RESUMO
A severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes mild-to-severe respiratory symptoms, including acute respiratory distress. Despite remarkable efforts to investigate the virological and pathological impacts of SARS-CoV-2, many of the characteristics of SARS-CoV-2 infection still remain unknown. The interferon-inducible ubiquitin-like protein ISG15 is covalently conjugated to several viral proteins to suppress their functions. It was reported that SARS-CoV-2 utilizes its papain-like protease (PLpro) to impede ISG15 conjugation, ISGylation. However, the role of ISGylation in SARS-CoV-2 infection remains unclear. We aimed to elucidate the role of ISGylation in SARS-CoV-2 replication. We observed that the SARS-CoV-2 nucleocapsid protein is a target protein for the HERC5 E3 ligase-mediated ISGylation in cultured cells. Site-directed mutagenesis reveals that the residue K374 within the C-terminal spacer B-N3 (SB/N3) domain is required for nucleocapsid-ISGylation, alongside conserved lysine residue in MERS-CoV (K372) and SARS-CoV (K375). We also observed that the nucleocapsid-ISGylation results in the disruption of nucleocapsid oligomerization, thereby inhibiting viral replication. Knockdown of ISG15 mRNA enhanced SARS-CoV-2 replication in the SARS-CoV-2 reporter replicon cells, while exogenous expression of ISGylation components partially hampered SARS-CoV-2 replication. Taken together, these results suggest that SARS-CoV-2 PLpro inhibits ISGylation of the nucleocapsid protein to promote viral replication by evading ISGylation-mediated disruption of the nucleocapsid oligomerization.IMPORTANCEISG15 is an interferon-inducible ubiquitin-like protein that is covalently conjugated to the viral protein via specific Lys residues and suppresses viral functions and viral propagation in many viruses. However, the role of ISGylation in SARS-CoV-2 infection remains largely unclear. Here, we demonstrated that the SARS-CoV-2 nucleocapsid protein is a target protein for the HERC5 E3 ligase-mediated ISGylation. We also found that the residue K374 within the C-terminal spacer B-N3 (SB/N3) domain is required for nucleocapsid-ISGylation. We obtained evidence suggesting that nucleocapsid-ISGylation results in the disruption of nucleocapsid-oligomerization, thereby suppressing SARS-CoV-2 replication. We discovered that SARS-CoV-2 papain-like protease inhibits ISG15 conjugation of nucleocapsid protein via its de-conjugating enzyme activity. The present study may contribute to gaining new insight into the roles of ISGylation-mediated anti-viral function in SARS-CoV-2 infection and may lead to the development of more potent and selective inhibitors targeted to SARS-CoV-2 nucleocapsid protein.
RESUMO
GTP-bound forms of Ras proteins (Rasâ¢GTP) assume two interconverting conformations, "inactive" state 1 and "active" state 2. Our previous study on the crystal structure of the state 1 conformation of H-Ras in complex with guanosine 5'-(ß, γ-imido)triphosphate (GppNHp) indicated that state 1 is stabilized by intramolecular hydrogen-bonding interactions formed by Gln61. Since Ras are constitutively activated by substitution mutations of Gln61, here we determine crystal structures of the state 1 conformation of H-Rasâ¢GppNHp carrying representative mutations Q61L and Q61H to observe the effect of the mutations. The results show that these mutations alter the mode of hydrogen-bonding interactions of the residue 61 with Switch II residues and induce conformational destabilization of the neighboring regions. In particular, Q61L mutation results in acquirement of state 2-like structural features. Moreover, the mutations are likely to impair an intramolecular structural communication between Switch I and Switch II. Molecular dynamics simulations starting from these structures support the above observations. These findings may give a new insight into the molecular mechanism underlying the aberrant activation of the Gln61 mutants.
