variety of stimuli, and this has been reliably correlated with enhanced NO production in cells (10, 11). Indeed, NO production is elevated in transgenic endothelium expressing an eNOS mutant containing an S1179D substitution, but not in tissue expressing an S1179A mutant (16). Shear stress or insulin treatment is correlated with Akt-catalyzed phosphorylation of Ser-1179 in endothelial cells, and this is correlated with increased NO production in the absence of extracellular Ca²⁺ (17–19). Akt-catalyzed phosphorylation or an S1179D substitution has also been correlated with increased synthase activity in cell extracts at low intracellular free [Ca²⁺] (17). Increased NO production has also been observed in cells expressing an eNOS mutant containing an S617D substitution, and physiological stimuli such as shear-stress, bradykinin, VEGF, and ATP appear to stimulate Akt-catalyzed phosphorylation of Ser-617 and Ser-1179 (12, 13, 20). Although S617D eNOS has been reported to have the same maximum activity in vitro as the wild type enzyme (20), in our hands an S617D substitution increases the maximal CaM-dependent synthase activity of purified mutant enzyme ∼2-fold, partially disinhibits reductase activity, and reduces the EC₅₀(Ca²⁺) values for CaM binding and enzyme activation (21).In this report, we describe the effects of a phosphomimetic Asp substitution at Ser-1179 in eNOS on the Ca²⁺ dependence of CaM binding and CaM-dependent activation of reductase and synthase activities. We also describe the effects on these properties of combining this substitution with one at Ser-617. Finally, we demonstrate that Akt-catalyzed phosphorylation and Asp substitutions at Ser-617 and Ser-1179 have similar functional effects. Our results suggest that phosphorylation of eNOS at Ser-617 and Ser-1179 can substantially increase synthase activity in cells at a typical basal free Ca²⁺ concentration of 50–100 nm, while single phosphorylations at these sites produce smaller activity increases, and can do so only at higher free Ca²⁺ concentrations.     

Familial FTDP-17 Missense Mutations Inhibit Microtubule Assembly-promoting Activity of Tau by Increasing Phosphorylation at Ser202 in Vitro

In Alzheimer disease (AD), frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) and other tauopathies, tau accumulates and forms paired helical filaments (PHFs) in the brain. Tau isolated from PHFs is phosphorylated at a number of sites, migrates as ∼60-, 64-, and 68-kDa bands on SDS-gel, and does not promote microtubule assembly. Upon dephosphorylation, the PHF-tau migrates as ∼50–60-kDa bands on SDS-gels in a manner similar to tau that is isolated from normal brain and promotes microtubule assembly. The site(s) that inhibits microtubule assembly-promoting activity when phosphorylated in the diseased brain is not known. In this study, when tau was phosphorylated by Cdk5 in vitro, its mobility shifted from ∼60-kDa bands to ∼64- and 68-kDa bands in a time-dependent manner. This mobility shift correlated with phosphorylation at Ser²⁰², and Ser²⁰² phosphorylation inhibited tau microtubule-assembly promoting activity. When several tau point mutants were analyzed, G272V, P301L, V337M, and R406W mutations associated with FTDP-17, but not nonspecific mutations S214A and S262A, promoted Ser²⁰² phosphorylation and mobility shift to a ∼68-kDa band. Furthermore, Ser²⁰² phosphorylation inhibited the microtubule assembly-promoting activity of FTDP-17 mutants more than of WT. Our data indicate that FTDP-17 missense mutations, by promoting phosphorylation at Ser²⁰², inhibit the microtubule assembly-promoting activity of tau in vitro, suggesting that Ser²⁰² phosphorylation plays a major role in the development of NFT pathology in AD and related tauopathies.Neurofibrillary tangles (NFTs)⁴ and senile plaques are the two characteristic neuropathological lesions found in the brains of patients suffering from Alzheimer disease (AD). The major fibrous component of NFTs are paired helical filaments (PHFs) (for reviews see Refs. 1–3). Initially, PHFs were found to be composed of a protein component referred to as “A68” (4). Biochemical analysis reveled that A68 is identical to the microtubule-associated protein, tau (4, 5). Some characteristic features of tau isolated from PHFs (PHF-tau) are that it is abnormally hyperphosphorylated (phosphorylated on more sites than the normal brain tau), does not bind to microtubules, and does not promote microtubule assembly in vitro. Upon dephosphorylation, PHF-tau regains its ability to bind to and promote microtubule assembly (6, 7). Tau hyperphosphorylation is suggested to cause microtubule instability and PHF formation, leading to NFT pathology in the brain (1–3).PHF-tau is phosphorylated on at least 21 proline-directed and non-proline-directed sites (8, 9). The individual contribution of these sites in converting tau to PHFs is not entirely clear. However, some sites are only partially phosphorylated in PHFs (8), whereas phosphorylation on specific sites inhibits the microtubule assembly-promoting activity of tau (6, 10). These observations suggest that phosphorylation on a few sites may be responsible and sufficient for causing tau dysfunction in AD.Tau purified from the human brain migrates as ∼50–60-kDa bands on SDS-gel due to the presence of six isoforms that are phosphorylated to different extents (2). PHF-tau isolated from AD brain, on the other hand, displays ∼60-, 64-, and 68 kDa-bands on an SDS-gel (4, 5, 11). Studies have shown that ∼64- and 68-kDa tau bands (the authors have described the ∼68-kDa tau band as an ∼69-kDa band in these studies) are present only in brain areas affected by NFT degeneration (12, 13). Their amount is correlated with the NFT densities at the affected brain regions. Moreover, the increase in the amount of ∼64- and 68-kDa band tau in the brain correlated with a decline in the intellectual status of the patient. The ∼64- and 68-kDa tau bands were suggested to be the pathological marker of AD (12, 13). Biochemical analyses determined that ∼64- and 68-kDa bands are hyperphosphorylated tau, which upon dephosphorylation, migrated as normal tau on SDS-gel (4, 5, 11). Tau sites involved in the tau mobility shift to ∼64- and 68-kDa bands were suggested to have a role in AD pathology (12, 13). It is not known whether phosphorylation at all 21 PHF-sites is required for the tau mobility shift in AD. However, in vitro the tau mobility shift on SDS-gel is sensitive to phosphorylation only on some sites (6, 14). It is therefore possible that in the AD brain, phosphorylation on some sites also causes a tau mobility shift. Identification of such sites will significantly enhance our knowledge of how NFT pathology develops in the brain.PHFs are also the major component of NFTs found in the brains of patients suffering from a group of neurodegenerative disorders collectively called tauopathies (2, 11). These disorders include frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17), corticobasal degeneration, progressive supranuclear palsy, and Pick disease. Each PHF-tau isolated from autopsied brains of patients suffering from various tauopathies is hyperphosphorylated, displays ∼60-, 64-, and 68-kDa bands on SDS-gel, and is incapable of binding to microtubules. Upon dephosphorylation, the above referenced PHF-tau migrates as a normal tau on SDS-gel, binds to microtubules, and promotes microtubule assembly (2, 11). These observations suggest that the mechanisms of NFT pathology in various tauopathies may be similar and the phosphorylation-dependent mobility shift of tau on SDS-gel may be an indicator of the disease. The tau gene is mutated in familial FTDP-17, and these mutations accelerate NFT pathology in the brain (15–18). Understanding how FTDP-17 mutations promote tau phosphorylation can provide a better understanding of how NFT pathology develops in AD and various tauopathies. However, when expressed in CHO cells, G272V, R406W, V337M, and P301L tau mutations reduce tau phosphorylation (19, 20). In COS cells, although G272V, P301L, and V337M mutations do not show any significant affect, the R406W mutation caused a reduction in tau phosphorylation (21, 22). When expressed in SH-SY5Y cells subsequently differentiated into neurons, the R406W, P301L, and V337M mutations reduce tau phosphorylation (23). In contrast, in hippocampal neurons, R406W increases tau phosphorylation (24). When phosphorylated by recombinant GSK3β in vitro, the P301L and V337M mutations do not have any effect, and the R406W mutation inhibits phosphorylation (25). However, when incubated with rat brain extract, all of the G272V, P301L, V337M, and R406W mutations stimulate tau phosphorylation (26). The mechanism by which FTDP-17 mutations promote tau phosphorylation leading to development of NFT pathology has remained unclear.Cyclin-dependent protein kinase 5 (Cdk5) is one of the major kinases that phosphorylates tau in the brain (27, 28). In this study, to determine how FTDP-17 missense mutations affect tau phosphorylation, we phosphorylated four FTDP-17 tau mutants (G272V, P301L, V337M, and R406W) by Cdk5. We have found that phosphorylation of tau by Cdk5 causes a tau mobility shift to ∼64- and 68 kDa-bands. Although the mobility shift to a ∼64-kDa band is achieved by phosphorylation at Ser^396/404 or Ser²⁰², the mobility shift to a 68-kDa band occurs only in response to phosphorylation at Ser²⁰². We show that in vitro, FTDP-17 missense mutations, by promoting phosphorylation at Ser²⁰², enhance the mobility shift to ∼64- and 68-kDa bands and inhibit the microtubule assembly-promoting activity of tau. Our data suggest that Ser²⁰² phosphorylation is the major event leading to NFT pathology in AD and related tauopathies.     

Structural Insights into Ca2+-dependent Regulation of Inositol 1,4,5-Trisphosphate Receptors by CaBP1

