RESUMEN
After the global spread of the SARS-CoV-2 Omicron BA.2, some BA.2 subvariants, including BA.2.9.1, BA.2.11, BA.2.12.1, BA.4, and BA.5, emerged in multiple countries. Our statistical analysis showed that the effective reproduction numbers of these BA.2 subvariants are greater than that of the original BA.2. Neutralization experiments revealed that the immunity induced by BA.1/2 infections is less effective against BA.4/5. Cell culture experiments showed that BA.2.12.1 and BA.4/5 replicate more efficiently in human alveolar epithelial cells than BA.2, and particularly, BA.4/5 is more fusogenic than BA.2. We further provided the structure of the BA.4/5 spike receptor-binding domain that binds to human ACE2 and considered how the substitutions in the BA.4/5 spike play roles in ACE2 binding and immune evasion. Moreover, experiments using hamsters suggested that BA.4/5 is more pathogenic than BA.2. Our multiscale investigations suggest that the risk of BA.2 subvariants, particularly BA.4/5, to global health is greater than that of original BA.2.
Asunto(s)
Enzima Convertidora de Angiotensina 2 , COVID-19 , Anticuerpos Antivirales , Humanos , Peptidil-Dipeptidasa A/genética , Peptidil-Dipeptidasa A/metabolismo , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismoRESUMEN
Soon after the emergence and global spread of the SARS-CoV-2 Omicron lineage BA.1, another Omicron lineage, BA.2, began outcompeting BA.1. The results of statistical analysis showed that the effective reproduction number of BA.2 is 1.4-fold higher than that of BA.1. Neutralization experiments revealed that immunity induced by COVID vaccines widely administered to human populations is not effective against BA.2, similar to BA.1, and that the antigenicity of BA.2 is notably different from that of BA.1. Cell culture experiments showed that the BA.2 spike confers higher replication efficacy in human nasal epithelial cells and is more efficient in mediating syncytia formation than the BA.1 spike. Furthermore, infection experiments using hamsters indicated that the BA.2 spike-bearing virus is more pathogenic than the BA.1 spike-bearing virus. Altogether, the results of our multiscale investigations suggest that the risk of BA.2 to global health is potentially higher than that of BA.1.
Asunto(s)
COVID-19 , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Animales , COVID-19/virología , Cricetinae , Células Epiteliales , Humanos , SARS-CoV-2/genética , SARS-CoV-2/patogenicidad , Glicoproteína de la Espiga del Coronavirus/genéticaRESUMEN
Type I Interferons (IFNs) are important cytokines for innate immunity against viruses and cancer. Sixteen human type I IFN variants signal through the same cell-surface receptors, IFNAR1 and IFNAR2, yet they can evoke markedly different physiological effects. The crystal structures of two human type I IFN ternary signaling complexes containing IFNα2 and IFNω reveal recognition modes and heterotrimeric architectures that are unique among the cytokine receptor superfamily but conserved between different type I IFNs. Receptor-ligand cross-reactivity is enabled by conserved receptor-ligand "anchor points" interspersed among ligand-specific interactions that "tune" the relative IFN-binding affinities, in an apparent extracellular "ligand proofreading" mechanism that modulates biological activity. Functional differences between IFNs are linked to their respective receptor recognition chemistries, in concert with a ligand-induced conformational change in IFNAR1, that collectively control signal initiation and complex stability, ultimately regulating differential STAT phosphorylation profiles, receptor internalization rates, and downstream gene expression patterns.
Asunto(s)
Interferón Tipo I/química , Interferón-alfa/química , Receptores de Interferón/metabolismo , Secuencia de Aminoácidos , Línea Celular Tumoral , Cristalografía por Rayos X , Humanos , Interferón Tipo I/metabolismo , Interferón-alfa/metabolismo , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
Interferons (IFNs) are essential for defense against viral infections but also drive recruitment of inflammatory cells to sites of infection, a key feature of severe COVID-19. Here, we explore the complexity of the IFN response in COVID-19, examine the effects of manipulating IFN on SARS-CoV-2 viral replication and pathogenesis, and highlight pre-clinical and clinical studies evaluating the therapeutic efficacy of IFN in limiting COVID-19 severity.
