Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 481
Filtrar
1.
Pathol Oncol Res ; 30: 1611643, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38515456

RESUMO

The increasing knowledge of molecular alterations in malignancies, including mutations and regulatory failures in the mTOR (mechanistic target of rapamycin) signaling pathway, highlights the importance of mTOR hyperactivity as a validated target in common and rare malignancies. This review summarises recent findings on the characterization and prognostic role of mTOR kinase complexes (mTORC1 and mTORC2) activity regarding differences in their function, structure, regulatory mechanisms, and inhibitor sensitivity. We have recently identified new tumor types with RICTOR (rapamycin-insensitive companion of mTOR) amplification and associated mTORC2 hyperactivity as useful potential targets for developing targeted therapies in lung cancer and other newly described malignancies. The activity of mTOR complexes is recommended to be assessed and considered in cancers before mTOR inhibitor therapy, as current first-generation mTOR inhibitors (rapamycin and analogs) can be ineffective in the presence of mTORC2 hyperactivity. We have introduced and proposed a marker panel to determine tissue characteristics of mTOR activity in biopsy specimens, patient materials, and cell lines. Ongoing phase trials of new inhibitors and combination therapies are promising in advanced-stage patients selected by genetic alterations, molecular markers, and/or protein expression changes in the mTOR signaling pathway. Hopefully, the summarized results, our findings, and the suggested characterization of mTOR activity will support therapeutic decisions.


Assuntos
Neoplasias Pulmonares , Serina-Treonina Quinases TOR , Humanos , Proteína Companheira de mTOR Insensível à Rapamicina/genética , Proteína Companheira de mTOR Insensível à Rapamicina/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Sirolimo/farmacologia , Fatores de Transcrição/metabolismo
2.
Endocrinology ; 165(4)2024 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-38325289

RESUMO

The mineralocorticoid receptor (MR) is a transcription factor for genes mediating diverse, cell-specific functions, including trophic effects as well as promoting fluid/electrolyte homeostasis. It was reported that in intercalated cells, phosphorylation of the MR at serine 843 (S843) by Unc-51-like kinase (ULK1) inhibits MR activation and that phosphorylation of ULK1 by mechanistic target of rapamycin (mTOR) inactivates ULK1, and thereby prevents MR inactivation. We extended these findings with studies in M1 mouse cortical collecting duct cells stably expressing the rat MR and a reporter gene. Pharmacological inhibition of ULK1 dose-dependently increased ligand-induced MR transactivation, while ULK1 activation had no effect. Pharmacological inhibition of mTOR and CRISPR/gRNA gene knockdown of rapamycin-sensitive adapter protein of mTOR (Raptor) or rapamycin-insensitive companion of mTOR (Rictor) decreased phosphorylated ULK1 and ligand-induced activation of the MR reporter gene, as well as transcription of endogenous MR-target genes. As predicted, ULK1 inhibition had no effect on aldosterone-mediated transcription in M1 cells with the mutated MR-S843A (alanine cannot be phosphorylated). In contrast, mTOR inhibition dose-dependently decreased transcription in the MR-S843A cells, though not as completely as in cells with the wild-type MR-S843. mTOR, Raptor, and Rictor coprecipitated with the MR and addition of aldosterone increased their phosphorylated, active state. These results suggest that mTOR significantly regulates MR activity in at least 2 ways: by suppressing MR inactivation by ULK1, and by a yet ill-defined mechanism that involves direct association with MR. They also provide new insights into the diverse functions of ULK1 and mTOR, 2 key enzymes that monitor the cell's energy status.


Assuntos
Aldosterona , Receptores de Mineralocorticoides , Animais , Camundongos , Ratos , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Ligantes , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Complexos Multiproteicos/metabolismo , Fosforilação , Proteína Companheira de mTOR Insensível à Rapamicina/metabolismo , Receptores de Mineralocorticoides/genética , Receptores de Mineralocorticoides/metabolismo , Proteína Regulatória Associada a mTOR , RNA Guia de Sistemas CRISPR-Cas , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição/metabolismo
3.
Behav Brain Res ; 463: 114888, 2024 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-38307148

RESUMO

Dysfunction of the mechanistic target of rapamycin (mTOR) signaling pathway is implicated in neuropsychiatric disorders including depression and anxiety. Most studies have been focusing on neurons, and the function of mTOR signaling pathway in astrocytes is less investigated. mTOR forms two distinct complexes, mTORC1 and mTORC2, with key scaffolding protein Raptor and Rictor, respectively. The ventral tegmental area (VTA), a vital component of the brain reward system, is enrolled in regulating both depression and anxiety. In the present study, we aimed to examine the regulation effect of VTA astrocytic mTOR signaling pathway on depression and anxiety. We specifically deleted Raptor or Rictor in VTA astrocytes in mice and performed a series of behavioral tests for depression and anxiety. Deletion of Raptor and Rictor both decreased the immobility time in the tail suspension test and the latency to eat in the novelty suppressed feeding test, and increased the horizontal activity and the movement time in locomotor activity. Deletion of Rictor decreased the number of total arm entries in the elevated plus-maze test and the vertical activity in locomotor activity. These data suggest that VTA astrocytic mTORC1 plays a role in regulating depression-related behaviors and mTORC2 is involved in both depression and anxiety-related behaviors. Our results indicate that VTA astrocytic mTOR signaling pathway might be new targets for the treatment of psychiatric disorders.


