The Lipid-binding Domain of Wild Type and Mutant α-Synuclein: COMPACTNESS AND INTERCONVERSION BETWEEN THE BROKEN AND EXTENDED HELIX FORMS*

α-Synuclein (αS) is linked to Parkinson disease through its deposition in an amyloid fibril form within Lewy Body deposits, and by the existence of three αS point mutations that lead to early onset autosomal dominant Parkinsonism. The normal function of αS is thought to be linked to the ability of the protein to bind to the surface of synaptic vesicles. Upon binding to vesicles, αS undergoes a structural reorganization from a dynamic and disordered ensemble to a conformation consisting of a long extended helix. In the presence of small spheroidal detergent micelles, however, this extended helix conformation can convert into a broken helix state, in which a region near the middle of the helix unwinds to form a linker between the two resulting separated helices. Membrane-bound conformations of αS likely mediate the function of the protein, but may also play a role in the aggregation and toxicity of the protein. Here we have undertaken a study of the effects of the three known PD-linked mutations on the detergent- and membrane-bound conformations of αS, as well as factors that govern the transition of the protein between the extended helix and broken helix states. Using pulsed dipolar ESR measurements of distances up to 8.7 nm, we show that all three PD-linked αS mutants retain the ability to transition from the broken helix to the extended helix conformation. In addition, we find that the ratio of protein to detergent, rather than just the absolute detergent concentration, determines whether the protein adopts the broken or extended helix conformation.     

Characterization of Semisynthetic and Naturally Nα-Acetylated α-Synuclein in Vitro and in Intact Cells: IMPLICATIONS FOR AGGREGATION AND CELLULAR PROPERTIES OF α-SYNUCLEIN

N-terminal acetylation is a very common post-translational modification, although its role in regulating protein physical properties and function remains poorly understood. α-Synuclein (α-syn), a protein that has been linked to the pathogenesis of Parkinson disease, is constitutively N(α)-acetylated in vivo. Nevertheless, most of the biochemical and biophysical studies on the structure, aggregation, and function of α-syn in vitro utilize recombinant α-syn from Escherichia coli, which is not N-terminally acetylated. To elucidate the effect of N(α)-acetylation on the biophysical and biological properties of α-syn, we produced N(α)-acetylated α-syn first using a semisynthetic methodology based on expressed protein ligation (Berrade, L., and Camarero, J. A. (2009) Cell. Mol. Life Sci. 66, 3909-3922) and then a recombinant expression strategy, to compare its properties to unacetylated α-syn. We demonstrate that both WT and N(α)-acetylated α-syn share a similar secondary structure and oligomeric state using both purified protein preparations and in-cell NMR on E. coli overexpressing N(α)-acetylated α-syn. The two proteins have very close aggregation propensities as shown by thioflavin T binding and sedimentation assays. Furthermore, both N(α)-acetylated and WT α-syn exhibited similar ability to bind synaptosomal membranes in vitro and in HeLa cells, where both internalized proteins exhibited prominent cytosolic subcellular distribution. We then determined the effect of attenuating N(α)-acetylation in living cells, first by using a nonacetylable mutant and then by silencing the enzyme responsible for α-syn N(α)-acetylation. Both approaches revealed similar subcellular distribution and membrane binding for both the nonacetylable mutant and WT α-syn, suggesting that N-terminal acetylation does not significantly affect its structure in vitro and in intact cells.     

Parkinson’s Protein α-Synuclein Binds Efficiently and with a Novel Conformation to Two Natural Membrane Mimics

Binding of human α-Synuclein, a protein associated with Parkinson’s disease, to natural membranes is thought to be crucial in relation to its pathological and physiological function. Here the binding of αS to small unilamellar vesicles mimicking the inner mitochondrial and the neuronal plasma membrane is studied in situ by continuous wave and pulsed electron paramagnetic resonance. Local binding information of αS spin labeled by MTSL at positions 56 and 69 respectively shows that also helix 2 (residues 50–100) binds firmly to both membranes. By double electron-electron resonance (DEER) on the mutant spin labeled at positions 27 and 56 (αS 27/56) a new conformation on the membrane is found with a distance of 3.6 nm/ 3.7 nm between residues 27 and 56. In view of the low negative charge density of these membranes, the strong interaction is surprising, emphasizing that function and pathology of αS could involve synaptic vesicles and mitochondria.     

