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1.
Life Sci Alliance ; 7(6)2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38499327

RESUMO

Cellular responses leading to development, proliferation, and differentiation depend on RAF/MEK/ERK signaling, which integrates and amplifies signals from various stimuli for downstream cellular responses. C-RAF activation has been reported in many types of tumor cell proliferation and developmental disorders, necessitating the discovery of potential C-RAF protein regulators. Here, we identify a novel and specific protein interaction between C-RAF among the RAF kinase paralogs, and SIRT4 among the mitochondrial sirtuin family members SIRT3, SIRT4, and SIRT5. Structurally, C-RAF binds to SIRT4 through the N-terminal cysteine-rich domain, whereas SIRT4 predominantly requires the C-terminus for full interaction with C-RAF. Interestingly, SIRT4 specifically interacts with C-RAF in a pre-signaling inactive (serine 259-phosphorylated) state. Consistent with this finding, the expression of SIRT4 in HEK293 cells results in an up-regulation of pS259-C-RAF levels and a concomitant reduction in MAPK signaling as evidenced by strongly decreased phospho-ERK signals. Thus, we propose an additional extra-mitochondrial function of SIRT4 as a cytosolic tumor suppressor of C-RAF-MAPK signaling, besides its metabolic tumor suppressor role of glutamate dehydrogenase and glutamate levels in mitochondria.


Assuntos
Sirtuínas , Humanos , Células HEK293 , Sirtuínas/genética , Sirtuínas/metabolismo , Transdução de Sinais , Mitocôndrias/metabolismo , Quinases raf/genética , Quinases raf/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo
2.
Angew Chem Int Ed Engl ; 63(13): e202316942, 2024 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-38305637

RESUMO

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.


Assuntos
Proteínas Proto-Oncogênicas p21(ras) , Transdução de Sinais , Humanos , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas ras/química , Quinases raf/metabolismo , Dimerização
3.
Signal Transduct Target Ther ; 8(1): 455, 2023 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-38105263

RESUMO

Metastatic dissemination of solid tumors, a leading cause of cancer-related mortality, underscores the urgent need for enhanced insights into the molecular and cellular mechanisms underlying metastasis, chemoresistance, and the mechanistic backgrounds of individuals whose cancers are prone to migration. The most prevalent signaling cascade governed by multi-kinase inhibitors is the mitogen-activated protein kinase (MAPK) pathway, encompassing the RAS-RAF-MAPK kinase (MEK)-extracellular signal-related kinase (ERK) pathway. RAF kinase is a primary mediator of the MAPK pathway, responsible for the sequential activation of downstream targets, such as MEK and the transcription factor ERK, which control numerous cellular and physiological processes, including organism development, cell cycle control, cell proliferation and differentiation, cell survival, and death. Defects in this signaling cascade are associated with diseases such as cancer. RAF inhibitors (RAFi) combined with MEK blockers represent an FDA-approved therapeutic strategy for numerous RAF-mutant cancers, including melanoma, non-small cell lung carcinoma, and thyroid cancer. However, the development of therapy resistance by cancer cells remains an important barrier. Autophagy, an intracellular lysosome-dependent catabolic recycling process, plays a critical role in the development of RAFi resistance in cancer. Thus, targeting RAF and autophagy could be novel treatment strategies for RAF-mutant cancers. In this review, we delve deeper into the mechanistic insights surrounding RAF kinase signaling in tumorigenesis and RAFi-resistance. Furthermore, we explore and discuss the ongoing development of next-generation RAF inhibitors with enhanced therapeutic profiles. Additionally, this review sheds light on the functional interplay between RAF-targeted therapies and autophagy in cancer.


Assuntos
Neoplasias Pulmonares , Melanoma , Humanos , Quinases de Proteína Quinase Ativadas por Mitógeno , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Quinases raf/genética , Quinases raf/metabolismo
4.
Zhen Ci Yan Jiu ; 48(10): 977-985, 2023 Oct 25.
Artigo em Inglês, Chinês | MEDLINE | ID: mdl-37879947

