RESUMEN
Serine phosphorylations on insulin receptor substrate 1 (IRS-1) by diverse kinases aoccur widely during obesity-, stress-, and inflammation-induced conditions in models of insulin resistance and type 2 diabetes. In this study, we define a region within the human IRS-1, which is directly C-terminal to the PTB domain encompassing numerous serine phosphorylation sites including Ser307 (mouse Ser302) and Ser312 (mouse 307) creating a phosphorylation insulin resistance (PIR) domain. We demonstrate that the IRS-1 PTB-PIR with its unphosphorylated serine residues interacts with the insulin receptor (IR) but loses the IR-binding when they are phosphorylated. Surface plasmon resonance studies further confirm that the PTB-PIR binds stronger to IR than just the PTB domain, and that phosphorylations at Ser307, Ser312, Ser315, and Ser323 within the PIR domain result in abrogating the binding. Insulin-responsive cells containing the mutant IRS-1 with all these four serines changed into glutamates to mimic phosphorylations show decreased levels of phosphorylations in IR, IRS-1, and AKT compared to the wild-type IRS-1. Hydrogen-deuterium exchange mass spectrometry experiments indicating the PIR domain interacting with the N-terminal lobe and the hinge regions of the IR kinase domain further suggest the possibility that the IRS-1 PIR domain protects the IR from the PTP1B-mediated dephosphorylation.
Asunto(s)
Diabetes Mellitus Tipo 2 , Resistencia a la Insulina , Ratones , Humanos , Animales , Fosforilación , Serina/metabolismo , Receptor de Insulina/metabolismo , Proteínas Sustrato del Receptor de Insulina/genética , Proteínas Sustrato del Receptor de Insulina/metabolismo , Línea Celular , Fosfoproteínas/metabolismo , Insulina/metabolismoRESUMEN
The phosphatidylinositol 3 kinase (PI3K) pathway regulates fundamental cellular processes such as metabolism, proliferation, and survival. A central component in this pathway is the p85α regulatory subunit, encoded by PIK3R1. Using whole-exome sequencing, we identified a heterozygous PIK3R1 mutation (c.1945C>T [p.Arg649Trp]) in two unrelated families affected by partial lipodystrophy, low body mass index, short stature, progeroid face, and Rieger anomaly (SHORT syndrome). This mutation led to impaired interaction between p85α and IRS-1 and reduced AKT-mediated insulin signaling in fibroblasts from affected subjects and in reconstituted Pik3r1-knockout preadipocytes. Normal PI3K activity is critical for adipose differentiation and insulin signaling; the mutated PIK3R1 therefore provides a unique link among lipodystrophy, growth, and insulin signaling.
Asunto(s)
Fosfatidilinositol 3-Quinasa Clase Ia/metabolismo , Trastornos del Crecimiento/enzimología , Hipercalcemia/enzimología , Enfermedades Metabólicas/enzimología , Nefrocalcinosis/enzimología , Transducción de Señal , Adipocitos/metabolismo , Adolescente , Adulto , Anciano , Secuencia de Aminoácidos , Índice de Masa Corporal , Diferenciación Celular , Fosfatidilinositol 3-Quinasa Clase Ia/genética , Análisis Mutacional de ADN , Activación Enzimática , Exoma , Femenino , Fibroblastos/metabolismo , Técnicas de Inactivación de Genes , Tamización de Portadores Genéticos , Predisposición Genética a la Enfermedad , Genética de Población/métodos , Trastornos del Crecimiento/patología , Humanos , Hipercalcemia/patología , Proteínas Sustrato del Receptor de Insulina/genética , Proteínas Sustrato del Receptor de Insulina/metabolismo , Masculino , Enfermedades Metabólicas/patología , Persona de Mediana Edad , Datos de Secuencia Molecular , Mutación , Nefrocalcinosis/patología , Linaje , Adulto Joven , Dominios Homologos srcRESUMEN
