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1.
PLoS One ; 15(1): e0227855, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31999741

RESUMO

The Src substrate Tks5 helps scaffold matrix-remodeling invadopodia in invasive cancer cells. Focus was directed here on how the five SH3 domains of Tks5 impact that activity. Mutations designed to inhibit protein-protein interactions were created in the individual SH3 domains of Tks5, and the constructs were introduced into the LNCaP prostate carcinoma cell line, a model system with intrinsically low Tks5 expression and which our lab had previously showed the dependence of Src-dependent Tks5 phosphorylation on invadopodia development. In LNCaP cells, acute increases in wild-type Tks5 led to increased gelatin matrix degradation. A similar result was observed when Tks5 was mutated in its 4th or 5th SH3 domains. This was in contrast to the 1st, 2nd, and 3rd SH3 domain mutations of Tks5 where each had a remarkable accentuating effect on gelatin degradation. Conversely, in the invadopodia-competent Src-3T3 model system, mutations in any one of the first three SH3 domains had a dominant negative effect that largely eliminated the presence of invadopodia, inhibited gelatin degradation activity, and redistributed both Src, cortactin, and Tks5 to what are likely endosomal compartments. A hypothesis involving Tks5 conformational states and the regulation of endosomal trafficking is presented as an explanation for these seemingly disparate results.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/genética , Carcinoma/genética , Neoplasias da Próstata/genética , Quinases da Família src/genética , Proteínas Adaptadoras de Transporte Vesicular/química , Carcinoma/metabolismo , Carcinoma/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Cortactina/genética , Fibroblastos/metabolismo , Fibroblastos/patologia , Gelatina/genética , Gelatina/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Masculino , Mutação/genética , Fosforilação , Podossomos/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Domínios e Motivos de Interação entre Proteínas/genética , Domínios de Homologia de src/genética
2.
Elife ; 82019 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-31464684

RESUMO

Endocytosis of transmembrane proteins is orchestrated by the AP2 clathrin adaptor complex. AP2 dwells in a closed, inactive state in the cytosol, but adopts an open, active conformation on the plasma membrane. Membrane-activated complexes are also phosphorylated, but the significance of this mark is debated. We recently proposed that NECAP negatively regulates AP2 by binding open and phosphorylated complexes (Beacham et al., 2018). Here, we report high-resolution cryo-EM structures of NECAP bound to phosphorylated AP2. The site of AP2 phosphorylation is directly coordinated by residues of the NECAP PHear domain that are predicted from genetic screens in C. elegans. Using membrane mimetics to generate conformationally open AP2, we find that a second domain of NECAP binds these complexes and cryo-EM reveals both domains of NECAP engaging closed, inactive AP2. Assays in vitro and in vivo confirm these domains cooperate to inactivate AP2. We propose that phosphorylation marks adaptors for inactivation.


Assuntos
Complexo 2 de Proteínas Adaptadoras/química , Complexo 2 de Proteínas Adaptadoras/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/química , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Processamento de Proteína Pós-Traducional , Microscopia Crioeletrônica , Fosforilação , Ligação Proteica
3.
Nano Lett ; 19(7): 4770-4778, 2019 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-31241342

RESUMO

Nanoporous surfaces are used in many applications in intracellular sensing and drug delivery. However, the effects of such nanostructures on cell membrane properties are still far from understood. Here, we use coarse-grained molecular dynamics simulations to show that nanoporous substrates can stimulate membrane-curvature effects and that this curvature-sensing effect is much more sensitive than previously thought. We define a series of design parameters for inducing a nanoscale membrane curvature and show that nanopore taper plays a key role in membrane deformation, elucidating a previously unexplored fabrication parameter applicable to many nanostructured biomaterials. We report significant changes in the membrane area per lipid and thickness at regions of curvature. Finally, we demonstrate that regions of the nanopore-induced membrane curvature act as local hotspots for an increased bioactivity. We show spontaneous binding and localization of the epsin N-terminal homology (ENTH) domain to the regions of curvature. Understanding this interplay between the membrane curvature and nanoporosity at the biointerface helps both explain recent biological results and suggests a pathway for developing the next generation of cell-active substrates.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/química , Membrana Celular/química , Bicamadas Lipídicas/química , Simulação de Dinâmica Molecular , Nanoporos , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Membrana Celular/metabolismo , Bicamadas Lipídicas/metabolismo
4.
Structure ; 27(7): 1103-1113.e3, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31104815

