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1.
Nat Commun ; 11(1): 5204, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-33060576

RESUMO

Toll-like receptor 7 (TLR7) recognizes both microbial and endogenous RNAs and nucleosides. Aberrant activation of TLR7 has been implicated in several autoimmune diseases including systemic lupus erythematosus (SLE). Here, by modifying potent TLR7 agonists, we develop a series of TLR7-specific antagonists as promising therapeutic agents for SLE. These compounds protect mice against lethal autoimmunity. Combining crystallography and cryo-electron microscopy, we identify the open conformation of the receptor and reveal the structural equilibrium between open and closed conformations that underlies TLR7 antagonism, as well as the detailed mechanism by which TLR7-specific antagonists bind to their binding pocket in TLR7. Our work provides small-molecule TLR7-specific antagonists and suggests the TLR7-targeting strategy for treating autoimmune diseases.


Assuntos
Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/química , Receptor 7 Toll-Like/antagonistas & inibidores , Receptor 7 Toll-Like/química , Animais , Doenças Autoimunes , Autoimunidade , Sítios de Ligação , Microscopia Crioeletrônica , Feminino , Ligantes , Lúpus Eritematoso Sistêmico , Camundongos , Camundongos Endogâmicos NZB , Modelos Moleculares , Conformação Proteica , Taxa de Sobrevida
2.
Nat Commun ; 11(1): 4017, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32782292

RESUMO

The thick mucus layer of the gut provides a barrier to infiltration of the underlying epithelia by both the normal microbiota and enteric pathogens. Some members of the microbiota utilise mucin glycoproteins as a nutrient source, but a detailed understanding of the mechanisms used to breakdown these complex macromolecules is lacking. Here we describe the discovery and characterisation of endo-acting enzymes from prominent mucin-degrading bacteria that target the polyLacNAc structures within oligosaccharide side chains of both animal and human mucins. These O-glycanases are part of the large and diverse glycoside hydrolase 16 (GH16) family and are often lipoproteins, indicating that they are surface located and thus likely involved in the initial step in mucin breakdown. These data provide a significant advance in our knowledge of the mechanism of mucin breakdown by the normal microbiota. Furthermore, we also demonstrate the potential use of these enzymes as tools to explore changes in O-glycan structure in a number of intestinal disease states.


Assuntos
Microbioma Gastrointestinal , Hexosaminidases/metabolismo , Glicoproteínas de Membrana/metabolismo , Mucinas/metabolismo , Animais , Bactérias/classificação , Bactérias/enzimologia , Bactérias/genética , Bactérias/metabolismo , Cristalografia por Raios X , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Hexosaminidases/química , Hexosaminidases/genética , Humanos , Glicoproteínas de Membrana/química , Estrutura Molecular , Mucinas/química , Filogenia , Polissacarídeos/química , Polissacarídeos/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato
3.
Nat Commun ; 11(1): 3569, 2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32678083

RESUMO

The clinically important MAM blood group antigen is present on haematopoietic cells of all humans except rare MAM-negative individuals. Its molecular basis is unknown. By whole-exome sequencing we identify EMP3, encoding epithelial membrane protein 3 (EMP3), as a candidate gene, then demonstrate inactivating mutations in ten known MAM-negative individuals. We show that EMP3, a purported tumour suppressor in various solid tumours, is expressed in erythroid cells. Disruption of EMP3 by CRISPR/Cas9 gene editing in an immortalised human erythroid cell line (BEL-A2) abolishes MAM expression. We find EMP3 to associate with, and stabilise, CD44 in the plasma membrane. Furthermore, cultured erythroid progenitor cells from MAM-negative individuals show markedly increased proliferation and higher reticulocyte yields, suggesting an important regulatory role for EMP3 in erythropoiesis and control of cell production. Our data establish MAM as a new blood group system and demonstrate an interaction of EMP3 with the cell surface signalling molecule CD44.


