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1.
Cell Mol Life Sci ; 81(1): 416, 2024 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-39367928

RESUMO

Neurons are dependent on efficient quality control mechanisms to maintain cellular homeostasis and function due to their polarization and long-life span. Autophagy is a lysosomal degradative pathway that provides nutrients during starvation and recycles damaged and/or aged proteins and organelles. In neurons, autophagosomes constitutively form in distal axons and at synapses and are trafficked retrogradely to the cell soma to fuse with lysosomes for cargo degradation. How the neuronal autophagy pathway is organized and controlled remains poorly understood. Several presynaptic endocytic proteins have been shown to regulate both synaptic vesicle recycling and autophagy. Here, by combining electron, fluorescence, and live imaging microscopy with biochemical analysis, we show that the neuron-specific protein APache, a presynaptic AP-2 interactor, functions in neurons as an important player in the autophagy process, regulating the retrograde transport of autophagosomes. We found that APache colocalizes and co-traffics with autophagosomes in primary cortical neurons and that induction of autophagy by mTOR inhibition increases LC3 and APache protein levels at synaptic boutons. APache silencing causes a blockade of autophagic flux preventing the clearance of p62/SQSTM1, leading to a severe accumulation of autophagosomes and amphisomes at synaptic terminals and along neurites due to defective retrograde transport of TrkB-containing signaling amphisomes along the axons. Together, our data identify APache as a regulator of the autophagic cycle, potentially in cooperation with AP-2, and hypothesize that its dysfunctions contribute to the early synaptic impairments in neurodegenerative conditions associated with impaired autophagy.


Assuntos
Autofagossomos , Autofagia , Transporte Axonal , Neurônios , Autofagossomos/metabolismo , Autofagia/fisiologia , Animais , Neurônios/metabolismo , Transporte Axonal/fisiologia , Camundongos , Células Cultivadas , Serina-Treonina Quinases TOR/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteína Sequestossoma-1/metabolismo , Receptor trkB/metabolismo , Transdução de Sinais , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , Terminações Pré-Sinápticas/metabolismo
2.
Biochemistry (Mosc) ; 89(1): 27-52, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38467544

RESUMO

Autophagy is a central process for degradation of intracellular components that do not operate correctly. Molecular mechanisms underlying this process are extremely difficult to study, since they involve a large number of participants. The main task of autophagy is redistribution of cellular resources in response to environmental changes, such as starvation. Recent studies show that autophagy regulation could be the key to achieve healthy longevity, as well as to create therapeutic agents for treatment of neurodegenerative diseases such as Parkinson's and Alzheimer's diseases. Thus, development of autophagy activators with established detailed mechanism of action is a really important area of research. Several commercial companies are at various stages of development of such molecules, and some of them have already begun to introduce autophagy activators to the market.


Assuntos
Doença de Alzheimer , Autofagia , Humanos , Autofagia/fisiologia , Doença de Alzheimer/metabolismo
3.
Biochemistry (Mosc) ; 89(7): 1300-1312, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-39218026

RESUMO

To date, the molecular mechanisms of the common neurodegenerative disorder Parkinson's disease (PD) are unknown and, as a result, there is no neuroprotective therapy that may stop or slow down the process of neuronal cell death. The aim of the current study was to evaluate the prospects of using the mTOR molecule as a potential target for PD therapy due to the dose-dependent effect of mTOR kinase activity inhibition on cellular parameters associated with, PD pathogenesis. The study used peripheral blood monocyte-derived macrophages and SH-SY5Y neuroblastoma cell line. As a result, we have for the first time showed that inhibition of mTOR by Torin1 only at a concentration of 100 nM affects the level of the lysosomal enzyme glucocerebrosidase (GCase), encoded by the GBA1 gene. Mutations in GBA1 are considered a high-risk factor for PD development. This concentration led a decrease in pathological phosphorylated alpha-synuclein (Ser129), an increase in its stable tetrameric form with no changes in the lysosomal enzyme activities and concentrations of lysosphingolipids. Our findings suggest that inhibition of the mTOR protein kinase could be a promising approach for developing therapies for PD, particularly for GBA1-associated PD.