RESUMO

Sortilin is a multifunctional neuronal receptor involved in sorting of neurotrophic factors and apoptosis signaling. So far, structural characterization of sortilin and its endogenous ligands has been limited to crystallographic studies of sortilin in complex with the neuropeptide neurotensin. Here, we use hydrogen/deuterium exchange mass spectrometry to investigate the conformational response of sortilin to binding biological ligands including the peptides neurotensin and the sortilin propeptide and the proteins progranulin and pro-nerve growth factor-ß. The results show that the ligands use two binding sites inside the cavity of the ß-propeller of sortilin. However, ligands have distinct differences in their conformational impact on the receptor. Interestingly, the protein ligands induce conformational stabilization in a remote membrane-proximal domain, hinting at an unknown conformational link between the ligand binding region and this membrane-proximal region of sortilin. Our findings improve our structural understanding of sortilin and how it mediates diverse ligand-dependent functions important in neurobiology.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/química , Fator de Crescimento Neural/química , Neurotensina/química , Progranulinas/química , Precursores de Proteínas/química , Proteínas Recombinantes de Fusão/química , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Glutationa Transferase/química , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Células HEK293 , Humanos , Espectrometria de Massa com Troca Hidrogênio-Deutério , Ligantes , Modelos Moleculares , Fator de Crescimento Neural/genética , Fator de Crescimento Neural/metabolismo , Neurotensina/genética , Neurotensina/metabolismo , Progranulinas/genética , Progranulinas/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
5.
Fish Shellfish Immunol ; 91: 108-121, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31091461

RESUMO

As an adaptor in Toll-like receptor (TLR) signaling pathway, Toll/interleukin-1 receptor (TIR) domain containing adaptor inducing interferon-ß (TRIF) mediates downstream signaling cascades and plays important roles in host innate immune responses. In the present study, a TRIF ortholog named Lc-TRIF was identified in large yellow croaker (Larimichthys crocea). Sequence comparison analysis revealed that Lc-TRIF has a conserved TIR domain but without TRAF6 binding motif. The genome structure of Lc-TRIF is conserved, with two exons and one intron. Syntenic comparison showed that the loci of fish TRIF was different from that in mammals or birds, and TRAM was absent in the genomes of fish, amphibians, and birds, but present in mammals and reptiles. Expression analysis revealed that Lc-TRIF was broadly expressed in examined organs/tissues, with the highest expression level in gill and weakest in brain, and could be up-regulated under poly I:C, LPS, PGN, and Pseudomonas plecoglossicida stimulation. Fluorescence microscopy results showed that Lc-TRIF exhibited a global localization throughout the entire cell including the nucleus in HEK 293T cells. Additionally, luciferase assays demonstrated that Lc-TRIF expression could significantly induce NF-κB, type I IFN, IRF3 as well as IRF7 promoter activation. These results collectively indicated that Lc-TRIF was function in host antiviral and antibacterial responses via NF-κB and IRF3/7 related signaling pathway.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/imunologia , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Perciformes/genética , Perciformes/imunologia , Proteínas Adaptadoras de Transporte Vesicular/química , Sequência de Aminoácidos , Animais , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Filogenia , Alinhamento de Sequência/veterinária
6.
Comput Biol Chem ; 80: 168-176, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30965174

