Bitopic membrane topology of the stable signal peptide in the tripartite Junín virus GP-C envelope glycoprotein complex

The stable signal peptide (SSP) of the GP-C envelope glycoprotein of the Junín arenavirus plays a critical role in trafficking of the GP-C complex to the cell surface and in its membrane fusion activity. SSP therefore may function on both sides of the lipid membrane. In this study, we have investigated the membrane topology of SSP by confocal microscopy of cells treated with the detergent digitonin to selectively permeabilize the plasma membrane. By using an affinity tag to mark the termini of SSP in the properly assembled GP-C complex, we find that both the N and C termini reside in the cytosol. Thus, SSP adopts a bitopic topology in which the C terminus is translocated from the lumen of the endoplasmic reticulum to the cytoplasm. This model is supported by (i) the presence of two conserved hydrophobic regions in SSP (hphi1 and hphi2) and (ii) our previous demonstration that lysine-33 in the ectodomain loop is essential for pH-dependent membrane fusion. Moreover, we demonstrate that the introduction of a charged side chain or single amino acid deletion in the membrane-spanning hphi2 region significantly diminishes SSP association in the GP-C complex and abolishes membrane fusion activity. Taken together, our results suggest that bitopic membrane insertion of SSP is centrally important in the assembly and function of the tripartite GP-C complex.     

Role of the stable signal peptide of Junín arenavirus envelope glycoprotein in pH-dependent membrane fusion

The envelope glycoprotein of the arenaviruses (GP-C) is unusual in that the mature complex retains the cleaved, 58-amino-acid signal peptide. Association of this stable signal peptide (SSP) has been shown to be essential for intracellular trafficking and proteolytic maturation of the GP-C complex. We identify here a specific and previously unrecognized role of SSP in pH-dependent membrane fusion. Amino acid substitutions that alter the positive charge at lysine K33 in SSP affect the ability of GP-C to mediate cell-cell fusion and the threshold pH at which membrane fusion is triggered. Based on the presumed location of K33 at or near the luminal domain of SSP, we postulate that SSP interacts with the membrane-proximal or transmembrane regions of the G2 fusion protein. This unique organization of the GP-C complex may suggest novel strategies for intervention in arenavirus infection.     

A novel zinc-binding domain is essential for formation of the functional Junín virus envelope glycoprotein complex

The envelope glycoprotein of the Junín arenavirus (GP-C) mediates entry into target cells through a pH-dependent membrane fusion mechanism. Unlike other class I viral fusion proteins, the mature GP-C complex retains a cleaved, 58-amino-acid signal peptide (SSP) as an essential subunit, required both for trafficking of GP-C to the cell surface and for the activation of membrane fusion. SSP has been shown to associate noncovalently in GP-C via the cytoplasmic domain (CTD) of the transmembrane fusion subunit G2. In this report we investigate the molecular basis for this intersubunit interaction. We identify an invariant series of six cysteine and histidine residues in the CTD of G2 that is essential for incorporation of SSP in the GP-C complex. Moreover, we show that a CTD peptide fragment containing His-447, His-449, and Cys-455 specifically binds Zn(2+) at subnanomolar concentrations. Together, these results suggest a zinc finger-like domain structure in the CTD of G2. We propose that the remaining residues in the series (His-459, Cys-467, and Cys-469) form an intersubunit zinc-binding center that incorporates Cys-57 of SSP. This unusual motif may act to retain SSP in the GP-C complex and position the ectodomain loop of SSP for its role in modulating membrane fusion activity. The unique tripartite organization of GP-C could provide novel molecular targets for therapeutic intervention in arenaviral disease.     