Calcium-binding protein 1 (CaBP1), a neuron-specific member of the calmodulin (CaM) superfamily, modulates Ca²⁺-dependent activity of inositol 1,4,5-trisphosphate receptors (InsP₃Rs). Here we present NMR structures of CaBP1 in both Mg²⁺-bound and Ca²⁺-bound states and their structural interaction with InsP₃Rs. CaBP1 contains four EF-hands in two separate domains. The N-domain consists of EF1 and EF2 in a closed conformation with Mg²⁺ bound at EF1. The C-domain binds Ca²⁺ at EF3 and EF4, and exhibits a Ca²⁺-induced closed to open transition like that of CaM. The Ca²⁺-bound C-domain contains exposed hydrophobic residues (Leu¹³², His¹³⁴, Ile¹⁴¹, Ile¹⁴⁴, and Val¹⁴⁸) that may account for selective binding to InsP₃Rs. Isothermal titration calorimetry analysis reveals a Ca²⁺-induced binding of the CaBP1 C-domain to the N-terminal region of InsP₃R (residues 1-587), whereas CaM and the CaBP1 N-domain did not show appreciable binding. CaBP1 binding to InsP₃Rs requires both the suppressor and ligand-binding core domains, but has no effect on InsP₃ binding to the receptor. We propose that CaBP1 may regulate Ca²⁺-dependent activity of InsP₃Rs by promoting structural contacts between the suppressor and core domains.Calcium ion (Ca²⁺) in the cell functions as an important messenger that controls neurotransmitter release, gene expression, muscle contraction, apoptosis, and disease processes (1). Receptor stimulation in neurons promotes large increases in intracellular Ca²⁺ levels controlled by Ca²⁺ release from intracellular stores through InsP₃Rs (2). The neuronal type-1 receptor (InsP₃R1)² is positively and negatively regulated by cytosolic Ca²⁺ (3-6), important for the generation of repetitive Ca²⁺ transients known as Ca²⁺ spikes and waves (1). Ca²⁺-dependent activation of InsP₃R1 contributes to the fast rising phase of Ca²⁺ signaling known as Ca²⁺-induced Ca²⁺ release (7). Ca²⁺-induced inhibition of InsP₃R1, triggered at higher cytosolic Ca²⁺ levels, coordinates the temporal decay of Ca²⁺ transients (6). The mechanism of Ca²⁺-dependent regulation of InsP₃Rs is complex (8, 9), and involves direct Ca²⁺ binding sites (5, 10) as well as remote sensing by extrinsic Ca²⁺-binding proteins such as CaM (11, 12), CaBP1 (13, 14), CIB1 (15), and NCS-1 (16).Neuronal Ca²⁺-binding proteins (CaBP1-5 (17)) represent a new sub-branch of the CaM superfamily (18) that regulate various Ca²⁺ channel targets. Multiple splice variants and isoforms of CaBPs are localized in different neuronal cell types (19-21) and perform specialized roles in signal transduction. CaBP1, also termed caldendrin (22), has been shown to modulate the Ca²⁺-sensitive activity of InsP₃Rs (13, 14). CaBP1 also regulates P/Q-type voltage-gated Ca²⁺ channels (23), L-type channels (24), and the transient receptor potential channel, TRPC5 (25). CaBP4 regulates Ca²⁺-dependent inhibition of L-type channels in the retina and may be genetically linked to retinal degeneration (26). Thus, the CaBP proteins are receiving increased attention as a family of Ca²⁺ sensors that control a variety of Ca²⁺ channel targets implicated in neuronal degenerative diseases.CaBP proteins contain four EF-hands, similar in sequence to those found in CaM and troponin C (18) (Fig. 1). By analogy to CaM (27), the four EF-hands are grouped into two domains connected by a central linker that is four residues longer in CaBPs than in CaM. In contrast to CaM, the CaBPs contain non-conserved amino acids within the N-terminal region that may confer target specificity. Another distinguishing property of CaBPs is that the second EF-hand lacks critical residues required for high affinity Ca²⁺ binding (17). CaBP1 binds Ca²⁺ only at EF3 and EF4, whereas it binds Mg²⁺ at EF1 that may serve a functional role (28). Indeed, changes in cytosolic Mg²⁺ levels have been detected in cortical neurons after treatment with neurotransmitter (29). Other neuronal Ca²⁺-binding proteins such as DREAM (30), CIB1 (31), and NCS-1 (32) also bind Mg²⁺ and exhibit Mg²⁺-induced physiological effects. Mg²⁺ binding in each of these proteins helps stabilize their Ca²⁺-free state to interact with signaling targets.Open in a separate windowFIGURE 1.Amino acid sequence alignment of human CaBP1 with CaM. Secondary structural elements (α-helices and β-strands) were derived from NMR analysis. The four EF-hands (EF1, EF2, EF3, and EF4) are highlighted green, red, cyan, and yellow. Residues in the 12-residue Ca²⁺-binding loops are underlined and chelating residues are highlighted bold. Non-conserved residues in the hydrophobic patch are colored red.Despite extensive studies on CaBP1, little is known about its structure and target binding properties, and regulation of InsP₃Rs by CaBP1 is somewhat controversial and not well understood. Here, we present the NMR solution structures of both Mg²⁺-bound and Ca²⁺-bound conformational states of CaBP1 and their structural interactions with InsP₃R1. These CaBP1 structures reveal important Ca²⁺-induced structural changes that control its binding to InsP₃R1. Our target binding analysis demonstrates that the C-domain of CaBP1 exhibits Ca²⁺-induced binding to the N-terminal cytosolic region of InsP₃R1. We propose that CaBP1 may regulate Ca²⁺-dependent channel activity in InsP₃Rs by promoting a structural interaction between the N-terminal suppressor and ligand-binding core domains that modulates Ca²⁺-dependent channel gating (8, 33, 34).     

The Neuromediator Glutamate, through Specific Substrate Interactions, Enhances Mitochondrial ATP Production and Reactive Oxygen Species Generation in Nonsynaptic Brain Mitochondria

The finding that upon neuronal activation glutamate is transported postsynaptically from synaptic clefts and increased lactate availability for neurons suggest that brain mitochondria (BM) utilize a mixture of substrates, namely pyruvate, glutamate, and the tricarboxylic acid cycle metabolites. We studied how glutamate affected oxidative phosphorylation and reactive oxygen species (ROS) production in rat BM oxidizing pyruvate + malate or succinate. Simultaneous oxidation of glutamate + pyruvate + malate increased state 3 and uncoupled respiration by 52 and 71%, respectively. The state 4 ROS generation increased 100% over BM oxidizing pyruvate + malate and 900% over that of BM oxidizing glutamate + malate. Up to 70% of ROS generation was associated with reverse electron transport. These effects of pyruvate + glutamate + malate were observed only with BM and not with liver or heart mitochondria. The effects of glutamate + pyruvate on succinate-supported respiration and ROS generation were not organ-specific and depended only on whether mitochondria were isolated with or without bovine serum albumin. With the non-bovine serum albumin brain and heart mitochondria oxidizing succinate, the addition of pyruvate and glutamate abrogated inhibition of Complex II by oxaloacetate. We conclude that (i) during neuronal activation, simultaneous oxidation of glutamate + pyruvate temporarily enhances neuronal mitochondrial ATP production, and (ii) intrinsic inhibition of Complex II by oxaloacetate is an inherent mechanism that protects against ROS generation during reverse electron transport.Recently, it has emerged that mitochondrial dysfunctions play an important role in the pathogenesis of degenerative diseases of the central nervous system (1–3). The processes underlying neuronal degeneration are complex, and some authors suggest that several genetic alterations are involved (4). However, another level of complexity may be derived from the fact that virtually all cellular activities depend upon energy metabolism in the cell (5). Alterations in energy metabolism processes within cells may also contribute to pathogenic mechanisms underlying neurodegenerative disease.A large body of evidence suggests that increased oxidative stress is an important pathogenic mechanism that promotes neurodegeneration (6). Because neurons have a long life span, and most neurodegenerative diseases have a clear association with age (7), it is important to understand mechanisms underlying reactive oxygen species (ROS)² production in neurons. Recently, Kudin et al. (8) analyzed the contribution of mitochondria to the total ROS production in brain tissue. They concluded that mitochondria are the major source of ROS and that at least 50% of ROS generated by brain mitochondria was associated with succinate-supported reverse electron transport (RET). Under conditions of normoxia, about 1% of the respiratory chain electron flow was redirected to form superoxide (8).Recently, we suggested that the organization of the respiratory chain complexes into supercomplexes that occurs in brain mitochondria (BM) (9) may represent one of the intrinsic mechanisms to prevent excessive ROS generation (10). In this paper, we put forward the hypothesis that inhibition of Complex II by oxaloacetate (OAA) represents another important intrinsic mechanism to prevent oxidative stress. We provide evidence that glutamate and pyruvate specifically exert control over the production of ROS at the level of Complex II. Below we present a brief account of published theoretical and experimental evidence that underlie our hypothesis.The neural processing of information is metabolically expensive (11). More than 80% of energy is spent postsynaptically to restore the ionic composition of neurons (11). When neurons are activated, reuptake of glutamate stimulates aerobic glycolysis in astroglial cells (12), thereby making lactate the major substrate for neuronal mitochondria (4, 13). However, rapid conversion of lactate to pyruvate in neurons requires activation of the malate-aspartate shuttle (MAS). The shuttle is the major pathway for cytosolic reducing equivalents from NADH to enter the mitochondria and be oxidized (14, 15). The key component of MAS is the mitochondrial aspartate/glutamate carrier (AGC) (16), and recent data suggest that the AGC is expressed mainly in neurons (14). Absence of the AGC from astrocytes in the brain implies a compartmentation of intermediary metabolism, with glycolysis taking place in astrocytes and lactate oxidation in neurons (13, 14, 17). Active operation of MAS requires that a certain amount of glutamate must be transported from synaptic clefts into activated neurons. In isolated BM, it has been shown that besides pyruvate, glutamate is also a good respiratory substrate (5, 18). In the presynaptic elements, the concentration of cytosolic glutamate is ∼10 mm at all times (19). Yudkoff et al. (18) have shown that synaptosomal mitochondria utilize glutamate and pyruvate as mitochondrial respiratory substrates. Glutamate is also oxidized by the astroglial mitochondria (13).Until recently, it was generally accepted that most of the glutamate is rapidly removed from the synaptic cleft by glutamate transporters EAAT1 and EAAT2 located on presynaptic termini and glial cells (20–24). However, recent data show that a significant fraction of glutamate is rapidly bound and transported by the glutamate transporter isoform, EAAT4, located juxtasynaptically in the membranes of spines and dendrites (20, 25–28). At the climbing fiber to Purkinje cell synapses in the cerebellum, about 17% (28) or more than 50% (29) of synaptically released glutamate may be removed by postsynaptic transporters. Besides the cerebellum, EAAT4 protein was found to be omnipresent throughout the fore- and midbrain regions (30). Moreover, it was shown that although most of the EAAT2 protein is astroglial, around 15% is distributed in nerve terminals and axons in hippocampal slices and that this protein may be responsible for more than half of the total uptake of glutamate from synaptic clefts (24). These data suggest that postsynaptic transport of glutamate into nerve terminals where mitochondria are located (31) may occur in all brain regions. According to calculations of Brasnjo and Otis (28), in a single synapse, EAAT4 (excitatory amino acid transporter 4) binds and transports postsynaptically about 1.3 ± 0.1 × 10⁶ glutamate molecules. In the brain, on average, 1 mm³ of tissue contains 1 × 10⁸ synapses (32, 33). Because of the high density of synaptic contacts, the neuronal cells may be exposed to mediators released from hundreds of firing synapses. Thus, in a narrow space of spines and dendrites, several million glutamate molecules postsynaptically transported from synaptic boutons may create local cytosolic concentration of glutamate in the low millimolar range. Consequently, neuronal mitochondria, particularly those located at the axonal or dendritic synaptic junctions, may, in addition to metabolizing pyruvate, temporarily metabolize glutamate and succinate formed during mitochondrial catabolism of γ-aminobutyric acid in postsynaptic cells (34).The purpose of this study was to examine how the neuromediator glutamate affects respiratory activity and ROS generation in nonsynaptic BM when combined with pyruvate and the tricarboxylic acid cycle intermediates succinate and malate. We show that with pyruvate + glutamate + malate, the rate of oxidative phosphorylation increased more than 50%, and in resting mitochondria the rate of ROS generation associated with the reverse electron transport increased severalfold. These effects were observed only with brain and spinal cord mitochondria, not with liver or heart mitochondria, suggesting that they may be restricted to neuronal cells.Taken together, the data presented support the hypothesis that in activated neurons, the neuromediator glutamate stimulates mitochondrial ATP production when energy demand is increased. However, in the absence of energy consumption, glutamate + pyruvate may increase the generation of ROS severalfold. We suggest that intrinsic inhibition of Complex II by oxaloacetate is an important natural protective mechanism against ROS associated with reverse electron transport.     