RESUMO

OBJECTIVES: To observe the effect of electroacupuncture (EA) on urodynamics and Raf/MEK/ERK signaling pathway in spine cord tissue of rats after suprasacral spinal cord injury (SSCI), so as to explore its possible mechanism in improving bladder function in rats with detrusor hyperreflexia after SSCI. METHODS: Female SD rats were randomly divided into blank, sham operation, model, EA and EA+PD98059 groups, with 12 rats in each group. Thorax (T) 10 spinal cord transection was performed by surgery. Rats in the EA group were given EA (10 Hz/50 Hz, 20 min) at "Ciliao" (BL32), "Zhongji" (CV3), "Sanyinjiao" (SP6) and "Dazhui" (GV14) once daily for 7 d. Rats of the EA+PD98059 group received intraperitoneal injection of PD98059 (5 mg/kg) 2 h before EA intervention. The urodyna-mics was used to measure the base pressure, leak point pressure, maximum pressure, maximum capacity and comp-liance of bladder, and the morphology of bladder detrusor tissue was observed with HE staining. The TUNEL staining was used to detect the cell apoptosis of the spinal cord tissue. The expression levels of exchange protein directly activated by cAMP 2 (Epac2), Rap, phosphorylated rapidly accelerated fibrosarcoma (p-Raf), phosphorylated mitogen-activated extracellular signal-regulated kinase (p-MEK), phosphorylated extracellular signal regulated kinase 1 and 2 (p-ERK1/2), B-cell lymphoma-2 (Bcl-2), and Bcl-2 associated X protein (Bax) were determined by Western blot. RESULTS: Compared with the sham operation group, the base pressure, leak point pressure and maximum pressure of bladder were significantly increased (P<0.01), the maximum bladder capacity and bladder compliance were decreased (P<0.01), the cell apoptosis rate of spinal cord tissue was increased (P<0.01), and the expression levels of Epac2, Rap, p-Raf, p-MEK, p-ERK1/2, and Bcl-2 protein in spinal cord tissue were decreased (P<0.01), while the expression level of Bax protein was increased (P<0.01) in the model group. After the treatment and compared with the model group, the base pressure, leak point pressure and maximum pressure of bladder, the cell apoptosis rate of spinal cord tissue, the expression level of Bax protein were decreased (P<0.05) in the EA group, while the maximum bladder capacity and bladder compliance, the expression levels of Epac2, Rap, p-Raf, p-MEK, p-ERK1/2, and Bcl-2 protein in spinal cord tissue were all increased (P<0.05, P<0.01). In comparison with the EA group, the base pressure, leak point pressure and maximum pressure of bladder, the cell apoptosis rate, the expression level of Bax protein were significantly increased (P<0.05), whereas the maximum bladder capacity, bladder compliance, and the expression levels of p-MEK, p-ERK1/2, and Bcl-2 protein were decreased (P<0.05) in the EA+PD98059 group. Results of HE staining showed disordered transitional epithelial cells and destroyed lamina propria in bladder detrusor tissue, with the infiltration of monocytes in the model group, which was obviously milder in both EA and EA+PD98059 groups, especially in the EA group. CONCLUSIONS: EA can improve the bladder function in detrusor hyperreflexia rats after SSCI, which may be related to its effect in up-regulating Epac2 and Rap, activating the Raf-MEK-ERK pathway, and reducing the cell apoptosis of spinal cord tissue.


Assuntos
Eletroacupuntura , Traumatismos da Medula Espinal , Animais , Feminino , Ratos , Proteína X Associada a bcl-2/metabolismo , Sistema de Sinalização das MAP Quinases , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Ratos Sprague-Dawley , Reflexo Anormal , Transdução de Sinais , Medula Espinal , Traumatismos da Medula Espinal/complicações , Traumatismos da Medula Espinal/genética , Traumatismos da Medula Espinal/terapia , Urodinâmica , Quinases raf/metabolismo
5.
Biochem Pharmacol ; 217: 115842, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37802240

RESUMO

RAS/RAF/MEK/ERK signaling pathway is one of the most important pathways of Mitogen-activated protein kinases (MAPK), which widely participate in regulating cell proliferation, differentiation, apoptosis and signaling transduction. Autophagy is an essential mechanism that maintains cellular homeostasis by degrading aged and damaged organelles. Recently, some studies revealed RAS/RAF/MEK/ERK signaling pathway is closely related to autophagy regulation and has a dual effect in tumor cells. However, the specific mechanism by which RAS/RAF/MEK/ERK signaling pathway participates in autophagy regulation is not fully understood. This article provides a comprehensive review of the research progress with regard to the RAS/RAF/MEK/ERK signaling pathway and autophagy, as well as their interplay in cancer therapy. The impact of small molecule inhibitors that target the RAS/RAF/MEK/ERK signaling pathway on autophagy is discussed in this study. The advantages and limitations of the clinical combination of these small molecule inhibitors with autophagy inhibitors are also explored. The findings from this study may provide additional perspectives for future cancer treatment strategies.