The signal peptide of the Junín arenavirus envelope glycoprotein is myristoylated and forms an essential subunit of the mature G1-G2 complex

Arenaviruses comprise a diverse family of rodent-borne viruses that are responsible for recurring and emerging outbreaks of viral hemorrhagic fevers worldwide. The Junín virus, a member of the New World arenaviruses, is endemic to the pampas grasslands of Argentina and is the etiologic agent of Argentine hemorrhagic fever. In this study, we have analyzed the assembly and function of the Junín virus envelope glycoproteins. The mature envelope glycoprotein complex is proteolytically processed from the GP-C precursor polypeptide and consists of three noncovalently associated subunits, G1, G2, and a stable 58-amino-acid signal peptide. This tripartite organization is found both on virions of the attenuated Candid 1 strain and in cells expressing the pathogenic MC2 strain GP-C gene. Replacement of the Junín virus GP-C signal peptide with that of human CD4 has little effect on glycoprotein assembly while abolishing the ability of the G1-G2 complex to mediate pH-dependent cell-cell fusion. In addition, we demonstrate that the Junín virus GP-C signal peptide subunit is myristoylated at its N-terminal glycine. Alanine substitution for the modified glycine residue in the GP-C signal peptide does not affect formation of the tripartite envelope glycoprotein complex but markedly reduces its membrane fusion activity. In contrast to the classical view that signal peptides act primarily in targeting nascent polypeptides to the endoplasmic reticulum, we suggest that the signal peptide of the arenavirus GP-C may serve additional functions in envelope glycoprotein structure and trafficking.     

A specific interaction of small molecule entry inhibitors with the envelope glycoprotein complex of the Junín hemorrhagic fever arenavirus

Arenaviruses are responsible for acute hemorrhagic fevers worldwide and are recognized to pose significant threats to public health and biodefense. Small molecule compounds have recently been discovered that inhibit arenavirus entry and protect against lethal infection in animal models. These chemically distinct inhibitors act on the tripartite envelope glycoprotein (GPC) through its unusual stable signal peptide subunit to stabilize the complex against pH-induced activation of membrane fusion in the endosome. Here, we report the production and characterization of the intact transmembrane GPC complex of Junín arenavirus and its interaction with these inhibitors. The solubilized GPC is antigenically indistinguishable from the native protein and forms a homogeneous trimer in solution. When reconstituted into a lipid bilayer, the purified complex interacts specifically with its cell-surface receptor transferrin receptor-1. We show that small molecule entry inhibitors specific to New World or Old World arenaviruses bind to the membrane-associated GPC complex in accordance with their respective species selectivities and with dissociation constants comparable with concentrations that inhibit GPC-mediated membrane fusion. Furthermore, competitive binding studies reveal that these chemically distinct inhibitors share a common binding pocket on GPC. In conjunction with previous genetic studies, these findings identify the pH-sensing interface of GPC as a highly vulnerable target for antiviral intervention. This work expands our mechanistic understanding of arenavirus entry and provides a foundation to guide the development of small molecule compounds for the treatment of arenavirus hemorrhagic fevers.     

Role of gelatinases in pathological and physiological processes involving the dystrophin–glycoprotein complex

Dystrophin is a cytosolic protein belonging to a membrane-spanning glycoprotein complex, called dystrophin–glycoprotein complex (DGC) that is expressed in many tissues, especially in skeletal muscle and in the nervous system. The DGC connects the cytoskeleton to the extracellular matrix and, although none of the proteins of the DGC displays kinase or phosphatase activity, it is involved in many signal transduction pathways. Mutations in some components of the DGC are linked to many forms of inherited muscular dystrophies. In particular, a mutation in the dystrophin gene, leading to a complete loss of the protein, provokes one of the most prominent muscular dystrophies, the Duchenne muscular dystrophy, which affects 1 out of 3500 newborn males. What is observed in these circumstances, is a dramatic alteration of the expression levels of a multitude of metalloproteinases (MMPs), a family of extracellular Zn²⁺-dependent endopeptidases, in particular of MMP-2 and MMP-9, also called gelatinases. Indeed, the enzymatic activity of MMP-2 and MMP-9 on dystroglycan, an important member of the DGC, plays a significant role also in physiological processes taking place in the central and peripheral nervous system. This mini-review discusses the role of MMP-2 and MMP-9, in physiological as well as pathological processes involving members of the DGC.     