Assuntos
Guanosina Trifosfato/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Cristalografia por Raios X , Guanosina Trifosfato/genética , Humanos , Conformação Molecular , Simulação de Dinâmica Molecular , Mutação , Proteínas Proto-Oncogênicas p21(ras)/genéticaRESUMO
The ras oncogene products (H-Ras, K-Ras, and N-Ras) have been regarded as some of the most promising targets for anticancer drug discovery because their activating mutations are frequently found in human cancers. Nonetheless, molecular targeted therapy for them is currently unavailable. Here, we report the discovery of a small-molecule compound carrying a naphthalene ring, named KBFM123, which binds to the GTP-bound form of H-Ras. The solution structure of its complex with the guanosine 5'-(ß,γ-imide) triphosphate-bound form of H-RasT35S (H-RasT35S·GppNHp) indicates that the naphthalene ring of KBFM123 interacts directly with a hydrophobic pocket located between switch I and switch II and allosterically inhibits the effector interaction by inducing conformational changes in switch I and its flanking region in strand ß2, which are directly involved in recognition of the effector molecules, including c-Raf-1. In particular, Asp38 of H-Ras, a crucial residue for the interaction with c-Raf-1 via the formation of a salt bridge with Arg89 of the Ras-binding domain (RBD) of c-Raf-1, shows a drastic conformational change: its side chain orients toward the opposite direction. Consistent with these results, KBFM123 exhibits an activity to inhibit, albeit weakly, the association of H-RasG12V·GppNHp with the c-Raf-1 RBD. The binding of the naphthalene ring to the hydrophobic pocket of H-RasT35S·GppNHp is further supported by nuclear magnetic resonance analyses showing that two other naphthalene-containing compounds with distinct structures also exhibit similar binding properties with KBFM123. These results indicate that the naphthalene ring could become a promising scaffold for the development of Ras inhibitors.
Assuntos
Guanosina Trifosfato/metabolismo , Naftalenos/química , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Sítios de Ligação , Catálise , Domínio Catalítico , Descoberta de Drogas , Humanos , Modelos Moleculares , Conformação ProteicaRESUMO
Mutational activation of the Ras oncogene products (H-Ras, K-Ras, and N-Ras) is frequently observed in human cancers, making them promising anticancer drug targets. Nonetheless, no effective strategy has been available for the development of Ras inhibitors, partly owing to the absence of well-defined surface pockets suitable for drug binding. Only recently, such pockets have been found in the crystal structures of a unique conformation of Rasâ GTP. Here we report the successful development of small-molecule Ras inhibitors by an in silico screen targeting a pocket found in the crystal structure of M-Rasâ GTP carrying an H-Ras-type substitution P40D. The selected compound Kobe0065 and its analog Kobe2602 exhibit inhibitory activity toward H-Rasâ GTP-c-Raf-1 binding both in vivo and in vitro. They effectively inhibit both anchorage-dependent and -independent growth and induce apoptosis of H-ras(G12V)-transformed NIH 3T3 cells, which is accompanied by down-regulation of downstream molecules such as MEK/ERK, Akt, and RalA as well as an upstream molecule, Son of sevenless. Moreover, they exhibit antitumor activity on a xenograft of human colon carcinoma SW480 cells carrying the K-ras(G12V) gene by oral administration. The NMR structure of a complex of the compound with H-Rasâ GTP(T35S), exclusively adopting the unique conformation, confirms its insertion into one of the surface pockets and provides a molecular basis for binding inhibition toward multiple Rasâ GTP-interacting molecules. This study proves the effectiveness of our strategy for structure-based drug design to target Rasâ GTP, and the resulting Kobe0065-family compounds may serve as a scaffold for the development of Ras inhibitors with higher potency and specificity.
