ABSTRACT
Microtubule-associated protein tau is a central factor in Alzheimer's disease and other tauopathies. However, the physiological functions of tau are unclear. Here, we used proximity-labelling proteomics to chart tau interactomes in primary neurons and mouse brains in vivo. Tau interactors map onto pathways of cytoskeletal, synaptic vesicle and postsynaptic receptor regulation and show significant enrichment for Parkinson's, Alzheimer's and prion disease. We find that tau interacts with and dose-dependently reduces the activity of N-ethylmaleimide sensitive fusion protein (NSF), a vesicular ATPase essential for AMPA-type glutamate receptor (AMPAR) trafficking. Tau-deficient (tau-/- ) neurons showed mislocalised expression of NSF and enhanced synaptic AMPAR surface levels, reversible through the expression of human tau or inhibition of NSF. Consequently, enhanced AMPAR-mediated associative and object recognition memory in tau-/- mice is suppressed by both hippocampal tau and infusion with an NSF-inhibiting peptide. Pathologic mutant tau from mouse models or Alzheimer's disease significantly enhances NSF inhibition. Our results map neuronal tau interactomes and delineate a functional link of tau with NSF in plasticity-associated AMPAR-trafficking and memory.
Subject(s)
Alzheimer Disease , Receptors, AMPA , tau Proteins/metabolism , Alzheimer Disease/genetics , Alzheimer Disease/metabolism , Animals , Hippocampus/metabolism , Humans , Memory , Mice , N-Ethylmaleimide-Sensitive Proteins/genetics , N-Ethylmaleimide-Sensitive Proteins/metabolism , Neurons/metabolism , Protein Transport , Receptors, AMPA/genetics , Receptors, AMPA/metabolismABSTRACT
Intracellular vesicle fusion is driven by the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and their cofactors, including Sec1/Munc18 (SM), α-SNAP, and NSF. α-SNAP and NSF play multiple layers of regulatory roles in the SNARE assembly, disassembling the cis-SNARE complex and the prefusion SNARE complex. How SM proteins coupled with NSF and α-SNAP regulate SNARE-dependent membrane fusion remains incompletely understood. Munc18c, an SM protein involved in the exocytosis of the glucose transporter GLUT4, binds and activates target (t-) SNAREs to accelerate the fusion reaction through a SNARE-like peptide (SLP). Here, using an in vitro reconstituted system, we discovered that α-SNAP blocks the GLUT4 SNAREs-mediated membrane fusion. Munc18c interacts with t-SNAREs to displace α-SNAP, which overcomes the fusion inhibition. Furthermore, Munc18c shields the trans-SNARE complex from NSF/α-SNAP-mediated disassembly and accelerates SNARE-dependent fusion kinetics in the presence of NSF and α-SNAP. The SLP in domain 3a is indispensable in Munc18c-assisted resistance to NSF and α-SNAP. Together, our findings demonstrate that Munc18c protects the prefusion SNARE complex from α-SNAP and NSF, promoting SNARE-dependent membrane fusion through its SLP.
Subject(s)
Membrane Fusion , Munc18 Proteins , SNARE Proteins , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins , Membrane Fusion/physiology , Munc18 Proteins/metabolism , N-Ethylmaleimide-Sensitive Proteins/genetics , N-Ethylmaleimide-Sensitive Proteins/metabolism , Organelles/metabolism , Peptides/metabolism , SNARE Proteins/metabolism , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins/genetics , Animals , MiceABSTRACT
ATPases associated with diverse cellular activities (AAA+ proteins) are a superfamily of proteins found throughout all domains of life. The hallmark of this family is a conserved AAA+ domain responsible for a diverse range of cellular activities. Typically, AAA+ proteins transduce chemical energy from the hydrolysis of ATP into mechanical energy through conformational change, which can drive a variety of biological processes. AAA+ proteins operate in a variety of cellular contexts with diverse functions including disassembly of SNARE proteins, protein quality control, DNA replication, ribosome assembly, and viral replication. This breadth of function illustrates both the importance of AAA+ proteins in health and disease and emphasizes the importance of understanding conserved mechanisms of chemo-mechanical energy transduction. This review is divided into three major portions. First, the core AAA+ fold is presented. Next, the seven different clades of AAA+ proteins and structural details and reclassification pertaining to proteins in each clade are described. Finally, two well-known AAA+ proteins, NSF and its close relative p97, are reviewed in detail.