Rapid emergence of new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has prompted an urgent need for the development of broadly applicable and potently neutralizing antibody platform against the SARS-CoV-2, which can be used for combatting the coronavirus disease 2019 (COVID-19). In this study, based on a noncompeting pair of phage display-derived human monoclonal antibodies (mAbs) specific to the receptor-binding domain (RBD) of SARS-CoV-2 isolated from human synthetic antibody library, we generated K202.B, a novel engineered bispecific antibody with an immunoglobulin G4-single-chain variable fragment design, with sub- or low nanomolar antigen-binding avidity. Compared with the parental mAbs or mAb cocktail, the K202.B antibody showed superior neutralizing potential against a variety of SARS-CoV-2 variants in vitro. Furthermore, structural analysis of bispecific antibody-antigen complexes using cryo-electron microscopy revealed the mode of action of K202.B complexed with a fully open three-RBD-up conformation of SARS-CoV-2 trimeric spike proteins by simultaneously interconnecting two independent epitopes of the SARS-CoV-2 RBD via inter-protomer interactions. Intravenous monotherapy using K202.B exhibited potent neutralizing activity in SARS-CoV-2 wild-type- and B.1.617.2 variant-infected mouse models, without significant toxicity in vivo. The results indicate that this novel approach of development of immunoglobulin G4-based bispecific antibody from an established human recombinant antibody library is likely to be an effective strategy for the rapid development of bispecific antibodies, and timely management against fast-evolving SARS-CoV-2 variants.
Asunto(s)
Anticuerpos Biespecíficos , COVID-19 , Animales , Ratones , Humanos , SARS-CoV-2/metabolismo , Anticuerpos Antivirales , Anticuerpos Biespecíficos/farmacología , Microscopía por Crioelectrón , Anticuerpos Neutralizantes , Glicoproteína de la Espiga del CoronavirusRESUMEN
We have solved the x-ray crystal structures of the RabGAP domains of human TBC1D1 and human TBC1D4 (AS160), at 2.2 and 3.5 Å resolution, respectively. Like the yeast Gyp1p RabGAP domain, whose structure was solved previously in complex with mouse Rab33B, the human TBC1D1 and TBC1D4 domains both have 16 α-helices and no ß-sheet elements. We expected the yeast Gyp1p RabGAP/mouse Rab33B structure to predict the corresponding interfaces between cognate mammalian RabGAPs and Rabs, but found that residues were poorly conserved. We further tested the relevance of this model by Ala-scanning mutagenesis, but only one of five substitutions within the inferred binding site of the TBC1D1 RabGAP significantly perturbed catalytic efficiency. In addition, substitution of TBC1D1 residues with corresponding residues from Gyp1p did not enhance catalytic efficiency. We hypothesized that biologically relevant RabGAP/Rab partners utilize additional contacts not described in the yeast Gyp1p/mouse Rab33B structure, which we predicted using our two new human TBC1D1 and TBC1D4 structures. Ala substitution of TBC1D1 Met(930), corresponding to a residue outside of the Gyp1p/Rab33B contact, substantially reduced catalytic activity. GLUT4 translocation assays confirmed the biological relevance of our findings. Substitutions with lowest RabGAP activity, including catalytically dead RK and Met(930) and Leu(1019) predicted to perturb Rab binding, confirmed that biological activity requires contacts between cognate RabGAPs and Rabs beyond those in the yeast Gyp1p RabGAP/mouse Rab33B structure.
