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1.
J Biol Chem ; 296: 100626, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33930461

RESUMEN

RAS effectors specifically interact with GTP-bound RAS proteins to link extracellular signals to downstream signaling pathways. These interactions rely on two types of domains, called RAS-binding (RB) and RAS association (RA) domains, which share common structural characteristics. Although the molecular nature of RAS-effector interactions is well-studied for some proteins, most of the RA/RB-domain-containing proteins remain largely uncharacterized. Here, we searched through human proteome databases, extracting 41 RA domains in 39 proteins and 16 RB domains in 14 proteins, each of which can specifically select at least one of the 25 members in the RAS family. We next comprehensively investigated the sequence-structure-function relationship between different representatives of the RAS family, including HRAS, RRAS, RALA, RAP1B, RAP2A, RHEB1, and RIT1, with all members of RA domain family proteins (RASSFs) and the RB-domain-containing CRAF. The binding affinity for RAS-effector interactions, determined using fluorescence polarization, broadly ranged between high (0.3 µM) and very low (500 µM) affinities, raising interesting questions about the consequence of these variable binding affinities in the regulation of signaling events. Sequence and structural alignments pointed to two interaction hotspots in the RA/RB domains, consisting of an average of 19 RAS-binding residues. Moreover, we found novel interactions between RRAS1, RIT1, and RALA and RASSF7, RASSF9, and RASSF1, respectively, which were systematically explored in sequence-structure-property relationship analysis, and validated by mutational analysis. These data provide a set of distinct functional properties and putative biological roles that should now be investigated in the cellular context.


Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Dominios y Motivos de Interacción de Proteínas , Proteínas Supresoras de Tumor/metabolismo , Proteínas ras/metabolismo , Proteínas Reguladoras de la Apoptosis/genética , Biología Computacional , Células HEK293 , Humanos , Unión Proteica , Transducción de Señal , Proteínas Supresoras de Tumor/genética , Proteínas ras/genética
2.
Am J Hum Genet ; 104(6): 1223-1232, 2019 06 06.
Artículo en Inglés | MEDLINE | ID: mdl-31130282

RESUMEN

Aberrant signaling through pathways controlling cell response to extracellular stimuli constitutes a central theme in disorders affecting development. Signaling through RAS and the MAPK cascade controls a variety of cell decisions in response to cytokines, hormones, and growth factors, and its upregulation causes Noonan syndrome (NS), a developmental disorder whose major features include a distinctive facies, a wide spectrum of cardiac defects, short stature, variable cognitive impairment, and predisposition to malignancies. NS is genetically heterogeneous, and mutations in more than ten genes have been reported to underlie this disorder. Despite the large number of genes implicated, about 10%-20% of affected individuals with a clinical diagnosis of NS do not have mutations in known RASopathy-associated genes, indicating that additional unidentified genes contribute to the disease, when mutated. By using a mixed strategy of functional candidacy and exome sequencing, we identify RRAS2 as a gene implicated in NS in six unrelated subjects/families. We show that the NS-causing RRAS2 variants affect highly conserved residues localized around the nucleotide binding pocket of the GTPase and are predicted to variably affect diverse aspects of RRAS2 biochemical behavior, including nucleotide binding, GTP hydrolysis, and interaction with effectors. Additionally, all pathogenic variants increase activation of the MAPK cascade and variably impact cell morphology and cytoskeletal rearrangement. Finally, we provide a characterization of the clinical phenotype associated with RRAS2 mutations.


Asunto(s)
Mutación con Ganancia de Función , Guanosina Trifosfato/metabolismo , Proteínas de la Membrana/genética , Proteínas de Unión al GTP Monoméricas/genética , Síndrome de Noonan/etiología , Adulto , Niño , Femenino , Estudios de Asociación Genética , Células HEK293 , Humanos , Lactante , Recién Nacido , Masculino , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Proteínas de Unión al GTP Monoméricas/química , Proteínas de Unión al GTP Monoméricas/metabolismo , Síndrome de Noonan/patología , Linaje , Conformación Proteica
3.
Nat Methods ; 15(9): 669-676, 2018 09.
Artículo en Inglés | MEDLINE | ID: mdl-30171252

RESUMEN

Single-molecule Förster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between ±0.02 and ±0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Our quantitative assessment of the reproducibility of intensity-based smFRET measurements and a unified correction procedure represents an important step toward the validation of distance networks, with the ultimate aim of achieving reliable structural models of biomolecular systems by smFRET-based hybrid methods.


Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Laboratorios/normas , Reproducibilidad de los Resultados
4.
Biochem Soc Trans ; 46(6): 1393-1406, 2018 12 17.
Artículo en Inglés | MEDLINE | ID: mdl-30381334

RESUMEN

RAF (rapidly accelerated fibrosarcoma) Ser/Thr kinases (ARAF, BRAF, and CRAF) link the RAS (rat sarcoma) protein family with the MAPK (mitogen-activated protein kinase) pathway and control cell growth, differentiation, development, aging, and tumorigenesis. Their activity is specifically modulated by protein-protein interactions, post-translational modifications, and conformational changes in specific spatiotemporal patterns via various upstream regulators, including the kinases, phosphatase, GTPases, and scaffold and modulator proteins. Dephosphorylation of Ser-259 (CRAF numbering) and dissociation of 14-3-3 release the RAF regulatory domains RAS-binding domain and cysteine-rich domain for interaction with RAS-GTP and membrane lipids. This, in turn, results in RAF phosphorylation at Ser-621 and 14-3-3 reassociation, followed by its dimerization and ultimately substrate binding and phosphorylation. This review focuses on structural understanding of how distinct binding partners trigger a cascade of molecular events that induces RAF kinase activation.


Asunto(s)
Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Proto-Oncogénicas A-raf/metabolismo , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas c-raf/metabolismo , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Animales , Humanos , Proteínas Quinasas Activadas por Mitógenos/genética , Fosforilación , Unión Proteica , Proteínas Proto-Oncogénicas A-raf/genética , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas c-raf/genética
5.
J Biol Chem ; 291(31): 16175-85, 2016 07 29.
Artículo en Inglés | MEDLINE | ID: mdl-27226581

RESUMEN

The N-methyl-d-aspartate (NMDA) receptors are heteromeric non-selective cation channels that require the binding of glycine and glutamate for gating. Based on crystal structures, the mechanism of partial agonism at the glycine-binding site is thought to be mediated by a shift in the conformational equilibrium between an open clamshell and a closed clamshell-like structure of the bilobed ligand-binding domain (LBD). Using single-molecule Förster resonance energy transfer (smFRET) and multiparameter fluorescence detection, which allows us to study the conformational states and dynamics in the submillisecond time scale, we show that there are at least three conformational states explored by the LBD: the low FRET, medium FRET, and high FRET states. The distance of the medium and low FRET states corresponds to what has been observed in crystallography structures. We show that the high FRET state, which would represent a more closed clamshell conformation than that observed in the crystal structure, is most likely the state initiating activation, as evidenced by the fact that the fraction of the protein in this state correlates well with the extent of activation. Furthermore, full agonist bound LBDs show faster dynamic motions between the medium and high FRET states, whereas they show slower dynamics when bound to weaker agonists or to antagonists.


Asunto(s)
Transferencia Resonante de Energía de Fluorescencia , Receptores de N-Metil-D-Aspartato/química , Animales , Línea Celular , Humanos , Dominios Proteicos , Ratas
7.
J Med Chem ; 67(15): 13252-13270, 2024 Aug 08.
Artículo en Inglés | MEDLINE | ID: mdl-39018123

RESUMEN

Mitochondria are cellular powerhouses and are crucial for cell function. However, they are vulnerable to internal and external perturbagens that may impair mitochondrial function and eventually lead to cell death. In particular, small molecules may impact mitochondrial function, and therefore, their influence on mitochondrial homeostasis is at best assessed early on in the characterization of biologically active small molecules and drug discovery. We demonstrate that unbiased morphological profiling by means of the cell painting assay (CPA) can detect mitochondrial stress coupled with the induction of an integrated stress response. This activity is common for compounds addressing different targets, is not shared by direct inhibitors of the electron transport chain, and enables prediction of mitochondrial stress induction for small molecules that are profiled using CPA.


Asunto(s)
Mitocondrias , Humanos , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Estrés Fisiológico/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Bibliotecas de Moléculas Pequeñas/química , Línea Celular Tumoral
8.
J Med Chem ; 67(11): 8862-8876, 2024 Jun 13.
Artículo en Inglés | MEDLINE | ID: mdl-38687818

RESUMEN

Screening for small-molecule modulators of disease-relevant targets and phenotypes is the first step on the way to new drugs. Large compound libraries have been synthesized by academia and, particularly, pharmaceutical companies to meet the need for novel chemical entities that are as diverse as possible. Screening of these compound libraries revealed a portion of small molecules that is inactive in more than 100 different assays and was therefore termed "dark chemical matter" (DCM). Deorphanization of DCM promises to yield very selective compounds as they are expected to have less off-target effects. We employed morphological profiling using the Cell Painting assay to detect bioactive DCM. Within the DCM collection, we identified bioactive compounds and confirmed several modulators of microtubules, DNA synthesis, and pyrimidine biosynthesis. Profiling approaches are, therefore, powerful tools to probe compound collections for bioactivity in an unbiased manner and are particularly suitable for deorphanization of DCM.


