RESUMO
Hsp90 is a molecular chaperone that acts on its clients through an ATP-dependent and conformationally dynamic functional cycle. The cochaperone Accelerator of Hsp90 ATPase, or Ahsa1, is the most potent stimulator of Hsp90 ATPase activity. Ahsa1 stimulates the rate of Hsp90 ATPase activity through a conserved motif, NxNNWHW. Metazoan Ahsa1, but not yeast, possesses an additional 20 amino acid peptide preceding the NxNNWHW motif that we have called the intrinsic chaperone domain (ICD). The ICD of Ahsa1 diminishes Hsp90 ATPase stimulation by interfering with the function of the NxNNWHW motif. Furthermore, the NxNNWHW modulates Hsp90's apparent affinity to Ahsa1 and ATP. Lastly, the ICD controls the regulated recruitment of Hsp90 in cells and its deletion results in the loss of interaction with Hsp90 and the glucocorticoid receptor. This work provides clues to how Ahsa1 conserved regions modulate Hsp90 kinetics and how they may be coupled to client folding status.
Assuntos
Adenosina Trifosfatases , Proteínas de Choque Térmico HSP90 , Proteínas de Choque Térmico HSP90/metabolismo , Adenosina Trifosfatases/metabolismo , Humanos , Ligação Proteica , Sequência Conservada , Motivos de Aminoácidos , Animais , Peptídeos/metabolismo , Peptídeos/farmacologia , Peptídeos/química , Sequência de Aminoácidos , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/genética , Trifosfato de Adenosina/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Receptores de Glucocorticoides/metabolismoRESUMO
In an effort to develop potent anti-influenza drugs that inhibit the activity of influenza virus RNA-dependent RNA polymerase (IAV RdRp), a database of nucleoside triphosphates with ~800 molecules were docked with the homology model of IAV RdRp from A/PR/8/34/H1N1 strain. Out of top 12 molecules that bind with higher affinities to the catalytic site of IAV RdRp above and below the PB1 priming loop, only seven molecules decreased the transcriptional activity of the viral RNA polymerase with an IC50 in the range of 0.09-3.58 µM. Molecular docking combining with experimental study indicated that the molecules with linear chain are more effective in inhibiting IAV RdRp replication than the molecules with V-shaped and are cyclic in nature. A correlation between ΔG and LogIC50 for these seven compounds resulted an R 2 value of 0.73. Overall, these newly developed seven nucleoside triphosphates lay a strong foundation for the future development of a new therapeutics that can satisfy the Lipinski's rule of five exhibiting high specificity to the catalytic site of influenza-A viruses.
RESUMO
Cytopathic effects are currently believed to contribute to hepatitis C virus (HCV)-induced liver injury and are readily observed in Huh7.5 cells infected with the JFH-1 HCV strain, manifesting as apoptosis highly correlated with growth arrest. Reactive oxygen species, which are induced by HCV infection, have recently emerged as activators of AMP-activated protein kinase. The net effect is ATP conservation via on/off switching of metabolic pathways that produce/consume ATP. Depending on the scenario, this can have either pro-survival or pro-apoptotic effects. We demonstrate reactive oxygen species-mediated activation of AMP-activated kinase in Huh7.5 cells during HCV (JFH-1)-induced growth arrest. Metabolic labeling experiments provided direct evidence that lipid synthesis is attenuated, and ß-oxidation is enhanced in these cells. A striking increase in nuclear peroxisome proliferator-activated receptor α, which plays a dominant role in the expression of ß-oxidation genes after ligand-induced activation, was also observed, and we provide evidence that peroxisome proliferator-activated receptor α is constitutively activated in these cells. The combination of attenuated lipid synthesis and enhanced ß-oxidation is not conducive to lipid accumulation, yet cellular lipids still accumulated during this stage of infection. Notably, the serum in the culture media was the only available source for polyunsaturated fatty acids, which were elevated (2-fold) in the infected cells, implicating altered lipid import/export pathways in these cells. This study also provided the first in vivo evidence for enhanced ß-oxidation during HCV infection because HCV-infected SCID/Alb-uPA mice accumulated higher plasma ketones while fasting than did control mice. Overall, this study highlights the reprogramming of hepatocellular lipid metabolism and bioenergetics during HCV infection, which are predicted to impact both the HCV life cycle and pathogenesis.