Asunto(s)
Proteínas Activadoras de GTPasa/química , Transportador de Glucosa de Tipo 4 , Modelos Moleculares , Animales , Cristalografía por Rayos X , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Humanos , Ratones , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Relación Estructura-Actividad , Proteínas de Unión al GTP rab/química , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismoRESUMEN
The pH-selective interaction between the immunoglobulin G (IgG) fragment crystallizable region (Fc region) and the neonatal Fc receptor (FcRn) is critical for prolonging the circulating half-lives of IgG molecules through intracellular trafficking and recycling. By using directed evolution, we successfully identified Fc mutations that improve the pH-dependent binding of human FcRn and prolong the serum persistence of a model IgG antibody and an Fc-fusion protein. Strikingly, trastuzumab-PFc29 and aflibercept-PFc29, a model therapeutic IgG antibody and an Fc-fusion protein, respectively, when combined with our engineered Fc (Q311R/M428L), both exhibited significantly higher serum half-lives in human FcRn transgenic mice than their counterparts with wild-type Fc. Moreover, in a cynomolgus monkey model, trastuzumab-PFc29 displayed a superior pharmacokinetic profile to that of both trastuzumab-YTE and trastuzumab-LS, which contain the well-validated serum half-life extension Fcs YTE (M252Y/S254T/T256E) and LS (M428L/N434S), respectively. Furthermore, the introduction of two identified mutations of PFc29 (Q311R/M428L) into the model antibodies enhanced both complement-dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity activity, which are triggered by the association between IgG Fc and Fc binding ligands and are critical for clearing cancer cells. In addition, the effector functions could be turned off by combining the two mutations of PFc29 with effector function-silencing mutations, but the antibodies maintained their excellent pH-dependent human FcRn binding profile. We expect our Fc variants to be an excellent tool for enhancing the pharmacokinetic profiles and potencies of various therapeutic antibodies and Fc-fusion proteins.
Asunto(s)
Antígenos de Histocompatibilidad Clase I , Inmunoglobulina G , Ratones , Animales , Humanos , Inmunoglobulina G/genética , Inmunoglobulina G/metabolismo , Macaca fascicularis/metabolismo , Semivida , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/química , Antígenos de Histocompatibilidad Clase I/metabolismo , Ratones Transgénicos , Mutación , Trastuzumab/uso terapéutico , Trastuzumab/genéticaRESUMEN
Protein dimerization or oligomerization resulting from swapping part of the protein between neighboring polypeptide chains is known to play a key role in the regulation of protein function and in the formation of protein aggregates. Glutaredoxin-1 from Clostridium oremlandii (cGrx1) was used as a model to explore the formation of multiple domain-swapped conformations, which were made possible by modulating several hinge-loop residues that can form a pivot for domain swapping. Specifically, two alternative domain-swapped structures were generated and analyzed using nuclear magnetic resonance (NMR), X-ray crystallography, circular-dichroism spectroscopy and hydrogen/deuterium-exchange (HDX) mass spectrometry. The first domain-swapped structure (ß3-swap) was formed by the hexameric cGrx1-cMsrA complex. The second domain-swapped structure (ß1-swap) was formed by monothiol cGrx1 (C16S) alone. In summary, the first domain-swapped structure of an oxidoreductase in a hetero-oligomeric complex is presented. In particular, a single point mutation of a key cysteine residue to serine led to the formation of an intramolecular disulfide bond, as opposed to an intermolecular disulfide bond, and resulted in modulation of the underlying free-energy landscape of protein oligomerization.
RESUMEN
TBC1D4 (also known as AS160) is a Rab·GTPase-activating protein (RabGAP) which functions in insulin signaling. TBC1D4 is critical for translocation of glucose transporter 4 (GLUT4), from an inactive, intracellular, vesicle-bound site to the plasma membrane, where it promotes glucose entry into cells. The TBC1D4 protein is structurally subdivided into two N-terminal phosphotyrosine-binding (PTB) domains, a C-terminal catalytic RabGAP domain, and a disordered segment in between containing potential Akt phosphorylation sites. Structural predictions further suggest that a region C-terminal to the RabGAP domain adopts a coiled-coil motif. We show that C-terminal region (CTR) region is largely α-helical and mediates TBC1D4 RabGAP dimerization. RabGAP catalytic activity and thermal stability appear to be independent of CTR-mediated dimerization.
