Aquaporin-2 in the ��-omics�� Era

Vasopressin controls renal water excretion largely through actions to regulate the water channel aquaporin-2 in collecting duct principal cells. Our knowledge of the mechanisms involved has increased markedly in recent years with the advent of methods for large-scale systems-level profiling such as protein mass spectrometry, yeast two-hybrid analysis, and oligonucleotide microarrays. Here we review this progress.Regulation of water excretion by the kidney is one of the most visible aspects of everyday physiology. An outdoor tennis game on a hot summer day can result in substantial water losses by sweating, and the kidneys respond by reducing water excretion. In contrast, excessive intake of water, a frequent occurrence in everyday life, results in excretion of copious amounts of clear urine. These responses serve to exact tight control on the tonicity of body fluids, maintaining serum osmolality in the range of 290–294 mosmol/kg of H₂O through the regulated return of water from the pro-urine in the renal collecting ducts to the bloodstream.The importance of this process is highlighted when the regulation fails. For example, polyuria (rapid uncontrolled excretion of water) is a sometimes devastating consequence of lithium therapy for bipolar disorder. On the other side of the coin are water balance disorders that result from excessive renal water retention causing systemic hypo-osmolality or hyponatremia. Hyponatremia due to excessive water retention can be seen with severe congestive heart failure, hepatic cirrhosis, and the syndrome of inappropriate antidiuresis.The chief regulator of water excretion is the peptide hormone AVP,² whereas the chief molecular target for regulation is the water channel AQP2. In this minireview, we describe new progress in the understanding of the molecular mechanisms involved in regulation of AQP2 by AVP in collecting duct cells, with emphasis on new information derived from “systems-level” approaches involving large-scale profiling and screening techniques such as oligonucleotide arrays, protein mass spectrometry, and yeast two-hybrid analysis. Most of the progress with these techniques is in the identification of individual molecules involved in AVP signaling and binding interactions with AQP2. Additional related issues are addressed in several recent reviews (1–4).     

An N-Glycosylation Site on the��-Propeller Domain of the Integrin ��5 Subunit Plays Key Roles in Both Its Function and Site-specific Modification by��1,4-N-Acetylglucosaminyltransferase III