Assuntos
Carcinoma Hepatocelular/metabolismo , Ácidos Graxos/metabolismo , Hepacivirus/fisiologia , Hepatite C/metabolismo , Lipídeos/biossíntese , Neoplasias Hepáticas/metabolismo , Mitocôndrias/metabolismo , Estresse Oxidativo , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/virologia , Hepatite C/virologia , Humanos , Fígado/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/virologia , Camundongos , Camundongos SCID , Mitocôndrias/genética , Oxirredução , PPAR alfa/genética , PPAR alfa/metabolismoRESUMO
Influenza is a major cause of morbidity and mortality in immunosuppressed persons, and vaccination often confers insufficient protection. IL-28B, a member of the interferon (IFN)-λ family, has variable expression due to single nucleotide polymorphisms (SNPs). While type-I IFNs are well known to modulate adaptive immunity, the impact of IL-28B on B- and T-cell vaccine responses is unclear. Here we demonstrate that the presence of the IL-28B TG/GG genotype (rs8099917, minor-allele) was associated with increased seroconversion following influenza vaccination (OR 1.99 pâ=â0.038). Also, influenza A (H1N1)-stimulated T- and B-cells from minor-allele carriers showed increased IL-4 production (4-fold) and HLA-DR expression, respectively. In vitro, recombinant IL-28B increased Th1-cytokines (e.g. IFN-γ), and suppressed Th2-cytokines (e.g. IL-4, IL-5, and IL-13), H1N1-stimulated B-cell proliferation (reduced 70%), and IgG-production (reduced>70%). Since IL-28B inhibited B-cell responses, we designed antagonistic peptides to block the IL-28 receptor α-subunit (IL28RA). In vitro, these peptides significantly suppressed binding of IFN-λs to IL28RA, increased H1N1-stimulated B-cell activation and IgG-production in samples from healthy volunteers (2-fold) and from transplant patients previously unresponsive to vaccination (1.4-fold). Together, these findings identify IL-28B as a key regulator of the Th1/Th2 balance during influenza vaccination. Blockade of IL28RA offers a novel strategy to augment vaccine responses.
Assuntos
Imunidade Adaptativa/efeitos dos fármacos , Linfócitos B/patologia , Vírus da Influenza A Subtipo H1N1/imunologia , Vacinas contra Influenza/farmacologia , Influenza Humana/patologia , Interleucinas/fisiologia , Linfócitos T/patologia , Imunidade Adaptativa/imunologia , Imunidade Adaptativa/fisiologia , Adulto , Idoso , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Proliferação de Células , Feminino , Antígenos HLA-DR/metabolismo , Humanos , Hospedeiro Imunocomprometido , Imunoglobulina G/metabolismo , Técnicas In Vitro , Vacinas contra Influenza/imunologia , Influenza Humana/metabolismo , Influenza Humana/prevenção & controle , Interferons , Interleucina-4/metabolismo , Interleucinas/genética , Interleucinas/farmacologia , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único/genética , Proteínas Recombinantes/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Células Th1/patologia , Células Th2/patologia , TransplantadosRESUMO
UNLABELLED: Although effective hepatitis C virus (HCV) antivirals are on the horizon, a global prophylactic vaccine for HCV remains elusive. The diversity of the virus is a major concern for vaccine development; there are 7 major genotypes of HCV found globally. Therefore, a successful vaccine will need to protect against HCV infection by all genotypes. Despite the diversity, many monoclonal antibodies (MAbs) with broadly cross-neutralizing activity have been described, suggesting the presence of conserved epitopes that can be targeted to prevent infection. Similarly, a vaccine comprising recombinant envelope glycoproteins (rE1E2) derived from the genotype 1a HCV-1 strain has been shown to be capable of eliciting cross-neutralizing antibodies in guinea pigs, chimpanzees, and healthy human volunteers. In order to investigate the basis for this cross-neutralization, epitope mapping of anti-E1E2 antibodies present within antisera from goats and humans immunized with HCV-1 rE1E2 was conducted through peptide mapping and competition studies with a panel of cross-neutralizing MAbs targeting various epitopes within E1E2. The immunized-goat antiserum was shown to compete with the binding of all MAbs tested (AP33, HC33.4, HC84.26, 1:7, AR3B, AR4A, AR5A, IGH526, and A4). Antisera showed the best competition against HC84.26 and AR3B and the weakest competition against AR4A. Furthermore, antisera from five immunized human vaccinees were shown to compete with five preselected MAbs (AP33, AR3B, AR4A, AR5A, and IGH526). These data show that immunization with HCV-1 rE1E2 elicits antibodies targeting multiple cross-neutralizing epitopes. Our results further support the use of such a vaccine antigen to induce cross-genotype neutralization. IMPORTANCE: An effective prophylactic vaccine for HCV is needed for optimal control of the disease burden. The high diversity