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1.  Structurally Similar Allosteric Modulators of α7 Nicotinic Acetylcholine Receptors Exhibit Five Distinct Pharmacological Effects* 
The Journal of Biological Chemistry  2014;290(6):3552-3562.
Background: Nicotinic receptors are activated by acetylcholine and have been implicated in several neurological disorders.
Results: Allosteric modulators, sharing close chemical similarity, exhibit five distinct pharmacological effects on α7 nicotinic receptors.
Conclusion: Small changes in chemical structure have profound effects on the pharmacological properties of allosteric modulators.
Significance: These findings may provide opportunities for novel approaches to therapeutic drug discovery.
Activation of nicotinic acetylcholine receptors (nAChRs) is associated with the binding of agonists such as acetylcholine to an extracellular site that is located at the interface between two adjacent receptor subunits. More recently, there has been considerable interest in compounds, such as positive and negative allosteric modulators (PAMs and NAMs), that are able to modulate nAChR function by binding to distinct allosteric sites. Here we examined a series of compounds differing only in methyl substitution of a single aromatic ring. This series of compounds includes a previously described α7-selective allosteric agonist, cis-cis-4-p-tolyl-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide (4MP-TQS), together with all other possible combinations of methyl substitution at a phenyl ring (18 additional compounds). Studies conducted with this series of compounds have revealed five distinct pharmacological effects on α7 nAChRs. These five effects can be summarized as: 1) nondesensitizing activation (allosteric agonists), 2) potentiation associated with minimal effects on receptor desensitization (type I PAMs), 3) potentiation associated with reduced desensitization (type II PAMs), 4) noncompetitive antagonism (NAMs), and 5) compounds that have no effect on orthosteric agonist responses but block allosteric modulation (silent allosteric modulators (SAMs)). Several lines of experimental evidence are consistent with all of these compounds acting at a common, transmembrane allosteric site. Notably, all of these chemically similar compounds that have been classified as nondesensitizing allosteric agonists or as nondesensitizing (type II) PAMs are cis-cis-diastereoisomers, whereas all of the NAMs, SAMs, and type I PAMs are cis-trans-diastereoisomers. Our data illustrate the remarkable pharmacological diversity of allosteric modulators acting on nAChRs.
PMCID: PMC4319022  PMID: 25516597
Cys Loop Receptor; Membrane Protein; Molecular Pharmacology; Neurobiology; Nicotinic Acetylcholine Receptors (nAChR); Receptor Structure-Function
2.  The B-type Channel Is a Major Route for Iron Entry into the Ferroxidase Center and Central Cavity of Bacterioferritin* 
The Journal of Biological Chemistry  2014;290(6):3732-3739.
Background: Routes of iron entry into prokaryote ferritins are unknown.
Results: Mutation of the B-type channel of E. coli bacterioferritin resulted in substantially decreased rates of Fe(II) oxidation and mineralization.
Conclusion: The B-type channel is an important route for iron entry into bacterioferritin.
Significance: B-type channels may be of general importance as routes of iron uptake in prokaryotic ferritins.
Bacterioferritin is a bacterial iron storage and detoxification protein that is capable of forming a ferric oxyhydroxide mineral core within its central cavity. To do this, iron must traverse the bacterioferritin protein shell, which is expected to occur through one or more of the channels through the shell identified by structural studies. The size and negative electrostatic potential of the 24 B-type channels suggest that they could provide a route for iron into bacterioferritin. Residues at the B-type channel (Asn-34, Glu-66, Asp-132, and Asp-139) of E. coli bacterioferritin were substituted to determine if they are important for iron core formation. A significant decrease in the rates of initial oxidation of Fe(II) at the ferroxidase center and subsequent iron mineralization was observed for the D132F variant. The crystal structure of this variant shows that substitution of residue 132 with phenylalanine caused a steric blockage of the B-type channel and no other material structural perturbation. We conclude that the B-type channel is a major route for iron entry into both the ferroxidase center and the iron storage cavity of bacterioferritin.
PMCID: PMC4319037  PMID: 25512375
Escherichia coli (E. coli); Ferritin; Iron; Site-directed Mutagenesis; X-ray Crystallography; Bacterioferritin, Iron Storage, Iron Core
3.  Complex Structure and Biochemical Characterization of the Staphylococcus aureus Cyclic Diadenylate Monophosphate (c-di-AMP)-binding Protein PstA, the Founding Member of a New Signal Transduction Protein Family* 
The Journal of Biological Chemistry  2014;290(5):2888-2901.
Background: PstA is a cyclic di-AMP receptor and PII-like signal transduction protein.
Results: Apo-PstA and complex crystal structures reveal a novel cyclic di-AMP-binding mode and induced conformational changes.
Conclusion: PstA has a similar fold but distinct signal transduction properties from classic PII proteins, which function in nitrogen metabolism.
Significance: Identification of common features allows for rational prediction of cyclic di-AMP-binding sites.
Signaling nucleotides are integral parts of signal transduction systems allowing bacteria to cope with and rapidly respond to changes in the environment. The Staphylococcus aureus PII-like signal transduction protein PstA was recently identified as a cyclic diadenylate monophosphate (c-di-AMP)-binding protein. Here, we present the crystal structures of the apo- and c-di-AMP-bound PstA protein, which is trimeric in solution as well as in the crystals. The structures combined with detailed bioinformatics analysis revealed that the protein belongs to a new family of proteins with a similar core fold but with distinct features to classical PII proteins, which usually function in nitrogen metabolism pathways in bacteria. The complex structure revealed three identical c-di-AMP-binding sites per trimer with each binding site at a monomer-monomer interface. Although distinctly different from other cyclic-di-nucleotide-binding sites, as the half-binding sites are not symmetrical, the complex structure also highlighted common features for c-di-AMP-binding sites. A comparison between the apo and complex structures revealed a series of conformational changes that result in the ordering of two anti-parallel β-strands that protrude from each monomer and allowed us to propose a mechanism on how the PstA protein functions as a signaling transduction protein.
PMCID: PMC4316997  PMID: 25505271
Bacterial Signal Transduction; Bioinformatics; Crystal Structure; Nucleotide; Staphylococcus aureus (S. aureus); Complex
4.  The H50Q Mutation Induces a 10-fold Decrease in the Solubility of α-Synuclein* 
The Journal of Biological Chemistry  2014;290(4):2395-2404.
Background: The basis of the pathogenicity of the H50Q variant α-synuclein is unknown.