Subject(s)
AAA Proteins , Adenosine Triphosphate , AAA Proteins/metabolism , Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/chemistry , N-Ethylmaleimide-Sensitive Proteins/chemistry , N-Ethylmaleimide-Sensitive Proteins/genetics , N-Ethylmaleimide-Sensitive Proteins/metabolism , SNARE Proteins/chemistry , SNARE Proteins/metabolismABSTRACT
The development of male and female gametophytes is a pre-requisite for successful reproduction of angiosperms. Factors mediating vesicular trafficking are among the key regulators controlling gametophytic development. Fusion between vesicles and target membranes requires the assembly of a fusogenic soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) complex, whose disassembly in turn ensures the recycle of individual SNARE components. The disassembly of post-fusion SNARE complexes is controlled by the AAA+ ATPase N-ethylmaleimide-sensitive factor (Sec18/NSF) and soluble NSF attachment protein (Sec17/α-SNAP) in yeast and metazoans. Although non-canonical α-SNAPs have been functionally characterized in soybeans, the biological function of canonical α-SNAPs has yet to be demonstrated in plants. We report here that the canonical α-SNAP in Arabidopsis is essential for male and female gametophytic development. Functional loss of the canonical α-SNAP in Arabidopsis results in gametophytic lethality by arresting the first mitosis during gametogenesis. We further show that Arabidopsis α-SNAP encodes two isoforms due to alternative splicing. Both isoforms interact with the Arabidopsis homolog of NSF whereas have distinct subcellular localizations. The presence of similar alternative splicing of human α-SNAP indicates that functional distinction of two α-SNAP isoforms is evolutionarily conserved.
Subject(s)
Arabidopsis/genetics , Gametogenesis/genetics , Plant Development/genetics , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins/genetics , ATPases Associated with Diverse Cellular Activities/genetics , Alternative Splicing/genetics , Arabidopsis/growth & development , Germ Cells, Plant/growth & development , Mitosis/genetics , N-Ethylmaleimide-Sensitive Proteins/genetics , Protein Isoforms/geneticsABSTRACT
In plants, the apoplast is a critical battlefield for plant-microbe interactions. Plants secrete defense-related proteins into the apoplast to ward off the invasion of pathogens. How microbial pathogens overcome plant apoplastic immunity remains largely unknown. In this study, we reported that an atypical RxLR effector PsAvh181 secreted by Phytophthora sojae, inhibits the secretion of plant defense-related apoplastic proteins. PsAvh181 localizes to plant plasma membrane and essential for P. sojae infection. By co-immunoprecipitation assay followed by liquid chromatography-tandem mass spectrometry analyses, we identified the soybean GmSNAP-1 as a candidate host target of PsAvh181. GmSNAP-1 encodes a soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein, which associates with GmNSF of the SNARE complex functioning in vesicle trafficking. PsAvh181 binds to GmSNAP-1 in vivo and in vitro. PsAvh181 interferes with the interaction between GmSNAP-1 and GmNSF, and blocks the secretion of apoplastic defense-related proteins, such as pathogenesis-related protein PR-1 and apoplastic proteases. Taken together, these data show that an atypical P. sojae RxLR effector suppresses host apoplastic immunity by manipulating the host SNARE complex to interfere with host vesicle trafficking pathway.
Subject(s)
Glycine max/parasitology , Host-Pathogen Interactions , Phytophthora infestans/physiology , Plant Diseases/parasitology , Plant Proteins/metabolism , Virulence Factors/metabolism , Virulence , N-Ethylmaleimide-Sensitive Proteins/genetics , N-Ethylmaleimide-Sensitive Proteins/metabolism , Plant Diseases/immunology , Plant Proteins/genetics , Protein Interaction Domains and Motifs , SNARE Proteins/genetics , SNARE Proteins/metabolism , Glycine max/immunology , Glycine max/metabolism , Virulence Factors/geneticsABSTRACT
Ariadne-1 (Ari-1) is an E3 ubiquitin-ligase essential for neuronal development, but whose neuronal substrates are yet to be identified. To search for putative Ari-1 substrates, we used an in vivo ubiquitin biotinylation strategy coupled to quantitative proteomics of Drosophila heads. We identified 16 candidates that met the established criteria: a significant change of at least twofold increase on ubiquitination, with at least two unique peptides identified. Among those candidates, we identified Comatose (Comt), the homologue of the N-ethylmaleimide sensitive factor (NSF), which is involved in neurotransmitter release. Using a pull-down approach that relies on the overexpression and stringent isolation of a GFP-fused construct, we validate Comt/NSF to be an ubiquitination substrate of Ari-1 in fly neurons, resulting in the preferential monoubiquitination of Comt/NSF. We tested the possible functional relevance of this modification using Ari-1 loss-of-function mutants, which displayed a lower rate of spontaneous neurotransmitter release due to failures at the presynaptic side. By contrast, evoked release in Ari-1 mutants was enhanced compared with controls in a Ca2+-dependent manner without modifications in the number of active zones, indicating that the probability of release per synapse is increased in these mutants. This phenotype distinction between spontaneous and evoked release suggests that NSF activity may discriminate between these two types of vesicle fusion. Our results thus provide a mechanism to regulate NSF activity in the synapse through Ari-1-dependent ubiquitination.