Recently we reported that N-glycans on the β-propeller domain of the integrin α5 subunit (S-3,4,5) are essential for α5β1 heterodimerization, expression, and cell adhesion. Herein to further investigate which N-glycosylation site is the most important for the biological function and regulation, we characterized the S-3,4,5 mutants in detail. We found that site-4 is a key site that can be specifically modified by N-acetylglucosaminyltransferase III (GnT-III). The introduction of bisecting GlcNAc into the S-3,4,5 mutant catalyzed by GnT-III decreased cell adhesion and migration on fibronectin, whereas overexpression of N-acetylglucosaminyltransferase V (GnT-V) promoted cell migration. The phenomenon is similar to previous observations that the functions of the wild-type α5 subunit were positively and negatively regulated by GnT-V and GnT-III, respectively, suggesting that the α5 subunit could be duplicated by the S-3,4,5 mutant. Interestingly GnT-III specifically modified the S-4,5 mutant but not the S-3,5 mutant. This result was confirmed by erythroagglutinating phytohemagglutinin lectin blot analysis. The reduction in cell adhesion was consistently observed in the S-4,5 mutant but not in the S-3,5 mutant cells. Furthermore mutation of site-4 alone resulted in a substantial decrease in erythroagglutinating phytohemagglutinin lectin staining and suppression of cell spread induced by GnT-III compared with that of either the site-3 single mutant or wild-type α5. These results, taken together, strongly suggest that N-glycosylation of site-4 on the α5 subunit is the most important site for its biological functions. To our knowledge, this is the first demonstration that site-specific modification of N-glycans by a glycosyltransferase results in functional regulation.Glycosylation is a crucial post-translational modification of most secreted and cell surface proteins (1). Glycosylation is involved in a variety of physiological and pathological events, including cell growth, migration, differentiation, and tumor invasion. It is well known that glycans play important roles in cell-cell communication, intracellular signal transduction, protein folding, and stability (2, 3).Integrins comprise a family of receptors that are important for cell adhesion. The major function of integrins is to connect cells to the extracellular matrix, activate intracellular signaling pathways, and regulate cytoskeletal formation (4). Integrin α5β1 is well known as a fibronectin (FN)³ receptor. The interaction between integrin α5 and FN is essential for cell migration, cell survival, and development (5–8). In addition, integrins are N-glycan carrier proteins. For example, α5β1 integrin contains 14 and 12 putative N-glycosylation sites on the α5 and β1 subunits, respectively. Several studies suggest that N-glycosylation is essential for functional integrin α5β1. When human fibroblasts were cultured in the presence of 1-deoxymannojirimycin, which prevents N-linked oligosaccharide processing, immature α5β1 integrin appeared on the cell surface, and FN-dependent adhesion was greatly reduced (9). Treatment of purified integrin α5β1 with N-glycosidase F, which cleaves between the innermost N-acetylglucosamine (GlcNAc) and asparagine N-glycan residues of N-linked glycoproteins, prevented the inherent association between subunits and blocked α5β1 binding to FN (10).A growing body of evidence indicates that the presence of the appropriate oligosaccharide can modulate integrin activation. N-Acetylglucosaminyltransferase III (GnT-III) catalyzes the addition of GlcNAc to mannose that is β1,4-linked to an underlying N-acetylglucosamine, producing what is known as a “bisecting” GlcNAc linkage as shown in Fig. 1B. GnT-III is generally regarded as a key glycosyltransferase in N-glycan biosynthetic pathways and contributes to inhibition of metastasis. The introduction of a bisecting GlcNAc catalyzed by GnT-III suppresses additional processing and elongation of N-glycans. These reactions, which are catalyzed in vitro by other glycosyltransferases, such as N-acetylglucosaminyltransferase V (GnT-V), which catalyzes the formation of β1,6 GlcNAc branching structures (Fig. 1B) and plays important roles in tumor metastasis, do not proceed because the enzymes cannot utilize the bisected N-glycans as a substrate. Introduction of the bisecting GlcNAc to integrin α5 by overexpression of GnT-III resulted in decreased in ligand binding and down-regulation of cell adhesion and migration (11–13). Contrary to the functions of GnT-III, overexpression of GnT-V promoted integrin α5β1-mediated cell migration on FN (14). These observations clearly demonstrate that the alteration of N-glycan structure affected the biological functions of integrin α5β1. Similarly characterization of the carbohydrate moieties in integrin α3β1 from non-metastatic and metastatic human melanoma cell lines showed that expression of β1,6 GlcNAc branched structures was higher in metastatic cells compared with non-metastatic cells, confirming the notion that the β1,6 GlcNAc branched structure confers invasive and metastatic properties to cancer cells. In fact, Partridge et al. (15) reported that GnT-V-modified N-glycans containing poly-N-acetyllactosamine, the preferred ligand for galectin-3, on surface receptors oppose their constitutive endocytosis, promoting intracellular signaling and consequently cell migration and tumor metastasis.Open in a separate windowFIGURE 1.Potential N-glycosylation sites on the α5 subunit and its modification by GnT-III and GnT-V. A, schematic diagram of potential N-glycosylation sites on the α5 subunit. Putative N-glycosylation sites are indicated by triangles, and point mutations are indicated by crosses (N84Q, N182Q, N297Q, N307Q, N316Q, N524Q, N530Q, N593Q, N609Q, N675Q, N712Q, N724Q, N773Q, and N868Q). B, illustration of the reaction catalyzed by GnT-III and GnT-V. Square, GlcNAc; circle, mannose. TM, transmembrane domain.In addition, sialylation on the non-reducing terminus of N-glycans of α5β1 integrin plays an important role in cell adhesion. Colon adenocarcinomas express elevated levels of α2,6 sialylation and increased activity of ST6GalI sialyltransferase. Elevated ST6GalI positively correlated with metastasis and poor survival. Therefore, ST6GalI-mediated hypersialylation likely plays a role in colorectal tumor invasion (16, 17). In fact, oncogenic ras up-regulated ST6GalI and, in turn, increased sialylation of β1 integrin adhesion receptors in colon epithelial cells (18). However, this is not always the case. The expression of hyposialylated integrin α5β1 was induced by phorbol esterstimulated differentiation in myeloid cells in which the expression of the ST6GalI was down-regulated by the treatment, increasing FN binding (19). A similar phenomenon was also observed in hematopoietic or other epithelial cells. In these cells, the increased sialylation of the β1 integrin subunit was correlated with reduced adhesiveness and metastatic potential (20–22). In contrast, the enzymatic removal of α2,8-linked oligosialic acids from the α5 integrin subunit inhibited cell adhesion to FN (23). Collectively these findings suggest that the interaction of integrin α5β1 with FN is dependent on its N-glycosylation and the processing status of N-glycans.Because integrin α5β1 contains multipotential N-glycosylation sites, it is important to determine the sites that are crucial for its biological function and regulation. Recently we found that N-glycans on the β-propeller domain (sites 3, 4, and 5) of the integrin α5 subunit are essential for α5β1 heterodimerization, cell surface expression, and biological function (24). In this study, to further investigate the underlying molecular mechanism of GnT-III-regulated biological functions, we characterized the N-glycans on the α5 subunit in detail using genetic and biochemical approaches and found that site-4 is a key site that can be specifically modified by GnT-III.     

ULK1��ATG13��FIP200 Complex Mediates mTOR Signaling and Is Essential for Autophagy