Assuntos
Antineoplásicos/farmacologia , Desenho de Fármacos , Proteínas ras/antagonistas & inibidores , Proteínas ras/metabolismo , Animais , Linhagem Celular Transformada , Linhagem Celular Tumoral , Biologia Computacional/métodos , Glutationa Transferase/metabolismo , Guanosina Trifosfato/química , Humanos , Camundongos , Camundongos Nus , Modelos Moleculares , Conformação Molecular , Mutação , Células NIH 3T3 , Transplante de Neoplasias , Ligação Proteica , Conformação Proteica , Transdução de SinaisRESUMO
Ras small GTPases undergo dynamic equilibrium of two interconverting conformations, state 1 and state 2, in the GTP-bound forms, where state 2 is recognized by effectors, whereas physiological functions of state 1 have been unknown. Limited information, such as static crystal structures and (31)P NMR spectra, was available for the study of the conformational dynamics. Here we determine the solution structure and dynamics of state 1 by multidimensional heteronuclear NMR analysis of an H-RasT35S mutant in complex with guanosine 5'-(ß, γ-imido)triphosphate (GppNHp). The state 1 structure shows that the switch I loop fluctuates extensively compared with that in state 2 or H-Ras-GDP. Also, backbone (1)H,(15)N signals for state 2 are identified, and their dynamics are studied by utilizing a complex with c-Raf-1. Furthermore, the signals for almost all the residues of H-Ras·GppNHp are identified by measurement at low temperature, and the signals for multiple residues are found split into two peaks corresponding to the signals for state 1 and state 2. Intriguingly, these residues are located not only in the switch regions and their neighbors but also in the rigidly structured regions, suggesting that global structural rearrangements occur during the state interconversion. The backbone dynamics of each state show that the switch loops in state 1 are dynamically mobile on the picosecond to nanosecond time scale, and these mobilities are significantly reduced in state 2. These results suggest that multiconformations existing in state 1 are mostly deselected upon the transition toward state 2 induced by the effector binding.
Assuntos
Guanosina Trifosfato/química , Proteínas Proto-Oncogênicas p21(ras)/química , Substituição de Aminoácidos , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/genética , Guanosina Trifosfato/metabolismo , Humanos , Mutação de Sentido Incorreto , Ressonância Magnética Nuclear Biomolecular , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-raf/química , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Relação Estrutura-AtividadeRESUMO
GTP-bound forms of Ras family small GTPases exhibit dynamic equilibrium between two interconverting conformations, "inactive" state 1 and "active" state 2. A great variation exists in their state distribution; H-Ras mainly adopts state 2, whereas M-Ras predominantly adopts state 1. Our previous studies based on comparison of crystal structures representing state 1 and state 2 revealed the importance of the hydrogen-bonding interactions of two flexible effector-interacting regions, switch I and switch II, with the γ-phosphate of GTP in establishing state 2 conformation. However, failure to obtain both state structures from a single protein hampered further analysis of state transition mechanisms. Here, we succeed in solving two crystal structures corresponding to state 1 and state 2 from a single Ras polypeptide, M-RasD41E, carrying an H-Ras-type substitution in residue 41, immediately preceding switch I, in complex with guanosine 5'-(ß,γ-imido)triphosphate. Comparison among the two structures and other state 1 and state 2 structures of H-Ras/M-Ras reveal two new structural features playing critical roles in state dynamics; interaction of residues 31/41 (H-Ras/M-Ras) with residues 29/39 and 30/40, which induces a conformational change of switch I favoring its interaction with the γ-phosphate, and the hydrogen-bonding interaction of switch II with its neighboring α-helix, α3-helix, which induces a conformational change of switch II favoring its interaction with the γ-phosphate. The importance of the latter interaction is proved by mutational analyses of the residues involved in hydrogen bonding. These results define the two novel functional regions playing critical roles during state transition.