Subject(s)
Drosophila Proteins/metabolism , N-Ethylmaleimide-Sensitive Proteins/metabolism , Animals , Carrier Proteins/metabolism , Drosophila Proteins/physiology , Drosophila melanogaster , Membrane Fusion , Mutation , N-Ethylmaleimide-Sensitive Proteins/genetics , Neurons/metabolism , Neurotransmitter Agents/metabolism , Phenotype , Synapses/metabolism , Synaptic Transmission/genetics , Synaptic Vesicles/metabolism , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Ubiquitination , Vesicular Transport Proteins/metabolismABSTRACT
Phagophore maturation is a key step in the macroautophagy pathway, which is critical in many important physiological and pathological processes. Here we identified Drosophila N-ethylmaleimide-sensitive fusion protein 2 (dNSF2) and soluble NSF attachment protein (Snap) as strong genetic modifiers of mutant CHMP2B, an ESCRT-III component that causes frontotemporal dementia and autophagosome accumulation. Among several SNAP receptor (SNARE) genes, Drosophila syntaxin 13 (syx13) exhibited a strong genetic interaction with mutant CHMP2B. Knockdown of syntaxin 13 (STX13) or its binding partner Vti1a in mammalian cells caused LC3-positive puncta to accumulate and blocks autophagic flux. STX13 was present on LC3-positive phagophores induced by rapamycin and was highly enriched on multilamellar structures induced by dysfunctional ESCRT-III. Loss of STX13 also caused the accumulation of Atg5-positive puncta and the formation of multilamellar structures. These results suggest that STX13 is a genetic modifier of ESCRT-III dysfunction and participates in the maturation of phagophores into closed autophagosomes.
Subject(s)
Autophagy , Drosophila Proteins/metabolism , Endosomal Sorting Complexes Required for Transport/metabolism , Phagosomes/metabolism , Qa-SNARE Proteins/metabolism , Vesicular Transport Proteins/metabolism , Animals , Blotting, Western , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Endosomal Sorting Complexes Required for Transport/genetics , Eye Abnormalities/genetics , Eye Abnormalities/metabolism , Frontotemporal Dementia/genetics , Frontotemporal Dementia/metabolism , HEK293 Cells , HeLa Cells , Humans , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Microscopy, Confocal , Microscopy, Immunoelectron , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Mutation , N-Ethylmaleimide-Sensitive Proteins/genetics , N-Ethylmaleimide-Sensitive Proteins/metabolism , Phagosomes/ultrastructure , Phenotype , Qa-SNARE Proteins/genetics , RNA Interference , Vesicular Transport Proteins/geneticsABSTRACT
N-ethylmaleimide sensitive factor (NSF) and α-soluble NSF attachment protein (α-SNAP) are essential eukaryotic housekeeping proteins that cooperatively function to sustain vesicular trafficking. The "resistance to Heterodera glycines 1" (Rhg1) locus of soybean (Glycine max) confers resistance to soybean cyst nematode, a highly damaging soybean pest. Rhg1 loci encode repeat copies of atypical α-SNAP proteins that are defective in promoting NSF function and are cytotoxic in certain contexts. Here, we discovered an unusual NSF allele (Rhg1-associated NSF on chromosome 07; NSFRAN07 ) in Rhg1+ germplasm. NSFRAN07 protein modeling to mammalian NSF/α-SNAP complex structures indicated that at least three of the five NSFRAN07 polymorphisms reside adjacent to the α-SNAP binding interface. NSFRAN07 exhibited stronger in vitro binding with Rhg1 resistance-type α-SNAPs. NSFRAN07 coexpression in planta was more protective against Rhg1 α-SNAP cytotoxicity, relative to WT NSFCh07 Investigation of a previously reported segregation distortion between chromosome 18 Rhg1 and a chromosome 07 interval now known to contain the Glyma.07G195900 NSF gene revealed 100% coinheritance of the NSFRAN07 allele with disease resistance Rhg1 alleles, across 855 soybean accessions and in all examined Rhg1+ progeny from biparental crosses. Additionally, we show that some Rhg1-mediated resistance is associated with depletion of WT α-SNAP abundance via selective loss of WT α-SNAP loci. Hence atypical coevolution of the soybean SNARE-recycling machinery has balanced the acquisition of an otherwise disruptive housekeeping protein, enabling a valuable disease resistance trait. Our findings further indicate that successful engineering of Rhg1-related resistance in plants will require a compatible NSF partner for the resistance-conferring α-SNAP.
Subject(s)
Disease Resistance/genetics , Gene Expression Regulation, Plant , Glycine max/growth & development , N-Ethylmaleimide-Sensitive Proteins/metabolism , Nematoda/physiology , Plants, Genetically Modified/growth & development , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins/metabolism , Animals , Host-Parasite Interactions , N-Ethylmaleimide-Sensitive Proteins/genetics , Plant Diseases/parasitology , Plants, Genetically Modified/genetics , Plants, Genetically Modified/parasitology , Polymorphism, Single Nucleotide , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins/genetics , Glycine max/genetics , Glycine max/parasitologyABSTRACT
NSF (N-ethylmaleimide sensitive factor) and its yeast counterpart Sec18 are highly conserved homohexameric proteins that play vital roles in eukaryotic membrane trafficking. Sec18 functions by disrupting SNARE complexes formed in cis, on the same membrane. However, the molecular mechanisms of this process are poorly understood, in large part due to the lack of selective, reversible inhibitors. A new study by Sparks et al. now reports a small molecule that appears to selectively inhibit Sec18 action in an in vitro assay. Their finding now paves the way to elucidate further details of Sec18-mediated SNARE priming.
