Agrin Regulation of ��3 Sodium-Potassium ATPase Activity Modulates Cardiac Myocyte Contraction

Drugs that inhibit Na,K-ATPases, such as digoxin and ouabain, alter cardiac myocyte contractility. We recently demonstrated that agrin, a protein first identified at the vertebrate neuromuscular junction, binds to and regulates the activity of α3 subunit-containing isoforms of the Na,K-ATPase in the mammalian brain. Both agrin and the α3 Na,K-ATPase are expressed in heart, but their potential for interaction and effect on cardiac myocyte function was unknown. Here we show that agrin binds to the α3 subunit of the Na,K-ATPase in cardiac myocyte membranes, inducing tyrosine phosphorylation and inhibiting activity of the pump. Agrin also triggers a rapid increase in cytoplasmic Na⁺ in cardiac myocytes, suggesting a role in cardiac myocyte function. Consistent with this hypothesis, spontaneous contraction frequencies of cultured cardiac myocytes prepared from mice in which agrin expression is blocked by mutation of the Agrn gene are significantly higher than in the wild type. The Agrn mutant phenotype is rescued by acute treatment with recombinant agrin. Furthermore, exposure of wild type myocytes to an agrin antagonist phenocopies the Agrn mutation. These data demonstrate that the basal frequency of myocyte contraction depends on endogenous agrin-α3 Na,K-ATPase interaction and suggest that agrin modulation of the α3 Na,K-ATPase is important in regulating heart function.Na,K-ATPases, or sodium pumps, are integral membrane enzymes found in all animal cells. Using energy from the hydrolysis of ATP they transport three Na⁺ ions out of the cell for every two K⁺ ions into the cell, resulting in a transmembrane chemical gradient that is reflected in the resting membrane potential and used to drive a variety of secondary transport processes. Each Na,K-ATPase is a heterodimer consisting of an α- and β-subunit. The α-subunit is the catalytic subunit and contains the binding sites for Na⁺ and K⁺. The β-subunit is required for pump function and targeting of the α-subunit to the plasma membrane. Four α- and three β-subunit genes have been identified. All combinations of α- and β-subunits form functional pumps, but developmental, cellular, and subcellular differences in expression suggest functional adaptation of the different isoforms (1).Na,K-ATPases play a central role in regulating the contractile activity of cardiac muscle (2). They are directly responsible for the Na⁺ gradient required for propagation of action potentials that initiate myocyte contraction. Moreover, because of the dependence of the Na⁺/Ca²⁺ exchanger (NCX)³ on the Na⁺ gradient as the source of counterions for transport of Ca²⁺ out of the cell, they play a critical role in Ca²⁺ homeostasis and excitation-contraction coupling. For example, inhibition of Na,K-ATPases by digoxin, ouabain, or other cardiac glycoside results in a decline of the Na⁺ gradient, reducing NCX activity and Ca²⁺ efflux. The inotropic effects of cardiac glycosides result from uptake of this “excess” cytoplasmic Ca²⁺ into the sarcoplasmic reticulum, raising the level of Ca²⁺ in intracellular stores, which, when released during excitation, enhances muscle contraction (3).In light of the importance of Na,K-ATPases for cardiac muscle function, it is not surprising that mechanisms have evolved to regulate their activity. Na,K-ATPases are susceptible to phosphorylation by either cAMP-dependent protein kinase or protein kinase C, and neurotransmitter- and peptide hormone-dependent activation of these cytoplasmic kinases have been shown to regulate pump activity (4). Other molecules exert their effects through direct interaction with the Na,K-ATPase. For example, phospholemman, a member of the FXYD family of membrane proteins expressed in heart, is tightly associated with the Na,K-ATPase and inhibits its function (5–7). Phosphorylation of phospholemman by either protein kinase C or cAMP-dependent protein kinase, however, relieves inhibition thereby restoring the activity of the pump (8, 9). Endogenous ouabain-like compounds have also been implicated in regulating Na,K-ATPase activity (10). Ouabain, or closely related molecules, is synthesized by the adrenal gland and hypothalamus, and increased circulating levels of these compounds observed in patients with congestive heart failure has been suggested as an adaptive response to improve heart function (11). Recent studies in the central nervous system have identified the protein agrin as a new endogenous ligand that regulates Na,K-ATPase function through interaction with its extracellular domains (12).Agrin was first identified as an extracellular matrix protein at the neuromuscular junction where, by signaling through a muscle-specific receptor tyrosine kinase called MuSK, it mediates the motor neuron-induced accumulation of acetylcholine receptors in the postsynaptic muscle fiber membrane (13). Agrin is also expressed in other tissues (14–16), but its function outside of the neuromuscular junction has been less well understood. Recently, however, we showed that agrin plays a role in regulating excitability of central nervous system neurons by binding to and inhibiting the activity of the α3 subunit-containing isoform of the Na,K-ATPase (12). Although both agrin (14, 16) and the α3 Na,K-ATPase (17) are expressed in heart, their potential interaction has not been explored. Here we show that the frequency of cardiac myocyte contraction is modulated by agrin regulation of α3 Na,K-ATPase activity.     