of HCV has posed a challenge for developing vaccines that elicit neutralizing antibodies for protection against infection. Despite this, we have previously shown that a vaccine comprising recombinant envelope glycoproteins derived from a single genotype 1a strain was capable of eliciting a cross-neutralizing antibody response in human volunteers. Here, we have used competition binding assays and peptide binding assays to show that antibodies present in the antisera from vaccinated goats and humans bind epitopes overlapping with those of a variety of well-characterized cross-neutralizing monoclonal antibodies. This provides a mechanism for the cross-neutralizing human antisera: antibodies present in the antisera bind to conserved regions associated with cross-neutralization. Importantly, this work provides further support for a vaccine comprising recombinant envelope glycoproteins, perhaps in a formulation with a vaccine component eliciting strong anti-HCV CD4(+) and CD8(+) T cell responses.
Assuntos
Anticorpos Neutralizantes/sangue , Reações Cruzadas , Hepacivirus/imunologia , Anticorpos Anti-Hepatite C/sangue , Proteínas do Envelope Viral/imunologia , Vacinas Virais/imunologia , Animais , Mapeamento de Epitopos , Epitopos/imunologia , Genótipo , Cabras , Hepacivirus/classificação , Hepacivirus/genética , Antígenos da Hepatite C/genética , Antígenos da Hepatite C/imunologia , Humanos , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Proteínas do Envelope Viral/genética , Vacinas Virais/administração & dosagem , Vacinas Virais/genéticaRESUMO
BACKGROUND: Feedback mechanisms between interferons α and λ (IFNs) may be affected by single nucleotide polymorphisms (SNP) in interleukin 28B (IL-28B; IFN-λ3) promoter region and may influence cytomegalovirus (CMV) replication. METHODS: We associated IL-28B SNPs with the risk of CMV replication after transplantation. Next, we examined the effect of IL-28B genotypes on IL-28B, and IFN-stimulated gene (ISG) expression, and CMV replication in human foreskin fibroblast (HFF) and peripheral blood mononuclear cells (PBMCs). RESULTS: Transplant recipients with an IL-28B SNP (rs8099917) had significantly less CMV replication (P = .036). Both HFF-cells and PBMCs with a SNP showed lower IL-28B expression during infection with CMV, but higher "antiviral" ISG expression (eg, OAS1). Fibroblasts with a SNP had a 3-log reduction of CMV replication at day 4 (P = .004). IL-28B pretreatment induced ISG expression in noninfected fibroblasts, but a relative decrease of ISG expression could be observed in CMV-infected fibroblasts. The inhibitory effects of IL-28B could be abolished by siRNA or antagonistic peptides against the IL-28 receptor. In fibroblasts, inhibition of IL-28 signaling resulted in an increase of ISG expression and 3-log reduction of CMV-replication (P = .01). CONCLUSIONS: We postulate that IL-28B may act as a key regulator of ISG expression during primary CMV infection. IL-28B SNPs may be associated with higher antiviral ISG expression, which results in better replication control.
Assuntos
Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/imunologia , Citomegalovirus/imunologia , Interleucinas/genética , Interleucinas/imunologia , Adulto , Idoso , Células Cultivadas , Citomegalovirus/fisiologia , Feminino , Fibroblastos/virologia , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Interferons , Leucócitos Mononucleares/virologia , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Transplante , Replicação ViralRESUMO
The proteolipid code determines how cytosolic proteins find and remodel membrane surfaces. Here, we investigate how this process works with sorting nexins Snx1 and Snx3. Both proteins form sorting machines by recognizing membrane zones enriched in phosphatidylinositol 3-phosphate (PI3P), phosphatidylserine (PS) and cholesterol. This co-localized combination forms a unique "lipid codon" or lipidon that we propose is responsible for endosomal targeting, as revealed by structures and interactions of their PX domain-based readers. We outline a membrane recognition and remodeling mechanism for Snx1 and Snx3 involving this code element alongside transmembrane pH gradients, dipole moment-guided docking and specific protein-protein interactions. This generates an initial membrane-protein assembly (memtein) that then recruits retromer and additional PX proteins to recruit cell surface receptors for sorting to the trans-Golgi network (TGN), lysosome and plasma membranes. Post-translational modification (PTM) networks appear to regulate how the sorting machines form and operate at each level. The commonalities and differences between these sorting nexins show how the proteolipid code orchestrates parallel flows of molecular information from ribosome emergence to organelle genesis, and illuminates a universally applicable model of the membrane.