Selective Transport of α-Mannosidase by Autophagic Pathways: STRUCTURAL BASIS FOR CARGO RECOGNITION BY Atg19 AND Atg34*

In the yeast Saccharomyces cerevisiae, a precursor form of aminopeptidase I (prApe1) and α-mannosidase (Ams1) are selectively transported to the vacuole through the cytoplasm-to-vacuole targeting pathway under vegetative conditions and through autophagy under starvation conditions. Atg19 plays a central role in these processes by linking Ams1 and prApe1 to Atg8 and Atg11. However, little is known about the molecular mechanisms of cargo recognition by Atg19. Here, we report structural and functional analyses of Atg19 and its paralog, Atg34. A protease-resistant domain was identified in the C-terminal region of Atg19, which was also conserved in Atg34. In vitro pulldown assays showed that the C-terminal domains of both Atg19 and Atg34 are responsible for Ams1 binding; these domains are hereafter referred to as Ams1-binding domains (ABDs). The transport of Ams1, but not prApe1, was blocked in atg19Δatg34Δ cells expressing Atg19^ΔABD, indicating that ABD is specifically required for Ams1 transport. We then determined the solution structures of the ABDs of Atg19 and Atg34 using NMR spectroscopy. Both ABD structures have a canonical immunoglobulin fold consisting of eight β-strands with highly conserved loops clustered at one side of the fold. These facts, together with the results of a mutational analysis, suggest that ABD recognizes Ams1 using these conserved loops.     

DNP-Enhanced MAS NMR: A Tool to Snapshot Conformational Ensembles of α-Synuclein in Different States

Intrinsically disordered proteins dynamically sample a wide conformational space and therefore do not adopt a stable and defined three-dimensional conformation. The structural heterogeneity is related to their proper functioning in physiological processes. Knowledge of the conformational ensemble is crucial for a complete comprehension of this kind of proteins. We here present an approach that utilizes dynamic nuclear polarization-enhanced solid-state NMR spectroscopy of sparsely isotope-labeled proteins in frozen solution to take snapshots of the complete structural ensembles by exploiting the inhomogeneously broadened line-shapes. We investigated the intrinsically disordered protein α-synuclein (α-syn), which plays a key role in the etiology of Parkinson’s disease, in three different physiologically relevant states. For the free monomer in frozen solution we could see that the so-called “random coil conformation” consists of α-helical and β-sheet-like conformations, and that secondary chemical shifts of neighboring amino acids tend to be correlated, indicative of frequent formation of secondary structure elements. Based on these results, we could estimate the number of disordered regions in fibrillar α-syn as well as in α-syn bound to membranes in different protein-to-lipid ratios. Our approach thus provides quantitative information on the propensity to sample transient secondary structures in different functional states. Molecular dynamics simulations rationalize the results.     

Dissecting the Mechanisms of Tissue Transglutaminase-induced Cross-linking of α-Synuclein: IMPLICATIONS FOR THE PATHOGENESIS OF PARKINSON DISEASE*S?