Assuntos
Simulação de Dinâmica Molecular , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Humanos , Camundongos , Proteínas Monoméricas de Ligação ao GTP/química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Homologia Estrutural de Proteína , Proteínas rasRESUMO
Ras family small GTPases assume two interconverting conformations, "inactive" state 1 and "active" state 2, in their GTP-bound forms. Here, to clarify the mechanism of state transition, we have carried out x-ray crystal structure analyses of a series of mutant H-Ras and M-Ras in complex with guanosine 5'-(beta,gamma-imido)triphosphate (GppNHp), representing various intermediate states of the transition. Crystallization of H-RasT35S-GppNHp enables us to solve the first complete tertiary structure of H-Ras state 1 possessing two surface pockets unseen in the state 2 or H-Ras-GDP structure. Moreover, determination of the two distinct crystal structures of H-RasT35S-GppNHp, showing prominent polysterism in the switch I and switch II regions, reveals a pivotal role of the guanine nucleotide-mediated interaction between the two switch regions and its rearrangement by a nucleotide positional change in the state 2 to state 1 transition. Furthermore, the (31)P NMR spectra and crystal structures of the GppNHp-bound forms of M-Ras mutants, carrying various H-Ras-type amino acid substitutions, also reveal the existence of a surface pocket in state 1 and support a similar mechanism based on the nucleotide-mediated interaction and its rearrangement in the state 1 to state 2 transition. Intriguingly, the conformational changes accompanying the state transition mimic those that occurred upon GDP/GTP exchange, indicating a common mechanistic basis inherent in the high flexibility of the switch regions. Collectively, these results clarify the structural features distinguishing the two states and provide new insights into the molecular basis for the state transition of Ras protein.
Assuntos
Guanosina Trifosfato/metabolismo , Proteínas Monoméricas de Ligação ao GTP/química , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Aminoácidos/metabolismo , Animais , Sítios de Ligação , Cristalografia por Raios X , Guanosina Difosfato/metabolismo , Guanilil Imidodifosfato/metabolismo , Humanos , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética , Camundongos , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Proteínas rasRESUMO
Bardet-Biedl syndrome (BBS) is a pleiotropically genetic disorder, whose etiology is linked to cilia. Mutations in the Arf/Arl-family GTPase Arl6 have been recently shown to be responsible for BBS type 3. Here we show that BBS mutations alter the guanine nucleotide-binding properties of Arl6. Specifically, substitution of 31st Threonine to Arginine selectively abrogates the GTP-binding ability of Arl6 without affecting GDP-binding/dissociating properties. Furthermore, all the BBS mutations in Arl6 result in low expression of the mutant proteins, which can be restored by the inhibition of the proteasome. These findings implicate that Arl6 mutants are destabilized and eliminated by the proteasome in cells, probably due to the altered nucleotide-binding properties.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Síndrome de Bardet-Biedl/enzimologia , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Mutação de Sentido Incorreto , Fatores de Ribosilação do ADP/genética , Animais , Síndrome de Bardet-Biedl/genética , Linhagem Celular , Cães , Estabilidade Enzimática/genética , Guanina/metabolismo , Humanos , Proteínas Monoméricas de Ligação ao GTP/genética , Complexo de Endopeptidases do Proteassoma/metabolismoRESUMO
Previous (31)P NMR studies revealed that small GTPases H-Ras and K-Ras in complex with GTP assume two interconverting conformational states, state 1 and state 2. While state 2 corresponds to an active conformation, little is known about the function of state 1, an inactive conformation incapable of effector binding. To address the biochemical properties of state 1, we measured the (31)P NMR spectra of five Ras family small GTPases; H-Ras, M-Ras, Rap1A, Rap2A and RalA, and find that they exhibit distinctive state 2/state 1 populations with the ratios ranging from 0.072 for M-Ras to 16 for Rap2A. Further, we show that GTPases with higher populations of state 1 exhibit higher dissociation and association rate constants for GTP. These results imply that GTP loading to the nucleotide-free small GTPases preferentially yields state 1, which is subsequently converted to state 2, rendering the GTP-bound form functional.