Autophagy is a degradative process that recycles long-lived and faulty cellular components. It is linked to many diseases and is required for normal development. ULK1, a mammalian serine/threonine protein kinase, plays a key role in the initial stages of autophagy, though the exact molecular mechanism is unknown. Here we report identification of a novel protein complex containing ULK1 and two additional protein factors, FIP200 and ATG13, all of which are essential for starvation-induced autophagy. Both FIP200 and ATG13 are critical for correct localization of ULK1 to the pre-autophagosome and stability of ULK1 protein. Additionally, we demonstrate by using both cellular experiments and a de novo in vitro reconstituted reaction that FIP200 and ATG13 can enhance ULK1 kinase activity individually but both are required for maximal stimulation. Further, we show that ATG13 and ULK1 are phosphorylated by the mTOR pathway in a nutrient starvation-regulated manner, indicating that the ULK1·ATG13·FIP200 complex acts as a node for integrating incoming autophagy signals into autophagosome biogenesis.Macroautophagy (herein referred to as autophagy) is a catabolic process whereby long-lived proteins and damaged organelles are shuttled to lysosomes for degradation. This process is conserved in all eukaryotes. Under normal growth conditions a housekeeping level of autophagy exists. Under stress, such as nutrient starvation, autophagy is strongly induced resulting in the engulfment of cytosolic components and organelles in specialized double-membrane structures termed autophagosomes. Following fusion of the outer autophagosomal membrane with lysosomes, the inner membrane and its cytoplasmic cargo are degraded and recycled (1–3). Recent work has implicated autophagy in many disease pathologies, including cancer, neurodegeneration, as well as in eliminating intracellular pathogens (4–8).The morphology of autophagy was first described in mammalian cells over 50 years ago (9). However, it is only recently through yeast genetic screens, that multiple autophagy-related (ATG) genes have been identified (10–12). The yeast ATG proteins have been classified into four major groups: the Atg1 protein kinase complex, the Vps34 phosphatidylinositol 3-phosphate kinase complex, the Atg8/Atg12 conjugation systems, and the Atg9 recycling complex (13). Even though many ATG genes are now known, most of which have functional homologs in mammalian cells (14, 15), the molecular mechanism by which they sense the initial triggers and subsequently dictate autophagy-specific intracellular membrane events is far from understood.In yeast, one of the earliest autophagy-specific events is believed to involve the Atg1 protein kinase complex. Atg1 is a serine/threonine protein kinase and a key autophagy-regulator (16). Atg1 is complexed to at least two other proteins during autophagy, Atg13 and Atg17, both of which are required for normal Atg1 function and autophagosome generation (17–19). Classical signaling pathways such as the cAMP-dependent kinase (PKA) pathway or the Tor kinase pathway appear to converge upon this complex, placing Atg1 at an early stage during autophagosome biogenesis (20–22). Atg1 phosphorylation by PKA blocks its association with the forming autophagosome (21), while the Tor pathway hyperphosphorylates Atg13 causing a reduced affinity of Atg13 for Atg1, resulting in repression of autophagy (17, 19). In contrast, nutrient starvation or inhibition of Tor leads to dephosphorylation of Atg13 thus increased Atg1 complex formation and kinase activity, resulting in stimulation of autophagy (19). Surprisingly, the physiological substrates of Atg1 kinase have not been identified; thus how Atg1 transduces upstream autophagic signaling is undefined. Recently, mammalian homologs of Atg1 have been identified as ULK1 and ULK2 (Unc-51-like kinase)² (23–25). ULK1 and ULK2 are ubiquitously expressed and localize to the isolation membrane, or forming autophagosome, upon nutrient starvation (25); RNAi-mediated depletion of ULK1 in HEK293 cells compromises autophagy (23, 24). The exact role of ULK1 versus ULK2 in autophagy is unclear, and it is possible some redundancy exists between the two isoforms (26).Given the conservation of autophagy from yeast to man, it is interesting to note that no mammalian counterpart to yeast Atg13 or Atg17 had been identified until very recently. The protein FIP200 (focal adhesion kinase family-interacting protein of 200 kDa) was identified as an autophagy-essential binding partner of both ULK1 and ULK2 (25), and it has been speculated that FIP200 might be the equivalent of yeast Atg17, despite low sequence similarity (25, 27).In this study, we delve deeper into the molecular regulation of ULK1 to gain a better insight into how mammalian signaling pathways affect autophagy initiation. We describe here the identification of a triple complex consisting of ULK1, FIP200, and the mammalian equivalent of Atg13. This complex is required not only for localization of ULK1 to the isolation membrane but also for maximal kinase activity. In addition, both ATG13 and ULK1 are kinase substrates in the mTOR pathway and thus might function to sense nutrient starvation. Therefore, this study defines the role of mammalian ULK1-ATG13-FIP200 complex in mediating the initial autophagic triggers and to transduce the signal to the core autophagic machinery.     

N-Glycosylation of the I-like Domain of ��1 Integrin Is Essential for ��1 Integrin Expression and Biological Function: IDENTIFICATION OF THE MINIMAL N-GLYCOSYLATION REQUIREMENT FOR ��5��1*

N-Glycosylation of integrin α5β1 plays a crucial role in cell spreading, cell migration, ligand binding, and dimer formation, but the detailed mechanisms by which N-glycosylation mediates these functions remain unclear. In a previous study, we showed that three potential N-glycosylation sites (α5S3–5) on the β-propeller of the α5 subunit are essential to the functional expression of the subunit. In particular, site 5 (α5S5) is the most important for its expression on the cell surface. In this study, the function of the N-glycans on the integrin β1 subunit was investigated using sequential site-directed mutagenesis to remove the combined putative N-glycosylation sites. Removal of the N-glycosylation sites on the I-like domain of the β1 subunit (i.e. the Δ4-6 mutant) decreased both the level of expression and heterodimeric formation, resulting in inhibition of cell spreading. Interestingly, cell spreading was observed only when the β1 subunit possessed these three N-glycosylation sites (i.e. the S4-6 mutant). Furthermore, the S4-6 mutant could form heterodimers with either α5S3-5 or α5S5 mutant of the α5 subunit. Taken together, the results of the present study reveal for the first time that N-glycosylation of the I-like domain of the β1 subunit is essential to both the heterodimer formation and biological function of the subunit. Moreover, because the α5S3-5/β1S4-6 mutant represents the minimal N-glycosylation required for functional expression of the β1 subunit, it might also be useful for the study of molecular structures.Integrin is a heterodimeric glycoprotein that consists of both an α and a β subunit (1). The interaction between integrin and the extracellular matrix is essential to both physiologic and pathologic events, such as cell migration, development, cell viability, immune homeostasis, and tumorigenesis (2, 3). Among the integrin superfamily, β1 integrin can combine with 12 distinct α subunits (α1–11, αv) to form heterodimers, thereby acquiring a wide variety of ligand specificity (1, 4). Integrins are thought to be regulated by inside-out signaling mechanisms that provoke conformational changes, which modulate the affinity of integrin for the ligand (5). However, an increasing body of evidence suggests that cell-surface carbohydrates mediate a variety of interactions between integrin and its extracellular environment, thereby affecting integrin activity and possibly tumor metastasis as well (6–8).Guo et al. (9) reported that an increase in β1–6-GlcNAc sugar chains on the integrin β1 subunit stimulated cell migration. In addition, elevated sialylation of the β1 subunit, because of Ras-induced STGal-I transferase activity, also induced cell migration (10, 11). Conversely, cell migration and spreading were reduced by the addition of a bisecting GlcNAc, which is a product of N-acetylglucosaminyltransferase III (GnT-III),² to the α5β1 and α3β1 integrins (12, 13). Alterations of N-glycans on integrins might also regulate their cis interactions with membrane-associated proteins, including the epidermal growth factor receptor, the galectin family, and the tetraspanin family of proteins (14–19).In addition to the positive and negative regulatory effects of N-glycan, several research groups have reported that N-glycans must be present on integrin α5β1 for the αβ heterodimer formation and proper integrin-matrix interactions. Consistent with this hypothesis, in the presence of the glycosylation inhibitor, tunicamycin, normal integrin-substrate binding and transport to the cell surface are inhibited (20). Moreover, treatment of purified integrin with N-glycosidase F blocked both the inherent association of the subunits and the interaction between integrin and fibronectin (FN) (21). These results suggest that N-glycosylation is essential to the functional expression of α5β1. However, because integrin α5β1 contains 26 potential N-linked glycosylation sites, 14 in the α subunit and 12 in the β subunit, identification of the sites that are essential to its biological functions is key to understanding the molecular mechanisms by which N-glycans alter integrin function. Recently, our group determined that N-glycosylation of the β-propeller domain on the α5 subunit is essential to both heterodimerization and biological functions of the subunit. Furthermore, we determined that sites 3–5 are the most important sites for α5 subunit-mediated cell spreading and migration on FN (22). The purpose of this study was to clarify the roles of N-glycosylation of the β1 subunit. Therefore, we performed combined substitutions in the putative N-glycosylation sites by replacement of asparagine residues with glutamine residues. We subsequently introduced these mutated genes into β1-deficient epithelial cells (GE11). The results of these mutation experiments revealed that the N-glycosylation sites on the I-like domain of the β1 subunit, sites number 4–6 (S4-6), are essential to both heterodimer formation and biological functions, such as cell spreading.     

