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1.
Sci Rep ; 13(1): 14632, 2023 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-37670025

RESUMO

The incidence of Clostridioides difficile infection (CDI) and associated mortality have increased rapidly worldwide in recent years. Therefore, it is critical to develop new therapies for CDI. Here we report on the development of mRNA-LNPs encoding camelid-derived VHH-based neutralizing agents (VNAs) targeting toxins A and/or B of C. difficile. In preclinical models, intravenous administration of the mRNA-LNPs provided serum VNA levels sufficient to confer protection of mice against severe disease progression following toxin challenge. Furthermore, we employed an mRNA-LNP encoded effector antibody, a molecular tool designed to specifically bind an epitopic tag linked to the VNAs, to prolong VNA serum half-life. Co-administration of VNA-encoding mRNA-LNPs and an effector antibody, either provided as recombinant protein or encoded by mRNA-LNP, increased serum VNA half-life in mice and in gnotobiotic piglets. Prolonged serum half-life was associated with higher concentrations of serum VNA and enhanced prophylactic protection of mice in challenge models.


Assuntos
Clostridioides difficile , Anticorpos de Domínio Único , Suínos , Animais , Camundongos , RNA , Meia-Vida , Anticorpos , RNA Mensageiro
2.
ACS Chem Biol ; 17(12): 3435-3449, 2022 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-36459441

RESUMO

While covalent drug discovery is reemerging as an important route to small-molecule therapeutic leads, strategies for the discovery and engineering of protein-based irreversible binding agents remain limited. Here, we describe the use of yeast display in combination with noncanonical amino acids (ncAAs) to identify irreversible variants of single-domain antibodies (sdAbs), also called VHHs and nanobodies, targeting botulinum neurotoxin light chain A (LC/A). Starting from a series of previously described, structurally characterized sdAbs, we evaluated the properties of antibodies substituted with reactive ncAAs capable of forming covalent bonds with nearby groups after UV irradiation (when using 4-azido-l-phenylalanine) or spontaneously (when using O-(2-bromoethyl)-l-tyrosine). Systematic evaluations in yeast display format of more than 40 ncAA-substituted variants revealed numerous clones that retain binding function while gaining either UV-mediated or spontaneous crosslinking capabilities. Solution-based analyses indicate that ncAA-substituted clones exhibit site-dependent target specificity and crosslinking capabilities uniquely conferred by ncAAs. Interestingly, not all ncAA substitution sites resulted in crosslinking events, and our data showed no apparent correlation between detected crosslinking levels and distances between sdAbs and LC/A residues. Our findings highlight the power of yeast display in combination with genetic code expansion in the discovery of binding agents that covalently engage their targets. This platform streamlines the discovery and characterization of antibodies with therapeutically relevant properties that cannot be accessed in the conventional genetic code.


Assuntos
Toxinas Botulínicas , Anticorpos de Domínio Único , Aminoácidos/química , Toxinas Botulínicas/imunologia , Código Genético , Saccharomyces cerevisiae/metabolismo , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/isolamento & purificação
3.
PLoS Pathog ; 18(1): e1010169, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34990480

RESUMO

Botulinum neurotoxins (BoNTs) are among the deadliest of bacterial toxins. BoNT serotype A and B in particular pose the most serious threat to humans because of their high potency and persistence. To date, there is no effective treatment for late post-exposure therapy of botulism patients. Here, we aim to develop single-domain variable heavy-chain (VHH) antibodies targeting the protease domains (also known as the light chain, LC) of BoNT/A and BoNT/B as antidotes for post-intoxication treatments. Using a combination of X-ray crystallography and biochemical assays, we investigated the structures and inhibition mechanisms of a dozen unique VHHs that recognize four and three non-overlapping epitopes on the LC of BoNT/A and BoNT/B, respectively. We show that the VHHs that inhibit the LC activity occupy the extended substrate-recognition exosites or the cleavage pocket of LC/A or LC/B and thus block substrate binding. Notably, we identified several VHHs that recognize highly conserved epitopes across BoNT/A or BoNT/B subtypes, suggesting that these VHHs exhibit broad subtype efficacy. Further, we identify two novel conformations of the full-length LC/A, that could aid future development of inhibitors against BoNT/A. Our studies lay the foundation for structure-based engineering of protein- or peptide-based BoNT inhibitors with enhanced potencies and cross-subtypes properties.