Engineered Protein Connectivity to Actin Mimics PDZ-dependent Recycling of G Protein-coupled Receptors but Not Its Regulation by Hrs

Many G protein-coupled receptors (GPCRs) recycle after agonist-induced endocytosis by a sequence-dependent mechanism, which is distinct from default membrane flow and remains poorly understood. Efficient recycling of the β2-adrenergic receptor (β2AR) requires a C-terminal PDZ (PSD-95/Discs Large/ZO-1) protein-binding determinant (PDZbd), an intact actin cytoskeleton, and is regulated by the endosomal protein Hrs (hepatocyte growth factor-regulated substrate). The PDZbd is thought to link receptors to actin through a series of protein interaction modules present in NHERF/EBP50 (Na⁺/H+ exchanger 3 regulatory factor/ezrin-binding phosphoprotein of 50 kDa) family and ERM (ezrin/radixin/moesin) family proteins. It is not known, however, if such actin connectivity is sufficient to recapitulate the natural features of sequence-dependent recycling. We addressed this question using a receptor fusion approach based on the sufficiency of the PDZbd to promote recycling when fused to a distinct GPCR, the δ-opioid receptor, which normally recycles inefficiently in HEK293 cells. Modular domains mediating actin connectivity promoted receptor recycling with similarly high efficiency as the PDZbd itself, and recycling promoted by all of the domains was actin-dependent. Regulation of receptor recycling by Hrs, however, was conferred only by the PDZbd and not by downstream interaction modules. These results suggest that actin connectivity is sufficient to mimic the core recycling activity of a GPCR-linked PDZbd but not its cellular regulation.G protein-coupled receptors (GPCRs)² comprise the largest family of transmembrane signaling receptors expressed in animals and transduce a wide variety of physiological and pharmacological information. While these receptors share a common 7-transmembrane-spanning topology, structural differences between individual GPCR family members confer diverse functional and regulatory properties (1-4). A fundamental mechanism of GPCR regulation involves agonist-induced endocytosis of receptors via clathrin-coated pits (4). Regulated endocytosis can have multiple functional consequences, which are determined in part by the specificity with which internalized receptors traffic via divergent downstream membrane pathways (5-7).Trafficking of internalized GPCRs to lysosomes, a major pathway traversed by the δ-opioid receptor (δOR), contributes to proteolytic down-regulation of receptor number and produces a prolonged attenuation of subsequent cellular responsiveness to agonist (8, 9). Trafficking of internalized GPCRs via a rapid recycling pathway, a major route traversed by the β2-adrenergic receptor (β2AR), restores the complement of functional receptors present on the cell surface and promotes rapid recovery of cellular signaling responsiveness (6, 10, 11). When co-expressed in the same cells, the δOR and β2AR are efficiently sorted between these divergent downstream membrane pathways, highlighting the occurrence of specific molecular sorting of GPCRs after endocytosis (12).Recycling of various integral membrane proteins can occur by default, essentially by bulk membrane flow in the absence of lysosomal sorting determinants (13). There is increasing evidence that various GPCRs, such as the β2AR, require distinct cytoplasmic determinants to recycle efficiently (14). In addition to requiring a cytoplasmic sorting determinant, sequence-dependent recycling of the β2AR differs from default recycling in its dependence on an intact actin cytoskeleton and its regulation by the conserved endosomal sorting protein Hrs (hepatocyte growth factor receptor substrate) (11, 14). Compared with the present knowledge regarding protein complexes that mediate sorting of GPCRs to lysosomes (15, 16), however, relatively little is known about the biochemical basis of sequence-directed recycling or its regulation.The β2AR-derived recycling sequence conforms to a canonical PDZ (PSD-95/Discs Large/ZO-1) protein-binding determinant (henceforth called PDZbd), and PDZ-mediated protein association(s) with this sequence appear to be primarily responsible for its endocytic sorting activity (17-20). Fusion of this sequence to the cytoplasmic tail of the δOR effectively re-routes endocytic trafficking of engineered receptors from lysosomal to recycling pathways, establishing the sufficiency of the PDZbd to function as a transplantable sorting determinant (18). The β2AR-derived PDZbd binds with relatively high specificity to the NHERF/EBP50 family of PDZ proteins (21, 22). A well-established biochemical function of NHERF/EBP50 family proteins is to associate integral membrane proteins with actin-associated cytoskeletal elements. This is achieved through a series of protein-interaction modules linking NHERF/EBP50 family proteins to ERM (ezrin-radixin-moesin) family proteins and, in turn, to actin filaments (23-26). Such indirect actin connectivity is known to mediate other effects on plasma membrane organization and function (23), however, and NHERF/EBP50 family proteins can bind to additional proteins potentially important for endocytic trafficking of receptors (23, 25). Thus it remains unclear if actin connectivity is itself sufficient to promote sequence-directed recycling of GPCRs and, if so, if such connectivity recapitulates the normal cellular regulation of sequence-dependent recycling. In the present study, we took advantage of the modular nature of protein connectivity proposed to mediate β2AR recycling (24, 26), and extended the opioid receptor fusion strategy used successfully for identifying diverse recycling sequences in GPCRs (27-29), to address these fundamental questions.Here we show that the recycling activity of the β2AR-derived PDZbd can be effectively bypassed by linking receptors to ERM family proteins in the absence of the PDZbd itself. Further, we establish that the protein connectivity network can be further simplified by fusing receptors to an interaction module that binds directly to actin filaments. We found that bypassing the PDZ-mediated interaction using either domain is sufficient to mimic the ability of the PDZbd to promote efficient, actin-dependent recycling of receptors. Hrs-dependent regulation, however, which is characteristic of sequence-dependent recycling of wild-type receptors, was recapitulated only by the fused PDZbd and not by the proposed downstream interaction modules. These results support a relatively simple architecture of protein connectivity that is sufficient to mimic the core recycling activity of the β2AR-derived PDZbd, but not its characteristic cellular regulation. Given that an increasing number of GPCRs have been shown to bind PDZ proteins that typically link directly or indirectly to cytoskeletal elements (17, 27, 30-32), the present results also suggest that actin connectivity may represent a common biochemical principle underlying sequence-dependent recycling of various GPCRs.     

Overexpression of E-cadherin on Melanoma Cells Inhibits Chemokine-promoted Invasion Involving p190RhoGAP/p120ctn-dependent Inactivation of RhoA

Melanoma cells express the chemokine receptor CXCR4 that confers high invasiveness upon binding to its ligand CXCL12. Melanoma cells at initial stages of the disease show reduction or loss of E-cadherin expression, but recovery of its expression is frequently found at advanced phases. We overexpressed E-cadherin in the highly invasive BRO lung metastatic cell melanoma cell line to investigate whether it could influence CXCL12-promoted cell invasion. Overexpression of E-cadherin led to defective invasion of melanoma cells across Matrigel and type I collagen in response to CXCL12. A decrease in individual cell migration directionality toward the chemokine and reduced adhesion accounted for the impaired invasion. A p190RhoGAP-dependent inhibition of RhoA activation was responsible for the impairment in chemokine-stimulated E-cadherin melanoma transfectant invasion. Furthermore, we show that p190RhoGAP and p120ctn associated predominantly on the plasma membrane of cells overexpressing E-cadherin, and that E-cadherin-bound p120ctn contributed to RhoA inactivation by favoring p190RhoGAP-RhoA association. These results suggest that melanoma cells at advanced stages of the disease could have reduced metastatic potency in response to chemotactic stimuli compared with cells lacking E-cadherin, and the results indicate that p190RhoGAP is a central molecule controlling melanoma cell invasion.Cadherins are a family of Ca²⁺-dependent adhesion molecules that mediate cell-cell contacts and are expressed in most solid tissues providing a tight control of morphogenesis (1, 2). Classical cadherins, such as epithelial (E) cadherin, are found in adherens junctions, forming core protein complexes with β-catenin, α-catenin, and p120 catenin (p120ctn). Both β-catenin and p120ctn directly interact with E-cadherin, whereas α-catenin associates with the complex through its binding to β-catenin, providing a link with the actin cytoskeleton (1, 2). E-cadherin is frequently lost or down-regulated in many human tumors, coincident with morphological epithelial to mesenchymal transition and acquisition of invasiveness (3-6).Although melanoma only accounts for 5% of skin cancers, when metastasis starts, it is responsible for 80% of deaths from skin cancers (7). Melanocytes express E-cadherin (8-10), but melanoma cells at early radial growth phase show a large reduction in the expression of this cadherin, and surprisingly, expression has been reported to be partially recovered by vertical growth phase and metastatic melanoma cells (9, 11, 12).Trafficking of cancer cells from primary tumor sites to intravasation into blood circulation and later to extravasation to colonize distant organs requires tightly regulated directional cues and cell migration and invasion that are mediated by chemokines, growth factors, and adhesion molecules (13). Solid tumor cells express chemokine receptors that provide guidance of these cells to organs where their chemokine ligands are expressed, constituting a homing model resembling the one used by immune cells to exert their immune surveillance functions (14). Most solid cancer cells express CXCR4, a receptor for the chemokine CXCL12 (also called SDF-1), which is expressed in lungs, bone marrow, and liver (15). Expression of CXCR4 in human melanoma has been detected in the vertical growth phase and on regional lymph nodes, which correlated with poor prognosis and increased mortality (16, 17). Previous in vivo experiments have provided evidence supporting a crucial role for CXCR4 in the metastasis of melanoma cells (18).Rho GTPases control the dynamics of the actin cytoskeleton during cell migration (19, 20). The activity of Rho GTPases is tightly regulated by guanine-nucleotide exchange factors (GEFs),⁴ which stimulate exchange of bound GDP by GTP, and inhibited by GTPase-activating proteins (GAPs), which promote GTP hydrolysis (21, 22), whereas guanine nucleotide dissociation inhibitors (GDIs) appear to mediate blocking of spontaneous activation (23). Therefore, cell migration is finely regulated by the balance between GEF, GAP, and GDI activities on Rho GTPases. Involvement of Rho GTPases in cancer is well documented (reviewed in Ref. 24), providing control of both cell migration and growth. RhoA and RhoC are highly expressed in colon, breast, and lung carcinoma (25, 26), whereas overexpression of RhoC in melanoma leads to enhancement of cell metastasis (27). CXCL12 activates both RhoA and Rac1 in melanoma cells, and both GTPases play key roles during invasion toward this chemokine (28, 29).Given the importance of the CXCL12-CXCR4 axis in melanoma cell invasion and metastasis, in this study we have addressed the question of whether changes in E-cadherin expression on melanoma cells might affect cell invasiveness. We show here that overexpression of E-cadherin leads to impaired melanoma cell invasion to CXCL12, and we provide mechanistic characterization accounting for the decrease in invasion.     