Tissue transglutaminase (tTG) has been implicated in the pathogenesis of Parkinson disease (PD). However, exactly how tTG modulates the structural and functional properties of α-synuclein (α-syn) and contributes to the pathogenesis of PD remains unknown. Using site-directed mutagenesis combined with detailed biophysical and mass spectrometry analyses, we sought to identify the exact residues involved in tTG-catalyzed cross-linking of wild-type α-syn and α-syn mutants associated with PD. To better understand the structural consequences of each cross-linking reaction, we determined the effect of tTG-catalyzed cross-linking on the oligomerization, fibrillization, and membrane binding of α-syn in vitro. Our findings show that tTG-catalyzed cross-linking of monomeric α-syn involves multiple cross-links (specifically 2-3). We subjected tTG-catalyzed cross-linked monomeric α-syn composed of either wild-type or Gln → Asn mutants to sequential proteolysis by multiple enzymes and peptide mapping by mass spectrometry. Using this approach, we identified the glutamine and lysine residues involved in tTG-catalyzed intramolecular cross-linking of α-syn. These studies demonstrate for the first time that Gln⁷⁹ and Gln¹⁰⁹ serve as the primary tTG reactive sites. Mutating both residues to asparagine abolishes tTG-catalyzed cross-linking of α-syn and tTG-induced inhibition of α-syn fibrillization in vitro. To further elucidate the sequence and structural basis underlying these effects, we identified the lysine residues that form isopeptide bonds with Gln⁷⁹ and Gln¹⁰⁹. This study provides mechanistic insight into the sequence and structural basis of the inhibitory effects of tTG on α-syn fibrillogenesis in vivo, and it sheds light on the potential role of tTG cross-linking on modulating the physiological and pathogenic properties of α-syn.Parkinson disease (PD)² is a progressive movement disorder that is caused by the loss of dopaminergic neurons in the substantia nigra, the part of the brain responsible for controlling movement. Clinically, PD is manifested in symptoms that include tremors, rigidity, and difficulty in initiating movement (bradykinesia). Pathologically, PD is characterized by the presence of intraneuronal, cytoplasmic inclusions known as Lewy bodies (LB), which are composed primarily of the protein “α-synuclein” (α-syn) (1) and are seen in the post-mortem brains of PD patients with the sporadic or familial forms of the disease (2). α-Syn is a presynaptic protein of 140 residues with a “natively” unfolded structure (3). Three missense point mutations in α-syn (A30P, E46K, and A53T) are associated with the early-onset, dominant, inherited form of PD (4, 5). Moreover, duplication or triplication of the α-syn gene has been linked to the familial form of PD, suggesting that an increase in α-syn expression is sufficient to cause PD. Together, these findings suggest that α-syn plays a central role in the pathogenesis of PD.The molecular and cellular determinants that govern α-syn oligomerization and fibrillogenesis in vivo remain poorly understood. In vitro aggregation studies have shown that the mutations associated with PD (A30P, E46K, and A53T) accelerate α-syn oligomerization, but only E46K and A53T α-syn show higher propensity to fibrillize than wild-type (WT) α-syn (6-8). This suggests that oligomerization, rather than fibrillization, is linked to early-onset familial PD (9). Our understanding of the molecular composition and biochemical state of α-syn in LBs has provided important clues about protein-protein interactions and post-translational modifications that may play a role in modulating oligomerization, fibrillogenesis, and LB formation of the protein. In addition to ubiquitination (10), phosphorylation (11, 12), nitration (13, 14), and C-terminal truncation (15, 16), analysis of post-mortem brain tissues from PD and Lewy bodies in dementia patients has confirmed the colocalization of tissue transglutaminase (tTG)-catalyzed cross-linked α-syn monomers and higher molecular aggregates in LBs within dopaminergic neurons (17, 18). Tissue transglutaminase