Asunto(s)
Antivirales , Tratamiento Farmacológico de COVID-19 , COVID-19 , Interferones , SARS-CoV-2 , Animales , Humanos , Antivirales/uso terapéutico , COVID-19/inmunología , COVID-19/virología , COVID-19/terapia , Interferones/uso terapéutico , Interferones/inmunología , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/inmunología , Replicación Viral/efectos de los fármacosRESUMEN
IMPORTANCE: Most studies investigating the characteristics of emerging SARS-CoV-2 variants have been focusing on mutations in the spike proteins that affect viral infectivity, fusogenicity, and pathogenicity. However, few studies have addressed how naturally occurring mutations in the non-spike regions of the SARS-CoV-2 genome impact virological properties. In this study, we proved that multiple SARS-CoV-2 Omicron BA.2 mutations, one in the spike protein and another downstream of the spike gene, orchestrally characterize this variant, shedding light on the importance of Omicron BA.2 mutations out of the spike protein.
Asunto(s)
Genoma Viral , Mutación , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Humanos , COVID-19/virología , SARS-CoV-2/genética , SARS-CoV-2/patogenicidad , SARS-CoV-2/fisiología , Glicoproteína de la Espiga del Coronavirus/genética , Genoma Viral/genéticaRESUMEN
Type I Interferons (IFN-Is) are a family of cytokines which play a major role in inhibiting viral infection. Resultantly, many viruses have evolved mechanisms in which to evade the IFN-I response. Here we tested the impact of expression of 27 different SARS-CoV-2 genes in relation to their effect on IFN production and activity using three independent experimental methods. We identified six gene products; NSP6, ORF6, ORF7b, NSP1, NSP5 and NSP15, which strongly (>10-fold) blocked MAVS-induced (but not TRIF-induced) IFNß production. Expression of the first three of these SARS-CoV-2 genes specifically blocked MAVS-induced IFNß-promoter activity, whereas all six genes induced a collapse in IFNß mRNA levels, corresponding with suppressed IFNß protein secretion. Five of these six genes furthermore suppressed MAVS-induced activation of IFNλs, however with no effect on IFNα or IFNγ production. In sharp contrast, SARS-CoV-2 infected cells remained extremely sensitive to anti-viral activity exerted by added IFN-Is. None of the SARS-CoV-2 genes were able to block IFN-I signaling, as demonstrated by robust activation of Interferon Stimulated Genes (ISGs) by added interferon. This, despite the reduced levels of STAT1 and phospho-STAT1, was likely caused by broad translation inhibition mediated by NSP1. Finally, we found that a truncated ORF7b variant that has arisen from a mutant SARS-CoV-2 strain harboring a 382-nucleotide deletion associating with mild disease (Δ382 strain identified in Singapore & Taiwan in 2020) lost its ability to suppress type I and type III IFN production. In summary, our findings support a multi-gene process in which SARS-CoV-2 blocks IFN-production, with ORF7b as a major player, presumably facilitating evasion of host detection during early infection. However, SARS-CoV-2 fails to suppress IFN-I signaling thus providing an opportunity to exploit IFN-Is as potential therapeutic antiviral drugs.
Asunto(s)
Interferón beta/metabolismo , SARS-CoV-2/inmunología , Proteínas Virales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Animales , Chlorocebus aethiops , Factor 2 Eucariótico de Iniciación/metabolismo , Células HEK293 , Humanos , Interferón beta/genética , Interferón beta/farmacología , SARS-CoV-2/efectos de los fármacos , Factor de Transcripción STAT1/metabolismo , Células Vero , Proteínas Virales/genéticaRESUMEN
[This corrects the article DOI: 10.1371/journal.ppat.1009800.].
RESUMEN
The formation of specific protein-protein interactions (PPIs) drive most biological processes. Malfunction of such interactions is the molecular driver of many diseases. Our ability to engineer existing PPIs or create new ones has become a vital research tool. In addition, engineered proteins with new or altered interactions are among the most critical drugs that have been developed in recent years. These include antibodies, cytokines, inhibitors, and others. Here, we provide a perspective on the current status of the methods used to engineer new or altered PPIs. The emergence of the COVID-19 pandemic, which resulted in a worldwide quest to develop specific PPI inhibitors as drugs, provided an up-to-date and state-of-the-art status report on the methodologies for engineering PPIs targeting the interaction of the viral spike protein with its cellular target, ACE2. Multiple, very high affinity binders were generated within a few months using in vitro evolution by itself, or in combination with computational design. The different experimental and computational methods used to block this interaction provide a road map for the future of PPI engineering.