Assuntos
Guanosina Trifosfato/química , Modelos Químicos , Modelos Moleculares , Proteínas Ativadoras de ras GTPase/química , Proteínas Ativadoras de ras GTPase/ultraestrutura , Sítios de Ligação , Simulação por Computador , Cinética , Ligação Proteica , Conformação ProteicaRESUMO
Metastasis stands as the major obstacle for the survival from cancers. Nonetheless most existing anti-cancer drugs inhibit only cell proliferation, and discovery of agents having both anti-proliferative and anti-metastatic properties would be more beneficial. We previously reported the discovery of small-molecule Ras inhibitors, represented by Kobe0065, that displayed anti-proliferative activity on xenografts of human colorectal cancer (CRC) cell line SW480 carrying the K-rasG12Vgene. Here we show that treatment of cancer cells carrying the activated ras genes with Kobe0065 or a siRNA targeting Ras downregulates the expression of lysyl oxidase (LOX), which has been implicated in metastasis. LOX expression is enhanced by co-expression of RasG12V through activation of phosphatidylinositol 3-kinase (PI3K)/Akt and concomitant accumulation of hypoxia-inducible factor (HIF)-1α. Furthermore, Kobe0065 effectively inhibits not only migration and invasion of cancer cells carrying the activated ras genes but also lung metastasis of human CRC cell line SW620 carrying the K-rasG12V gene. Collectively, these results indicate that Kobe0065 prevents metastasis through inhibition of the Ras-PI3K-Akt-HIF-1α-LOX signaling and suggest that Ras inhibitors in general might exhibit both anti-proliferative and anti-metastatic properties toward cancer cells carrying the activated ras genes.
Assuntos
Antineoplásicos/farmacologia , Movimento Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Pulmonares/prevenção & controle , Neoplasias Pancreáticas/tratamento farmacológico , Fosfatidilinositol 3-Quinase/metabolismo , Proteína-Lisina 6-Oxidase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Animais , Relação Dose-Resposta a Droga , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundário , Células MCF-7 , Camundongos , Camundongos Nus , Mutação , Células NIH 3T3 , Invasividade Neoplásica , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Interferência de RNA , Transdução de Sinais/efeitos dos fármacos , Transfecção , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The state transitions of solvated H-Ras protein with GTP were theoretically analyzed through molecular dynamics (MD) simulations. To accelerate the structural changes associated with the locations of two switch regions (I and II), the Parallel Cascade Selection MD (PaCS-MD) method was employed in this study. The interconversions between the State 1 and State 2 were thus studied in atomic details, leading to a reasonable agreement with experimental observations and consequent scenarios concerning the transition mechanism that would be essential for the development of Ras inhibitors as anti-cancer agents. Furthermore, the state-transition-based local network entropy (SNE) was calculated for the transition process from State 1 to State 2, by which the temporal evolution of information entropy associated with the dynamical behavior of hydrogen bond network composed of hydration water molecules was described. The calculated results of SNE thus proved to provide a good indicator to detect the dynamical state transition of solvated Ras protein system (and probably more general systems) from a viewpoint of nonequilibrium statistical thermodynamics.
Assuntos
Conformação Proteica , Proteínas Proto-Oncogênicas p21(ras)/química , Termodinâmica , Entropia , Humanos , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , Água/químicaRESUMO
Rasâ¢GTP adopts two interconverting conformational states, state 1 and state 2, corresponding to inactive and active forms, respectively. However, analysis of the mechanism for state transition was hampered by the lack of the structural information on wild-type Ras state 1 despite its fundamental nature conserved in the Ras superfamily. Here we solve two new crystal structures of wild-type H-Ras, corresponding to state 1 and state 2. The state 2 structure seems to represent an intermediate of state transition and, intriguingly, the state 1 crystal is successfully derived from this state 2 crystal by regulating the surrounding humidity. Structural comparison enables us to infer the molecular mechanism for state transition, during which a wide range of hydrogen-bonding networks across Switch I, Switch II and the α3-helix interdependently undergo gross rearrangements, where fluctuation of Tyr32, translocation of Gln61, loss of the functional water molecules and positional shift of GTP play major roles. The NMR-based hydrogen/deuterium exchange experiments also support this transition mechanism. Moreover, the unveiled structural features together with the results of the biochemical study provide a new insight into the physiological role of state 1 as a stable pool of Rasâ¢GTP in the GDP/GTP cycle of Ras.