Assuntos
Astrócitos , Área Tegmentar Ventral , Humanos , Camundongos , Animais , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Área Tegmentar Ventral/metabolismo , Astrócitos/metabolismo , Depressão , Complexos Multiproteicos/metabolismo , Proteína Companheira de mTOR Insensível à Rapamicina/metabolismo , Proteínas de Transporte/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteína Regulatória Associada a mTOR/metabolismo , Fatores de Transcrição/metabolismo , Ansiedade
4.
Cell Signal ; 116: 111065, 2024 04.
Artigo em Inglês | MEDLINE | ID: mdl-38281616

RESUMO

Cardiomyocyte apoptosis and cardiac fibrosis are the leading causes of mortality in patients with ischemic heart disease. As such, these processes represent potential therapeutic targets to treat heart failure resulting from ischemic insult. We previously demonstrated that the mitochondrial acetyltransferase protein GCN5L1 regulates cardiomyocyte cytoprotective signaling in ischemia-reperfusion injury in vivo and hypoxia-reoxygenation injury in vitro. The current study investigated the mechanism underlying GCN5L1-mediated regulation of the Akt/mTORC2 cardioprotective signaling pathway. Rictor protein levels in cardiac tissues from human ischemic heart disease patients were significantly decreased relative to non-ischemic controls. Rictor protein levels were similarly decreased in cardiac AC16 cells following hypoxic stress, while mRNA levels remained unchanged. The reduction in Rictor protein levels after hypoxia was enhanced by the knockdown of GCN5L1, and was blocked by GCN5L1 overexpression. These findings correlated with changes in Rictor lysine acetylation, which were mediated by GCN5L1 acetyltransferase activity. Rictor degradation was regulated by proteasomal activity, which was antagonized by increased Rictor acetylation. Finally, we found that GCN5L1 knockdown restricted cytoprotective Akt signaling, in conjunction with decreased mTOR abundance and activity. In summary, these studies suggest that GCN5L1 promotes cardioprotective Akt/mTORC2 signaling by maintaining Rictor protein levels through enhanced lysine acetylation.


Assuntos
Isquemia Miocárdica , Proteínas Proto-Oncogênicas c-akt , Humanos , Acetilação , Acetiltransferases/genética , Acetiltransferases/metabolismo , Hipóxia/metabolismo , Lisina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Proteínas Mitocondriais/metabolismo , Isquemia Miocárdica/metabolismo , Miócitos Cardíacos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína Companheira de mTOR Insensível à Rapamicina/genética , Fatores de Transcrição/metabolismo
5.
J Transl Med ; 21(1): 919, 2023 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-38110956

RESUMO

BACKGROUND: Mutations in TP53 gene is considered a main driver of hepatocellular carcinoma (HCC). While TP53 mutations are the leading cause of p53 dysfunction, their occurrence rates may drop to approximately 10% in cohorts without hepatitis B virus and aflatoxin exposure. This observation suggests that the deactivation of wild-type p53 (p53wt) may be a critical factor in the majority of HCC cases. However, the mechanism undermining p53wt activity in the liver remains unclear. METHODS: Microarray analysis and luciferase assay were utilized to confirm target associations. Gain- and/or loss-of-function methods were employed to assess alterations in signaling pathways. Protein interactions were analyzed by molecular immunological methods and further visualized by confocal microscopy. Bioinformatic analysis was performed to analyze clinical significance. Tumor xenograft nude mice were used to validate the findings in vivo. RESULTS: Our study highlights the oncogenic role of Rictor, a key component of the mammalian target of rapamycin complex 2 (mTORC2), in hepatocytes. Rictor exerts its oncogenic function by binding to p53wt and subsequently blocking p53wt activity based on p53 status, requiring the involvement of mTOR. Moreover, we observed a dynamic nucleocytoplasmic distribution pattern of Rictor, characterized by its translocation from the nucleus (in precancerous lesions) to the cytoplasm (in HCCs) during malignant transformation. Notably, Rictor is directly targeted by the liver-enriched microRNA miR-192, and the disruption of the miR-192-Rictor-p53-miR-192 signaling axis was consistently observed in both human and rat HCC models. Clinical analysis associated lower miR-192/higher Rictor with shorter overall survival and more advanced clinical stages (P < 0.05). In mice, xenograft tumors overexpressing miR-192 exhibited lower Rictor expression levels, leading to higher p53 activity, and these tumors displayed slower growth compared to untreated HCC cells. CONCLUSIONS: Rictor dynamically shuttles between the nucleus and cytoplasm during HCC development. Its pivotal oncogenic role involves binding and inhibiting p53wt activity within the nucleus in early hepatocarcinogenesis. Targeting Rictor presents a promising strategy for HCC based on p53 status.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , MicroRNAs , Proteína Companheira de mTOR Insensível à Rapamicina , Animais , Humanos , Camundongos , Ratos , Carcinogênese/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Regulação Neoplásica da Expressão Gênica , Genes p53 , Hepatócitos/patologia , Neoplasias Hepáticas/patologia , Camundongos Nus , MicroRNAs/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteína Companheira de mTOR Insensível à Rapamicina/metabolismo
6.
Sci Rep ; 13(1): 19610, 2023 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-37949943