Subject(s)
Adenosine Triphosphatases/chemistry , N-Ethylmaleimide-Sensitive Proteins/chemistry , SNARE Proteins/genetics , Saccharomyces cerevisiae Proteins/chemistry , Small Molecule Libraries/chemistry , Vesicular Transport Proteins/chemistry , Adenosine Triphosphatases/genetics , Membrane Fusion/genetics , N-Ethylmaleimide-Sensitive Proteins/genetics , Protein Binding/genetics , Protein Transport/genetics , SNARE Proteins/chemistry , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Small Molecule Libraries/pharmacology , Vesicular Transport Proteins/geneticsABSTRACT
The homeostasis of most organelles requires membrane fusion mediated by soluble N -ethylmaleimide-sensitive factor (NSF) attachment protein receptors (SNAREs). SNAREs undergo cycles of activation and deactivation as membranes move through the fusion cycle. At the top of the cycle, inactive cis-SNARE complexes on a single membrane are activated, or primed, by the hexameric ATPase associated with the diverse cellular activities (AAA+) protein, N-ethylmaleimide-sensitive factor (NSF/Sec18), and its co-chaperone α-SNAP/Sec17. Sec18-mediated ATP hydrolysis drives the mechanical disassembly of SNAREs into individual coils, permitting a new cycle of fusion. Previously, we found that Sec18 monomers are sequestered away from SNAREs by binding phosphatidic acid (PA). Sec18 is released from the membrane when PA is hydrolyzed to diacylglycerol by the PA phosphatase Pah1. Although PA can inhibit SNARE priming, it binds other proteins and thus cannot be used as a specific tool to further probe Sec18 activity. Here, we report the discovery of a small-molecule compound, we call IPA (inhibitor of priming activity), that binds Sec18 with high affinity and blocks SNARE activation. We observed that IPA blocks SNARE priming and competes for PA binding to Sec18. Molecular dynamics simulations revealed that IPA induces a more rigid NSF/Sec18 conformation, which potentially disables the flexibility required for Sec18 to bind to PA or to activate SNAREs. We also show that IPA more potently and specifically inhibits NSF/Sec18 activity than does N-ethylmaleimide, requiring the administration of only low micromolar concentrations of IPA, demonstrating that this compound could help to further elucidate SNARE-priming dynamics.
Subject(s)
Adenosine Triphosphatases/genetics , Ethylmaleimide/metabolism , Phosphatidic Acids/chemistry , Saccharomyces cerevisiae Proteins/genetics , Small Molecule Libraries/chemistry , Vesicular Transport Proteins/genetics , ATPases Associated with Diverse Cellular Activities/chemistry , ATPases Associated with Diverse Cellular Activities/genetics , Adenosine Triphosphatases/chemistry , Membrane Fusion/drug effects , Membrane Fusion/genetics , Membrane Lipids/chemistry , Membrane Lipids/genetics , Membrane Transport Proteins/chemistry , Membrane Transport Proteins/genetics , Molecular Dynamics Simulation , N-Ethylmaleimide-Sensitive Proteins/chemistry , N-Ethylmaleimide-Sensitive Proteins/genetics , Phosphatidic Acids/antagonists & inhibitors , SNARE Proteins/chemistry , SNARE Proteins/genetics , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/chemistry , Small Molecule Libraries/pharmacology , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins/chemistry , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins/genetics , Vacuoles/genetics , Vesicular Transport Proteins/chemistryABSTRACT
Axon ensheathment by specialized glial cells is an important process for fast propagation of action potentials. The rapid electrical conduction along myelinated axons is mainly due to its saltatory nature characterized by the accumulation of ion channels at the nodes of Ranvier. However, how these ion channels are transported and anchored along axons is not fully understood. We have identified N-myc downstream-regulated gene 4, ndrg4, as a novel factor that regulates sodium channel clustering in zebrafish. Analysis of chimeric larvae indicates that ndrg4 functions autonomously within neurons for sodium channel clustering at the nodes. Molecular analysis of ndrg4 mutants shows that expression of snap25 and nsf are sharply decreased, revealing a role of ndrg4 in controlling vesicle exocytosis. This uncovers a previously unknown function of ndrg4 in regulating vesicle docking and nodes of Ranvier organization, at least through its ability to finely tune the expression of the t-SNARE/NSF machinery.