RESUMO

The alarm is rang for friendly fire; Saccharomyces cerevisiae (S. cerevisiae) newfound as a fungal pathogen with an individual feature. S. cerevisiae has food safety and is not capable of producing infection but, when the host defenses are weakened, there is room for opportunistic S. cerevisiae strains to cause a health issues. Fungal diseases are challenging to treat because, unlike bacteria, the fungal are eukaryotes. Antibiotics only target prokaryotic cells, whereas compounds that kill fungi also harm the mammalian host. Small differences between mammalian and fungal cells regarding genes and proteins sequence and function make finding a drug target more challenging. Recently, Chitin synthase has been considered as a promising target for antifungal drug development as it is absent in mammals. In S. cerevisiae, CHS3, a class IV chitin synthase, produces 90% of the chitin and essential for cell growth. CHS3 from the trans-Golgi network to the plasma membrane requires assembly of the exomer complex (including proteins cargo such as CHS5, CHS6, Bach1, and Arf1). In this work, we performed SELEX (Systematic Evolution of Ligands by EXponential enrichment) as high throughput virtual screening of the RCSB data bank to find an aptamer as potential inhibit of the class IV chitin synthase of S. cerevisiae. Among all the candidates, G-rich VEGF (GVEGF) aptamer (PDB code: 2M53) containing locked sugar parts was observed as potential inhibitor of the assembly of CHS5-CHS6 exomer complex a subsequently block the chitin biosynthesis pathway as an effective anti-fungal. It was suggested from the simulation that an assembly of exomer core should begin CHS5-CHS6, not from CHS5-Bach1. It is notable that secondary structures of CHS6 and Bach1 was observed very similar, but they have only 25% identity at the amino acid sequence that exhibited different features in exomer assembly.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Aptâmeros de Nucleotídeos/metabolismo , Quitina Sintase/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Multimerização Proteica/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/metabolismo , Fator A de Crescimento do Endotélio Vascular/química , Proteínas Adaptadoras de Transporte Vesicular/química , Sequência de Aminoácidos , Antifúngicos/metabolismo , Aptâmeros de Nucleotídeos/genética , Sítios de Ligação , Quitina Sintase/química , Quadruplex G , Peptídeos e Proteínas de Sinalização Intracelular/química , Proteínas de Membrana/química , Simulação de Acoplamento Molecular , Ligação Proteica , Técnica de Seleção de Aptâmeros , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/química , Alinhamento de Sequência
7.
Structure ; 27(6): 977-987.e5, 2019 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-31031201

RESUMO

The scaffolding protein intersectin 1 plays important roles in clathrin-mediated endocytosis and in the replenishment of release-ready synaptic vesicles (SV). Two splice variants of intersectin's SH3A domain are expressed in the brain, and association of the neuron-specific variant with synapsin I has been shown to enable sustained neurotransmission and to be regulated by an adjacent C-terminal motif. Here, we demonstrate that the ubiquitously expressed short SH3A variant of intersectin 1 interacts with an N-terminal intramolecular sequence that operates synergistically with the C-terminal motif. NMR spectroscopic investigations show that the five-amino acid insertion into the ß strand 2 of the neuronal SH3A variant introduces conformational plasticity incompatible with binding of the N-terminal sequence. The difference in the autoregulatory mechanism of the domain's variants differentially affects its synaptic binding partners, thereby establishing alternative splicing in conjunction with autoinhibitory motif variation as a mechanism to regulate protein interaction networks.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/genética , Processamento Alternativo , Endocitose/genética , Éxons/genética , Proteínas Adaptadoras de Transporte Vesicular/química , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Animais , Células Cultivadas , Regulação da Expressão Gênica , Camundongos Knockout , Neurônios/citologia , Neurônios/metabolismo , Ligação Proteica , Conformação Proteica , Mapas de Interação de Proteínas/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transmissão Sináptica , Domínios de Homologia de src
8.
Acta Crystallogr D Struct Biol ; 75(Pt 2): 234-241, 2019 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-30821711