Membrane localization of Junín virus glycoproteins requires cholesterol and cholesterol rich membranes

Arenavirus morphogenesis and budding occurs at cellular plasma membrane; however, the nature of membrane assembly sites remains poorly understood. In this study we examined the effect of different cholesterol-lowering agents on Junín virus (JUNV) multiplication. We found that cholesterol cell depletion reduced JUNV glycoproteins (GPs) membrane expression and virus budding. Analysis of membrane protein insolubility in Triton X-100 suggested that JUNV GPs associate with cholesterol enriched membranes. Rafts dissociation conditions as warm detergent extraction and cholesterol removal by methyl-β-cyclodextrin compound showed to impair GPs cholesterol enriched membrane association. Analysis of GPs transfected cells showed similar results suggesting that membrane raft association is independent of other viral proteins.     

Junín virus infection activates the type I interferon pathway in a RIG-I-dependent manner

Junín virus (JUNV), an arenavirus, is the causative agent of Argentine hemorrhagic fever, an infectious human disease with 15-30% case fatality. The pathogenesis of AHF is still not well understood. Elevated levels of interferon and cytokines are reported in AHF patients, which might be correlated to the severity of the disease. However the innate immune response to JUNV infection has not been well evaluated. Previous studies have suggested that the virulent strain of JUNV does not induce IFN in human macrophages and monocytes, whereas the attenuated strain of JUNV was found to induce IFN response in murine macrophages via the TLR-2 signaling pathway. In this study, we investigated the interaction between JUNV and IFN pathway in human epithelial cells highly permissive to JUNV infection. We have determined the expression pattern of interferon-stimulated genes (ISGs) and IFN-β at both mRNA and protein levels during JUNV infection. Our results clearly indicate that JUNV infection activates the type I IFN response. STAT1 phosphorylation, a downstream marker of activation of IFN signaling pathway, was readily detected in JUNV infected IFN-competent cells. Our studies also demonstrated for the first time that RIG-I was required for IFN production during JUNV infection. IFN activation was detected during infection by either the virulent or attenuated vaccine strain of JUNV. Curiously, both virus strains were relatively insensitive to human IFN treatment. Our studies collectively indicated that JUNV infection could induce host type I IFN response and provided new insights into the interaction between JUNV and host innate immune system, which might be important in future studies on vaccine development and antiviral treatment.     

Ultrastructure of Junín Virus in Mouse Whole Brain and Mouse Brain Tissue Cultures

Comparative ultrastructural studies were performed on the development of Junín virus in mouse brain and in cerebellum explants and brain monolayers of the same animal. In mouse brain, neurons and astrocytes released virus particles by a budding mechanism identical to that previously described for this virus. In the neurons, the viral multiplication took place in the perikarion as well as in the cytoplasmic processes, including areas near synapses. Viral particles were observed emerging from pericapillary neurons and astrocytes. In the explants, the budding also occurred in neurons and astrocytes. In the monolayers, however, the virus originated in astrocytes and cells of fibroblastic appearance, which were the two cell types that developed in this substrate. These results indicate that the characteristics of the development of Junín virus in mouse brain are faithfully reproduced in cerebellum explants from the same animal, thus allowing some extrapolation of data from one system to the other. The explant proved to be a better model than the monolayer, not only because it reproduced the structural complexity of nervous tissue better, but also because it contains neurons and astrocytes, i.e., the two cell types that release the virus in the in vivo system.     

Watching Every Step of the Way: Junín Virus Attenuation Markers in the Vaccine Lineage

The Arenaviridae family includes several hemorrhagic fever viruses which are important emerging pathogens. Junín virus, a member of this family, is the etiological agent of Argentine Hemorrhagic Fever (AHF). A collaboration between the Governments of Argentina and the USA rendered the attenuated Junín virus vaccine strain Candid#1. Arenaviruses are enveloped viruses with genomes consisting of two single-stranded RNA species (L and S), each carrying two coding regions separated by a stably structured, non-coding intergenic region. Molecular characterization of the vaccine strain and of its more virulent ancestors, XJ13 (prototype) and XJ#44, allows a systematic approach for the discovery of key elements in virulence attenuation. We show comparisons of sequence information for the S RNA of the strains XJ13, XJ#44 and Candid#1 of Junín virus, along with other strains from the vaccine lineage and a set of Junín virus field strains collected at the AHF endemic area. Comparisons of nucleotide and amino acid sequences revealed different point mutations which might be linked to the attenuated phenotype. The majority of changes are consistent with a progressive attenuation of virulence between XJ13, XJ#44 and Candid#1. We propose that changes found in genomic regions with low natural variation frequencies are more likely to be associated with the virulence attenuation process. We partially sequenced field strains to analyze the genomic variability naturally occurring for Junín virus. This information, together with the sequence analysis of strains with intermediate virulence, will serve as a starting point to study the molecular bases for viral attenuation.     