Subject(s)
Muscle Proteins/genetics , N-Ethylmaleimide-Sensitive Proteins/biosynthesis , Ranvier's Nodes/genetics , Synaptosomal-Associated Protein 25/biosynthesis , Zebrafish Proteins/biosynthesis , Zebrafish Proteins/genetics , Zebrafish/genetics , Animals , Axons/metabolism , Exocytosis/genetics , Gene Expression Regulation , Humans , Muscle Proteins/metabolism , N-Ethylmaleimide-Sensitive Proteins/genetics , Neuroglia/metabolism , Neurons/metabolism , Ranvier's Nodes/metabolism , Schwann Cells , Sodium Channels/genetics , Sodium Channels/metabolism , Synaptic Transmission/genetics , Synaptosomal-Associated Protein 25/genetics , Zebrafish/metabolism , Zebrafish Proteins/metabolismABSTRACT
Melatonin (N-acetyl-5-methoxytryptamine) is involved in many developmental processes and responses to various abiotic stresses in plants. Most of the studies on melatonin focus on its functions and physiological responses in plants, while its regulation mechanism remains unknown. Caffeic acid 3-O-methyltransferase (COMT) functions at a key step of the biosynthesis process of melatonin. In this study, a COMT-like gene, TaCOMT (Traes_1AL_D9035D5E0.1) was identified in common wheat (Triticum aestivum L.). Transient transformation in wheat protoplasts determined that TaCOMT is localized in cytoplasm. TaCOMT in wheat was induced by drought stress, gibberellin (GA)3 and 3-Indoleacetic acid (IAA), but not by ABA. In TaCOMT transgenic Arabidopsis, melatonin contents were higher than that in wild type (WT) plants. Under D-Mannitol treatment, the fresh weight of the transgenic Arabidopsis was significantly higher than WT, and transgenic lines had a stronger root system compared to WT. Drought tolerance assays in pots showed that the survival rate of TaCOMT-overexpression lines was significantly higher than that of WT lines. this phenotype was similar to that the WT lines treated with melatonin under drought condition. In addition, the TaCOMT transgenic lines had higher proline content and lower malondialdehyde (MDA) content compared to WT lines after drought treatment. These results indicated that overexpression of the wheat TaCOMT gene enhances drought tolerance and increases the content of melatonin in transgenic Arabidopsis. It could be one of the potential genes for agricultural applications.
Subject(s)
Adaptation, Biological , Arabidopsis/genetics , Arabidopsis/metabolism , Droughts , Gene Expression , Melatonin/biosynthesis , N-Ethylmaleimide-Sensitive Proteins/genetics , Amino Acid Sequence , N-Ethylmaleimide-Sensitive Proteins/chemistry , N-Ethylmaleimide-Sensitive Proteins/metabolism , Plants, Genetically Modified , Signal Transduction , Stress, Physiological/genetics , Triticum/genetics , Triticum/metabolismABSTRACT
The Golgi apparatus is a key station of glycosylation and membrane traffic. It consists of stacked cisternae in most eukaryotes. However, the mechanisms how the Golgi stacks are formed and maintained are still obscure. The model plant Arabidopsis thaliana provides a nice system to observe Golgi structures by light microscopy, because the Golgi in A. thaliana is in the form of mini-stacks that are distributed throughout the cytoplasm. To obtain a clue to understand the molecular basis of Golgi morphology, we took a forward-genetic approach to isolate A. thaliana mutants that show abnormal structures of the Golgi under a confocal microscope. In the present report, we describe characterization of one of such mutants, named #46-3. The #46-3 mutant showed pleiotropic Golgi phenotypes. The Golgi size was in majority smaller than the wild type, but varied from very small ones, sometimes without clear association of cis and trans cisternae, to abnormally large ones under a confocal microscope. At the ultrastructual level by electron microscopy, queer-shaped large Golgi stacks were occasionally observed. By positional mapping, genome sequencing, and complementation and allelism tests, we linked the mutant phenotype to the missense mutation D374N in the NSF gene, encoding the N-ethylmaleimide-sensitive factor (NSF), a key component of membrane fusion. This residue is near the ATP-binding site of NSF, which is very well conserved in eukaryotes, suggesting that the biochemical function of NSF is important for maintaining the normal morphology of the Golgi.Key words: Golgi morphology, N-ethylmaleimide-sensitive factor (NSF), Arabidopsis thaliana.
Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis/metabolism , Golgi Apparatus/metabolism , N-Ethylmaleimide-Sensitive Proteins/genetics , Adenosine Triphosphatases/metabolism , Amino Acid Sequence , Animals , Arabidopsis/genetics , Arabidopsis/growth & development , Arabidopsis Proteins/metabolism , Binding Sites , Golgi Apparatus/pathology , Golgi Apparatus/ultrastructure , Humans , Membrane Fusion , Microscopy, Confocal , Microscopy, Electron , Mutation, Missense , N-Ethylmaleimide-Sensitive Proteins/metabolism , Phenotype , Sequence AlignmentABSTRACT
Defects in vesicular trafficking underlie a wide variety of human diseases. Genetic disruption of leucine-rich repeat kinase 2 (LRRK2) in rodents results in epithelial vesicular trafficking errors that can also be induced by treatment of animals with LRRK2 kinase inhibitors. Here we demonstrate that defects in human renal cells lacking LRRK2 phenocopy those seen in the kidneys of Lrrk2 knockout mice, characterized by accumulation of intracellular waste vesicles and fragmentation of the Golgi apparatus. This phenotype can be recapitulated by knockdown of N-ethylmaleimide-sensitive factor, which physically associates with LRRK2 in renal cells. Deficiency in either protein leads to a defect in trans-Golgi to lysosome protein trafficking, which compromises the capacity of lysosomes to degrade endocytic and autophagic cargo. In contrast, neither bulk endocytosis nor autophagic flux are impaired when LRRK2 is acutely knocked down in normal immortalized human kidney (HK2) cells. These data collectively suggest that the primary renal defect caused by LRRK2 deficiency is in protein trafficking between the Golgi apparatus and late endosome/lysosome, which leads to progressive impairments in lysosomal function.