catalyzes a calcium-dependent transamidating reaction involving glutamine and lysine residues, which results in the formation of a covalent cross-link via ε-(γ-glutamyl) lysine bonds (Fig. 2F). To date, seven different isoforms of tTGs have been reported, of which only tTG2 seems to be expressed in the human brain (19), whereas tTG1 and tTG3 are more abundantly found in stratified squamous epithelia (20). Subsequent immuno-histochemical, colocalization, and immunoprecipitation studies have shown that the levels of tTG and cross-linked α-syn species are increased in the substantia nigra of PD brains (17). These findings, combined with the known role of tTG in cross-linking and stabilizing bimolecular assemblies, led to the hypothesis that tTG plays an important role in the initiation and propagation of α-syn fibril formation and that it contributes to fibril stability in LBs. This hypothesis was initially supported by in vitro studies demonstrating that tTG catalyzes the polymerization of the α-syn-derived non-amyloid component (NAC) peptide via intermolecular covalent cross-linking of residues Gln⁷⁹ and Lys⁸⁰ (21) and by other studies suggesting that tTG promotes the fibrillization of amyloidogenic proteins implicated in the pathogenesis of other neurodegenerative diseases such as Alzheimer disease, supranuclear palsy, Huntington disease, and other polyglutamine diseases (22-24). However, recent in vitro studies with full-length α-syn have shown that tTG catalyzes intramolecular cross-linking of monomeric α-syn and inhibits, rather than promotes, its fibrillization in vitro (25, 26). The structural basis of this inhibitory effect and the exact residues involved in tTG-mediated cross-linking of α-syn, as well as structural and functional consequences of these modifications, remain poorly understood.Open in a separate windowFIGURE 2.tTG-catalyzed cross-linking of α-syn involves one to three intramolecular cross-links. A-C, MALDI-TOF/TOF analysis of native (—) and cross-linked (- - -) α-syn, showing that most tTG-catalyzed cross-linking products of WT or disease-associated mutant forms of α-syn are intramolecularly linked (predominant peak with two cross-links), and up to three intramolecular cross-links can occur (left shoulder). The abbreviations M and m/cl are used to designate native and cross-linked α-synuclein, respectively. D and E, kinetic analysis of α-syn (A30P) cross-linking monitored by MALDI-TOF and SDS-PAGE. F, schematic depiction of the tTG-catalyzed chemical reaction (isodipeptide formation) between glutamine and lysine residues.In this study, we have identified the primary glutamine and lysine residues involved in tTG-catalyzed, intramolecularly cross-linked monomeric α-syn and investigated how cross-linking these residues affects the oligomerization, fibrillization, and membrane binding of α-syn in vitro. Using single-site mutagenesis and mass spectrometry applied to exhaustive proteolytic digests of native and cross-linked monomeric α-syn, we identified Gln¹⁰⁹ and Gln⁷⁹ as the major tTG substrates. We demonstrate that the altered electrophoretic mobility of the intramolecularly cross-linked α-syn in SDS-PAGE occurs as a result of tTG-catalyzed cross-linking of Gln¹⁰⁹ to lysine residues in the N terminus of α-syn, which leads to the formation of more compact monomers. Consistent with previous studies, we show that intramolecularly cross-linked α-syn forms off-pathway oligomers that are distinct from those formed by the wild-type protein and that do not convert to fibrils within the time scale of our experiments (3-5 days). We also show that membrane-bound α-syn is a substrate of tTG and that intramolecular cross-linking does not interfere with the ability of monomeric α-syn to adopt an α-helical conformation upon binding to synthetic membranes. These studies provide novel mechanistic insight into the sequence and structural basis of events that allow tTG to inhibit α-syn fibrillogenesis, and they shed light on the potential role of tTG-catalyzed cross-linking in modulating the physiological and pathogenic properties of α-syn.     