Identification of structural motifs in the E2 glycoprotein of Chikungunya involved in virus–host interaction

Chikungunya fever is one of the reemerging vector-borne diseases. It has become a major global health problem especially in the developing countries. There are no vaccines or specific antiviral drugs available to date. This study reports small molecule inhibitors of envelope glycoprotein 2 (E2 glycoprotein) which are predicted based on Chikungunya virus–host interactions. E2 glycoprotein of Chikungunya virus interacts at 216 residue of the host receptor protein which plays a vital role in initiating infection. Understanding the structural aspects of E2 glycoprotein is crucial to develop specific inhibitors to prevent the virus binding from host receptors. In silico method was adopted to predict the sequence motifs of envelope protein, as the method like yeast two hybrid system is laborious, time consuming, and costly. The E2 glycoprotein structure of the Indian isolate was modeled using two templates (2XFC and 3JOC) and then validated. The class III PDZ domain binding motif was found to be identified at 213–216 amino acids. The corresponding peptide structures which recognize the PDZ domain binding motif were identified by the literature search and were used for generating five point pharmacophore model (ADDDR) containing acceptor, donor and aromatic ring features. Databases such as Asinex, TosLab and Maybridge were searched for the matches for the predicted pharmacophore model. Two compounds were identified as lead molecules as their glide score is?>?5?kcal/mol. Since the pharmacophore model is developed based on Chikungunya virus–host interaction, it can be used for designing promising antiviral lead compounds for the treatment of Chikungunya fever.An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:21     

Role of Ca2+ and calmodulin-dependent enzymes in the regulation of glycine transport in Müller glia

Glycine (Gly) is considered an obligatory co-agonist at NMDA receptors. Müller glia from the retina harbor functional NMDA receptors, as well as low and high affinity Gly transporters, the later identified as GLYT1. We here studied the regulation of Gly transport in primary cultures of Müller glia, as this process could contribute to the modulation of NMDA receptor activity at glutamatergic synapses in the retina. We demonstrate that neither glutamate stimulation nor the activation or inhibition of protein kinases A or C modify transport. In order to assess a function for Ca2+ and calmodulin (CaM)-dependent processes in the regulation of Gly transport, we explored the participation of Ca2+ concentration, CaM and Ca2+/CaM-dependent enzymes on Gly transporter activity. ATP and carbachol, known to induce Ca2+ waves in Müller cells, as well as caffeine-induced Ca2+ release from intracellular stores stimulated transport, whereas Ca2+ chelation by BAPTA-AM markedly reduced transport. CaM inhibitors W-7, ophiobolin A, R-24571 and trifluoperazine, induced a specific dose-dependent inhibition of transport. The inhibition of CaMKII by the autocamtide-2-related inhibitory peptide or by KN62 caused a decrease in transport which, in the case of KN62, was due to the abolition of the high affinity component, ascribed to GLYT1. Our results further suggest that Gly transport is under cytoskeletal control, as activation of calpain by major increases in [Ca2+]i induced by ionophores, as well as actin destabilization clearly inhibit uptake. We here demonstrate for the first time the participation of CaM, CaMKII and the actin cytoskeleton in the regulation of Gly transport in glia. Ca2+ waves are induced in Müller cells by distinct neuroactive compounds released by neurons and glia, hence the regulation of [Gly] by this system may be of physiological relevance in the control of retinal excitability.     