Assuntos
Lisossomos , Macrófagos , Doença de Parkinson , Serina-Treonina Quinases TOR , alfa-Sinucleína , Humanos , alfa-Sinucleína/metabolismo , Lisossomos/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Serina-Treonina Quinases TOR/antagonistas & inibidores , Doença de Parkinson/metabolismo , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/patologia , Linhagem Celular Tumoral , Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Neuroblastoma/metabolismo , Neuroblastoma/tratamento farmacológico , Neuroblastoma/patologia , Relação Dose-Resposta a Droga , Glucosilceramidase/metabolismo , Glucosilceramidase/antagonistas & inibidores , Naftiridinas
4.
Ann Hematol ; 102(9): 2317-2327, 2023 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-37421506

RESUMO

B cell hyper-function plays an important role in the pathogenesis of immune thrombocytopenia (ITP), but the molecular mechanisms underlying such changes remain unclear. We sought to identify regulators of B cell dysfunction in ITP patients through transcriptome sequencing and the use of inhibitors. B cells were isolated from PBMC of 25 ITP patients for B cell function test and transcriptome sequencing. For the potential regulatory factors identified by transcriptome sequencing, the corresponding protein inhibitors were used to explore the regulatory effect of the regulatory factors on B cell dysfunction in vitro. In this study, increased antibody production, enhanced terminal differentiation and highly expressed costimulatory molecules CD80 and CD86 were found in B cells of patients with ITP. In addition, RNA sequencing revealed highly activated mTOR pathway in these pathogenic B cells, indicating that the mTOR pathway may be involved in B cell hyper-function. Furthermore, mTOR inhibitors rapamycin or Torin1 effectively blocked the activation of mTORC1 in B cells, resulting in reduce antibody secretion, impaired differentiation of B cells into plasmablasts and downregulation of costimulatory molecules. Interestingly, as an unspecific inhibitor of mTORC2 besides mTORC1, Torin1 did not show a stronger capacity to modulate B cell function than rapamycin, suggesting that the regulation of B cells by Torin1 may depend on blockade of mTORC1 rather than mTORC2 pathway. These results indicated that the activation of mTORC1 pathway is involved in B cell dysfunction in patients with ITP, and inhibition of mTORC1 pathway might be a potential therapeutic approach for ITP.


Assuntos
Púrpura Trombocitopênica Idiopática , Trombocitopenia , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Transdução de Sinais , Púrpura Trombocitopênica Idiopática/genética , Leucócitos Mononucleares/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Sirolimo , Alvo Mecanístico do Complexo 2 de Rapamicina/genética , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Fatores de Transcrição
5.
Cell Commun Signal ; 21(1): 120, 2023 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-37226246

RESUMO

Extracellular vesicles (EVs) are important mediators of intercellular communication in the tumour microenvironment. Many studies suggest that cancer cells release higher amounts of EVs exposing phosphatidylserine (PS) at the surface. There are lots of interconnections between EVs biogenesis and autophagy machinery. Modulation of autophagy can probably affect not only the quantity of EVs but also their content, which can deeply influence the resulting pro-tumourigenic or anticancer effect of autophagy modulators. In this study, we found that autophagy modulators autophinib, CPD18, EACC, bafilomycin A1 (BAFA1), 3-hydroxychloroquine (HCQ), rapamycin, NVP-BEZ235, Torin1, and starvation significantly alter the composition of the protein content of phosphatidylserine-positive EVs (PS-EVs) produced by cancer cells. The greatest impact had HCQ, BAFA1, CPD18, and starvation. The most abundant proteins in PS-EVs were proteins typical for extracellular exosomes, cytosol, cytoplasm, and cell surface involved in cell adhesion and angiogenesis. PS-EVs protein content involved mitochondrial proteins and signalling molecules such as SQSTM1 and TGFß1 pro-protein. Interestingly, PS-EVs contained no commonly determined cytokines, such as IL-6, IL-8, GRO-α, MCP-1, RANTES, and GM-CSF, which indicates that secretion of these cytokines is not predominantly mediated through PS-EVs. Nevertheless, the altered protein content of PS-EVs can still participate in the modulation of the fibroblast metabolism and phenotype as p21 was accumulated in fibroblasts influenced by EVs derived from CPD18-treated FaDu cells. The altered protein content of PS-EVs (data are available via ProteomeXchange with identifier PXD037164) also provides information about the cellular compartments and processes that are affected by the applied autophagy modulators. Video Abstract.