Asunto(s)
Enzima Convertidora de Angiotensina 2/metabolismo , Ingeniería de Proteínas/métodos , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Enzima Convertidora de Angiotensina 2/genética , Anticuerpos Neutralizantes/metabolismo , Anticuerpos Antivirales/metabolismo , Antivirales/uso terapéutico , COVID-19/metabolismo , COVID-19/prevención & control , COVID-19/virología , Biología Computacional , Humanos , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Glicoproteína de la Espiga del Coronavirus/genéticaRESUMEN
Chronic activation of the immune system in HIV infection is one of the strongest predictors of morbidity and mortality. As such, approaches that reduce immune activation have received considerable interest. Previously, we demonstrated that administration of a type I interferon receptor antagonist (IFN-1ant) during acute SIV infection of rhesus macaques results in increased virus replication and accelerated disease progression. Here, we administered a long half-life PASylated IFN-1ant to ART-treated and ART-naïve macaques during chronic SIV infection and measured expression of interferon stimulated genes (ISG) by RNA sequencing, plasma viremia, plasma cytokines, T cell activation and exhaustion as well as cell-associated virus in CD4 T cell subsets sorted from peripheral blood and lymph nodes. Our study shows that IFN-1ant administration in both ART-suppressed and ART-untreated chronically SIV-infected animals successfully results in reduction of IFN-I-mediated inflammation as defined by reduced expression of ISGs but had no effect on plasma levels of IL-1ß, IL-1ra, IL-6 and IL-8. Unlike in acute SIV infection, we observed no significant increase in plasma viremia up to 25 weeks after IFN-1ant administration or up to 15 weeks after ART interruption. Likewise, cell-associated virus measured by SIV gag DNA copies was similar between IFN-1ant and placebo groups. In addition, evaluation of T cell activation and exhaustion by surface expression of CD38, HLA-DR, Ki67, LAG-3, PD-1 and TIGIT, as well as transcriptome analysis showed no effect of IFN-I blockade. Thus, our data show that blocking IFN-I signaling during chronic SIV infection suppresses IFN-I-related inflammatory pathways without increasing virus replication, and thus may constitute a safe therapeutic intervention in chronic HIV infection.
Asunto(s)
Antirretrovirales/farmacología , Inflamación/prevención & control , Interferón Tipo I/antagonistas & inhibidores , Síndrome de Inmunodeficiencia Adquirida del Simio , Linfocitos T/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Animales , Antirretrovirales/uso terapéutico , Enfermedad Crónica , Inflamación/inmunología , Inflamación/virología , Interferón Tipo I/metabolismo , Activación de Linfocitos/efectos de los fármacos , Macaca mulatta , Receptores de Interferón/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/tratamiento farmacológico , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/efectos de los fármacos , Virus de la Inmunodeficiencia de los Simios/inmunología , Virus de la Inmunodeficiencia de los Simios/fisiología , Linfocitos T/inmunologíaRESUMEN
Inflammation in HIV infection is predictive of non-AIDS morbidity and death, higher set point plasma virus load and virus acquisition; thus, therapeutic agents are in development to reduce its causes and consequences. However, inflammation may simultaneously confer both detrimental and beneficial effects. This dichotomy is particularly applicable to type I interferons (IFN-I) which, while contributing to innate control of infection, also provide target cells for the virus during acute infection, impair CD4 T-cell recovery, and are associated with disease progression. Here we manipulated IFN-I signalling in rhesus macaques (Macaca mulatta) during simian immunodeficiency virus (SIV) transmission and acute infection with two complementary in vivo interventions. We show that blockade of the IFN-I receptor caused reduced antiviral gene expression, increased SIV reservoir size and accelerated CD4 T-cell depletion with progression to AIDS despite decreased T-cell activation. In contrast, IFN-α2a administration initially upregulated expression of antiviral genes and prevented systemic infection. However, continued IFN-α2a treatment induced IFN-I desensitization and decreased antiviral gene expression, enabling infection with increased SIV reservoir size and accelerated CD4 T-cell loss. Thus, the timing of IFN-induced innate responses in acute SIV infection profoundly affects overall disease course and outweighs the detrimental consequences of increased immune activation. Yet, the clinical consequences of manipulation of IFN signalling are difficult to predict in vivo and therapeutic interventions in human studies should be approached with caution.
