RESUMEN
RAS GTPases associate with the biological membrane where they function as molecular switches to regulate cell growth. Recent studies indicate that RAS proteins oligomerize on membranes, and disrupting these assemblies represents an alternative therapeutic strategy. However, conflicting reports on RAS assemblies, ranging in size from dimers to nanoclusters, have brought to the fore key questions regarding the stoichiometry and parameters that influence oligomerization. Here, we probe three isoforms of RAS [Kirsten Rat Sarcoma viral oncogene (KRAS), Harvey Rat Sarcoma viral oncogene (HRAS), and Neuroblastoma oncogene (NRAS)] directly from membranes using mass spectrometry. We show that KRAS on membranes in the inactive state (GDP-bound) is monomeric but forms dimers in the active state (GTP-bound). We demonstrate that the small molecule BI2852 can induce dimerization of KRAS, whereas the binding of effector proteins disrupts dimerization. We also show that RAS dimerization is dependent on lipid composition and reveal that oligomerization of NRAS is regulated by palmitoylation. By monitoring the intrinsic GTPase activity of RAS, we capture the emergence of a dimer containing either mixed nucleotides or GDP on membranes. We find that the interaction of RAS with the catalytic domain of Son of Sevenless (SOScat) is influenced by membrane composition. We also capture the activation and monomer to dimer conversion of KRAS by SOScat. These results not only reveal the stoichiometry of RAS assemblies on membranes but also uncover the impact of critical factors on oligomerization, encompassing regulation by nucleotides, lipids, and palmitoylation.
Asunto(s)
Membrana Celular , Multimerización de Proteína , Proteínas Proto-Oncogénicas p21(ras) , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/química , Humanos , Membrana Celular/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , GTP Fosfohidrolasas/metabolismo , GTP Fosfohidrolasas/química , GTP Fosfohidrolasas/genética , Lipoilación , Proteínas ras/metabolismo , Proteínas ras/química , Guanosina Trifosfato/metabolismo , Guanosina Difosfato/metabolismoRESUMEN
RAS proteins are conserved guanosine triphosphate (GTP) hydrolases (GTPases) that act as molecular binary switches and play vital roles in numerous cellular processes. Upon GTP binding, RAS GTPases adopt an active conformation and interact with specific proteins termed RAS effectors that contain a conserved ubiquitin-like domain, thereby facilitating downstream signaling. Over 50 effector proteins have been identified in the human proteome, and many have been studied as potential mediators of RAS-dependent signaling pathways. Biochemical and structural analyses have provided mechanistic insights into these effectors, and studies using model organisms have complemented our understanding of their role in physiology and disease. Yet, many critical aspects regarding the dynamics and biological function of RAS-effector complexes remain to be elucidated. In this review, we discuss the mechanisms and functions of known RAS effector proteins, provide structural perspectives on RAS-effector interactions, evaluate their significance in RAS-mediated signaling, and explore their potential as therapeutic targets.
Asunto(s)
Transducción de Señal , Proteínas ras , Humanos , Proteínas ras/metabolismo , Proteínas ras/química , Animales , Unión Proteica , Modelos Moleculares , Relación Estructura-Actividad , Conformación Proteica , Guanosina Trifosfato/metabolismoRESUMEN
Protein-protein interactions (PPIs) are central in cell metabolism but research tools for the structural and functional characterization of these PPIs are often missing. Here we introduce broadly applicable immunization (Cross-link PPIs and immunize llamas, ChILL) and selection strategies (Display and co-selection, DisCO) for the discovery of diverse nanobodies that either stabilize or disrupt PPIs in a single experiment. We apply ChILL and DisCO to identify competitive, connective, or fully allosteric nanobodies that inhibit or facilitate the formation of the SOS1â¢RAS complex and modulate the nucleotide exchange rate on this pivotal GTPase in vitro as well as RAS signalling in cellulo. One of these connective nanobodies fills a cavity that was previously identified as the binding pocket for a series of therapeutic lead compounds. The long complementarity-determining region (CDR3) that penetrates this binding pocket serves as pharmacophore for extending the repertoire of potential leads.