RESUMO

Processing X-ray free-electron laser (XFEL) diffraction images poses challenges, as an XFEL pulse is powerful enough to destroy or damage the diffracting volume and thereby yields only one diffraction image per volume. Moreover, the crystal is stationary during the femtosecond pulse, so reflections are generally only partially recorded. Therefore, each XFEL diffraction image must be scaled individually and, ideally, corrected for partiality prior to merging. An additional complication may arise owing to indexing ambiguities when the symmetry of the Bravais lattice is higher than that of the space group, or when the unit-cell dimensions are similar to each other. Here, an automated method is presented that diagnoses these indexing ambiguities based on the Brehm-Diederichs algorithm [Brehm & Diederichs (2014), Acta Cryst. D70, 101-109] and produces a consistent indexing choice for the large majority of diffraction images. This method was applied to an XFEL diffraction data set measured from crystals of the neuronal SNARE-complexin-1-synaptotagmin-1 complex. After correcting the indexing ambiguities, substantial improvements were observed in the merging statistics and the atomic model refinement R values. This method should be a useful addition to the arsenal of tools for the processing of XFEL diffraction data sets.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/química , Coleta de Dados/métodos , Proteínas do Tecido Nervoso/química , Proteínas SNARE/química , Sinaptotagmina I/química , Difração de Raios X/métodos , Algoritmos , Animais , Elétrons , Lasers , Modelos Moleculares , Ratos , Síncrotrons
9.
Elife ; 82019 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-30657450

RESUMO

Neurotransmitter release requires formation of trans-SNARE complexes between the synaptic vesicle and plasma membranes, which likely underlies synaptic vesicle priming to a release-ready state. It is unknown whether Munc18-1, Munc13-1, complexin-1 and synaptotagmin-1 are important for priming because they mediate trans-SNARE complex assembly and/or because they prevent trans-SNARE complex disassembly by NSF-αSNAP, which can lead to de-priming. Here we show that trans-SNARE complex formation in the presence of NSF-αSNAP requires both Munc18-1 and Munc13-1, as proposed previously, and is facilitated by synaptotagmin-1. Our data also show that Munc18-1, Munc13-1, complexin-1 and likely synaptotagmin-1 contribute to maintaining assembled trans-SNARE complexes in the presence of NSF-αSNAP. We propose a model whereby Munc18-1 and Munc13-1 are critical not only for mediating vesicle priming but also for precluding de-priming by preventing trans-SNARE complex disassembly; in this model, complexin-1 also impairs de-priming, while synaptotagmin-1 may assist in priming and hinder de-priming.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/química , Proteínas Munc18/química , Proteínas Sensíveis a N-Etilmaleimida/química , Proteínas do Tecido Nervoso/química , Proteína 25 Associada a Sinaptossoma/química , Sinaptotagminas/química , Animais , Células CHO , Cálcio/química , Cricetinae , Cricetulus , Microscopia Crioeletrônica , Citoplasma/química , Transferência Ressonante de Energia de Fluorescência , Cinética , Mutação , Proteínas R-SNARE/química , Ratos , Sintaxina 1/química
10.
Sci Rep ; 9(1): 54, 2019 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-30631134

RESUMO

Sterile alpha motif (SAM) domains are protein interaction modules that are involved in a diverse range of biological functions such as transcriptional and translational regulation, cellular signalling, and regulation of developmental processes. SH3 domain-containing protein expressed in lymphocytes 1 (SLy1) is involved in immune regulation and contains a SAM domain of unknown function. In this report, the structure of the SLy1 SAM domain was solved and revealed that this SAM domain forms a symmetric homodimer through a novel interface. The interface consists primarily of the two long C-terminal helices, α5 and α5', of the domains packing against each other. The dimerization is characterized by a dissociation constant in the lower micromolar range. A SLy1 SAM domain construct with an extended N-terminus containing five additional amino acids of the SLy1 sequence further increases the stability of the homodimer, making the SLy1 SAM dimer two orders of magnitude more stable than previously studied SAM homodimers, suggesting that the SLy1 SAM dimerization is of functional significance. The SLy1 SAM homodimer contains an exposed mid-loop surface on each monomer, which may provide a scaffold for mediating interactions with other SAM domain-containing proteins via a typical mid-loop-end-helix interface.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/química , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Multimerização Proteica , Motivo Estéril alfa , Conformação Proteica
11.
Comput Biol Chem ; 78: 282-289, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30602138