Results: The critical concentration of α-synuclein is decreased by 10-fold by the H50Q mutation, and its aggregation is modulated by the wild-type isoform.
Conclusion: Key effects of the H50Q mutation on the aggregation of α-synuclein can be quantified.
Significance: Our data provide insights into the mechanism of Lewy body formation in vivo.
The conversion of α-synuclein from its intrinsically disordered monomeric state into the fibrillar cross-β aggregates characteristically present in Lewy bodies is largely unknown. The investigation of α-synuclein variants causative of familial forms of Parkinson disease can provide unique insights into the conditions that promote or inhibit aggregate formation. It has been shown recently that a newly identified pathogenic mutation of α-synuclein, H50Q, aggregates faster than the wild-type. We investigate here its aggregation propensity by using a sequence-based prediction algorithm, NMR chemical shift analysis of secondary structure populations in the monomeric state, and determination of thermodynamic stability of the fibrils. Our data show that the H50Q mutation induces only a small increment in polyproline II structure around the site of the mutation and a slight increase in the overall aggregation propensity. We also find, however, that the H50Q mutation strongly stabilizes α-synuclein fibrils by 5.0 ± 1.0 kJ mol−1, thus increasing the supersaturation of monomeric α-synuclein within the cell, and strongly favors its aggregation process. We further show that wild-type α-synuclein can decelerate the aggregation kinetics of the H50Q variant in a dose-dependent manner when coaggregating with it. These last findings suggest that the precise balance of α-synuclein synthesized from the wild-type and mutant alleles may influence the natural history and heterogeneous clinical phenotype of Parkinson disease.
PMCID: PMC4303689  PMID: 25505181
alpha-Synuclein (a-synuclein); Amyloid; Fibril; Parkinson Disease; Protein Aggregation; Aggregation Propensity; Fibrils Thermodynamic Stability; Polyproline II Structure
5.  Atypical Ubiquitylation in Yeast Targets Lysine-less Asi2 for Proteasomal Degradation* 
The Journal of Biological Chemistry  2014;290(4):2489-2495.
Background: Atypical degradative polyubiquitylation on non-lysine residues has only been reported in metazoans.
Results: Lysine-less mutant of Asi2 inner nuclear membrane protein is ubiquitylated and targeted to proteasomes in a Doa10-, Ubc6-, and Ubc7-dependent manner.
Conclusion: Well characterized enzymes of the endoplasmic reticulum-associated degradation pathway can catalyze atypical ubiquitylation.
Significance: Atypical degradative ubiquitylation is not restricted to metazoans and represents an unexplored process in yeast.
Proteins are typically targeted for proteasomal degradation by the attachment of a polyubiquitin chain to ϵ-amino groups of lysine residues. Non-lysine ubiquitylation of proteasomal substrates has been considered an atypical and rare event limited to complex eukaryotes. Here we report that a fully functional lysine-less mutant of an inner nuclear membrane protein in yeast, Asi2, is polyubiquitylated and targeted for proteasomal degradation. Efficient degradation of lysine-free Asi2 requires E3-ligase Doa10 and E2 enzymes Ubc6 and Ubc7, components of the endoplasmic reticulum-associated degradation pathway. Together, our data suggest that non-lysine ubiquitylation may be more prevalent than currently considered.
PMCID: PMC4303697  PMID: 25492870
Endoplasmic Reticulum-associated Protein Degradation (ERAD); Nuclear Envelope; Proteasome; Protein Degradation; Saccharomyces cerevisiae; Ubiquitin; E2 Ubiquitin-conjugating Enzymes (Ubc6, Ubc7); E3 Ubiquitin Ligase (Doa10); Inner Nuclear Membrane; Polytopic Membrane Proteins
6.  A Revised Mechanism for the Activation of Complement C3 to C3b 
The Journal of Biological Chemistry  2014;290(4):2334-2350.
Background: An understanding of the solution structure of complement C3b is essential to understand its reactivity.
Results: Ultracentrifugation and scattering revealed compact C3b structures in low salt and extended ones in physiological salt.
Conclusion: The two conformations reflect Arg102–Glu1032 salt bridge formation only in low salt.
Significance: The functional differences between the major C3S (Arg102) and C3F (Gly102) allotypes are explained.
The solution structure of complement C3b is crucial for the understanding of complement activation and regulation. C3b is generated by the removal of C3a from C3. Hydrolysis of the C3 thioester produces C3u, an analog of C3b. C3b cleavage results in C3c and C3d (thioester-containing domain; TED). To resolve functional questions in relation to C3b and C3u, analytical ultracentrifugation and x-ray and neutron scattering studies were used with C3, C3b, C3u, C3c, and C3d, using the wild-type allotype with Arg102. In 50 mm NaCl buffer, atomistic scattering modeling showed that both C3b and C3u adopted a compact structure, similar to the C3b crystal structure in which its TED and macroglobulin 1 (MG1) domains were connected through the Arg102–Glu1032 salt bridge. In physiological 137 mm NaCl, scattering modeling showed that C3b and C3u were both extended in structure, with the TED and MG1 domains now separated by up to 6 nm. The importance of the Arg102–Glu1032 salt bridge was determined using surface plasmon resonance to monitor the binding of wild-type C3d(E1032) and mutant C3d(A1032) to immobilized C3c. The mutant did not bind, whereas the wild-type form did. The high conformational variability of TED in C3b in physiological buffer showed that C3b is more reactive than previously thought. Because the Arg102-Glu1032 salt bridge is essential for the C3b-Factor H complex during the regulatory control of C3b, the known clinical associations of the major C3S (Arg102) and disease-linked C3F (Gly102) allotypes of C3b were experimentally explained for the first time.
PMCID: PMC4303685  PMID: 25488663
Analytical Ultracentrifugation; Inflammation; Molecular Modeling; Neutron Scattering; Surface Plasmon Resonance (SPR); X-ray Scattering; Complement C3
7.  A+-Helix of Protein C Inhibitor (PCI) Is a Cell-penetrating Peptide That Mediates Cell Membrane Permeation of PCI* 
The Journal of Biological Chemistry  2014;290(5):3081-3091.
Background: Extracellular protein C inhibitor (PCI) can cross the cellular plasma membrane.
Results: Testisin (fluid-phase and cell membrane-anchored) cleaves PCI close to its N terminus. N-terminally truncated PCI can no longer be internalized by cells.
Conclusion: Testisin removes helix A+, a cell-penetrating peptide, which mediates cell membrane permeation of PCI.
Significance: Testisin or other proteases could regulate PCI internalization by removing its N terminus.