Assuntos
Antígenos de Grupos Sanguíneos/genética , Proliferação de Células , Células Eritroides/citologia , Glicoproteínas de Membrana/genética , Antígenos de Grupos Sanguíneos/química , Antígenos de Grupos Sanguíneos/metabolismo , Plaquetas/metabolismo , Células Cultivadas , Membrana Eritrocítica/metabolismo , Células Eritroides/metabolismo , Humanos , Receptores de Hialuronatos/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Modelos Moleculares , Mutação , Fenótipo , Ligação Proteica , Sequenciamento Completo do Exoma
4.
Nat Commun ; 11(1): 2140, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32358586

RESUMO

The trans-synaptic interaction of the cell-adhesion molecules teneurins (TENs) with latrophilins (LPHNs/ADGRLs) promotes excitatory synapse formation when LPHNs simultaneously interact with FLRTs. Insertion of a short alternatively-spliced region within TENs abolishes the TEN-LPHN interaction and switches TEN function to specify inhibitory synapses. How alternative-splicing regulates TEN-LPHN interaction remains unclear. Here, we report the 2.9 Å resolution cryo-EM structure of the TEN2-LPHN3 complex, and describe the trimeric TEN2-LPHN3-FLRT3 complex. The structure reveals that the N-terminal lectin domain of LPHN3 binds to the TEN2 barrel at a site far away from the alternatively spliced region. Alternative-splicing regulates the TEN2-LPHN3 interaction by hindering access to the LPHN-binding surface rather than altering it. Strikingly, mutagenesis of the LPHN-binding surface of TEN2 abolishes the LPHN3 interaction and impairs excitatory but not inhibitory synapse formation. These results suggest that a multi-level coincident binding mechanism mediated by a cryptic adhesion complex between TENs and LPHNs regulates synapse specificity.


Assuntos
Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Sinapses/metabolismo , Processamento Alternativo/genética , Processamento Alternativo/fisiologia , Sítios de Ligação/genética , Células HEK293 , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/genética , Mutação/genética , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Ligação Proteica/genética , Estrutura Secundária de Proteína , Receptores Acoplados a Proteínas-G/química , Receptores Acoplados a Proteínas-G/genética , Receptores de Peptídeos/química , Receptores de Peptídeos/genética , Receptores de Peptídeos/metabolismo , Sinapses/fisiologia
5.
J Food Sci ; 85(5): 1438-1449, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32339270

RESUMO

Because of the structural multiplicity of yeast mannoproteins, they have shown a great interest as food ingredients for a wide range of applications. The yields and the structural properties of mannoproteins varied depending on the isolation methods and their sources (baker's and brewer's Saccharomyces cerevisiae yeasts). Noncovalently bound mannoproteins (6.5 kDa) with a mannan/protein ratio of 0.63 and 2.78 were recovered upon the heat treatment of brewer's and baker's yeasts, respectively, whereas sodium dodecyl sulfate treatment led mainly to the release of nonglycosylated proteins. The highest yield of mannoproteins was achieved upon the enzymatic isolation with Zymolyase® from Arthrobacter luteus. The recovered covalently bound mannoproteins were characterized by a higher mannan/protein ratio (13.1 to 42.7) and a wider molecular weight distribution (5 to 10 kDa; 10 to 100 kDa; 100 to 400 kDa). Predictive models were developed to understand and modulate the effects of isolation parameters on yield, the mannoproteins content, and the mannan/protein ratio. The enzyme concentration was the most significant parameter affecting the yield, whereas the reaction time was the most significant parameter affecting mannan/protein ratio. Comparison of predicted and experimental values validated the established predicted models for the isolation of well-defined mannoproteins from yeast for targeted food applications. PRACTICAL APPLICATION: The increasing demand for clean label health-promoting foods has fueled the development of highly functional ingredients that offer both techno-functionalities and health-promoting properties. This study reveals the efficiency of whole inactivated yeasts as sources of mannoproteins. Given the dependence of the techno-functional and health-promoting properties of mannoproteins on their molecular properties, the investigation of the effects of the yeast sources and the type of isolation methods on the structural properties of mannoproteins would allow the modulation of their properties. Furthermore, the developed predictive models for the enzymatic process are expected to enhance the isolation efficiency of mannoproteins with well-defined structures.