Assuntos
MAP Quinases Reguladas por Sinal Extracelular , Neoplasias , Idoso , Humanos , Autofagia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Sistema de Sinalização das MAP Quinases , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Neoplasias/tratamento farmacológico , Quinases raf/metabolismo , Transdução de Sinais , Proteínas Proto-Oncogênicas p21(ras)/metabolismo
6.
Elife ; 122023 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-37823369

RESUMO

RAF kinase inhibitors can, under certain conditions, increase RAF kinase signaling. This process, which is commonly referred to as 'paradoxical activation' (PA), is incompletely understood. We use mathematical and computational modeling to investigate PA and derive rigorous analytical expressions that illuminate the underlying mechanism of this complex phenomenon. We find that conformational autoinhibition modulation by a RAF inhibitor could be sufficient to create PA. We find that experimental RAF inhibitor drug dose-response data that characterize PA across different types of RAF inhibitors are best explained by a model that includes RAF inhibitor modulation of three properties: conformational autoinhibition, dimer affinity, and drug binding within the dimer (i.e., negative cooperativity). Overall, this work establishes conformational autoinhibition as a robust mechanism for RAF inhibitor-driven PA based solely on equilibrium dynamics of canonical interactions that comprise RAF signaling and inhibition.


Assuntos
Transdução de Sinais , Quinases raf , Quinases raf/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/química , Conformação Molecular , Proteínas Proto-Oncogênicas B-raf/metabolismo
7.
Protein Sci ; 32(10): e4767, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37615343

RESUMO

RAS GTPases are proto-oncoproteins that regulate cell growth, proliferation, and differentiation in response to extracellular signals. The signaling functions of RAS, and other small GTPases, are dependent on their ability to cycle between GDP-bound and GTP-bound states. Structural analyses suggest that GTP hydrolysis catalyzed by HRAS can be regulated by an allosteric site located between helices 3, 4, and loop 7. Here we explore the relationship between intrinsic GTP hydrolysis on HRAS and the position of helix 3 and loop 7 through manipulation of the allosteric site, showing that the two sites are functionally connected. We generated several hydrophobic mutations in the allosteric site of HRAS to promote shifts in helix 3 relative to helix 4. By combining crystallography and enzymology to study these mutants, we show that closure of the allosteric site correlates with increased hydrolysis of GTP on HRAS in solution. Interestingly, binding to the RAS binding domain of RAF kinase (RAF-RBD) inhibits GTP hydrolysis in the mutants. This behavior may be representative of a cluster of mutations found in human tumors, which potentially cooperate with RAF complex formation to stabilize the GTP-bound state of RAS.


Assuntos
Quinases raf , Proteínas ras , Humanos , Sítio Alostérico , Hidrólise , Quinases raf/química , Quinases raf/genética , Quinases raf/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismo , Guanosina Trifosfato/metabolismo
8.
Fish Shellfish Immunol ; 137: 108772, 2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-37100311

RESUMO

Glyphosate is an herbicide commonly used worldwide, and its substantial use causes widespread pollution with runoff. However, research on glyphosate toxicity has mostly remained at the embryonic level and existing studies are limited. In the present study, we investigated whether glyphosate can induce autophagy in hepatic L8824 cells by regulating energy metabolism and rat sarcoma (RAS)/rapidly accelerated fibrosarcoma (RAF)/mitogen-activated extracellular signal-regulated kinase (MEK)/extracellular regulated protein kinases (ERK) signaling by activating nitric oxide (NO). First, we selected 0, 50, 200, and 500 µg/mL as the challenge doses, according to the inhibitory concentration of 50% (IC50) of glyphosate. The results showed that glyphosate exposure increased the enzyme activity of inducible nitric oxide synthase (iNOS), which in turn increased the NO content. The activity and expression of enzymes related to energy metabolism, such as hexokinase (HK)1, HK2, phosphofructokinase (PFK), phosphokinase (PK), succinate dehydrogenase (SDH), and nicotinamide adenine dinucleotide with hydrogen (NADH), were inhibited, and the RAS/RAF/MEK/ERK signaling pathway was activated. This led to the negative expression of mammalian target of rapamycin (mTOR) and P62 in hepatic L8824 cells and the activation of the autophagy marker genes microtubule-associated proteins light chain 3 (LC3) and Beclin1 to induce autophagy. The above results were dependent on glyphosate concentration. To verify whether autophagy can be excited by the RAS/RAF/MEK/ERK signaling pathway, we treated L8824 cells with the ERK inhibitor U0126 and found that the autophagy gene LC3 was reduced due to the inhibition of ERK, thus demonstrating the reliability of the results. In conclusion, our results demonstrate that glyphosate can induce autophagy in hepatic L8824 cells by activating NO, thus regulating energy metabolism and the RAS/RAF/MEK/ERK signaling pathway.