RESUMO

Brain derived neurotrophic factor (BDNF) has a critical role in the neurogenesis, differentiation, survival of the neurons, regulation of the appetite, and energy homeostasis. Two key proteins, Huntingtin associated protein-1 (HAP1) and sortilin1, regulate the intracellular trafficking and stabilization of the precursor proBDNF through interaction with its prodomain region and mark it for secretion. Evidence suggests that the most frequent single nucleotide polymorphism (SNP) of BDNF gene (rs6265) has been associated with different psychiatric, neurodegenerative and eating disorders. In this study, structural bioinformatics and molecular dynamics (MD) simulations were applied, in order to get precise insights into the impact of Val66Met polymorphism on the proBDNF structure and its interaction with HAP1 and Sortilin1. Homology modeling, structure validation, refinement and also protein-protein docking were performed using appropriate servers. The stability, the fluctuations and the compactness of protein complexes were measured by MD simulation parameters including root mean square deviation (RMSD), root mean square fluctuation (RMSF) and Radius of gyration (Rg), respectively. The mutant proBDNF complexes with HAP1 and Sortilin1 revealed higher RMSD and RMSF values and also variable Rg over time compared with wild-type proBDNF. These computational results indicated that, wild-type proBDNF possessed more stable structure in binding with HAP1 and Sortilin1 compared with its mutant form. Therefore, Val66Met SNP could be deleterious due to making structural changes. It may cause a decrease in proBDNF secretion, which could possibly lead to different psychiatric, neurodegenerative and eating disorders. Further experimental lab studies are required for a more accurate conclusion.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/química , Fator Neurotrófico Derivado do Encéfalo/química , Fator Neurotrófico Derivado do Encéfalo/genética , Proteínas do Tecido Nervoso/química , Polimorfismo Genético/genética , Humanos , Modelos Moleculares , Conformação Proteica
12.
Methods Mol Biol ; 1860: 3-13, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30317495

RESUMO

Molecular dynamics (MD) simulations enable in silico investigations of the dynamic behavior of proteins and protein complexes. Here, we describe MD simulations of the SNARE complex and its interactions with the neuronal protein complexin. Complexin is an effector of neuronal secretion that inhibits spontaneous fusion and is thought to clamp the fusion process via the interactions with the SNARE complex. We describe MD simulations of the SNARE complex alone and bound to complexin. The MD simulations under external forces imitating the repulsion between lipid bilayers enabled us to investigate unraveling and assembly of the SNARE complex.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Modelos Biológicos , Simulação de Dinâmica Molecular , Proteínas SNARE/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/química , Cristalografia por Raios X , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Bicamadas Lipídicas/metabolismo , Ligação Proteica , Proteínas SNARE/química , Homologia de Sequência de Aminoácidos
13.
J Cell Biol ; 218(2): 664-682, 2019 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-30504247

RESUMO

Cellular membranes are continuously remodeled. The crescent-shaped bin-amphiphysin-rvs (BAR) domains remodel membranes in multiple cellular pathways. Based on studies of isolated BAR domains in vitro, the current paradigm is that BAR domain-containing proteins polymerize into cylindrical scaffolds that stabilize lipid tubules. But in nature, proteins that contain BAR domains often also contain large intrinsically disordered regions. Using in vitro and live cell assays, here we show that full-length BAR domain-containing proteins, rather than stabilizing membrane tubules, are instead surprisingly potent drivers of membrane fission. Specifically, when BAR scaffolds assemble at membrane surfaces, their bulky disordered domains become crowded, generating steric pressure that destabilizes lipid tubules. More broadly, we observe this behavior with BAR domains that have a range of curvatures. These data suggest that the ability to concentrate disordered domains is a key driver of membrane remodeling and fission by BAR domain-containing proteins.