Protein C inhibitor (PCI) is a serpin with broad protease reactivity. It binds glycosaminoglycans and certain phospholipids that can modulate its inhibitory activity. PCI can penetrate through cellular membranes via binding to phosphatidylethanolamine. The exact mechanism of PCI internalization and the intracellular role of the serpin are not well understood. Here we showed that testisin, a glycosylphosphatidylinositol-anchored serine protease, cleaved human PCI and mouse PCI (mPCI) at their reactive sites as well as at sites close to their N terminus. This cleavage was observed not only with testisin in solution but also with cell membrane-anchored testisin on U937 cells. The cleavage close to the N terminus released peptides rich in basic amino acids. Synthetic peptides corresponding to the released peptides of human PCI (His1–Arg11) and mPCI (Arg1–Ala18) functioned as cell-penetrating peptides. Because intact mPCI but not testisin-cleaved mPCI was internalized by Jurkat T cells, a truncated mPCI mimicking testisin-cleaved mPCI was created. The truncated mPCI lacking 18 amino acids at the N terminus was not taken up by Jurkat T cells. Therefore our model suggests that testisin or other proteases could regulate the internalization of PCI by removing its N terminus. This may represent one of the mechanisms regulating the intracellular functions of PCI.
PMCID: PMC4317013  PMID: 25488662
Cell Permeabilization; Cell-penetrating Peptide (CPP); Nuclear Translocation; Peptides; Serpin; Protein C Inhibitor; Testisin
8.  Lysosomal Two-pore Channel Subtype 2 (TPC2) Regulates Skeletal Muscle Autophagic Signaling* 
The Journal of Biological Chemistry  2014;290(6):3377-3389.
Background: The endolysosomal TPC2 ion channel interacts with mTOR to regulate cellular energy utilization.
Results: Mice lacking TPC2 display muscle atrophy phenotype with reduced muscle endurance, altered autophagy, and lysosomal enzymatic activities.
Conclusion: TPC2 regulates autophagic signaling in skeletal muscle.
Significance: TPC2 impacts protein turnover via regulating autophagy signaling in the process of tissue homeostasis and aging.
Postnatal skeletal muscle mass is regulated by the balance between anabolic protein synthesis and catabolic protein degradation, and muscle atrophy occurs when protein homeostasis is disrupted. Autophagy has emerged as critical in clearing dysfunctional organelles and thus in regulating protein turnover. Here we show that endolysosomal two-pore channel subtype 2 (TPC2) contributes to autophagy signaling and protein homeostasis in skeletal muscle. Muscles derived from Tpcn2−/− mice exhibit an atrophic phenotype with exacerbated autophagy under starvation. Compared with wild types, animals lacking TPC2 demonstrated an enhanced autophagy flux characterized by increased accumulation of autophagosomes upon combined stress induction by starvation and colchicine treatment. In addition, deletion of TPC2 in muscle caused aberrant lysosomal pH homeostasis and reduced lysosomal protease activity. Association between mammalian target of rapamycin and TPC2 was detected in skeletal muscle, allowing for appropriate adjustments to cellular metabolic states and subsequent execution of autophagy. TPC2 therefore impacts mammalian target of rapamycin reactivation during the process of autophagy and contributes to maintenance of muscle homeostasis.
PMCID: PMC4319008  PMID: 25480788
Autophagy; Calcium Channel; Lysosome; Muscle Atrophy; Protein Turnover
9.  Identification and Pharmacological Inactivation of the MYCN Gene Network as a Therapeutic Strategy for Neuroblastic Tumor Cells* 
The Journal of Biological Chemistry  2014;290(4):2198-2212.
Background: Neuroblastic tumors are often addicted to the MYCN protooncogene.
Results: Using a genome wide shRNA screen, we have identified key MYCN synthetic lethal genes.
Conclusion: Chemical inhibition of the newly identified MYCN synthetic lethal genes selectively kills MYCN-amplified cell lines.
Significance: Decoding the MYCN gene network will help to develop drugs for the treatment of neuroblastic tumors with activated MYCN.
The MYC family of transcription factors consists of three well characterized members, c-MYC, L-MYC, and MYCN, deregulated in the majority of human cancers. In neuronal tumors such as neuroblastoma, MYCN is frequently activated by gene amplification, and reducing its expression by RNA interference has been shown to promote growth arrest and apoptosis of tumor cells. From a clinical perspective, RNA interference is not yet a viable option, and small molecule inhibitors of transcription factors are difficult to develop. We therefore planned to identify, at the global level, the genes interacting functionally with MYCN required to promote fitness of tumor cells facing oncogenic stress. To find genes whose inactivation is synthetically lethal to MYCN, we implemented a genome-wide approach in which we carried out a drop-out shRNA screen using a whole genome library that was delivered into isogenic neuroblastoma cell lines expressing or not expressing MYCN. After the screen, we selected for in-depth analysis four shRNAs targeting AHCY, BLM, PKMYT1, and CKS1B. These genes were chosen because they are directly regulated by MYC proteins, associated with poor prognosis of neuroblastoma patients, and inhibited by small molecule compounds. Mechanistically, we found that BLM and PKMYT1 are required to limit oncogenic stress and promote stabilization of the MYCN protein. Cocktails of small molecule inhibitors of CKS1B, AHCY, BLM, and PKMYT1 profoundly affected the growth of all neuroblastoma cell lines but selectively caused death of MYCN-amplified cells. Our findings suggest that drugging the MYCN network is a promising avenue for the treatment of high risk, neuroblastic cancers.
PMCID: PMC4303671  PMID: 25477524
Anticancer Drug; Cancer Biology; Cancer Therapy; Myc (c-Myc); Neuroblastoma; MYCN; shRNA Screen; Synthetic Lethality
10.  Using Fluorescent Myosin to Directly Visualize Cooperative Activation of Thin Filaments*♦ 
The Journal of Biological Chemistry  2014;290(4):1915-1925.
Background: How calcium regulates thin filament activation is uncertain.
Results: Single molecule imaging is used to report on thin filament activation across relevant solution conditions.
Conclusion: Two myosin heads are required to activate a thin filament, which occurs as three states, and myosin binding opens up 11 sites for subsequent binders.
Significance: This first direct visualization of cooperativity in action exposes how complex systems function.