The Serine Protease Plasmin Cleaves the Amino-terminal Domain of the NR2A Subunit to Relieve Zinc Inhibition of the N-Methyl-d-aspartate Receptors

Zinc is hypothesized to be co-released with glutamate at synapses of the central nervous system. Zinc binds to NR1/NR2A N-methyl-d-aspartate (NMDA) receptors with high affinity and inhibits NMDAR function in a voltage-independent manner. The serine protease plasmin can cleave a number of substrates, including protease-activated receptors, and may play an important role in several disorders of the central nervous system, including ischemia and spinal cord injury. Here, we demonstrate that plasmin can cleave the native NR2A amino-terminal domain (NR2A_ATD), removing the functional high affinity Zn²⁺ binding site. Plasmin also cleaves recombinant NR2A_ATD at lysine 317 (Lys³¹⁷), thereby producing a ∼40-kDa fragment, consistent with plasmin-induced NR2A cleavage fragments observed in rat brain membrane preparations. A homology model of the NR2A_ATD predicts that Lys³¹⁷ is near the surface of the protein and is accessible to plasmin. Recombinant expression of NR2A with an amino-terminal deletion at Lys³¹⁷ is functional and Zn²⁺ insensitive. Whole cell voltage-clamp recordings show that Zn²⁺ inhibition of agonist-evoked NMDA receptor currents of NR1/NR2A-transfected HEK 293 cells and cultured cortical neurons is significantly reduced by plasmin treatment. Mutating the plasmin cleavage site Lys³¹⁷ on NR2A to alanine blocks the effect of plasmin on Zn²⁺ inhibition. The relief of Zn²⁺ inhibition by plasmin occurs in PAR1^-/- cortical neurons and thus is independent of interaction with protease-activated receptors. These results suggest that plasmin can directly interact with NMDA receptors, and plasmin may increase NMDA receptor responses through disruption or removal of the amino-terminal domain and relief of Zn²⁺ inhibition.N-Methyl-d-aspartate (NMDA)² receptors are one of three types of ionotropic glutamate receptors that play critical roles in excitatory neurotransmission, synaptic plasticity, and neuronal death (1–3). NMDA receptors are comprised of glycine-binding NR1 subunits in combination with at least one type of glutamate-binding NR2 subunit (1, 4). Each subunit contains three transmembrane domains, one cytoplasmic re-entrant membrane loop, one bi-lobed domain that forms the ligand binding site, and one bi-lobed amino-terminal domain (ATD), thought to share structural homology to periplasmic amino acid-binding proteins (4–6). Activation of NMDA receptors requires combined stimulation by glutamate and the co-agonist glycine in addition to membrane depolarization to overcome voltage-dependent Mg²⁺ block of the ion channel (7). The activity of NMDA receptors is negatively modulated by a variety of extracellular ions, including Mg²⁺, polyamines, protons, and Zn²⁺ ions, which can exert tonic inhibition under physiological conditions (1, 4). Several extracellular modulators such as Zn²⁺ and ifenprodil are thought to act at the ATD of the NMDA receptor (8–14).Zinc is a transition metal that plays key roles in both catalytic and structural capacities in all mammalian cells (15). Zinc is required for normal growth and survival of cells. In addition, neuronal death in hypoxia-ischemia and epilepsy has been associated with Zn²⁺ (16–18). Abnormal metabolism of zinc may contribute to induction of cytotoxicity in neurodegenerative diseases, such as Alzheimer''s disease, Parkinson''s disease, and amyotrophic lateral sclerosis (19). Zinc is co-released with glutamate at excitatory presynaptic terminals and inhibits native NMDA receptor activation (20, 21). Zn²⁺ inhibits NMDA receptor function through a dual mechanism, which includes voltage-dependent block and voltage-independent inhibition (22–24). Voltage-independent Zn²⁺ inhibition at low nanomolar concentrations (IC₅₀, 20 nm) is observed for NR2A-containing NMDA receptors (25–28). Evidence has accumulated that the amino-terminal domain of the NR2A subunit controls high-affinity Zn²⁺ inhibition of NMDA receptors, and several histidine residues in this region may constitute part of an NR2A-specific Zn²⁺ binding site (8, 9, 11, 12). For the NR2A subunit, several lines of evidence suggest that Zn²⁺ acts by enhancing proton inhibition (8, 11, 29, 30).Serine proteases present in the circulation, mast cells, and elsewhere signal directly to cells by cleaving protease-activated receptors (PARs), members of a subfamily of G-protein-coupled receptors. Cleavage exposes a tethered ligand domain that binds to and activates the cleaved receptors (31, 32). Protease receptor activation has been studied extensively in relation to coagulation and thrombolysis (33). In addition to their circulation in the bloodstream, some serine proteases and PARs are expressed in the central nervous system, and have been suggested to play roles in physiological conditions (e.g. long-term potentiation or memory) and pathophysiological states such as glial scarring, edema, seizure, and neuronal death (31, 34–36).Functional interactions between proteases and NMDA receptors have previously been suggested. Earlier studies reported that the blood-derived serine protease thrombin potentiates NMDA receptor response more than 2-fold through activation of PAR1 (37). Plasmin, another serine protease, similarly potentiates NMDA receptor response (38). Tissue-plasminogen activator (tPA), which catalyzes the conversion of the zymogen precursor plasminogen to plasmin and results in PAR1 activation, also interacts with and cleaves the ATD of the NR1 subunit of the NMDA receptor (39, 40). This raises the possibility that plasmin may also interact directly with the NMDA receptor subunits to modulate receptor response. We therefore investigated the ability of plasmin to cleave the NR2A NMDA receptor subunit. We found that nanomolar concentrations of plasmin can cleave within the ATD, a region that mediates tonic voltage-independent Zn²⁺ inhibition of NR2A-containing NMDA receptors. We hypothesized that plasmin cleavage reduces the Zn²⁺-mediated inhibition of NMDA receptors by removing the Zn²⁺ binding domain. In the present study, we have demonstrated that Zn²⁺ inhibition of agonist-evoked NMDA currents is decreased significantly by plasmin treatment in recombinant NR1/NR2A-transfected HEK 293 cells and cultured cortical neurons. These concentrations of plasmin may be pathophysiologically relevant in situations in which the blood-brain barrier is compromised, which could allow blood-derived plasmin to enter brain parenchyma at concentrations in excess of these that can cleave NR2A. Thus, ability of plasmin to potentiate NMDA function through the relief of the Zn²⁺ inhibition could exacerbate the harmful actions of NMDA receptor overactivation in pathological situations. In addition, if newly cleaved NR2A_ATD enters the bloodstream during ischemic injury, it could serve as a biomarker of central nervous system injury.     

Galectin-8 Induces Apoptosis in Jurkat T Cells by Phosphatidic Acid-mediated ERK1/2 Activation Supported by Protein Kinase A Down-regulation

Galectins have been implicated in T cell homeostasis playing complementary pro-apoptotic roles. Here we show that galectin-8 (Gal-8) is a potent pro-apoptotic agent in Jurkat T cells inducing a complex phospholipase D/phosphatidic acid signaling pathway that has not been reported for any galectin before. Gal-8 increases phosphatidic signaling, which enhances the activity of both ERK1/2 and type 4 phosphodiesterases (PDE4), with a subsequent decrease in basal protein kinase A activity. Strikingly, rolipram inhibition of PDE4 decreases ERK1/2 activity. Thus Gal-8-induced PDE4 activation releases a negative influence of cAMP/protein kinase A on ERK1/2. The resulting strong ERK1/2 activation leads to expression of the death factor Fas ligand and caspase-mediated apoptosis. Several conditions that decrease ERK1/2 activity also decrease apoptosis, such as anti-Fas ligand blocking antibodies. In addition, experiments with freshly isolated human peripheral blood mononuclear cells, previously stimulated with anti-CD3 and anti-CD28, show that Gal-8 is pro-apoptotic on activated T cells, most likely on a subpopulation of them. Anti-Gal-8 autoantibodies from patients with systemic lupus erythematosus block the apoptotic effect of Gal-8. These results implicate Gal-8 as a novel T cell suppressive factor, which can be counterbalanced by function-blocking autoantibodies in autoimmunity.Glycan-binding proteins of the galectin family have been increasingly studied as regulators of the immune response and potential therapeutic agents for autoimmune disorders (1). To date, 15 galectins have been identified and classified according with the structural organization of their distinctive monomeric or dimeric carbohydrate recognition domain for β-galactosides (2, 3). Galectins are secreted by unconventional mechanisms and once outside the cells bind to and cross-link multiple glycoconjugates both at the cell surface and at the extracellular matrix, modulating processes as diverse as cell adhesion, migration, proliferation, differentiation, and apoptosis (4–10). Several galectins have been involved in T cell homeostasis because of their capability to kill thymocytes, activated T cells, and T cell lines (11–16). Pro-apoptotic galectins might contribute to shape the T cell repertoire in the thymus by negative selection, restrict the immune response by eliminating activated T cells at the periphery (1), and help cancer cells to escape the immune system by eliminating cancer-infiltrating T cells (17). They have also a promising therapeutic potential to eliminate abnormally activated T cells and inflammatory cells (1). Studies on the mostly explored galectins, Gal-1, -3, and -9 (14, 15, 18–20), as well as in Gal-2 (13), suggest immunosuppressive complementary roles inducing different pathways to apoptosis. Galectin-8 (Gal-8)⁴ is one of the most widely expressed galectins in human tissues (21, 22) and cancerous cells (23, 24). Depending on the cell context and mode of presentation, either as soluble stimulus or extracellular matrix, Gal-8 can promote cell adhesion, spreading, growth, and apoptosis (6, 7, 9, 10, 22, 25). Its role has been mostly studied in relation to tumor malignancy (23, 24). However, there is some evidence regarding a role for Gal-8 in T cell homeostasis and autoimmune or inflammatory disorders. For instance, the intrathymic expression and pro-apoptotic effect of Gal-8 upon CD4^highCD8^high thymocytes suggest a role for Gal-8 in shaping the T cell repertoire (16). Gal-8 could also modulate the inflammatory function of neutrophils (26), Moreover Gal-8-blocking agents have been detected in chronic autoimmune disorders (10, 27, 28). In rheumatoid arthritis, Gal-8 has an anti-inflammatory action, promoting apoptosis of synovial fluid cells, but can be counteracted by a specific rheumatoid version of CD44 (CD44vRA) (27). In systemic lupus erythematosus (SLE), a prototypic autoimmune disease, we recently described function-blocking autoantibodies against Gal-8 (10, 28). Thus it is important to define the role of Gal-8 and the influence of anti-Gal-8 autoantibodies in immune cells.In Jurkat T cells, we previously reported that Gal-8 interacts with specific integrins, such as α1β1, α3β1, and α5β1 but not α4β1, and as a matrix protein promotes cell adhesion and asymmetric spreading through activation of the extracellular signal-regulated kinases 1 and 2 (ERK1/2) (10). These early effects occur within 5–30 min. However, ERK1/2 signaling supports long term processes such as T cell survival or death, depending on the moment of the immune response. During T cell activation, ERK1/2 contributes to enhance the expression of interleukin-2 (IL-2) required for T cell clonal expansion (29). It also supports T cell survival against pro-apoptotic Fas ligand (FasL) produced by themselves and by other previously activated T cells (30, 31). Later on, ERK1/2 is required for activation-induced cell death, which controls the extension of the immune response by eliminating recently activated and restimulated T cells (32, 33). In activation-induced cell death, ERK1/2 signaling contributes to enhance the expression of FasL and its receptor Fas/CD95 (32, 33), which constitute a preponderant pro-apoptotic system in T cells (34). Here, we ask whether Gal-8 is able to modulate the intensity of ERK1/2 signaling enough to participate in long term processes involved in T cell homeostasis.The functional integration of ERK1/2 and PKA signaling (35) deserves special attention. cAMP/PKA signaling plays an immunosuppressive role in T cells (36) and is altered in SLE (37). Phosphodiesterases (PDEs) that degrade cAMP release the immunosuppressive action of cAMP/PKA during T cell activation (38, 39). PKA has been described to control the activity of ERK1/2 either positively or negatively in different cells and processes (35). A little explored integration among ERK1/2 and PKA occurs via phosphatidic acid (PA) and PDE signaling. Several stimuli activate phospholipase D (PLD) that hydrolyzes phosphatidylcholine into PA and choline. Such PLD-generated PA plays roles in signaling interacting with a variety of targeting proteins that bear PA-binding domains (40). In this way PA recruits Raf-1 to the plasma membrane (41). It is also converted by phosphatidic acid phosphohydrolase (PAP) activity into diacylglycerol (DAG), which among other functions, recruits and activates the GTPase Ras (42). Both Ras and Raf-1 are upstream elements of the ERK1/2 activation pathway (43). In addition, PA binds to and activates PDEs of the type 4 subfamily (PDE4s) leading to decreased cAMP levels and PKA down-regulation (44). The regulation and role of PA-mediated control of ERK1/2 and PKA remain relatively unknown in T cell homeostasis, because it is also unknown whether galectins stimulate the PLD/PA pathway.Here we found that Gal-8 induces apoptosis in Jurkat T cells by triggering cross-talk between PKA and ERK1/2 pathways mediated by PLD-generated PA. Our results for the first time show that a galectin increases the PA levels, down-regulates the cAMP/PKA system by enhancing rolipram-sensitive PDE activity, and induces an ERK1/2-dependent expression of the pro-apoptotic factor FasL. The enhanced PDE activity induced by Gal-8 is required for the activation of ERK1/2 that finally leads to apoptosis. Gal-8 also induces apoptosis in human peripheral blood mononuclear cells (PBMC), especially after activating T cells with anti-CD3/CD28. Therefore, Gal-8 shares with other galectins the property of killing activated T cells contributing to the T cell homeostasis. The pathway involves a particularly integrated signaling context, engaging PLD/PA, cAMP/PKA, and ERK1/2, which so far has not been reported for galectins. The pro-apoptotic function of Gal-8 also seems to be unique in its susceptibility to inhibition by anti-Gal-8 autoantibodies.     