Asunto(s)
Unión Proteica , Proteína SOS1 , Anticuerpos de Dominio Único , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/metabolismo , Proteína SOS1/metabolismo , Proteína SOS1/química , Proteína SOS1/genética , Proteína SOS1/inmunología , Humanos , Animales , Regulación Alostérica , Proteínas ras/metabolismo , Proteínas ras/química , Regiones Determinantes de Complementariedad/química , Regiones Determinantes de Complementariedad/inmunología , Sitios de Unión , Camélidos del Nuevo Mundo/inmunología , Inmunización , Transducción de Señal , Modelos MolecularesRESUMEN
RAF family protein kinases are a key node in the RAS/RAF/MAP kinase pathway, the signaling cascade that controls cellular proliferation, differentiation, and survival in response to engagement of growth factor receptors on the cell surface. Over the past few years, structural and biochemical studies have provided new understanding of RAF autoregulation, RAF activation by RAS and the SHOC2 phosphatase complex, and RAF engagement with HSP90-CDC37 chaperone complexes. These studies have important implications for pharmacologic targeting of the pathway. They reveal RAF in distinct regulatory states and show that the functional RAF switch is an integrated complex of RAF with its substrate (MEK) and a 14-3-3 dimer. Here we review these advances, placing them in the context of decades of investigation of RAF regulation. We explore the insights they provide into aberrant activation of the pathway in cancer and RASopathies (developmental syndromes caused by germline mutations in components of the pathway).
Asunto(s)
Transducción de Señal , Quinasas raf , Proteínas ras , Humanos , Proteínas ras/metabolismo , Proteínas ras/genética , Proteínas ras/química , Quinasas raf/metabolismo , Quinasas raf/genética , Animales , Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/patología , Proteínas 14-3-3/metabolismo , Proteínas 14-3-3/genéticaRESUMEN
Self-assemblies (i.e., nanoclusters) of the RAS GTPase on the membrane act as scaffolds that activate downstream RAF kinases and drive MAPK signaling for cell proliferation and tumorigenesis. However, the mechanistic details of nanoclustering remain largely unknown. Here, size-tunable nanodisc platforms and paramagnetic relaxation enhancement (PRE) analyses revealed the structural basis of the cooperative assembly processes of fully processed KRAS, mutated in a quarter of human cancers. The cooperativity is modulated by the mutation and nucleotide states of KRAS and the lipid composition of the membrane. Notably, the oncogenic mutants assemble in nonsequential pathways with two mutually cooperative 'α/α' and 'α/ß' interfaces, while α/α dimerization of wild-type KRAS promotes the secondary α/ß interaction sequentially. Mutation-based interface engineering was used to selectively trap the oligomeric intermediates of KRAS and probe their favorable interface interactions. Transiently exposed interfaces were available for the assembly. Real-time NMR demonstrated that higher-order oligomers retain higher numbers of active GTP-bound protomers in KRAS GTPase cycling. These data provide a deeper understanding of the nanocluster-enhanced signaling in response to the environment. Furthermore, our methodology is applicable to assemblies of many other membrane GTPases and lipid nanoparticle-based formulations of stable protein oligomers with enhanced cooperativity.
Asunto(s)
Proteínas Proto-Oncogénicas p21(ras) , Transducción de Señal , Humanos , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas ras/química , Quinasas raf/metabolismo , DimerizaciónRESUMEN
BACKGROUND: Mutations in the K-Ras gene are among the most frequent genetic alterations in various cancers, and inhibiting RAS signaling has shown promising results in treating solid tumors. However, finding effective drugs that can bind to the RAS protein remains challenging. This drove us to explore new compounds that could inhibit tumor growth, particularly in cancers that harbor K-Ras mutations. METHODS: Our study used bioinformatic techniques such as E-pharmacophore virtual screening, molecular simulation, principal component analysis (PCA), extra precision (XP) docking, and ADMET analyses to identify potential inhibitors for K-Ras mutants G12C and G12D. RESULTS: In our study, we discovered that inhibitors such as afatinib, osimertinib, and hydroxychloroquine strongly inhibit the G12C mutant. Similarly, hydroxyzine, zuclopenthixol, fluphenazine, and doxapram were potent inhibitors for the G12D mutant. Notably, all six of these molecules exhibit a high binding affinity for the H95 cryptic groove present in the mutant structure. These molecules exhibited a unique affinity mechanism at the molecular level, which was further enhanced by hydrophobic interactions. Molecular simulations and PCA revealed the formation of stable complexes within switch regions I and II. This was particularly evident in three complexes: G12C-osimertinib, G12D-fluphenazine, and G12D-zuclopenthixol. Despite the dynamic nature of switches I and II in K-Ras, the interaction of inhibitors remained stable. According to QikProp results, the properties and descriptors of the selected molecules fell within an acceptable range compared to sotorasib. CONCLUSIONS: We have successfully identified potential inhibitors of the K-Ras protein, laying the groundwork for the development of targeted therapies for cancers driven by K-Ras mutations.