Proinsulin and the Genetics of Diabetes Mellitus

Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.     

Rescue of a Trafficking Defective Human Pacemaker Channel via a Novel Mechanism: ROLES OF Src, Fyn, AND Yes TYROSINE KINASES*

Therapeutic strategies such as using channel blockers and reducing culture temperature have been used to rescue some long QT-associated voltage-gated potassium Kv trafficking defective mutant channels. A hyperpolarization-activated cyclic nucleotide-gated HCN4 pacemaker channel mutant (D553N) has been recently found in a patient associated with cardiac arrhythmias including long QT. D553N showed the defective trafficking to the cell surface, leading to little ionic current expression (loss-of-function). We show in this report that enhanced tyrosine phosphorylation mediated by Src, Fyn, and Yes kinases was able to restore the surface expression of D553N for normal current expression. Src or Yes, but not Fyn, significantly increased the current density and surface expression of D553N. Fyn accelerated the activation kinetics of the rescued D553N. Co-expression of D553N with Yes exhibited the slowest activation kinetics of D553N. Src, Fyn, and Yes significantly enhanced the tyrosine phosphorylation of D553N. A combination of Src, Fyn, and Yes rescued the current expression and the gating of D553N comparable with those of wild-type HCN4. In conclusion, we demonstrate a novel mechanism using three endogenous Src kinases to rescue a trafficking defective HCN4 mutant channel (D553N) by enhancing the tyrosine phosphorylation of the mutant channel protein.Defective trafficking leading to the reduced surface expression of ion channels is one of the mechanisms responsible for a loss-of-function of the ion channel on the plasma membrane (1). Several methods have been developed to rescue the voltage-gated potassium Kv trafficking defective channels: reducing the culture temperature, applying the channel blockers, altering the molar ratio of glycerol, and using the sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase inhibitor thapsigargin (2–6).Hyperpolarizing-activated cyclic nucleotide-gated (HCN)³ pacemaker channels generate time- and voltage-dependent inward currents, named I_h in neurons or I_f in the heart (7). They are important in various cell functions including excitability, synapse transmission, and rhythmic activity (7). The most well studied regulation of I_f is its response to autonomic stimulation. β-Adrenergic receptor activation increases and acetylcholine receptor activation decreases the intracellular cAMP levels, which in turn increases/decreases I_f by binding to the cyclic nucleotide-binding domain of the HCN channels, respectively (7). Other important mechanisms for the modulation of I_f/HCN channels have recently been found including β-subunit (8), lipids (9, 10), and p38 mitogen-activated protein kinase (11).Accumulating evidence has revealed tyrosine phosphorylation as an important mechanism for modulation of HCN channel properties (12–16). An acute increase in tyrosine phosphorylation of I_f or HCN channels increases the channel activity, including an increase in the current amplitude, a positive shift of the voltage-dependent activation, an acceleration of activation kinetics, and an increase in whole cell conductance (12–15). Recently, we discovered that the cell surface expression of HCN2 channels can be remarkably inhibited by tyrosine dephosphorylation mediated by receptor-like protein tyrosine phosphatase α (RPTPα) and increased by tyrosine phosphorylation via Src kinase after long term treatment (17).D553N, a missense HCN4 mutant, was recently identified in a patient with cardiac arrhythmia associated with depressed HCN gating properties (18). Functional and structural assays revealed that D553N expresses little ionic currents, which is possibly due to the defective channel trafficking so that the channels cannot reach the plasma membrane for normal functions (18).The Src kinase family has nine members (19). They are closely related and share the same regulatory function. Three of them, Src, Fyn, and Yes, are ubiquitously expressed in a variety of tissues including neurons and myocytes (19, 20). Without stimulation, they are inactive. However, mutation of key tyrosine residue results in the constitutively active form of the kinase, SrcY529F, FynY531F, and YesY537F, respectively (15, 21, 22). Using these Src kinases, we show in this report a novel approach that can restore the surface expression of D553N for normal current expression via tyrosine phosphorylation.     

