Publications by authors named "Shenyan Gu"

20 Publications

  • Page 1 of 1

Functional α6β4 acetylcholine receptor expression enables pharmacological testing of nicotinic agonists with analgesic properties.

J Clin Invest 2020 11;130(11):6158-6170

The α6β4 nicotinic acetylcholine receptor (nAChR) is enriched in dorsal root ganglia neurons and is an attractive non-opioid therapeutic target for pain. However, difficulty expressing human α6β4 receptors in recombinant systems has precluded drug discovery. Here, genome-wide screening identified accessory proteins that enable reconstitution of human α6β4 nAChRs. BARP, an auxiliary subunit of voltage-dependent calcium channels, promoted α6β4 surface expression while IRE1α, an unfolded protein response sensor, enhanced α6β4 receptor assembly. Effects on α6β4 involve BARP's N-terminal region and IRE1α's splicing of XBP1 mRNA. Furthermore, clinical efficacy of nicotinic agents in relieving neuropathic pain best correlated with their activity on α6β4. Finally, BARP-knockout, but not NACHO-knockout mice lacked nicotine-induced antiallodynia, highlighting the functional importance of α6β4 in pain. These results identify roles for IRE1α and BARP in neurotransmitter receptor assembly and unlock drug discovery for the previously elusive α6β4 receptor.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/JCI140311DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7598046PMC
November 2020

Hair cell α9α10 nicotinic acetylcholine receptor functional expression regulated by ligand binding and deafness gene products.

Proc Natl Acad Sci U S A 2020 09 14;117(39):24534-24544. Epub 2020 Sep 14.

Neuroscience Discovery, Janssen Pharmaceutical Companies of Johnson & Johnson, San Diego, CA 92121

Auditory hair cells receive olivocochlear efferent innervation, which refines tonotopic mapping, improves sound discrimination, and mitigates acoustic trauma. The olivocochlear synapse involves α9α10 nicotinic acetylcholine receptors (nAChRs), which assemble in hair cells only coincident with cholinergic innervation and do not express in recombinant mammalian cell lines. Here, genome-wide screening determined that assembly and surface expression of α9α10 require ligand binding. Ion channel function additionally demands an auxiliary subunit, which can be transmembrane inner ear (TMIE) or TMEM132e. Both of these single-pass transmembrane proteins are enriched in hair cells and underlie nonsyndromic human deafness. Inner hair cells from TMIE mutant mice show altered postsynaptic α9α10 function and retain α9α10-mediated transmission beyond the second postnatal week associated with abnormally persistent cholinergic innervation. Collectively, this study provides a mechanism to link cholinergic input with α9α10 assembly, identifies unexpected functions for human deafness genes TMIE/TMEM132e, and enables drug discovery for this elusive nAChR implicated in prevalent auditory disorders.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.2013762117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7533656PMC
September 2020

NACHO Engages N-Glycosylation ER Chaperone Pathways for α7 Nicotinic Receptor Assembly.

Cell Rep 2020 08;32(6):108025

Neuroscience Discovery, Janssen Pharmaceutical Companies of Johnson & Johnson, 3210 Merryfield Row, San Diego, CA 92121, USA. Electronic address:

The α7 nicotinic acetylcholine receptor participates in diverse aspects of brain physiology and disease. Neurons tightly control α7 assembly, which relies upon NACHO, an endoplasmic reticulum (ER)-localized integral membrane protein. By constructing α7 chimeras and mutants, we find that NACHO requires the α7 ectodomain to promote receptor assembly and surface trafficking. Also critical are two amino acids in the α7 second transmembrane domain. NACHO-mediated assembly is independent and separable from that induced by cholinergic ligands or RIC-3 protein, the latter of which acts on the large α7 intracellular loop. Proteomics indicates that NACHO associates with the ER oligosaccharyltransferase machinery and with calnexin. Accordingly, NACHO-mediated effects on α7 assembly and channel function require N-glycosylation and calnexin chaperone activity. These studies identify ER pathways that mediate α7 assembly by NACHO and provide insights into novel pharmacological strategies for these crucial nicotinic receptors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2020.108025DOI Listing
August 2020

NACHO Engages N-Glycosylation ER Chaperone Pathways for α7 Nicotinic Receptor Assembly.