Contraction of striated muscle is tightly regulated by the release and sequestration of calcium within myocytes. At the molecular level, calcium modulates myosin's access to the thin filament. Once bound, myosin is hypothesized to potentiate the binding of further myosins. Here, we directly image single molecules of myosin binding to and activating thin filaments. Using this approach, the cooperative binding of myosin along thin filaments has been quantified. We have found that two myosin heads are required to laterally activate a regulatory unit of thin filament. The regulatory unit is found to be capable of accommodating 11 additional myosins. Three thin filament activation states possessing differential myosin binding capacities are also visible. To describe this system, we have formulated a simple chemical kinetic model of cooperative activation that holds across a wide range of solution conditions. The stochastic nature of activation is strongly highlighted by data obtained in sub-optimal activation conditions where the generation of activation waves and their catastrophic collapse can be observed. This suggests that the thin filament has the potential to be turned fully on or off in a binary fashion.
PMCID: PMC4303648  PMID: 25429108
Actin; Cooperativity; Myosin; Single Molecule Biophysics; Tropomyosin; Troponin; Muscle Regulation
11.  Acceleration of α-Synuclein Aggregation by Exosomes* 
The Journal of Biological Chemistry  2014;290(5):2969-2982.
Background: Cell-to-cell transmission of α-syn via exosomes has been proposed to propagate Parkinson disease pathology.
Results: Exosomes contain gangliosides, several other lipid classes, and proteins. Exosomes and ganglioside vesicles accelerate α-syn aggregation. Vesicles made of other membrane lipids do not.
Conclusion: Exosomes provide catalytic environments for nucleation of α-syn aggregation.
Significance: Revealing factors that promote α-syn aggregation may provide insight into Parkinson disease pathogenesis.
Exosomes are small vesicles released from cells into extracellular space. We have isolated exosomes from neuroblastoma cells and investigated their influence on the aggregation of α-synuclein, a protein associated with Parkinson disease pathology. Using cryo-transmission electron microscopy of exosomes, we found spherical unilamellar vesicles with a significant protein content, and Western blot analysis revealed that they contain, as expected, the proteins Flotillin-1 and Alix. Using thioflavin T fluorescence to monitor aggregation kinetics, we found that exosomes catalyze the process in a similar manner as a low concentration of preformed α-synuclein fibrils. The exosomes reduce the lag time indicating that they provide catalytic environments for nucleation. The catalytic effects of exosomes derived from naive cells and cells that overexpress α-synuclein do not differ. Vesicles prepared from extracted exosome lipids accelerate aggregation, suggesting that the lipids in exosomes are sufficient for the catalytic effect to arise. Using mass spectrometry, we found several phospholipid classes in the exosomes, including phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, and the gangliosides GM2 and GM3. Within each class, several species with different acyl chains were identified. We then prepared vesicles from corresponding pure lipids or defined mixtures, most of which were found to retard α-synuclein aggregation. As a striking exception, vesicles containing ganglioside lipids GM1 or GM3 accelerate the process. Understanding how α-synuclein interacts with biological membranes to promote neurological disease might lead to the identification of novel therapeutic targets.
PMCID: PMC4317028  PMID: 25425650
{alpha}-Synuclein; Amyloid; Exosome; Fibril; Fluorescence; Lipid; Mass Spectrometry (MS); Membrane; Parkinson Disease; Protein Aggregation
12.  Microseconds Simulations Reveal a New Sodium-binding Site and the Mechanism of Sodium-coupled Substrate Uptake by LeuT* 
The Journal of Biological Chemistry  2014;290(1):544-555.
Background: LeuT is a homologue of neurotransmitter transporters.
Results: Microseconds simulations disclose multiple translocation sites for Na+, including a new site for initial binding.
Conclusion: Coupled Na+- and substrate-binding events are accompanied by local and global (interhelical) rearrangements in the outward facing structure.
Significance: New insights are gained into Na+ and substrate uptake/efflux mechanisms of LeuT.
The bacterial sodium-coupled leucine/alanine transporter LeuT is broadly used as a model system for studying the transport mechanism of neurotransmitters because of its structural and functional homology to mammalian transporters such as serotonin, dopamine, or norepinephrine transporters, and because of the resolution of its structure in different states. Although the binding sites (S1 for substrate, and Na1 and Na2 for two co-transported sodium ions) have been resolved, we still lack a mechanistic understanding of coupled Na+- and substrate-binding events. We present here results from extensive (>20 μs) unbiased molecular dynamics simulations generated using the latest computing technology. Simulations show that sodium binds initially the Na1 site, but not Na2, and, consistently, sodium unbinding/escape to the extracellular (EC) region first takes place at Na2, succeeded by Na1. Na2 diffusion back to the EC medium requires prior dissociation of substrate from S1. Significantly, Na+ binding (and unbinding) consistently involves a transient binding to a newly discovered site, Na1″, near S1, as an intermediate state. A robust sequence of substrate uptake events coupled to sodium bindings and translocations between those sites assisted by hydration emerges from the simulations: (i) bindings of a first Na+ to Na1″, translocation to Na1, a second Na+ to vacated Na1″ and then to Na2, and substrate to S1; (ii) rotation of Phe253 aromatic group to seclude the substrate from the EC region; and (iii) concerted tilting of TM1b and TM6a toward TM3 and TM8 to close the EC vestibule.
PMCID: PMC4281755  PMID: 25381247
Biophysics; Membrane Transport; Neurotransmitter Transport; Sodium Transport; Transport; LeuT; NSS; Mechanism of Binding; Sodium-coupled
13.  Phosphorylation of Rat Melanopsin at Ser-381 and Ser-398 by Light/Dark and Its Importance for Intrinsically Photosensitive Ganglion Cells (ipRGCs) Cellular Ca2+ Signaling* 
The Journal of Biological Chemistry  2014;289(51):35482-35493.
Background: Melanopsin is a retinal light-sensitive receptor involved in non-image forming functions including circadian timing.
Results: The intracellular C-terminal part of melanopsin was shown to be phosphorylated at distinct functional sites in response to light and dark.
Conclusion: Phosphorylation status of melanopsin influences its functions.
Significance: Distinct sites of phosphorylation are important in determining the signaling properties of melanopsin.