Asunto(s)
Neoplasias , Proteínas Proto-Oncogénicas p21(ras) , Humanos , Unión Proteica , Proteínas Proto-Oncogénicas p21(ras)/genética , Farmacóforo , Clopentixol , Reposicionamiento de Medicamentos , Flufenazina , Detección Precoz del Cáncer , Proteínas ras/genética , Proteínas ras/química , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Simulación de Dinámica MolecularRESUMEN
Ras is a central cellular hub protein controlling multiple cell fates. How Ras interacts with a variety of potential effector proteins is relatively unexplored, with only some key effectors characterized in great detail. Here, we have used homology modeling based on X-ray and AlphaFold2 templates to build structural models for 54 Ras-effector complexes. These models were used to estimate binding affinities using a supervised learning regressor. Furthermore, we systematically introduced Ras "branch-pruning" (or branchegetic) mutations to identify 200 interface mutations that affect the binding energy with at least one of the model structures. The impacts of these branchegetic mutants were integrated into a mathematical model to assess the potential for rewiring interactions at the Ras hub on a systems level. These findings have provided a quantitative understanding of Ras-effector interfaces and their impact on systems properties of a key cellular hub.
Asunto(s)
Proteínas , Proteínas ras , Unión Proteica , Proteínas ras/genética , Proteínas ras/química , Proteínas ras/metabolismo , Mutación , Proteínas/metabolismo , Simulación de Dinámica MolecularRESUMEN
Mutations highly affect the structural flexibility of two switch domains in M-RAS considered an important target of anticancer drug design. Gaussian accelerated molecular dynamics (GaMD) simulations were applied to probe the effect of mutations P40D, D41E, and P40D/D41E/L51R on the conformational transition of the switch domains from the GTP-bound M-RAS. The analyses of free energy landscapes (FELs) not only reveal that three mutations induce less energetic states than the wild-type (WT) M-RAS but also verify that the switch domains are extremely disordered. Principal component analysis (PCA) and dynamics analysis suggest that three mutations greatly affect collective motions and structural flexibility of the switch domains that mostly overlap with binding regions of M-RAS to its effectors, which in turn disturbs the activity of M-RAS. The analyses of the interaction network between GTP and M-RAS show that the high instability in hydrogen bonding interactions (HBIs) of GTP with residue 41 and Y42 in the switch domain I drives the disordered states of the switch domains. This work is expected to provide a molecular mechanism for deeply understanding the function of M-RAS and future drug design towards the treatment of cancers.
Asunto(s)
Simulación de Dinámica Molecular , Proteínas ras , Proteínas ras/genética , Proteínas ras/química , Proteínas ras/metabolismo , Conformación Proteica , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , MutaciónRESUMEN
RAS proteins regulate most aspects of cellular physiology. They are mutated in 30% of human cancers and 4% of developmental disorders termed Rasopathies. They cycle between active GTP-bound and inactive GDP-bound states. When active, they can interact with a wide range of effectors that control fundamental biochemical and biological processes. Emerging evidence suggests that RAS proteins are not simple on/off switches but sophisticated information processing devices that compute cell fate decisions by integrating external and internal cues. A critical component of this compute function is the dynamic regulation of RAS activation and downstream signaling that allows RAS to produce a rich and nuanced spectrum of biological outputs. We discuss recent findings how the dynamics of RAS and its downstream signaling is regulated. Starting from the structural and biochemical properties of wild-type and mutant RAS proteins and their activation cycle, we examine higher molecular assemblies, effector interactions and downstream signaling outputs, all under the aspect of dynamic regulation. We also consider how computational and mathematical modeling approaches contribute to analyze and understand the pleiotropic functions of RAS in health and disease.