Structure and Orientation of Troponin in the Thin Filament

The troponin complex on the thin filament plays a crucial role in the regulation of muscle contraction. However, the precise location of troponin relative to actin and tropomyosin remains uncertain. We have developed a method of reconstructing thin filaments using single particle analysis that does not impose the helical symmetry of actin and is independent of a starting model. We present a single particle three-dimensional reconstruction of the thin filament. Atomic models of the F-actin filament were fitted into the electron density maps and troponin and tropomyosin located. The structure provides evidence that the globular head region of troponin labels the two strands of actin with a 27.5-Å axial stagger. The density attributed to troponin appears tapered with the widest point toward the barbed end. This leads us to interpret the polarity of the troponin complex in the thin filament as reversed with respect to the widely accepted model.Regulation of actin filament function is a fundamental biological process with implications ranging from cell migration to muscle contraction. Skeletal and cardiac muscle thin filaments consist of actin and the regulatory proteins troponin and tropomyosin. Contraction is initiated by release of Ca²⁺ into the sarcomere and the consequent binding of Ca²⁺ to regulatory sites on troponin. Troponin is believed to undergo a conformational change leading to an azimuthal movement of tropomyosin, which allows myosin heads to interact with actin, hydrolyze ATP, and generate force. The molecular basis by which troponin acts to regulate muscle contraction is only partly understood. It is essential that the structure of troponin in the thin filament at high and low Ca²⁺ is determined to properly understand the mechanism of regulation.The basic structure of the thin filament was described by Ebashi in 1972 (1). In this structure each tropomyosin molecule covers seven actin monomers, and there is a 27.5-Å stagger between troponin molecules. The 7-Å tropomyosin structure (2), the atomic model of F-actin (3), and the troponin “core domain” (4) have recently been used to generate atomic models of the thin filament in low and high Ca²⁺ states (5). While the position of troponin in these models was constrained by known distance measurements between filament components, the exact arrangement of the complex on the filament has not been determined a priori. Although recently published crystal structures of partial troponin complexes (4, 6) have provided valuable insights into the arrangement of the globular head or core domain, the complex in its entirety has not been crystallized.Troponin is believed to consist of a globular core domain with an extended tail (7). The globular core contains the Ca²⁺-binding subunit (TnC),² the inhibitory subunit (TnI), and the C-terminal part (residues 156–262) of the tropomyosin-binding subunit (TnT). The extended tail consists of the N-terminal part of TnT (residues 1–155). A structural rearrangement associated with Ca²⁺ dissociation from the troponin core has been observed (4) such that the helix connecting the two domains of TnC collapses, releasing the TnI inhibitory segment. It is postulated that the TnI inhibitory segment then becomes able to bind actin, in so doing biasing tropomyosin (8). To understand properly how Ca²⁺ binding to TnC leads to movement of tropomyosin, it is necessary to determine a high resolution structure of troponin attached to the thin filament, allowing unambiguous docking of the available crystal structures and direct observation of any changes at a molecular level caused by Ca²⁺ binding.Direct visualization of the thin filament is possible using electron microscopy. Tropomyosin strands have been resolved in the low and high Ca²⁺ states confirming the movement of tropomyosin and the steric blocking model (9, 10). Until recently the actin helical repeat has been imposed in the majority of reconstructions of the thin filament causing artifacts. Helical averaging using the actin repeat spreads troponin density over every actin monomer, which prevents the detailed position and shape of the troponin complex from being found (11). It is possible to avoid this effect by applying a single particle approach. Individual filament images are divided into segments and each segment treated as a particle. Three-dimensional reconstruction may then be carried out by single particle techniques of alignment, classification (12, 13), Euler angle assignment (14–16) and exact filter back-projection (17, 18).Two forms of single particle analysis have emerged: helical single particle analysis (19), where the determined helical symmetry is applied to the final reconstruction, and non-helical single particle analysis, which treats the complex as a truly asymmetric particle. Helical single particle analysis has been used to successfully reconstruct a myosin containing invertebrate thick filament to a resolution of 25 Å (20), and non-helical single particle analysis has been applied to the vertebrate skeletal muscle thick filament allowing azimuthal perturbations of the myosin heads to be observed (21).Model-based single particle image processing methods have recently been applied to the structural analysis of the vertebrate (5, 22, 23) and the insect thin filament (24). We have deliberately avoided starting with a model and any potential model bias by using a reference-free alignment procedure. The adaptation of conventional procedures and their application to the structural study of the muscle thin filament has been documented (25).     

A Highly Conserved Motif within the NH2-terminal Coiled-coil Domain of Angiopoietin-like Protein 4 Confers Its Inhibitory Effects on Lipoprotein Lipase by Disrupting the Enzyme Dimerization

Lipoprotein lipase (LPL) is a principal enzyme responsible for the clearance of chylomicrons and very low density lipoproteins from the bloodstream. Two members of the Angptl (angiopoietin-like protein) family, namely Angptl3 and Angptl4, have been shown to inhibit LPL activity in vitro and in vivo. Here, we further investigated the structural basis underlying the LPL inhibition by Angptl3 and Angptl4. By multiple sequence alignment analysis, we have identified a highly conserved 12-amino acid consensus motif that is present within the coiled-coil domain (CCD) of both Angptl3 and Angptl4, but not other members of the Angptl family. Substitution of the three polar amino acid residues (His⁴⁶, Gln⁵⁰, and Gln⁵³) within this motif with alanine abolishes the inhibitory effect of Angptl4 on LPL in vitro and also abrogates the ability of Angptl4 to elevate plasma triglyceride levels in mice. The CCD of Angptl4 interacts with LPL and converts the catalytically active dimers of LPL to its inactive monomers, whereas the mutant protein with the three polar amino acids being replaced by alanine loses such a property. Furthermore, a synthetic peptide consisting of the 12-amino acid consensus motif is sufficient to inhibit LPL activity, although the potency is much lower than the recombinant CCD of Angptl4. In summary, our data suggest that the 12-amino acid consensus motif within the CCD of Angptl4, especially the three polar residues within this motif, is responsible for its interaction with and inhibition of LPL by blocking the enzyme dimerization.Lipoprotein lipase (LPL)³ is an endothelium-bound enzyme that catalyzes the hydrolysis of plasma triglyceride (TG) associated with chylomicrons and very low density lipoproteins (1, 2). This enzyme plays a major role in maintaining lipid homeostasis by promoting the clearance of TG-rich lipoproteins from the bloodstream. Abnormality in LPL functions has been associated with a number of pathological conditions, including atherosclerosis, dyslipidemia associated with diabetes, and Alzheimer disease (1).LPL is expressed in a wide variety of cell types, particularly in adipocytes and myocytes (2). As a rate-limiting enzyme for clearance of TG-rich lipoproteins, the activity of LPL is tightly modulated by multiple mechanisms in a tissue-specific manner in response to nutritional changes (3, 4). The enzymatic activity of LPL in adipose tissue is enhanced after feeding to facilitate the storage of TG, whereas it is down-regulated during fasting to increase the utilization of TG by other tissues (5). The active form of LPL is a noncovalent homodimer with the subunits associated in a head-to-tail manner, and the dissociation of its dimeric form leads to the formation of a stable inactive monomeric conformation and irreversible enzyme inactivation (6). At the post-translational level, the LPL activity is regulated by numerous apolipoprotein co-factors. For instance, apoCII, a small apolipoprotein consisting of 79 amino acid residues in human, activates LPL by directly binding to the enzyme (7, 8). By contrast, several other apolipoproteins such as apoCI, apo-CIII, and apoE have been shown to inhibit the LPL activity in vitro (3).Angiopoietin-like proteins (Angptl) are a family of secreted proteins consisting of seven members, Angptl1 to Angptl7 (9, 10). All the members of the Angptl family share a similar domain organization to those of angiopoietins, with an NH₂-terminal coiled-coil domain (CCD) and a COOH-terminal fibrinogen-like domain. Among the seven family members, only Angptl3 and Angptl4 have been shown to be involved in regulating triglyceride metabolism (10, 11). The biological functions of Angptl3 in lipid metabolism were first discovered by Koishi et al. (12) in their positional cloning of the recessive mutation gene responsible for the hypolipidemia phenotype in a strain of obese mouse KK/snk. Subsequent studies have demonstrated that Angptl3 increases plasma TG levels by inhibiting the LPL enzymatic activity (13–15). Angptl4, also known as fasting-induced adipocyte factor, hepatic fibrinogen/angiopoietin-related protein, or peroxisome proliferator-activated receptor-γ angiopoietin-related, is a secreted glycoprotein abundantly expressed in adipocyte, liver, and placenta (16–18). In addition to its role in regulating angiogenesis, a growing body of evidence demonstrated that Angptl4 is an important player of lipid metabolism (10, 11). Elevation of circulating Angptl4 by transgenic or adenoviral overexpression, or by direct supplementation of recombinant protein, leads to a marked elevation in the levels of plasma TG and low density lipoprotein cholesterol in mice (19–22). By contrast, Angptl4 knock-out mice exhibit much lower plasma TG and cholesterol levels compared with the wild type littermates (19, 20). Notably, treatment of several mouse models (such as C57BL/6J, ApoE^–/–, LDLR^–/–, and db/db obese/diabetic mice) with a neutralizing antibody against Angptl4 recapitulate the lipid phenotype found in Angptl4 knock-out mice (19). The role of Angptl4 as a physiological inhibitor of LPL is also supported by the finding that its expression levels in adipose tissue change rapidly during the fed-to-fasting transitions and correlate inversely with LPL activity (23). In humans, a genetic variant of the ANGPTL4 gene (E40K) has been found to be associated with significantly lower plasma TG levels and higher high density lipoprotein cholesterol concentrations in several ethnic groups (24–26).Angptl3 and Angptl4 share many common biochemical and functional properties (10). In both humans and rodents, Angptl3 and Angptl4 are proteolytically cleaved at the linker region and circulate in plasma as two truncated fragments, including NH₂-terminal CCD and COOH-terminal fibrinogen-like domain (14, 27–29). The effects of both Angptl3 and Angptl4 on elevating plasma TG levels are mediated exclusively by their NH₂-terminal CCDs (15, 22, 23, 27, 30). The CCDs of Angptl3 and Angptl4 have been shown to inhibit the LPL activity in vitro as well as in mice (23,30,31). Angptl4 inhibits LPL by promoting the conversion of the catalytically active LPL dimers into catalytically inactive LPL monomers, thereby leading to the inactivation of LPL (23, 31). However, the detailed structural and molecular basis underlying the LPL inhibition by Angptl3 and Angptl4 remain poorly characterized at this stage.In this study, we analyzed all known amino acid sequences of Angptl3 and Angptl4 from various species and found a short motif, LAXGLLXLGXGL (where X represents polar amino acid residues), which corresponds to amino acid residues 46–57 and 44–55 of human Angptl3 and Angptl4, respectively, is highly conserved despite the low degree of their overall homology (∼30%). Using both in vitro and in vivo approaches, we demonstrated that this 12-amino acid sequence motif, in particular the three polar amino acid residue within this motif, is essential for mediating the interactions between LPL and Angpt4, which in turn disrupts the dimerization of the enzyme.     