Subject(s)
Endocytosis , Epithelial Cells/enzymology , Golgi Apparatus/enzymology , Kidney Tubules, Proximal/enzymology , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/deficiency , Lysosomes/enzymology , Autophagy , Cell Line , Cell Proliferation , Epithelial Cells/pathology , Gene Knockdown Techniques , Genotype , Golgi Apparatus/pathology , Humans , Kidney Tubules, Proximal/pathology , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/genetics , Lysosomes/pathology , N-Ethylmaleimide-Sensitive Proteins/genetics , N-Ethylmaleimide-Sensitive Proteins/metabolism , Phenotype , Protein Transport , ProteolysisABSTRACT
In eukaryotic cells, the soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) proteins comprise the minimal machinery that triggers fusion of transport vesicles with their target membranes. Comparative studies revealed that genes encoding the components of the SNARE system are highly conserved in yeast, insect, and human genomes. Upon infection of insect cells by the virus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the transcript levels of most SNARE genes initially were upregulated. We found that overexpression of dominant-negative (DN) forms of NSF or knockdown of the expression of NSF, the key regulator of the SNARE system, significantly affected infectious AcMNPV production. In cells expressing DN NSF, entering virions were trapped in the cytoplasm or transported to the nucleus with low efficiency. The presence of DN NSF also moderately reduced trafficking of the viral envelope glycoprotein GP64 to the plasma membrane but dramatically inhibited production of infectious budded virions (BV). Transmission electron microscopy analysis of infections in cells expressing DN NSF revealed that progeny nucleocapsids were retained in a perinuclear space surrounded by inner and outer nuclear membranes. Several baculovirus conserved (core) proteins (Ac76, Ac78, GP41, Ac93, and Ac103) that are important for infectious budded virion production were found to associate with NSF, and NSF was detected within the assembled BV. Together, these data indicate that the cellular SNARE system is involved in AcMNPV infection and that NSF is required for efficient entry and nuclear egress of budded virions of AcMNPV.IMPORTANCE Little is known regarding the complex interplay between cellular factors and baculoviruses during viral entry and egress. Here, we examined the cellular SNARE system, which mediates the fusion of vesicles in healthy cells, and its relation to baculovirus infection. Using a DN approach and RNA interference knockdown, we demonstrated that a general disruption of the SNARE machinery significantly inhibited the production of infectious BV of AcMNPV. The presence of a DN NSF protein resulted in low-efficiency entry of BV and the retention of progeny nucleocapsids in the perinuclear space during egress. Combined with these effects, we also found that several conserved (core) baculovirus proteins closely associate with NSF, and these results suggest their involvement in the egress of BV. Our findings are the first to demonstrate that the SNARE system is required for efficient entry of BV and nuclear egress of progeny nucleocapsids of baculoviruses.
Subject(s)
N-Ethylmaleimide-Sensitive Proteins/genetics , N-Ethylmaleimide-Sensitive Proteins/metabolism , Nucleopolyhedroviruses/physiology , SNARE Proteins/metabolism , Virus Internalization , Virus Release , Active Transport, Cell Nucleus , Animals , Cell Line , Cell Nucleus/virology , Cytoplasm/virology , Humans , Microscopy, Electron, Transmission , N-Ethylmaleimide-Sensitive Proteins/deficiency , Nucleocapsid/metabolism , Nucleopolyhedroviruses/ultrastructure , RNA Interference , SNARE Proteins/genetics , Sf9 Cells , Spodoptera/cytology , Spodoptera/virology , Viral Envelope Proteins/metabolism , Virion , Virus Assembly , Yeasts/metabolismABSTRACT
BACKGROUND: Dementia with Lewy bodies is characterized by transient clinical features, including fluctuating cognition and visual hallucinations, implicating dysfunction of cerebral hub regions, such as the pulvinar nuclei of the thalamus. However, the pulvinar is typically only mildly affected by Lewy body pathology in dementia with Lewy bodies, suggesting additional factors may account for its proposed dysfunction. METHODS: We conducted a comprehensive analysis of postmortem pulvinar tissue using whole-transcriptome RNA sequencing, protein expression analysis, and histological evaluation. RESULTS: We identified 321 transcripts as significantly different between dementia with Lewy bodies cases and neurologically normal controls, with gene ontology pathway analysis suggesting the enrichment of transcripts related to synapses and positive regulation of immune functioning. At the protein level, proteins related to synaptic efficiency were decreased, and general synaptic markers remained intact. Analysis of glial subpopulations revealed astrogliosis without activated microglia, which was associated with synaptic changes but not neurodegenerative pathology. DISCUSSION: These results indicate that the pulvinar, a region with relatively low Lewy body pathological burden, manifests changes at the molecular level that differ from previous reports in a more severely affected region. We speculate that these alterations result from neurodegenerative changes in regions connected to the pulvinar and likely contribute to a variety of cognitive changes resulting from decreased cortical synchrony in dementia with Lewy bodies. © 2018 International Parkinson and Movement Disorder Society.