The G protein-coupled light-sensitive receptor melanopsin is involved in non-image-forming light responses including circadian timing. The predicted secondary structure of melanopsin indicates a long cytoplasmic tail with many potential phosphorylation sites. Using bioinformatics, we identified a number of amino acids with a high probability of being phosphorylated. We generated antibodies against melanopsin phosphorylated at Ser-381 and Ser-398, respectively. The antibody specificity was verified by immunoblotting and immunohistochemical staining of HEK-293 cells expressing rat melanopsin mutated in Ser-381 or Ser-398. Using the antibody recognizing phospho-Ser-381 melanopsin, we demonstrated by immunoblotting and immunohistochemical staining in HEK-293 cells expressing rat melanopsin that the receptor is phosphorylated in this position during the dark and dephosphorylated when light is turned on. On the contrary, we found that melanopsin at Ser-398 was unphosphorylated in the dark and became phosphorylated after light stimulation. The light-induced changes in phosphorylation at both Ser-381 and Ser-398 were rapid and lasted throughout the 4-h experimental period. Furthermore, phosphorylation at Ser-381 and Ser-398 was independent of each other. The changes in phosphorylation were confirmed in vivo by immunohistochemical staining of rat retinas during light and dark. We further demonstrated that mutation of Ser-381 and Ser-398 in melanopsin-expressing HEK-293 cells affected the light-induced Ca2+ response, which was significantly reduced as compared with wild type. Examining the light-evoked Ca2+ response in a melanopsin Ser-381 plus Ser-398 double mutant provided evidence that the phosphorylation events were independent.
PMCID: PMC4271233  PMID: 25378407
Calcium Imaging; G Protein-coupled Receptor (GPCR); Mutant; Phosphorylation; Photoreceptor; Retinal Ganglion Cells
14.  Multiple Drugs Compete for Transport via the Plasmodium falciparum Chloroquine Resistance Transporter at Distinct but Interdependent Sites* 
The Journal of Biological Chemistry  2014;289(52):36336-36351.
Background: Mutations in the chloroquine resistance transporter (PfCRT) change the susceptibility of Plasmodium falciparum to diverse antimalarial drugs.
Results: In addition to chloroquine, PfCRT transports quinine, quinidine, and verapamil, which bind to distinct but antagonistically interacting sites.
Conclusion: PfCRT is a multidrug carrier with a polyspecific drug-binding cavity.
Significance: These findings could be used to develop high affinity inhibitors of PfCRT.
Mutations in the “chloroquine resistance transporter” (PfCRT) are a major determinant of drug resistance in the malaria parasite Plasmodium falciparum. We have previously shown that mutant PfCRT transports the antimalarial drug chloroquine away from its target, whereas the wild-type form of PfCRT does not. However, little is understood about the transport of other drugs via PfCRT or the mechanism by which PfCRT recognizes different substrates. Here we show that mutant PfCRT also transports quinine, quinidine, and verapamil, indicating that the protein behaves as a multidrug resistance carrier. Detailed kinetic analyses revealed that chloroquine and quinine compete for transport via PfCRT in a manner that is consistent with mixed-type inhibition. Moreover, our analyses suggest that PfCRT accepts chloroquine and quinine at distinct but antagonistically interacting sites. We also found verapamil to be a partial mixed-type inhibitor of chloroquine transport via PfCRT, further supporting the idea that PfCRT possesses multiple substrate-binding sites. Our findings provide new mechanistic insights into the workings of PfCRT, which could be exploited to design potent inhibitors of this key mediator of drug resistance.
PMCID: PMC4276893  PMID: 25378409
Drug Resistance; Malaria; Membrane Transport; Parasitology; Transporter; Chloroquine; Mixed-type Inhibition; Quinine; Verapamil
15.  β2-Microglobulin Amyloid Fibrils Are Nanoparticles That Disrupt Lysosomal Membrane Protein Trafficking and Inhibit Protein Degradation by Lysosomes* 
The Journal of Biological Chemistry  2014;289(52):35781-35794.
Background: The causative agents and pathological mechanisms of amyloid disease are poorly understood.
Results: β2-Microglobulin amyloid fibrils display length-dependent internalization, alter trafficking of lysosomal membrane proteins, and inhibit the degradation of proteins by lysosomes.
Conclusion: Amyloid fibrils act as nanoparticles that disrupt membrane trafficking and protein degradation.
Significance: Fibril length, by determining access to intracellular compartments, may contribute to amyloid disease.
Fragmentation of amyloid fibrils produces fibrils that are reduced in length but have an otherwise unchanged molecular architecture. The resultant nanoscale fibril particles inhibit the cellular reduction of the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), a substrate commonly used to measure cell viability, to a greater extent than unfragmented fibrils. Here we show that the internalization of β2-microglobulin (β2m) amyloid fibrils is dependent on fibril length, with fragmented fibrils being more efficiently internalized by cells. Correspondingly, inhibiting the internalization of fragmented β2m fibrils rescued cellular MTT reduction. Incubation of cells with fragmented β2m fibrils did not, however, cause cell death. Instead, fragmented β2m fibrils accumulate in lysosomes, alter the trafficking of lysosomal membrane proteins, and inhibit the degradation of a model protein substrate by lysosomes. These findings suggest that nanoscale fibrils formed early during amyloid assembly reactions or by the fragmentation of longer fibrils could play a role in amyloid disease by disrupting protein degradation by lysosomes and trafficking in the endolysosomal pathway.
PMCID: PMC4276847  PMID: 25378395
Amyloid; Fibril; Lysosome; Membrane Trafficking; Protein Degradation
16.  Dendritic Cell IL-1α and IL-1β Are Polyubiquitinated and Degraded by the Proteasome* 
The Journal of Biological Chemistry  2014;289(51):35582-35592.
Background: Interleukin-1 secretion is an important process in inflammation and thus, the intracellular regulation of these cytokines is of interest.
Results: Inhibition of the proteasome in dendritic cells inhibits interleukin-1 degradation and leads to an accumulation of polyubiquitinated interleukin-1.
Conclusion: Interleukin-1 cytokines are regulated by polyubiquitination and proteasomal degradation.
Significance: Polyubiquitination and degradation are important processes in the intracellular regulation of interleukin-1.
IL-1α and β are key players in the innate immune system. The secretion of these cytokines by dendritic cells (DC) is integral to the development of proinflammatory responses. These cytokines are not secreted via the classical secretory pathway. Instead, 2 independent processes are required; an initial signal to induce up-regulation of the precursor pro-IL-1α and -β, and a second signal to drive cleavage and consequent secretion. Pro-IL-1α and -β are both cytosolic and thus, are potentially subject to post-translational modifications. These modifications may, in turn, have a functional outcome in the context of IL-1α and -β secretion and hence inflammation. We report here that IL-1α and -β were degraded intracellularly in murine bone marrow-derived DC and that this degradation was dependent on active cellular processes. In addition, we demonstrate that degradation was ablated when the proteasome was inhibited, whereas autophagy did not appear to play a major role. Furthermore, inhibition of the proteasome led to an accumulation of polyubiquitinated IL-1α and -β, indicating that IL-1α and -β were polyubiquitinated prior to proteasomal degradation. Finally, our investigations suggest that polyubiquitination and proteasomal degradation are not continuous processes but instead are up-regulated following DC activation. Overall, these data highlight that IL-1α and -β polyubiquitination and proteasomal degradation are central mechanisms in the regulation of intracellular IL-1 levels in DC.