Assuntos
Proteínas de Transporte , Proteínas de Transporte Vesicular , Proteínas de Transporte/química , Proteínas de Transporte Vesicular/metabolismo , Nexinas de Classificação/metabolismo , Transporte Proteico , Proteolipídeos/metabolismoRESUMO
Growth differentiation factor 15 (GDF15) is a peptide with utility in obesity, as it decreases appetite and promotes weight loss. Because obesity increases the risk for type 2 diabetes (T2D) and cardiovascular disease, it is imperative to understand the cardiovascular actions of GDF15, especially since elevated GDF15 levels are an established biomarker for heart failure. As weight loss should be encouraged in the early stages of obesity-related prediabetes/T2D, where diabetic cardiomyopathy is often present, we assessed whether treatment with GDF15 influences its pathology. We observed that GDF15 treatment alleviates diastolic dysfunction in mice with T2D independent of weight loss. This cardioprotection was associated with a reduction in cardiac inflammation, which was likely mediated via indirect actions, as direct treatment of adult mouse cardiomyocytes and differentiated THP-1 human macrophages with GDF15 failed to alleviate lipopolysaccharide-induced inflammation. Therapeutic manipulation of GDF15 action may thus have utility for both obesity and diabetic cardiomyopathy.
Assuntos
Cardiomiopatias Diabéticas , Fator 15 de Diferenciação de Crescimento , Miócitos Cardíacos , Fator 15 de Diferenciação de Crescimento/metabolismo , Animais , Cardiomiopatias Diabéticas/metabolismo , Cardiomiopatias Diabéticas/patologia , Cardiomiopatias Diabéticas/tratamento farmacológico , Camundongos , Humanos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Camundongos Endogâmicos C57BL , Masculino , Diástole/efeitos dos fármacos , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/complicações , Inflamação/patologia , Inflamação/metabolismo , Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Células THP-1 , Obesidade/metabolismo , Lipopolissacarídeos/farmacologiaRESUMO
Endoplasmic reticulum (ER) stress is a homeostatic mechanism, which is used by cells to adapt to intercellular and intracellular changes. Moreover, ER stress is closely linked to inflammatory pathways. We hypothesized that ER stress is an integral component of neuroinflammation and contributes to the development of neurological diseases. In autopsied brain specimens from multiple sclerosis (MS) and non-MS patients, XBP-1 spliced variant (XBP-1/s) was increased in MS brains (p < 0.05) and was correlated with the expression of the human endogenous retrovirus-W envelope transcript, which encodes the glycoprotein, Syncytin-1 (p < 0.05). In primary human fetal astrocytes transfected with a Syncytin-1-expressing plasmid, XBP-1/s, BiP, and NOS2 were induced, which was suppressed by crocin treatment (p < 0.05). Crocin also protected oligodendrocytes exposed to cytotoxic supernatants derived from Syncytin-1-expressing astrocytes (p < 0.05) and NO-mediated oligodendrocytotoxicity (p < 0.05). During experimental autoimmune encephalomyelitis (EAE), the transcript levels of the ER stress genes XBP-1/s, BiP, PERK, and CHOP were increased in diseased spinal cords compared with healthy littermates (p < 0.05), although CHOP expression was not involved in the EAE disease phenotype. Daily treatment with crocin starting on day 7 post-EAE induction suppressed ER stress and inflammatory gene expression in spinal cords (p < 0.05), which was accompanied by preserved myelination and axonal density, together with reduced T cell infiltration and macrophage activation. EAE-associated neurobehavioral deficits were also ameliorated by crocin treatment (p < 0.05). These findings underscored the convergent roles of pathogenic ER stress and immune pathways in neuroinflammatory disease and point to potential therapeutic applications for crocin.