Insulin Resistance in Striated Muscle-specific Integrin Receptor ��1-deficient Mice

Integrin receptor plays key roles in mediating both inside-out and outside-in signaling between cells and the extracellular matrix. We have observed that the tissue-specific loss of the integrin β1 subunit in striated muscle results in a near complete loss of integrin β1 subunit protein expression concomitant with a loss of talin and to a lesser extent, a reduction in F-actin content. Muscle-specific integrin β1-deficient mice had no significant difference in food intake, weight gain, fasting glucose, and insulin levels with their littermate controls. However, dynamic analysis of glucose homeostasis using euglycemichyperinsulinemic clamps demonstrated a 44 and 48% reduction of insulin-stimulated glucose infusion rate and glucose clearance, respectively. The whole body insulin resistance resulted from a specific inhibition of skeletal muscle glucose uptake and glycogen synthesis without any significant effect on the insulin suppression of hepatic glucose output or insulin-stimulated glucose uptake in adipose tissue. The reduction in skeletal muscle insulin responsiveness occurred without any change in GLUT4 protein expression levels but was associated with an impairment of the insulin-stimulated protein kinase B/Akt serine 473 phosphorylation but not threonine 308. The inhibition of insulin-stimulated serine 473 phosphorylation occurred concomitantly with a decrease in integrin-linked kinase expression but with no change in the mTOR·Rictor·LST8 complex (mTORC2). These data demonstrate an in vivo crucial role of integrin β1 signaling events in mediating cross-talk to that of insulin action.Integrin receptors are a large family of integral membrane proteins composed of a single α and β subunit assembled into a heterodimeric complex. There are 19 α and 8 β mammalian subunit isoforms that combine to form 25 distinct α,β heterodimeric receptors (1-5). These receptors play multiple critical roles in conveying extracellular signals to intracellular responses (outside-in signaling) as well as altering extracellular matrix interactions based upon intracellular changes (inside-out signaling). Despite the large overall number of integrin receptor complexes, skeletal muscle integrin receptors are limited to seven α subunit subtypes (α1, α3, α4, α5, α6, α7, and αν subunits), all associated with the β1 integrin subunit (6, 7).Several studies have suggested an important cross-talk between extracellular matrix and insulin signaling. For example, engagement of β1 subunit containing integrin receptors was observed to increase insulin-stimulated insulin receptor substrate (IRS)² phosphorylation, IRS-associated phosphatidylinositol 3-kinase, and activation of protein kinase B/Akt (8-11). Integrin receptor regulation of focal adhesion kinase was reported to modulate insulin stimulation of glycogen synthesis, glucose transport, and cytoskeleton organization in cultured hepatocytes and myoblasts (12, 13). Similarly, the integrin-linked kinase (ILK) was suggested to function as one of several potential upstream kinases that phosphorylate and activate Akt (14-18). In this regard small interfering RNA gene silencing of ILK in fibroblasts and conditional ILK gene knockouts in macrophages resulted in a near complete inhibition of insulin-stimulated Akt serine 473 (Ser-473) phosphorylation concomitant with an inhibition of Akt activity and phosphorylation of Akt downstream targets (19). However, a complex composed of mTOR·Rictor·LST8 (termed mTORC2) has been identified in several other studies as the Akt Ser-473 kinase (20, 21). In addition to Ser-473, Akt protein kinase activation also requires phosphorylation on threonine 308 Thr-30 by phosphoinositide-dependent protein kinase, PDK1 (22-24).In vivo, skeletal muscle is the primary tissue responsible for postprandial (insulin-stimulated) glucose disposal that results from the activation of signaling pathways leading to the translocation of the insulin-responsive glucose transporter, GLUT4, from intracellular sites to the cell surface membranes (25, 26). Dysregulation of any step of this process in skeletal muscle results in a state of insulin resistance, thereby predisposing an individual for the development of diabetes (27-33). Although studies described above have utilized a variety of tissue culture cell systems to address the potential involvement of integrin receptor signaling in insulin action, to date there has not been any investigation of integrin function on insulin action or glucose homeostasis in vivo. To address this issue, we have taken advantage of Cre-LoxP technology to inactivate the β1 integrin receptor subunit gene in striated muscle. We have observed that muscle creatine kinase-specific integrin β1 knock-out (MCKItgβ1 KO) mice display a reduction of insulin-stimulated glucose infusion rate and glucose clearance. The impairment of insulin-stimulated skeletal muscle glucose uptake and glycogen synthesis resulted from a decrease in Akt Ser-473 phosphorylation concomitant with a marked reduction in ILK expression. Together, these data demonstrate an important cross-talk between integrin receptor function and insulin action and suggests that ILK may function as an Akt Ser-473 kinase in skeletal muscle.     