The Power of Two: ARGININE 51 AND ARGININE 239* FROM A NEIGHBORING SUBUNIT ARE ESSENTIAL FOR CATALYSIS IN α-AMINO-β-CARBOXYMUCONATE-?-SEMIALDEHYDE DECARBOXYLASE*

Although the crystal structure of α-amino-β-carboxymuconate-ϵ-semialdehyde decarboxylase from Pseudomonas fluorescens was solved as a dimer, this enzyme is a mixture of monomer, dimer, and higher order structures in solution. In this work, we found that the dimeric state, not the monomeric state, is the functionally active form. Two conserved arginine residues are present in the active site: Arg-51 and an intruding Arg-239* from the neighboring subunit. In this study, they were each mutated to alanine and lysine, and all four mutants were catalytically inactive. The mutants were also incapable of accommodating pyridine-2,6-dicarboxylic acid, a competitive inhibitor of the native enzyme, suggesting that the two Arg residues are involved in substrate binding. It was also observed that the decarboxylase activity was partially recovered in a heterodimer hybridization experiment when inactive R51(A/K) and R239(A/K) mutants were mixed together. Of the 20 crystal structures obtained from mixing inactive R51A and R239A homodimers that diffracted to a resolution lower than 3.00 Å, two structures are clearly R51A/R239A heterodimers and belong to the C2 space group. They were refined to 1.80 and 2.00 Å resolutions, respectively. Four of the remaining crystals are apparently single mutants and belong to the P4₂2₁2 space group. In the heterodimer structures, one active site is shown to contain dual mutation of Ala-51 and Ala-239*, whereas the other contains the native Arg-51 and Arg-239* residues, identical to the wild-type structure. Thus, these observations provide the foundation for a molecular mechanism by which the oligomerization state of α-amino-β-carboxymuconate-ϵ-semialdehyde decarboxylase could regulate the enzyme activity.     

