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1.
Nat Commun ; 12(1): 648, 2021 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-33510170

RESUMO

Controlling nanocarrier interactions with the immune system requires a thorough understanding of the surface properties that modulate protein adsorption in biological fluids, since the resulting protein corona redefines cellular interactions with nanocarrier surfaces. Albumin is initially one of the dominant proteins to adsorb to nanocarrier surfaces, a process that is considered benign or beneficial by minimizing opsonization or inflammation. Here, we demonstrate the surface chemistry of a model nanocarrier can be engineered to stabilize or denature the three-dimensional conformation of adsorbed albumin, which respectively promotes evasion or non-specific clearance in vivo. Interestingly, certain common chemistries that have long been considered to convey stealth properties denature albumin to promote nanocarrier recognition by macrophage class A1 scavenger receptors, providing a means for their eventual removal from systemic circulation. We establish that the surface chemistry of nanocarriers can be specified to modulate adsorbed albumin structure and thereby tune clearance by macrophage scavenger receptors.


Assuntos
Macrófagos/metabolismo , Nanopartículas/química , Dobramento de Proteína , Soroalbumina Bovina/química , Adsorção , Animais , Bovinos , Microscopia Crioeletrônica , Humanos , Cinética , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Nanopartículas/ultraestrutura , Coroa de Proteína/química , Coroa de Proteína/metabolismo , Células RAW 264.7 , Receptores Depuradores/química , Receptores Depuradores/metabolismo , Soroalbumina Bovina/metabolismo , Propriedades de Superfície
2.
Sci Rep ; 9(1): 4218, 2019 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-30862948

RESUMO

Intestinal absorption of heme has remained enigmatic for years, even though heme provides the most bioavailable form of iron. The salmon louse, Lepeophtheirus salmonis, is a heme auxotrophic ectoparasite feeding on large quantities of blood from its host, the salmon. Here we show that a scavenging CD36-like receptor is a potential mediator of heme absorption in the intestine of the salmon louse. The receptor was characterized by a heme binding assay using recombinantly expressed protein, in situ hybridization and immunohistochemistry, as well as functional knockdown studies in the louse. A computational structural model of the receptor predicted a binding pocket for heme, as also supported by in silico docking. The mRNA and protein were expressed exclusively in the intestine of the louse. Further, knocking down the transcript resulted in lower heme levels in the adult female louse, production of shorter egg strings, and an overall lower hatching success of the eggs. Finally, starving the lice caused the transcript expression of the receptor to decrease. To our knowledge, this is the first time a CD36-like protein has been suggested to be an intestinal heme receptor.


Assuntos
Proteínas de Artrópodes , Copépodes , Absorção Intestinal , Intestinos , Simulação de Acoplamento Molecular , Receptores Depuradores , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/metabolismo , Sítios de Ligação , Copépodes/química , Copépodes/metabolismo , Heme , Receptores Depuradores/química , Receptores Depuradores/metabolismo
3.
Nat Microbiol ; 4(3): 414-419, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30531980

RESUMO

Enterovirus 71 (EV71) is a common cause of hand, foot and mouth disease-a disease endemic especially in the Asia-Pacific region1. Scavenger receptor class B member 2 (SCARB2) is the major receptor of EV71, as well as several other enteroviruses responsible for hand, foot and mouth disease, and plays a key role in cell entry2. The isolated structures of EV71 and SCARB2 are known3-6, but how they interact to initiate infection is not. Here, we report the EV71-SCARB2 complex structure determined at 3.4 Å resolution using cryo-electron microscopy. This reveals that SCARB2 binds EV71 on the southern rim of the canyon, rather than across the canyon, as predicted3,7,8. Helices 152-163 (α5) and 183-193 (α7) of SCARB2 and the viral protein 1 (VP1) GH and VP2 EF loops of EV71 dominate the interaction, suggesting an allosteric mechanism by which receptor binding might facilitate the low-pH uncoating of the virus in the endosome/lysosome. Remarkably, many residues within the binding footprint are not conserved across SCARB2-dependent enteroviruses; however, a conserved proline and glycine seem to be key residues. Thus, although the virus maintains antigenic variability even within the receptor-binding footprint, the identification of binding 'hot spots' may facilitate the design of receptor mimic therapeutics less likely to quickly generate resistance.