Assuntos
Toxinas Botulínicas/antagonistas & inibidores , Peptídeo Hidrolases/química , Anticorpos de Domínio Único , Animais , Toxinas Botulínicas/química , Inibidores de Proteases/farmacologia , Domínios Proteicos/efeitos dos fármacos , Anticorpos de Domínio Único/farmacologia , Relação Estrutura-Atividade
4.
Cell Rep ; 35(1): 108951, 2021 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-33826884

RESUMO

Somatodendritic dopamine (DA) release from midbrain DA neurons activates D2 autoreceptors on these cells to regulate their activity. However, the source of autoregulatory DA remains controversial. Here, we test the hypothesis that D2 autoreceptors on a given DA neuron in the substantia nigra pars compacta (SNc) are activated primarily by DA released from that same cell, rather than from its neighbors. Voltage-clamp recording allows monitoring of evoked D2-receptor-mediated inhibitory currents (D2ICs) in SNc DA neurons as an index of DA release. Single-cell application of antibodies to Na+ channels via the recording pipette decreases spontaneous activity of recorded neurons and attenuates evoked D2ICs; antibodies to SNAP-25, a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein, also decrease D2IC amplitude. Evoked D2ICs are nearly abolished by the light chain of botulinum neurotoxin A, which cleaves SNAP-25, whereas synaptically activated GABAB-receptor-mediated currents are unaffected. Thus, somatodendritic DA release in the SNc autoinhibits the neuron that releases it.


Assuntos
Dendritos/metabolismo , Dopamina/metabolismo , Substância Negra/metabolismo , Animais , Anticorpos/metabolismo , Estimulação Elétrica , Potenciais Pós-Sinápticos Inibidores , Cinética , Masculino , Camundongos Endogâmicos C57BL , Receptores de Dopamina D2/metabolismo , Análise de Célula Única , Proteína 25 Associada a Sinaptossoma/metabolismo , Canais de Sódio Disparados por Voltagem/metabolismo , Ácido gama-Aminobutírico/metabolismo
5.
Sci Transl Med ; 13(575)2021 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-33408188

RESUMO

Botulism is caused by a potent neurotoxin that blocks neuromuscular transmission, resulting in death by asphyxiation. Currently, the therapeutic options are limited and there is no antidote. Here, we harness the structural and trafficking properties of an atoxic derivative of botulinum neurotoxin (BoNT) to transport a function-blocking single-domain antibody into the neuronal cytosol where it can inhibit BoNT serotype A (BoNT/A1) molecular toxicity. Post-symptomatic treatment relieved toxic signs of botulism and rescued mice, guinea pigs, and nonhuman primates after lethal BoNT/A1 challenge. These data demonstrate that atoxic BoNT derivatives can be harnessed to deliver therapeutic protein moieties to the neuronal cytoplasm where they bind and neutralize intracellular targets in experimental models. The generalizability of this platform might enable delivery of antibodies and other protein-based therapeutics to previously inaccessible intraneuronal targets.