Conformational Properties of ��-PrP

Prion propagation involves a conformational transition of the cellular form of prion protein (PrP^C) to a disease-specific isomer (PrP^Sc), shifting from a predominantly α-helical conformation to one dominated by β-sheet structure. This conformational transition is of critical importance in understanding the molecular basis for prion disease. Here, we elucidate the conformational properties of a disulfide-reduced fragment of human PrP spanning residues 91–231 under acidic conditions, using a combination of heteronuclear NMR, analytical ultracentrifugation, and circular dichroism. We find that this form of the protein, which similarly to PrP^Sc, is a potent inhibitor of the 26 S proteasome, assembles into soluble oligomers that have significant β-sheet content. The monomeric precursor to these oligomers exhibits many of the characteristics of a molten globule intermediate with some helical character in regions that form helices I and III in the PrP^C conformation, whereas helix II exhibits little evidence for adopting a helical conformation, suggesting that this region is a likely source of interaction within the initial phases of the transformation to a β-rich conformation. This precursor state is almost as compact as the folded PrP^C structure and, as it assembles, only residues 126–227 are immobilized within the oligomeric structure, leaving the remainder in a mobile, random-coil state.Prion diseases, such as Creutzfeldt-Jacob and Gerstmann-Sträussler-Scheinker in humans, scrapie in sheep, and bovine spongiform encephalopathy in cattle, are fatal neurological disorders associated with the deposition of an abnormally folded form of a host-encoded glycoprotein, prion (PrP)² (1). These diseases may be inherited, arise sporadically, or be acquired through the transmission of an infectious agent (2, 3). The disease-associated form of the protein, termed the scrapie form or PrP^Sc, differs from the normal cellular form (PrP^C) through a conformational change, resulting in a significant increase in the β-sheet content and protease resistance of the protein (3, 4). PrP^C, in contrast, consists of a predominantly α-helical structured domain and an unstructured N-terminal domain, which is capable of binding a number of divalent metals (5–12). A single disulfide bond links two of the main α-helices and forms an integral part of the core of the structured domain (13, 14).According to the protein-only hypothesis (15), the infectious agent is composed of a conformational isomer of PrP (16) that is able to convert other isoforms to the infectious isomer in an autocatalytic manner. Despite numerous studies, little is known about the mechanism of conversion of PrP^C to PrP^Sc. The most coherent and general model proposed thus far is that PrP^C fluctuates between the dominant native state and minor conformations, one or a set of which can self-associate in an ordered manner to produce a stable supramolecular structure composed of misfolded PrP monomers (3, 17). This stable, oligomeric species can then bind to, and stabilize, rare non-native monomer conformations that are structurally complementary. In this manner, new monomeric chains are recruited and the system can propagate.In view of the above model, considerable effort has been devoted to generating and characterizing alternative, possibly PrP^Sc-like, conformations in the hope of identifying common properties or features that facilitate the formation of amyloid oligomers. This has been accomplished either through PrP^Sc-dependent conversion reactions (18–20) or through conversion of PrP^C in the absence of a PrP^Sc template (21–25). The latter approach, using mainly disulfide-oxidized recombinant PrP, has generated a wide range of novel conformations formed under non-physiological conditions where the native state is relatively destabilized. These conformations have ranged from near-native (14, 26, 27), to those that display significant β-sheet content (21, 23, 28–33). The majority of these latter species have shown a high propensity for aggregation, although not all are on-pathway to the formation of amyloid. Many of these non-native states also display some of the characteristics of PrP^Sc, such as increased β-sheet content, protease resistance, and a propensity for oligomerization (28, 29, 31) and some have been claimed to be associated with the disease process (34).One such PrP folding intermediate, termed β-PrP, differs from the majority of studied PrP intermediate states in that it is formed by refolding the PrP molecule from the native α-helical conformation (here termed α-PrP), at acidic pH in a reduced state, with the disulfide bond broken (22, 35). Although no covalent differences between the PrP^C and PrP^Sc have been consistently identified to date, the role of the disulfide bond in prion propagation remains disputed (25, 36–39). β-PrP is rich in β-sheet structure (22, 35), and displays many of the characteristics of a PrP^Sc-like precursor molecule, such as partial resistance to proteinase K digestion, and the ability to form amyloid fibrils in the presence of physiological concentrations of salts (40).The β-PrP species previously characterized, spanning residues 91–231 of PrP, was soluble at low ionic strength buffers and monomeric, according to elution volume on gel filtration (22). NMR analysis showed that it displayed radically different spectra to those of α-PrP, with considerably fewer observable peaks and markedly reduced chemical shift dispersion. Data from circular dichroism experiments showed that fixed side chain (tertiary) interactions were lost, in contrast to the well defined β-sheet secondary structure, and thus in conjunction with the NMR data, indicated that β-PrP possessed a number of characteristics associated with a “molten globule” folding intermediate (22). Such states have been proposed to be important in amyloid and fibril formation (41). Indeed, antibodies raised against β-PrP (e.g. ICSM33) are capable of recognizing native PrP^Sc (but not PrP^C) (42–44). Subsequently, a related study examining the role of the disulfide bond in PrP folding confirmed that a monomeric molten globule-like form of PrP was formed on refolding the disulfide-reduced protein at acidic pH, but reported that, under their conditions, the circular dichroism response interpreted as β-sheet structure was associated with protein oligomerization (45). Indeed, atomic force microscopy on oligomeric full-length β-PrP (residues 23–231) shows small, round particles, showing that it is capable of formation of oligomers without forming fibrils (35). Notably, however, salt-induced oligomeric β-PrP has been shown to be a potent inhibitor of the 26 S proteasome, in a similar manner to PrP^Sc (46). Impairment of the ubiquitin-proteasome system in vivo has been linked to prion neuropathology in prion-infected mice (46).Although the global properties of several PrP intermediate states have been determined (30–32, 35), no information on their conformational properties on a sequence-specific basis has been obtained. Their conformational properties are considered important, as the elucidation of the chain conformation may provide information on the way in which these chains pack in the assembly process, and also potentially provide clues on the mechanism of amyloid assembly and the phenomenon of prion strains. As the conformational fluctuations and heterogeneity of molten globule states give rise to broad NMR spectra that preclude direct observation of their conformational properties by NMR (47–50), here we use denaturant titration experiments to determine the conformational properties of β-PrP, through the population of the unfolded state that is visible by NMR. In addition, we use circular dichroism and analytical ultracentrifugation to examine the global structural properties, and the distribution of multimeric species that are formed from β-PrP.     