Assuntos
Carotenoides/uso terapêutico , Doenças Desmielinizantes/prevenção & controle , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/patologia , Mediadores da Inflamação/uso terapêutico , Doenças Neurodegenerativas/prevenção & controle , Animais , Carotenoides/administração & dosagem , Células Cultivadas , Doenças Desmielinizantes/patologia , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/patologia , Retículo Endoplasmático/efeitos dos fármacos , Feminino , Sequestradores de Radicais Livres/uso terapêutico , Lobo Frontal/efeitos dos fármacos , Lobo Frontal/metabolismo , Lobo Frontal/patologia , Humanos , Mediadores da Inflamação/administração & dosagem , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Doenças Neurodegenerativas/patologia , Ratos , Ratos Sprague-DawleyRESUMO
Variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are emerging rapidly and offer surfaces that are optimized for recognition of host cell membranes while also evading antibodies arising from vaccinations and previous infections. Host cell infection is a multi-step process in which spike heads engage lipid bilayers and one or more angiotensin-converting enzyme 2 (ACE-2) receptors. Here, the membrane binding surfaces of Omicron subvariants are compared using cryo-electron microscopy (cEM) structures of spike trimers from BA.2, BA.2.12.1, BA.2.13, BA.2.75, BA.3, BA.4, and BA.5 viruses. Despite significant differences around mutated sites, they all maintain strong membrane binding propensities that first appeared in BA.1. Both their closed and open states retain elevated membrane docking capacities, although the presence of more closed than open states diminishes opportunities to bind receptors while enhancing membrane engagement. The electrostatic dipoles are generally conserved. However, the BA.2.75 spike dipole is compromised, and its ACE-2 affinity is increased, and BA.3 exhibits the opposite pattern. We propose that balancing the functional imperatives of a stable, readily cleavable spike that engages both lipid bilayers and receptors while avoiding host defenses underlies betacoronavirus evolution. This provides predictive criteria for rationalizing future pandemic waves and COVID-19 transmissibility while illuminating critical sites and strategies for simultaneously combating multiple variants.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Microscopia Crioeletrônica , Bicamadas Lipídicas , Anticorpos , Membrana CelularRESUMO
Structures can now be predicted for any protein using programs like AlphaFold and Rosetta, which rely on a foundation of experimentally determined structures of architecturally diverse proteins. The accuracy of such artificial intelligence and machine learning (AI/ML) approaches benefits from the specification of restraints which assist in navigating the universe of folds to converge on models most representative of a given protein's physiological structure. This is especially pertinent for membrane proteins, with structures and functions that depend on their presence in lipid bilayers. Structures of proteins in their membrane environments could conceivably be predicted from AI/ML approaches with user-specificized parameters that describe each element of the architecture of a membrane protein accompanied by its lipid environment. We propose the Classification Of Membrane Proteins based On Structures Engaging Lipids (COMPOSEL), which builds on existing nomenclature types for monotopic, bitopic, polytopic and peripheral membrane proteins as well as lipids. Functional and regulatory elements are also defined in the scripts, as shown with membrane fusing synaptotagmins, multidomain PDZD8 and Protrudin proteins that recognize phosphoinositide (PI) lipids, the intrinsically disordered MARCKS protein, caveolins, the ß barrel assembly machine (BAM), an adhesion G-protein coupled receptor (aGPCR) and two lipid modifying enzymes - diacylglycerol kinase DGKε and fatty aldehyde dehydrogenase FALDH. This demonstrates how COMPOSEL communicates lipid interactivity as well as signaling mechanisms and binding of metabolites, drug molecules, polypeptides or nucleic acids to describe the operations of any protein. Moreover COMPOSEL can be scaled to express how genomes encode membrane structures and how our organs are infiltrated by pathogens such as SARS-CoV-2.