Assuntos
Toxinas Botulínicas Tipo A , Botulismo , Anticorpos de Domínio Único , Animais , Botulismo/tratamento farmacológico , Cobaias , Camundongos , Modelos Animais , Neurotoxinas
6.
Toxins (Basel) ; 12(10)2020 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-32987745

RESUMO

Botulinum neurotoxin (BoNT) serotype E is one of three serotypes that cause the preponderance of human botulism cases and is a Tier 1 Select Agent. BoNT/E is unusual among BoNT serotypes for its rapid onset and short duration of intoxication. Here we report two large panels of unique, unrelated camelid single-domain antibodies (VHHs) that were selected for their ability to bind to BoNT/E holotoxin and/or to the BoNT/E light chain protease domain (LC/E). The 19 VHHs which bind to BoNT/E were characterized for their subunit specificity and 8 VHHs displayed the ability to neutralize BoNT/E intoxication of neurons. Heterodimer antitoxins consisting of two BoNT/E-neutralizing VHHs, including one heterodimer designed using structural information for simultaneous binding, were shown to protect mice against co-administered toxin challenges of up to 500 MIPLD50. The 22 unique VHHs which bind to LC/E were characterized for their binding properties and 9 displayed the ability to inhibit LC/E protease activity. Surprisingly, VHHs selected on plastic-coated LC/E were virtually unable to recognize soluble or captured LC/E while VHHs selected on captured LC/E were poorly able to recognize LC/E coated to a plastic surface. This panel of anti-LC/E VHHs offer insight into BoNT/E function, and some may have value as components of therapeutic antidotes that reverse paralysis following BoNT/E exposures.


Assuntos
Anticorpos Neutralizantes/farmacologia , Toxinas Botulínicas/antagonistas & inibidores , Botulismo/prevenção & controle , Camelídeos Americanos/imunologia , Neurônios/efeitos dos fármacos , Peptídeo Hidrolases , Inibidores de Proteases/farmacologia , Anticorpos de Domínio Único/farmacologia , Animais , Anticorpos Neutralizantes/imunologia , Especificidade de Anticorpos , Sítios de Ligação de Anticorpos , Toxinas Botulínicas/administração & dosagem , Toxinas Botulínicas/imunologia , Botulismo/imunologia , Botulismo/microbiologia , Células Cultivadas , Modelos Animais de Doenças , Imunização , Masculino , Camundongos , Neurônios/metabolismo , Neurônios/patologia , Peptídeo Hidrolases/administração & dosagem , Peptídeo Hidrolases/imunologia , Inibidores de Proteases/imunologia , Ratos , Anticorpos de Domínio Único/imunologia
7.
Sci Rep ; 7: 42923, 2017 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-28220863

RESUMO

Botulinum neurotoxin (BoNT) binds to and internalizes its light chain into presynaptic compartments with exquisite specificity. While the native toxin is extremely lethal, bioengineering of BoNT has the potential to eliminate toxicity without disrupting neuron-specific targeting, thereby creating a molecular vehicle capable of delivering therapeutic cargo into the neuronal cytosol. Building upon previous work, we have developed an atoxic derivative (ad) of BoNT/C1 through rationally designed amino acid substitutions in the metalloprotease domain of wild type (wt) BoNT/C1. To test if BoNT/C1 ad retains neuron-specific targeting without concomitant toxic host responses, we evaluated the localization, activity, and toxicity of BoNT/C1 ad in vitro and in vivo. In neuronal cultures, BoNT/C1 ad light chain is rapidly internalized into presynaptic compartments, but does not cleave SNARE proteins nor impair spontaneous neurotransmitter release. In mice, systemic administration resulted in the specific co-localization of BoNT/C1 ad with diaphragmatic motor nerve terminals. The mouse LD50 of BoNT/C1 ad is 5 mg/kg, with transient neurological symptoms emerging at sub-lethal doses. Given the low toxicity and highly specific neuron-targeting properties of BoNT/C1 ad, these data suggest that BoNT/C1 ad can be useful as a molecular vehicle for drug delivery to the neuronal cytoplasm.