Assuntos
Exossomos , Vesículas Extracelulares , Fosfatidilserinas , Autofagia , Citocinas
6.
Arch Toxicol ; 97(5): 1385-1396, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-36826473

RESUMO

Bortezomib (BTZ) is a proteasome inhibitor serves as a first-line drug for multiple myeloma treatment. BTZ-induced peripheral neuropathy (BIPN) is the most common adverse effect of BTZ with an incidence as high as 40-60%. However, the pathological mechanisms underlying BIPN remain largely unclear. BTZ leads to dramatic Schwann cell demyelination in sciatic nerves. Previous studies implied that myelin debris was predominantly degraded via autophagy-lysosome pathway in Schwann cells. However, the association of autophagy with BIPN has not been made. Mice were treated with BTZ (2 mg/kg, i.v.) on Day1 and Day4 each week for continuous 4 weeks. BTZ-treated mice showed enhanced mechanical hyperalgesia, decreased tail nerve conduction and sciatic nerve demyelination. Unexpectedly, BTZ led to the accumulation of autophagic vesicles, LC3-II and p62 in the sciatic nerve. Moreover, BTZ blocked autophagic flux in RSC96 Schwann cells as determined by mcherry-GFP-LC3 assay, suggesting BTZ may impair lysosomal function rather than inducing autophagy in Schwann cells. BTZ significantly reduced the lysosomal activity in Schwann cells as determined by reduced LysoTracker Red and DQ-Red-BSA staining and increased the level of immature Cathepsin B (CTSB). Remarkably, lysosomal activators PP242 and Torin1, significantly reversed the blockage of autophagic flux by BTZ. We further verified that Torin1 rescued the demyelination, nerve conduction and reduced the mechanical hyperalgesia in BIPN mice. Additionally, Torin1 did not compromise the efficacy of BTZ in suppressing multiple myeloma RPMI8226 cell. Taken together, we identified that lysosomal dysfunction in Schwann cells caused by BTZ is involved in the BIPN pathology. Improved lysosomal function in Schwann cells can be a promising strategy for BIPN treatment.


Assuntos
Doenças Desmielinizantes , Mieloma Múltiplo , Síndromes Neurotóxicas , Camundongos , Animais , Bortezomib/efeitos adversos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/patologia , Hiperalgesia/induzido quimicamente , Células de Schwann/patologia , Síndromes Neurotóxicas/patologia , Doenças Desmielinizantes/induzido quimicamente , Doenças Desmielinizantes/patologia
7.
Biol Reprod ; 107(1): 76-84, 2022 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-35552649

RESUMO

Coordinated development of the germline and the somatic compartments within a follicle is an essential prerequisite for creating a functionally normal oocyte. Bi-directional communication between the oocyte and the granulosa cells enables the frequent interchange of metabolites and signals that support the development and functions of both compartments. Mechanistic target of rapamycin (MTOR), a conserved serine/threonine kinase and a widely recognized integrator of signals and pathways key for cellular metabolism, proliferation, and differentiation, is emerging as a major player that regulates many facets of oocyte and follicle development. Here, we summarized our recent observations on the role of oocyte- and granulosa cell-expressed MTOR in the control of the oocyte's and granulosa cell's own development, as well as the development of one another, and provided new data that further strengthen the role of cumulus cell-expressed MTOR in synchronizing oocyte and follicle development. Inhibition of MTOR induced oocyte meiotic resumption in cultured large antral follicles, as well as cumulus expansion and the expression of cumulus expansion-related transcripts in cumulus-oocyte complexes in vitro. In vivo, the activity of MTOR in cumulus cells was diminished remarkably by 4 h after hCG administration. These results thus suggest that activation of MTOR in cumulus cells contributes to the maintenance of oocyte meiotic arrest before the LH surge. Based on the observations made by us here and previously, we propose that MTOR is an essential mediator of the bi-directional communication between the oocyte and granulosa cells that regulates the development and function of both compartments.