Assuntos
COVID-19 , Proteínas de Membrana , Humanos , Proteínas de Membrana/química , Lipídeos de Membrana , Inteligência Artificial , Modelos Moleculares , SARS-CoV-2/metabolismo , Bicamadas Lipídicas/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismoRESUMO
During Plasmodium falciparum infection in pregnancy, VAR2CSA is expressed on the surface of infected erythrocytes (IEs) and mediates their sequestration in the placenta. As a result, antibodies to VAR2CSA are largely restricted to women who were infected during pregnancy. However, we discovered that VAR2CSA antibodies can also be elicited by P. vivax Duffy binding protein (PvDBP). We proposed that infection with P. vivax in non-pregnant individuals can generate antibodies that cross-react with VAR2CSA. To better understand the specificity of these antibodies, we took advantage of a mouse monoclonal antibody (3D10) raised against PvDBP that cross-reacts with VAR2CSA and identified the epitopes targeted by this antibody. We screened two peptide arrays that span the ectodomain of VAR2CSA from the FCR3 and NF54 alleles. Based on the top epitope recognized by 3D10, we designed a 34-amino acid synthetic peptide, which we call CRP1, that maps to a highly conserved region in DBL3X. Specific lysine residues are critical for 3D10 recognition, and these same amino acids are within a previously defined chondroitin sulfate A (CSA) binding site in DBL3X. We showed by isothermal titration calorimetry that the CRP1 peptide can bind directly to CSA, and antibodies to CRP1 raised in rats significantly blocked the binding of IEs to CSA in vitro. In our Colombian cohorts of pregnant and non-pregnant individuals, at least 45% were seroreactive to CRP1. Antibody reactivities to CRP1 and the 3D10 natural epitope in PvDBP region II, subdomain 1 (SD1), were strongly correlated in both cohorts. These findings suggest that antibodies arising from PvDBP may cross-react with VAR2CSA through the epitope in CRP1 and that CRP1 could be a potential vaccine candidate to target a distinct CSA binding site in VAR2CSA.
Assuntos
Malária Falciparum , Malária Vivax , Gravidez , Camundongos , Feminino , Ratos , Animais , Plasmodium vivax , Epitopos , Plasmodium falciparum/química , Anticorpos Antiprotozoários , Antígenos de Protozoários , Malária Falciparum/metabolismo , Placenta , Sulfatos de Condroitina/metabolismo , Eritrócitos , Ligação ProteicaRESUMO
Endoplasmic reticulum (ER) homeostasis requires molecular regulators that tailor mitochondrial bioenergetics to the needs of protein folding. For instance, calnexin maintains mitochondria metabolism and mitochondria-ER contacts (MERCs) through reactive oxygen species (ROS) from NADPH oxidase 4 (NOX4). However, induction of ER stress requires a quick molecular rewiring of mitochondria to adapt to new energy needs. This machinery is not characterized. We now show that the oxidoreductase ERO1⺠covalently interacts with protein kinase RNA-like ER kinase (PERK) upon treatment with tunicamycin. The PERK-ERO1⺠interaction requires the C-terminal active site of ERO1⺠and cysteine 216 of PERK. Moreover, we show that the PERK-ERO1⺠complex promotes oxidization of MERC proteins and controls mitochondrial dynamics. Using proteinaceous probes, we determined that these functions improve ER-mitochondria Ca2+ flux to maintain bioenergetics in both organelles, while limiting oxidative stress. Therefore, the PERK-ERO1⺠complex is a key molecular machinery that allows quick metabolic adaptation to ER stress.
Assuntos
Mitocôndrias , Oxirredutases , Oxirredutases/metabolismo , Mitocôndrias/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Retículo Endoplasmático/metabolismo , Estresse OxidativoRESUMO
Human endogenous retroviruses (HERVs) constitute 5-8% of human genomic DNA and are replication incompetent despite expression of individual HERV genes from different chromosomal loci depending on the specific tissue. Several HERV genes have been detected as transcripts and proteins in the central nervous system, frequently in the context of neuroinflammation. The HERV-W family has received substantial attention in large part because of associations with diverse syndromes including multiple sclerosis (MS) and several psychiatric disorders. A HERV-W-related retroelement, multiple sclerosis retrovirus (MSRV), has been reported in MS patients to be both a biomarker as well as an effector of aberrant immune responses. HERV-H and HERV-K have also been implicated in MS and other neurological diseases but await delineation of their contributions to disease. The HERV-W envelope-encoded glycosylated protein, syncytin-1, is encoded by chromosome 7q21 and exhibits increased glial expression within MS lesions. Overexpression of syncytin-1 in glia induces endoplasmic reticulum stress leading to neuroinflammation and the induction of free radicals, which damage proximate cells. Syncytin-1's receptor, ASCT1 is a neutral amino acid transporter expressed on glia and is suppressed in white matter of MS patients. Of interest, antioxidants ameliorate syncytin-1's neuropathogenic effects raising the possibility of using these agents as therapeutics for neuroinflammatory diseases. Given the multiple insertion sites of HERV genes as complete and incomplete open reading frames, together with their differing capacity to be expressed and the complexities of individual HERVs as both disease markers and bioactive effectors, HERV biology is a compelling area for understanding neuropathogenic mechanisms and developing new therapeutic strategies.