Lipids as Tumoricidal Components of Human α-Lactalbumin Made Lethal to Tumor Cells (HAMLET): UNIQUE AND SHARED EFFECTS ON SIGNALING AND DEATH*

Long-chain fatty acids are internalized by receptor-mediated mechanisms or receptor-independent diffusion across cytoplasmic membranes and are utilized as nutrients, building blocks, and signaling intermediates. Here we describe how the association of long-chain fatty acids to a partially unfolded, extracellular protein can alter the presentation to target cells and cellular effects. HAMLET (human α-lactalbumin made lethal to tumor cells) is a tumoricidal complex of partially unfolded α-lactalbumin and oleic acid (OA). As OA lacks independent tumoricidal activity at concentrations equimolar to HAMLET, the contribution of the lipid has been debated. We show by natural abundance ¹³C NMR that the lipid in HAMLET is deprotonated and by chromatography that oleate rather than oleic acid is the relevant HAMLET constituent. Compared with HAMLET, oleate (175 μm) showed weak effects on ion fluxes and gene expression. Unlike HAMLET, which causes metabolic paralysis, fatty acid metabolites were less strongly altered. The functional overlap increased with higher oleate concentrations (500 μm). Cellular responses to OA were weak or absent, suggesting that deprotonation favors cellular interactions of fatty acids. Fatty acids may thus exert some of their essential effects on host cells when in the deprotonated state and when presented in the context of a partially unfolded protein.     

High Resolution NMR-based Model for the Structure of a scFv-IL-1�� Complex: POTENTIAL FOR NMR AS A KEY TOOL IN THERAPEUTIC ANTIBODY DESIGN AND DEVELOPMENT*

Monoclonal antibodies have recently started to deliver on their promise as highly specific and active drugs; however, a more effective, knowledge-based approach to the selection, design, and optimization of potential therapeutic antibodies is currently limited by the surprising lack of detailed structural information for complexes formed with target proteins. Here we show that complexes formed with minimal antigen binding single chain variable fragments (scFv) reliably reflect all the features of the binding interface present in larger Fab fragments, which are commonly used as therapeutics, and report the development of a robust, reliable, and relatively rapid approach to the determination of high resolution models for scFv-target protein complexes. This NMR spectroscopy-based approach combines experimental determination of the interaction surfaces and relative orientations of the scFv and target protein, with NMR restraint-driven, semiflexible docking of the proteins to produce a reliable and highly informative model of the complex. Experience with scFvs and Fabs targeted at a number of secreted regulatory proteins suggests that the approach will be applicable to many therapeutic antibodies targeted at proteins, and its application is illustrated for a potential therapeutic antibody targeted at the cytokine IL-1β. The detailed structural information that can be obtained by this approach has the potential to have a major impact on the rational design and development of an increasingly important class of biological pharmaceuticals.     

Effect of α-Synuclein on Amyloid β-Induced Toxicity: Relevance to Lewy Body Variant of Alzheimer Disease

Alzheimer’s disease, the most prevalent age-related neurodegenerative disease, is characterized by the presence of extracellular senile plaques composed of amyloid-beta (Aβ) peptide and intracellular neurofibrillary tangles. More than 50 % of Alzheimer’s disease (AD) patients also exhibit abundant accumulation of α-synuclein (α-Syn)-positive Lewy bodies. This Lewy body variant of AD (LBV-AD) is associated with accelerated cognitive dysfunction and progresses more rapidly than pure AD. In addition, it has been suggested that Aβ and α-Syn can directly interact. In this study we investigated the effect of α-Syn on Aβ-induced toxicity in cortical neurons. In order to mimic the intracellular accumulation of α-Syn observed in the brain of LBV-AD patients, we used valproic acid (VPA) to increase its endogenous expression levels. The release of α-Syn from damaged presynaptic terminals that occurs during the course of the disease was simulated by challenging cells with recombinant α-Syn. Our results showed that either VPA-induced α-Syn upregulation or addition of recombinant α-Syn protect primary cortical neurons from soluble Aβ1-42 decreasing the caspase-3-mediated cell death. It was also found that neuroprotection against Aβ-induced toxicity mediated by α-Syn overexpression involves the PI3K/Akt cell survival pathway. Furthermore, recombinant α-Syn was shown to directly interact with Aβ1-42 and to decrease the levels of Aβ1-42 oligomers, which might explain its neuroprotective effect. In conclusion, we demonstrate that either endogenous or exogenous α-Syn can be neuroprotective against Aβ-induced cell death, suggesting a cell defence mechanism during the initial stages of the mixed pathology.     