Assuntos
Toxinas Botulínicas/metabolismo , Portadores de Fármacos/química , Sequência de Aminoácidos , Animais , Toxinas Botulínicas/genética , Toxinas Botulínicas/toxicidade , Células Cultivadas , Dimerização , Feminino , Dose Letal Mediana , Camundongos , Microscopia Confocal , Células-Tronco Embrionárias Murinas/citologia , Neurônios/citologia , Neurônios/metabolismo , Transmissão Sináptica/efeitos dos fármacos , Proteína 25 Associada a Sinaptossoma/metabolismo , Sintaxina 1/metabolismo
8.
Sci Rep ; 6: 30429, 2016 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-27484492

RESUMO

Cyto-012 is a recombinant derivative of Botulinum neurotoxin Type A (BoNT/A). It primarily differs from wild type (wt) BoNT/A1 in that it incorporates two amino acid substitutions in the catalytic domain of the light chain (LC) metalloprotease (E224 > A and Y366 > A), designed to provide a safer clinical profile. Cyto-012 is specifically internalized into rat cortical and hippocampal neurons, and cleaves Synaptosomal-Associated Protein 25 (SNAP-25), the substrate of wt BoNT/A, but exhibits slower cleavage kinetics and therefore requires a higher absolute dose to exhibit pharmacologic activity. The pharmacodynamics of Cyto-012 and wt BoNT/A have similar onset and duration of action using the Digital Abduction Assay (DAS). Intramuscular LD50 values for Cyto-012 and wt BoNT/A respectively, were 0.63 ug (95% CI = 0.61, 0.66) and 6.22 pg (95% CI = 5.42, 7.02). ED50 values for Cyto-012 and wt BoNT/A were respectively, 0.030 ug (95% CI = 0.026, 0.034) and 0.592 pg (95% CI = 0.488, 0.696). The safety margin (intramuscular LD50/ED50 ratio) for Cyto-012 was found to be improved 2-fold relative to wt BoNT/A (p < 0.001). The DAS response to Cyto-012 was diminished when a second injection was administered 32 days after the first. These data suggest that the safety margin of BoNT/A can be improved by modulating their activity towards SNAP-25.


Assuntos
Toxinas Botulínicas Tipo A/toxicidade , Fármacos Neuromusculares/toxicidade , Animais , Toxinas Botulínicas Tipo A/farmacocinética , Células Cultivadas , Feminino , Dose Letal Mediana , Camundongos , Músculo Esquelético/metabolismo , Fármacos Neuromusculares/farmacocinética , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/toxicidade
9.
PLoS One ; 9(10): e111238, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25337697

RESUMO

Botulinum neurotoxin type A1 (BoNT/A1) is a potent protein toxin responsible for the potentially fatal human illness botulism. Notwithstanding, the long-lasting flaccid muscle paralysis caused by BoNT/A has led to its utility as a powerful and versatile bio-pharmaceutical. The flaccid paralysis is due to specific cleavage of neuronal SNAREs by BoNTs. However, actions of BoNTs on intoxicated neurons besides the cleavage of SNAREs have not been studied in detail. In this study we investigated by microarray analysis the effects of BoNT/A and a catalytically inactive derivative (BoNT/A ad) on the transcriptome of human induced pluripotent stem cell (hiPSC)-derived neurons at 2 days and 2 weeks after exposure. While there were only minor changes in expression levels at 2 days post exposure, at 2 weeks post exposure 492 genes were differentially expressed more than 2-fold in BoNT/A1-exposed cells when compared to non-exposed populations, and 682 genes were differentially expressed in BoNT/A ad-exposed cells. The vast majority of genes were similarly regulated in BoNT/A1 and BoNT/A ad-exposed neurons, and the few genes differentially regulated between BoNT/A1 and BoNT/A ad-exposed neurons were differentially expressed less than 3.5 fold. These data indicate a similar response of neurons to BoNT/A1 and BoNT/A ad exposure. The most highly regulated genes in cells exposed to either BoNT/A1 or BoNT/A ad are involved in neurite outgrowth and calcium channel sensitization.