Asunto(s)
Bibliotecas de Moléculas Pequeñas , Humanos , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , ADN/química , Pirimidinas/química , Pirimidinas/farmacología , Pirimidinas/síntesis química , Línea Celular Tumoral
9.
Cells ; 11(3)2022 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-35159317

RESUMEN

Embryonic stem cell-expressed Ras (ERas) is an atypical constitutively active member of the Ras family and controls distinct signaling pathways, which are critical, for instance, for the maintenance of quiescent hepatic stellate cells (HSCs). Unlike classical Ras paralogs, ERas has a unique N-terminal extension (Nex) with as yet unknown function. In this study, we employed affinity pull-down and quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses and identified 76 novel binding proteins for human and rat ERas Nex peptides, localized in different subcellular compartments and involved in various cellular processes. One of the identified Nex-binding proteins is the nonmitochondrial, cytosolic arginase 1 (ARG1), a key enzyme of the urea cycle and involved in the de novo synthesis of polyamines, such as spermidine and spermine. Here, we show, for the first time, a high-affinity interaction between ERas Nex and purified ARG1 as well as their subcellular colocalization. The inhibition of ARG1 activity strikingly accelerates the activation of HSCs ex vivo, suggesting a central role of ARG1 activity in the maintenance of HSC quiescence.


Asunto(s)
Arginasa , Células Estrelladas Hepáticas , Proteína Oncogénica p21(ras) , Animales , Arginasa/metabolismo , Cromatografía Liquida , Células Madre Embrionarias/metabolismo , Células Estrelladas Hepáticas/metabolismo , Humanos , Proteína Oncogénica p21(ras)/metabolismo , Ratas , Espectrometría de Masas en Tándem
10.
Nat Commun ; 9(1): 3724, 2018 09 13.
Artículo en Inglés | MEDLINE | ID: mdl-30214057

RESUMEN

Previous studies of the N-terminal PDZ tandem from PSD-95 produced divergent models and failed to identify interdomain contacts stabilizing the structure. We used ensemble and single-molecule FRET along with replica-exchange molecular dynamics to fully characterize the energy landscape. Simulations and experiments identified two conformations: an open-like conformation with a small contact interface stabilized by salt bridges, and a closed-like conformation with a larger contact interface stabilized by surface-exposed hydrophobic residues. Both interfaces were confirmed experimentally. Proximity of interdomain contacts to the binding pockets may explain the observed coupling between conformation and binding. The low-energy barrier between conformations allows submillisecond dynamics, which were time-averaged in previous NMR and FRET studies. Moreover, the small contact interfaces were likely overridden by lattice contacts as crystal structures were rarely sampled in simulations. Our hybrid approach can identify transient interdomain interactions, which are abundant in multidomain proteins yet often obscured by dynamic averaging.


Asunto(s)
Homólogo 4 de la Proteína Discs Large/química , Dominios PDZ , Animales , Simulación por Computador , Disulfuros , Escherichia coli/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Interacciones Hidrofóbicas e Hidrofílicas , Ligandos , Espectroscopía de Resonancia Magnética , Simulación de Dinámica Molecular , Fotones , Unión Proteica , Ratas , Factores de Transcripción
11.
J Vis Exp ; (123)2017 05 13.
Artículo en Inglés | MEDLINE | ID: mdl-28570518

RESUMEN

A protocol on how to perform high-precision interdye distance measurements using Förster resonance energy transfer (FRET) at the single-molecule level in multiparameter fluorescence detection (MFD) mode is presented here. MFD maximizes the usage of all "dimensions" of fluorescence to reduce photophysical and experimental artifacts and allows for the measurement of interdye distance with an accuracy up to ~1 Å in rigid biomolecules. This method was used to identify three conformational states of the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor to explain the activation of the receptor upon ligand binding. When comparing the known crystallographic structures with experimental measurements, they agreed within less than 3 Å for more dynamic biomolecules. Gathering a set of distance restraints that covers the entire dimensionality of the biomolecules would make it possible to provide a structural model of dynamic biomolecules.


Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Receptores de N-Metil-D-Aspartato/química , Fluorescencia , Ligandos , Conformación Molecular , Unión Proteica , Dominios Proteicos , Receptores de N-Metil-D-Aspartato/metabolismo
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