Assuntos
Enterovirus/metabolismo , Interações entre Hospedeiro e Microrganismos , Glicoproteínas de Membrana Associadas ao Lisossomo/química , Receptores Depuradores/química , Proteínas Virais/química , Microscopia Crioeletrônica , Enterovirus/ultraestrutura , Humanos , Ligação Viral
4.
Infect Immun ; 86(7)2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29685986

RESUMO

The high-resolution structure of glucan binding protein C (GbpC) at 1.14 Å, a sucrose-dependent virulence factor of the dental caries pathogen Streptococcus mutans, has been determined. GbpC shares not only structural similarities with the V regions of AgI/II and SspB but also functional adherence to salivary agglutinin (SAG) and its scavenger receptor cysteine-rich domains (SRCRs). This is not only a newly identified function for GbpC but also an additional fail-safe binding mechanism for S. mutans Despite the structural similarities with S. mutans antigen I/II (AgI/II) and SspB of Streptococcus gordonii, GbpC remains unique among these surface proteins in its propensity to adhere to dextran/glucans. The complex crystal structure of GbpC with dextrose (ß-d-glucose; Protein Data Bank ligand BGC) highlights exclusive structural features that facilitate this interaction with dextran. Targeted deletion mutant studies on GbpC's divergent loop region in the vicinity of a highly conserved calcium binding site confirm its role in biofilm formation. Finally, we present a model for adherence to dextran. The structure of GbpC highlights how artfully microbes have engineered the lectin-like folds to broaden their functional adherence repertoire.


Assuntos
Aderência Bacteriana , Proteínas de Transporte/fisiologia , Lectinas/fisiologia , Streptococcus mutans/fisiologia , Sacarose/farmacologia , Biofilmes , Proteínas de Ligação ao Cálcio , Proteínas de Transporte/química , Cristalografia , Proteínas de Ligação a DNA , Dextranos/química , Lectinas/química , Receptores de Superfície Celular/química , Receptores Depuradores/química , Proteínas Supressoras de Tumor
5.
Acta Virol ; 62(1): 50-57, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29521103

RESUMO

There is still no effective clinical antiviral drug against human enterovirus 71 (EV71) infection, which causes hand, foot and mouth disease (HFMD) in children. Scavenger receptor class B member 2 (SCARB2) is an important receptor of EV71 as it plays a vital role in the early steps of viral infection. In this study, recombinant SCARB2 protein was expressed and purified in a prokaryotic expression system, and was identified by western blot with a monoclonal antibody and mass spectrometry analysis. Detection of the sera from mice immunized with the recombinant SCARB2 protein using ELISA and western blot showed good immunogenicity of the recombinant protein. Furthermore, in the neutralization test cytopathic effect was significantly decreased when EV71 was incubated with the immune sera before infection. In summary, the SCARB2 protein was expressed successfully, and the immune sera showed obvious antiviral effect against EV71. This study provides useful information about the interaction mechanism between SCARB2 and EV71, and is also helpful for further clinical treatment research of HFMD.


Assuntos
Enterovirus Humano A/metabolismo , Glicoproteínas de Membrana Associadas ao Lisossomo/imunologia , Receptores Depuradores/imunologia , Animais , Anticorpos Antivirais , Sequência de Bases , Linhagem Celular Tumoral , Enterovirus Humano A/química , Enterovirus Humano A/imunologia , Ensaio de Imunoadsorção Enzimática , Doença de Mão, Pé e Boca/metabolismo , Humanos , Glicoproteínas de Membrana Associadas ao Lisossomo/química , Glicoproteínas de Membrana Associadas ao Lisossomo/metabolismo , Camundongos , Testes de Neutralização , Ligação Proteica , Receptores Depuradores/química , Receptores Depuradores/metabolismo , Proteínas Recombinantes , Vacinas Virais/imunologia
6.
Fish Shellfish Immunol ; 74: 141-151, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29305330

