RESUMEN
The activation level of RAS can be determined by GTP hydrolysis rate (khy) and GDP-GTP exchange rates (kex). Either impaired GTP hydrolysis or enhanced GDP-GTP exchange causes the aberrant activation of RAS in oncogenic mutants. Therefore, it is important to quantify the khy and kex for understanding the mechanisms of RAS oncogenesis and drug development. Conventional methods have individually measured the kex and khy of RAS. However, within the intracellular environment, GTP hydrolysis and GDP-GTP exchange reactions occur simultaneously under conditions where GTP concentration is kept constant. In addition, the intracellular activity of RAS is influenced by endogenous regulatory proteins, such as RAS GTPase activating proteins (GAPs) and the guanine-nucleotide exchange factors (GEFs). Here, we describe the in vitro and in-cell NMR methods to estimate the khy and kex simultaneously by measuring the time-dependent changes of the fraction of GTP-bound ratio under the condition of constant GTP concentration.
Asunto(s)
Factores de Intercambio de Guanina Nucleótido , Proteínas Activadoras de ras GTPasa , Guanosina Trifosfato/metabolismo , Proteínas Activadoras de ras GTPasa/metabolismo , Hidrólisis , Factores de Intercambio de Guanina Nucleótido/metabolismo , Espectroscopía de Resonancia Magnética , Guanosina Difosfato/metabolismoRESUMEN
HDM2 negatively regulates the activity of the tumor suppressor p53. Previous NMR studies have shown that apo-HDM2 interconverts between an "open" state in which the N-terminal "lid" is disordered and a "closed" state in which the lid covers the p53-binding site in the core region. Molecular dynamics (MD) simulation studies have been performed to elucidate the conformational dynamics of HDM2, but the direct relevance of the experimental and computational analyses is unclear. In addition, how the phosphorylation of S17 in the lid contributes to the inhibition of p53 binding remains controversial. Here, we used both NMR and MD simulations to investigate the conformational dynamics of apo-HDM2. The NMR analysis revealed that apo-HDM2 exists in a fast-exchanging equilibrium within two closed states, closed 1 and closed 2, in addition to a previously demonstrated slow-exchanging "open-closed" equilibrium. MD simulations visualized two characteristic closed states, where the spatial orientation of the key residues corresponds well to the chemical shift changes of the NMR spectra. Furthermore, the phosphorylation of S17 induced an equilibrium shift toward closed 1, thereby suppressing the binding of p53 to HDM2. This study reveals a multi-state equilibrium of apo-HDM2 and provides new insights into the regulation mechanism of HDM2-p53 interactions.
Asunto(s)
Simulación de Dinámica Molecular , Proteína p53 Supresora de Tumor , Proteína p53 Supresora de Tumor/química , Proteínas Proto-Oncogénicas c-mdm2/química , Unión Proteica , Espectroscopía de Resonancia MagnéticaRESUMEN
KRAS mutations are major drivers of various cancers. Recently, allele-specific inhibitors of the KRAS G12C mutant were developed that covalently modify the thiol of Cys12, thereby trapping KRAS in an inactive GDP-bound state. To study the mechanism of action of the covalent inhibitors in both in vitro and intracellular environments, we used real-time NMR to simultaneously observe GTP hydrolysis and inhibitor binding. In vitro NMR experiments showed that the rate constant of ARS-853 modification is identical to that of GTP hydrolysis, indicating that GTP hydrolysis is the rate-limiting step for ARS-853 modification. In-cell NMR analysis revealed that the ARS-853 reaction proceeds significantly faster than that in vitro, reflecting acceleration of GTP hydrolysis by endogenous GTPase proteins. This study demonstrated that the KRAS covalent inhibitor is as effective in the cell as in vitro and that in-cell NMR is a valuable validation tool for assessing the pharmacological properties of the drug in the intracellular context.
Asunto(s)
Neoplasias , Proteínas Proto-Oncogénicas p21(ras) , Humanos , Proteínas Proto-Oncogénicas p21(ras)/genética , Neoplasias/genética , Mutación , Espectroscopía de Resonancia Magnética , Guanosina Trifosfato/químicaRESUMEN
Open reading frame 6 (ORF6), the accessory protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that suppresses host type-I interferon signaling, possesses amyloidogenic sequences. ORF6 amyloidogenic peptides self-assemble to produce cytotoxic amyloid fibrils. Currently, the molecular properties of the ORF6 remain elusive. Here, we investigate the structural dynamics of the full-length ORF6 protein in a near-physiological environment using high-speed atomic force microscopy. ORF6 oligomers were ellipsoidal and readily assembled into ORF6 protofilaments in either a circular or a linear pattern. The formation of ORF6 protofilaments was enhanced at higher temperatures or on a lipid substrate. ORF6 filaments were sensitive to aliphatic alcohols, urea, and SDS, indicating that the filaments were predominantly maintained by hydrophobic interactions. In summary, ORF6 self-assembly could be necessary to sequester host factors and causes collateral damage to cells via amyloid aggregates. Nanoscopic imaging unveiled the innate molecular behavior of ORF6 and provides insight into drug repurposing to treat amyloid-related coronavirus disease 2019 complications.
Asunto(s)
Sistemas de Lectura Abierta , SARS-CoV-2 , Proteínas Virales , Amiloide , Péptidos , SARS-CoV-2/genética , Transducción de Señal , Proteínas Virales/genéticaRESUMEN
The small guanosine triphosphatase (GTPase) RAS serves as a molecular switch in signal transduction, and its mutation and aberrant activation are implicated in tumorigenesis. Here, we perform real-time, in-cell nuclear magnetic resonance (NMR) analyses of non-farnesylated RAS to measure time courses of the fraction of the active GTP-bound form (fGTP) within cytosol of live mammalian cells. The observed intracellular fGTP is significantly lower than that measured in vitro for wild-type RAS as well as oncogenic mutants, due to both decrease of the guanosine diphosphate (GDP)-GTP exchange rate (kex) and increase of GTP hydrolysis rate (khy). In vitro reconstitution experiments show that highly viscous environments promote a reduction of kex, whereas the increase of khy is stimulated by unidentified cytosolic proteins. This study demonstrates the power of in-cell NMR to directly detect the GTP-bound levels of RAS in mammalian cells, thereby revealing that the khy and kex of RAS are modulated by various intracellular factors.
Asunto(s)
Guanosina Trifosfato/metabolismo , Espectroscopía de Resonancia Magnética/métodos , HumanosRESUMEN
To understand how intracellular proteins respond to oxidative stresses, the redox status of the target protein, as well as the intracellular redox potential ( EGSH), which is defined by the concentrations of reduced and oxidized glutathione, should be observed simultaneously within living cells. In this study, we developed a method that can monitor the redox status of thioredoxin (Trx) and EGSH by direct NMR observation of Trx and glutathione within living cells. Unlike the midpoint potential of Trx measured in vitro (â¼â¯-300 mV), the intracellular Trx exhibited the redox transition at EGSH between -250 and -200 mV, the range known to trigger the oxidative stress-mediated signalings. Furthermore, we quantified the contribution of Trx reductase to the redox status of Trx, demonstrating that the redox profile of Trx is determined by the interplay between the elevation of EGSH and the reduction by Trx reductase and other endogenous molecules.