A Novel Signaling Pathway: FIBROBLAST NICOTINIC RECEPTOR ��1 BINDS UROKINASE AND PROMOTES RENAL FIBROSIS*

The nicotinic acetylcholine receptor α1 (nAChRα1) was investigated as a potential fibrogenic molecule in the kidney, given reports that it may be an alternative urokinase (urokinase plasminogen activator; uPA) receptor in addition to the classical receptor uPAR. In a mouse obstructive uropathy model of chronic kidney disease, interstitial fibroblasts were identified as the primary cell type that bears nAChRα1 during fibrogenesis. Silencing of the nAChRα1 gene led to significantly fewer interstitial αSMA⁺ myofibroblasts (2.8 times decreased), reduced interstitial cell proliferation (2.6 times decreased), better tubular cell preservation (E-cadherin 14 times increased), and reduced fibrosis severity (24% decrease in total collagen). The myofibroblast-inhibiting effect of nAChRα1 silencing in uPA-sufficient mice disappeared in uPA-null mice, suggesting that a uPA-dependent fibroblastic nAChRα1 pathway promotes renal fibrosis. To further establish this possible ligand-receptor relationship and to identify downstream signaling pathways, in vitro studies were performed using primary cultures of renal fibroblasts. ³⁵S-Labeled uPA bound to nAChRα1 with a K_d of 1.6 × 10⁻⁸ m, which was displaced by the specific nAChRα1 inhibitor d-tubocurarine in a dose-dependent manner. Pre-exposure of uPA to the fibroblasts inhibited [³H]nicotine binding. The uPA binding induced a cellular calcium influx and an inward membrane current that was entirely prevented by d-tubocurarine preincubation or nAChRα1 silencing. By mass spectrometry phosphoproteome analyses, uPA stimulation phosphorylated nAChRα1 and a complex of signaling proteins, including calcium-binding proteins, cytoskeletal proteins, and a nucleoprotein. This signaling pathway appears to regulate the expression of a group of genes that transform renal fibroblasts into more active myofibroblasts characterized by enhanced proliferation and contractility. This new fibrosis-promoting pathway may also be relevant to disorders that extend beyond chronic kidney disease.Urokinase was first isolated from human urine in 1955 and identified as an activator of plasminogen (urokinase plasminogen activator (uPA)²) (1). This serine protease is abundantly produced by kidney tubular cells and secreted across the apical membrane into the urinary space. Other cellular sources include monocytes/macrophages, fibroblasts, and myofibroblasts (2). Despite high uPA levels, its primary physiological function in the kidney remains unknown. Suggested roles have been an inhibitor of kidney stone formation and urinary tract infections due to its proteolytic activity and endogenous antibiotic function, respectively (3, 4). Increased uPA activity has been reported in several pathological conditions, such as chronic kidney disease (2), atherosclerosis, and malignant tumors (5, 6). Endogenous plasma uPA levels may be elevated 2–4-fold in patients with chronic kidney disease due to increased uPA released from damaged kidneys (7, 8).Since its identification as a mediator of fibrin/fibrinogen degradation, uPA has been used in clinical settings as a fibrinolytic agent. The classical cellular urokinase receptor (uPAR) was first discovered on the surface of monocytes in 1985. Since then, a diverse array of biological functions triggered by uPA-uPAR interactions has been elucidated and shown to have important effects on cellular behavior during embryogenesis, angiogenesis, wound healing, and metastases (9–11). The specific role of uPA in fibrotic disorders appears to be organ-specific. uPA deficiency worsened bleomycin-induced lung fibrosis and reduced fibrosis in hearts damaged by viral myocarditis or left ventricular pressure overload, whereas there was no net effect on the severity of renal fibrosis induced by ureteral obstruction (UUO), although uPAR deficiency worsened fibrosis in that model (2). Despite its association with a broad repertoire of activities, the uPA mechanism of action remains incompletely understood. In particular, there is accumulating evidence that uPA may have protease- and uPAR-independent cellular effects. For example, macrophage uPA overexpression causes cardiac inflammation and fibrosis. Of particular note, this effect is independent of the classic uPA receptor uPAR and can be abrogated using a calcium channel blocker (12).Recent evidence suggests that additional uPA receptor(s) may exist (13–15). We reported that urokinase initiates renal fibroblast signaling via the MAPK/ERK pathway (16). This response appears to be mediated, at least in part, by an alternative urokinase receptor, since uPA can initiate mitogenesis in uPAR^−/− fibroblasts. Using phage display technology, Liang et al. (17) reported putative uPA-binding consensus sequences in 12 transmembrane receptors and suggested them as candidate alternative uPA receptor(s). Of these candidate receptors, several are already known as uPAR co-receptors: low density lipoprotein receptor-related protein, gp130, integrin αv, uPAR-associated protein (also known as Endo180 and Mrc2), and the insulin-like growth factor II/mannose 6-phosphate receptor. It is also possible that different uPA domains might simultaneously bind to uPAR and one of its co-receptors (18). The muscle type nicotinic receptor α1 (nAChRα1) was among the receptor candidates. The muscle type nAChR is a ligand-gated ion channel known to mediate signal transduction at the neuromuscular junction (19). This receptor is a pentametric glycoprotein comprising five membrane-spanning subunits (two α1, β1, γ, and δ) that form a ligand-gated ion channel. The currently known nAChRα1 ligands are nicotine and acetylcholine. The ligand-binding domain (interface of α1/γ or α1/δ) involves the two α1 chains, which form a specialized pocket of aromatic and hydrophobic residues structurally similar to uPAR (20, 21). Upon ligation, the receptor changes its conformation and becomes permeable to sodium and calcium ions. Receptor function is regulated by tyrosine phosphorylation and dephosphorylation by kinases and phosphatases, respectively (22). The nAChRα1 is expressed and activated during muscle differentiation during embryonic development and following mature muscle denervation (23). Vascular endothelium, macrophages, and fibroblasts are also known to express certain nAChR subtypes (24). We observed that nAChRα1 expression was significantly higher in the kidneys of uPAR-deficient mice that develop worse scarring during UUO (supplemental Fig. S1a).Evidence that nAChRα1 might function as an alternative uPA receptor was suggested by our microarray data that compared uPAR^−/− and uPAR^+/+ renal fibroblasts. The nAChRα1 was the only one of the 12 receptor candidates identified by Liang et al. (17) that was significantly up-regulated on the uPAR^−/− fibroblasts (n = 5, p < 0.01 by analysis of variance (ANOVA), >2-fold change) (supplemental Fig. S1b). Based on the assumption that the receptor may be up-regulated in damaged kidneys in the absence of uPAR and contribute to the development of more severe fibrosis, this study was designed to determine if a ligand-receptor relationship exists between uPA and nAChRα1 and to investigate its functional role in fibroblast growth and renal fibrosis. In vivo functional knockdown of the nAChRα1 was shown to significantly attenuate fibrosis after ureteral obstruction, an effect that was uPA-dependent. In vitro studies provided additional evidence that nAChRα1 was an uPA signaling receptor for fibroblasts, activating a complex of signaling proteins by tyrosine phosphorylation and calcium influx to stimulate proliferation and enhance contractility.     