Assuntos
Retrovirus Endógenos/patogenicidade , Esclerose Múltipla/etiologia , Sequência de Aminoácidos , Animais , Encefalomielite Autoimune Experimental/etiologia , Retrovirus Endógenos/classificação , Retrovirus Endógenos/genética , Retículo Endoplasmático/metabolismo , Feminino , Produtos do Gene env/genética , Produtos do Gene env/metabolismo , Humanos , Masculino , Camundongos , Modelos Biológicos , Dados de Sequência Molecular , Esclerose Múltipla/genética , Esclerose Múltipla/patologia , Esclerose Múltipla/virologia , Filogenia , Gravidez , Proteínas da Gravidez/genética , Proteínas da Gravidez/metabolismo , Receptores Virais/metabolismo , Homologia de Sequência de Aminoácidos , Estresse Fisiológico , Resposta a Proteínas não DobradasRESUMO
The human microbiome is comprised of commensal and pathogenic microorganisms, which exert diverse effects in close proximity to the site of intection as well as in remote tissues through immune-mediated mechanisms. Multiple infectious agents have been implicated in the pathogenesis of multiple sclerosis (MS) with variable findings depending on the agent, techniques, and disease phenotype. Herein, the contributions of individual infectious agents to MS and their effects on the immune and nervous systems are reviewed, focusing on herpes viruses, coronaviruses, retroviruses, and synchronic infections. While infectious agents are often assumed to be pathogenic, their effects might also be beneficial to the host in the long-term, depending on age and the type of immunogen/pathogen exposure, as proposed by the hygiene hypothesis. The human microbiome has potential impact on future diagnostic and therapeutic issues in MS.
Assuntos
Viroses do Sistema Nervoso Central/complicações , Sistema Nervoso Central , Metagenoma , Esclerose Múltipla/microbiologia , Animais , Sistema Nervoso Central/imunologia , Sistema Nervoso Central/microbiologia , Sistema Nervoso Central/patologia , Humanos , Metagenoma/genética , Modelos Biológicos , Esclerose Múltipla/genética , Esclerose Múltipla/patologiaRESUMO
Invariant natural killer T (iNKT) cells are innate lymphocytes whose functions are regulated by self and foreign glycolipid antigens presented by the antigen-presenting molecule CD1d. Activation of iNKT cells in vivo results in rapid release of copious amounts of effector cytokines and chemokines with which they regulate innate and adaptive immune responses to pathogens, certain types of cancers and self-antigens. The nature of CD1d-restricted antigens, the manner in which they are recognised and the unique effector functions of iNKT cells suggest an innate immunoregulatory role for this subset of T cells. Their ability to respond fast and our ability to steer iNKT cell cytokine response to altered lipid antigens make them an important target for vaccine design and immunotherapies against autoimmune diseases. This review summarises our current understanding of CD1d-restricted antigen presentation, the recognition of such antigens by an invariant T-cell receptor on iNKT cells, and the functional consequences of these interactions.
Assuntos
Antígenos CD1/fisiologia , Antígenos/química , Glicolipídeos/química , Animais , Apresentação de Antígeno , Antígenos CD1/imunologia , Antígenos CD1d , Autoantígenos/química , Doenças Autoimunes/metabolismo , Membrana Celular/metabolismo , Humanos , Sistema Imunitário , Células Matadoras Naturais/metabolismo , Modelos Biológicos , Neoplasias/metabolismo , Fenótipo , Receptores de Antígenos de Linfócitos T/metabolismoRESUMO
The 5S subunit of transcarboxylase was expressed and purified. Recent methods of NMR spectroscopy as transferred NOESY, INPHARMA and Saturation Transfer Difference (STD) NMR were used to investigate ligand binding of free biotin to the 5S protein. The binding epitope for biotin is very similar to that obtained at the 12S subunit of transcarboxylase, however no common binding site for pyruvate and biotin exists.