Asunto(s)
Neoplasias , Transducción de Señal , Humanos , Proteínas ras/química , Guanosina Trifosfato/metabolismoRESUMEN
Ras family GTPases (H/K/N-Ras) modulate numerous effectors, including the lipid kinase PI3K (phosphatidylinositol-3-kinase) that generates growth signal lipid PIP3 (phosphatidylinositol-3,4,5-triphosphate). Active GTP-Ras binds PI3K with high affinity, thereby stimulating PIP3 production. We hypothesize the affinity of this binding interaction could be significantly increased or decreased by Ras mutations at PI3K contact positions, with clinical implications since some Ras mutations at PI3K contact positions are disease-linked. To enable tests of this hypothesis, we have developed an approach combining UV spectral deconvolution, HPLC, and microscale thermophoresis to quantify the KD for binding. The approach measures the total Ras concentration, the fraction of Ras in the active state, and the affinity of active Ras binding to its docking site on PI3K Ras binding domain (RBD) in solution. The approach is illustrated by KD measurements for the binding of active H-Ras and representative mutants, each loaded with GTP or GMPPNP, to PI3Kγ RBD. The findings demonstrate that quantitation of the Ras activation state increases the precision of KD measurements, while also revealing that Ras mutations can increase (Q25L), decrease (D38E, Y40C), or have no effect (G13R) on PI3K binding affinity. Significant Ras affinity changes are predicted to alter PI3K regulation and PIP3 growth signals.
Asunto(s)
Fosfatidilinositol 3-Quinasas , Proteínas ras , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/química , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas ras/química , Unión Proteica , Guanosina Trifosfato/metabolismo , FosfatidilinositolesRESUMEN
Motile cells have developed a large array of molecular machineries to actively change their direction of movement in response to spatial cues from their environment. In this process, small GTPases act as molecular switches and work in tandem with regulators and sensors of their guanine nucleotide status (GAP, GEF, GDI and effectors) to dynamically polarize the cell and regulate its motility. In this review, we focus on Myxococcus xanthus as a model organism to elucidate the function of an atypical small Ras GTPase system in the control of directed cell motility. M. xanthus cells direct their motility by reversing their direction of movement through a mechanism involving the redirection of the motility apparatus to the opposite cell pole. The reversal frequency of moving M. xanthus cells is controlled by modular and interconnected protein networks linking the chemosensory-like frizzy (Frz) pathway - that transmits environmental signals - to the downstream Ras-like Mgl polarity control system - that comprises the Ras-like MglA GTPase protein and its regulators. Here, we discuss how variations in the GTPase interactome landscape underlie single-cell decisions and consequently, multicellular patterns.
Asunto(s)
Proteínas Bacterianas , Movimiento Celular , Myxococcus xanthus , Proteínas ras , Myxococcus xanthus/citología , Myxococcus xanthus/enzimología , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Transducción de Señal , Proteínas ras/química , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Modelos BiológicosRESUMEN
Receptor tyrosine kinase (RTK)-RAS signalling through the downstream mitogen-activated protein kinase (MAPK) cascade regulates cell proliferation and survival. The SHOC2-MRAS-PP1C holophosphatase complex functions as a key regulator of RTK-RAS signalling by removing an inhibitory phosphorylation event on the RAF family of proteins to potentiate MAPK signalling1. SHOC2 forms a ternary complex with MRAS and PP1C, and human germline gain-of-function mutations in this complex result in congenital RASopathy syndromes2-5. However, the structure and assembly of this complex are poorly understood. Here we use cryo-electron microscopy to resolve the structure of the SHOC2-MRAS-PP1C complex. We define the biophysical principles of holoenzyme interactions, elucidate the assembly order of the complex, and systematically interrogate the functional consequence of nearly all of the possible missense variants of SHOC2 through deep mutational scanning. We show that SHOC2 binds PP1C and MRAS through the concave surface of the leucine-rich repeat region and further engages PP1C through the N-terminal disordered region that contains a cryptic RVXF motif. Complex formation is initially mediated by interactions between SHOC2 and PP1C and is stabilized by the binding of GTP-loaded MRAS. These observations explain how mutant versions of SHOC2 in RASopathies and cancer stabilize the interactions of complex members to enhance holophosphatase activity. Together, this integrative structure-function model comprehensively defines key binding interactions within the SHOC2-MRAS-PP1C holophosphatase complex and will inform therapeutic development .