Assuntos
Fibrossarcoma , MAP Quinase Quinase Quinases , Animais , Óxido Nítrico , Reprodutibilidade dos Testes , Quinases raf/genética , Transdução de Sinais , MAP Quinases Reguladas por Sinal Extracelular , Linhagem Celular , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Metabolismo Energético , Autofagia , Sistema de Sinalização das MAP Quinases , Mamíferos/metabolismo
9.
J Chem Inf Model ; 63(8): 2483-2494, 2023 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-37022803

RESUMO

The ERK pathway is one of the most important signaling cascades involved in tumorigenesis. So far, eight noncovalent inhibitors of RAF and MEK kinases in the ERK pathway have been approved by the FDA for the treatment of cancers; however, their efficacies are limited due to various resistance mechanisms. There is an urgent need to develop novel targeted covalent inhibitors. Here we report a systematic study of the covalent ligandabilities of the ERK pathway kinases (ARAF, BRAF, CRAF, KSR1, KSR2, MEK1, MEK2, ERK1, and ERK2) using constant pH molecular dynamics titration and pocket analysis. Our data revealed that the hinge GK (gate keeper)+3 cysteine in RAF family kinases (ARAF, BRAF, CRAF, KSR1, and KSR2) and the back loop cysteine in MEK1 and MEK2 are reactive and ligandable. Structure analysis suggests that the type II inhibitors belvarafenib and GW5074 may be used as scaffolds for designing pan-RAF or CRAF-selective covalent inhibitors directed at the GK+3 cysteine, while the type III inhibitor cobimetinib may be modified to label the back loop cysteine in MEK1/2. The reactivities and ligandabilities of the remote cysteine in MEK1/2 and the DFG-1 cysteine in MEK1/2 and ERK1/2 are also discussed. Our work provides a starting point for medicinal chemists to design novel covalent inhibitors of the ERK pathway kinases. The computational protocol is general and can be applied to the systematic evaluation of covalent ligandabilities of the human cysteinome.


Assuntos
MAP Quinase Quinase Quinases , Sistema de Sinalização das MAP Quinases , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , MAP Quinase Quinase Quinases/metabolismo , Proteínas Proto-Oncogênicas B-raf/química , Proteínas Proto-Oncogênicas B-raf/metabolismo , Cisteína/metabolismo , Transdução de Sinais , Quinases raf/metabolismo
10.
Mol Cell ; 83(8): 1210-1215, 2023 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-36990093

RESUMO

One of the open questions in RAS biology is the existence of RAS dimers and their role in RAF dimerization and activation. The idea of RAS dimers arose from the discovery that RAF kinases function as obligate dimers, which generated the hypothesis that RAF dimer formation might be nucleated by G-domain-mediated RAS dimerization. Here, we review the evidence for RAS dimerization and describe a recent discussion among RAS researchers that led to a consensus that the clustering of two or more RAS proteins is not due to the stable association of G-domains but, instead, is a consequence of RAS C-terminal membrane anchors and the membrane phospholipids with which they interact.


Assuntos
Quinases raf , Proteínas ras , Dimerização , Consenso , Proteínas ras/genética , Proteínas ras/metabolismo , Quinases raf/genética , Quinases raf/metabolismo , Lipídeos , Proteínas Proto-Oncogênicas c-raf/metabolismo
11.
Elife ; 112022 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-36458814