Asunto(s)
Progresión de la Enfermedad , Interferón-alfa/uso terapéutico , Macaca mulatta/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio , Virus de la Inmunodeficiencia de los Simios/inmunología , Animales , Antivirales/farmacología , Antivirales/uso terapéutico , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Regulación de la Expresión Génica/efectos de los fármacos , Inmunidad Innata/efectos de los fármacos , Interferón-alfa/farmacología , Estimación de Kaplan-Meier , Transducción de Señal/efectos de los fármacos , Síndrome de Inmunodeficiencia Adquirida del Simio/tratamiento farmacológico , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/prevención & controlRESUMEN
Protein-protein interactions occur via well-defined interfaces on the protein surface. Whereas the location of homologous interfaces is conserved, their composition varies, suggesting that multiple solutions may support high-affinity binding. In this study, we examined the plasticity of the interface of TEM1 ß-lactamase with its protein inhibitor BLIP by low-stringency selection of a random TEM1 library using yeast surface display. Our results show that most interfacial residues could be mutated without a loss in binding affinity, protein stability, or enzymatic activity, suggesting plasticity in the interface composition supporting high-affinity binding. Interestingly, many of the selected mutations promoted faster association. Further selection for faster binders was achieved by drastically decreasing the library-ligand incubation time to 30 s. Preequilibrium selection as suggested here is a novel methodology for specifically selecting faster-associating protein complexes.
Asunto(s)
Proteínas Bacterianas/química , Mutación , Inhibidores de beta-Lactamasas/química , beta-Lactamasas/química , Secuencia de Aminoácidos , Biblioteca de Genes , Unión Proteica , Conformación Proteica , Mapeo de Interacción de Proteínas , Análisis de Secuencia de ADN , Técnicas del Sistema de Dos HíbridosRESUMEN
Protein-protein interactions mediate the vast majority of cellular processes. Though protein interactions obey basic chemical principles also within the cell, the in vivo physiological environment may not allow for equilibrium to be reached. Thus, in vitro measured thermodynamic affinity may not provide a complete picture of protein interactions in the biological context. Binding kinetics composed of the association and dissociation rate constants are relevant and important in the cell. Therefore, changes in protein-protein interaction kinetics have a significant impact on the in vivo activity of the proteins. The common protocol for the selection of tighter binders from a mutant library selects for protein complexes with slower dissociation rate constants. Here we describe a method to specifically select for variants with faster association rate constants by using pre-equilibrium selection, starting from a large random library. Toward this end, we refine the selection conditions of a TEM1-ß-lactamase library against its natural nanomolar affinity binder ß-lactamase inhibitor protein (BLIP). The optimal selection conditions depend on the ligand concentration and on the incubation time. In addition, we show that a second sort of the library helps to separate signal from noise, resulting in a higher percent of faster binders in the selected library. Fast associating protein variants are of particular interest for drug development and other biotechnological applications.