Cell Rep 2020 08;32(6):108025

Neuroscience Discovery, Janssen Pharmaceutical Companies of Johnson & Johnson, 3210 Merryfield Row, San Diego, CA 92121, USA. Electronic address:

The α7 nicotinic acetylcholine receptor participates in diverse aspects of brain physiology and disease. Neurons tightly control α7 assembly, which relies upon NACHO, an endoplasmic reticulum (ER)-localized integral membrane protein. By constructing α7 chimeras and mutants, we find that NACHO requires the α7 ectodomain to promote receptor assembly and surface trafficking. Also critical are two amino acids in the α7 second transmembrane domain. NACHO-mediated assembly is independent and separable from that induced by cholinergic ligands or RIC-3 protein, the latter of which acts on the large α7 intracellular loop. Proteomics indicates that NACHO associates with the ER oligosaccharyltransferase machinery and with calnexin. Accordingly, NACHO-mediated effects on α7 assembly and channel function require N-glycosylation and calnexin chaperone activity. These studies identify ER pathways that mediate α7 assembly by NACHO and provide insights into novel pharmacological strategies for these crucial nicotinic receptors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2020.108025DOI Listing
August 2020

α7 nicotinic acetylcholine receptor upregulation by anti-apoptotic Bcl-2 proteins.

Nat Commun 2019 06 21;10(1):2746. Epub 2019 Jun 21.

Neuroscience Discovery, Janssen Pharmaceutical Companies of Johnson & Johnson, 3210 Merryfield Row, San Diego, CA, 92121, USA.

Nicotinic acetylcholine receptors (nAChRs) mediate and modulate synaptic transmission throughout the brain, and contribute to learning, memory, and behavior. Dysregulation of α7-type nAChRs in neuropsychiatric as well as immunological and oncological diseases makes them attractive targets for pharmaceutical development. Recently, we identified NACHO as an essential chaperone for α7 nAChRs. Leveraging the robust recombinant expression of α7 nAChRs with NACHO, we utilized genome-wide cDNA library screening and discovered that several anti-apoptotic Bcl-2 family proteins further upregulate receptor assembly and cell surface expression. These effects are mediated by an intracellular motif on α7 that resembles the BH3 binding domain of pro-apoptotic Bcl-2 proteins, and can be blocked by BH3 mimetic Bcl-2 inhibitors. Overexpression of Bcl-2 member Mcl-1 in neurons enhanced surface expression of endogenous α7 nAChRs, while a combination of chemotherapeutic Bcl2-inhibitors suppressed neuronal α7 receptor assembly. These results demonstrate that Bcl-2 proteins link α7 nAChR assembly to cell survival pathways.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-019-10723-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6588605PMC
June 2019

α6-Containing Nicotinic Acetylcholine Receptor Reconstitution Involves Mechanistically Distinct Accessory Components.

Cell Rep 2019 01;26(4):866-874.e3

Neuroscience Discovery, Janssen Pharmaceutical Companies of Johnson & Johnson, 3210 Merryfield Row, San Diego, CA 92121, USA. Electronic address:

Acetylcholine gates a large family of nicotinic receptor cation channels that control neuronal excitation and neurotransmitter release. These receptors are key targets for neuropsychiatric disorders; however, difficulties in expressing nicotinic acetylcholine (nACh) receptors hamper elaboration of their pharmacology and obscure elucidation of their biological functions. Particularly intriguing are α6-containing nACh receptors, which mediate nicotine-induced dopamine release in striatum-nucleus accumbens. Using genome-wide cDNA screening, we identify three accessory proteins, β-anchoring and -regulatory protein (BARP), lysosomal-associated membrane protein 5 (LAMP5), and SULT2B1, that complement the nACh receptor chaperone NACHO to reconstitute α6β2β3 channel function. Whereas NACHO mediates α6β2β3 assembly, BARP primarily enhances channel gating and LAMP5 and SULT2B1 promote receptor surface trafficking. BARP knockout mice show perturbations in presynaptic striatal nACh receptors that are consistent with BARP modulation of receptor desensitization. These studies unravel the molecular complexity of α6β2β3 biogenesis and enable physiological studies of this crucial neuropharmacological target.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2018.12.103DOI Listing
January 2019

Getting a Handle on Neuropharmacology by Targeting Receptor-Associated Proteins.