RESUMO

Alterations in mTOR signalling molecules, including RICTOR amplification, have been previously described in many cancers, particularly associated with poor prognosis. In this study, RICTOR copy number variation (CNV) results of diagnostic next-generation sequencing (NGS) were analysed in 420 various human malignant tissues. RICTOR amplification was tested by Droplet Digital PCR (ddPCR) and validated using the "gold standard" fluorescence in situ hybridisation (FISH). Additionally, the consequences of Rictor protein expression were also studied by immunohistochemistry. RICTOR amplification was presumed in 37 cases with CNV ≥ 3 by NGS, among these, 16 cases (16/420; 3.8%) could be validated by FISH, however, ddPCR confirmed only 11 RICTOR-amplified cases with lower sensitivity. Based on these, neither NGS nor ddPCR could replace traditional FISH in proof of RICTOR amplification. However, NGS could be beneficial to highlight potential RICTOR-amplified cases. The obtained results of the 14 different tumour types with FISH-validated RICTOR amplification demonstrate the importance of RICTOR amplification in a broad spectrum of tumours. The newly described RICTOR-amplified entities could initiate further collaborative studies with larger cohorts to analyse the prevalence of RICTOR amplification in rare diseases. Finally, our and further work could help to improve and expand future therapeutic opportunities for mTOR-targeted therapies.


Assuntos
Variações do Número de Cópias de DNA , Neoplasias , Humanos , Neoplasias/genética , Serina-Treonina Quinases TOR/genética , Proteína Companheira de mTOR Insensível à Rapamicina/genética , Sequenciamento de Nucleotídeos em Larga Escala , Amplificação de Genes
7.
Int J Mol Sci ; 24(14)2023 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-37511253

RESUMO

The mechanistic target of rapamycin (mTOR) kinase is a central regulator of cell growth and metabolism. It is the catalytic subunit of two distinct large protein complexes, mTOR complex 1 (mTORC1) and mTORC2. mTOR activity is subjected to tight regulation in response to external nutrition and growth factor stimulation. As an important mechanism of signaling transduction, the 'second messenger' cyclic nucleotides including cAMP and cGMP and their associated cyclic nucleotide-dependent kinases, including protein kinase A (PKA) and protein kinase G (PKG), play essential roles in mediating the intracellular action of a variety of hormones and neurotransmitters. They have also emerged as important regulators of mTOR signaling in various physiological and disease conditions. However, the mechanism by which cAMP and cGMP regulate mTOR activity is not completely understood. In this review, we will summarize the earlier work establishing the ability of cAMP to dampen mTORC1 activation in response to insulin and growth factors and then discuss our recent findings demonstrating the regulation of mTOR signaling by the PKA- and PKG-dependent signaling pathways. This signaling framework represents a new non-canonical regulation of mTOR activity that is independent of AKT and could be a novel mechanism underpinning the action of a variety of G protein-coupled receptors that are linked to the mTOR signaling network. We will further review the implications of these signaling events in the context of cardiometabolic disease, such as obesity, non-alcoholic fatty liver disease, and cardiac remodeling. The metabolic and cardiac phenotypes of mouse models with targeted deletion of Raptor and Rictor, the two essential components for mTORC1 and mTORC2, will be summarized and discussed.


Assuntos
Doenças Cardiovasculares , Complexos Multiproteicos , Sirolimo , Serina-Treonina Quinases TOR , Animais , Camundongos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Complexos Multiproteicos/metabolismo , Nucleotídeos Cíclicos/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína Companheira de mTOR Insensível à Rapamicina/metabolismo , Serina-Treonina Quinases TOR/metabolismo
8.
Acta Pharmacol Sin ; 44(11): 2243-2252, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37407703