RESUMO

Macrophage receptor with collagenous structure (MARCO) plays essential roles in phagocytic cell-mediated innate immune responses. However, studies regarding MARCO, especially its functions, are limited in teleost species. In this study, we identified a MARCO molecule (PaMARCO) from ayu (Plecoglossus altivelis). PaMARCO shared conserved functional domains with its mammalian counterparts. Sequence analysis showed that PaMARCO was most closely related to its rainbow trout (Oncorhynchus mykiss) counterpart. PaMARCO expression was upregulated in all tested immune tissues and monocytes/macrophages (MO/MΦ) upon Vibrio anguillarum infection, and blocking its function significantly decreased the immune responses of MO/MΦ during infection. PaMARCO could bind to the tested gram-positive and -negative bacteria in a Ca2+-dependent manner in vitro. Furthermore, the phagocytosis and bacterial killing activities of MO/MΦ were significantly decreased upon PaMARCO blockade using anti-PaMARCO IgG. PaMARCO was also involved in the polarization processes of ayu MO/MΦ. The upregulated expression of representative cytokines in LPS-induced M1 type (TNF-α, IL-1ß) or cAMP-induced M2 type (TGF-ß, IL-10) were inhibited in the anti-PaMARCO IgG-treated group, indicating that PaMARCO may be involved in the regulation of both inflammation priming and inflammation resolution of MO/MΦ. In conclusion, our results implicate that PaMARCO has essential regulatory roles for bacterial binding, clearance, and the polarization processes of ayu MO/MΦ.


Assuntos
Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Osmeriformes/genética , Osmeriformes/imunologia , Receptores Imunológicos/genética , Receptores Imunológicos/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica , Macrófagos/imunologia , Monócitos/imunologia , Filogenia , Receptores Imunológicos/química , Receptores Depuradores/química , Receptores Depuradores/genética , Receptores Depuradores/imunologia , Alinhamento de Sequência/veterinária , Vibrio/fisiologia , Vibrioses/imunologia
7.
Cell Mol Immunol ; 15(6): 563-574, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29375122

RESUMO

Circulating immunoglobulin M (IgM) exists in a pentameric form, possessing a polyreactive nature that responds not only to foreign antigens but also to autoantigens; thus, it is involved in both beneficial and detrimental immune responses, including protection from infection and the progression of autoimmunity. On the other hand, IgM also behaves as a carrier of the apoptosis inhibitor of macrophage (AIM) protein, storing a large amount of the inactivated form of AIM in the blood through this association. Under different disease conditions, AIM can dissociate from IgM locally or systemically to exert its function, inducing the removal of various biological debris such as excess fat, bacteria, cancer cells or dead cell debris. Most typically, upon induction of acute kidney injury (AKI), IgM-free AIM is filtered by the glomerulus in the kidney, which stimulates the clearance of intraluminal dead cells debris at the obstructed proximal tubules, thereby facilitating the repair of kidney injury. Interestingly, cats exhibit a deficiency in AIM release from IgM, which may increase their susceptibility to renal failure. Conversely, association with AIM inhibits IgM binding to the Fcα/µ receptor on follicular dendritic cells at the splenic germinal center, thereby protecting the IgM immune complex from Fcα/µ receptor-mediated internalization, which supports IgM-dependent antigen presentation to B cells and stimulates high-affinity IgG antibody production. The regulation of AIM-IgM binding, resulting from the discovery of reciprocal actions between AIM and IgM, could lead to the development of novel therapies against different diseases.


Assuntos
Imunoglobulina M/metabolismo , Receptores Depuradores/metabolismo , Lesão Renal Aguda/sangue , Lesão Renal Aguda/imunologia , Lesão Renal Aguda/patologia , Lesão Renal Aguda/urina , Animais , Autoanticorpos/sangue , Progressão da Doença , Humanos , Imunoglobulina M/sangue , Imunoglobulina M/química , Imunoglobulina M/urina , Receptores Depuradores/química
8.
Artigo em Inglês | MEDLINE | ID: mdl-29342974

RESUMO

Trypsin is a serine protease, which has been proved to be a novel superoxide scavenger. The burst of superoxide induced by polychlorinated biphenyls can be impeded by trypsin in both wild type and sod knockout mutants of Escherichia coli. The experimental results demonstrated that the activities of superoxide scavenging of trypsin were significantly accelerated by Cu ions. Also, with the addition of Cu ions, a new ß-sheet (ß7) transited from a random coil in the Cu(II)-trypsin (TP) system, which was favorable for the formation of more contacts with other sheets of trypsin. Residue-residue network analysis and the porcupine plots proved that the Cu ion in trypsin strengthened some native interactions among residues, which ultimately resulted in much greater stability of the Cu(II)-TP system. Moreover, compact and stable trypsin structures with Cu ions might be responsible for significantly provoking the activity of superoxide scavenging.