RESUMO
Myxofibrosarcoma is a common mesenchymal malignancy with complex genomics and heterogeneous clinical outcomes. Through gene-expression profiling of 64 primary high-grade myxofibrosarcomas, we defined an expression signature associated with clinical outcome. The gene most significantly associated with disease-specific death and distant metastasis was ITGA10 (integrin-α10). Functional studies revealed that myxofibrosarcoma cells strongly depended on integrin-α10, whereas normal mesenchymal cells did not. Integrin-α10 transmitted its tumor-specific signal via TRIO and RICTOR, two oncoproteins that are frequently co-overexpressed through gene amplification on chromosome 5p. TRIO and RICTOR activated RAC/PAK and AKT/mTOR to promote sarcoma cell survival. Inhibition of these proteins with EHop-016 (RAC inhibitor) and INK128 (mTOR inhibitor) had antitumor effects in tumor-derived cell lines and mouse xenografts, and combining the drugs enhanced the effects. Our results demonstrate the importance of integrin-α10/TRIO/RICTOR signaling for driving myxofibrosarcoma progression and provide the basis for promising targeted treatment strategies for patients with high-risk disease. SIGNIFICANCE: Identifying the molecular pathogenesis for myxofibrosarcoma progression has proven challenging given the highly complex genomic alterations in this tumor type. We found that integrin-α10 promotes tumor cell survival through activation of TRIO-RAC-RICTOR-mTOR signaling, and that inhibitors of RAC and mTOR have antitumor effects in vivo, thus identifying a potential treatment strategy for patients with high-risk myxofibrosarcoma. Cancer Discov; 6(10); 1148-65. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 1069.
Assuntos
Proteínas de Transporte/genética , Fibrossarcoma/genética , Cadeias alfa de Integrinas/genética , Proteínas rac de Ligação ao GTP/genética , Animais , Linhagem Celular Tumoral , Fibrossarcoma/tratamento farmacológico , Fibrossarcoma/metabolismo , Fibrossarcoma/patologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Cadeias alfa de Integrinas/metabolismo , Camundongos , Terapia de Alvo Molecular , Gradação de Tumores , Transplante de Neoplasias , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteína Companheira de mTOR Insensível à Rapamicina , Transdução de Sinais , Serina-Treonina Quinases TOR/genética , Regulação para CimaRESUMO
Despite the importance of ras as driver genes in many cancers, clinically effective anti-cancer drugs targeting their products, Ras, have been unavailable so far, which was in part ascribable to the apparently 'undruggable' nature of their tertiary structures. Nonetheless, recent studies in academia and industry have identified novel surface pockets accepting small-molecule ligands in both their active GTP-bound and inactive GDP-bound forms (Rasâ¢GTP and Rasâ¢GDP, respectively), which has led to a surge of investigations into the discovery of Ras-specific inhibitors particularly by utilizing their structural information for structure-based drug design (SBDD). We have been developing Ras inhibitors by SBDD targeting a novel conformation of Rasâ¢GTP called state 1, possessing 'druggable' surface pockets, which emerges from the conformational dynamics. In this article, we will survey Ras functions from the structural biological point of view and summarize the current status of the development of Ras inhibitors including our own.
Assuntos
Inibidores Enzimáticos/farmacologia , Proteínas ras/antagonistas & inibidores , Animais , Humanos , Modelos Moleculares , Ligação Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Proteínas ras/química , Proteínas ras/metabolismoRESUMO
Genetic studies on Schizosaccharomyces pombe adenylyl cyclase (cyr1) have shown that its activity is positively regulated by a heterotrimetric G protein a subunit gpa2 and that the resulting increase in intracellular cAMP concentration causes inhibition of sexual development including mating and meiosis. However, molecular mechanism underlying this gpa2-dependent regulation of cyr1 remains to be clarified. Here, we show that gpa2 exhibits a direct and GTP-dependent binding to the Ras-associating domain (RAD) of cyr1, which is identified by a computer algorithm-based search of the cyr1 amino acid sequence. Overexpression of this RAD results in acceleration of the sexual development of fission yeast cells presumably by competitive sequestration of gpa2. Furthermore, cyr1 is activated in vitro by the addition of purified gpa2, which is converted to the active state by treatment with AlF4-. These results indicate a crucial role of the RAD as a direct binding site of gpa2 in activation of cyr1. Thus, RADs, which have been defined as a conserved motif shared among the Ras-family small G protein-associating domains, are for the first time shown to exhibit a functional association with a member of the heterotrimeric G proteins.