PMCID: PMC4271241  PMID: 25371210
Dendritic Cell; Inflammation; Interleukin 1 (IL-1); Proteasome; Ubiquitylation (Ubiquitination); IL-1α IL-1β
17.  Evaluating the Capacity to Generate and Preserve Nitric Oxide Bioactivity in Highly Purified Earthworm Erythrocruorin 
Background: Earthworm hemoglobin (LtHb) is a potential blood substitute.
Results: LtHb can generate nitric oxide (NO) and preserve NO bioactivity.
Conclusion: LtHb reactions with nitrite and NO are indicative of therapeutic possibilities.
Significance: The results further highlight the potential role of hemoglobins in NO homeostasis.
The giant extracellular hemoglobin (erythrocruorin) from the earth worm (Lumbricus terrestris) has shown promise as a potential hemoglobin-based oxygen carrier (HBOC) in in vivo animal studies. An important beneficial characteristic of this hemoglobin (LtHb) is the large number of heme-based oxygen transport sites that helps overcome issues of osmotic stress when attempting to provide enough material for efficient oxygen delivery. A potentially important additional property is the capacity of the HBOC either to generate nitric oxide (NO) or to preserve NO bioactivity to compensate for decreased levels of NO in the circulation. The present study compares the NO-generating and NO bioactivity-preserving capability of LtHb with that of human adult hemoglobin (HbA) through several reactions including the nitrite reductase, reductive nitrosylation, and still controversial nitrite anhydrase reactions. An assignment of a heme-bound dinitrogen trioxide as the stable intermediate associated with the nitrite anhydrase reaction in both LtHb and HbA is supported based on functional and EPR spectroscopic studies. The role of the redox potential as a factor contributing to the NO-generating activity of these two proteins is evaluated. The results show that LtHb undergoes the same reactions as HbA and that the reduced efficacy for these reactions for LtHb relative to HbA is consistent with the much higher redox potential of LtHb. Evidence of functional heterogeneity in LtHb is explained in terms of the large difference in the redox potential of the isolated subunits.
PMCID: PMC4281771  PMID: 25371199
Hemoglobin; Myoglobin; Nitric Oxide; Oxidation-Reduction (Redox); Reductase; Dinitrogen Trioxide; Erythrocruorin; Nitrite Anhydrase; Nitrite Reductase
18.  Syk and Src Family Kinases Regulate C-type Lectin Receptor 2 (CLEC-2)-mediated Clustering of Podoplanin and Platelet Adhesion to Lymphatic Endothelial Cells* 
The Journal of Biological Chemistry  2014;289(52):35695-35710.
Background: The interaction of platelet CLEC-2 with Podoplanin is critical for development of the lymphatics.
Results: CLEC-2 forms a central cluster upon engagement with Podoplanin, which clusters Podoplanin. Clustering is dependent on Syk and is critical for adhesion.
Conclusion: Clustering regulates the interaction of platelets with lymphatic endothelial cells.
Significance: These findings account for the similar lymphatic phenotype of CLEC-2- and Syk-deficient mice.
The interaction of C-type lectin receptor 2 (CLEC-2) on platelets with Podoplanin on lymphatic endothelial cells initiates platelet signaling events that are necessary for prevention of blood-lymph mixing during development. In the present study, we show that CLEC-2 signaling via Src family and Syk tyrosine kinases promotes platelet adhesion to primary mouse lymphatic endothelial cells at low shear. Using supported lipid bilayers containing mobile Podoplanin, we further show that activation of Src and Syk in platelets promotes clustering of CLEC-2 and Podoplanin. Clusters of CLEC-2-bound Podoplanin migrate rapidly to the center of the platelet to form a single structure. Fluorescence lifetime imaging demonstrates that molecules within these clusters are within 10 nm of one another and that the clusters are disrupted by inhibition of Src and Syk family kinases. CLEC-2 clusters are also seen in platelets adhered to immobilized Podoplanin using direct stochastic optical reconstruction microscopy. These findings provide mechanistic insight by which CLEC-2 signaling promotes adhesion to Podoplanin and regulation of Podoplanin signaling, thereby contributing to lymphatic vasculature development.
PMCID: PMC4276840  PMID: 25368330
Endothelial Cell; Lipid Bilayer; Platelet; Receptor; Tyrosine-Protein Kinase (Tyrosine Kinase); CLEC-2; ITAM; Podoplanin; Src Family Kinase; Syk
19.  Small Molecules Dorsomorphin and LDN-193189 Inhibit Myostatin/GDF8 Signaling and Promote Functional Myoblast Differentiation* 
The Journal of Biological Chemistry  2014;290(6):3390-3404.
Background: GDF8/myostatin suppresses myogenic differentiation.
Results: The small molecule inhibitors dorsomorphin and LDN-193189 bind to and inhibit the GDF8 receptor ActRII and ALK4.
Conclusion: Dorsomorphin and LDN-193189 promote myogenesis in vitro.
Significance: Detailed molecular characterization of small molecule inhibitors targeting the GDF8/myostatin pathway demonstrates their potential and risk when applied to promote muscle development.
GDF8, or myostatin, is a member of the TGF-β superfamily of secreted polypeptide growth factors. GDF8 is a potent negative regulator of myogenesis both in vivo and in vitro. We found that GDF8 signaling was inhibited by the small molecule ATP competitive inhibitors dorsomorphin and LDN-193189. These compounds were previously shown to be potent inhibitors of BMP signaling by binding to the BMP type I receptors ALK1/2/3/6. We present the crystal structure of the type II receptor ActRIIA with dorsomorphin and demonstrate that dorsomorphin or LDN-193189 target GDF8 induced Smad2/3 signaling and repression of myogenic transcription factors. As a result, both inhibitors rescued myogenesis in myoblasts treated with GDF8. As revealed by quantitative live cell microscopy, treatment with dorsomorphin or LDN-193189 promoted the contractile activity of myotubular networks in vitro. We therefore suggest these inhibitors as suitable tools to promote functional myogenesis.