Ligand-specific Conformational Changes in the ��1 Glycine Receptor Ligand-binding Domain

Understanding the activation mechanism of Cys loop ion channel receptors is key to understanding their physiological and pharmacological properties under normal and pathological conditions. The ligand-binding domains of these receptors comprise inner and outer β-sheets and structural studies indicate that channel opening is accompanied by conformational rearrangements in both β-sheets. In an attempt to resolve ligand-dependent movements in the ligand-binding domain, we employed voltage-clamp fluorometry on α1 glycine receptors to compare changes mediated by the agonist, glycine, and by the antagonist, strychnine. Voltage-clamp fluorometry involves labeling introduced cysteines with environmentally sensitive fluorophores and inferring structural rearrangements from ligand-induced fluorescence changes. In the inner β-sheet, we labeled residues in loop 2 and in binding domain loops D and E. At each position, strychnine and glycine induced distinct maximal fluorescence responses. The pre-M1 domain responded similarly; at each of four labeled positions glycine produced a strong fluorescence signal, whereas strychnine did not. This suggests that glycine induces conformational changes in the inner β-sheet and pre-M1 domain that may be important for activation, desensitization, or both. In contrast, most labeled residues in loops C and F yielded fluorescence changes identical in magnitude for glycine and strychnine. A notable exception was H201C in loop C. This labeled residue responded differently to glycine and strychnine, thus underlining the importance of loop C in ligand discrimination. These results provide an important step toward mapping the domains crucial for ligand discrimination in the ligand-binding domain of glycine receptors and possibly other Cys loop receptors.Glycine receptor (GlyR)3 chloride channels are pentameric Cys loop receptors that mediate fast synaptic transmission in the nervous system (1, 2). This family also includes nicotinic acetylcholine receptors (nAChRs), γ-aminobutyric acid type A and type C receptors, and serotonin type 3 receptors. Individual subunits comprise a large ligand-binding domain (LBD) and a transmembrane domain consisting of four α-helices (M1–M4). The LBD consists of a 10-strand β-sandwich made of an inner β-sheet with six strands and an outer β-sheet with four strands (3). The ligand-binding site is situated at the interface of adjacent subunits and is formed by loops A–C from one subunit and loops D–F from the neighboring subunit (3).The activation mechanism of Cys loop receptors is currently the subject of intense investigation because it is key to understanding receptor function under normal and pathological conditions (4, 5). Based on structural analysis of Torpedo nAChRs, Unwin and colleagues (6, 7) originally proposed that agonist binding induced the inner β-sheet to rotate, whereas the outer β-sheet tilted slightly upwards with loop C clasping around the agonist. These movements were thought to be transmitted to the transmembrane domain via a differential movement of loop 2 (β1-β2) and loop 7 (β6-β7) (both part of the inner β-sheet) and the pre-M1 domain (which is linked via a β-strand to the loop C in the outer sheet). The idea of large loop C movements accompanying agonist binding is supported by structural and functional data (3, 8–13). However, a direct link between loop C movements and channel gating has proved more difficult to establish. Although computational modeling studies have suggested that this loop may be a major component of the channel opening mechanism (14–18), experimental support for this model is not definitive. Similarly, loop F is also thought to move upon ligand binding, although there is as yet no consensus as to whether these changes represent local or global conformational changes (11, 19–21). Recently, a comparison of crystal structures of bacterial Cys loop receptors in the closed and open states revealed that although both the inner and outer β-sheets exhibit different conformations in closed and open states, the pre-M1 domain remains virtually stationary (22, 23). It is therefore relevant to question whether loop C, loop F, and pre-M1 movements are essential for Cys loop receptor activation.Strychnine is a classical competitive antagonist of GlyRs (24, 25), and to date there is no evidence that it can produce LBD structural changes. In this study we use voltage-clamp fluorometry (VCF) to compare glycine- and strychnine-induced conformational changes in the GlyR loops 2, C, D, E, and F and the pre-M1 domain in an attempt to determine whether they signal ligand-binding events, local conformational changes, or conformational changes associated with receptor activation.In a typical VCF experiment, a domain of interest is labeled with an environmentally sensitive fluorophore, and current and fluorescence are monitored simultaneously during ligand application. VCF is ideally suited for identifying ligand-specific conformational changes because it can report on electrophysiologically silent conformational changes (26), such as those induced by antagonists. Indeed, VCF has recently provided valuable insights into the conformational rearrangements of various Cys loop receptors (19, 21, 27–33).     