Assuntos
Adenilil Ciclases/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/fisiologia , Subunidades alfa de Proteínas de Ligação ao GTP/fisiologia , Proteínas de Schizosaccharomyces pombe/fisiologia , Schizosaccharomyces/fisiologia , Sequência de Aminoácidos , Sítios de Ligação , Ativação Enzimática , Regulação Fúngica da Expressão Gênica , Genes ras/fisiologia , Dados de Sequência Molecular , Schizosaccharomyces/enzimologia , Schizosaccharomyces/genética , Diferenciação Sexual/genéticaRESUMO
Ras proteins, particularly their active GTP-bound forms (Ras·GTP), were thought "undruggable" owing to the absence of apparent drug-accepting pockets in their crystal structures. Only recently, such pockets have been found in the crystal structures representing a novel Ras·GTP conformation. We have conducted an in silico docking screen targeting a pocket in the crystal structure of M-Ras(P40D)·GTP and obtained Kobe0065, which, along with its analogue Kobe2602, inhibits binding of H-Ras·GTP to c-Raf-1. They inhibit the growth of H-rasG12V-transformed NIH3T3 cells, which are accompanied by downregulation of not only MEK/ERK but also Akt, RalA, and Sos, indicating the blockade of interaction with multiple effectors. Moreover, they exhibit antitumor activity on a xenograft of human colon carcinoma carrying K-rasG12V. The nuclear magnetic resonance structure of a complex of the compound with H-Ras(T35S)·GTP confirms its insertion into the surface pocket. Thus, these compounds may serve as a novel scaffold for the development of Ras inhibitors with higher potency and specificity.
Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Guanosina Trifosfato/metabolismo , Neoplasias/tratamento farmacológico , Proteínas ras/antagonistas & inibidores , Animais , Descoberta de Drogas , Humanos , Neoplasias/metabolismo , Bibliotecas de Moléculas PequenasRESUMO
GTP-bound Ras adopts two interconverting conformations, "inactive" state 1 and "active" state 2. However, the tertiary structure of wild-type (WT) state 1 remains unsolved. Here we solve the state 1 crystal structures of H-Ras WT together with its oncogenic G12V and Q61L mutants. They assume open structures characterized by impaired interactions of both Thr-35 in switch I and Gly-60 in switch II with the γ-phosphate of GTP and possess two surface pockets of mutually different shapes unseen in state 2, a potential target for selective inhibitor development. Furthermore, they provide a structural basis for the low GTPase activity of state 1.