Assuntos
Glicoproteínas de Membrana/isolamento & purificação , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Saccharomyces cerevisiae/química , Ingredientes de Alimentos/análise , Alimento Funcional , Mananas , Glicoproteínas de Membrana/química , Peso Molecular , Proteínas de Saccharomyces cerevisiae/química
6.
Science ; 368(6489)2020 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-32327568

RESUMO

Misfolded luminal endoplasmic reticulum (ER) proteins undergo ER-associated degradation (ERAD-L): They are retrotranslocated into the cytosol, polyubiquitinated, and degraded by the proteasome. ERAD-L is mediated by the Hrd1 complex (composed of Hrd1, Hrd3, Der1, Usa1, and Yos9), but the mechanism of retrotranslocation remains mysterious. Here, we report a structure of the active Hrd1 complex, as determined by cryo-electron microscopy analysis of two subcomplexes. Hrd3 and Yos9 jointly create a luminal binding site that recognizes glycosylated substrates. Hrd1 and the rhomboid-like Der1 protein form two "half-channels" with cytosolic and luminal cavities, respectively, and lateral gates facing one another in a thinned membrane region. These structures, along with crosslinking and molecular dynamics simulation results, suggest how a polypeptide loop of an ERAD-L substrate moves through the ER membrane.


Assuntos
Proteínas de Transporte/química , Degradação Associada com o Retículo Endoplasmático , Glicoproteínas de Membrana/química , Proteínas de Membrana/química , Complexos Multiproteicos/química , Proteólise , Proteínas de Saccharomyces cerevisiae/química , Ubiquitina-Proteína Ligases/química , Proteínas de Transporte/metabolismo , Microscopia Crioeletrônica , Retículo Endoplasmático/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Simulação de Dinâmica Molecular , Complexos Multiproteicos/metabolismo , Domínios Proteicos , Dobramento de Proteína , Proteínas de Saccharomyces cerevisiae/metabolismo
7.
J Virol ; 94(12)2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32269117

RESUMO

West Nile virus (WNV), a member of the Flavivirus genus and currently one of the most common arboviruses worldwide, is associated with severe neurological disease in humans. Its high potential to reemerge and rapidly disseminate makes it a bona fide global public health problem. The surface membrane glycoprotein (M) has been associated with Flavivirus-induced pathogenesis. Here, we identified a key amino acid residue at position 36 of the M protein whose mutation impacts WNV secretion and promotes viral attenuation. We also identified a compensatory site at position M-43 whose mutation stabilizes M-36 substitution both in vitro and in vivo Moreover, we found that introduction of the two mutations together confers a full attenuation phenotype and protection against wild-type WNV lethal challenge, eliciting potent neutralizing-antibody production in mice. Our study thus establishes the M protein as a new viral target for rational design of attenuated WNV strains.IMPORTANCE West Nile virus (WNV) is a worldwide (re)emerging mosquito-transmitted Flavivirus causing fatal neurological diseases in humans. However, no human vaccine has been yet approved. One of the most effective live-attenuated vaccines was empirically obtained by serial passaging of wild-type yellow fever Flavivirus However, such an approach is not acceptable nowadays, and the development of a rationally designed vaccine is necessary. Generating molecular infectious clones and mutating specific residues known to be involved in Flavivirus virulence constitute a powerful tool to promote viral attenuation. WNV membrane glycoprotein is thought to carry such essential determinants. Here, we identified two residues of this protein whose substitutions are key to the full and stable attenuation of WNV in vivo, most likely through inhibition of secretion and possible alteration of morphology. Applied to other flaviviruses, this approach should help in designing new vaccines against these viruses, which are an increasing threat to global human health.