RESUMO

Group 3 innate lymphoid cells (ILC3s) are mediators of intestinal immunity and barrier function. Recent studies have investigated the role of the mammalian target of rapamycin complex (mTOR) in ILC3s, whereas the mTORC1-related mechanisms and crosstalk between mTORC1 and mTORC2 involved in regulating ILC3 homeostasis remain unknown. In this study, we found that mTORC1 but not mTORC2 was critical in ILC3 development, IL-22 production, and ILC3-mediated intestinal homeostasis. Single-cell RNA sequencing revealed that mTORC1 deficiency led to disruption of ILC3 heterogeneity, showing an increase in differentiation into ILC1-like phenotypes. Mechanistically, mTORC1 deficiency decreased the expression of NFIL3, which is a critical transcription factor responsible for ILC3 development. The activities of both mTORC1 and mTORC2 were increased in wild-type ILC3s after activation by IL-23, whereas inhibition of mTORC1 by Raptor deletion or rapamycin treatment resulted in increased mTORC2 activity. Previous studies have demonstrated that S6K, the main downstream target of mTORC1, can directly phosphorylate Rictor to dampen mTORC2 activity. Our data found that inhibition of mTORC1 activity by rapamycin reduced Rictor phosphorylation in ILC3s. Reversing the increased mTORC2 activity via heterozygous or homozygous knockout of Rictor in Raptor-deleted ILC3s resulted in severe ILC3 loss and complete susceptibility to intestinal infection in mice with mTORC1 deficiency (100% mortality). Thus, mTORC1 acts as a rheostat of ILC3 heterogeneity, and mTORC2 protects ILC3s from severe loss of cells and immune activity against intestinal infection when mTORC1 activity is diminished.


Assuntos
Imunidade Inata , Linfócitos , Camundongos , Animais , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Proteína Companheira de mTOR Insensível à Rapamicina/metabolismo , Proteína Regulatória Associada a mTOR/genética , Fatores de Transcrição/metabolismo , Sirolimo/farmacologia , Mamíferos/metabolismo
9.
Genes (Basel) ; 14(6)2023 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-37372460

RESUMO

The importance of the network defined by phosphatidylinositol-3-kinase (PI3K), AKT and mammalian target of rapamycin (mTOR) downstream of Receptor Tyrosine Kinase (RTK) has been recognized for many years. However, the central role of RICTOR (rapamycin-insensitive companion of mTOR) in this pathway has only recently come to light. The function of RICTOR in pan-cancer still needs to be systematically elucidated. In this study, we examined RICTOR's molecular characteristics and clinical prognostic value by pan-cancer analysis. Our findings indicate that RICTOR was overexpressed in twelve cancer types, and a high RICTOR expression was linked to poor overall survival. Moreover, the CRISPR Achilles' knockout analysis revealed that RICTOR was a critical gene for the survival of many tumor cells. Function analysis revealed that RICTOR-related genes were mainly involved in TOR signaling and cell growth. We further demonstrated that the RICTOR expression was significantly influenced by genetic alteration and DNA-methylation in multiple cancer types. Additionally, we found a positive relationship between RICTOR expression and the immune infiltration of macrophages and cancer-associated fibroblasts in Colon adenocarcinoma and Head and Neck squamous cell carcinoma. Finally, we validated the ability of RICTOR in sustaining tumor growth and invasion in the Hela cell line using cell-cycle analysis, the cell proliferation assay, and wound-healing assay. Our pan-cancer analysis highlights the critical role of RICTOR in tumor progression and its potential as a prognostic marker for various cancer types.


Assuntos
Adenocarcinoma , Neoplasias do Colo , Humanos , Células HeLa , Proteína Companheira de mTOR Insensível à Rapamicina/genética , Proteína Companheira de mTOR Insensível à Rapamicina/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição/metabolismo , Sirolimo , Prognóstico
10.
Oncogene ; 42(21): 1763-1776, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-37037900

RESUMO

The mTORC2 pathway plays a critical role in promoting tumor progression in human colorectal cancer (CRC). The regulatory mechanisms for this signaling pathway are only partially understood. We previously identified UBXN2A as a novel tumor suppressor protein in CRCs and hypothesized that UBXN2A suppresses the mTORC2 pathway, thereby inhibiting CRC growth and metastasis. We first used murine models to show that haploinsufficiency of UBXN2A significantly increases colon tumorigenesis. Induction of UBXN2A reduces AKT phosphorylation downstream of the mTORC2 pathway, which is essential for a plethora of cellular processes, including cell migration. Meanwhile, mTORC1 activities remain unchanged in the presence of UBXN2A. Mechanistic studies revealed that UBXN2A targets Rictor protein, a key component of the mTORC2 complex, for 26S proteasomal degradation. A set of genetic, pharmacological, and rescue experiments showed that UBXN2A regulates cell proliferation, apoptosis, migration, and colon cancer stem cells (CSCs) in CRC. CRC patients with a high level of UBXN2A have significantly better survival, and high-grade CRC tissues exhibit decreased UBXN2A protein expression. A high level of UBXN2A in patient-derived xenografts and tumor organoids decreases Rictor protein and suppresses the mTORC2 pathway. These findings provide new insights into the functions of an ubiquitin-like protein by inhibiting a dominant oncogenic pathway in CRC.


