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.
Amino Acid Sequence ; Animals ; Antibodies, Monoclonal ; chemistry ; genetics ; metabolism ; Binding Sites ; Cell Line ; Crystallography, X-Ray ; Enterovirus A, Human ; drug effects ; genetics ; growth & development ; immunology ; Fibroblasts ; drug effects ; virology ; Gene Expression ; HEK293 Cells ; Humans ; Immunoglobulin Fab Fragments ; chemistry ; genetics ; metabolism ; Lysosome-Associated Membrane Glycoproteins ; chemistry ; genetics ; immunology ; Mice ; Models, Molecular ; Protein Binding ; Protein Conformation, alpha-Helical ; Protein Conformation, beta-Strand ; Protein Interaction Domains and Motifs ; Receptors, Scavenger ; chemistry ; genetics ; immunology ; Receptors, Virus ; chemistry ; genetics ; immunology ; Recombinant Fusion Proteins ; chemistry ; genetics ; immunology ; Sequence Alignment ; Sequence Homology, Amino Acid ; Sf9 Cells ; Spodoptera ; Thermodynamics

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.
Hand, Foot and Mouth Disease ; genetics ; Humans ; Lysosome-Associated Membrane Glycoproteins ; chemistry ; genetics ; physiology ; Myoclonic Epilepsies, Progressive ; genetics ; Parkinson Disease ; genetics ; Receptors, Scavenger ; chemistry ; genetics ; physiology

Uch37 is a de-ubiquitinating enzyme that is activated by Rpn13 and involved in the proteasomal degradation of proteins. The full-length Uch37 was shown to exhibit low iso-peptidase activity and is thought to be auto-inhibited. Structural comparisons revealed that within a homo-dimer of Uch37, each of the catalytic domains was blocking the other's ubiquitin (Ub)-binding site. This blockage likely prevented Ub from entering the active site of Uch37 and might form the basis of auto-inhibition. To understand the mode of auto-inhibition clearly and shed light on the activation mechanism of Uch37 by Rpn13, we investigated the Uch37-Rpn13 complex using a combination of mutagenesis, biochemical, NMR, and small-angle X-ray scattering (SAXS) techniques. Our results also proved that Uch37 oligomerized in solution and had very low activity against the fluorogenic substrate ubiquitin-7-amino-4-methylcoumarin (Ub-AMC) of de-ubiquitinating enzymes. Uch37Δ(Hb,Hc,KEKE), a truncation removal of the C-terminal extension region (residues 256-329) converted oligomeric Uch37 into a monomeric form that exhibited iso-peptidase activity comparable to that of a truncation-containing the Uch37 catalytic domain only. We also demonstrated that Rpn13C (Rpn13 residues 270-407) could disrupt the oligomerization of Uch37 by sequestering Uch37 and forming a Uch37-Rpn13 complex. Uch37 was activated in such a complex, exhibiting 12-fold-higher activity than Uch37 alone. Time-resolved SAXS (TR-SAXS) and FRET experiments supported the proposed mode of auto-inhibition and the activation mechanism of Uch37 by Rpn13. Rpn13 activated Uch37 by forming a 1:1 stoichiometric complex in which the active site of Uch37 was accessible to Ub.
Binding Sites ; Catalytic Domain ; Chromatography, Gel ; Crystallography, X-Ray ; Humans ; Membrane Glycoproteins ; chemistry ; genetics ; metabolism ; Nuclear Magnetic Resonance, Biomolecular ; Protein Binding ; Protein Conformation ; Protein Multimerization ; Scattering, Small Angle ; Ubiquitin Thiolesterase ; chemistry ; genetics ; metabolism ; Ultracentrifugation

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.
Acids ; chemistry ; Amino Acid Sequence ; Animals ; Capsid Proteins ; chemistry ; genetics ; metabolism ; Enterovirus A, Human ; genetics ; metabolism ; physiology ; HEK293 Cells ; Host-Pathogen Interactions ; Humans ; Hydrogen-Ion Concentration ; Lysosome-Associated Membrane Glycoproteins ; chemistry ; genetics ; metabolism ; Molecular Docking Simulation ; Molecular Sequence Data ; Protein Binding ; Protein Conformation ; Protein Interaction Mapping ; Protein Structure, Tertiary ; RNA, Viral ; genetics ; metabolism ; Receptors, Scavenger ; chemistry ; genetics ; metabolism ; Sequence Homology, Amino Acid ; Sf9 Cells ; Static Electricity ; Virion ; genetics ; metabolism ; Virus Attachment

Chronic granulomatous disease (CGD) is a rare genetic disease, which is caused by defects in the NADPH oxidase complex (gp91phox, p22phox, p40phox, p47phox, and p67phox) of phagocytes. This defect results in impaired production of superoxide anions and other reactive oxygen species (ROS), which are necessary for killing bacterial and fungal microorganisms and leads to recurrent, life-threatening bacterial and fungal infections and granulomatous inflammation. The dihydrorhodamine (DHR) flow cytometry assay is a useful diagnostic tool for CGD that can detect absent or reduced NADPH oxidase activity in stimulated phagocytes. We report a patient with X-linked CGD carrying a novel mutation of the CYBB gene whose chimerism status following hematopoietic stem cell transplantation (HSCT) has been rapidly determined using the DHR assay. The level of DHR activity correlates well with short tandem repeat PCR analysis. Considering the advantages of this simple, rapid, and cost-effective procedure, serial measurement of DHR assay would facilitate the rapid determination of a patient's engraftment status, as a supplementary monitoring tool of chimerism status following HSCT.
Base Sequence ; *Chimerism ; DNA Mutational Analysis ; Flow Cytometry ; Granulomatous Disease, Chronic/*diagnosis/*enzymology/genetics/surgery ; *Hematopoietic Stem Cell Transplantation ; Homozygote ; Humans ; Infant, Newborn ; Male ; Membrane Glycoproteins/chemistry/*genetics ; Mutation ; NADPH Oxidase/chemistry/*genetics ; Polymerase Chain Reaction ; Rhodamines/chemistry/metabolism