Assuntos
Anticorpos Neutralizantes/biossíntese , Anticorpos Antivirais/biossíntese , Glicoproteínas de Membrana/genética , Mutação , Febre do Nilo Ocidental/virologia , Vírus do Nilo Ocidental/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Chlorocebus aethiops , Modelos Animais de Doenças , Feminino , Expressão Gênica , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Neurônios/imunologia , Neurônios/virologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Análise de Sobrevida , Células Vero , Proteínas Virais , Febre do Nilo Ocidental/imunologia , Febre do Nilo Ocidental/mortalidade , Febre do Nilo Ocidental/patologia , Vírus do Nilo Ocidental/crescimento & desenvolvimento , Vírus do Nilo Ocidental/imunologia
8.
EBioMedicine ; 52: 102645, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32014819

RESUMO

BACKGROUND: TLRs are some of the actively pursued drug-targets in immune disorders. Owing to a recent surge in the cognizance of TLR structural biology and signalling pathways, numerous therapeutic modulators, ranging from low-molecular-weight organic compounds to polypeptides and nucleic acid agents have been developed. METHODS: A penetratin-conjugated small peptide (TIP3), derived from the core ß-sheet of TIRAP, was evaluated in vitro by monitoring the TLR-mediated cytokine induction and quantifying the protein expression using western blot. The therapeutic potential of TIP3 was further evaluated in TLR-dependent in vivo disease models. FINDINGS: TIP3 blocks the TLR4-mediated cytokine production through both the MyD88- and TRIF-dependent pathways. A similar inhibitory-effect was exhibited for TLR3 but not on other TLRs. A profound therapeutic effect was observed in vivo, where TIP3 successfully alleviated the inflammatory response in mice model of collagen-induced arthritis and ameliorated the disease symptoms in psoriasis and SLE models. INTERPRETATION: Our data suggest that TIP3 may be a potential lead candidate for the development of effective therapeutics against TLR-mediated autoimmune disorders. FUNDING: This work was supported by the National Research Foundation of Korea (NRF-2019M3A9A8065098, 2019M3D1A1078940 and 2019R1A6A1A11051471). The funders did not have any role in the design of the present study, data collection, data analysis, interpretation, or the writing of the manuscript.


Assuntos
Glicoproteínas de Membrana/química , Peptídeos/química , Peptídeos/farmacologia , Conformação Proteica em Folha beta , Receptores de Interleucina-1/química , Receptor 4 Toll-Like/química , Sequência de Aminoácidos , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Autoimunidade , Linhagem Celular , Citocinas/metabolismo , Modelos Animais de Doenças , Fatores Imunológicos/química , Fatores Imunológicos/farmacologia , Inflamação/tratamento farmacológico , Inflamação/etiologia , Inflamação/metabolismo , Inflamação/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Glicoproteínas de Membrana/metabolismo , Camundongos , Modelos Moleculares , Óxido Nítrico/metabolismo , Peptídeos/metabolismo , Psoríase/tratamento farmacológico , Psoríase/imunologia , Psoríase/metabolismo , Psoríase/patologia , Espécies Reativas de Oxigênio/metabolismo , Receptores de Interleucina-1/metabolismo , Transdução de Sinais , Relação Estrutura-Atividade , Receptor 4 Toll-Like/metabolismo , Receptores Toll-Like/metabolismo
9.
Sci Rep ; 10(1): 3663, 2020 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-32107424

RESUMO

Recently, the critical roles played by genetic variants of TREM2 (Triggering Receptor Expressed on Myeloid cells 2) in Alzheimer's disease have been aggressively highlighted. However, few studies have focused on the deleterious roles of Nasu-Hakola disease (NHD) associated TREM2 variants. In order to get insights into the contributions made by these variants to neurodegeneration, we investigated the influences of four NHD associated TREM2 mutations (Y38C, W50C, T66M, and V126G) on loss-of-function, and followed this with in silico prediction and conventional molecular dynamics simulation. NHD mutations were predicted to be highly deleterious by eight different in silico bioinformatics tools and found to induce conformational changes by molecular dynamics simulation. As compared with the wild-type, the four variants produced substantial differences in the collective motions of loop regions, which not only promoted structural remodeling in the CDR2 (complementarity-determining region 2) loop but also in the CDR1 loop, by changing inter- and intra-loop hydrogen bonding networks. In addition, structural studies in a free energy landscape analysis showed that Y38, T66, and V126 are crucial for maintaining the structural features of CDR1 and CDR2 loops, and that mutations in these positions produced steric clashes and loss of ligand binding. These results showed the presence of mutations in the TREM2 ectodomain induced flexibility and caused structural alterations. Dynamical scenarios, as provided by the present study, may be critical to our understanding of the roles of these TREM2 mutations in neurodegenerative diseases.