Neuron 2017 Dec;96(5):989-1001

Neuroscience Discovery, Janssen Pharmaceutical Companies of Johnson & Johnson, 3210 Merryfield Row, San Diego, CA 92121, USA. Electronic address:

Targeted therapy for neuropsychiatric disorders requires selective modulation of dysfunctional neuronal pathways. Receptors relevant to CNS disorders typically have associated proteins discretely expressed in specific neuronal pathways; these accessory proteins provide a new dimension for drug discovery. Recent studies show that targeting a TARP auxiliary subunit of AMPA receptors selectively modulates neuronal excitability in specific forebrain pathways relevant to epilepsy. Other medicinally important ion channels, gated by glutamate, γ-aminobutyric acid (GABA), and acetylcholine, also have associated proteins, which may be druggable. This emerging pharmacology of receptor-associated proteins provides a new approach for improving drug efficacy while mitigating side effects.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.neuron.2017.10.001DOI Listing
December 2017

NACHO Mediates Nicotinic Acetylcholine Receptor Function throughout the Brain.

Cell Rep 2017 04;19(4):688-696

Neuroscience Discovery, Janssen Pharmaceutical Companies of Johnson & Johnson, 3210 Merryfield Row, San Diego, CA 92121, USA. Electronic address:

Neuronal nicotinic acetylcholine receptors (nAChRs) participate in diverse aspects of brain function and mediate behavioral and addictive properties of nicotine. Neuronal nAChRs derive from combinations of α and β subunits, whose assembly is tightly regulated. NACHO was recently identified as a chaperone for α7-type nAChRs. Here, we find NACHO mediates assembly of all major classes of presynaptic and postsynaptic nAChR tested. NACHO acts at early intracellular stages of nAChR subunit assembly and then synergizes with RIC-3 for receptor surface expression. NACHO knockout mice show profound deficits in binding sites for α-bungarotoxin, epibatidine, and conotoxin MII, illustrating essential roles for NACHO in proper assembly of α7-, α4β2-, and α6-containing nAChRs, respectively. By contrast, GABA receptors are unaffected consistent with NACHO specifically modulating nAChRs. NACHO knockout mice show abnormalities in locomotor and cognitive behaviors compatible with nAChR deficiency and underscore the importance of this chaperone for physiology and disease associated with nAChRs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2017.04.008DOI Listing
April 2017

A Genome-Wide Arrayed cDNA Screen to Identify Functional Modulators of α7 Nicotinic Acetylcholine Receptors.

SLAS Discov 2017 02 28;22(2):155-165. Epub 2016 Oct 28.

1 Discovery Sciences, Janssen Research and Development LLC, La Jolla, CA, USA.

Cellular signaling is in part regulated by the composition and subcellular localization of a series of protein interactions that collectively form a signaling complex. Using the α7 nicotinic acetylcholine receptor (α7nAChR) as a proof-of-concept target, we developed a platform to identify functional modulators (or auxiliary proteins) of α7nAChR signaling. The Broad cDNA library was transiently cotransfected with α7nAChR cDNA in HEK293T cells in a high-throughput fashion. Using this approach in combination with a functional assay, we identified positive modulators of α7nAChR activity. We identified known positive modulators/auxiliary proteins present in the cDNA library that regulate α7nAChR signaling, in addition to identifying novel modulators of α7nAChR signaling. These included NACHO, SPDYE11, TCF4, and ZC3H12A, all of which increased PNU-120596-mediated nicotine-dependent calcium flux. Importantly, these auxiliary proteins did not modulate GluR1(o)-mediated Ca flux. To elucidate a possible mechanism of action, we employed an α7nAChR-HA surface staining assay. NACHO enhanced α7nAChR surface expression; however, the mechanism responsible for the SPDYE11-, TCF4-, and ZC3H12A-dependent modulation of α7nAChR has yet to be defined. This report describes the development and validation of a high-throughput, genome-wide cDNA screening platform coupled to FLIPR functional assays in order to identify functional modulators of α7nAChR signaling.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1177/1087057116676086DOI Listing
February 2017

Brain α7 Nicotinic Acetylcholine Receptor Assembly Requires NACHO.

Neuron 2016 Mar 11;89(5):948-55. Epub 2016 Feb 11.

Neuroscience Discovery, Janssen Pharmaceutical Companies of Johnson & Johnson, 3210 Merryfield Row, San Diego, CA 92121, USA. Electronic address:

Nicotine exerts its behavioral and additive actions through a family of brain nicotinic acetylcholine receptors (nAChRs). Enhancing α7-type nAChR signaling improves symptoms in Alzheimer's disease and schizophrenia. The pharmaceutical study of α7 receptors is hampered because these receptors do not form their functional pentameric structure in cell lines, and mechanisms that underlie α7 receptor assembly in neurons are not understood. Here, a genomic screening strategy solves this long-standing puzzle and identifies NACHO, a transmembrane protein of neuronal endoplasmic reticulum that mediates assembly of α7 receptors. NACHO promotes α7 protein folding, maturation through the Golgi complex, and expression at the cell surface. Knockdown of NACHO in cultured hippocampal neurons or knockout of NACHO in mice selectively and completely disrupts α7 receptor assembly and abolishes α7 channel function. This work identifies NACHO as an essential, client-specific chaperone for nAChRs and has implications for physiology and disease associated with these widely distributed neurotransmitter receptors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.neuron.2016.01.018DOI Listing
March 2016

Structural basis of the pH-dependent assembly of a botulinum neurotoxin complex.