Assuntos
Membrana Celular/metabolismo , Proteínas Intrinsicamente Desordenadas/metabolismo , Bicamadas Lipídicas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/química , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Linhagem Celular , Membrana Celular/química , Humanos , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Bicamadas Lipídicas/química , Modelos Moleculares , Proteínas Monoméricas de Montagem de Clatrina/química , Proteínas Monoméricas de Montagem de Clatrina/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Domínios Proteicos , Ratos , Relação Estrutura-Atividade
14.
Adv Exp Med Biol ; 1111: 55-76, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-29774507

RESUMO

The AP180 N-terminal homology (ANTH) and Epsin N-terminal homology (ENTH) domains are crucially involved in membrane budding processes. All the ANTH/ENTH-containing proteins share the phosphoinositide-binding activity and can interact with clathrin or its related proteins via multiple binding motifs. Their function also include promotion of clathrin assembly, induction of membrane curvature, and recruitment of various effector proteins, such as those involved in membrane fission. Furthermore, they play a role in the sorting of specific cargo proteins, thereby enabling the cargos to be accurately transported and function at their appropriate locations. As the structural bases underlying these functions are clarified, contrary to their apparent similarity, the mechanisms by which these proteins recognize lipids and proteins have unexpectedly been found to differ from each other. In addition, studies using knockout mice have suggested that their physiological roles may be more complicated than merely supporting membrane budding processes. In this chapter, we review the current knowledge on the biochemical features of ANTH/ENTH domains, their functions predicted from the phenotypes of animals deficient in these domain-containing proteins, and recent findings on the structural basis enabling specific recognition of their ligands. We also discuss the association of these domains with human diseases. Here we focus on CALM, a protein containing an ANTH domain, which is implicated in the pathogenesis of blood cancers and Alzheimer disease, and discuss how alteration of CALM function is involved in these diseases.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/química , Doença de Alzheimer , Neoplasias Hematológicas , Proteínas Monoméricas de Montagem de Clatrina/química , Domínios Proteicos , Animais , Membrana Celular/metabolismo , Clatrina/química , Clatrina/metabolismo , Humanos , Transporte Proteico
15.
Sci Rep ; 8(1): 16499, 2018 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-30405210

RESUMO

The current study was conducted to assess the effects of microinjection of different dosages of guide RNA (gRNA)/Cas9 protein on the mutation rate, embryo survival, embryonic development, hatchability and early fry survival in channel catfish, Ictalurus punctatus. Guide RNAs targeting two of the channel catfish immune-related genes, toll/interleukin 1 receptor domain-containing adapter molecule (TICAM 1) and rhamnose binding lectin (RBL) genes, were designed and prepared. Three dosages of gRNA/Cas9 protein (low, 2.5 ng gRNA/7.5 ng Cas9, medium, 5 ng gRNA/15 ng Cas9 and high, 7.5 ng gRNA/22.5 ng Cas9) were microinjected into the yolk of one-cell embryos. Mutation rate increased with higher dosages (p < 0.05). Higher dosages increased the mutation frequency in individual embryos where biallelic mutations were detected. For both genes, microinjection procedures increased the embryo mortality (p < 0.05). Increasing the dosage of gRNA/Cas9 protein increased the embryo mortality and reduced the hatching percent (p < 0.05). Embryonic development was delayed when gRNAs targeting RBL gene were injected. Means of fry survival time were similar for different dosages (p > 0.05). The current results lay the foundations for designing gene editing experiments in channel catfish and can be used as a guide for other fish species.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/genética , Sistemas CRISPR-Cas , Proteínas Cromossômicas não Histona/genética , Desenvolvimento Embrionário/genética , Ictaluridae/fisiologia , Taxa de Mutação , Mutação , Proteínas Adaptadoras de Transporte Vesicular/química , Animais , Sequência de Bases , Proteínas Cromossômicas não Histona/química , Anormalidades Congênitas/diagnóstico , Anormalidades Congênitas/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Mortalidade , Fases de Leitura Aberta , Fenótipo , Reprodução/genética
16.
Methods Enzymol ; 611: 227-286, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30471689