Assuntos
Toxinas Botulínicas Tipo A/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Células Cultivadas , Análise por Conglomerados , Perfilação da Expressão Gênica , Humanos , Neurônios/citologia , Reprodutibilidade dos Testes , Fatores de Tempo
10.
PLoS One ; 9(1): e85517, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24465585

RESUMO

We have previously described genetic constructs and expression systems that enable facile production of recombinant derivatives of botulinum neurotoxins (BoNTs) that retain the structural and trafficking properties of wt BoNTs. In this report we describe the properties of one such derivative, BoNT/A ad, which was rendered atoxic by introducing two amino acid mutations to the light chain (LC) of wt BoNT/A, and which is being developed as a molecular vehicle for delivering drugs to the neuronal cytoplasm. The neuronal binding, internalization, and intracellular trafficking of BoNT/A ad in primary hippocampal cultures was evaluated using three complimentary techniques: flow cytometry, immunohistochemistry, and Western blotting. Neuronal binding of BoNT ad was significantly increased when neurons were incubated in depolarizing medium. Flow cytometry demonstrated that BoNT/A ad internalized into neurons but not glia. After 24 hours, the majority of the neuron-bound BoNT/A ad became internalized, as determined by its resistance to pronase E-induced proteolytic degradation of proteins associated with the plasma membrane of intact cells. Significant amounts of the atoxic LC accumulated in a Triton X-100-extractable fraction of the neurons, and persisted as such for at least 11 days with no evidence of degradation. Immunocytochemical analysis demonstrated that the LC of BoNT/A ad was translocated to the neuronal cytoplasm after uptake and was specifically targeted to SNARE proteins. The atoxic LC consistently co-localized with synaptic markers SNAP-25 and VAMP-2, but was rarely co-localized with markers for early or late endosomes. These data demonstrate that BoNT/A ad mimics the trafficking properties of wt BoNT/A, confirming that our platform for designing and expressing BoNT derivatives provides an accessible system for elucidating the molecular details of BoNT trafficking, and can potentially be used to address multiple medical and biodefense needs.


Assuntos
Toxinas Botulínicas Tipo A/administração & dosagem , Toxinas Botulínicas Tipo A/farmacocinética , Endocitose , Neurônios/metabolismo , Animais , Western Blotting , Toxinas Botulínicas Tipo A/genética , Células Cultivadas , Citoplasma/metabolismo , Feminino , Citometria de Fluxo , Hipocampo/citologia , Hipocampo/embriologia , Hipocampo/metabolismo , Microscopia Confocal , Mutação , Neurotoxinas/administração & dosagem , Neurotoxinas/genética , Neurotoxinas/farmacocinética , Ligação Proteica , Transporte Proteico , Ratos , Ratos Sprague-Dawley , Fatores de Tempo , Proteína 2 Associada à Membrana da Vesícula/metabolismo
11.
Biochem Biophys Res Commun ; 405(4): 673-7, 2011 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-21284937

RESUMO

Non-toxic derivatives of botulinum neurotoxin A (BoNT/A) have potential use as neuron-targeting delivery vehicles, and as reagents to study intracellular trafficking. We have designed and expressed an atoxic derivative of BoNT/A (BoNT/A ad) as a full-length 150 kDa molecule consisting of a 50 kDa light chain (LC) and a 100 kDa heavy chain (HC) joined by a disulfide bond and rendered atoxic through the introduction of metalloprotease-inactivating point mutations in the light chain. Studies in neuronal cultures demonstrated that BoNT/A ad cannot cleave synaptosomal-associated protein 25 (SNAP25), the substrate of wt BoNT/A, and that it effectively competes with wt BoNT/A for binding to endogenous neuronal receptors. In vitro and in vivo studies indicate accumulation of BoNT/A ad at the neuromuscular junction of the mouse diaphragm. Immunoprecipitation studies indicate that the LC of BoNT/A ad forms a complex with SNAP25 present in the neuronal cytosolic fraction, demonstrating that the atoxic LC retains the SNAP25 binding capability of the wt toxin. Toxicity of BoNT/A ad was found to be reduced approximately 100,000-fold relative to wt BoNT/A.