Subject(s)
Gene Expression/physiology , Lewy Body Disease/pathology , Lewy Body Disease/physiopathology , Pulvinar/metabolism , Pulvinar/pathology , Acetyltransferases/genetics , Acetyltransferases/metabolism , Chitinase-3-Like Protein 1/genetics , Chitinase-3-Like Protein 1/metabolism , Cohort Studies , Diagnosis , Dynamins/genetics , Dynamins/metabolism , Female , Gene Ontology , Glial Fibrillary Acidic Protein/genetics , Glial Fibrillary Acidic Protein/metabolism , Glutamate Decarboxylase/genetics , Glutamate Decarboxylase/metabolism , HSP70 Heat-Shock Proteins/genetics , HSP70 Heat-Shock Proteins/metabolism , Hallucinations/etiology , Humans , Male , N-Ethylmaleimide-Sensitive Proteins/genetics , N-Ethylmaleimide-Sensitive Proteins/metabolism , RNA, Messenger/metabolism , Synaptophysin/genetics , Synaptophysin/metabolism , Syntaxin 1/genetics , Syntaxin 1/metabolism , alpha-Synuclein/genetics , alpha-Synuclein/metabolismABSTRACT
Dopamine receptors regulate exocytosis via protein-protein interactions (PPIs) as well as via adenylyl cyclase transduction pathways. Evidence has been obtained for PPIs in inner ear hair cells coupling D1A to soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE)-related proteins snapin, otoferlin, N-ethylmaleimide-sensitive factor (NSF), and adaptor-related protein complex 2, mu 1 (AP2mu1), dependent on [Ca2+] and phosphorylation. Specifically, the carboxy terminus of dopamine D1A was found to directly bind t-SNARE-associated protein snapin in teleost and mammalian hair cell models by yeast two-hybrid (Y2H) and pull-down assays, and snapin directly interacts with hair cell calcium-sensor otoferlin. Surface plasmon resonance (SPR) analysis, competitive pull-downs, and co-immunoprecipitation indicated that these interactions were promoted by Ca2+ and occur together. D1A was also found to separately interact with NSF, but with an inverse dependence on Ca2+ Evidence was obtained, for the first time, that otoferlin domains C2A, C2B, C2D, and C2F interact with NSF and AP2mu1, whereas C2C or C2E do not bind to either protein, representing binding characteristics consistent with respective inclusion or omission in individual C2 domains of the tyrosine motif YXXΦ. In competitive pull-down assays, as predicted by KD values from SPR (+Ca2+), C2F pulled down primarily NSF as opposed to AP2mu1. Phosphorylation of AP2mu1 gave rise to a reversal: an increase in binding by C2F to phosphorylated AP2mu1 was accompanied by a decrease in binding to NSF, consistent with a molecular switch for otoferlin from membrane fusion (NSF) to endocytosis (AP2mu1). An increase in phosphorylated AP2mu1 at the base of the cochlear inner hair cell was the observed response elicited by a dopamine D1A agonist, as predicted.
Subject(s)
Calcium Signaling/physiology , Fish Proteins , Hair Cells, Vestibular/metabolism , N-Ethylmaleimide-Sensitive Proteins , Receptors, Dopamine D1 , Synapses , Trout , Animals , Fish Proteins/genetics , Fish Proteins/metabolism , Mice , N-Ethylmaleimide-Sensitive Proteins/genetics , N-Ethylmaleimide-Sensitive Proteins/metabolism , Rats , Receptors, Dopamine D1/genetics , Receptors, Dopamine D1/metabolism , SNARE Proteins/genetics , SNARE Proteins/metabolism , Synapses/genetics , Synapses/metabolism , Trout/genetics , Trout/metabolismABSTRACT
The physical act of eating or feeding involves the coordinated action of several organs like eyes and jaws, and associated neural networks. Moreover, the activity of the neural networks controlling jaw movements (branchiomotor circuits) is regulated by the visual, olfactory, gustatory and hypothalamic systems, which are largely well characterized at the physiological level. By contrast, the behavioral output of the branchiomotor circuits and the functional consequences of disruption of these circuits by abnormal neural development are poorly understood. To begin to address these questions, we sought to evaluate the feeding ability of zebrafish larvae, a direct output of the branchiomotor circuits, and developed a qualitative assay for measuring food intake in zebrafish larvae at 7 days post-fertilization. We validated the assay by examining the effects of ablating the branchiomotor neurons. Metronidazole-mediated ablation of nitroreductase-expressing branchiomotor neurons resulted in a predictable reduction in food intake without significantly affecting swimming ability, indicating that the assay is robust. Laser-mediated ablation of trigeminal motor neurons resulted in a significant decrease in food intake, indicating that the assay is sensitive. Importantly, in larvae of a genetic mutant with severe loss of branchiomotor neurons, food intake was abolished. These studies establish a foundation for dissecting the neural circuits driving a motor behavior essential for survival.