Assuntos
Lipodistrofia , Glicoproteínas de Membrana/química , Simulação de Dinâmica Molecular , Mutação de Sentido Incorreto , Osteocondrodisplasias , Receptores Imunológicos/química , Panencefalite Esclerosante Subaguda , Substituição de Aminoácidos , Humanos , Glicoproteínas de Membrana/genética , Domínios Proteicos , Estrutura Secundária de Proteína , Receptores Imunológicos/genética
10.
Int J Mol Sci ; 21(3)2020 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-32019241

RESUMO

Tyrosinase-related protein 1 (TYRP1) is one of the three human melanogenic enzymes involved in the biosynthesis of melanin, a pigment responsible for the color of the skin, hair, and eyes. It shares high sequence identity with tyrosinase, but has two zinc ions in its active site rather than two copper ions as in tyrosinase. Typical tyrosinase inhibitors do not directly coordinate to the zinc ions of TYRP1. Here, we show, from an X-ray crystal structure determination, that phenylthiourea, a highly potent tyrosinase inhibitor, does neither coordinate the active site zinc ions, but binds differently from other structurally characterized TYRP1-inhibitor complexes. Its aromatic ring is directed outwards from the active site, apparently as a result from the absence of polar oxygen substituents that can take the position of water molecules bound in the active site. The compound binds via hydrophobic interactions, thereby blocking substrate access to the active site.


Assuntos
Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Oxirredutases/química , Oxirredutases/metabolismo , Feniltioureia/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Humanos , Modelos Moleculares , Conformação Proteica
11.
Proc Natl Acad Sci U S A ; 117(1): 388-394, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31848245

RESUMO

Surface layers (S-layers) are crystalline protein coats surrounding microbial cells. S-layer proteins (SLPs) regulate their extracellular self-assembly by crystallizing when exposed to an environmental trigger. However, molecular mechanisms governing rapid protein crystallization in vivo or in vitro are largely unknown. Here, we demonstrate that the Caulobacter crescentus SLP readily crystallizes into sheets in vitro via a calcium-triggered multistep assembly pathway. This pathway involves 2 domains serving distinct functions in assembly. The C-terminal crystallization domain forms the physiological 2-dimensional (2D) crystal lattice, but full-length protein crystallizes multiple orders of magnitude faster due to the N-terminal nucleation domain. Observing crystallization using a time course of electron cryo-microscopy (Cryo-EM) imaging reveals a crystalline intermediate wherein N-terminal nucleation domains exhibit motional dynamics with respect to rigid lattice-forming crystallization domains. Dynamic flexibility between the 2 domains rationalizes efficient S-layer crystal nucleation on the curved cellular surface. Rate enhancement of protein crystallization by a discrete nucleation domain may enable engineering of kinetically controllable self-assembling 2D macromolecular nanomaterials.


Assuntos
Proteínas de Bactérias/metabolismo , Caulobacter crescentus/metabolismo , Membrana Celular/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/ultraestrutura , Cálcio/metabolismo , Caulobacter crescentus/genética , Caulobacter crescentus/ultraestrutura , Membrana Celular/química , Membrana Celular/ultraestrutura , Microscopia Crioeletrônica , Cristalização , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/ultraestrutura , Mutagênese
12.
Appl Radiat Isot ; 155: 108936, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31655351

RESUMO

68Ga-PSMA-11 is currently one of the most investigated PET agents for imaging both recurrent prostate cancer and relevant metastases; however, the production and distribution of 68Ga-PSMA-11 is limited to a supply of only a few daily doses when using a commercially available 68Ge/68Ga generator. 68Ge/68Ga generators deliver only a modest amount of activity, up to 1850 MBq (50 mCi), when new, but it decreases with time. Additionally, the production of 68Ga/68Ge generators has not been able to meet the increasing demand of 68Ga radiotracers. In response to the need for a more economically viable alternative, the focus of this study was to provide a simple and efficient method for producing 68Ga-PSMA-11, using cyclotron-produced 68Ga that is ready for routine clinical practice.