Assuntos
Cobre/química , Escherichia coli/química , Íons/química , Receptores Depuradores/química , Superóxido Dismutase/metabolismo , Superóxidos/química , Tripsina/química , Sítios de Ligação , Simulação de Dinâmica Molecular , Tripsinogênio
9.
J Mol Graph Model ; 77: 189-199, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28869863

RESUMO

Scavenger receptor A (SRA), as an immune regulator, has been shown to play important roles in lipid metabolism, cardiovascular diseases, and pathogen recognition. Several natural product inhibitors of SRA have been studied for their potential application in modulating SRA functions. To understand the binding mode of these inhibitors on SRA, we conducted systematic molecular modeling studies in order to identify putative binding domain(s) that may be responsible for their recognition to the receptor as well as their inhibitory activity. Treatment of SRA with one of the natural product inhibitors, rhein, led to significant dissociation of SRA oligomers to its trimer and dimer forms, which further supported our hypothesis on their putative mechanism of action. Such information is believed to shed light on design of more potent inhibitors for the receptor in order to develop potential therapeutics through immune system modulation.


Assuntos
Antraquinonas/química , Modelos Moleculares , Receptores Depuradores/química , Antraquinonas/farmacologia , Sítios de Ligação , Humanos , Ligação Proteica , Multimerização Proteica/efeitos dos fármacos , Receptores Depuradores/antagonistas & inibidores
10.
Fish Shellfish Immunol ; 70: 426-436, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28916359

RESUMO

Scavenger receptors (SRs) are important pattern recognition receptors (PRRs), which play significant roles in host defense against pathogens by identifying pathogen-associated molecular patterns (PAMPs). In this study, we report the cloning and characterization of a SR from Eriocheir sinensis (EsSR-B1) which is a 500 amino acid protein encoded by a gene comprised of 2726 nucleotides with a 1503 bp open reading frame. The domains of EsSR-B1 were found to be evolutionarily conserved. EsSR-B1 was widely detected in different tissues of E. sinensis and significantly up-regulated in hemocytes after stimulation by Staphyloccocus aureus or Vibrio parahaemolyticus. Recombinant EsSR-B1 protein could bind to bacteria and promote phagocytosis upon bacterial stimulation. Moreover, antimicrobial peptide expression was reduced in EsSR-B1-silenced hemocytes after challenge by S. aureus or V. parahaemolyticus. Thus, EsSR-B1 has a critical role in the binding of bacteria and subsequent promotion of hemocyte phagocytosis.


Assuntos
Braquiúros/genética , Braquiúros/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Receptores Depuradores/genética , Receptores Depuradores/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Sequência de Bases , Feminino , Perfilação da Expressão Gênica , Hemócitos/metabolismo , Masculino , Fagocitose , Filogenia , Distribuição Aleatória , Receptores Depuradores/química , Alinhamento de Sequência , Staphylococcus aureus/fisiologia , Vibrio parahaemolyticus/fisiologia
11.
Molecules ; 22(8)2017 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-28805750

RESUMO

In this study, the characterization of chemical constituents and biological activity of the roots of Taraxacum coreanum (Asteraceae) was attempted. Phytochemical investigation of the roots of T. coreanum led to the isolation of two new inositol derivatives, taraxinositols A (1) and B (2), and a new phenolic compound, taraxinol (16), together with twenty known compounds including four inositol derivatives, neo-inositol-1,4-bis (4-hydroxybenzeneacetate) (3), chiro-inositol-1,5-bis(4- hydroxybenzeneacetate) (4), chiro-inositol-2,3-bis (4-hydroxybenzeneacetate) (5) and chiro-inositol- 1,2,3-tris (4-hydroxybenzeneacetate) (6), nine phenolic compounds: p-hydroxybenzaldehyde (7), vanillin (8), syringaldehyde (9), vanillic acid (10), 4-methoxyphenylacetic acid (11), 4-hydroxy- phenylacetic acid methyl ester (12), optivanin (13), isoferulic acid (14) and dihydroconiferyl alcohol (15), four coumarins: nodakenetin (17), decursinol (18), prangol (19) and isobyakangelicin (20), and three lignans: syringaresinol-4'-O-ß-d-glucoside (21), syringaresinol (22), and pinoresinol (23). The structures of isolated compounds were determined on the basis of spectroscopic analysis. Among the isolated compounds, vanillic acid, isoferulic acid and syringaresinol showed radical scavenging activity with IC50 values ranging from 30.4 to 75.2 µM.