J Mol Biol 2014 Nov 18;426(22):3773-3782. Epub 2014 Sep 18.

Department of Physiology and Biophysics, University of California, Irvine, CA 92697, USA. Electronic address:

Botulinum neurotoxins (BoNTs) are among the most poisonous biological substances known. They assemble with non-toxic non-hemagglutinin (NTNHA) protein to form the minimally functional progenitor toxin complexes (M-PTC), which protects BoNT in the gastrointestinal tract and releases it upon entry into the circulation. Here we provide molecular insight into the assembly between BoNT/A and NTNHA-A using small-angle X-ray scattering. We found that the free form BoNT/A maintains a pH-independent conformation with limited domain flexibility. Intriguingly, the free form NTNHA-A adopts pH-dependent conformational changes due to a torsional motion of its C-terminal domain. Once forming a complex at acidic pH, they each adopt a stable conformation that is similar to that observed in the crystal structure of the M-PTC. Our results suggest that assembly of the M-PTC depends on the environmental pH and that the complex form of BoNT/A is induced by interacting with NTNHA-A at acidic pH.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jmb.2014.09.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4252799PMC
November 2014

Molecular basis for disruption of E-cadherin adhesion by botulinum neurotoxin A complex.

Science 2014 Jun;344(6190):1405-10

Department of Physiology and Biophysics, University of California, Irvine, CA 92697, USA.

How botulinum neurotoxins (BoNTs) cross the host intestinal epithelial barrier in foodborne botulism is poorly understood. Here, we present the crystal structure of a clostridial hemagglutinin (HA) complex of serotype BoNT/A bound to the cell adhesion protein E-cadherin at 2.4 angstroms. The HA complex recognizes E-cadherin with high specificity involving extensive intermolecular interactions and also binds to carbohydrates on the cell surface. Binding of the HA complex sequesters E-cadherin in the monomeric state, compromising the E-cadherin-mediated intercellular barrier and facilitating paracellular absorption of BoNT/A. We reconstituted the complete 14-subunit BoNT/A complex using recombinantly produced components and demonstrated that abolishing either E-cadherin- or carbohydrate-binding of the HA complex drastically reduces oral toxicity of BoNT/A complex in vivo. Together, these studies establish the molecular mechanism of how HAs contribute to the oral toxicity of BoNT/A.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/science.1253823DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4164303PMC
June 2014

Botulinum neurotoxin A complex recognizes host carbohydrates through its hemagglutinin component.

Toxins (Basel) 2014 Feb 12;6(2):624-35. Epub 2014 Feb 12.

Department of Physiology and Biophysics, University of California, Irvine, CA 92697, USA.

Botulinum neurotoxins (BoNTs) are potent bacterial toxins. The high oral toxicity of BoNTs is largely attributed to the progenitor toxin complex (PTC), which is assembled from BoNT and nontoxic neurotoxin-associated proteins (NAPs) that are produced together with BoNT in bacteria. Here, we performed ex vivo studies to examine binding of the highly homogeneous recombinant NAPs to mouse small intestine. We also carried out the first comprehensive glycan array screening with the hemagglutinin (HA) component of NAPs. Our data confirmed that intestinal binding of the PTC is partly mediated by the HA moiety through multivalent interactions between HA and host carbohydrates. The specific HA-carbohydrate recognition could be inhibited by receptor-mimicking saccharides.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/toxins6020624DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3942755PMC
February 2014

Structure of a bimodular botulinum neurotoxin complex provides insights into its oral toxicity.

PLoS Pathog 2013 10;9(10):e1003690. Epub 2013 Oct 10.

Department of Physiology and Biophysics, University of California, Irvine, California, United States of America.