Subject(s)
Eating/physiology , Larva/physiology , Motor Neurons/physiology , Movement/physiology , Analysis of Variance , Animals , Animals, Genetically Modified , Eating/genetics , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HSP70 Heat-Shock Proteins/genetics , HSP70 Heat-Shock Proteins/metabolism , LIM-Homeodomain Proteins/genetics , LIM-Homeodomain Proteins/metabolism , Larva/cytology , Laser Therapy/methods , Locomotion/genetics , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , N-Ethylmaleimide-Sensitive Proteins/genetics , N-Ethylmaleimide-Sensitive Proteins/metabolism , Nerve Net/physiology , Transcription Factors/genetics , Transcription Factors/metabolism , Trigeminal Ganglion/cytology , Zebrafish , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolism , Red Fluorescent ProteinABSTRACT
Dysbindin is a schizophrenia susceptibility factor and subunit of the biogenesis of lysosome-related organelles complex 1 (BLOC-1) required for lysosome-related organelle biogenesis, and in neurons, synaptic vesicle assembly, neurotransmission, and plasticity. Protein networks, or interactomes, downstream of dysbindin/BLOC-1 remain partially explored despite their potential to illuminate neurodevelopmental disorder mechanisms. Here, we conducted a proteome-wide search for polypeptides whose cellular content is sensitive to dysbindin/BLOC-1 loss of function. We identified components of the vesicle fusion machinery as factors downregulated in dysbindin/BLOC-1 deficiency in neuroectodermal cells and iPSC-derived human neurons, among them the N-ethylmaleimide-sensitive factor (NSF). Human dysbindin/BLOC-1 coprecipitates with NSF and vice versa, and both proteins colocalized in a Drosophila model synapse. To test the hypothesis that NSF and dysbindin/BLOC-1 participate in a pathway-regulating synaptic function, we examined the role for NSF in dysbindin/BLOC-1-dependent synaptic homeostatic plasticity in Drosophila. As previously described, we found that mutations in dysbindin precluded homeostatic synaptic plasticity elicited by acute blockage of postsynaptic receptors. This dysbindin mutant phenotype is fully rescued by presynaptic expression of either dysbindin or Drosophila NSF. However, neither reduction of NSF alone or in combination with dysbindin haploinsufficiency impaired homeostatic synaptic plasticity. Our results demonstrate that dysbindin/BLOC-1 expression defects result in altered cellular content of proteins of the vesicle fusion apparatus and therefore influence synaptic plasticity.
Subject(s)
Drosophila Proteins/metabolism , Dystrophin-Associated Proteins/metabolism , Gene Expression Regulation/physiology , N-Ethylmaleimide-Sensitive Proteins/metabolism , Neuronal Plasticity/physiology , Synapses/physiology , Animals , Animals, Genetically Modified , Cell Line, Tumor , Drosophila , Drosophila Proteins/genetics , Dysbindin , Dystrophin-Associated Proteins/genetics , Gene Expression Regulation/genetics , Humans , Melanoma/pathology , N-Ethylmaleimide-Sensitive Proteins/genetics , Nerve Net/physiology , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neuroblastoma/pathology , Neuromuscular Junction/genetics , Neuromuscular Junction/metabolism , Neuronal Plasticity/drug effects , Neuronal Plasticity/genetics , SNARE Proteins/metabolism , Synapses/genetics , Synaptic Vesicles/genetics , Synaptic Vesicles/metabolismABSTRACT
Interactions between GluR2 and N-ethylmaleimide-sensitive factor (NSF) mediate AMPA receptors trafficking. This might be linked with molecular mechanisms related with memory formation. Previous research has shown basolateral amygdala (BLA) dependent activity changes in the perirhinal cortex (PRh) during the formation of taste memory. In the present experiments we investigate both the behavioral performance and the expression profile of NSF and GluR2 genes in several brain areas, including PRh, BLA, and hippocampus. Twenty-one naïve male Wistar rats were exposed to a saccharin solution (0.4%) during the first (novel), the second (Familiar I), and the sixth presentation (Familiar II). Total RNA was extracted and gene expression was measured by quantitative PCR (qPCR) using TaqMan gene expression assays. In addition the expression of the synaptic plasticity related immediate early genes, Homer 1 and Narp, was also assessed. We have found increased expression of NSF gene in BLA and PRh in Group Familiar I in comparison with Familiar II. No changes in the expression of GluR2, Homer 1, and Narp genes were found. The results suggest the relevance of a potential network in the temporal lobe for taste recognition memory and open new possibilities for understanding the molecular mechanisms mediating the impact of sensory experience on brain circuit function.