Assuntos
Neoplasias do Colo , Humanos , Camundongos , Animais , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Proteína Companheira de mTOR Insensível à Rapamicina/genética , Proteína Companheira de mTOR Insensível à Rapamicina/metabolismo , Neoplasias do Colo/patologia , Linhagem Celular Tumoral , Células-Tronco Neoplásicas/patologia , Transdução de Sinais , Fatores de Transcrição/genética , Carcinogênese/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ubiquitinas/metabolismo
11.
Ecotoxicol Environ Saf ; 257: 114914, 2023 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-37084658

RESUMO

Ingestion of arsenic interferes with spermatogenesis and increases the risk of male infertility, but the underlying mechanism remines unclear. In this study, we investigated spermatogenic injury with a focus on blood-testis barrier (BTB) disruption by administrating 5 mg/L and 15 mg/L arsenic orally to adult male mice for 60 d. Our results showed that arsenic exposure reduced sperm quality, altered testicular architecture, and impaired Sertoli cell junctions at the BTB. Analysis of BTB junctional proteins revealed that arsenic intake downregulated Claudin-11 expression and increased protein levels of ß-catenin, N-cadherin, and Connexin-43. Aberrant localization of these membrane proteins was also observed in arsenic-treated mice. Meanwhile, arsenic exposure altered the components of Rictor/mTORC2 pathway in mouse testis, including inhibition of Rictor expression, reduced phosphorylation of protein kinase Cα (PKCα) and protein kinase B (PKB), and elevated matrix metalloproteinase-9 (MMP-9) levels. Furthermore, arsenic also induced testicular lipid peroxidative damage, inhibited antioxidant enzyme (T-SOD) activity, and caused glutathione (GSH) depletion. Our findings suggest that disruption of BTB integrity is one of the main factors responsible for the decline in sperm quality caused by arsenic. PKCα-mediated rearrangement of actin filaments and PKB/MMP-9-increased barrier permeability jointly contribute to arsenic-induced BTB disruption.


Assuntos
Arsênio , Camundongos , Masculino , Animais , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Arsênio/toxicidade , Arsênio/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Proteína Quinase C-alfa/metabolismo , Barreira Hematotesticular/metabolismo , Sêmen , Testículo/metabolismo , Espermatogênese , Fatores de Transcrição/metabolismo , Proteína Companheira de mTOR Insensível à Rapamicina/metabolismo
12.
PLoS Genet ; 19(2): e1010629, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36787291

RESUMO

Pharmacological vitamin C (VC) is a potential natural compound for cancer treatment. However, the mechanism underlying its antitumor effects remains unclear. In this study, we found that pharmacological VC significantly inhibits the mTOR (including mTORC1 and mTORC2) pathway activation and promotes GSK3-FBXW7-mediated Rictor ubiquitination and degradation by increasing the cellular ROS. Moreover, we identified that HMOX1 is a checkpoint for pharmacological-VC-mediated mTOR inactivation, and the deletion of FBXW7 or HMOX1 suppresses the regulation of pharmacological VC on mTOR activation, cell size, cell viability, and autophagy. More importantly, it was observed that the inhibition of mTOR by pharmacological VC supplementation in vivo produces positive therapeutic responses in tumor growth, while HMOX1 deficiency rescues the inhibitory effect of pharmacological VC on tumor growth. These results demonstrate that VC influences cellular activities and tumor growth by inhibiting the mTOR pathway through Rictor and HMOX1, which may have therapeutic potential for cancer treatment.


Assuntos
Ácido Ascórbico , Neoplasias , Humanos , Proteína 7 com Repetições F-Box-WD/metabolismo , Ácido Ascórbico/farmacologia , Quinase 3 da Glicogênio Sintase/metabolismo , Proteína Companheira de mTOR Insensível à Rapamicina/genética , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/genética , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Fatores de Transcrição/metabolismo , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo
13.
Int J Mol Sci ; 23(24)2022 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-36555556

RESUMO

The existing studies by our team demonstrated the pro-recovery effect of 3-Acetyl-11-keto-beta-boswellic acid (AKBA) on a sciatic nerve injury. To further investigate the role of AKBA in peripheral nerve injury repair, The TMT quantitative proteomics technique was used to obtain differentially significant proteins in a Sham group, Model group, and AKBA group. After that, three time points (5, 14, and 28 d) and four groups (Sham + AKBA, Sham, Model, and AKBA) were set up, and immunoblotting, immunofluorescence, and cellular assays were applied to investigate the expression of CDC42, Rac1, RhoA, and Rictor in the sciatic nerve at different time points for each group in more depth. The results showed that AKBA enriched the cellular components of the myelin sheath and axon regeneration after a sciatic nerve injury and that AKBA upregulated CDC42 and Rac1 and downregulated RhoA expression 5 d after a sciatic nerve injury, promoting axon regeneration and improving the repair of a sciatic nerve injury in rats. Rictor is regulated by AKBA and upregulated in PC12 cells after AKBA action. Our findings provide a new basis for AKBA treatment of a peripheral nerve injury.