Regulation of Intracellular Cholesterol Distribution by Na/K-ATPase

Recent studies have ascribed many non-pumping functions to the Na/K-ATPase. We show here that graded knockdown of cellular Na/K-ATPase α1 subunit produces a parallel decrease in both caveolin-1 and cholesterol in light fractions of LLC-PK1 cell lysates. This observation is further substantiated by imaging analyses, showing redistribution of cholesterol from the plasma membrane to intracellular compartments in the knockdown cells. Moreover, this regulation is confirmed in α1^+/– mouse liver. Functionally, the knockdown-induced redistribution appears to affect the cholesterol sensing in the endoplasmic reticulum, because it activates the sterol regulatory element-binding protein pathway and increases expression of hydroxymethylglutaryl-CoA reductase and low density lipoprotein receptor in the liver. Consistently, we detect a modest increase in hepatic cholesterol as well as a reduction in the plasma cholesterol. Mechanistically, α1^+/– livers show increases in cellular Src and ERK activity and redistribution of caveolin-1. Although activation of Src is not required in Na/K-ATPase-mediated regulation of cholesterol distribution, the interaction between the Na/K-ATPase and caveolin-1 is important for this regulation. Taken together, our new findings demonstrate a novel function of the Na/K-ATPase in control of the plasma membrane cholesterol distribution. Moreover, the data also suggest that the plasma membrane Na/K-ATPase-caveolin-1 interaction may represent an important sensing mechanism by which the cells regulate the sterol regulatory element-binding protein pathway.The Na/K-ATPase, also called the sodium pump, is an ion transporter that mediates active transport of Na⁺ and K⁺ across the plasma membrane by hydrolyzing ATP (1, 2). The functional sodium pump is mainly composed of α and β subunits. The α subunit is the catalytic component of the holoenzyme; it contains both the nucleotide and the cation binding sites (3). So far, four isoforms of α subunit have been discovered, and each one shows a distinct tissue distribution pattern (4, 5). Interestingly, studies during the past few years have uncovered many non-pumping functions of Na/K-ATPase (6–10). Recently, we have demonstrated that more than half of the Na/K-ATPase may actually perform cellular functions other than ion pumping at least in LLC-PK1 cells (11). Moreover, the non-pumping pool of Na/K-ATPase mainly resides in caveolae and interacts with a variety of proteins such as Src, inositol 1,4,5-trisphosphate receptor, and caveolin-1 (12–14). While the interaction between Na/K-ATPase and inositol 1,4,5-trisphosphate receptor facilitates Ca²⁺ signaling (13) the dynamic association between Na/K-ATPase and Src appears to be an essential step for ouabain to stimulate cellular kinases (15). More recently, we report that the interaction between the Na/K-ATPase and caveolin-1 plays an important role for the membrane trafficking of caveolin-1. Knockdown of the Na/K-ATPase leads to altered subcellular distribution of caveolin-1 and increases the mobility of caveolin-1-containing vesicles (16).Caveolin is a protein marker for caveolae (17). Caveolae are flask-shaped vesicular invaginations of plasma membrane and are enriched in cholesterol, glycosphingolipids, and sphingomyelin (18). There are three genes and six isoforms of caveolin. Caveolin-1 is a 22-kDa protein and is expressed in many types of cells, including epithelial and endothelial cells. In addition to their role in biogenesis of caveolae (19), accumulating evidence has implicated caveolin proteins in cellular cholesterol homeostasis (20). For instance, caveolin-1 directly binds to cholesterol in a 1:1 ratio (21). It was also found to be an integral member of the intracellular cholesterol trafficking machinery between internal membranes and plasma membrane (22, 23). The expression of caveolin-1 appears to be under control of SREBPs,² the master regulators of intracellular cholesterol level (24). Furthermore, knockout of caveolin-1 significantly affected cholesterol metabolism in mouse embryonic fibroblasts and mouse peritoneal macrophages (25). Because we found that the Na/K-ATPase regulates cellular distribution of caveolin-1, we propose that it may also affect intracellular cholesterol distribution and metabolism. To test our hypothesis, we have investigated whether sodium pump α1 knockdown affects cholesterol distribution and metabolism both in vitro and in vivo. Our results indicate that sodium pump α1 expression level plays a role in the proper distribution of intracellular cholesterol. Down-regulation of sodium pump α1 not only redistributes cholesterol between the plasma membrane and cytosolic compartments, but also alters cholesterol metabolism in mice.     