Copper��Dopamine Complex Induces Mitochondrial Autophagy Preceding Caspase-independent Apoptotic Cell Death

Parkinsonism is one of the major neurological symptoms in Wilson disease, and young workers who worked in the copper smelting industry also developed Parkinsonism. We have reported the specific neurotoxic action of copper·dopamine complex in neurons with dopamine uptake. Copper·dopamine complex (100 μm) induces cell death in RCSN-3 cells by disrupting the cellular redox state, as demonstrated by a 1.9-fold increase in oxidized glutathione levels and a 56% cell death inhibition in the presence of 500 μm ascorbic acid; disruption of mitochondrial membrane potential with a spherical shape and well preserved morphology determined by transmission electron microscopy; inhibition (72%, p < 0.001) of phosphatidylserine externalization with 5 μm cyclosporine A; lack of caspase-3 activation; formation of autophagic vacuoles containing mitochondria after 2 h; transfection of cells with green fluorescent protein-light chain 3 plasmid showing that 68% of cells presented autophagosome vacuoles; colocalization of positive staining for green fluorescent protein-light chain 3 and Rhod-2AM, a selective indicator of mitochondrial calcium; and DNA laddering after 12-h incubation. These results suggest that the copper·dopamine complex induces mitochondrial autophagy followed by caspase-3-independent apoptotic cell death. However, a different cell death mechanism was observed when 100 μm copper·dopamine complex was incubated in the presence of 100 μm dicoumarol, an inhibitor of NAD(P)H quinone:oxidoreductase (EC 1.6.99.2, also known as DT-diaphorase and NQ01), because a more extensive and rapid cell death was observed. In addition, cyclosporine A had no effect on phosphatidylserine externalization, significant portions of compact chromatin were observed within a vacuolated nuclear membrane, DNA laddering was less pronounced, the mitochondria morphology was more affected, and the number of cells with autophagic vacuoles was a near 4-fold less.A possible role of copper in the neurodegeneration of dopaminergic neurons is supported by the fact that patients with neurological Wilson disease, a copper deposition disorder, display a number of extrapyramidal motor symptoms, including Parkinsonism. The cerebral manifestations in neurological Wilson disease are expressed as bradykinesia, rigidity, tremor, dyskinesia, and dysarthria (1). It has been proposed that neurological Wilson disease can be assigned to the group of secondary Parkinsonian syndromes (2). Interestingly, young workers who worked in the copper smelting industry also developed Parkinsonism (3).Studies performed in rats showed copper (Cu²⁺)-induced degeneration of dopaminergic neurons in the nigrostriatal system. Likewise, it was described that copper neurotoxicity in rat substantia nigra and striatum is dependent on NAD(P)H dehydrogenase inhibition (4, 5). All of these results support a possible role for copper in the neurodegeneration of dopaminergic neurons.The general mechanism of toxicity, induced by the reduced form of copper (Cu⁺) catalyzing the formation of hydroxyl radicals in the presence of hydrogen peroxide through the Fenton reaction, cannot explain the specific degeneration of dopaminergic neurons in Parkinsonism induced in neurological Wilson disease, or in miners working in the copper smelting industry. The selective action of copper can be explained by the ability of copper to form a complex with dopamine, allowing this metal to be transported by cells that have the ability to take up dopamine (6). This specific neurotoxic action of copper in neurons with dopamine uptake is dependent on (i) the ability of copper to form a complex with dopamine (Cu·DA)² (6, 7), (ii) uptake of Cu·DA complex by dopamine transporters, (iii) oxidation of dopamine to aminochrome, and (iv) one-electron reduction of aminochrome by inhibiting NAD(P)H dehydrogenase (6). These findings may explain the selective neurotoxic action of copper in the brain, but they do not explain the cell death mechanism.Currently, cell death is divided into three categories: apoptosis, autophagy, and necrosis. At the current time, only apoptosis and autophagic cell death are generally accepted as being legitimate forms of programmed cell death. Alternative models of cell death have therefore been proposed, including para-apoptosis, mitotic catastrophe, oncosis, and pyroptosis (8–12). Necrosis is characterized mostly by the absence of caspase activation, cytochrome c release, and DNA oligonucleosomal fragmentation. Apoptotic cells are characterized by the formation of blebs, chromatin condensation, DNA oligonucleosomal fragmentation, and exposure of phosphatidylserine on the external membrane. This mode of cell death can be dependent or independent of activation of caspases (13). On the other hand, autophagy can be distinguished from apoptosis by sequestration of bulk cytoplasm and organelles in double or multimembrane autophagic vacuoles that then fuse with the lysosomal system. Some of these described mechanisms are related to neurological diseases such as Parkinson disease (14, 15). Cells can use different methods to activate their own destruction, and more than one death program may be activated at the same time (16, 17).The purpose of this study was to examine the Cu·DA complex-induced cell death mechanism in RCSN-3 cells, a cell line that expresses dopamine, norepinephrine, and serotonin transporters (18, 19).     