Asunto(s)
Proteínas Portadoras/metabolismo , Inhibidores de beta-Lactamasas/metabolismo , beta-Lactamasas/metabolismo , Cinética , Unión Proteica , Conformación Proteica , TermodinámicaRESUMEN
Type I interferons (IFN-1) are cytokines that affect the expression of thousands of genes, resulting in profound cellular changes. IFN-1 activates the cell by dimerizing its two-receptor chains, IFNAR1 and IFNAR2, which are expressed on all nucleated cells. Despite a similar mode of binding, the different IFN-1s activate a spectrum of activities. The causes for differential activation may stem from differences in IFN-1-binding affinity, duration of binding, number of surface receptors, induction of feedbacks, and cell type-specific variations. All together these will alter the signal that is transmitted from the extracellular domain inward. The intracellular domain binds, directly or indirectly, different effector proteins that transmit signals. The composition of effector molecules deviates between different cell types and tissues, inserting an additional level of complexity to the system. Moreover, IFN-1s do not act on their own, and clearly there is much cross-talk between the activated effector molecules by IFN-1 and other cytokines. The outcome generated by all of these factors (processing step) is an observed phenotype, which can be the transformation of the cell to an antiviral state, differentiation of the cell to a specific immune cell, senescence, apoptosis, and many more. IFN-1 activities can be divided into robust and tunable. Antiviral activity, which is stimulated by minute amounts of IFN-1 and is common to all cells, is termed robust. The other activities, which we term tunable, are cell type-specific and often require more stringent modes of activation. In this review, I summarize the current knowledge on the mode of activation and processing that is initiated by IFN-1, in perspective of the resulting phenotypes.
Asunto(s)
Interferón-alfa/inmunología , Receptor de Interferón alfa y beta/inmunología , Transducción de Señal/inmunología , Animales , Humanos , Interferón-alfa/genética , Ratones , Fosforilación/genética , Fosforilación/inmunología , Receptor de Interferón alfa y beta/genética , Factores de Transcripción STAT/genética , Factores de Transcripción STAT/inmunología , Transducción de Señal/genéticaRESUMEN
For over a century, enzymatic activity has been studied in vitro, assuming similar activity in the crowded cellular milieu. Here, we determined in real time the catalytic activity of TEM1-ß-lactamase inside living cells and compared the values to those obtained in vitro We found the apparent in vivo catalytic efficiency, kcat/Km , to be lower than in vitro, with significant cell-to-cell variability. Surprisingly, the results show that inside the cell the apparent catalytic efficiency decreases, and Km increases with increasing enzyme concentration. To rationalize these findings, we measured enzyme and substrate diffusion rates in the cell and found the latter to be slower than expected. Simulations showed that for attenuated diffusion the substrate flux becomes rate-limiting, explaining why reaction rates in vivo can be independent on enzyme concentrations. The octanol/water partition of the substrate is 4.5, which is in the range of Food and Drug Administration-approved drugs. This suggests substrate-limited reaction rates to be common. These findings indicate that in vitro data cannot be simply extrapolated to the crowded in vivo environment.
Asunto(s)
Antígenos CD/metabolismo , Antígenos de Neoplasias/metabolismo , Pruebas de Enzimas/métodos , Antígenos CD/genética , Antígenos de Neoplasias/genética , Biocatálisis , Supervivencia Celular , Células HeLa , Humanos , Mutagénesis , MutaciónRESUMEN
Type I interferons (IFNs) are best known for their role in innate immunity, but they are also involved in other functions including immunomodulation, restricting proliferation, cancer surveillance, and the regulation of the adaptive immune response. All these responses are mediated through the interaction with a single cell surface receptor, albeit at different ligand and receptor concentrations, ligand subtypes, and time of activation. Here we review the functional plasticity of IFN signaling from a quantitative perspective, showing how variations in different ingredients of the system lead to differential IFN responses and how cells tune the system to maximize efficiency while minimizing detrimental effects. We present a basic model wherein the integrated action of different feedback mechanisms can provide sufficient temporal control to differentially drive cellular decisions.