Assuntos
Células da Granulosa , Meiose , Oócitos , Serina-Treonina Quinases TOR , Animais , Feminino , Células da Granulosa/metabolismo , Camundongos , Oócitos/metabolismo , Folículo Ovariano/metabolismo , Serina-Treonina Quinases TOR/metabolismo
8.
J Biol Chem ; 295(52): 18459-18473, 2020 12 25.
Artigo em Inglês | MEDLINE | ID: mdl-33082139

RESUMO

The amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD)-linked RNA-binding protein called FUS (fused in sarcoma) has been implicated in several aspects of RNA regulation, including mRNA translation. The mechanism by which FUS affects the translation of polyribosomes has not been established. Here we show that FUS can associate with stalled polyribosomes and that this association is sensitive to mTOR (mammalian target of rapamycin) kinase activity. Specifically, we show that FUS association with polyribosomes is increased by Torin1 treatment or when cells are cultured in nutrient-deficient media, but not when cells are treated with rapamycin, the allosteric inhibitor of mTORC1. Moreover, we report that FUS is necessary for efficient stalling of translation because deficient cells are refractory to the inhibition of mTOR-dependent signaling by Torin1. We also show that ALS-linked FUS mutants R521G and P525L associate abundantly with polyribosomes and decrease global protein synthesis. Importantly, the inhibitory effect on translation by FUS is impaired by mutations that reduce its RNA-binding affinity. These findings demonstrate that FUS is an important RNA-binding protein that mediates translational repression through mTOR-dependent signaling and that ALS-linked FUS mutants can cause a toxic gain of function in the cytoplasm by repressing the translation of mRNA at polyribosomes.


Assuntos
Esclerose Lateral Amiotrófica/patologia , Mutação , Polirribossomos/metabolismo , Proteína FUS de Ligação a RNA/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Citoplasma/metabolismo , Células HEK293 , Humanos , Corpos de Inclusão , Polirribossomos/genética , Biossíntese de Proteínas , Proteína FUS de Ligação a RNA/genética , Serina-Treonina Quinases TOR/genética
9.
J Virol ; 94(22)2020 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-32878890