Enzymatic Basis for N-Glycan Sialylation: STRUCTURE OF RAT α2,6-SIALYLTRANSFERASE (ST6GAL1) REVEALS CONSERVED AND UNIQUE FEATURES FOR GLYCAN SIALYLATION*

Glycan structures on glycoproteins and glycolipids play critical roles in biological recognition, targeting, and modulation of functions in animal systems. Many classes of glycan structures are capped with terminal sialic acid residues, which contribute to biological functions by either forming or masking glycan recognition sites on the cell surface or secreted glycoconjugates. Sialylated glycans are synthesized in mammals by a single conserved family of sialyltransferases that have diverse linkage and acceptor specificities. We examined the enzymatic basis for glycan sialylation in animal systems by determining the crystal structures of rat ST6GAL1, an enzyme that creates terminal α2,6-sialic acid linkages on complex-type N-glycans, at 2.4 Å resolution. Crystals were obtained from enzyme preparations generated in mammalian cells. The resulting structure revealed an overall protein fold broadly resembling the previously determined structure of pig ST3GAL1, including a CMP-sialic acid-binding site assembled from conserved sialylmotif sequence elements. Significant differences in structure and disulfide bonding patterns were found outside the sialylmotif sequences, including differences in residues predicted to interact with the glycan acceptor. Computational substrate docking and molecular dynamics simulations were performed to predict and evaluate the CMP-sialic acid donor and glycan acceptor interactions, and the results were compared with kinetic analysis of active site mutants. Comparisons of the structure with pig ST3GAL1 and a bacterial sialyltransferase revealed a similar positioning of donor, acceptor, and catalytic residues that provide a common structural framework for catalysis by the mammalian and bacterial sialyltransferases.     

The LIMP-2/SCARB2 Binding Motif on Acid β-Glucosidase: BASIC AND APPLIED IMPLICATIONS FOR GAUCHER DISEASE AND ASSOCIATED NEURODEGENERATIVE DISEASES*

The acid β-glucosidase (glucocerbrosidase (GCase)) binding sequence to LIMP-2 (lysosomal integral membrane protein 2), the receptor for intracellular GCase trafficking to the lysosome, has been identified. Heterologous expression of deletion constructs, the available GCase crystal structures, and binding and co-localization of identified peptides or mutant GCases were used to identify and characterize a highly conserved 11-amino acid sequence, DSPIIVDITKD, within human GCase. The binding to LIMP-2 is not dependent upon a single amino acid, but the interactions of GCase with LIMP-2 are heavily influenced by Asp³⁹⁹ and the di-isoleucines, Ile⁴⁰² and Ile⁴⁰³. A single alanine substitution at any of these decreases GCase binding to LIMP-2 and alters its pH-dependent binding as well as diminishing the trafficking of GCase to the lysosome and significantly increasing GCase secretion. Enterovirus 71 also binds to LIMP-2 (also known as SCARB2) on the external surface of the plasma membrane. However, the LIMP-2/SCARB2 binding sequences for enterovirus 71 and GCase are not similar, indicating that LIMP-2/SCARB2 may have multiple or overlapping binding sites with differing specificities. These findings have therapeutic implications for the production of GCase and the distribution of this enzyme that is delivered to various organs.     

A Novel Inhibitor of Amyloid β (Aβ) Peptide Aggregation: FROM HIGH THROUGHPUT SCREENING TO EFFICACY IN AN ANIMAL MODEL OF ALZHEIMER DISEASE*

Compelling evidence indicates that aggregation of the amyloid β (Aβ) peptide is a major underlying molecular culprit in Alzheimer disease. Specifically, soluble oligomers of the 42-residue peptide (Aβ42) lead to a series of events that cause cellular dysfunction and neuronal death. Therefore, inhibiting Aβ42 aggregation may be an effective strategy for the prevention and/or treatment of disease. We describe the implementation of a high throughput screen for inhibitors of Aβ42 aggregation on a collection of 65,000 small molecules. Among several novel inhibitors isolated by the screen, compound D737 was most effective in inhibiting Aβ42 aggregation and reducing Aβ42-induced toxicity in cell culture. The protective activity of D737 was most significant in reducing the toxicity of high molecular weight oligomers of Aβ42. The ability of D737 to prevent Aβ42 aggregation protects against cellular dysfunction and reduces the production/accumulation of reactive oxygen species. Most importantly, treatment with D737 increases the life span and locomotive ability of flies in a Drosophila melanogaster model of Alzheimer disease.