Hepatitis C Virus NS5A Protein Is a Substrate for the Peptidyl-prolyl cis/trans Isomerase Activity of Cyclophilins A and B

We report here a biochemical and structural characterization of domain 2 of the nonstructural 5A protein (NS5A) from the JFH1 Hepatitis C virus strain and its interactions with cyclophilins A and B (CypA and CypB). Gel filtration chromatography, circular dichroism spectroscopy, and finally NMR spectroscopy all indicate the natively unfolded nature of this NS5A-D2 domain. Because mutations in this domain have been linked to cyclosporin A resistance, we used NMR spectroscopy to investigate potential interactions between NS5A-D2 and cellular CypA and CypB. We observed a direct molecular interaction between NS5A-D2 and both cyclophilins. The interaction surface on the cyclophilins corresponds to their active site, whereas on NS5A-D2, it proved to be distributed over the many proline residues of the domain. NMR heteronuclear exchange spectroscopy yielded direct evidence that many proline residues in NS5A-D2 form a valid substrate for the enzymatic peptidyl-prolyl cis/trans isomerase (PPIase) activity of CypA and CypB.Hepatitis C virus (HCV)⁴ is a small, positive strand, RNA-enveloped virus belonging to the Flaviviridae family and the genus Hepacivirus. With 120–180 million chronically infected individuals worldwide, hepatitis C virus infection represents a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (1). The HCV viral genome (∼9.6 kb) codes for a unique polyprotein of ∼3000 amino acids (recently reviewed in Refs. 2–4). Following processing via viral and cellular proteases, this polyprotein gives rise to at least 10 viral proteins, divided into structural (core, E1, and E2 envelope glycoproteins) and nonstructural proteins (p7, NS2, NS3, NS4A, NS4B, NS5A, NS5B). Nonstructural proteins are involved in polyprotein processing and viral replication. The set composed of NS3, NS4A, NS4B, NS5A, and NS5B constitutes the minimal protein component required for viral replication (5).Cyclophilins are cellular proteins that have been identified first as CsA-binding proteins (6). As FK506-binding proteins (FKBP) and parvulins, cyclophilins are peptidyl-prolyl cis/trans isomerases (PPIase) that catalyze the cis/trans isomerization of the peptide linkage preceding a proline (6, 7). Several subtypes of cyclophilins are present in mammalian cells (8). They share a high sequence homology and a well conserved three-dimensional structure but display significant differences in their primary cellular localization and in abundance (9). CypA, the most abundant of the cyclophilins, is primarily cytoplasmic, whereas CypB is directed to the endoplasmic reticulum lumen or the secretory pathway. CypD, on the other hand, is the mitochondrial cyclophilin. Cyclophilins are involved in numerous physiological processes such as protein folding, immune response, and apoptosis and also in the replication cycle of viruses including vaccinia virus, vesicular stomatitis virus, severe acute respiratory syndrome (SARS)-coronavirus, and human immunodeficiency virus (HIV) (for review see Ref. 10). For HIV, CypA has been shown to interact with the capsid domain of the HIV Gag precursor polyprotein (11). CypA thereby competes with capsid domain/TRIM5 interaction, resulting in a loss of the antiviral protective effect of the cellular restriction factor TRIM5α (12, 13). Moreover, it has been shown that CypA catalyzes the cis/trans isomerization of Gly²²¹-Pro²²² in the capsid domain and that it has functional consequences for HIV replication efficiency (14–16). For HCV, Watashi et al. (17) have described a molecular and functional interaction between NS5B, the viral RNA-dependent RNA polymerase (RdRp), and cyclophilin B (CypB). CypB may be a key regulator in HCV replication by modulating the affinity of NS5B for RNA. This regulation is abolished in the presence of cyclosporin A (CsA), an inhibitor of cyclophilins (6). These results provided for the first time a molecular mechanism for the early-on observed anti-HCV activity of CsA (18–20). Although this initial report suggests that only CypB would be involved in the HCV replication process (17), a growing number of studies have recently pointed out a role for other cyclophilins (21–25).In vitro selection of CsA-resistant HCV mutants indicated the importance of two HCV nonstructural proteins, NS5B and NS5A (26), with a preponderant effect for mutations in the C-terminal half of NS5A. NS5A is a large phosphoprotein (49 kDa), indispensable for HCV replication and particle assembly (27–29), but for which the exact function(s) in the HCV replication cycle remain to be elucidated. This nonstructural protein is anchored to the cytoplasmic leaflet of the endoplasmic reticulum membrane via an N-terminal amphipathic α-helix (residues 1–27) (30, 31). Its cytoplasmic sequence can be divided into three domains: D1 (residues 27–213), D2 (residues 250–342), and D3 (residues 356–447), all connected by low complexity sequences (32). D1, a zinc-binding domain, adopts a dimeric claw-shaped structure, which is proposed to interact with RNA (33, 34). NS5A-D2 is essential for HCV replication, whereas NS5A-D3 is a key determinant for virus infectious particle assembly (27, 35). NS5A-D2 and -D3, for which sequence conservation among HCV genotypes is significantly lower than for D1, have been proposed to be natively unfolded domains (28, 32). Molecular and structural characterization of NS5A-D2 from HCV genotype 1a has confirmed the disordered nature of this domain (36, 37).As it is still not clear which cyclophilins are cofactors for HCV replication, and as mutations in HCV NS5A protein have been associated with CsA resistance, we decided to examine the interaction between both CypA and CypB and domain 2 of the HCV NS5A protein. We first characterized, at the molecular level, NS5A-D2 from the HCV JFH1 infectious strain (genotype 2a) and showed by NMR spectroscopy that this natively unfolded domain indeed interacts with both cyclophilin A and cyclophilin B. Our NMR chemical shift mapping experiments indicated that the interaction occurs at the level of the cyclophilin active site, whereas it lacks a precise localization on NS5A-D2. A peptide derived from the only well conserved amino acid motif in NS5A-D2 did interact with cyclophilin A but only with a 10-fold lower affinity than the full domain. We concluded from this that the many proline residues form multiple anchoring points, especially when they adopt the cis conformation. NMR exchange spectroscopy further demonstrated that NS5A-D2 is a substrate for the PPIase activities of both CypA and CypB. Both the NS5A/cyclophilin interaction and the PPIase activity of the cyclophilins on NS5A-D2 were abolished by CsA, underscoring the specificity of the interaction.     

Secreted Trefoil Factor 2 Activates the CXCR4 Receptor in Epithelial and Lymphocytic Cancer Cell Lines

The secreted trefoil factor family 2 (TFF2) protein contributes to the protection of the gastrointestinal mucosa from injury by strengthening and stabilizing mucin gels, stimulating epithelial restitution, and restraining the associated inflammation. Although trefoil factors have been shown to activate signaling pathways, no cell surface receptor has been directly linked to trefoil peptide signaling. Here we demonstrate the ability of TFF2 peptide to activate signaling via the CXCR4 chemokine receptor in cancer cell lines. We found that both mouse and human TFF2 proteins (at ∼0.5 μm) activate Ca²⁺ signaling in lymphoblastic Jurkat cells that could be abrogated by receptor desensitization (with SDF-1α) or pretreatment with the specific antagonist AMD3100 or an anti-CXCR4 antibody. TFF2 pretreatment of Jurkat cells decreased Ca²⁺ rise and chemotactic response to SDF-1α. In addition, the CXCR4-negative gastric epithelial cell line AGS became highly responsive to TFF2 treatment upon expression of the CXCR4 receptor. TFF2-induced activation of mitogen-activated protein kinases in gastric and pancreatic cancer cells, KATO III and AsPC-1, respectively, was also dependent on the presence of the CXCR4 receptor. Finally we demonstrate a distinct proliferative effect of TFF2 protein on an AGS gastric cancer cell line that expresses CXCR4. Overall these data identify CXCR4 as a bona fide signaling receptor for TFF2 and suggest a mechanism through which TFF2 may modulate immune and tumorigenic responses in vivo.Trefoil factor 2 (TFF2),² previously known as spasmolytic polypeptide, is a unique member of the trefoil family that is expressed primarily in gastric mucous neck cells and is up-regulated in the setting of chronic inflammation. Experimental induction of ulceration in the rat stomach leads to rapid up-regulation of TFF2 expression with high levels observed 30 min after ulceration with persistence for up to 10 days (1). TFF2 is secreted into the mucus layer of the gastrointestinal tract of mammals where it stabilizes the mucin gel layer and stimulates migration of epithelial cells (2–4), suggesting an important role in restitution and in maintenance of the integrity of the gut. Exogenous administration of recombinant TFF2, either orally or intravenously, provides mucosal protection in several rodent models of acute gastric or intestinal injury (5, 6). A TFF2^-/- knock-out mouse model has confirmed the importance of TFF2 in the protection of gastrointestinal mucosa against chronic injury (7).It is widely accepted that trefoil factors exert their biological action through a cell surface receptor. This suggestion comes from studies on binding of ¹²⁵I-labeled TFF2 that demonstrated specific binding sites in the gastric glands, intestine, and colon that could be displaced by non-radioactive TFF2 (6, 8–10). Structural studies have revealed potential binding sites for receptors for all members of the trefoil factor family (11, 12). In concordance with this hypothesis, several membrane proteins were found to interact with TFF2. First it was shown that recombinant human TFF2 (and TFF3) could bind to a 28-kDa peptide from membrane fractions of rat jejunum and two human adenocarcinoma cell lines, MCF-7 and Colony-29 (13). Later it was found that recombinant TFF3 fused with biotin selectively bound with a 50-kDa protein from the membrane of rat small intestinal cells (14). However, these 28- and 50-kDa proteins were characterized only by their molecular size without further identification. Two TFF2-binding proteins that have been characterized include a 140-kDa protein, the β subunit of the fibronectin receptor, and a 224-kDa protein called muclin (15). Another TFF2-binding protein was isolated by probing two-dimensional blots of mouse stomach with a murine TFF2 fusion protein, leading to the identification of the gastric foveolar protein blottin, a murine homolog of the human peptide TFIZ1(16). Although these three proteins have now been well characterized, none of them has been shown to mediate responses to TFF2, and no activated signaling cascades have been shown.Despite the absence of an identified cell surface receptor for TFF2, there is nevertheless clear evidence that TFF2 and TFF3 rapidly activate signal transduction pathways (17, 18). TFF3 prevents cell death via activation of the serine/threonine kinase AKT in colon cancer cell lines (19). The TFF3 protein also activates STAT3 signaling in human colorectal cancer cells, thus providing cells with invasion potential (20). TFF3 treatment leads to EGF receptor activation and β-catenin phosphorylation in HT-29 cells (21) and to transient phosphorylation of ERK1/2 in oral keratinocytes (22). With respect to TFF2, recombinant peptide enhances the migration of human bronchial epithelial cell line BEAS-2B (4). TFF2 has been shown to induce phosphorylation of c-Jun NH₂-terminal kinase (JNK) and ERK1/2. Consistent with this observation, the motogenic effect of TFF2 is significantly inhibited by antagonists of ERK kinases and protein kinase C but not by inhibitors of p38 mitogen-activated protein kinase (MAPK). It is believed that the motogenic effect of trefoil factors and of TFF2 in particular, could contribute to in vivo restitution of gastric epithelium by enhancing cell migration.Although previous studies have suggested that TFF2 functions primarily in cytoprotection, accumulating evidence now suggests that TFF2 may also play a role in the regulation of host immunity. For example, recombinant TFF2 reduces inflammation in rat and mouse models of colitis (23, 24). In addition, TFF2 was detected in rat lymphoid tissues (spleen, lymph nodes, and bone marrow) (25). Recently we and others found TFF2 mRNA expression in primary and secondary lymphopoietic organs (26, 27). These data suggest that TFF2 may play some function in the immune system. In concordance with these findings, we detected an exacerbated inflammatory response to acute injury in TFF2 knock-out animals (27, 28). These observations prompted us to look at the possible function of TFF2 in immune cells. Unexpectedly we found that TFF2 modulates Ca²⁺ and AKT signaling in lymphoblastic Jurkat cells and that these effects appear to be mediated through the CXCR4 receptor.     