Printor, a Novel TorsinA-interacting Protein Implicated in Dystonia Pathogenesis

Early onset generalized dystonia (DYT1) is an autosomal dominant neurological disorder caused by deletion of a single glutamate residue (torsinA ΔE) in the C-terminal region of the AAA⁺ (ATPases associated with a variety of cellular activities) protein torsinA. The pathogenic mechanism by which torsinA ΔE mutation leads to dystonia remains unknown. Here we report the identification and characterization of a 628-amino acid novel protein, printor, that interacts with torsinA. Printor co-distributes with torsinA in multiple brain regions and co-localizes with torsinA in the endoplasmic reticulum. Interestingly, printor selectively binds to the ATP-free form but not to the ATP-bound form of torsinA, supporting a role for printor as a cofactor rather than a substrate of torsinA. The interaction of printor with torsinA is completely abolished by the dystonia-associated torsinA ΔE mutation. Our findings suggest that printor is a new component of the DYT1 pathogenic pathway and provide a potential molecular target for therapeutic intervention in dystonia.Early onset generalized torsion dystonia (DYT1) is the most common and severe form of hereditary dystonia, a movement disorder characterized by involuntary movements and sustained muscle spasms (1). This autosomal dominant disease has childhood onset and its dystonic symptoms are thought to result from neuronal dysfunction rather than neurodegeneration (2, 3). Most DYT1 cases are caused by deletion of a single glutamate residue at positions 302 or 303 (torsinA ΔE) of the 332-amino acid protein torsinA (4). In addition, a different torsinA mutation that deletes amino acids Phe³²³–Tyr³²⁸ (torsinA Δ323–328) was identified in a single family with dystonia (5), although the pathogenic significance of this torsinA mutation is unclear because these patients contain a concomitant mutation in another dystonia-related protein, ϵ-sarcoglycan (6). Recently, genetic association studies have implicated polymorphisms in the torsinA gene as a genetic risk factor in the development of adult-onset idiopathic dystonia (7, 8).TorsinA contains an N-terminal endoplasmic reticulum (ER)³ signal sequence and a 20-amino acid hydrophobic region followed by a conserved AAA⁺ (ATPases associated with a variety of cellular activities) domain (9, 10). Because members of the AAA⁺ family are known to facilitate conformational changes in target proteins (11, 12), it has been proposed that torsinA may function as a molecular chaperone (13, 14). TorsinA is widely expressed in brain and multiple other tissues (15) and is primarily associated with the ER and nuclear envelope (NE) compartments in cells (16–20). TorsinA is believed to mainly reside in the lumen of the ER and NE (17–19) and has been shown to bind lamina-associated polypeptide 1 (LAP1) (21), lumenal domain-like LAP1 (LULL1) (21), and nesprins (22). In addition, recent evidence indicates that a significant pool of torsinA exhibits a topology in which the AAA⁺ domain faces the cytoplasm (20). In support of this topology, torsinA is found in the cytoplasm, neuronal processes, and synaptic terminals (2, 3, 15, 23–26) and has been shown to bind cytosolic proteins snapin (27) and kinesin light chain 1 (20). TorsinA has been proposed to play a role in several cellular processes, including dopaminergic neurotransmission (28–31), NE organization and dynamics (17, 22, 32), and protein trafficking (27, 33). However, the precise biological function of torsinA and its regulation remain unknown.To gain insights into torsinA function, we performed yeast two-hybrid screens to search for torsinA-interacting proteins in the brain. We report here the isolation and characterization of a novel protein named printor (protein interactor of torsinA) that interacts selectively with wild-type (WT) torsinA but not the dystonia-associated torsinA ΔE mutant. Our data suggest that printor may serve as a cofactor of torsinA and provide a new molecular target for understanding and treating dystonia.     