PMCID: PMC4319009  PMID: 25368322
Bone Morphogenetic Protein (BMP); Myogenesis; Myostatin; Serine/Threonine Protein Kinase; Small Molecule
20.  Cellulose Surface Degradation by a Lytic Polysaccharide Monooxygenase and Its Effect on Cellulase Hydrolytic Efficiency* 
The Journal of Biological Chemistry  2014;289(52):35929-35938.
Background: Lytic polysaccharide monooxygenase (LPMO) has recently been discovered to depolymerize cellulose.
Results: Dynamic imaging was applied to reveal the effects of LPMO and cellulase activity on solid cellulose surface.
Conclusion: Critical features of surface morphology for LPMO synergy with cellulases are recognized.
Significance: Direct insights into cellulose deconstruction by LPMO alone and in synergy with cellulases are obtained.
Lytic polysaccharide monooxygenase (LPMO) represents a unique principle of oxidative degradation of recalcitrant insoluble polysaccharides. Used in combination with hydrolytic enzymes, LPMO appears to constitute a significant factor of the efficiency of enzymatic biomass depolymerization. LPMO activity on different cellulose substrates has been shown from the slow release of oxidized oligosaccharides into solution, but an immediate and direct demonstration of the enzyme action on the cellulose surface is lacking. Specificity of LPMO for degrading ordered crystalline and unordered amorphous cellulose material of the substrate surface is also unknown. We show by fluorescence dye adsorption analyzed with confocal laser scanning microscopy that a LPMO (from Neurospora crassa) introduces carboxyl groups primarily in surface-exposed crystalline areas of the cellulosic substrate. Using time-resolved in situ atomic force microscopy we further demonstrate that cellulose nano-fibrils exposed on the surface are degraded into shorter and thinner insoluble fragments. Also using atomic force microscopy, we show that prior action of LPMO enables cellulases to attack otherwise highly resistant crystalline substrate areas and that it promotes an overall faster and more complete surface degradation. Overall, this study reveals key characteristics of LPMO action on the cellulose surface and suggests the effects of substrate morphology on the synergy between LPMO and hydrolytic enzymes in cellulose depolymerization.
PMCID: PMC4276861  PMID: 25361767
Atomic Force Microscopy (AFM); Biofuel; Cellulase; Cellulose; Copper Monooxygenase; GH61-AA9; Lytic Polysaccharide Monooxygenase (LPMO); Oxidative Cellulose Surface Degradation; Synergy
21.  Heparan Sulfate Inhibits Hematopoietic Stem and Progenitor Cell Migration and Engraftment in Mucopolysaccharidosis I* 
The Journal of Biological Chemistry  2014;289(52):36194-36203.
Background: Hematopoietic stem cell transplant in mucopolysaccharidosis I (MPSI) patients often results in graft failure.
Results: In mice with MPSI we link reduced hematopoietic engraftment post-transplant to accumulated overly-sulfated extracellular heparan sulfate.
Conclusion: Excess extracellular heparan sulfate alters cytokine gradient formation, restricting stem cell migration.
Significance: This provides a mechanistic insight into the observed engraftment difficulties seen in patients.
Mucopolysaccharidosis I Hurler (MPSI-H) is a pediatric lysosomal storage disease caused by genetic deficiencies in IDUA, coding for α-l-iduronidase. Idua−/− mice share similar clinical pathology with patients, including the accumulation of the undegraded glycosaminoglycans (GAGs) heparan sulfate (HS), and dermatan sulfate (DS), progressive neurodegeneration, and dysostosis multiplex. Hematopoietic stem cell transplantation (HSCT) is the most effective treatment for Hurler patients, but reduced intensity conditioning is a risk factor in transplantation, suggesting an underlying defect in hematopoietic cell engraftment. HS is a co-receptor in the CXCL12/CXCR4 axis of hematopoietic stem and progenitor cell (HSPC) migration to the bone marrow (BM), but the effect of HS alterations on HSPC migration, or the functional role of HS in MPSI-H are unknown. We demonstrate defective WT HSPC engraftment and migration in Idua−/− recipient BM, particularly under reduced intensity conditioning. Both intra- but especially extracellular Idua−/− BM HS was significantly increased and abnormally sulfated. Soluble heparinase-sensitive GAGs from Idua−/− BM and specifically 2-O-sulfated HS, elevated in Idua−/− BM, both inhibited CXCL12-mediated WT HSPC transwell migration, while DS had no effect. Thus we have shown that excess overly sulfated extracellular HS binds, and sequesters CXCL12, limiting hematopoietic migration and providing a potential mechanism for the limited scope of HSCT in Hurler disease.
PMCID: PMC4276882  PMID: 25359774
Animal Model; Bone Marrow; Hematopoietic Stem Cells; Heparan Sulfate; Lysosomal Storage Disease; Migration; Bone Marrow Transplant; CXCL12; Mucopolysaccharidosis I; Hurler
22.  A Coiled-coil Clamp Controls Both Conformation and Clustering of Stromal Interaction Molecule 1 (STIM1)* 
The Journal of Biological Chemistry  2014;289(48):33231-33244.
Background: STIM1 and Orai1 are key players in the store-operated Ca2+ entry.
Results: The activation state of STIM1 is precisely controlled by heteromeric interaction between coiled-coil domains.
Conclusion: A coiled-coil clamp provides control over STIM1 conformation and clustering.
Significance: Understanding of the STIM1 C-terminal switching mechanism is crucial for the control of Orai1 activation.
Store-operated Ca2+ entry, essential for the adaptive immunity, is initiated by the endoplasmic reticulum (ER) Ca2+ sensor STIM1. Ca2+ entry occurs through the plasma membrane resident Ca2+ channel Orai1 that directly interacts with the C-terminal STIM1 domain, named SOAR/CAD. Depletion of the ER Ca2+ store controls this STIM1/Orai1 interaction via transition to an extended STIM1 C-terminal conformation, exposure of the SOAR/CAD domain, and STIM1/Orai1 co-clustering. Here we developed a novel approach termed FRET-derived Interaction in a Restricted Environment (FIRE) in an attempt to dissect the interplay of coiled-coil (CC) interactions in controlling STIM1 quiescent as well as active conformation and cluster formation. We present evidence of a sequential activation mechanism in the STIM1 cytosolic domains where the interaction between CC1 and CC3 segment regulates both SOAR/CAD exposure and CC3-mediated higher-order oligomerization as well as cluster formation. These dual levels of STIM1 auto-inhibition provide efficient control over the coupling to and activation of Orai1 channels.