Asunto(s)
Microscopía por Crioelectrón , Péptidos y Proteínas de Señalización Intracelular , Complejos Multiproteicos , Proteína Fosfatasa 1 , Proteínas ras , Secuencias de Aminoácidos , Sitios de Unión , Guanosina Trifosfato/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Sistema de Señalización de MAP Quinasas , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Complejos Multiproteicos/ultraestructura , Mutación Missense , Fosforilación , Unión Proteica , Proteína Fosfatasa 1/química , Proteína Fosfatasa 1/metabolismo , Proteína Fosfatasa 1/ultraestructura , Estabilidad Proteica , Quinasas raf , Proteínas ras/química , Proteínas ras/metabolismo , Proteínas ras/ultraestructuraRESUMEN
RAS-MAPK signalling is fundamental for cell proliferation and is altered in most human cancers1-3. However, our mechanistic understanding of how RAS signals through RAF is still incomplete. Although studies revealed snapshots for autoinhibited and active RAF-MEK1-14-3-3 complexes4, the intermediate steps that lead to RAF activation remain unclear. The MRAS-SHOC2-PP1C holophosphatase dephosphorylates RAF at serine 259, resulting in the partial displacement of 14-3-3 and RAF-RAS association3,5,6. MRAS, SHOC2 and PP1C are mutated in rasopathies-developmental syndromes caused by aberrant MAPK pathway activation6-14-and SHOC2 itself has emerged as potential target in receptor tyrosine kinase (RTK)-RAS-driven tumours15-18. Despite its importance, structural understanding of the SHOC2 holophosphatase is lacking. Here we determine, using X-ray crystallography, the structure of the MRAS-SHOC2-PP1C complex. SHOC2 bridges PP1C and MRAS through its concave surface and enables reciprocal interactions between all three subunits. Biophysical characterization indicates a cooperative assembly driven by the MRAS GTP-bound active state, an observation that is extendible to other RAS isoforms. Our findings support the concept of a RAS-driven and multi-molecular model for RAF activation in which individual RAS-GTP molecules recruit RAF-14-3-3 and SHOC2-PP1C to produce downstream pathway activation. Importantly, we find that rasopathy and cancer mutations reside at protein-protein interfaces within the holophosphatase, resulting in enhanced affinities and function. Collectively, our findings shed light on a fundamental mechanism of RAS biology and on mechanisms of clinically observed enhanced RAS-MAPK signalling, therefore providing the structural basis for therapeutic interventions.
Asunto(s)
Cristalografía por Rayos X , Péptidos y Proteínas de Señalización Intracelular , Complejos Multiproteicos , Proteína Fosfatasa 1 , Proteínas ras , Proteínas 14-3-3 , Guanosina Trifosfato/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Sistema de Señalización de MAP Quinasas , Complejos Multiproteicos/química , Mutación , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Proteína Fosfatasa 1/química , Proteína Fosfatasa 1/genética , Proteína Fosfatasa 1/metabolismo , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Quinasas raf , Proteínas ras/química , Proteínas ras/metabolismoRESUMEN
The RAS-RAF pathway is one of the most commonly dysregulated in human cancers1-3. Despite decades of study, understanding of the molecular mechanisms underlying dimerization and activation4 of the kinase RAF remains limited. Recent structures of inactive RAF monomer5 and active RAF dimer5-8 bound to 14-3-39,10 have revealed the mechanisms by which 14-3-3 stabilizes both RAF conformations via specific phosphoserine residues. Prior to RAF dimerization, the protein phosphatase 1 catalytic subunit (PP1C) must dephosphorylate the N-terminal phosphoserine (NTpS) of RAF11 to relieve inhibition by 14-3-3, although PP1C in isolation lacks intrinsic substrate selectivity. SHOC2 is as an essential scaffolding protein that engages both PP1C and RAS to dephosphorylate RAF NTpS11-13, but the structure of SHOC2 and the architecture of the presumptive SHOC2-PP1C-RAS complex remain unknown. Here we present a cryo-electron microscopy structure of the SHOC2-PP1C-MRAS complex to an overall resolution of 3 Å, revealing a tripartite molecular architecture in which a crescent-shaped SHOC2 acts as a cradle and brings together PP1C and MRAS. Our work demonstrates the GTP dependence of multiple RAS isoforms for complex formation, delineates the RAS-isoform preference for complex assembly, and uncovers how the SHOC2 scaffold and RAS collectively drive specificity of PP1C for RAF NTpS. Our data indicate that disease-relevant mutations affect complex assembly, reveal the simultaneous requirement of two RAS molecules for RAF activation, and establish rational avenues for discovery of new classes of inhibitors to target this pathway.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular , Proteína Fosfatasa 1 , Transducción de Señal , Proteínas ras , Microscopía por Crioelectrón , Guanosina Trifosfato/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Complejos Multiproteicos/ultraestructura , Mutación , Fosfoserina , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/ultraestructura , Proteína Fosfatasa 1/química , Proteína Fosfatasa 1/genética , Proteína Fosfatasa 1/metabolismo , Proteína Fosfatasa 1/ultraestructura , Especificidad por Sustrato , Quinasas raf/metabolismo , Proteínas ras/química , Proteínas ras/genética , Proteínas ras/metabolismo , Proteínas ras/ultraestructuraRESUMEN