RESUMO

Zika virus (ZIKV), a mosquito-transmitted flavivirus, is linked to microcephaly and other neurological defects in neonates and Guillain-Barré syndrome in adults. The molecular mechanisms regulating ZIKV infection and pathogenic outcomes are incompletely understood. Signaling by the mechanistic (mammalian) target of rapamycin (mTOR) kinase is important for cell survival and proliferation, and viruses are known to hijack this pathway for their replication. Here, we show that in human neuronal precursors and glial cells in culture, ZIKV infection activates both mTOR complex 1 (mTORC1) and mTORC2. Inhibition of mTOR kinase by Torin1 or rapamycin results in reduction in ZIKV protein expression and progeny production. Depletion of Raptor, the defining subunit of mTORC1, by small interfering RNA (siRNA) negatively affects ZIKV protein expression and viral replication. Although depletion of Rictor, the unique subunit of mTORC2, or the mTOR kinase itself also inhibits the viral processes, the extent of inhibition is less pronounced. Autophagy is transiently induced early by ZIKV infection, and impairment of autophagosome elongation by the class III phosphatidylinositol 3-kinase (PI3K) inhibitor 3-methyladenine (3-MA) enhances viral protein accumulation and progeny production. mTOR phosphorylates and inactivates ULK1 (S757) at later stages of ZIKV infection, suggesting a link between autophagy inhibition and mTOR activation by ZIKV. Accordingly, inhibition of ULK1 (by MRT68921) or autophagy (by 3-MA) reversed the effects of mTOR inhibition, leading to increased levels of ZIKV protein expression and progeny production. Our results demonstrate that ZIKV replication requires the activation of both mTORC1 and mTORC2, which negatively regulates autophagy to facilitate ZIKV replication.IMPORTANCE The re-emergence of Zika virus (ZIKV) and its association with neurological complications necessitates studies on the molecular mechanisms that regulate ZIKV pathogenesis. The mTOR signaling cascade is tightly regulated and central to normal neuronal development and survival. Disruption of mTOR signaling can result in neurological abnormalities. In the studies reported here, we demonstrate for the first time that ZIKV infection results in activation of both mTORC1 and mTORC2 to promote virus replication. Although autophagy is activated early in infection to counter virus replication, it is subsequently suppressed by mTOR. These results reveal critical roles of mTOR signaling and autophagy in ZIKV infection and point to a possible mechanism underlying ZIKV-induced pathogenesis. Elucidating the role of mTOR signaling in ZIKV infection will provide insights into the mechanisms of ZIKV-induced neurological complications and potential targets for therapeutic approaches.


Assuntos
Autofagia/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologia , Replicação Viral/efeitos dos fármacos , Zika virus/efeitos dos fármacos , Linhagem Celular , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Virais , Zika virus/fisiologia , Infecção por Zika virus/virologia
10.
Exp Eye Res ; 203: 108421, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33359326

RESUMO

Glaucoma, the second leading cause of blindness worldwide, is characterized by aberrant elevations of intraocular pressure (IOP), which can damage the optic nerve. IOP reduction is the only effective therapy for prevention of visual impairment and blindness in both hypertensive and normotensive individuals, and in some cases, trabeculectomy is a major surgical procedure that can lower IOP in patients with glaucoma. No matter how surgical technique and postoperative care advances, excessive scarring and tissue fibrosis could result from increased human conjunctival fibroblast (HCF) proliferation and extracellular matrix (ECM) deposition of the subconjunctival tissue and scleral flaps would persist after trabeculectomy. And these issues are major impediments to IOP reduction and filtering of bleb formations, so the modulation of the factors which can induce fibrosis could used as a novel strategy to control scarring after trabeculectomy. In this study, we examined the effects of mammalian target of rapamycin (mTOR) inhibitors (rapamycin or Torin1) on the fibrotic response induced by transforming growth factor-beta 1 (TGF-ß1) in cultured human conjunctival fibroblast (HCF) cells. The study also examined the effects of mTOR inhibitor on fibrosis after trabeculectomy in rabbit eyes. In in vitro studies, we stimulated HCFs with TGF-ß1, and confirmed that the expression levels of fibronectin, collagen type I alpha 1 chain (COL1A1), and α-smooth muscle actin (SMA) were significantly upregulated in HCFs with TGF-ß1, by means of quantitative real-time polymerase chain reaction and immunocytochemistry. And those TGF-ß1-induced changes were significantly attenuated with mTOR inhibitors, rapamycin or Torin1. Additionally the migration rate of HCFs was examined under conditions of TGF-ß1 induction, TGF-ß1-induced changes were significantly attenuated with mTOR inhibitors. A rabbit model of trabeculectomy was examined in vivo, and the effects of topical mTOR inhibitor were also examined, and found that topical treatment with mTOR inhibitor significantly suppressed collagen deposition in rabbit eyes after trabeculectomy. These results have demonstrated that mTOR inhibitors may provide a novel treatment modality for reducing the fibrotic response in HCFs and improving bleb scarring after filtration surgery.