RESUMO

Undruggability of RAS proteins has necessitated alternative strategies for the development of effective inhibitors. In this respect, phosphorylation has recently come into prominence as this reversible post-translational modification attenuates sensitivity of RAS towards RAF. As such, in this study, we set out to unveil the impact of phosphorylation on dynamics of HRASWT and aim to invoke similar behavior in HRASG12D mutant by means of small therapeutic molecules. To this end, we performed molecular dynamics (MD) simulations using phosphorylated HRAS and showed that phosphorylation of Y32 distorted Switch I, hence the RAS/RAF interface. Consequently, we targeted Switch I in HRASG12D by means of approved therapeutic molecules and showed that the ligands enabled detachment of Switch I from the nucleotide-binding pocket. Moreover, we demonstrated that displacement of Switch I from the nucleotide-binding pocket was energetically more favorable in the presence of the ligand. Importantly, we verified computational findings in vitro where HRASG12D/RAF interaction was prevented by the ligand in HEK293T cells that expressed HRASG12D mutant protein. Therefore, these findings suggest that targeting Switch I, hence making Y32 accessible might open up new avenues in future drug discovery strategies that target mutant RAS proteins.


Assuntos
Quinases raf , Proteínas ras , Humanos , Células HEK293 , Ligantes , Nucleotídeos/metabolismo , Fosforilação , Proteínas ras/metabolismo , Proteínas Mutantes , Quinases raf/metabolismo
12.
Mol Cancer ; 21(1): 213, 2022 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-36476495

RESUMO

BACKGROUND: Inactivation of the Hippo pathway promotes Yap nuclear translocation, enabling execution of a transcriptional program that induces tissue growth. Genetic lesions of Hippo intermediates only identify a minority of cancers with illegitimate YAP activation. Yap has been implicated in resistance to targeted therapies, but the mechanisms by which YAP may impact adaptive resistance to MAPK inhibitors are unknown. METHODS: We screened 52 thyroid cancer cell lines for illegitimate nuclear YAP localization by immunofluorescence and fractionation of cell lysates. We engineered a doxycycline (dox)-inducible thyroid-specific mouse model expressing constitutively nuclear YAPS127A, alone or in combination with endogenous expression of either HrasG12V or BrafV600E. We also generated cell lines expressing dox-inducible sh-miR-E-YAP and/or YAPS127A. We used cell viability, invasion assays, immunofluorescence, Western blotting, qRT-PCRs, flow cytometry and cell sorting, high-throughput bulk RNA sequencing and in vivo tumorigenesis to investigate YAP dependency and response of BRAF-mutant cells to vemurafenib. RESULTS: We found that 27/52 thyroid cancer cell lines had constitutively aberrant YAP nuclear localization when cultured at high density (NU-YAP), which rendered them dependent on YAP for viability, invasiveness and sensitivity to the YAP-TEAD complex inhibitor verteporfin, whereas cells with confluency-driven nuclear exclusion of YAP (CYT-YAP) were not. Treatment of BRAF-mutant thyroid cancer cells with RAF kinase inhibitors resulted in YAP nuclear translocation and activation of its transcriptional output. Resistance to vemurafenib in BRAF-mutant thyroid cells was driven by YAP-dependent NRG1, HER2 and HER3 activation across all isogenic human and mouse thyroid cell lines tested, which was abrogated by silencing YAP and relieved by pan-HER kinase inhibitors. YAP activation induced analogous changes in BRAF melanoma, but not colorectal cells. CONCLUSIONS: YAP activation in thyroid cancer generates a dependency on this transcription factor. YAP governs adaptive resistance to RAF kinase inhibitors and induces a gene expression program in BRAFV600E-mutant cells encompassing effectors in the NRG1 signaling pathway, which play a central role in the insensitivity to MAPK inhibitors in a lineage-dependent manner. HIPPO pathway inactivation serves as a lineage-dependent rheostat controlling the magnitude of the adaptive relief of feedback responses to MAPK inhibitors in BRAF-V600E cancers.


Assuntos
Neoplasias da Glândula Tireoide , Humanos , Animais , Camundongos , Neoplasias da Glândula Tireoide/tratamento farmacológico , Neoplasias da Glândula Tireoide/genética , Quinases raf
13.
Viruses ; 14(9)2022 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-36146864

RESUMO

Influenza virus (IV) infections pose a burden on global public health with significant morbidity and mortality. The limited range of currently licensed IV antiviral drugs is susceptible to the rapid rise of resistant viruses. In contrast, FDA-approved kinase inhibitors can be repurposed as fast-tracked host-targeted antivirals with a higher barrier of resistance. Extending our recent studies, we screened 21 FDA-approved small-molecule kinase inhibitors (SMKIs) and identified seven candidates as potent inhibitors of pandemic and seasonal IV infections. These SMKIs were further validated in a biologically and clinically relevant ex vivo model of human precision-cut lung slices. We identified steps of the virus infection cycle affected by these inhibitors (entry, replication, egress) and found that most SMKIs affected both entry and egress. Based on defined and overlapping targets of these inhibitors, the candidate SMKIs target receptor tyrosine kinase (RTK)-mediated activation of Raf/MEK/ERK pathways to limit influenza A virus infection. Our data and the established safety profiles of these SMKIs support further clinical investigations and repurposing of these SMKIs as host-targeted influenza therapeutics.