Assuntos
Biotina/metabolismo , Carboxil e Carbamoil Transferases/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Biotina/química , Carboxil e Carbamoil Transferases/química , Carboxil e Carbamoil Transferases/genética , Estrutura Molecular , Propionibacterium/enzimologia , Ligação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
INTRODUCTION: To date, various therapeutic strategies identified numerous anti-prion compounds and antibodies that stabilize PrPC, block the conversion of PrPC-PrPSc and increased effect on PrPSc clearance. However, no suitable drug has been identified clinically so far due to the poor oral absorption, low blood-brain-barrier [BBB] penetration, and high toxicity. Although some of the drugs were proven to be effective in prion-infected cell culture and whole animal models, none of them increased the rate of survival compared to placebo. Areas covered: In this review, the authors highlight the importance of in silico approaches like molecular docking, virtual screening, pharmacophore analysis, molecular dynamics, QSAR, CoMFA and CoMSIA applied to detect molecular mechanisms of prion inhibition and conversion from PrPC-PrPSc. Expert opinion: Several in silico approaches combined with experimental studies have provided many structural and functional clues on the stability and physiological activity of prion mutants. Further, various studies of in silico and in vivo approaches were also shown to identify several new small organic anti-scrapie compounds to decrease the accumulation of PrPres in cell culture, inhibit the aggregation of a PrPC peptide, and possess pharmacokinetic characteristics that confirm the drug-likeness of these compounds.
Assuntos
Simulação por Computador , Desenho de Fármacos , Doenças Priônicas/tratamento farmacológico , Animais , Barreira Hematoencefálica/metabolismo , Modelos Animais de Doenças , Humanos , Simulação de Acoplamento Molecular , Proteínas PrPC/efeitos dos fármacos , Proteínas PrPC/metabolismo , Proteínas PrPSc/antagonistas & inibidores , Proteínas PrPSc/efeitos dos fármacos , Proteínas PrPSc/metabolismo , Taxa de Sobrevida , Distribuição TecidualRESUMO
Type III interferons (IFN-lambdas) are important antiviral cytokines that also modulate immune responses acting through a unique IFN-λR1/IL-10R2 heterodimeric receptor. Conflicting data has been reported for which cells express the IFN-λR1 subunit and directly respond to IFN-λs. In this study we developed a novel method to measure IFN-λ3 binding to IFN-λR1/IL-10R2 on the surface of cells and relate this to a functional readout of interferon stimulated gene (ISG) activity in various cell lines. We show that Huh7.5 hepatoma cells bind IFN-λ3 at the highest levels with the lowest Kd(app), translating to the highest induction of various ISGs. Raji and Jurkat cell lines, representing B and T cells, respectively, moderately bind IFN-λ3 and have lower ISG responses. U937 cells, representing monocytes, did not bind IFN-λ3 well and therefore, did not have any ISG induction. Importantly, knockdown of IFNLR1 in Huh7.5 cells decreased our binding signal proportionally and reduced ISG induction by up to 93%. IFN-λ3 responsiveness increased over time with maximal ISG responses seen at 24h for all but one gene. These data confirm our new IFN-λ3 binding assay can be used to quantify IFN-λ receptor surface expression on a variety of cell types and reflects IFN-λ3 responsiveness.
Assuntos
Citometria de Fluxo , Interleucinas/análise , Receptores de Interferon/genética , Sítios de Ligação , Linhagem Celular Tumoral , Humanos , Interferons , Interleucinas/imunologia , Receptores de Interferon/imunologiaRESUMO
Acquired cardiac long QT syndrome (LQTS) is a frequent drug-induced toxic event that is often caused through blocking of the human ether-á-go-go-related (hERG) K(+) ion channel. This has led to the removal of several major drugs post-approval and is a frequent cause of termination of clinical trials. We report here a computational atomistic model derived using long molecular dynamics that allows sensitive prediction of hERG blockage. It identified drug-mediated hERG blocking activity of a test panel of 18 compounds with high sensitivity and specificity and was experimentally validated using hERG binding assays and patch clamp electrophysiological assays. The model discriminates between potent, weak, and non-hERG blockers and is superior to previous computational methods. This computational model serves as a powerful new tool to predict hERG blocking thus rendering drug development safer and more efficient. As an example, we show that a drug that was halted recently in clinical development because of severe cardiotoxicity is a potent inhibitor of hERG in two different biological assays which could have been predicted using our new computational model.