Assuntos
Toxinas Botulínicas Tipo A/metabolismo , Neurônios/metabolismo , Proteínas Recombinantes/metabolismo , Animais , Bioensaio , Toxinas Botulínicas Tipo A/genética , Toxinas Botulínicas Tipo A/toxicidade , Citosol/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos ICR , Junção Neuromuscular/metabolismo , Neurônios/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/genética , Proteínas Recombinantes/toxicidade , Proteína 25 Associada a Sinaptossoma/metabolismo
12.
Protein Expr Purif ; 71(1): 62-73, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20045734

RESUMO

Work from multiple laboratories has clarified how the structural domains of botulinum neurotoxin A (BoNT/A) disable neuronal exocytosis, but important questions remain unanswered. Because BoNT/A intoxication disables its own uptake, light chain (LC) does not accumulate in neurons at detectable levels. We have therefore designed, expressed and purified a series of BoNT/A atoxic derivatives (ad) that retain the wild type features required for native trafficking. BoNT/A1ad(ek) and BoNT/A1ad(tev) are full length derivatives rendered atoxic through double point mutations in the LC protease (E(224)>A; Y(366)>A). DeltaLC-peptide-BoNT/A(tev) and DeltaLC-GFP-BoNT/A(tev) are derivatives wherein the catalytic portion of the LC is replaced with a short peptide or with GFP plus the peptide. In all four derivatives, we have fused the S6 peptide sequence GDSLSWLLRLLN to the N-terminus of the proteins to enable site-specific attachment of cargo using Sfp phosphopantetheinyl transferase. Cargo can be attached in a manner that provides a homogeneous derivative population rather than a polydisperse mixture of singly and multiply-labeled molecular species. All four derivatives contain an introduced cleavage site for conversion into disulfide-bonded heterodimers. These constructs were expressed in a baculovirus system and the proteins were secreted into culture medium and purified to homogeneity in yields ranging from 1 to 30 mg per liter. These derivatives provide unique tools to study toxin trafficking in vivo, and to assess how the structure of cargo linked to the heavy chain (HC) influences delivery to the neuronal cytosol. Moreover, they create the potential to engineer BoNT-based molecular vehicles that can target therapeutic agents to the neuronal cytoplasm.


Assuntos
Toxinas Botulínicas Tipo A/metabolismo , Neurônios/metabolismo , Engenharia de Proteínas/métodos , Sequência de Aminoácidos , Toxinas Botulínicas Tipo A/química , Toxinas Botulínicas Tipo A/isolamento & purificação , Corantes Fluorescentes/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Peptídeos/química , Peptídeos/isolamento & purificação , Processamento de Proteína Pós-Traducional , Transporte Proteico , Proteínas Recombinantes de Fusão/isolamento & purificação , Espectrometria de Massas por Ionização por Electrospray
13.
Science ; 298(5602): 2353-8, 2002 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-12459547

RESUMO

The low-density lipoprotein receptor mediates cholesterol homeostasis through endocytosis of lipoproteins. It discharges its ligand in the endosome at pH < 6. In the crystal structure at pH = 5.3, the ligand-binding domain (modules R2 to R7) folds back as an arc over the epidermal growth factor precursor homology domain (the modules A, B, beta propeller, and C). The modules R4 and R5, which are critical for lipoprotein binding, associate with the beta propeller via their calcium-binding loop. We propose a mechanism for lipoprotein release in the endosome whereby the beta propeller functions as an alternate substrate for the ligand-binding domain, binding in a calcium-dependent way and promoting lipoprotein release.