Allosteric mechanisms are pervasive in nature, but human-designed allosteric perturbagens are rare. The history of KRASG12C inhibitor development suggests that covalent chemistry may be a key to expanding the armamentarium of allosteric inhibitors. In that effort, irreversible targeting of a cysteine converted a non-deal allosteric binding pocket and low affinity ligands into a tractable drugging strategy. Here we examine the feasibility of expanding this approach to other allosteric pockets of RAS and kinase family members, given that both protein families are regulators of vital cellular processes that are often dysregulated in cancer and other human diseases. Moreover, these heavily studied families are the subject of numerous drug development campaigns that have resulted, sometimes serendipitously, in the discovery of allosteric inhibitors. We consequently conducted a comprehensive search for cysteines, a commonly targeted amino acid for covalent drugs, using AlphaFold-generated structures of those families. This new analysis presents potential opportunities for allosteric targeting of validated and understudied drug targets, with an emphasis on cancer therapy.
Asunto(s)
Antineoplásicos , Terapia Molecular Dirigida , Neoplasias , Inhibidores de Proteínas Quinasas , Proteínas Quinasas , Proteínas ras , Antineoplásicos/química , Antineoplásicos/farmacología , Cisteína/metabolismo , Humanos , Neoplasias/enzimología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/química , Proteínas ras/antagonistas & inhibidores , Proteínas ras/químicaRESUMEN
As the hub of major signaling pathways, Ras proteins are implicated in 19% of tumor-caused cancers due to perturbations in their conformational and/or catalytic properties. Despite numerous studies, the functions of the conformational substates for the most important isoform, KRas, remain elusive. In this work, we perform an extensive simulation analysis on the conformational landscape of KRas in its various chemical states during the GTP hydrolysis cycle: the reactant state KRasGTP·Mg2+, the intermediate state KRasGDP·Pi·Mg2+ and the product state KRasGDP·Mg2+. The results from enhanced sampling simulations reveal that State 1 of KRasGTP·Mg2+ has multiple stable substates in solution, one of which might account for interacting with GEFs. State 2 of KRasGTP·Mg2+ features two substates "Tyr32in" and "Tyr32out", which are poised to interact with effectors and GAPs, respectively. For the intermediate state KRasGDP·Pi·Mg2+, Gln61 and Pi are found to assume a broad set of conformations, which might account for the weak oncogenic effect of Gln61 mutations in KRas in contrast to the situation in HRas and NRas. Finally, the product state KRasGDP·Mg2+ has more than two stable substates in solution, pointing to a conformation-selection mechanism for complexation with GEFs. Based on these results, some specific inhibition strategies for targeting the binding sites of the high-energy substates of KRas during GTP hydrolysis are discussed.
Asunto(s)
Proteínas ras , Sitios de Unión , Guanosina Trifosfato/química , Hidrólisis , Mutación , Proteínas ras/químicaRESUMEN
KRAS is the most frequently mutated RAS protein in cancer patients, and it is estimated that about 20% of the cancer patients in the United States carried mutant RAS proteins. To accelerate therapeutic development, structures and dynamics of RAS proteins had been extensively studied by various biophysical techniques for decades. Although 31P NMR studies revealed population equilibrium of the two major states in the active GMPPNP-bound form, more complex conformational dynamics in RAS proteins and oncogenic mutants subtly modulate the interactions with their downstream effectors. We established a set of customized NMR relaxation dispersion techniques to efficiently and systematically examine the ms-µs conformational dynamics of RAS proteins. This method allowed us to observe varying synchronized motions that connect the effector and allosteric lobes in KRAS. We demonstrated the role of conformational dynamics of KRAS in controlling its interaction with the Ras-binding domain of the downstream effector RAF1, the first kinase in the MAPK pathway. This allows one to explain, as well as to predict, the altered binding affinities of various KRAS mutants, which was neither previously reported nor apparent from the structural perspective.