Asunto(s)
Proteínas Activadoras de GTPasa/química , Multimerización de Proteína , Humanos , Dominios Proteicos , Estabilidad Proteica , Estructura Cuaternaria de Proteína , TemperaturaRESUMEN
Rationale: Signal transducer and activator of transcription-3 (STAT3) plays a pivotal role in cancer biology. Many small-molecule inhibitors that target STAT3 have been developed as potential anticancer drugs. While designing small-molecule inhibitors that target the SH2 domain of STAT3 remains the leading focus for drug discovery, there has been a growing interest in targeting the DNA-binding domain (DBD) of the protein. Methods: We demonstrated the potential antitumor activity of a novel, small-molecule (E)-2-methoxy-4-(3-(4-methoxyphenyl)prop-1-en-1-yl)phenol (MMPP) that directly binds to the DBD of STAT3, in patient-derived non-small cell lung cancer (NSCLC) xenograft model as well as in NCI-H460 cell xenograft model in nude mice. Results: MMPP effectively inhibited the phosphorylation of STAT3 and its DNA binding activity in vitro and in vivo. It induced G1-phase cell cycle arrest and apoptosis through the regulation of cell cycle- and apoptosis-regulating genes by directly binding to the hydroxyl residue of threonine 456 in the DBD of STAT3. Furthermore, MMPP showed a similar or better antitumor activity than that of docetaxel or cisplatin. Conclusion: MMPP is suggested to be a potential candidate for further development as an anticancer drug that targets the DBD of STAT3.
Asunto(s)
Antineoplásicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Ácidos Ftálicos/farmacología , Factor de Transcripción STAT3/metabolismo , Células A549 , Animales , Apoptosis/efectos de los fármacos , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Neoplasias Pulmonares/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
To find a more effective chemical reagent for improved monoclonal antibody (mAb) production, eight chemical reagents (curcumin, quercein, DL-sulforaphane, thymidine, valeric acid, phenyl butyrate, valproic acid, and lithium chloride) known to induce cell cycle arrest were examined individually as chemical additives to recombinant CHO (rCHO) cell cultures producing mAb. Among these chemical additives, valeric acid showed the best production performance. Valeric acid decreased specific growth rate (µ), but increased culture longevity and specific mAb productivity (qmAb ) in a dose-dependent manner. The beneficial effect of valeric acid on culture longevity and qmAb outweighed its detrimental effect on µ, resulting in 2.9-fold increase in the maximum mAb concentration when 1.5 mM valeric acid was added to the cultures. Furthermore, valeric acid did not negatively affect the mAb quality attributes with regard to aggregation, charge variation, and galactosylation. Unexpectedly, galactosylation of the mAb increased by the 1.5 mM valeric acid addition. Taken together, the results obtained here demonstrate that valeric acid is an effective chemical reagent to increase mAb production in rCHO cells.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Células CHO/efectos de los fármacos , Puntos de Control de la Fase G1 del Ciclo Celular , Ácidos Pentanoicos/farmacología , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/genética , Células CHO/citología , Técnicas de Cultivo de Célula/métodos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cricetinae , Cricetulus , Medios de Cultivo/química , Medios de Cultivo/farmacología , Relación Dosis-Respuesta a Droga , Galactosa/metabolismo , Glicosilación , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Piperlongumine has anti-cancer activity in numerous cancer cell lines via various signaling pathways. But there has been no study regarding the mechanisms of PL on the lung cancer yet. Thus, we evaluated the anti-cancer effects and possible mechanisms of PL on non-small cell lung cancer (NSCLC) cells in vivo and in vitro. Our findings showed that PL induced apoptotic cell death and suppressed the DNA binding activity of NF-κB in a concentration dependent manner (0-15 µM) in NSCLC cells. Docking model and pull down assay showed that PL directly binds to the DNA binding site of nuclear factor-κB (NF-κB) p50 subunit, and surface plasmon resonance (SPR) analysis showed that PL binds to p50 concentration-dependently. Moreover, co-treatment of PL with NF-κB inhibitor phenylarsine oxide (0.1 µM) or p50 siRNA (100 nM) augmented PL-induced inhibitory effect on cell growth and activation of Fas and DR4. Notably, co-treatment of PL with p50 mutant plasmid (C62S) partially abolished PL-induced cell growth inhibition and decreased the enhanced expression of Fas and DR4. In xenograft mice model, PL (2.5-5 mg/kg) suppressed tumor growth of NSCLC dose-dependently. Therefore, these results indicated that PL could inhibit lung cancer cell growth via inhibition of NF-κB signaling pathway in vitro and in vivo.