Assuntos
Inositol/química , Fenol/química , Extratos Vegetais/química , Raízes de Plantas/química , Taraxacum/química , Antioxidantes/química , Antioxidantes/farmacologia , Cromatografia Líquida de Alta Pressão/métodos , Cumarínicos/química , Furanos/química , Glucosídeos/química , Humanos , Concentração Inibidora 50 , Inositol/isolamento & purificação , Lignanas/química , Espectroscopia de Ressonância Magnética/métodos , Fenol/isolamento & purificação , Extratos Vegetais/isolamento & purificação , Receptores Depuradores/química , Receptores Depuradores/metabolismo
12.
Fish Shellfish Immunol ; 67: 254-262, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28602682

RESUMO

Scavenger receptors (SRs) comprise a large family of structurally diverse glycoproteins located on the cell membrane and function as pattern-recognition receptors (PRRs) participating in innate immunity in different species. Class C scavenger receptor (SRC) has been only identified in invertebrates and its biological functions still need to be researched. In this study, we characterized the anti-bacterial function of a SRC from kuruma shrimp Marsupenaeus japonicus (MjSRC). The mRNA level of MjSRC was up-regulated significantly in hemocytes of kuruma shrimp challenged by Vibrio anguillarum or Staphylococcus aureus. The recombinant extracellular domains (MAM and CCP domains) of MjSRC have the ability of binding different bacteria and glycans in vitro. After knockdown of MjSRC, the bacterial clearance ability and phagocytic rate of hemocyte decreased significantly in vivo. Meanwhile, overexpression of MjSRC in shrimp enhanced the clearance ability and phagocytic rate of hemocytes. Further study found that MjSRC could regulate the expression of several antimicrobial peptides (AMPs). All these results indicate that MjSRC plays important roles in antibacterial immunity in kuruma shrimp by enhancing hemocyte phagocytosis and AMP expression.


Assuntos
Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Penaeidae/genética , Penaeidae/imunologia , Receptores Depuradores/genética , Receptores Depuradores/imunologia , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/imunologia , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Sequência de Bases , Hemócitos/imunologia , Fagocitose , Filogenia , Polissacarídeos/farmacologia , Receptores Depuradores/química , Alinhamento de Sequência/veterinária , Staphylococcus aureus/fisiologia , Vibrio/fisiologia
13.
Immunogenetics ; 69(6): 401-407, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28364129

RESUMO

The Scavenger Receptor Cysteine-Rich (SRCR) proteins are an archaic group of proteins characterized by the presence of multiple SRCR domains. They are membrane-bound or secreted proteins, which are generally related to host defense systems in animals. Deleted in Malignant Brain Tumors 1 (DMBT1) is a SRCR protein which is secreted in mucosal fluids and involved in host defense by pathogen binding by its SRCR domains. Genetic polymorphism within DMBT1 leads to DMBT1-alleles giving rise to polypeptides with interindividually different numbers of SRCR domains, ranging from 8 SRCR domains (encoded by 6 kb DMBT1 variant) to 13 SRCR domains (encoded by the 8 kb DMBT1 variant). In the present study, we have investigated whether reduction from 13 to 8 amino-terminal SRCR domains leads to reduction of bacterial binding. The 6 kb variant bound ~20-45% less bacteria compared to the 8 kb variant. These results support the hypothesis that genetic variation in DMBT1 may influence microbial defense.