Dynamic ��-Helix Structure of Micelle-bound Human Amylin

Amylin is an endocrine hormone that regulates metabolism. In patients afflicted with type 2 diabetes, amylin is found in fibrillar deposits in the pancreas. Membranes are thought to facilitate the aggregation of amylin, and membrane-bound oligomers may be responsible for the islet β-cell toxicity that develops during type 2 diabetes. To better understand the structural basis for the interactions between amylin and membranes, we determined the NMR structure of human amylin bound to SDS micelles. The first four residues in the structure are constrained to form a hairpin loop by the single disulfide bond in amylin. The last nine residues near the C terminus are unfolded. The core of the structure is an α-helix that runs from about residues 5–28. A distortion or kink near residues 18–22 introduces pliancy in the angle between the N- and C-terminal segments of the α-helix. Mobility, as determined by ¹⁵N relaxation experiments, increases from the N to the C terminus and is strongly correlated with the accessibility of the polypeptide to spin probes in the solution phase. The spin probe data suggest that the segment between residues 5 and 17 is positioned within the hydrophobic lipid environment, whereas the amyloidogenic segment between residues 20 and 29 is at the interface between the lipid and solvent. This orientation may direct the aggregation of amylin on membranes, whereas coupling between the two segments may mediate the transition to a toxic structure.Type 2 diabetes affects over 100 million people worldwide (1) and is thought to cost upward of $130 billion dollars a year to treat in the United States alone (2). The endocrine hormone amylin (also known as islet amyloid polypeptide) appears to have key roles in diabetes pathology (3–5). The normal functions of amylin include the inhibition of glucagon secretion, slowing down the emptying of the stomach, and inducing a feeling of satiety through the actions of the hormone on neurons of the hypothalamus in the brain (5). The effects of amylin are exerted in concert with those of insulin and reduce the level of glucose in the blood (3, 5). Circulating amylin levels increase in a number of pathological conditions, including obesity, syndrome X, pancreatic cancer, and renal failure (3). Amylin levels together with insulin are raised initially in type 2 diabetes but fall as the disease progresses to a stage where the pancreatic islets of Langerhans β-cells that synthesize amylin no longer function (3).One of the hallmarks of type 2 diabetes, found in 90% of patients, is the formation of extracellular amyloid aggregates composed of amylin (3–5). The amyloid deposits accumulate in the interstitial fluid between islet cells and are usually juxtaposed with the β-cell membranes (3). Aggregates of amylin are toxic when added to cultures of β-cells, so that the amyloid found in situ may be responsible for β-cell death as type 2 diabetes progresses (6, 7). Genetic evidence that amylin is directly involved in pathology includes a familial S20G mutation that leads to early onset of the disease (8) and produces an amylin variant that aggregates more readily (9).As with all amyloids it is unclear whether fibrillar structures or soluble oligomers are responsible for pathology. A recurrent theme for amyloidogenic proteins is that toxicity appears to be exerted through membrane-bound oligomers that form pores and disrupt ion balance across membranes (4, 10–13). Experimental evidence for such oligomers has been found for the amyloid-β (Aβ)² peptides (14), which cause Alzheimer disease, and for α-synuclein (αS), the protein involved in Parkinson disease (15), a particular interest of our laboratory. The similar toxic effects exerted by these amyloidogenic molecules may have a common structural and physical basis. Detailed structural models are available for Aβ (16) and αS (17) bound to SDS micelle mimetics of membranes. For amylin there are models of peptide fragments 1–19 (18), 20–29 (19), and 17–29 (20) bound to micelles but as of yet no model of the complete hormone. This turns out to be particularly important as the interplay between structure and dynamics in amylin only comes to light when considering the whole molecule.Here we report the solution structure of human amylin bound to SDS micelles. We complement the structure with information on dynamics and on the immersion of amylin into micelles.     