Botulinum neurotoxins (BoNTs) are produced by Clostridium botulinum and cause the fatal disease botulism, a flaccid paralysis of the muscle. BoNTs are released together with several auxiliary proteins as progenitor toxin complexes (PTCs) to become highly potent oral poisons. Here, we report the structure of a ∼760 kDa 14-subunit large PTC of serotype A (L-PTC/A) and reveal insight into its absorption mechanism. Using a combination of X-ray crystallography, electron microscopy, and functional studies, we found that L-PTC/A consists of two structurally and functionally independent sub-complexes. A hetero-dimeric 290 kDa complex protects BoNT, while a hetero-dodecameric 470 kDa complex facilitates its absorption in the harsh environment of the gastrointestinal tract. BoNT absorption is mediated by nine glycan-binding sites on the dodecameric sub-complex that forms multivalent interactions with carbohydrate receptors on intestinal epithelial cells. We identified monosaccharides that blocked oral BoNT intoxication in mice, which suggests a new strategy for the development of preventive countermeasures for BoNTs based on carbohydrate receptor mimicry.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.ppat.1003690DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3795040PMC
May 2014

Assembly and function of the botulinum neurotoxin progenitor complex.

Curr Top Microbiol Immunol 2013 ;364:21-44

Center for Neuroscience, Aging and Stem Cell Research, Sanford-Burnham Medical Research Institute, La Jolla, CA 92037, USA.

Botulinum neurotoxins (BoNTs) are among the most poisonous substances known to man, but paradoxically, BoNT-containing medicines and cosmetics have been used with great success in the clinic. Accidental BoNT poisoning mainly occurs through oral ingestion of food contaminated with Clostridium botulinum. BoNTs are naturally produced in the form of progenitor toxin complexes (PTCs), which are high molecular weight (up to ~900 kDa) multiprotein complexes composed of BoNT and several non-toxic neurotoxin-associated proteins (NAPs). NAPs protect the inherently fragile BoNTs against the hostile environment of the gastrointestinal (GI) tract and help BoNTs pass through the intestinal epithelial barrier before they are released into the general circulation. These events are essential for ingested BoNTs to gain access to motoneurons, where they inhibit neurotransmitter release and cause muscle paralysis. In this review, we discuss the structural basis for assembly of NAPs and BoNT into the PTC that protects BoNT and facilitate its delivery into the bloodstream.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-3-642-33570-9_2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3875173PMC
April 2013

Botulinum neurotoxin is shielded by NTNHA in an interlocked complex.

Science 2012 Feb;335(6071):977-81

Center for Neuroscience, Aging and Stem Cell Research, Sanford-Burnham Medical Research Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA.

Botulinum neurotoxins (BoNTs) are highly poisonous substances that are also effective medicines. Accidental BoNT poisoning often occurs through ingestion of Clostridium botulinum-contaminated food. Here, we present the crystal structure of a BoNT in complex with a clostridial nontoxic nonhemagglutinin (NTNHA) protein at 2.7 angstroms. Biochemical and functional studies show that NTNHA provides large and multivalent binding interfaces to protect BoNT from gastrointestinal degradation. Moreover, the structure highlights key residues in BoNT that regulate complex assembly in a pH-dependent manner. Collectively, our findings define the molecular mechanisms by which NTNHA shields BoNT in the hostile gastrointestinal environment and releases it upon entry into the circulation. These results will assist in the design of small molecules for inhibiting oral BoNT intoxication and of delivery vehicles for oral administration of biologics.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/science.1214270DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3545708PMC
February 2012

Structural basis of agrin-LRP4-MuSK signaling.

Genes Dev 2012 Feb;26(3):247-58

Center for Neuroscience, Aging, and Stem Cell Research, Sanford-Burnham Medical Research Institute, La Jolla, California 92037, USA.

Synapses are the fundamental units of neural circuits that enable complex behaviors. The neuromuscular junction (NMJ), a synapse formed between a motoneuron and a muscle fiber, has contributed greatly to understanding of the general principles of synaptogenesis as well as of neuromuscular disorders. NMJ formation requires neural agrin, a motoneuron-derived protein, which interacts with LRP4 (low-density lipoprotein receptor-related protein 4) to activate the receptor tyrosine kinase MuSK (muscle-specific kinase). However, little is known of how signals are transduced from agrin to MuSK. Here, we present the first crystal structure of an agrin-LRP4 complex, consisting of two agrin-LRP4 heterodimers. Formation of the initial binary complex requires the z8 loop that is specifically present in neuronal, but not muscle, agrin and that promotes the synergistic formation of the tetramer through two additional interfaces. We show that the tetrameric complex is essential for neuronal agrin-induced acetylcholine receptor (AChR) clustering. Collectively, these results provide new insight into the agrin-LRP4-MuSK signaling cascade and NMJ formation and represent a novel mechanism for activation of receptor tyrosine kinases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/gad.180885.111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3278892PMC
February 2012

Crystal structure of the glutamate receptor GluA1 N-terminal domain.