Asunto(s)
Inmunidad Innata , Interferón Tipo I/inmunología , Interleucinas/inmunología , Receptor de Interferón alfa y beta/inmunología , Receptores de Citocinas/inmunología , Inmunidad Adaptativa , Retroalimentación Fisiológica , Regulación de la Expresión Génica/inmunología , Humanos , Vigilancia Inmunológica , Inmunomodulación , Interferón Tipo I/clasificación , Interferón Tipo I/genética , Interleucinas/clasificación , Interleucinas/genética , Linfocitos/inmunología , Receptor de Interferón alfa y beta/genética , Receptores de Citocinas/genética , Transducción de SeñalRESUMEN
Type I interferons serve as the first line of defense against pathogen invasion. Binding of IFNs to its receptors, IFNAR1 and IFNAR2, is leading to activation of the IFN response. To determine whether structural perturbations observed during binding are propagated to the cytoplasmic domain, multiple mutations were introduced into the transmembrane helix and its surroundings. Insertion of one to five alanine residues near either the N or C terminus of the transmembrane domain (TMD) likely promotes a rotation of 100° and a translation of 1.5 Å per added residue. Surprisingly, the added alanines had little effect on the binding affinity of IFN to the cell surface receptors, STAT phosphorylation, or gene induction. Similarly, substitution of the juxtamembrane residues of the TMD with alanines, or replacement of the TMD of IFNAR1 with that of IFNAR2, did not affect IFN binding or activity. Finally, only the addition of 10 serine residues (but not 2 or 4) between the extracellular domain of IFNAR1 and the TMD had some effect on signaling. Bioinformatic analysis shows a correlation between high sequence conservation of TMDs of cytokine receptors and the ability to transmit structural signals. Sequence conservation near the TMD of IFNAR1 is low, suggesting limited functional importance for this region. Our results suggest that IFN binding to the extracellular domains of IFNAR1 and IFNAR2 promotes proximity between the intracellular domains and that differential signaling is a function of duration of activation and affinity of binding rather than specific conformational changes transmitted from the outside to the inside of the cell.
Asunto(s)
Interferón-alfa/metabolismo , Modelos Moleculares , Receptor de Interferón alfa y beta/agonistas , Transducción de Señal , Secuencia de Aminoácidos , Línea Celular , Biología Computacional , Secuencia Conservada , Técnicas de Inactivación de Genes , Humanos , Cinética , Mutagénesis Insercional , Proteínas Mutantes/agonistas , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Fragmentos de Péptidos/agonistas , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Terciaria de Proteína , Receptor de Interferón alfa y beta/química , Receptor de Interferón alfa y beta/genética , Receptor de Interferón alfa y beta/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
We analysed gene expression microarray data from whole blood samples from 228 multiple sclerosis (MS) patients either untreated or treated with one of three alternative commonly used interferon beta (IFNß) disease modifying drugs: Avonex (×1 weekly), Betaseron (every second day) or Rebif (×3 weekly). Patient injections were not timed to coordinate sample collections, thus providing a global transcriptomic profile for each population of patients studied. Three hundred and fifty one genes were significantly differentially expressed by at least one of the IFNß drugs. Despite the different drug sources with distinct injection and dosage protocols, a striking similarity was found in the identity and functional classes of the differentially expressed genes induced. Using the 25 most-upregulated genes, we defined a robust IFNß gene expression signature that quantifies the IFN activation state per blood sample collected irrespective of the type of IFNß therapy. This 25-gene signature also defined basal IFN activation states among untreated MS patients, which differed among individuals but remained relatively constant per patient with time. The maximum drug-induced IFN-activation state was similar for all three drugs despite a 1.7-2.0-fold diminished average effect for Avonex. This and a more erratic effect of Avonex per patient across longitudinal measurements is likely a result of its reduced injection frequency. In summary, we have defined a robust blood-derived type I IFN gene signature from MS patients. This signature could potentially serve to generically quantify the systemic Type I IFN activation status for any other clinical manifestation, inclusive of other autoimmune diseases.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Interferón beta-1a/farmacología , Interferon beta-1b/farmacología , Esclerosis Múltiple/sangre , Transcriptoma/efectos de los fármacos , Adyuvantes Inmunológicos/uso terapéutico , Humanos , Interferón beta-1a/uso terapéutico , Interferon beta-1b/uso terapéutico , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/patologíaRESUMEN
Type I interferons (IFNs) form a network of homologous cytokines that bind to a shared, heterodimeric cell surface receptor and engage signaling pathways that activate innate and adaptive immune responses. The ability of IFNs to mediate differential responses through the same cell surface receptor has been subject of a controversial debate and has important medical implications. During the past decade, a comprehensive insight into the structure, energetics, and dynamics of IFN recognition by its two-receptor subunits, as well as detailed correlations with their functional properties on the level of signal activation, gene expression, and biological responses were obtained. All type I IFNs bind the two-receptor subunits at the same sites and form structurally very similar ternary complexes. Differential IFN activities were found to be determined by different lifetimes and ligand affinities toward the receptor subunits, which dictate assembly and dynamics of the signaling complex in the plasma membrane. We present a simple model, which explains differential IFN activities based on rapid endocytosis of signaling complexes and negative feedback mechanisms interfering with ternary complex assembly. More insight into signaling pathways as well as endosomal signaling and trafficking will be required for a comprehensive understanding, which will eventually lead to therapeutic applications of IFNs with increased efficacy.