Assuntos
Traumatismos dos Nervos Periféricos , Neuropatia Ciática , Animais , Ratos , Axônios , Regeneração Nervosa/fisiologia , Proteína Companheira de mTOR Insensível à Rapamicina , Nervo Isquiático/lesões , Neuropatia Ciática/tratamento farmacológico
14.
J Biol Chem ; 298(10): 102379, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35973513

RESUMO

Mechanistic target of rapamycin (mTOR) complex 2 (mTORC2) regulates metabolism, cell proliferation, and cell survival. mTORC2 activity is stimulated by growth factors, and it phosphorylates the hydrophobic motif site of the AGC kinases AKT, SGK, and PKC. However, the proteins that interact with mTORC2 to control its activity and localization remain poorly defined. To identify mTORC2-interacting proteins in living cells, we tagged endogenous RICTOR, an essential mTORC2 subunit, with the modified BirA biotin ligase BioID2 and performed live-cell proximity labeling. We identified 215 RICTOR-proximal proteins, including proteins with known mTORC2 pathway interactions, and 135 proteins (63%) not previously linked to mTORC2 signaling, including nuclear and cytoplasmic proteins. Our imaging and cell fractionation experiments suggest nearly 30% of RICTOR is in the nucleus, hinting at potential nuclear functions. We also identified 29 interactors containing RICTOR-dependent, insulin-stimulated phosphorylation sites, thus providing insight into mTORC2-dependent insulin signaling dynamics. Finally, we identify the endogenous ADP ribosylation factor 1 (ARF1) GTPase as an mTORC2-interacting protein. Through gain-of-function and loss-of-function studies, we provide functional evidence that ARF1 may negatively regulate mTORC2. In summary, we present a new method of studying endogenous mTORC2, a resource of RICTOR/mTORC2 protein interactions in living cells, and a potential mechanism of mTORC2 regulation by the ARF1 GTPase.


Assuntos
Fator 1 de Ribosilação do ADP , Mapas de Interação de Proteínas , Proteína Companheira de mTOR Insensível à Rapamicina , Serina-Treonina Quinases TOR , Humanos , Fator 1 de Ribosilação do ADP/metabolismo , Insulina/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína Companheira de mTOR Insensível à Rapamicina/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Mapeamento de Interação de Proteínas/métodos
15.
J Biol Chem ; 298(9): 102288, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35926713

RESUMO

Mechanistic target of rapamycin complex 2 (mTORC2) is a multi-subunit kinase complex, central to multiple essential signaling pathways. Two core subunits, Rictor and mSin1, distinguish it from the related mTORC1 and support context-dependent phosphorylation of its substrates. mTORC2 structures have been determined previously; however, important questions remain, particularly regarding the structural determinants mediating substrate specificity and context-dependent activity. Here, we used cryo-EM to obtain high-resolution structures of the human mTORC2 apo-complex in the presence of substrates Akt and SGK1. Using functional assays, we then tested predictions suggested by substrate-induced structural changes in mTORC2. For the first time, we visualized in the apo-state the side chain interactions between Rictor and mTOR that sterically occlude recruitment of mTORC1 substrates and confer resistance to the mTORC1 inhibitor rapamycin. Also in the apo-state, we observed that mSin1 formed extensive contacts with Rictor via a pair of short α-helices nestled between two Rictor helical repeat clusters, as well as by an extended strand that makes multiple weak contacts with Rictor helical cluster 1. In co-complex structures, we found that SGK1, but not Akt, markedly altered the conformation of the mSin1 N-terminal extended strand, disrupting multiple weak interactions while inducing a large rotation of mSin1 residue Arg-83, which then interacts with a patch of negatively charged residues within Rictor. Finally, we demonstrate mutation of Arg-83 to Ala selectively disrupts mTORC2-dependent phosphorylation of SGK1, but not of Akt, supporting context-dependent substrate selection. These findings provide new structural and functional insights into mTORC2 specificity and context-dependent activity.