Assuntos
Ciclotrons , Glicoproteínas de Membrana/química , Compostos Organometálicos/química , Automação , Linhagem Celular Tumoral , Humanos , Masculino
13.
Dev Comp Immunol ; 102: 103493, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31499098

RESUMO

The existence of pattern recognition receptors (PRRs) on immune cells was discussed in 1989 by Charles Janeway, Jr., who proposed a general concept of the ability of PRRs to recognize and bind conserved molecular structures of microorganisms known as pathogen-associated molecular patterns (PAMPs). Upon PAMP engagement, PRRs trigger intracellular signaling cascades resulting in the expression of various proinflammatory molecules. These recognition molecules represent an important and efficient innate immunity tool of all organisms. As invertebrates lack the instruments of the adaptive immune system, based on "true" lymphocytes and functional antibodies, the importance of PRRs are even more fundamental. In the present review, the structure, specificity, and expression profiles of PRRs characterized in annelids are discussed, and their role in innate defense is suggested.


Assuntos
Anelídeos/imunologia , Imunidade Inata , Receptores de Reconhecimento de Padrão/metabolismo , Proteínas da Fase Aguda/química , Proteínas da Fase Aguda/genética , Proteínas da Fase Aguda/metabolismo , Animais , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Regulação da Expressão Gênica , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Padrões Moleculares Associados a Patógenos/imunologia , Padrões Moleculares Associados a Patógenos/metabolismo , Receptores de Reconhecimento de Padrão/química , Receptores de Reconhecimento de Padrão/genética , Transdução de Sinais/imunologia , Distribuição Tecidual , Receptores Toll-Like/química , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo
14.
PLoS One ; 14(12): e0225797, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31851670

RESUMO

Measuring transport properties like diffusion and directional flow is essential for understanding dynamics within heterogeneous systems including living cells and novel materials. Fluorescent molecules traveling within these inhomogeneous environments under the forces of Brownian motion and flow exhibit fluctuations in their concentration, which are directly linked to the transport properties. We present a method utilizing single photon interference and fluorescence correlation spectroscopy (FCS) to simultaneously measure transport of fluorescent molecules within aqueous samples. Our method, within seconds, measures transport in thousands of homogenous voxels (100 nm)3 and under certain conditions, eliminates photo-physical artifacts associated with blinking of fluorescent molecules. A comprehensive theoretical framework is presented and validated by measuring transport of quantum dots, associated with VSV-G receptor along cellular membranes as well as within viscous gels.


Assuntos
Membrana Celular/química , Glicoproteínas de Membrana/química , Pontos Quânticos/química , Espectrometria de Fluorescência/métodos , Proteínas do Envelope Viral/química , Difusão , Células HeLa , Humanos , Interferometria/métodos , Luz , Movimento (Física)
15.
Proc Natl Acad Sci U S A ; 116(50): 25278-25286, 2019 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-31767763

RESUMO

Surface protein layers (S-layers) often form the only structural component of the archaeal cell wall and are therefore important for cell survival. S-layers have a plethora of cellular functions including maintenance of cell shape, osmotic, and mechanical stability, the formation of a semipermeable protective barrier around the cell, and cell-cell interaction, as well as surface adhesion. Despite the central importance of S-layers for archaeal life, their 3-dimensional (3D) architecture is still poorly understood. Here we present detailed 3D electron cryomicroscopy maps of archaeal S-layers from 3 different Sulfolobus strains. We were able to pinpoint the positions and determine the structure of the 2 subunits SlaA and SlaB. We also present a model describing the assembly of the mature S-layer.