Asunto(s)
Retroalimentación Fisiológica , Interferón Tipo I/química , Subunidades de Proteína/química , Receptor de Interferón alfa y beta/química , Linfocitos T/inmunología , Sitios de Unión , Endocitosis/inmunología , Humanos , Interferón Tipo I/inmunología , Interferón Tipo I/metabolismo , Modelos Moleculares , Unión Proteica , Multimerización de Proteína , Estructura Terciaria de Proteína , Subunidades de Proteína/inmunología , Subunidades de Proteína/metabolismo , Receptor de Interferón alfa y beta/inmunología , Receptor de Interferón alfa y beta/metabolismo , Transducción de Señal , Linfocitos T/metabolismo , TermodinámicaRESUMEN
IFNß is a common therapeutic option to treat multiple sclerosis. It is unique among the family of type I IFNs in that it binds to the interferon receptors with high affinity, conferring exceptional biological properties. We have previously reported the generation of an interferon superagonist (dubbed YNSα8) that is built on the backbone of a low affinity IFNα but modified to exhibit higher receptor affinity than even for IFNß. Here, YNSα8 was fused with a 600-residue hydrophilic, unstructured N-terminal polypeptide chain comprising proline, alanine, and serine (PAS) to prolong its plasma half-life via "PASylation." PAS-YNSα8 exhibited a 10-fold increased half-life in both pharmacodynamic and pharmacokinetic assays in a transgenic mouse model harboring the human receptors, notably without any detectable loss in biological potency or bioavailability. This long-lived superagonist conferred significantly improved protection from MOG35-55-induced experimental autoimmune encephalomyelitis compared with IFNß, despite being injected with a 4-fold less frequency and at an overall 16-fold lower dosage. These data were corroborated by FACS measurements showing a decrease of CD11b(+)/CD45(hi) myeloid lineage cells detectable in the CNS, as well as a decrease in IBA(+) cells in spinal cord sections determined by immunohistochemistry for PAS-YNSα8-treated animals. Importantly, PAS-YNSα8 did not induce antibodies upon repeated administration, and its biological efficacy remained unchanged after 21 days of treatment. A striking correlation between increased levels of CD274 (PD-L1) transcripts from spleen-derived CD4(+) cells and improved clinical response to autoimmune encephalomyelitis was observed, indicating that, at least in this mouse model of multiple sclerosis, CD274 may serve as a biomarker to predict the effectiveness of IFN therapy to treat this complex disease.
Asunto(s)
Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Interferón Tipo I/agonistas , Interferón Tipo I/farmacología , Péptidos/química , Animales , Separación Celular , Encefalomielitis Autoinmune Experimental/metabolismo , Femenino , Citometría de Flujo , Humanos , Interferón beta/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Esclerosis Múltiple/tratamiento farmacológico , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/química , Resonancia por Plasmón de Superficie , Resultado del TratamientoRESUMEN
Historically, rate constants were determined in vitro and it was unknown whether they were valid for in vivo biological processes. Here, we bridge this gap by measuring binding dynamics between a pair of proteins in living HeLa cells. Binding of a ß-lactamase to its protein inhibitor was initiated by microinjection and monitored by Förster resonance energy transfer. Association rate constants for the wild-type and an electrostatically optimized mutant were only 25% and 50% lower than in vitro values, whereas no change in the rate constant was observed for a slower binding mutant. These changes are much smaller than might be anticipated considering the high macromolecular crowding within the cell. Single-cell analyses of association rate constants and fluorescence recovery after photobleaching reveals a naturally occurring variation in cell density, which is translated to an up to a twofold effect on binding rate constants. The data show that for this model protein interaction the intracellular environment had only a small effect on the association kinetics, justifying the extrapolation of in vitro data to processes in the cell.