Thermodynamic Analysis Reveals a Temperature-dependent Change in the Catalytic Mechanism of Bacillus stearothermophilus Tyrosyl-tRNA Synthetase

Catalysis of tRNA^Tyr aminoacylation by tyrosyl-tRNA synthetase can be divided into two steps. In the first step, tyrosine is activated by ATP to form the tyrosyl-adenylate intermediate. In the second step, the tyrosyl moiety is transferred to the 3′ end of tRNA. To investigate the roles that enthalpic and entropic contributions play in catalysis by Bacillus stearothermophilus tyrosyl-tRNA synthetase (TyrRS), the temperature dependence for the activation of tyrosine and subsequent transfer to tRNA^Tyr has been determined using single turnover kinetic methods. A van''t Hoff plot for binding of ATP to the TyrRS·Tyr complex reveals three distinct regions. Particularly striking is the change occurring at 25 °C, where the values of ΔH⁰ and ΔS⁰ go from –144 kJ/mol and –438 J/mol K below 25 °C to +137.9 kJ/mol and +507 J/mol K above 25 °C. Nonlinear Eyring and van''t Hoff plots are also observed for formation of the TyrRS·[Tyr-ATP]^‡ and TyrRS·Tyr-AMP complexes. Comparing the van''t Hoff plots for the binding of ATP to tyrosyl-tRNA synthetase in the absence and presence of saturating tyrosine concentrations indicates that the temperature-dependent changes in ΔH⁰ and ΔS⁰ for the binding of ATP only occur when tyrosine is bound to the enzyme. Previous investigations revealed a similar synergistic interaction between the tyrosine and ATP substrates when the “KMSKS” signature sequence is deleted or replaced by a nonfunctional sequence. We propose that the temperature-dependent changes in ΔH⁰ and ΔS⁰ are because of the KMSKS signature sequence being conformationally constrained and unable to disrupt this synergistic interaction below 25 °C.Aminoacyl-tRNA synthetases catalyze the transfer of amino acids to the 3′ end of tRNA in a two-step reaction shown as Reactions 1 and 2, REACTION 1 REACTION 2 where aaRS,² AA, and tRNA^AA represent the aminoacyl-tRNA synthetase, its amino acid substrate, and the cognate tRNA, respectively, and “·” and “–” represent noncovalent and covalent interactions, respectively. In general, the first step of the reaction (the activation of the amino acid) does not require the binding of tRNA to the enzyme (1–5). This allows the two steps in the tRNA aminoacylation reaction to be run independently of each other.The aminoacyl-tRNA synthetases can be separated into two classes that are structurally distinct (6–10). Class I aminoacyl-tRNA synthetases are characterized by an amino-terminal Rossmann fold domain containing the active site and two signature sequences, “HIGH” and “KMSKS” (6, 7, 10–15). These sequences stabilize the transition state for the amino acid activation step of the reaction (16–24). In class II aminoacyl-tRNA synthetases, the active site domain consists of a seven-stranded β-sheet surrounded by three α-helices (25–33). With the exception of the tyrosyl- and tryptophanyl-tRNA synthetases, which are functional homodimers, all of the class I aminoacyl-tRNA synthetases are functional monomers (34). In contrast, all of the class II aminoacyl-tRNA synthetases are functional dimers (34).The class I aminoacyl-tRNA synthetases contain an insertion domain, known as the CP1 domain, between the two halves of the Rossmann fold (10, 11, 35). In tyrosyl-tRNA synthetase (and the structurally related tryptophanyl-tRNA synthetase), the CP1 domains of the two monomers form the dimer interface. Although tyrosyl-tRNA synthetase (TyrRS) is composed of two identical monomers, in solution it displays an extreme form of negative cooperativity, known as “half-of-the-sites” reactivity, with respect to tyrosine binding and tyrosyl-adenylate formation (36, 37).Kinetic analysis of the tyrosine activation reaction supports a random order mechanism for the binding of tyrosine and ATP to tyrosyl-tRNA synthetase (38–40). In contrast, initial analysis of the Bacillus stearothermophilus tyrosyl-tRNA synthetase crystal structure suggested that the tyrosine binding pocket is blocked when ATP is bound to the enzyme (6, 7). This apparent contradiction between the kinetic and structural results was initially resolved by invoking a virtual equilibrium for the binding of tyrosine to the TyrRS·ATP complex (41). More recently, analysis of the structurally related tryptophanyl-tRNA synthetase and molecular dynamics simulations of tyrosyl-tRNA synthetase suggest that the enzyme·ATP complex exists in an open conformation that allows access to the amino acid binding pocket (42, 43). This model is consistent with a random order mechanism for substrate binding to tyrosyl-tRNA synthetase.In general, interactions between tyrosyl-tRNA synthetase and the tyrosine substrate form on the initial binding of tyrosine and do not change in strength throughout the course of the reaction (41). In contrast, the initial binding of ATP is relatively weak (K′ ^ATP_d = 4.7 mm for B. stearothermophilus tyrosyl-tRNA synthetase; where K′ ^ATP_d indicates the dissociation of ATP from the TyrRS·Tyr·ATP complex), with most of the interactions between the enzyme and ATP being formed in the transition state of the reaction (41). In other words, tyrosyl-tRNA synthetase uses tyrosine binding energy to increase the specificity of the active site and ATP binding energy to catalyze the activation of tyrosine. In addition, tyrosyl-tRNA synthetase variants, in which the KMSKS signature sequence has been deleted or made nonfunctional through mutagenesis, display a 20-fold increase in ATP binding affinity relative to that of the wild-type enzyme (19, 22). This increased affinity for ATP is dependent on the binding of tyrosine to the enzyme (22). These observations indicate that there is a synergistic interaction between the tyrosine and ATP substrates that occurs in the TyrRS·Tyr·ATP complex when the KMSKS sequence is absent or nonfunctional. One of the functions of the KMSKS sequence is to disrupt this synergistic interaction during the initial binding of ATP, allowing it to instead be used to stabilize the transition state of the amino acid activation step of the reaction (22).In this study, we investigate the role that enthalpy and entropy play in catalysis of tRNA^Tyr aminoacylation by B. stearothermophilus tyrosyl-tRNA synthetase using single turnover conditions. Although the standard free energy for this reaction is not significantly affected by increasing temperatures, there is a dramatic shift in both the van''t Hoff and Eyring plots at ∼25 °C for the tyrosine activation reaction. The hypothesis that a synergistic interaction between tyrosine and ATP is responsible for this temperature-dependent change is tested.     

Strain-induced Proliferation Requires the Phosphatidylinositol 3-Kinase/AKT/Glycogen Synthase Kinase Pathway

The intestinal epithelium is repetitively deformed by shear, peristalsis, and villous motility. Such repetitive deformation stimulates the proliferation of intestinal epithelial cells on collagen or laminin substrates via ERK, but the upstream mediators of this effect are poorly understood. We hypothesized that the phosphatidylinositol 3-kinase (PI3K)/AKT cascade mediates this mitogenic effect. PI3K, AKT, and glycogen synthase kinase-3β (GSK-3β) were phosphorylated by 10 cycles/min strain at an average 10% deformation, and pharmacologic blockade of these molecules or reduction by small interfering RNA (siRNA) prevented the mitogenic effect of strain in Caco-2 or IEC-6 intestinal epithelial cells. Strain MAPK activation required PI3K but not AKT. AKT isoform-specific siRNA transfection demonstrated that AKT2 but not AKT1 is required for GSK-3β phosphorylation and the strain mitogenic effect. Furthermore, overexpression of AKT1 or an AKT chimera including the PH domain and hinge region of AKT2 and the catalytic domain and C-tail of AKT1 prevented strain activation of GSK-3β, but overexpression of AKT2 or a chimera including the PH domain and hinge region of AKT1 and the catalytic domain and C-tail of AKT2 did not. These data delineate a role for PI3K, AKT2, and GSK-3β in the mitogenic effect of strain. PI3K is required for both ERK and AKT2 activation, whereas AKT2 is sequentially required for GSK-3β. Furthermore, AKT2 specificity requires its catalytic domain and tail region. Manipulating this pathway may prevent mucosal atrophy and maintain the mucosal barrier in conditions such as ileus, sepsis, and prolonged fasting when peristalsis and villous motility are decreased and the mucosal barrier fails.Mechanical forces are part of the normal intestinal epithelial environment. Numerous different forces deform these cells including shear stress from endoluminal chyme, bowel peristalsis, and villous motility (1, 2). During normal bowel function the mucosa is subjected to injury that must be repaired to maintain the mucosal barrier (3, 4). Deformation patterns of the bowel are altered in conditions such as prolonged fasting, post-surgical ileus, and sepsis states, resulting in profoundly reduced mucosal deformation. When such states are prolonged, proliferation slows, the mucosa becomes atrophic, and bacterial translocation may ensue as the mucosal barrier of the gut breaks down (5–7).In vitro, repetitive deformation is trophic for intestinal epithelial cells (8) cultured on type I or type IV collagen or laminin. Human Caco-2 intestinal epithelial cells (9), non-transformed rat IEC-6 intestinal epithelial cells (10), and primary human intestinal epithelial cells isolated from surgical specimens (11) proliferate more rapidly in response to cyclic strain (12) unless substantial quantities of fibronectin are added to the media or matrix (11) to mimic the acute phase reaction of acute or chronic inflammation and injury. Cyclic strain also stimulates proliferation in HCT 116 colon cancer cells (13) and differentiation of Caco-2 cells cultured on a collagen substrate (9). This phenomenon has also been observed in vivo (14). Thus, repetitive deformation may help to maintain the normal homeostasis of the gut mucosa under non-inflammatory conditions. Previous work in our laboratory has implicated Src, focal adhesion kinase, and the mitogen-activated protein kinase (MAPK)² extracellular signal-related kinase (ERK) in the mitogenic effect of strain (10). Although p38 is also activated in Caco-2 cells subjected to cyclic strain on a collagen matrix, its activity is not required for the mitogenic effect of strain (12).Although often the PI3K/AKT pathway is thought of as a parallel pathway to the MAPK, this is not always the case. Protein kinase C isoenzymes differentially modulate thrombin effect on MAPK-dependent retinal pigment epithelial cell (RPE) proliferation, and it has been shown that PI3K or AKT inhibition prevented thrombin-induced ERK activation and RPE proliferation (15).PI3K, AKT, and glycogen synthase kinase (GSK), a downstream target of AKT (16), have been implemented in intestinal epithelial cell proliferation in numerous cell systems not involving strain (17–19) including uncontrolled proliferation in gastrointestinal cancers (20–22). Mechanical forces activate this pathway as well. PI3K and AKT are required for increased extracellular pressure to stimulate colon cancer cell adhesion (23), although the pathway by which pressure stimulates colon cancer cells in suspension differs from the response of adherent intestinal epithelial cells to repetitive deformation (24), and GSK is not involved in this effect.³ Repetitive strain also stimulates vascular endothelial cell proliferation via PI3K and AKT (25, 26), whereas respiratory strain stimulates angiogenic responses via PI3K (27). We, therefore, hypothesized that the PI3K/AKT/GSK axis would be involved in the mitogenic effects of repetitive deformation on a collagen matrix.To test this hypothesis, we used the Flexcell apparatus to rhythmically deform Caco-2 intestinal epithelial cells. IEC-6 cells were used to confirm key results. A frequency of 10 cycles per min was used, which is similar in order of magnitude to the frequency that the intestinal mucosa might be deformed by peristalsis or villous motility in vivo (28, 29). Mechanical forces such as repetitive deformation are likely cell-type and frequency-specific, as different cell types respond to different frequencies. Vascular endothelial cells respond to frequencies of 60–80 cycles/min (25), whereas intestinal epithelial cells may actually decrease proliferation in response to frequencies of 5 cycles/min (30). We characterized PI3K, AKT, and GSK phosphorylation with strain, blocked these molecules pharmacologically or by siRNA, and delineated the specificity of the AKT effect using isozyme-specific siRNA and transfection of AKT1/2 chimeras. We also characterized the interaction of this pathway with the activation of ERK by strain, which has previously been implicated in the mitogenic response (12).     