Assuntos
Proteínas Imediatamente Precoces , Proteínas Monoméricas de Ligação ao GTP , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas c-akt , Proteína Companheira de mTOR Insensível à Rapamicina , Humanos , Proteínas Imediatamente Precoces/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína Companheira de mTOR Insensível à Rapamicina/genética , Proteína Companheira de mTOR Insensível à Rapamicina/metabolismo , Sirolimo/farmacologia , Fatores de Transcrição/metabolismo
16.
Int J Biol Sci ; 18(13): 4869-4883, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35982899

RESUMO

Non-small cell lung cancer (NSCLC) is one of the deadliest cancers in the world. Metastasis is considered one of the leading causes of treatment failure and death in NSCLC patients. A crucial factor of promoting metastasis in epithelium-derived carcinoma has been considered as epithelial-mesenchymal transition (EMT). Rictor, one of the components of mTORC2, has been reportedly involved in EMT and metastasis of human malignancies. However, the regulatory mechanisms of Rictor, Rictor-mediated EMT and metastasis in cancers remain unknown. Our present study indicates that Rictor is highly expressed in human NSCLC cell lines and tissues and is regulated, at least partially, at the transcriptional level. Knockdown of Rictor expression causes phenotype alterations through EMT, which is accompanied by the impairment of migration and invasion ability in NSCLC cells. Additionally, we have cloned and identified the human Rictor core promoter for the first time and confirmed that transcription factor KLF4 directly binds to the Rictor promoter and transcriptionally upregulated Rictor expression. Knockdown of KLF4 results in Rictor's downregulation accompanied by a series of characteristic changes of mesenchymal-epithelial transition (MET) and significantly decreases migration, invasion as well as metastasis of NSCLC cells. Re-introducing Rictor in KLF4-knockdown NSCLC cells partially reverses the epithelial phenotype to the mesenchymal phenotype and attenuates the inhibition of cell migration and invasion caused by KLF4 knocking down. Knockdown of KLF4 prevents mTOR/Rictor interaction and metastasis of NSCLC in vivo. The understanding of the regulator upstream of Rictor may provide an opportunity for the development of new inhibitors and the rational design of combination regimens based on different metastasis-related molecular targets and finally prevents cancer metastasis.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Proteína Companheira de mTOR Insensível à Rapamicina , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Movimento Celular , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Humanos , Fator 4 Semelhante a Kruppel/genética , Neoplasias Pulmonares/patologia , Proteína Companheira de mTOR Insensível à Rapamicina/genética , Proteína Companheira de mTOR Insensível à Rapamicina/metabolismo , Fatores de Transcrição/metabolismo
17.
Diabetes ; 71(10): 2123-2135, 2022 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-35877180

RESUMO

Long-term glucagon receptor (GCGR) agonism is associated with hyperglycemia and glucose intolerance, while acute GCGR agonism enhances whole-body insulin sensitivity and hepatic AKTSer473 phosphorylation. These divergent effects establish a critical gap in knowledge surrounding GCGR action. mTOR complex 2 (mTORC2) is composed of seven proteins, including RICTOR, which dictates substrate binding and allows for targeting of AKTSer473. We used a liver-specific Rictor knockout mouse (RictorΔLiver) to investigate whether mTORC2 is necessary for insulin receptor (INSR) and GCGR cross talk. RictorΔLiver mice were characterized by impaired AKT signaling and glucose intolerance. Intriguingly, RictorΔLiver mice were also resistant to GCGR-stimulated hyperglycemia. Consistent with our prior report, GCGR agonism increased glucose infusion rate and suppressed hepatic glucose production during hyperinsulinemic-euglycemic clamp of control animals. However, these benefits to insulin sensitivity were ablated in RictorΔLiver mice. We observed diminished AKTSer473 and GSK3α/ßSer21/9 phosphorylation in RictorΔLiver mice, whereas phosphorylation of AKTThr308 was unaltered in livers from clamped mice. These signaling effects were replicated in primary hepatocytes isolated from RictorΔLiver and littermate control mice, confirming cell-autonomous cross talk between GCGR and INSR pathways. In summary, our study reveals the necessity of RICTOR, and thus mTORC2, in GCGR-mediated enhancement of liver and whole-body insulin action.


Assuntos
Intolerância à Glucose , Hiperglicemia , Resistência à Insulina , Animais , Glucose/metabolismo , Intolerância à Glucose/metabolismo , Homeostase , Hiperglicemia/metabolismo , Insulina/metabolismo , Insulina/farmacologia , Insulina Regular Humana , Fígado/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína Companheira de mTOR Insensível à Rapamicina , Receptor de Insulina/metabolismo , Receptores de Glucagon/metabolismo , Serina-Treonina Quinases TOR/metabolismo
18.
Toxicol Appl Pharmacol ; 449: 116135, 2022 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-35732230

RESUMO

Polychlorinated biphenyls (PCBs) are a typical type of persistent organic pollutant. PCB exposure is associated to the occurrence and development of osteoarthritis (OA); however, the involved mechanisms have yet to be elucidated. Here, we investigated the pro-osteoarthritic effect of 2, 2', 4, 4', 5, 5'-hexachlorobiphenyl (PCB153), and the involvement of the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/ mammalian target of rapamycin (mTOR) and the RICTOR/Akt/mTOR signaling pathways. PCB153 of 20 and 30 µM increased the expression of MMP13 and decreased the expression of type II collagen, in a concentration-dependent manner. PCB153 treatment reduced the expression of Beclin 1 and LC3B, but increased the expression of p62 by upregulating miR-155 levels. PCB153 treatment activated the PI3K/Akt/mTOR signaling pathway by upregulating miR-155 levels. RICTOR was involved in activating the Akt/mTOR signaling pathway, and was also regulated by miR-155. In conclusion, PCB153 could promote the degradation of the extracellular matrix of chondrocytes by upregulating miR-155 via a mechanism related to the activation of the PI3K/Akt/mTOR and RICTOR/Akt/mTOR signaling pathway, which suppressed autophagy and facilitated the development of OA. MiR-155 may represent potential therapeutic targets to alleviate the development of OA.