Assuntos
Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/ultraestrutura , Sulfolobus/metabolismo , Microscopia Crioeletrônica , Dimerização , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Sulfolobus/química , Sulfolobus/genética , Sulfolobus/ultraestrutura
16.
Nat Commun ; 10(1): 4670, 2019 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-31604943

RESUMO

The mechanisms by which many human cytomegalovirus (HCMV)-encoded proteins help the virus to evade immune surveillance remain poorly understood. In particular, it is unknown whether HCMV proteins arrest Toll-like receptor (TLR) signaling pathways required for antiviral defense. Here, we report that US7 and US8 as key suppressors that bind both TLR3 and TLR4, facilitating their destabilization by distinct mechanisms. US7 exploits the ER-associated degradation components Derlin-1 and Sec61, promoting ubiquitination of TLR3 and TLR4. US8 not only disrupts the TLR3-UNC93B1 association but also targets TLR4 to the lysosome, resulting in rapid degradation of the TLR. Accordingly, a mutant HCMV lacking the US7-US16 region has an impaired ability to hinder TLR3 and TLR4 activation, and the impairment is reversed by the introduction of US7 or US8. Our findings reveal an inhibitory effect of HCMV on TLR signaling, which contributes to persistent avoidance of the host antiviral response to achieve viral latency.


Assuntos
Citomegalovirus/patogenicidade , Imunidade Inata , Glicoproteínas de Membrana/fisiologia , Receptores Toll-Like/metabolismo , Proteínas Virais/fisiologia , Linhagem Celular , Humanos , Glicoproteínas de Membrana/química , Complexo de Endopeptidases do Proteassoma/fisiologia , Domínios Proteicos , Proteólise , Transdução de Sinais , Receptores Toll-Like/genética , Receptores Toll-Like/fisiologia , Ubiquitina/metabolismo , Proteínas Virais/química
17.
ACS Chem Biol ; 14(10): 2141-2147, 2019 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-31584261

RESUMO

Neu5Ac, Neu5Gc, and KDN are three forms of sialic acids in vertebrates that possess distinct biological functions. Herein, we report the synthesis and metabolic incorporation of the 9-azido analogues of three sialic acid forms in mammalian cells. The incorporated sialic acid analogues enable fluorescent imaging of cell-surface sialoglycans and proteomic profiling of sialoglycoproteins. Furthermore, we apply them to metabolically engineer cell surfaces with sialoglycans terminated with distinct sialic acids or their 9-azido analogues. The remodeled cells expressing specific cell-surface sialoglycoforms show distinct binding affinity toward subtilase cytotoxin (SubAB), a toxin secreted by Shiga toxigenic Escherichia coli. The 9-azido analogues of sialic acid forms developed in this work provide a versatile tool for metabolic remodeling of cell-surface properties and modulating pathogen-host interactions.


Assuntos
Azidas/metabolismo , Glicoproteínas de Membrana/metabolismo , Ácidos Siálicos/metabolismo , Sialoglicoproteínas/metabolismo , Animais , Células CHO , Engenharia Celular/métodos , Linhagem Celular Tumoral , Chlorocebus aethiops , Cricetulus , Proteínas de Escherichia coli/metabolismo , Humanos , Glicoproteínas de Membrana/química , Proteômica , Sialoglicoproteínas/química , Subtilisinas/metabolismo , Células Vero
18.
Matrix Biol ; 83: 97-115, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31479698

RESUMO

Colon cancer is one of the first tumor types where a functional link between inflammation and tumor onset has been described; however, the microenvironmental cues affecting colon cancer progression are poorly understood. Here we demonstrate that the expression of the ECM molecule EMILIN-1 halts the development of AOM-DSS induced tumors. In fact, upon AOM-DSS treatment the Emilin1-/- (E1-/-) mice were characterized by a higher tumor incidence, bigger adenomas and less survival. Similar results were obtained with the E933A EMILIN-1 (E1-E933A) transgenic mouse model, expressing a mutant EMILIN-1 unable to interact with α4/α9ß1 integrins. Interestingly, upon chronic treatment with DSS, E1-/- and E1-E933A mice were characterized by the presence of increased inflammatory infiltrates, higher colitis scores and more severe mucosal injury respect to the wild type (E1+/+) mice. Since alterations of the intestinal lymphatic network are a well-established feature of human inflammatory bowel disease and EMILIN-1 is a key structural element in the maintenance of the integrity of lymphatic vessels, we assessed the lymphatic vasculature in this context. The analyses revealed that both E1-/- and E1-E933A mice displayed a higher density of LYVE-1 positive vessels; however, their functionality was severely compromised after colitis induction. Taken together, these results suggest that the loss of EMILIN-1 expression may cause the reduction of the inflammatory resolution during colon cancer progression due to a decreased lymph flow and impaired inflammatory cell drainage.