Assuntos
Vírus da Influenza A , Influenza Humana , Infecções por Orthomyxoviridae , Antivirais/farmacologia , Antivirais/uso terapêutico , Linhagem Celular , Humanos , Influenza Humana/tratamento farmacológico , Quinases de Proteína Quinase Ativadas por Mitógeno/farmacologia , Quinases de Proteína Quinase Ativadas por Mitógeno/uso terapêutico , Infecções por Orthomyxoviridae/tratamento farmacológico , Infecções por Orthomyxoviridae/metabolismo , Receptores Proteína Tirosina Quinases , Estados Unidos , United States Food and Drug Administration , Replicação Viral , Quinases raf/metabolismo
14.
Nat Struct Mol Biol ; 29(10): 966-977, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36175670

RESUMO

SHOC2 acts as a strong synthetic lethal interactor with MEK inhibitors in multiple KRAS cancer cell lines. SHOC2 forms a heterotrimeric complex with MRAS and PP1C that is essential for regulating RAF and MAPK-pathway activation by dephosphorylating a specific phosphoserine on RAF kinases. Here we present the high-resolution crystal structure of the SHOC2-MRAS-PP1C (SMP) complex and apo-SHOC2. Our structures reveal that SHOC2, MRAS, and PP1C form a stable ternary complex in which all three proteins synergistically interact with each other. Our results show that dephosphorylation of RAF substrates by PP1C is enhanced upon interacting with SHOC2 and MRAS. The SMP complex forms only when MRAS is in an active state and is dependent on SHOC2 functioning as a scaffolding protein in the complex by bringing PP1C and MRAS together. Our results provide structural insights into the role of the SMP complex in RAF activation and how mutations found in Noonan syndrome enhance complex formation, and reveal new avenues for therapeutic interventions.


Assuntos
Síndrome de Noonan , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Sistema de Sinalização das MAP Quinases/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Síndrome de Noonan/genética , Síndrome de Noonan/metabolismo , Fosfosserina/metabolismo , Proteína Fosfatase 1 , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Quinases raf/genética , Quinases raf/metabolismo , Proteínas ras/metabolismo
15.
Circ Res ; 131(7): 620-636, 2022 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-36052698

RESUMO

BACKGROUND: Heart failure is the leading cause of mortality, morbidity, and health care expenditures worldwide. Numerous studies have implicated GSK-3 (glycogen synthase kinase-3) as a promising therapeutic target for cardiovascular diseases. GSK-3 isoforms seem to play overlapping, unique and even opposing functions in the heart. Previously, we have shown that of the 2 isoforms of GSK-3, cardiac fibroblast GSK-3ß acts as a negative regulator of myocardial fibrosis in the ischemic heart. However, the role of cardiac fibroblast-GSK-3α in the pathogenesis of cardiac diseases is completely unknown. METHODS: To define the role of cardiac fibroblast-GSK-3α in myocardial fibrosis and heart failure, GSK-3α was deleted from fibroblasts or myofibroblasts with tamoxifen-inducible Tcf21- or Postn-promoter-driven Cre recombinase. Control and GSK-3α KO mice were subjected to cardiac injury and heart parameters were evaluated. The fibroblast kinome mapping was carried out to delineate molecular mechanism followed by in vivo and in vitro analysis. RESULTS: Fibroblast-specific GSK-3α deletion restricted fibrotic remodeling and preserved function of the injured heart. We observed reductions in cell migration, collagen gel contraction, α-SMA protein levels, and expression of ECM genes in TGFß1-treated KO fibroblasts, indicating that GSK-3α is required for myofibroblast transformation. Surprisingly, GSK-3α deletion did not affect SMAD3 activation, suggesting the profibrotic role of GSK-3α is SMAD3 independent. The molecular studies confirmed decreased ERK signaling in GSK-3α-KO CFs. Conversely, adenovirus-mediated expression of a constitutively active form of GSK-3α (Ad-GSK-3αS21A) in fibroblasts increased ERK activation and expression of fibrogenic proteins. Importantly, this effect was abolished by ERK inhibition. CONCLUSIONS: GSK-3α-mediated MEK-ERK activation is a critical profibrotic signaling circuit in the injured heart, which operates independently of the canonical TGF-ß1-SMAD3 pathway. Therefore, strategies to inhibit the GSK-3α-MEK-ERK signaling circuit could prevent adverse fibrosis in diseased hearts.