A model of O·2 - generation in the complex III of the electron transport chain

Oxidation of semiquinone by O₂ in the Q cycle is known to be one of the sources of superoxide anion (O·₂ ^-) in aerobic cells. In this paper, such a phenomenon was analyzed using the chemical kinetics model of electron transfer from succinate to cytochrome c, including coenzyme Q, the complex III non-heme iron protein FeS_III and cytochromes b₁, b_h and c₁. Electron transfers from QH₂ to FeS_III and cytochrome b₁ were assumed to occur according to direct transfer mechanism (dynamic channelling) involving the formation of FeS^red _III -Q·^- and Q·^--cytochrome b₁ complexes. For oxidation/reduction reactions involving cytochromes b_h and b₁, the dependence of the equilibrium and elementary rate constants on the membrane potential () was taken into consideration. The rate of O·₂ ^- generation was found to increase dramatically with increase in above the values found in State 3. On the other hand, the rate of cytochrome c reduction decreased sharply at the same values of the membrane potential. This explains experimental data that the O·2^- generation at State 4 appears to be very much faster than at State 3. A mild uncoupling in State 4 can markedly decrease the superoxide generation due to a decrease in below the above mentioned critical level. pH appears to be equally effective as in stimulation of superoxide production which depends, in fact, upon the ^- _H + level.     

Screening of integrin-binding peptides from the laminin α4 and α5 chain G domain peptide library

Laminins, a multifunctional protein family of extracellular matrix, interact with various types of integrin. Here, integrin-mediated cell adhesive peptides have been systematically screened in the laminin α4 and α5 chain G domain peptide library consisting of 211 peptides by both the peptide-coated plastic plates and peptide-conjugated Sepharose bead assays using human dermal fibroblasts. Thirteen peptides promoted cell spreading and the activity was specifically inhibited by EDTA. Cell attachment to 11 peptides was inhibited by anti-integrin β1 antibody. Additionally, cell attachment to the A5G81 (AGQWHRVSVRWG) and A5G84 (TWSQKALHHRVP) peptides was specifically inhibited by anti-integrin α3 and α6 antibodies. These results suggest that the A5G81 and A5G84 peptides promote integrin α3β1- and α6β1-mediated cell attachment. Further, most of the integrin-mediated cell adhesive peptides are located in the loop regions in the G domains, suggesting that structure is important for the integrin specific recognition. Integrin binding peptides are useful for understanding laminin functions and have a potential to use for biomaterials and drug development.     

Function of the syndecan-4 cytoplasmic domain in oligomerization and association with α-actinin in turkey muscle satellite cells

Syndecan-4 (S4) is a cell membrane heparan sulfate proteoglycan that plays a role in satellite cell mediated myogenesis. S4 modulates the proliferation of myogenic satellite cells, but the mechanism of how S4 functions during myogenesis is not well understood. In other cell systems, S4 has been shown to form oligomers in the cell membrane and interact through its cytoplasmic domain with the cytoskeletal protein α-actinin. This study addressed if S4 forms oligomers and interacts with α-actinin in muscle. The S4 cytoplasmic domain was found to interact with α-actinin in a phosphatidylinositol-4,5-bisphosphate dependent manner, but did not associate with vinculin. Through confocal microscopy, both S4 and syndecan-4 without the cytoplasmic domain were localized to the cell membrane. Although the cytoplasmic domain was necessary for the interaction with α-actinin, S4 oligomer formation occurred in the absence of the cytoplasmic domain. These data indicated that S4 function in skeletal muscle is mediated through the formation of oligomers and interaction with the cytoskeletal protein α-actinin.     

Pivotal Role of Extended Linker 2 in the Activation of Gα by G Protein-coupled Receptor

G protein-coupled receptors (GPCRs) relay extracellular signals mainly to heterotrimeric G-proteins (Gαβγ) and they are the most successful drug targets. The mechanisms of G-protein activation by GPCRs are not well understood. Previous studies have revealed a signal relay route from a GPCR via the C-terminal α5-helix of Gα to the guanine nucleotide-binding pocket. Recent structural and biophysical studies uncover a role for the opening or rotating of the α-helical domain of Gα during the activation of Gα by a GPCR. Here we show that β-adrenergic receptors activate eight Gα_s mutant proteins (from a screen of 66 Gα_s mutants) that are unable to bind Gβγ subunits in cells. Five of these eight mutants are in the αF/Linker 2/β2 hinge region (extended Linker 2) that connects the Ras-like GTPase domain and the α-helical domain of Gα_s. This extended Linker 2 is the target site of a natural product inhibitor of G_q. Our data show that the extended Linker 2 is critical for Gα activation by GPCRs. We propose that a GPCR via its intracellular loop 2 directly interacts with the β₂/β₃ loop of Gα to communicate to Linker 2, resulting in the opening and closing of the α-helical domain and the release of GDP during G-protein activation.     