Assuntos
Colite/complicações , Colite/genética , Neoplasias do Colo/genética , Integrina beta1/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Animais , Azoximetano/efeitos adversos , Linhagem Celular Tumoral , Proliferação de Células , Colite/induzido quimicamente , Colite/metabolismo , Neoplasias do Colo/etiologia , Neoplasias do Colo/metabolismo , Sulfato de Dextrana/efeitos adversos , Modelos Animais de Doenças , Humanos , Integrina beta1/química , Glicoproteínas de Membrana/química , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Camundongos Knockout , Ligação Proteica
19.
Cells ; 8(8)2019 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-31430955

RESUMO

Receptor tyrosine kinases are believed to be activated through ligand-induced dimerization. We now demonstrate that in cultured neurons, a substantial amount of endogenous TrkB, the receptor for brain-derived neurotrophic factor (BDNF), exists as an inactive preformed dimer, and the application of BDNF activates the pre-existing dimer. Deletion of the extracellular juxtamembrane motif (EJM) of TrkB increased the amount of preformed dimer, suggesting an inhibitory role of EJM on dimer formation. Further, binding of an agonistic antibody (MM12) specific to human TrkB-EJM activated the full-length TrkB and unexpectedly also truncated TrkB lacking ECD (TrkBdelECD365), suggesting that TrkB is activated by attenuating the inhibitory effect of EJM through MM12 binding-induced conformational changes. Finally, in cells co-expressing rat and human TrkB, MM12 could only activate TrkB human-human dimer but not TrkB human-rat TrkB dimer, indicating that MM12 binding to two TrkB monomers is required for activation. Our results support a model that TrkB preforms as an inactive dimer and BDNF induces TrkB conformation changes leading to its activation.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Glicoproteínas de Membrana/química , Neurônios/metabolismo , Receptor trkB/química , Motivos de Aminoácidos , Animais , Células CHO , Membrana Celular/metabolismo , Cricetulus , Neurônios/citologia , Células PC12 , Multimerização Proteica , Ratos
20.
Colloids Surf B Biointerfaces ; 183: 110284, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31421406

RESUMO

Bacterial surface layer proteins (S-layer) possess unique binding properties for metal ions. By combining the binding capability of S-layer proteins with the optical properties of gold nanoparticles (AuNP), namely plasmonic resonance, a colorimetric detection system for metal and metalloid ions in water was developed. Eight S-layer proteins from different bacteria species were used for the functionalization of AuNP. The thus developed biohybrid systems, AuNP functionalized with S-layer proteins, were tested with different metal salt solutions, e.g. Indium(III)-chloride, Yttrium(III)-chloride or Nickel(II)-chloride, to determine their selective and sensitive binding to ionic analytes. All tested S-layer proteins displayed unique binding affinities for the different metal ions. For each S-layer and metal ion combination markedly different reaction patterns and differences in concentration range and absorption spectra were detected by UV/vis spectroscopy. In this way, the selective detection of tested metal ions was achieved by differentiated analysis of a colorimetric screening assay of these biohybrid systems. A highly selective and sensitive detection of yttrium ions down to a concentration of 1.67 × 10-5 mol/l was achieved with S-layer protein SslA functionalized AuNP. The presented biohybrid systems can thus be used as a sensitive and fast sensor system for metal and metalloid ions in aqueous systems.


Assuntos
Colorimetria/métodos , Índio/isolamento & purificação , Níquel/isolamento & purificação , Poluentes Químicos da Água/isolamento & purificação , Ítrio/isolamento & purificação , Bacillaceae/química , Ouro/química , Humanos , Glicoproteínas de Membrana/química , Nanopartículas Metálicas/química , Ligação Proteica , Sporosarcina/química , Ressonância de Plasmônio de Superfície/métodos , Água/química
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