Amino Acid Position-specific Contributions to Amyloid ��-Protein Oligomerization

Understanding the structural and assembly dynamics of the amyloid β-protein (Aβ) has direct relevance to the development of therapeutic agents for Alzheimer disease. To elucidate these dynamics, we combined scanning amino acid substitution with a method for quantitative determination of the Aβ oligomer frequency distribution, photo-induced cross-linking of unmodified proteins (PICUP), to perform “scanning PICUP.” Tyr, a reactive group in PICUP, was substituted at position 1, 10, 20, 30, or 40 (for Aβ40) or 42 (for Aβ42). The effects of these substitutions were probed using circular dichroism spectroscopy, thioflavin T binding, electron microscopy, PICUP, and mass spectrometry. All peptides displayed a random coil → α/β → β transition, but substitution-dependent alterations in assembly kinetics and conformer complexity were observed. Tyr¹-substituted homologues of Aβ40 and Aβ42 assembled the slowest and yielded unusual patterns of oligomer bands in gel electrophoresis experiments, suggesting oligomer compaction had occurred. Consistent with this suggestion was the observation of relatively narrow [Tyr¹]Aβ40 fibrils. Substitution of Aβ40 at the C terminus decreased the population conformational complexity and substantially extended the highest order of oligomers observed. This latter effect was observed in both Aβ40 and Aβ42 as the Tyr substitution position number increased. The ability of a single substitution (Tyr¹) to alter Aβ assembly kinetics and the oligomer frequency distribution suggests that the N terminus is not a benign peptide segment, but rather that Aβ conformational dynamics and assembly are affected significantly by the competition between the N and C termini to form a stable complex with the central hydrophobic cluster.Alzheimer disease (AD)⁴ is the most common cause of late-life dementia (1) and is estimated to afflict more than 27 million people worldwide (2). An important etiologic hypothesis is that amyloid β-protein (Aβ) oligomers are the proximate neurotoxins in AD. Substantial in vivo and in vitro evidence supports this hypothesis (3–12). Neurotoxicity studies have shown that Aβ assemblies are potent neurotoxins (5, 13–20), and the toxicity of some oligomers can be greater than that of the corresponding fibrils (21). Soluble Aβ oligomers inhibit hippocampal long term potentiation (4, 5, 13, 15, 17, 18, 22) and disrupt cognitive function (23). Compounds that bind and disrupt the formation of oligomers have been shown to block the neurotoxicity of Aβ (24, 25). Importantly, recent studies in higher vertebrates (dogs) have shown that substantial reduction in amyloid deposits in the absence of decreases in oligomer concentration has little effect on recovery of neurological function (26).Recent studies of Aβ oligomers have sought to correlate oligomer size and biological activity. Oligomers in the supernatants of fibril preparations centrifuged at 100,000 × g caused sustained calcium influx in rat hippocampal neurons, leading to calpain activation and dynamin 1 degradation (27). Aβ-derived diffusible ligand-like Aβ42 oligomers induced inflammatory responses in cultured rat astrocytes (28). A 90-kDa Aβ42 oligomer (29) has been shown to activate ERK1/2 in rat hippocampal slices (30) and bind avidly to human cortical neurons (31), in both cases causing apoptotic cell death. A comparison of the time dependence of the toxic effects of the 90-kDa assembly with that of Aβ-derived diffusible ligands revealed a 5-fold difference, Aβ-derived diffusible ligands requiring more time for equivalent effects (31). A 56-kDa oligomer, “Aβ*56,” was reported to cause memory impairment in middle-aged transgenic mice expressing human amyloid precursor protein (32). A nonamer also had adverse effects. Impaired long term potentiation in rat brain slices has been attributed to Aβ trimers identified in media from cultured cells expressing human amyloid precursor protein (33). Dimers and trimers from this medium also have been found to cause progressive loss of synapses in organotypic rat hippocampal slices (10). In mice deficient in neprilysin, an enzyme that has been shown to degrade Aβ in vivo (34), impairment in neuronal plasticity and cognitive function correlated with significant increases in Aβ dimer levels and synapse-associated Aβ oligomers (35).The potent pathologic effects of Aβ oligomers provide a compelling reason for elucidating the mechanism(s) of their formation. This has been a difficult task because of the metastability and polydispersity of Aβ assemblies (36). To obviate these problems, we introduced the use of the method of photo-induced cross-linking of unmodified proteins (PICUP) to rapidly (<1 s) and covalently stabilize oligomer mixtures (for reviews see Refs. 37, 38). Oligomers thus stabilized no longer exist in equilibrium with monomers or each other, allowing determination of oligomer frequency distributions by simple techniques such as SDS-PAGE (37). Recently, to obtain population-average information on contributions to fibril formation of amino acid residues at specific sites in Aβ, we employed a scanning intrinsic fluorescence approach (39). Tyr was used because it is a relatively small fluorophore, exists natively in Aβ, and possesses the side chain most reactive in the PICUP chemistry (40). Using this approach, we found that the central hydrophobic cluster region (Leu¹⁷–Ala²¹) was particularly important in controlling fibril formation of Aβ40, whereas the C terminus was the predominant structural element controlling Aβ42 assembly (39). Here we present results of studies in which key strategic features of the two methods have been combined to enable execution of “scanning PICUP” and the consequent revelation of site-specific effects on Aβ oligomerization.     