Assuntos
Mutação em Linhagem Germinativa , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores Depuradores/genética , Receptores Depuradores/metabolismo , Deleção de Sequência , Aderência Bacteriana/genética , Proteínas de Ligação ao Cálcio , Proteínas de Ligação a DNA , Humanos , Polimorfismo Genético , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Receptores de Superfície Celular/química , Receptores Depuradores/química , Proteínas Supressoras de Tumor
14.
Protein Cell ; 8(8): 590-600, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28447294

RESUMO

Entero virus 71 (EV71) causes hand, foot, and mouth disease (HFMD) and occasionally leads to severe neurological complications and even death. Scavenger receptor class B member 2 (SCARB2) is a functional receptor for EV71, that mediates viral attachment, internalization, and uncoating. However, the exact binding site of EV71 on SCARB2 is unknown. In this study, we generated a monoclonal antibody (mAb) that binds to human but not mouse SCARB2. It is named JL2, and it can effectively inhibit EV71 infection of target cells. Using a set of chimeras of human and mouse SCARB2, we identified that the region containing residues 77-113 of human SCARB2 contributes significantly to JL2 binding. The structure of the SCARB2-JL2 complex revealed that JL2 binds to the apical region of SCARB2 involving α-helices 2, 5, and 14. Our results provide new insights into the potential binding sites for EV71 on SCARB2 and the molecular mechanism of EV71 entry.


Assuntos
Anticorpos Monoclonais/química , Enterovirus Humano A/efeitos dos fármacos , Fragmentos Fab das Imunoglobulinas/química , Glicoproteínas de Membrana Associadas ao Lisossomo/química , Receptores Depuradores/química , Receptores Virais/química , Proteínas Recombinantes de Fusão/química , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Sítios de Ligação , Linhagem Celular , Cristalografia por Raios X , Enterovirus Humano A/genética , Enterovirus Humano A/crescimento & desenvolvimento , Enterovirus Humano A/imunologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/virologia , Expressão Gênica , Células HEK293 , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/metabolismo , Glicoproteínas de Membrana Associadas ao Lisossomo/genética , Glicoproteínas de Membrana Associadas ao Lisossomo/imunologia , Camundongos , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Receptores Depuradores/genética , Receptores Depuradores/imunologia , Receptores Virais/genética , Receptores Virais/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Células Sf9 , Spodoptera , Termodinâmica
15.
Anal Chem ; 89(10): 5373-5381, 2017 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-28414218

RESUMO

Enterovirus 71 (EV71) is a major public health threat that requires rapid point-of-care detection. Here, we developed a surface-enhanced Raman spectroscopy (SERS)-based scheme that utilized protein-induced aggregation of colloidal gold nanostars (AuNS) to rapidly detect EV71 without the need for fabricating a solid substrate, Raman labels or complicated sample handling. We used AuNS (hydrodynamic diameter, DH of 105.12 ± 1.13 nm) conjugated to recombinant scavenger receptor class B, member 2 (SCARB2) protein with known affinity to EV71. In the absence of EV71, AuNS-SCARB2 aggregated in biological media and produced four enhanced Raman peaks at 390, 510, 670, and 910 cm-1. In the presence of EV71, the three peaks at 510, 670, and 910 cm-1 disappeared, while the peak at 390 cm-1 diminished in intensity as the virus bound to AuNS-SCARB2 and prevented them from aggregation. These three peaks (510, 670, and 910 cm-1) were potential markers for specific detection of EV71 as their disappearance was not observable with a different dengue virus (DENV) as our control. Furthermore, the Raman measurements from colloidal SERS were more sensitive in probing the aggregation of AuNS-SCARB2 for detecting the presence of EV71 in protein-rich samples compared to UV-vis spectrum measurements. With this facile "anti-aggregation" approach, we were able to detect EV71 in protein-rich biological medium within 15 min with reasonable sensitivity of 107 pfu/mL and minimal sample preparation, making this translatable for point-of-care applications.