RESUMO

Complexins play a critical role in the regulation of neurotransmission by regulating SNARE-mediated exocytosis of synaptic vesicles. Complexins can exert either a facilitatory or an inhibitory effect on neurotransmitter release, depending on the context, and different complexin domains contribute differently to these opposing roles. Structural characterization of the central helix domain of complexin bound to the assembled SNARE bundle provided key insights into the functional mechanism of this domain of complexin, which is critical for both complexin activities, but many questions remain, particularly regarding the roles and mechanisms of other complexin domains. Recent progress has clarified the structural properties of these additional domains, and has led to various proposals regarding how they contribute to complexin function. This chapter describes spectroscopic approaches used in our laboratory and others, primarily involving circular dichroism and solution-state NMR spectroscopy, to characterize structure within complexins when isolated or when bound to interaction partners. The ability to characterize complexin structure enables structure/function studies employing in vitro or in vivo assays of complexin function. More generally, these types of approaches can be used to study the binding of other intrinsically disordered proteins or protein regions to membrane surfaces or for that matter to other large physiological binding partners.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Dicroísmo Circular/métodos , Proteínas Intrinsicamente Desordenadas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas Adaptadoras de Transporte Vesicular/química , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Escherichia coli/genética , Expressão Gênica , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Camundongos , Mutagênese , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Ligação Proteica , Mapeamento de Interação de Proteínas/métodos
17.
FEBS Lett ; 592(15): 2647-2657, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29972886

RESUMO

Sortilin is a multifunctional sorting receptor involved in cytokine production in immune cells. To understand the mechanism of Sortilin-mediated cytokine trafficking, we determined the 2.45-Å structure of the dimerized Sortilin ectodomain (sSortilin or the Vps10-domain) crystallized at acidic pH. Substantial conformational changes upon dimerization lead to the intermolecular hydrophobic interaction between the conserved E455 and F137. Analysis of the electrostatic surface and size-exclusion chromatography revealed that sSortilin dimerization occurs due to an increase in hydrophobic interactions at the neutral dimer interface at acidic pH. The N682-attached N-glycan in the vicinity of the dimer interface implies its involvement in the dimerization. The disruption of Sortilin dimerization by mutations impairs efficient interferon-alpha secretion from cells. These results suggest the functional importance of Sortilin dimerization in cytokine trafficking.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/química , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Ácidos/farmacologia , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Células CHO , Cricetinae , Cricetulus , Cristalografia por Raios X , Humanos , Concentração de Íons de Hidrogênio , Interferon-alfa/metabolismo , Ligantes , Modelos Moleculares , Mutagênese Sítio-Dirigida , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Domínios e Motivos de Interação entre Proteínas/genética , Multimerização Proteica/efeitos dos fármacos , Multimerização Proteica/genética , Estrutura Quaternária de Proteína/genética , Transporte Proteico/genética
18.
Elife ; 72018 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-30044227