Assuntos
Cardiomiopatias , Insuficiência Cardíaca , Animais , Cardiomiopatias/metabolismo , Colágeno/metabolismo , MAP Quinases Reguladas por Sinal Extracelular , Fibroblastos/metabolismo , Fibrose , Quinase 3 da Glicogênio Sintase/metabolismo , Quinase 3 da Glicogênio Sintase/farmacologia , Glicogênio Sintase Quinase 3 beta/metabolismo , Insuficiência Cardíaca/metabolismo , Sistema de Sinalização das MAP Quinases , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/farmacologia , Miofibroblastos/metabolismo , Tamoxifeno/farmacologia , Fator de Crescimento Transformador beta1/metabolismo , Quinases raf
16.
Mol Cell ; 82(18): 3438-3452.e8, 2022 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-36055235

RESUMO

RAF kinases are RAS-activated enzymes that initiate signaling through the MAPK cascade to control cellular proliferation, differentiation, and survival. Here, we describe the structure of the full-length RAF1 protein in complex with HSP90 and CDC37 obtained by cryoelectron microscopy. The reconstruction reveals a RAF1 kinase with an unfolded N-lobe separated from its C-lobe. The hydrophobic core of the N-lobe is trapped in the HSP90 dimer, while CDC37 wraps around the chaperone and interacts with the N- and C-lobes of the kinase. The structure indicates how CDC37 can discriminate between the different members of the RAF family. Our structural analysis also reveals that the folded RAF1 assembles with 14-3-3 dimers, suggesting that after folding RAF1 follows a similar activation as B-RAF. Finally, disruption of the interaction between CDC37 and the DFG segment of RAF1 unveils potential vulnerabilities in attempting the pharmacological degradation of RAF1 for therapeutic purposes.


Assuntos
Proteínas de Ciclo Celular , Chaperoninas , Proteínas de Ciclo Celular/metabolismo , Chaperoninas/química , Microscopia Crioeletrônica , Proteínas de Choque Térmico HSP90/metabolismo , Chaperonas Moleculares/metabolismo , Ligação Proteica , Quinases raf/metabolismo
17.
J Transl Med ; 20(1): 310, 2022 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-35794555

RESUMO

BACKGROUND: Breast cancer (BC) is one of the most common malignant tumors with the highest mortality in the world. Modern pharmacological studies have shown that Syringin has an inhibitory effect on many tumors, but its anti-BC efficacy and mechanism are still unclear. METHODS: First, Syringin was isolated from Acanthopanax senticosus (Rupr. & Maxim.) Harms (ASH) by systematic solvent extraction and silica gel chromatography column. The plant name is composed of genus epithet, species additive words and the persons' name who give its name. Then, the hub targets of Syringin against BC were revealed by bioinformatics. To provide a more experimental basis for later research, the hub genes which could be candidate biomarkers of BC and a ceRNA network related to them were obtained. And the potential mechanism of Syringin against BC was proved in vitro experiments. RESULTS: Syringin was obtained by liquid chromatography-mass spectrometry (LC-MS), nuclear magnetic resonance (NMR), and high-performance liquid chromatography (HPLC). Bioinformatics results showed that MAP2K1, PIK3CA, HRAS, EGFR, Caspase3, and PTGS2 were the hub targets of Syringin against BC. And PIK3CA and HRAS were related to the survival and prognosis of BC patients, the PIK3CA-hsa-mir-139-5p-LINC01278 and PIK3CA-hsa-mir-375 pathways might be closely related to the mechanism of Syringin against BC. In vitro experiments confirmed that Syringin inhibited the proliferation and migration and promoted apoptosis of BC cells through the above hub targets. CONCLUSIONS: Syringin against BC via PI3K-AKT-PTGS2 and EGFR-RAS-RAF-MEK-ERK pathways, and PIK3CA and HRAS are hub genes for adjuvant treatment of BC.