Detecting Morphologically Distinct Oligomeric Forms of ��-Synuclein

Neuropathologic and genetics studies as well as transgenic animal models have provided strong evidence linking misfolding and aggregation of α-synuclein to the progression of Parkinson disease (PD) and other related disorders. A growing body of evidence implicates various oligomeric forms of α-synuclein as the toxic species responsible for neurodegeneration and neuronal cell death. Although numerous different oligomeric forms of α-synuclein have been identified in vitro, it is not known which forms are involved in PD or how, when, and where different forms contribute to the progression of PD. Reagents that can interact with specific aggregate forms of α-synuclein would be very useful not only as tools to study how different aggregate forms affect cell function, but also as potential diagnostic and therapeutic agents for PD. Here we show that a single chain antibody fragment (syn-10H scFv) isolated from a phage display antibody library binds to a larger, later stage oligomeric form of α-synuclein than a previously reported oligomeric specific scFv isolated in our laboratory. The scFv described here inhibits aggregation of α-synuclein in vitro, blocks extracellular α-synuclein-induced toxicity in both undifferentiated and differentiated human neuroblastoma cell lines (SH-SY5Y), and specifically recognizes naturally occurring aggregates in PD but not in healthy human brain tissue.Parkinson disease (PD)² is the second most common neurodegenerative disorder of the elderly, affecting more than 500,000 people in the United States (1), with 50,000 new cases reported each year at an annual cost estimated at 10 billion dollars per year. Pathologically, PD is characterized by the progressive loss of dopaminergic neurons in the substantia nigra and formation of fibrillar cytoplasmic inclusions known as Lewy bodies and Lewy neurites (2, 3). The protein α-synuclein has been strongly linked to PD (4, 5) and other related neurodegenerative disorders (6, 7) by several lines of evidence. 1) It is the major component of the hallmark Lewy body aggregates associated with PD. 2) Mutations (A53T, A30P, and E46K, where A30P is human A30P α-synuclein; A53T is human A53T α-synuclein; E46K is human E46K α-synuclein) or multiplication in the α-synuclein gene have been linked to familial PD (8–10). 3) Overexpression of α-synuclein in transgenic mice and Drosophila has been shown to induce the formation of PD-like pathological phenotypes and behavior, although the animal models do not in general replicate neuronal loss patterns (11, 12).α-Synuclein is a small protein (14 kDa) expressed mainly in brain tissues and is primarily localized at the presynaptic terminals of neurons (13). The primary structure of α-synuclein consists of three distinct regions. The N-terminal region of α-synuclein includes the mutation sites associated with familial PD (A53T, A30P, and E46K) and contains six imperfectly conserved repeats (KTKEGV) that may facilitate protein-protein binding. This repeat section is predicted to form amphipathic α-helices, typical of the lipid-binding domain of apolipoproteins (14). The central region, non-amyloid component, is extremely hydrophobic and includes a 12-residue stretch (VTGVTAVAQKTV) that is essential for aggregation (15). The C-terminal region is enriched with acidic glutamate and aspartate residues and is responsible for the chaperone function of α-synuclein (16).α-Synuclein normally exists as an unfolded protein, but it can adopt several different folded conformations depending on the environment, including small aggregates or oligomers, spherical and linear protofibrils, as well as the fibrillar structure found in Lewy bodies (14, 15). A growing body of evidence implicates the oligomeric forms of α-synuclein as the toxic species responsible for neurodegeneration and neuronal cell death (16–18). Several different oligomeric forms of α-synuclein including spherical, annular (19), pore-like (20), and dopamine-stabilized structures have been identified in vitro (21).α-Synuclein is considered a cytosolic protein, and consequently its pathogenic effect was assumed to be limited to the cytoplasm of single cells. However, recent studies have suggested that α-synuclein also has extracellular pathogenic effects (22–25). α-Synuclein was detected in blood plasma and cerebrospinal fluid in both monomeric and oligomeric forms (22–25), and the presence of significantly elevated levels of oligomeric species of α-synuclein has been reported extracellularly in plasma and cerebrospinal fluid samples from patients with PD (23). Furthermore, various studies have shown that aggregated α-synuclein added extracellularly to the culture medium is cytotoxic (26–32).Despite all these studies, it is still not clear how the various aggregate forms of α-synuclein are involved in the progression of PD. Therefore, reagents that can interact with specific aggregate forms of α-synuclein would be very useful not only for fundamental studies of how α-synuclein aggregates affect cell function but also as potential diagnostic and therapeutic agents for PD.Recently, we reported inhibition of both aggregation and extracellular toxicity of α-synuclein in vitro by a single chain variable domain antibody fragment (scFv) that specifically recognized an oligomeric form of α-synuclein (32). In this study, we describe a second scFv (syn-10H) that binds a larger later stage oligomeric form of α-synuclein than the previously reported scFv. The syn-10H scFv neutralizes α-synuclein-induced toxicity in both undifferentiated and differentiated SH-SY5Y human neuroblastoma cell line and inhibits α-synuclein aggregation in vitro. The syn-10H scFv reacts specifically with homogenized PD brain tissue but does not cross-react with similarly treated samples taken from Alzheimer disease (AD) or healthy brain samples. Such scFvs therefore have potential value as diagnostic reagents to identify the presence of specific oligomeric species in PD tissue and fluid samples. The scFvs also have value as therapeutic agents as they can be used either extracellularly or expressed intracellularly (intrabodies) to prevent formation of toxic aggregates in vivo whether inside or outside of cells. Intrabodies have been used efficiently to neutralize toxic effects of different pathogenic agents, including α-synuclein (33–36). Moreover, immunization studies in mouse models of PD have shown that extracellular antibodies can reduce accumulation of intracellular aggregates of α-synuclein (37), thereby providing precedent for the use of scFvs in potential passive vaccination strategies for treating PD.     

Proper Restoration of Excitation-Contraction Coupling in the Dihydropyridine Receptor ��1-null Zebrafish Relaxed Is an Exclusive Function of the ��1a Subunit