Asunto(s)
Neoplasias , Proteínas Proto-Oncogénicas p21(ras) , Fenómenos Fisiológicos Celulares , Humanos , Conformación Molecular , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas ras/químicaRESUMEN
The lipid-anchored RAS (Rat sarcoma) small GTPases (guanosine triphosphate hydrolases) are highly prevalent in human cancer. Traditional strategies of targeting the enzymatic activities of RAS have been shown to be difficult. Alternatively, RAS function and pathology are mostly restricted to nanoclusters on the plasma membrane (PM). Lipids are important structural components of these signaling platforms on the PM. However, how RAS nanoclusters selectively enrich distinct lipids in the PM, how different lipids contribute to RAS signaling and oncogenesis and whether the selective lipid sorting of RAS nanoclusters can be targeted have not been well-understood. Latest advances in quantitative super-resolution imaging and molecular dynamic simulations have allowed detailed characterization RAS/lipid interactions. In this review, we discuss the latest findings on the select lipid composition (with headgroup and acyl chain specificities) within RAS nanoclusters, the specific mechanisms for the select lipid sorting of RAS nanoclusters on the PM and how perturbing lipid compositions within RAS nanoclusters impacts RAS function and pathology. We also describe different strategies of manipulating lipid composition within RAS nanoclusters on the PM.
Asunto(s)
Lípidos/química , Nanopartículas/química , Proteínas ras/metabolismo , Animales , Membrana Celular/metabolismo , Humanos , Modelos Biológicos , Transducción de Señal , Proteínas ras/químicaRESUMEN
S-Palmitoylation is a reversible post-translational lipid modification that regulates protein trafficking and signaling. The enzymatic depalmitoylation of proteins is inhibited by the beta-lactones Palmostatin M and B, which have been found to target several serine hydrolases. In efforts to better understand the mechanism of action of Palmostatin M, we describe herein the synthesis, chemical proteomic analysis, and functional characterization of analogs of this compound. We identify Palmostatin M analogs that maintain inhibitory activity in N-Ras depalmitoylation assays while displaying complementary reactivity across the serine hydrolase class as measured by activity-based protein profiling. Active Palmostatin M analogs inhibit the recently characterized ABHD17 subfamily of depalmitoylating enzymes, while sparing other candidate depalmitoylases such as LYPLA1 and LYPLA2. These findings improve our understanding of the structure-activity relationship of Palmostatin M and refine the set of serine hydrolase targets relevant to the compound's effects on N-Ras palmitoylation dynamics.
Asunto(s)
Lactonas/análisis , Propiolactona/análogos & derivados , Proteómica , Sulfonas/análisis , Proteínas ras/metabolismo , Humanos , Lactonas/metabolismo , Lactonas/farmacología , Estructura Molecular , Propiolactona/análisis , Propiolactona/metabolismo , Propiolactona/farmacología , Sulfonas/metabolismo , Sulfonas/farmacología , Proteínas ras/antagonistas & inhibidores , Proteínas ras/químicaRESUMEN
RAS proteins (HRAS, KRAS, NRAS) participate in many physiological signal transduction processes related to cell growth, division, and survival. The RAS proteins are small (188/189 amino acid residues) and they function as GTPases. These proteins toggle between inactive and functional forms; the conversion of inactive RAS-GDP to active RAS-GTP as mediated by guanine nucleotide exchange factors (GEFs) turns the switch on and the intrinsic RAS-GTPase activity stimulated by the GTPase activating proteins (GAPs) turns the switch off. RAS is upstream to the RAS-RAF-MEK-ERK and the PI3-kinase-AKT signaling modules. Importantly, the overall incidence of RAS mutations in all cancers is about 19% and RAS mutants have been a pharmacological target for more than three decades. About 84% of all RAS mutations involve KRAS. Except for the GTP/GDP binding site, the RAS proteins lack other deep surface pockets thereby hindering efforts to identify high-affinity antagonists; thus, they have been considered to be undruggable. KRAS mutations frequently occur in lung, colorectal, and pancreatic cancers, the three most deadly cancers in the United States. Studies within the last decade demonstrated that the covalent modification of KRAS C12, which accounts for about 10% of all RAS mutations, led to the discovery of an adjacent pocket (called the switch II pocket) that accommodated a portion of the drug. This led to the development of sotorasib as a second-line treatment of KRASG12C-mutant non-small cell lung cancer. Considerable effort also has been expended to develop MAP kinase and PI3-kinase pathway inhibitors as indirect RAS antagonists.