Biochem J 2011 Sep;438(2):255-63

Fujian Provincial Key Laboratory of Neurodegenerative Disease and Aging Research, College of Medicine, Xiamen University, Fujian, China.

The AMPA (α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) subfamily of iGluRs (ionotropic glutamate receptors) is essential for fast excitatory neurotransmission in the central nervous system. The malfunction of AMPARs (AMPA receptors) has been implicated in many neurological diseases, including Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis. The active channels of AMPARs and other iGluR subfamilies are tetramers formed exclusively by assembly of subunits within the same subfamily. It has been proposed that the assembly process is controlled mainly by the extracellular ATD (N-terminal domain) of iGluR. In addition, ATD has also been implicated in synaptogenesis, iGluR trafficking and trans-synaptic signalling, through unknown mechanisms. We report in the present study a 2.5 Å (1 Å=0.1 nm) resolution crystal structure of the ATD of GluA1. Comparative analyses of the structure of GluA1-ATD and other subunits sheds light on our understanding of how ATD drives subfamily-specific assembly of AMPARs. In addition, analysis of the crystal lattice of GluA1-ATD suggests a novel mechanism by which the ATD might participate in inter-tetramer AMPAR clustering, as well as in trans-synaptic protein-protein interactions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1042/BJ20110801DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3296483PMC
September 2011

The biological activity of botulinum neurotoxin type C is dependent upon novel types of ganglioside binding sites.

Mol Microbiol 2011 Jul 2;81(1):143-56. Epub 2011 Jun 2.

Institut für Toxikologie, OE 5340 Institut für Biochemie, OE 4310, Medizinische Hochschule Hannover, Carl-Neuberg-Str 1, 30625 Hannover, Germany.

The seven botulinum neurotoxins (BoNT) cause muscle paralysis by selectively cleaving core components of the vesicular fusion machinery. Their extraordinary activity primarily relies on highly specific entry into neurons. Data on BoNT/A, B, E, F and G suggest that entry follows a dual receptor interaction with complex gangliosides via an established ganglioside binding region and a synaptic vesicle protein. Here, we report high resolution crystal structures of the BoNT/C cell binding fragment alone and in complex with sialic acid. The WY-motif characteristic of the established ganglioside binding region was located on an exposed loop. Sialic acid was co-ordinated at a novel position neighbouring the binding pocket for synaptotagmin in BoNT/B and G and the sialic acid binding site in BoNT/D and TeNT respectively. Employing synaptosomes and immobilized gangliosides binding studies with BoNT/C mutants showed that the ganglioside binding WY-loop, the newly identified sialic acid-co-ordinating pocket and the area corresponding to the established ganglioside binding region of other BoNTs are involved in ganglioside interaction. Phrenic nerve hemidiaphragm activity tests employing ganglioside deficient mice furthermore evidenced that the biological activity of BoNT/C depends on ganglioside interaction with at least two binding sites. These data suggest a unique cell binding and entry mechanism for BoNT/C among clostridial neurotoxins.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/j.1365-2958.2011.07682.xDOI Listing
July 2011

Crystal structure and association behaviour of the GluR2 amino-terminal domain.

EMBO J 2009 Jun 21;28(12):1812-23. Epub 2009 May 21.

Center for Neuroscience, Aging, and Stem Cell Research, Burnham Institute for Medical Research, La Jolla, CA, USA.

Fast excitatory neurotransmission is mediated largely by ionotropic glutamate receptors (iGluRs), tetrameric, ligand-gated ion channel proteins comprised of three subfamilies, AMPA, kainate and NMDA receptors, with each subfamily sharing a common, modular-domain architecture. For all receptor subfamilies, active channels are exclusively formed by assemblages of subunits within the same subfamily, a molecular process principally encoded by the amino-terminal domain (ATD). However, the molecular basis by which the ATD guides subfamily-specific receptor assembly is not known. Here we show that AMPA receptor GluR1- and GluR2-ATDs form tightly associated dimers and, by the analysis of crystal structures of the GluR2-ATD, propose mechanisms by which the ATD guides subfamily-specific receptor assembly.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/emboj.2009.140DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2699365PMC
June 2009