Assuntos
Neoplasias da Mama , Glucosídeos , MicroRNAs , Fenilpropionatos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Ciclo-Oxigenase 2/metabolismo , Receptores ErbB/metabolismo , Feminino , Glucosídeos/farmacologia , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Fenilpropionatos/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Quinases raf/metabolismo , Proteínas ras/metabolismo
18.
Biophys J ; 121(19): 3616-3629, 2022 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-35794829

RESUMO

HRas, KRas, and NRas are GTPases with a common set of effectors that control many cell-signaling pathways, including proliferation through Raf kinase. Their G-domains are nearly identical in sequence, with a few isoform-specific residues that have an effect on dynamics and biochemical properties. Here, we use accelerated molecular dynamics (aMD) simulations consistent with solution x-ray scattering experiments to elucidate mechanisms through which isoform-specific residues associated with each Ras isoform affects functionally important regions connected to the active site. HRas-specific residues cluster in loop 8 to stabilize the nucleotide-binding pocket, while NRas-specific residues on helix 3 directly affect the conformations of switch I and switch II. KRas, the most globally flexible of the isoforms, shows greatest fluctuations in the switch regions enhanced by a KRas-specific residue in loop 7 and a highly dynamic loop 8 region. The analysis of isoform-specific residue effects on Ras proteins is supported by NMR experiments and is consistent with previously published biochemical data.


Assuntos
Nucleotídeos , Proteínas ras , Guanosina Trifosfato/metabolismo , Mutação , Nucleotídeos/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Quinases raf/metabolismo , Proteínas ras/metabolismo
19.
Gene ; 842: 146757, 2022 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-35907565

RESUMO

BACKGROUND: Aldehyde dehydrogenase 6 family member A1 (ALDH6A1) is associated with multiple diseases, but its pathogenesis in colon cancer (CC) is ambiguous and needs further study so that this research explores the function of ALDH6A1 in CC. METHODS: The level of ALDH6A1 in colon adenocarcinoma (COAD), CC tissues, and cells was measured by starBase v2.0, quantitative real-time polymerase chain reaction (qRT-PCR), and western blot. Post transfection with overexpressed (oe)-ALDH6A1, cell biological behaviors, as well as apoptosis-, matrix metalloproteinase (MMP)-, and rat sarcoma virus (RAS)/rapidly accelerated fibrosarcoma (RAF)/mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway-related markers were measured by cell function experiments, qRT-PCR, and western blot. Next, the effects of small interfering RNA targeting ALDH6A1 (si-ALDH6A1) and RAS/RAF inhibitor (MCP110) on cell biological behaviors, as well as apoptosis-, MMP-, and RAS/RAF/MEK/ERK pathway-related markers were detected again. RESULTS: ALDH6A1 was low-expressed in COAD, CC tissues, and cells. Oe-ALDH6A1 weakened cell vitality, migration and invasionbut facilitated apoptosis; while it reduced expression levels of Bcl-2, MMP-2, MMP-9 and the RAS/RAF/MEK/ERK pathway-related markers but promoted Bax level. However, the regulation of si-ALDH6A1 on cell biological behaviors and related genes was opposite to that of oe-ALDH6A1. Moreover, MCP110 rescued the regulation of si-ALDH6A1 on cell biological behaviors, expressions of apoptosis- MMP- as well as RAS/RAF/MEK/ERK pathway-related markers. To sum up, ALDH6A1 attenuated CC progression by down-regulating the expressions of RAS/RAF/MEK/ERK pathway-related markers.


Assuntos
Adenocarcinoma , Neoplasias do Colo , Fibrossarcoma , Linhagem Celular , Neoplasias do Colo/patologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Quinases raf/genética , Quinases raf/metabolismo
20.
Nature ; 609(7926): 416-423, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35830882

RESUMO

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.


Assuntos
Cristalografia por Raios X , Peptídeos e Proteínas de Sinalização Intracelular , Complexos Multiproteicos , Proteína Fosfatase 1 , Proteínas ras , Proteínas 14-3-3 , Guanosina Trifosfato/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Sistema de Sinalização das MAP Quinases , Complexos Multiproteicos/química , Mutação , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Proteína Fosfatase 1/química , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Quinases raf , Proteínas ras/química , Proteínas ras/metabolismo
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