The spike (S) glycoprotein of HCoV-NL63 is a major target in the development of diagnostic assays and vaccines, but its antigenic and immunogenic properties remain unclear. Four fragments coding spike proteins (S1, S2, RL and RS) from HCoV-NL63 were amplified and cloned into the expression vector derived from vaccinia virus (Tiantan strain), and recombinant vaccinia viruses expressing four segments of spike proteins were generated (vJSC1175-S1; vJSC1175-S2; vJSC1175-RL; vJSC1175-RS), respectively. Their expression location in cell and level were characterized using indirect immune fluorescence assay (IFA) and Western-Blot, respectively. The expressions of four segments of spike proteins in recombinant vaccinia viruses were showed at appropriate level and with posttranslational modification (glycosylation), and S1, RL and RS were mainly distributed in the cell membrane, while the S2 was mainly distributed in the cytoplasm. Our results provide a basis for further exploring diagnostic role and vaccine development of different spike segments from HCoV-NL63.
Base Sequence ; Blotting, Western ; Coronavirus NL63, Human ; chemistry ; Fluorescent Antibody Technique, Indirect ; Humans ; Membrane Glycoproteins ; biosynthesis ; genetics ; Molecular Sequence Data ; Plasmids ; Recombinant Proteins ; biosynthesis ; Spike Glycoprotein, Coronavirus ; Vaccinia virus ; genetics ; Viral Envelope Proteins ; biosynthesis ; genetics

Animals ; Antigens, CD ; chemistry ; genetics ; metabolism ; Antiviral Agents ; chemistry ; metabolism ; GPI-Linked Proteins ; HIV Infections ; metabolism ; virology ; HIV-1 ; genetics ; physiology ; Human Immunodeficiency Virus Proteins ; genetics ; metabolism ; Humans ; Membrane Glycoproteins ; chemistry ; genetics ; metabolism ; Protein Binding ; Viral Regulatory and Accessory Proteins ; genetics ; metabolism ; Virus Shedding

Phagocytes such as neutrophils play a vital role in host defense against microbial pathogens. The anti-microbial function of neutrophils is based on the production of superoxide anion (O2(.-)), which generates other microbicidal reactive oxygen species (ROS) and release of antimicrobial peptides and proteins. The enzyme responsible for O2(.-) production is called the NADPH oxidase or respiratory burst oxidase. This multicomponent enzyme system is composed of two transmembrane proteins (p22phox and gp91phox, also called NOX2, which together form the cytochrome b(558)) and four cytosolic proteins (p47phox, p67phox, p40phox and a GTPase Rac1 or Rac2), which assemble at membrane sites upon cell activation. NADPH oxidase activation in phagocytes can be induced by a large number of soluble and particulate agents. This process is dependent on the phosphorylation of the cytosolic protein p47phox. p47phox is a 390 amino acids protein with several functional domains: one phox homology (PX) domain, two src homology 3 (SH3) domains, an auto-inhibitory region (AIR), a proline rich domain (PRR) and has several phosphorylated sites located between Ser303 and Ser379. In this review, we will describe the structure of p47phox, its phosphorylation and discuss how these events regulate NADPH oxidase activation.
*Disease ; Enzyme Activation ; Humans ; Membrane Glycoproteins/chemistry/*metabolism ; NADPH Oxidase/chemistry/genetics/*metabolism ; Phagocytes/cytology/*metabolism ; Phosphorylation ; Protein Conformation

Carrier Proteins ; genetics ; Cloning, Molecular ; DNA, Complementary ; Humans ; Liver ; chemistry ; Membrane Glycoproteins ; genetics

The S1 gene hypervariable region I (HVR I) of 22 infectious bronchitis virus (IBV) strains isolated in Guangxi during the period of 1985-2007 were sequenced and compared to that of the other IBV reference strains and the pigeon coronavirus isolates. A phylogenetic tree based on nucleotide sequences of HVR I of all the IBV showed that they were classified into 5 distinct Clusters. 16 out of 22 IBV isolates were grouped into Cluster I, and had higher homology with pigeon coronavirus isolates but lower homology with the Massachusetts (Mass) type vaccine strains. There were 4 and 3 amino-acid residues inserted at the sites of 33-34 and 34-35 respectively within HVR I in 15 isolates, except in isolate GX-NN6 there had 4 amino-acid residues inserted at the both sites; isolates GX-YL1 and GX-NN2 had close relationship with Mass type vaccine strains, and they shared Cluster II; isolates GX-G and GX-XD of Cluster III had close relationship with the Japanese strain JP Miyazaki 89 which was isolated at the same period; isolates GX-YL6 and GX-NN7 of Cluster V had close relationship with the European strain 4/91. The results showed that there were high phylogenetic diversity among the IBVs prevailed in the field in Guangxi resulting from the commonly occurred mutation or insertion within the S1 gene HVR I of the viruses, and majority of the isolates had lower homology with the commonly used Mass type vaccine strains. There was much higher homology among viruses isolated in the same period of time, but without distinct difference in geographical origins.
Amino Acid Sequence ; Animals ; Chickens ; virology ; Genetic Variation ; Infectious bronchitis virus ; classification ; genetics ; isolation & purification ; Membrane Glycoproteins ; chemistry ; genetics ; Molecular Sequence Data ; Phylogeny ; Spike Glycoprotein, Coronavirus ; Viral Envelope Proteins ; chemistry ; genetics