The paralyzed zebrafish strain relaxed carries a null mutation for the skeletal muscle dihydropyridine receptor (DHPR) β_1a subunit. Lack of β_1a results in (i) reduced membrane expression of the pore forming DHPR α_1S subunit, (ii) elimination of α_1S charge movement, and (iii) impediment of arrangement of the DHPRs in groups of four (tetrads) opposing the ryanodine receptor (RyR1), a structural prerequisite for skeletal muscle-type excitation-contraction (EC) coupling. In this study we used relaxed larvae and isolated myotubes as expression systems to discriminate specific functions of β_1a from rather general functions of β isoforms. Zebrafish and mammalian β_1a subunits quantitatively restored α_1S triad targeting and charge movement as well as intracellular Ca²⁺ release, allowed arrangement of DHPRs in tetrads, and most strikingly recovered a fully motile phenotype in relaxed larvae. Interestingly, the cardiac/neuronal β_2a as the phylogenetically closest, and the ancestral housefly β_M as the most distant isoform to β_1a also completely recovered α_1S triad expression and charge movement. However, both revealed drastically impaired intracellular Ca²⁺ transients and very limited tetrad formation compared with β_1a. Consequently, larval motility was either only partially restored (β_2a-injected larvae) or not restored at all (β_M). Thus, our results indicate that triad expression and facilitation of 1,4-dihydropyridine receptor (DHPR) charge movement are common features of all tested β subunits, whereas the efficient arrangement of DHPRs in tetrads and thus intact DHPR-RyR1 coupling is only promoted by the β_1a isoform. Consequently, we postulate a model that presents β_1a as an allosteric modifier of α_1S conformation enabling skeletal muscle-type EC coupling.Excitation-contraction (EC)³ coupling in skeletal muscle is critically dependent on the close interaction of two distinct Ca²⁺ channels. Membrane depolarizations of the myotube are sensed by the voltage-dependent 1,4-dihydropyridine receptor (DHPR) in the sarcolemma, leading to a rearrangement of charged amino acids (charge movement) in the transmembrane segments S4 of the pore-forming DHPR α_1S subunit (1, 2). This conformational change induces via protein-protein interaction (3, 4) the opening of the sarcoplasmic type-1 ryanodine receptor (RyR1) without need of Ca²⁺ influx through the DHPR (5). The release of Ca²⁺ from the sarcoplasmic reticulum via RyR1 consequently induces muscle contraction. The protein-protein interaction mechanism between DHPR and RyR1 requires correct ultrastructural targeting of both channels. In Ca²⁺ release units (triads and peripheral couplings) of the skeletal muscle, groups of four DHPRs (tetrads) are coupled to every other RyR1 and hence are geometrically arranged following the RyR-specific orthogonal arrays (6).The skeletal muscle DHPR is a heteromultimeric protein complex, composed of the voltage-sensing and pore-forming α_1S subunit and auxiliary subunits β_1a, α₂δ-1, and γ₁ (7). While gene knock-out of the DHPR γ₁ subunit (8, 9) and small interfering RNA knockdown of the DHPR α₂δ-1 subunit (10-12) have indicated that neither subunit is essential for coupling of the DHPR with RyR1, the lack of the α_1S or of the intracellular β_1a subunit is incompatible with EC coupling and accordingly null model mice die perinatally due to asphyxia (13, 14). β subunits of voltage-gated Ca²⁺ channels were repeatedly shown to be responsible for the facilitation of α₁ membrane insertion and to be potent modulators of α₁ current kinetics and voltage dependence (15, 16). Whether the loss of EC coupling in β₁-null mice was caused by decreased DHPR membrane expression or by the lack of a putative specific contribution of the β subunit to the skeletal muscle EC coupling apparatus (17, 18) was not clearly resolved. Recently, other β-functions were identified in skeletal muscle using the β₁-null mutant zebrafish relaxed (19, 20). Like the β₁-knock-out mouse (14) zebrafish relaxed is characterized by complete paralysis of skeletal muscle (21, 22). While β₁-knock-out mouse pups die immediately after birth due to respiratory paralysis (14), larvae of relaxed are able to survive for several days because of oxygen and metabolite diffusion via the skin (23). Using highly differentiated myotubes that are easy to isolate from these larvae, the lack of EC coupling could be described by quantitative immunocytochemistry as a moderate ∼50% reduction of α_1S membrane expression although α_1S charge movement was nearly absent, and, most strikingly, as the complete lack of the arrangement of DHPRs in tetrads (19). Thus, in skeletal muscle the β subunit enables EC coupling by (i) enhancing α_1S membrane targeting, (ii) facilitating α_1S charge movement, and (iii) enabling the ultrastructural arrangement of DHPRs in tetrads.The question arises, which of these functions are specific for the skeletal muscle β_1a and which ones are rather general properties of Ca²⁺ channel β subunits. Previous reconstitution studies made in the β₁-null mouse system (24, 25) using different β subunit constructs (26) did not allow differentiation between β-induced enhancement of non-functional α_1S membrane expression and the facilitation of α_1S charge movement, due to the lack of information on α_1S triad expression levels. Furthermore, the β-induced arrangement of DHPRs in tetrads was not detected as no ultrastructural information was obtained.In the present study, we established zebrafish mutant relaxed as an expression system to test different β subunits for their ability to restore skeletal muscle EC coupling. Using isolated myotubes for in vitro experiments (19, 27) and complete larvae for in vivo expression studies (28-31) and freeze-fracture electron microscopy, a clear differentiation between the major functional roles of β subunits was feasible in the zebrafish system. The cloned zebrafish β_1a and a mammalian (rabbit) β_1a were shown to completely restore all parameters of EC coupling when expressed in relaxed myotubes and larvae. However, the phylogenetically closest β subunit to β_1a, the cardiac/neuronal isoform β_2a from rat, as well as the ancestral β_M isoform from the housefly (Musca domestica), could recover functional α_1S membrane insertion, but led to very restricted tetrad formation when compared with β_1a, and thus to impaired DHPR-RyR1 coupling. This impairment caused drastic changes in skeletal muscle function.The present study shows that the enhancement of functional α_1S membrane expression is a common function of all the tested β subunits, from β_1a to even the most distant β_M, whereas the effective formation of tetrads and thus proper skeletal muscle EC coupling is an exclusive function of the skeletal muscle β_1a subunit. In context with previous studies, our results suggest a model according to which β_1a acts as an allosteric modifier of α_1S conformation. Only in the presence of β_1a, the α_1S subunit is properly folded to allow RyR1 anchoring and thus skeletal muscle-type EC coupling.