Deorphanization of GPR109B as a Receptor for the ��-Oxidation Intermediate 3-OH-octanoic Acid and Its Role in the Regulation of Lipolysis

The orphan G-protein-coupled receptor GPR109B is the result of a recent gene duplication of the nicotinic acid and ketone body receptor GPR109A being found in humans but not in rodents. Like GPR109A, GPR109B is predominantly expressed in adipocytes and is supposed to mediate antilipolytic effects. Here we show that GPR109B serves as a receptor for the β-oxidation intermediate 3-OH-octanoic acid, which has antilipolytic activity on human but not on murine adipocytes. GPR109B is coupled to G_i-type G-proteins and is activated by 2- and 3-OH-octanoic acid with EC₅₀ values of about 4 and 8 μm, respectively. Interestingly, 3-OH-octanoic acid plasma concentrations reach micromolar concentrations under conditions of increased β-oxidation rates, like in diabetic ketoacidosis or under a ketogenic diet. These data suggest that the ligand receptor pair 3-OH-octanoic acid/GPR109B mediates in humans a negative feedback regulation of adipocyte lipolysis to counteract prolipolytic influences under conditions of physiological or pathological increases in β-oxidation rates.Triacylglycerols stored in the white adipose tissue serve as the major energy reserve in higher eukaryotes (1). Although they are constantly turned over by lipolysis and re-esterification, their mobilization and storage are precisely balanced by various hormones and other factors depending on the nutritional state (2). The net rate of lipolysis is increased during fasting or periods of increased energy demand. Fatty acids generated via lipolysis undergo β-oxidation in the muscle and liver to serve directly as a source of energy or as a precursor for ketone bodies (3). The major intracellular regulator of lipolysis is cyclic AMP, which stimulates cAMP-dependent kinase to activate lipolytic enzymes (2, 4–6). This lipolytic pathway is induced, for example, via β-adrenergic receptors that couple to the G-protein G_s and thereby stimulate adenylyl cyclase (7, 8). To adjust lipolysis at the appropriate rate, the effects of prolipolytic stimuli are balanced by various antilipolytic influences. Besides insulin, which promotes the degradation of cAMP via activation of phosphodiesterase 3B (2, 5, 7), several antilipolytic stimuli decrease cAMP levels by activation of G_i-coupled receptors, which mediate an inhibition of adenylyl cyclase (5, 8). One of these receptors, GPR109A, has recently been shown to mediate the anti-lipolytic effects of high concentrations of the ketone body 3-OH-butyrate thereby providing a negative feedback mechanism during fasting (9, 10). GPR109A also binds nicotinic acid (11–13) and mediates the anti-lipolytic effects of this anti-dyslipidemic drug (12).GPR109B, a close relative of GPR109A, is the result of a recent gene duplication being present in humans but not in rodents and most other mammals (14). GPR109B differs from GPR109A in an extended C-terminal tail as well as in 16 amino acids (11, 13). Despite its high homology to GPR109A, GPR109B does not bind nicotinic acid or 3-OH-butyrate with reasonable affinity (10, 11, 13). Because GPR109A and GPR109B have very similar expression patterns (11, 13, 15) and are likely to have the same basic signaling properties, agonists of GPR109B are expected to have physiological and pharmacological effects comparable with those of the GPR109A agonist 3-OH-butyrate and nicotinic acid, respectively. Recently, several synthetic compounds as well as various aromatic d-amino acids have been shown to be selective agonists at GPR109B (16–18). However, endogenous physiological anti-lipolytic ligands of GPR109B are unknown.In this study we tested endogenous carboxylic acids for their ability to activate GPR109B. We found that the fatty acid β-oxidation intermediate 3-OH-octanoic acid is a highly specific agonist of GPR109B. 3-OH-octanoic acid has anti-lipolytic activity, and its plasma concentration in humans reflects the β-oxidation flux. Our data suggest that 3-OH-octanoic acid and GPR109B mediate a negative feedback regulation of adipocyte lipolysis.