Assuntos
Enterovirus Humano A/isolamento & purificação , Doença de Mão, Pé e Boca/diagnóstico , Nanoestruturas/química , Análise Espectral Raman , Enterovirus Humano A/química , Ouro/química , Doença de Mão, Pé e Boca/virologia , Humanos , Glicoproteínas de Membrana Associadas ao Lisossomo/química , Glicoproteínas de Membrana Associadas ao Lisossomo/genética , Glicoproteínas de Membrana Associadas ao Lisossomo/metabolismo , Ligação Proteica , Receptores Depuradores/química , Receptores Depuradores/genética , Receptores Depuradores/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectrofotometria
16.
Biochim Biophys Acta ; 1860(6): 1118-28, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26922829

RESUMO

BACKGROUND: C-reactive protein (CRP) is a plasma pentraxin family protein that is massively induced as part of the innate immune response to infection and tissue injury. CRP and other pentraxin proteins can activate a complement pathway through C1q, collectins, or on microbe surfaces. It has been found that a lectin-like oxidized LDL receptor 1 (LOX-1), which is an endothelial scavenger receptor (SR) having a C-type lectin-like domain, interacts with CRP to activate the complement pathway using C1q. However it remains elusive whether other lectins or SRs are involved in CRP-mediated complement activation and the downstream effect of the complement activation is also unknown. METHODS: We prepared CHO/ldlA7 cells expressing collectin placenta-1 (CL-P1) and studied the interaction of CRP with cells. We further used ELISA for testing binding between proteins. We tested for C3 fragment deposition and terminal complement complex (TCC) formation on HEK293 cells expressing CL-P1. RESULTS: Here, we demonstrated that CL-P1 bound CRP in a charge dependent manner and the interaction of CRP with CL-P1 mediated a classical complement activation pathway through C1q and additionally drove an amplification pathway using properdin. However, CRP also recruits complement factor H (CFH) on CL-P1 expressing cell surfaces, to inhibit the formation of a terminal complement complex in normal complement serum conditions. GENERAL SIGNIFICANCE: The interaction of collectin CL-P1 with CFH might be key for preventing attack on "self" as a result of complement activation induced by the CL-P1 and CRP interaction.


Assuntos
Proteína C-Reativa/química , Colectinas/química , Ativação do Complemento , Receptores Depuradores/química , Animais , Proteína C-Reativa/fisiologia , Células CHO , Colectinas/fisiologia , Fator H do Complemento/química , Cricetulus , Células HEK293 , Humanos , Receptores Depuradores/fisiologia
17.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 32(5): 723-7, 2015 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-26419000

RESUMO

SCARB2 (scavenger receptor class B, member 2) is a lysosomal membrane glucoprotein, which is encoded by SCARB2 gene. It takes vital parts in the physiological and pathological processes including the transportation of beta-glucocerebrosidase to the lysosome, infection of EV71 and load-induced cardiac myocyte hypertrophy. This article has reviewed the molecular structure and functions of SCARB2 gene and its protein, as well as their relationship with diseases.


Assuntos
Glicoproteínas de Membrana Associadas ao Lisossomo/fisiologia , Receptores Depuradores/fisiologia , Doença de Mão, Pé e Boca/genética , Humanos , Glicoproteínas de Membrana Associadas ao Lisossomo/química , Glicoproteínas de Membrana Associadas ao Lisossomo/genética , Epilepsias Mioclônicas Progressivas/genética , Doença de Parkinson/genética , Receptores Depuradores/química , Receptores Depuradores/genética
18.
J Drug Target ; 23(6): 538-51, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25766080

RESUMO

Recent studies have demonstrated that the carboxyterminal third domain of alpha-fetoprotein (AFP-CD) binds with various ligands and receptors. Reports within the last decade have established that AFP-CD contains a large fragment of amino acids that interact with several different receptor types. Using computer software specifically designed to identify protein-to-protein interaction at amino acid sequence docking sites, the computer searches identified several types of scavenger-associated receptors and their amino acid sequence locations on the AFP-CD polypeptide chain. The scavenger receptors (SRs) identified were CD36, CD163, Stabilin, SSC5D, SRB1 and SREC; the SR-associated receptors included the mannose, low-density lipoprotein receptors, the asialoglycoprotein receptor, and the receptor for advanced glycation endproducts (RAGE). Interestingly, some SR interaction sites were localized on the AFP-derived Growth Inhibitory Peptide (GIP) segment at amino acids #480-500. Following the detection studies, a structural subdomain analysis of both the receptor and the AFP-CD revealed the presence of epidermal growth factor (EGF) repeats, extracellular matrix-like protein regions, amino acid-rich motifs and dimerization subdomains. For the first time, it was reported that EGF-like sequence repeats were identified on each of the three domains of AFP. Thereafter, the localization of receptors on specific cell types were reviewed and their functions were discussed.