Assuntos
Guanosina Trifosfato/química , Mutação de Sentido Incorreto , Proteínas Proto-Oncogênicas p21(ras)/química , Sítios de Ligação , Cristalografia por Raios X , Humanos , Ligação de Hidrogênio , Modelos Moleculares , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas p21(ras)/genéticaAssuntos
Proteínas ras/fisiologia , Animais , Sítios de Ligação , Humanos , Metabolismo dos Lipídeos , Ligação Proteica , Conformação Proteica , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas Proto-Oncogênicas c-raf/química , Proteínas Proto-Oncogênicas c-raf/fisiologia , Transdução de Sinais/genética , Fator ral de Troca do Nucleotídeo Guanina/química , Fator ral de Troca do Nucleotídeo Guanina/fisiologia , Proteínas rap de Ligação ao GTP/química , Proteínas rap de Ligação ao GTP/fisiologia , Proteínas rap1 de Ligação ao GTP/química , Proteínas rap1 de Ligação ao GTP/fisiologia , Proteínas ras/química , Proteínas ras/metabolismoRESUMO
Although some members of Ras family small GTPases, including M-Ras, share the primary structure of their effector regions with Ras, they exhibit vastly different binding properties to Ras effectors such as c-Raf-1. We have solved the crystal structure of M-Ras in the GDP-bound and guanosine 5'-(beta,gamma-imido)triphosphate (Gpp(NH)p)-bound forms. The overall structure of M-Ras resembles those of H-Ras and Rap2A, except that M-Ras-Gpp(NH)p exhibits a distinctive switch I conformation, which is caused by impaired intramolecular interactions between Thr-45 (corresponding to Thr-35 of H-Ras) of the effector region and the gamma-phosphate of Gpp(NH)p. Previous 31P NMR studies showed that H-Ras-Gpp(NH)p exists in two interconverting conformations, states 1 and 2. Whereas state 2 is a predominant form of H-Ras and corresponds to the "on" conformation found in the complex with effectors, state 1 is thought to represent the "off" conformation, whose tertiary structure remains unknown. 31P NMR analysis shows that free M-Ras-Gpp(NH)p predominantly assumes the state 1 conformation, which undergoes conformational transition to state 2 upon association with c-Raf-1. These results indicate that the solved structure of M-Ras-Gp-p(NH)p corresponds to the state 1 conformation. The predominance of state 1 in M-Ras is likely to account for its weak binding ability to the Ras effectors, suggesting the importance of the tertiary structure factor in small GTPase-effector interaction. Further, the first determination of the state 1 structure provides a molecular basis for developing novel anti-cancer drugs as compounds that hold Ras in the state 1 "off" conformation.
Assuntos
Guanosina Trifosfato/metabolismo , Proteínas Monoméricas de Ligação ao GTP/química , Estrutura Terciária de Proteína , Proteínas ras/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cristalografia por Raios X , Guanosina Difosfato/química , Guanosina Difosfato/metabolismo , Guanilil Imidodifosfato/química , Guanilil Imidodifosfato/metabolismo , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Ressonância Magnética Nuclear Biomolecular , Proteínas Proto-Oncogênicas c-raf/química , Proteínas Proto-Oncogênicas c-raf/metabolismo , Alinhamento de Sequência , Proteínas rap de Ligação ao GTP/química , Proteínas rap de Ligação ao GTP/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
Mammalian candidate effectors of the small GTPase Ras, such as RalGDS, afadin/AF-6, Rin1, and phospholipase Cepsilon, have been shown to share structurally conserved modules termed Ras-associating (RA) domains at their Ras-binding sites. The Ras-binding domains of Raf-1 and phosphoinositide 3-kinase gamma (other Ras effectors) also share a similar tertiary structure with the RA domains. On the other hand, the primary Ras-binding site of Saccharomyces cerevisiae adenylyl cyclase, the best characterized Ras effector, has been mapped by mutational studies to the leucine-rich repeats (LRR) domain (amino acids 674-1300), whose structure apparently bears no resemblance to the RA domains. By a computer algorithm-based search we have unexpectedly found an RA domain in the N-terminal 81 amino acid residues (676) of the LRR domain. The purified RA-domain polypeptide exhibits an ability to bind directly to Ras in a GTP-dependent manner and to competitively inhibit Ras-dependent activation of adenylyl cyclase in vitro, with an affinity comparable with that of the whole LRR domain. The specificity of binding of the RA domain to various Ras effector region mutants is indistinguishable from that of the full-length adenylyl cyclase. The activated RAS2 (RAS2(Val-19))-dependent heat shock sensitivity of yeast cells is suppressed by overexpression of the RA domain polypeptide. Further, mutations of the RA domain abolish its Ras binding activity, and adenylyl cyclase molecules carrying these mutations are rendered unactivatable by Ras in vitro. This RA domain bears highest similarity to the Ras-binding domain of Raf-1 based on comparison of its primary and predicted secondary structures with those of other Ras effectors. These results indicate that the RA domain is a primary Ras-binding site for activation of adenylyl cyclase, implicating RA domains as universal modules for interaction of effectors with Ras, conserved from yeast to mammals.