PMCID: PMC4246082  PMID: 25342749
Calcium Release-activated Calcium Channel Protein 1 (ORAI1); Fluorescence; Patch Clamp; Signal Transduction; Stromal Interaction Molecule 1 (STIM1)
23.  A Negatively Charged Residue Stabilizes the Tropoelastin N-terminal Region for Elastic Fiber Assembly* 
The Journal of Biological Chemistry  2014;289(50):34815-34826.
Background: Negative residues are rare in human tropoelastin, and their contributions to protein structure and function are unknown.
Results: Mutating aspartate 72 reduces coacervation, cross-linking, and elastogenesis and alters the N-terminal conformation.
Conclusion: Aspartate 72 residue stabilizes the N-terminal structure for functional assembly into elastic fibers.
Significance: This work provides the first direct evidence of the importance of the tropoelastin N-terminal structure in elastic fiber assembly.
Tropoelastin is an extracellular matrix protein that assembles into elastic fibers that provide elasticity and strength to vertebrate tissues. Although the contributions of specific tropoelastin regions during each stage of elastogenesis are still not fully understood, studies predominantly recognize the central hinge/bridge and C-terminal foot as the major participants in tropoelastin assembly, with a number of interactions mediated by the abundant positively charged residues within these regions. However, much less is known about the importance of the rarely occurring negatively charged residues and the N-terminal coil region in tropoelastin assembly. The sole negatively charged residue in the first half of human tropoelastin is aspartate 72. In contrast, the same region comprises 17 positively charged residues. We mutated this aspartate residue to alanine and assessed the elastogenic capacity of this novel construct. We found that D72A tropoelastin has a decreased propensity for initial self-association, and it cross-links aberrantly into denser, less porous hydrogels with reduced swelling properties. Although the mutant can bind cells normally, it does not form elastic fibers with human dermal fibroblasts and forms fewer atypical fibers with human retinal pigmented epithelial cells. This impaired functionality is associated with conformational changes in the N-terminal region. Our results strongly point to the role of the Asp-72 site in stabilizing the N-terminal segment of human tropoelastin and the importance of this region in facilitating elastic fiber assembly.
PMCID: PMC4263881  PMID: 25342751
Aspartate (Aspartic Acid); Elastin; Protein Self-assembly; Small Angle X-ray Scattering; Tertiary Structure; Domain 6; Elastic Fiber Assembly; N-terminal Region; Tropoelastin
24.  Dramatic Potentiation of the Antiviral Activity of HIV Antibodies by Cholesterol Conjugation* 
The Journal of Biological Chemistry  2014;289(50):35015-35028.
Background: Some HIV-neutralizing antibodies have an antigen-binding site with dual specificity, for the plasma membrane and a viral epitope.
Results: We engineered membrane affinity outside the antigen-binding site by conjugating cholesterol, with concomitantly increased antiviral potency.
Conclusion: A natural mechanism dependent on affinity maturation is mimicked by conjugation of a lipid.
Significance: This is a general strategy to boost the potency of antiviral antibodies.
The broadly neutralizing antibodies HIV 2F5 and 4E10, which bind to overlapping epitopes in the membrane-proximal external region of the fusion protein gp41, have been proposed to use a two-step mechanism for neutralization; first, they bind and preconcentrate at the viral membrane through their long, hydrophobic CDRH3 loops, and second, they form a high affinity complex with the protein epitope. Accordingly, mutagenesis of the CDRH3 can abolish their neutralizing activity, with no change in the affinity for the peptide epitope. We show here that we can mimic this mechanism by conjugating a cholesterol group outside of the paratope of an antibody. Cholesterol-conjugated antibodies bind to lipid raft domains on the membrane, and because of this enrichment, they show increased antiviral potency. In particular, we find that cholesterol conjugation (i) rescues the antiviral activity of CDRH3-mutated 2F5, (ii) increases the antiviral activity of WT 2F5, (iii) potentiates the non-membrane-binding HIV antibody D5 10–100-fold (depending on the virus strain), and (iv) increases synergy between 2F5 and D5. Conjugation can be made at several positions, including variable and constant domains. Cholesterol conjugation therefore appears to be a general strategy to boost the potency of antiviral antibodies, and, because membrane affinity is engineered outside of the antibody paratope, it can complement affinity maturation strategies.
PMCID: PMC4263897  PMID: 25342747
Antibody Engineering; Cholesterol; Human Immunodeficiency Virus (HIV); Lipid Raft; Membrane Fusion; Molecular Evolution; Viral Immunology; Virus Entry
25.  Oligomer Formation of Tau Protein Hyperphosphorylated in Cells* 
The Journal of Biological Chemistry  2014;289(49):34389-34407.
Background: The causal relationship between Tau hyperphosphorylation and aggregation in neuropathology is still under debate.
Results: Tau highly phosphorylated in cells increases oligomerization without pronounced aggregation. Oligomers cause reduction of dendritic spines but not cell death.
Conclusion: Hyperphosphorylation does not drive Tau fibrillization but contributes to synaptotoxicity.
Significance: Pathways and effects of Tau hyperphosphorylation are distinct from those of aggregation.
Abnormal phosphorylation (“hyperphosphorylation”) and aggregation of Tau protein are hallmarks of Alzheimer disease and other tauopathies, but their causative connection is still a matter of debate. Tau with Alzheimer-like phosphorylation is also present in hibernating animals, mitosis, or during embryonic development, without leading to pathophysiology or neurodegeneration. Thus, the role of phosphorylation and the distinction between physiological and pathological phosphorylation needs to be further refined. So far, the systematic investigation of highly phosphorylated Tau was difficult because a reliable method of preparing reproducible quantities was not available. Here, we generated full-length Tau (2N4R) in Sf9 cells in a well defined phosphorylation state containing up to ∼20 phosphates as judged by mass spectrometry and Western blotting with phospho-specific antibodies. Despite the high concentration in living Sf9 cells (estimated ∼230 μm) and high phosphorylation, the protein was not aggregated. However, after purification, the highly phosphorylated protein readily formed oligomers, whereas fibrils were observed only rarely. Exposure of mature primary neuronal cultures to oligomeric phospho-Tau caused reduction of spine density on dendrites but did not change the overall cell viability.
PMCID: PMC4256367  PMID: 25339173
Fluorescence Anisotropy; Neuron; Phosphorylation; Synapse; Tau Protein (Tau); Oligomers; Time-correlated Single Photon Counting; Toxicity

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