RESUMO

ComplexinII (CpxII) inhibits non-synchronized vesicle fusion, but the underlying mechanisms have remained unclear. Here, we provide evidence that the far C-terminal domain (CTD) of CpxII interferes with SNARE assembly, thereby arresting tonic exocytosis. Acute infusion of a CTD-derived peptide into mouse chromaffin cells enhances synchronous release by diminishing premature vesicle fusion like full-length CpxII, indicating a direct, inhibitory function of the CTD that sets the magnitude of the primed vesicle pool. We describe a high degree of structural similarity between the CpxII CTD and the SNAP25-SN1 domain (C-terminal half) and show that the CTD peptide lowers the rate of SDS-resistant SNARE complex formation in vitro. Moreover, corresponding CpxII:SNAP25 chimeras do restore complexin's function and even 'superclamp' tonic secretion. Collectively, these results support a so far unrecognized clamping mechanism wherein the CpxII C-terminus hinders spontaneous SNARE complex assembly, enabling the build-up of a release-ready pool of vesicles for synchronized Ca2+-triggered exocytosis.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/química , Exocitose/genética , Proteínas do Tecido Nervoso/química , Vesículas Sinápticas/química , Proteína 25 Associada a Sinaptossoma/química , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Cálcio/química , Membrana Celular/química , Membrana Celular/genética , Fusão de Membrana/genética , Camundongos , Proteínas do Tecido Nervoso/genética , Ligação Proteica , Domínios Proteicos/genética , Proteínas SNARE/química , Proteínas SNARE/genética , Vesículas Sinápticas/genética , Proteína 25 Associada a Sinaptossoma/genética
19.
FEBS Lett ; 592(13): 2259-2267, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29851086

RESUMO

At present, the role of ubiquitination of cargoes internalized from the plasma membrane is better understood than the consequences of ubiquitination of proteins comprising the endocytic machinery. Here, we show that the E3 ubiquitin ligase AIP4/ITCH contributes to the differential ubiquitination of isoforms of the endocytic scaffold protein intersectin1 (ITSN1). The major isoform ITSN1-s is monoubiquitinated, whereas the minor one, ITSN1-22a undergoes a combination of mono- and oligoubiquitination. The monoubiquitination is required for ITSN1-s stability, whereas the oligoubiquitination of ITSN1-22a causes its proteasomal degradation. This explains the observed low abundance of the minor isoform in cells. Thus, different modes of ubiquitination regulated by AIP4 have opposite effects on ITSN1 isoform stability.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas Repressoras/fisiologia , Ubiquitina-Proteína Ligases/fisiologia , Ubiquitinação , Proteínas Adaptadoras de Transporte Vesicular/química , Sequência de Aminoácidos , Células HEK293 , Humanos , Isoformas de Proteínas/metabolismo , Estabilidade Proteica , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
20.
Cell ; 174(2): 325-337.e14, 2018 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-29887380

RESUMO

Multiple proteins act co-operatively in mammalian clathrin-mediated endocytosis (CME) to generate endocytic vesicles from the plasma membrane. The principles controlling the activation and organization of the actin cytoskeleton during mammalian CME are, however, not fully understood. Here, we show that the protein FCHSD2 is a major activator of actin polymerization during CME. FCHSD2 deletion leads to decreased ligand uptake caused by slowed pit maturation. FCHSD2 is recruited to endocytic pits by the scaffold protein intersectin via an unusual SH3-SH3 interaction. Here, its flat F-BAR domain binds to the planar region of the plasma membrane surrounding the developing pit forming an annulus. When bound to the membrane, FCHSD2 activates actin polymerization by a mechanism that combines oligomerization and recruitment of N-WASP to PI(4,5)P2, thus promoting pit maturation. Our data therefore describe a molecular mechanism for linking spatiotemporally the plasma membrane to a force-generating actin platform guiding endocytic vesicle maturation.


Assuntos
Citoesqueleto de Actina/fisiologia , Proteínas de Transporte/metabolismo , Clatrina/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/química , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Membrana Celular/química , Membrana Celular/metabolismo , Vesículas Revestidas por Clatrina/metabolismo , Endocitose , Células HeLa , Humanos , Lipossomos/química , Lipossomos/metabolismo , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Microscopia de Fluorescência , Modelos Moleculares , Mutagênese Sítio-Dirigida , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteína Neuronal da Síndrome de Wiskott-Aldrich/química , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo , Domínios de Homologia de src
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