Assuntos
Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Receptores Depuradores/química , Receptores Depuradores/metabolismo , alfa-Fetoproteínas/química , alfa-Fetoproteínas/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Humanos , Modelos Moleculares
19.
Biochem Biophys Res Commun ; 457(3): 334-40, 2015 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-25576872

RESUMO

The lysosomal integral membrane protein type-2 (LIMP-2/SCARB2) has been identified as a receptor for enterovirus 71 uptake and mannose-6-phosphate-independent lysosomal trafficking of the acid hydrolase ß-glucocerebrosidase. Here we show that LIMP-2 undergoes proteolytic cleavage mediated by lysosomal cysteine proteases. Heterologous expression and in vitro studies suggest that cathepsin-F is mainly responsible for the lysosomal processing of wild-type LIMP-2. Furthermore, examination of purified lysosomes revealed that LIMP-2 undergoes proteolysis in vivo. Mutations in the gene encoding cathepsin-F (CTSF) have recently been associated with type-B-Kufs-disease, an adult form of neuronal ceroid-lipofuscinosis. In this study we show that disease-causing cathepsin-F mutants fail to cleave LIMP-2. Our findings provide evidence that LIMP-2 represents an in vivo substrate of cathepsin-F with relevance for understanding the pathophysiology of type-B-Kufs-disease.


Assuntos
Catepsina F/genética , Catepsina F/metabolismo , Glicoproteínas de Membrana Associadas ao Lisossomo/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Lipofuscinoses Ceroides Neuronais/genética , Lipofuscinoses Ceroides Neuronais/metabolismo , Receptores Depuradores/metabolismo , Animais , Antígenos CD36/química , Antígenos CD36/genética , Antígenos CD36/metabolismo , Linhagem Celular , Células HEK293 , Humanos , Glicoproteínas de Membrana Associadas ao Lisossomo/química , Glicoproteínas de Membrana Associadas ao Lisossomo/genética , Lisossomos/metabolismo , Camundongos , Modelos Moleculares , Conformação Proteica , Estrutura Secundária de Proteína , Proteólise , Receptores Depuradores/química , Receptores Depuradores/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
20.
Protein Cell ; 5(9): 692-703, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24986489

RESUMO

Unlike the well-established picture for the entry of enveloped viruses, the mechanism of cellular entry of non-enveloped eukaryotic viruses remains largely mysterious. Picornaviruses are representative models for such viruses, and initiate this entry process by their functional receptors. Here we present the structural and functional studies of SCARB2, a functional receptor of the important human enterovirus 71 (EV71). SCARB2 is responsible for attachment as well as uncoating of EV71. Differences in the structures of SCARB2 under neutral and acidic conditions reveal that SCARB2 undergoes a pivotal pH-dependent conformational change which opens a lipid-transfer tunnel to mediate the expulsion of a hydrophobic pocket factor from the virion, a pre-requisite for uncoating. We have also identified the key residues essential for attachment to SCARB2, identifying the canyon region of EV71 as mediating the receptor interaction. Together these results provide a clear understanding of cellular attachment and initiation of uncoating for enteroviruses.


Assuntos
Enterovirus Humano A/metabolismo , Glicoproteínas de Membrana Associadas ao Lisossomo/metabolismo , Receptores Depuradores/metabolismo , Vírion/metabolismo , Ligação Viral , Ácidos/química , Sequência de Aminoácidos , Animais , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Enterovirus Humano A/genética , Enterovirus Humano A/fisiologia , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Concentração de Íons de Hidrogênio , Glicoproteínas de Membrana Associadas ao Lisossomo/química , Glicoproteínas de Membrana Associadas ao Lisossomo/genética , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , RNA Viral/genética , RNA Viral/metabolismo , Receptores Depuradores/química , Receptores Depuradores/genética , Homologia de Sequência de Aminoácidos , Células Sf9 , Eletricidade Estática , Vírion/genética
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