Structure and function of the C-terminal domain of MrpA in the Bacillus subtilis Mrp-antiporter complex – The evolutionary progenitor of the long horizontal helix in complex I

MrpA and MrpD are homologous to NuoL, NuoM and NuoN in complex I over the first 14 transmembrane helices. In this work, the C-terminal domain of MrpA, outside this conserved area, was investigated. The transmembrane orientation was found to correspond to that of NuoJ in complex I. We have previously demonstrated that the subunit NuoK is homologous to MrpC. The function of the MrpA C-terminus was tested by expression in a previously used Bacillus subtilis model system. At neutral pH, the truncated MrpA still worked, but at pH 8.4, where Mrp-complex formation is needed for function, the C-terminal domain of MrpA was absolutely required.     

Conformational transitions regulate the exposure of a DNA-binding domain in the RuvBL1–RuvBL2 complex

RuvBL1 and RuvBL2, also known as Pontin and Reptin, are AAA+ proteins essential in small nucleolar ribonucloprotein biogenesis, chromatin remodelling, nonsense-mediated messenger RNA decay and telomerase assembly, among other functions. They are homologous to prokaryotic RuvB, forming single- and double-hexameric rings; however, a DNA binding domain II (DII) is inserted within the AAA+ core. Despite their biological significance, questions remain regarding their structure. Here, we report cryo-electron microscopy structures of human double-ring RuvBL1–RuvBL2 complexes at ∼15 Å resolution. Significantly, we resolve two coexisting conformations, compact and stretched, by image classification techniques. Movements in DII domains drive these conformational transitions, extending the complex and regulating the exposure of DNA binding regions. DII domains connect with the AAA+ core and bind nucleic acids, suggesting that these conformational changes could impact the regulation of RuvBL1–RuvBL2 containing complexes. These findings resolve some of the controversies in the structure of RuvBL1–RuvBL2 by revealing a mechanism that extends the complex by adjustments in DII.     

Role of A β and the α 7 nicotinic acetylcholine receptor in regulating synaptic plasticity in Alzheimer's disease

Alzheimer's disease (AD) is caused by the accumulation of β-amyloid protein (Aβ) in the brain. The aggregation of β-amyloid protein to higher molecular weight fibrillar forms is also considered to be an important step in the pathogenesis of the disease. The memory problems associated with AD are likely to be caused by changes in synaptic plasticity. Recent studies suggest that Aβ binds to the α 7 nicotinic acetylcholine receptor (α 7 nAChR), which plays an important role in synaptic plasticity and memory. A loop domain localized towards the C-terminus of the extracellular region of the receptor has been identified as forming part of a putative Aβ-binding site. In cell culture experiments, the binding of Aβ to the α 7 nAChR has been found to cause an increase in the level of acetylcholinesterase, which is also increased around amyloid plaques in the AD brain. These studies indicate that the Aβ-binding site on the α 7 nAChR receptor is an important new target for therapeutic development in AD.     

Dissociation of the α-subunit Calf-2 domain and the β-subunit I-EGF4 domain in integrin activation and signaling

Integrin conformational changes mediate integrin activation and signaling triggered by intracellular molecules or extracellular ligands. Even though it is known that αβ transmembrane domain separation is required for integrin signaling, it is still not clear how this signal is transmitted from the transmembrane domain through two long extracellular legs to the ligand-binding headpiece. This study addresses whether the separation of the membrane-proximal extracellular αβ legs is critical for integrin activation and outside-in signaling. Using a disulfide bond to restrict dissociation of the α-subunit Calf-2 domain and β-subunit I-EGF4 domain, we were able to abolish integrin inside-out activation and outside-in signaling. In contrast, disrupting the interface by introducing a glycosylation site into either subunit activated integrins for ligand binding through a global conformational change. Our results suggest that the interface of the Calf-2 domain and the I-EGF4 domain is critical for integrin bidirectional signaling.