Assuntos
MicroRNAs , Osteoartrite , Bifenilos Policlorados , Animais , Ratos , Autofagia , Condrócitos , Mamíferos/metabolismo , MicroRNAs/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Bifenilos Policlorados/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína Companheira de mTOR Insensível à Rapamicina/genética , Proteína Companheira de mTOR Insensível à Rapamicina/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Regulação para Cima
19.
J Mol Neurosci ; 72(6): 1243-1258, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35618880

RESUMO

Neuronal hyperactivation of the mTOR signaling pathway may play a role in driving the pathological sequelae that follow status epilepticus. Animal studies using pharmacological tools provide support for this hypothesis, however, systemic inhibition of mTOR-a growth pathway active in every mammalian cell-limits conclusions on cell type specificity. To circumvent the limitations of pharmacological approaches, we developed a viral/genetic strategy to delete Raptor or Rictor, inhibiting mTORC1 or mTORC2, respectively, from excitatory hippocampal neurons after status epilepticus in mice. Raptor or Rictor was deleted from roughly 25% of hippocampal granule cells, with variable involvement of other hippocampal neurons, after pilocarpine status epilepticus. Status epilepticus induced the expected loss of hilar neurons, sprouting of granule cell mossy fiber axons and reduced c-Fos activation. Gene deletion did not prevent these changes, although Raptor loss reduced the density of c-Fos-positive granule cells overall relative to Rictor groups. Findings demonstrate that mTOR signaling can be effectively modulated with this approach and further reveal that blocking mTOR signaling in a minority (25%) of granule cells is not sufficient to alter key measures of status epilepticus-induced pathology. The approach is suitable for producing higher deletion rates, and altering the timing of deletion, which may lead to different outcomes.


Assuntos
Epilepsia do Lobo Temporal , Aves Predatórias , Estado Epiléptico , Animais , Modelos Animais de Doenças , Epilepsia do Lobo Temporal/metabolismo , Hipocampo/metabolismo , Mamíferos , Camundongos , Fibras Musgosas Hipocampais/patologia , Fibras Musgosas Hipocampais/fisiologia , Pilocarpina , Proteína Companheira de mTOR Insensível à Rapamicina/genética , Proteína Companheira de mTOR Insensível à Rapamicina/metabolismo , Aves Predatórias/metabolismo , Estado Epiléptico/genética , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo
20.
J Clin Invest ; 132(15)2022 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-35579957

RESUMO

The encoding of noxious stimuli into action potential firing is largely mediated by nociceptive free nerve endings. Tissue inflammation, by changing the intrinsic properties of the nociceptive endings, leads to nociceptive hyperexcitability and thus to the development of inflammatory pain. Here, we showed that tissue inflammation-induced activation of the mammalian target of rapamycin complex 2 (mTORC2) triggers changes in the architecture of nociceptive terminals and leads to inflammatory pain. Pharmacological activation of mTORC2 induced elongation and branching of nociceptor peripheral endings and caused long-lasting pain hypersensitivity. Conversely, nociceptor-specific deletion of the mTORC2 regulatory protein rapamycin-insensitive companion of mTOR (Rictor) prevented inflammation-induced elongation and branching of cutaneous nociceptive fibers and attenuated inflammatory pain hypersensitivity. Computational modeling demonstrated that mTORC2-mediated structural changes in the nociceptive terminal tree are sufficient to increase the excitability of nociceptors. Targeting mTORC2 using a single injection of antisense oligonucleotide against Rictor provided long-lasting alleviation of inflammatory pain hypersensitivity. Collectively, we showed that tissue inflammation-induced activation of mTORC2 causes structural plasticity of nociceptive free nerve endings in the epidermis and inflammatory hyperalgesia, representing a therapeutic target for inflammatory pain.


Assuntos
Dor Crônica , Nociceptores , Humanos , Hiperalgesia/genética , Hiperalgesia/metabolismo , Inflamação/induzido quimicamente , Inflamação/genética , Alvo Mecanístico do Complexo 2 de Rapamicina/genética , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Nociceptores/fisiologia , Proteína Companheira de mTOR Insensível à Rapamicina/genética , Proteína Companheira de mTOR Insensível à Rapamicina/metabolismo , Sirolimo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...