Asunto(s)
Antineoplásicos Fitogénicos/administración & dosificación , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Dioxolanos/administración & dosificación , Neoplasias Pulmonares/tratamiento farmacológico , FN-kappa B/metabolismo , Células A549 , Animales , Antineoplásicos Fitogénicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Dioxolanos/farmacología , Relación Dosis-Respuesta a Droga , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Neoplasias Pulmonares/metabolismo , Modelos Moleculares , Simulación del Acoplamiento Molecular , FN-kappa B/química , Unión Proteica/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Obesity-induced inflammation mediated by immune cells in adipose tissue appears to participate in the pathogenesis of insulin resistance. We show that natural killer (NK) cells in adipose tissue play an important role. High-fat diet (HFD) increases NK cell numbers and the production of proinflammatory cytokines, notably TNFα, in epididymal, but not subcutaneous, fat depots. When NK cells were depleted either with neutralizing antibodies or genetic ablation in E4bp4(+/-) mice, obesity-induced insulin resistance improved in parallel with decreases in both adipose tissue macrophage (ATM) numbers, and ATM and adipose tissue inflammation. Conversely, expansion of NK cells following IL-15 administration or reconstitution of NK cells into E4bp4(-/-) mice increased both ATM numbers and adipose tissue inflammation and exacerbated HFD-induced insulin resistance. These results indicate that adipose NK cells control ATMs as an upstream regulator potentially by producing proinflammatory mediators, including TNFα, and thereby contribute to the development of obesity-induced insulin resistance.
Asunto(s)
Tejido Adiposo/patología , Inflamación/complicaciones , Resistencia a la Insulina , Células Asesinas Naturales/patología , Macrófagos/patología , Obesidad/complicaciones , Tejido Adiposo/inmunología , Animales , Células Cultivadas , Dieta Alta en Grasa/efectos adversos , Inflamación/inmunología , Inflamación/patología , Células Asesinas Naturales/inmunología , Macrófagos/inmunología , Ratones Endogámicos C57BL , Obesidad/inmunología , Obesidad/patologíaRESUMEN
It is increasingly accepted that chronic inflammation participates in obesity-induced insulin resistance and type 2 diabetes (T2D). Salicylates and thiazolidinediones (TZDs) both have anti-inflammatory and anti-hyperglycemic properties. The present study compared the effects of these drugs on obesity-induced inflammation in adipose tissue (AT) and AT macrophages (ATMs), as well as the metabolic and immunological phenotypes of the animal models. Both drugs improved high fat diet (HFD)-induced insulin resistance. However, salicylates did not affect AT and ATM inflammation, whereas Pioglitazone improved these parameters. Interestingly, HFD and the drug treatments all modulated systemic inflammation as assessed by changes in circulating immune cell numbers and activation states. HFD increased the numbers of circulating white blood cells, neutrophils, and a pro-inflammatory monocyte subpopulation (Ly6C(hi)), whereas salicylates and Pioglitazone normalized these cell numbers. The drug treatments also decreased circulating lymphocyte numbers. These data suggest that obesity induces systemic inflammation by regulating circulating immune cell phenotypes and that anti-diabetic interventions suppress systemic inflammation by normalizing circulating immune phenotypes.