Calmodulin Binds to Extracellular Sites on the Plasma Membrane of Plant Cells and Elicits a Rise in Intracellular Calcium Concentration

Calmodulin (CaM) is a highly conserved intracellular calcium sensor. In plants, CaM also appears to be present in the apoplasm, and application of exogenous CaM has been shown to influence a number of physiological functions as a polypeptide signal; however, the existence and localization of its corresponding apoplasmic binding sites remain controversial. To identify the site(s) of action, a CaM-conjugated quantum dot (QD) system was employed for single molecule level detection at the surface of plant cells. Using this approach, we show that QD-CaM binds selectively to sites on the outer surface of the plasma membrane, which was further confirmed by high resolution transmission electron microscopy. Measurements of Ca²⁺ fluxes across the plasma membrane, using ion-selective microelectrodes, demonstrated that exogenous CaM induces a net influx into protoplasts. Consistent with these flux studies, calcium-green-dextran and FRET experiments confirmed that applied CaM/QD-CaM elicited an increase in cytoplasmic Ca²⁺ levels. These results support the hypothesis that apoplasmic CaM can act as a signaling agent. These findings are discussed in terms of CaM acting as an apoplasmic peptide ligand to mediate transmembrane signaling in the plant kingdom.Calmodulin (CaM)² is a conserved multifunctional calcium sensor that mediates intracellular Ca²⁺ signaling and regulates diverse cellular processes by interacting with calmodulin-binding proteins (1–3). Interestingly, in both animals and plants, CaM may also act as an extracellular agent to regulate physiological events (4). Consistent with this notion, extracellular CaM has been detected within the cell walls of a broad range of plant species (4, 5).Functional studies have established that exogenously applied CaM can stimulate the proliferation of suspension-cultured plant cells (6) as well as affect intracellular activities of heterotrimeric G proteins and phospholipases in protoplasts (7, 8). Based on these findings, it has been proposed that, in plants, extracellular CaM may function as a signaling agent involved in the regulation of cell growth and development (4). However, as a 17-kDa hydrophilic protein, exogenously applied CaM could well be retrieved from the apoplasmic space and then exert its effects on components within the cytoplasm. Evidence against this hypothesis was provided by studies with Arabidopsis thaliana suspension-cultured cells in which it was shown that 24 h of incubation in exogenous CaM did not result in protein uptake or degradation (4).To exert an effect from the apoplasm, it would seem logical to assume that a protein(s) within the plant plasma membrane would have a CaM-binding site exposed to the apoplasm. Although a number of studies have addressed the molecular mechanism(s) by which extracellular CaM might act as a signal (6, 9) and attempts have been made to identify extracellular CaM-binding proteins (4, 6), currently there is no direct evidence in support of the hypothesis that specific CaM-binding sites exist at the surface of plant cells.To address this question, a CaM-conjugated quantum dot (QD) system was employed for single molecule level detection (10–13) at the surface of plant cells. These nanoparticles have several advantages over conventional fluorophores for light microscopic imaging, including their higher brightness and photostability (14, 15). In addition, because of their electron dense nature, QDs can be used for single labeling studies at the transmission electron microscope level (16, 17). Using this QD-CaM system, we demonstrate that QD-CaM binds selectively to sites on the outer surface of the plant plasma membrane. We also show by three independent methods that applied CaM can modulate Ca²⁺ fluxes across the plasma membrane, leading to alterations in cytoplasmic Ca²⁺ status. These findings support the hypothesis that, in plants, apoplasmic CaM can act as a signaling agent.     

Effects of Differential Glycosylation of Glycodelins on Lymphocyte Survival

Glycodelin is a human glycoprotein with four reported glycoforms, namely glycodelin-A (GdA), glycodelin-F (GdF), glycodelin-C (GdC), and glycodelin-S (GdS). These glycoforms have the same protein core and appear to differ in their N-glycosylation. The glycosylation of GdA is completely different from that of GdS. GdA inhibits proliferation and induces cell death of T cells. However, the glycosylation and immunomodulating activities of GdF and GdC are not known. This study aimed to use ultra-high sensitivity mass spectrometry to compare the glycomes of GdA, GdC, and GdF and to study the relationship between the immunological activity and glycosylation pattern among glycodelin glycoforms. Using MALDI-TOF strategies, the glycoforms were shown to contain an enormous diversity of bi-, tri-, and tetra-antennary complex-type glycans carrying Galβ1–4GlcNAc (lacNAc) and/or GalNAcβ1–4GlcNAc (lacdiNAc) antennae backbones with varying levels of fucose and sialic acid substitution. Interestingly, they all carried a family of Sda (NeuAcα2–3(GalNAcβ1–4)Gal)-containing glycans, which were not identified in the earlier study because of less sensitive methodologies used. Among the three glycodelins, GdA is the most heavily sialylated. Virtually all the sialic acid on GdC is located on the Sda antennae. With the exception of the Sda epitope, the GdC N-glycome appears to be the asialylated counterpart of the GdA/GdF glycomes. Sialidase activity, which may be responsible for transforming GdA/GdF to GdC, was detected in cumulus cells. Both GdA and GdF inhibited the proliferation, induced cell death, and suppressed interleukin-2 secretion of Jurkat cells and peripheral blood mononuclear cells. In contrast, no immunosuppressive effect was observed for GdS and GdC.Glycodelin is a member of the lipocalin family. It consists of 180 amino acid residues (1) with two sites of N-linked glycosylation. There are four reported glycodelin isoforms, namely glycodelin-A (amniotic fluid isoform, GdA),⁴ glycodelin-F (follicular fluid, GdF), glycodelin-C (cumulus matrix, GdC) and glycodelin-S (seminal plasma, GdS) (2–5). Among the four glycodelin isoforms, only the N-glycan structures of GdA and GdS have been previously determined. This was achieved using fast atom bombardment mass spectrometry (6, 7). The glycan structures of GdA and GdS are completely different. In GdA, the Asn-28 site carries high mannose, hybrid, and complex-type structures, whereas the second Asn-63 site is exclusively occupied by complex-type glycans (6). The major non-reducing epitopes characterized in the complex-type glycans are Galβ1–4GlcNAc (lacNAc), GalNAcβ1–4GlcNAc (lacdiNAc), NeuAcα2–6Galβ1–4GlcNAc (sialylated lacNAc), NeuAcα2–6GalNAcβ1–4GlcNAc (sialylated lacdiNAc), Galβ1–4(Fucα1–3)GlcNAc (Lewis-x), and GalNAcβ1–4(Fucα1–3)GlcNAc (lacdiNAc analog of the blood group substance Lewis-x) (6). Many of these oligosaccharides are rare in other human glycoproteins. GdS glycans are unusually fucose-rich, and the major complex type glycan structures are bi-antennary glycans with Lewis-x and Lewis-y antennae. Glycosylation of GdS is highly site-specific. Asn-28 contains only high mannose structures, whereas Asn-63 contains only complex type glycans. More than 80% of the complex glycans have 3–5 fucose residues/glycan, and none of the glycans is sialylated, which is unusual for a secreted human glycoprotein (7). The glycan structures of GdF and GdC are not known, although they differ in lectin-binding properties and isoelectric point from the other two glycodelin isoforms (5).Glycans are involved in various intracellular, intercellular, and cell-matrix recognition events (8, 9). Glycosylation determines the biological activities of the glycodelin isoforms (2, 10). For example, both GdA and GdF inhibit the spermatozoa-zona pellucida binding (11) via fucosyltransferase-5 (12), but only the latter inhibits progesterone-induced acrosome reaction, thus preventing a premature acrosome reaction of the spermatozoa. There is evidence that cumulus cells can convert exogenous GdA and -F to GdC, the physicochemical properties of which suggest that it is differently glycosylated compared with GdA/F (5). Moreover, GdC stimulated spermatozoa-zona pellucida binding in a dose-dependent manner, and it effectively displaced sperm-bound GdA and -F (4, 5). GdS suppresses capacitation probably via its inhibitory activity on cholesterol efflux from spermatozoa (13).Except for the effects on fertilization, GdA is involved in fetomaternal defense. This glycodelin isoform suppresses proliferation and induces apoptosis of T cells (2) and inhibits natural killer cell (14) and B-cell (15) activities. Glycosylation is involved in the binding of GdA to receptors on T cells (16). The sialic acid of GdA contributes to the apoptotic activity in T cells (17, 18) and binding to CD45, a potential GdA receptor (16). The importance of glycosylation in glycodelin is further shown by the absence of immunosuppressive activities in GdS with different glycosylation (18). The immunomodulating activities of GdF and GdC are unknown.Our previous work showed that glycans are indispensable for the different glycodelins to exhibit their binding activities and biological effects (13, 19, 20). The present study aims to identify the effect of all four glycodelin isoforms on lymphocyte viability, cell death, and interleukin-2 (IL-2) secretion and to correlate these bioactivities with their glycosylation patterns determined by mass spectrometry.