Assuntos
Endossomos/metabolismo , Lipoproteínas LDL/metabolismo , Estrutura Terciária de Proteína , Receptores de LDL/química , Receptores de LDL/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cálcio/metabolismo , Cristalização , Cristalografia por Raios X , Fator de Crescimento Epidérmico/química , Humanos , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Modelos Biológicos , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Precursores de Proteínas/química , Estrutura Secundária de Proteína , Receptores de LDL/genética , Sequências Repetitivas de Aminoácidos
14.
J Biol Chem ; 277(48): 46518-26, 2002 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-12270923

RESUMO

The calcium-independent receptor of alpha-latrotoxin (CIRL), a neuronal cell surface receptor implicated in the regulation of exocytosis, is a natural chimera of the cell adhesion protein and the G protein-coupled receptor (GPCR). In contrast with canonic GPCRs, CIRL consists of two heterologous non-covalently bound subunits, p120 and p85, due to endogenous proteolytic processing of the receptor precursor in the endoplasmic reticulum. Extracellularly oriented p120 contains hydrophilic cell adhesion domains, whereas p85 resembles a generic GPCR. We determined that the site of the CIRL cleavage is located within a juxtamembrane Cys- and Trp-rich domain of the N-terminal extracellular region of CIRL. Mutations in this domain make CIRL resistant to the cleavage and impair its trafficking. Therefore, we have named it GPS for G protein-coupled receptor proteolysis site. The GPS motif is found in homologous adhesion GPCRs and thus defines a novel receptor family. We postulate that the proteolytic processing and two-subunit structure is a common characteristic feature in the family of GPS-containing adhesion GPCRs.


Assuntos
Adesão Celular , Proteínas de Ligação ao GTP/metabolismo , Processamento de Proteína Pós-Traducional , Receptores de Superfície Celular/metabolismo , Receptores de Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS , Primers do DNA , Humanos , Hidrólise , Proteínas de Membrana , Dados de Sequência Molecular , Receptores de Superfície Celular/química , Receptores Acoplados a Proteínas G , Receptores de Peptídeos/química , Proteínas Recombinantes de Fusão/química , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
15.
J Biol Chem ; 277(39): 35887-95, 2002 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-12110683

RESUMO

Receptor-like protein-tyrosine phosphatase sigma (PTPvarsigma) is essential for neuronal development and function. Here we report that PTPvarsigma is a target of alpha-latrotoxin, a strong stimulator of neuronal exocytosis. alpha-Latrotoxin binds to the cell adhesion-like extracellular region of PTPvarsigma. This binding results in the stimulation of exocytosis. The toxin-binding site is located in the C-terminal part of the PTPvarsigma ectodomain and includes two fibronectin type III repeats. The intracellular catalytic domains of PTPvarsigma are not required for the alpha-latrotoxin binding and secretory response triggered by the toxin in chromaffin cells. These features of PTPvarsigma resemble two other previously described alpha-latrotoxin receptors, neurexin and CIRL. Thus, alpha-latrotoxin represents an unusual example of the neurotoxin that has three independent, equally potent, and yet structurally distinct targets. The known structural and functional characteristics of PTPvarsigma, neurexin, and CIRL suggest that they define a functional family of neuronal membrane receptors with complementary or converging roles in presynaptic function via a mechanism that involves cell-to-cell and cell-to-matrix interaction.


Assuntos
Proteínas Tirosina Fosfatases/química , Proteínas Tirosina Fosfatases/fisiologia , Receptores de Peptídeos/química , Animais , Sítios de Ligação , Western Blotting , Encéfalo/metabolismo , Células COS , Cálcio/metabolismo , Catálise , Membrana Celular/metabolismo , Células Cromafins/metabolismo , Reagentes de Ligações Cruzadas/farmacologia , Detergentes/farmacologia , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Exocitose , Deleção de Genes , Glicoproteínas , Hormônio do Crescimento Humano/farmacologia , Humanos , Ligantes , Espectrometria de Massas , Proteínas de Membrana , Modelos Genéticos , Mutagênese , Mutação , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Neuropeptídeos , Plasmídeos/metabolismo , Testes de Precipitina , Ligação Proteica , Estrutura Terciária de Proteína , Ratos , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores , Receptores Acoplados a Proteínas G , Receptores de Peptídeos/isolamento & purificação , Receptores de Peptídeos/metabolismo , Proteínas Recombinantes/metabolismo , Sefarose/farmacologia , Coloração pela Prata , Transfecção
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