Assuntos
Túnica Conjuntiva/patologia , Naftiridinas/farmacologia , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Trabeculectomia , Actinas/genética , Actinas/metabolismo , Animais , Western Blotting , Células Cultivadas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadeia alfa 1 do Colágeno Tipo I , Túnica Conjuntiva/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibronectinas/genética , Fibronectinas/metabolismo , Fibrose , Humanos , Imuno-Histoquímica , Masculino , RNA Mensageiro/genética , Coelhos , Reação em Cadeia da Polimerase em Tempo Real , Fator de Crescimento Transformador beta1/farmacologia
11.
New Phytol ; 221(1): 247-260, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30040123

RESUMO

Target of rapamycin (TOR) kinase is a conserved regulator of cell growth whose activity is modulated in response to nutrients, energy and stress. Key proteins involved in the pathway are conserved in the model photosynthetic microalga Chlamydomonas reinhardtii, but the substrates of TOR kinase and downstream signaling network have not been elucidated. Our study provides a new resource for investigating the phosphorylation networks governed by the TOR kinase pathway in Chlamydomonas. We used quantitative phosphoproteomics to investigate the effects of inhibiting Chlamydomonas TOR kinase on dynamic protein phosphorylation. Wild-type and AZD-insensitive Chlamydomonas strains were treated with TOR-specific chemical inhibitors (rapamycin, AZD8055 and Torin1), after which differentially affected phosphosites were identified. Our quantitative phosphoproteomic dataset comprised 2547 unique phosphosites from 1432 different proteins. Inhibition of TOR kinase caused significant quantitative changes in phosphorylation at 258 phosphosites, from 219 unique phosphopeptides. Our results include Chlamydomonas homologs of TOR signaling-related proteins, including a site on RPS6 with a decrease in phosphorylation. Additionally, phosphosites on proteins involved in translation and carotenoid biosynthesis were identified. Follow-up experiments guided by these phosphoproteomic findings in lycopene beta/epsilon cyclase showed that carotenoid levels are affected by TORC1 inhibition and carotenoid production is under TOR control in algae.


Assuntos
Chlamydomonas reinhardtii/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Plantas/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Carotenoides/metabolismo , Chlamydomonas reinhardtii/efeitos dos fármacos , Chlamydomonas reinhardtii/genética , Análise por Conglomerados , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Morfolinas , Mutação , Naftiridinas , Fosforilação/efeitos dos fármacos , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/metabolismo
12.
Invest New Drugs ; 37(5): 902-911, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-30542835

RESUMO

Chaetoglobosin K (ChK) is a natural product that has been shown to promote F-actin capping, inhibit growth, arrest cell cycle G2 phase, and induce apoptosis. ChK also has been shown to downregulate two important kinases involved in oncogenic pathways, Akt and JNK. This report investigates how ChK is involved in the receptor tyrosine kinase pathway (RTK/PI3K/mTORC2/Akt) to the centrally located protein kinase, Akt. Studies have reported that ChK does not inhibit PI3K comparable to wortmannin and does not affect PDK1 activation. PDK1 is responsible for phosphorylation on Akt T308, while mTORC2 phosphorylates Akt S473. Yet, Akt's two activation sites, T308 and S473, are known to be affected by ChK treatment. It was our hypothesis that ChK acts on the mTORC2 complex to inhibit the phosphorylation seen at Akt S473. This inhibition at mTORC2 should decrease phosphorylation at both these proteins, Akt and mTORC2 complex, compared to a known mTOR specific inhibitor, Torin1. Human lung adenocarcinoma H1299 and H2009 cells were treated with IGF-1 or calyculin A to increase phosphorylation at complex mTORC2 and Akt. Pretreatment with ChK was able to significantly decrease phosphorylation at Akt S473 similarly to Torin1 with either IGF-1 or calyculin A treatment. Moreover, the autophosphorylation site on complex mTORC2, S2481, was also significantly reduced with ChK pretreatment, similar to Torin1. This is the first report to illustrate that ChK has a significant effect at mTORC2 S2481 and Akt S473 comparable to Torin1, indicating that it may be a mTOR inhibitor.


Assuntos
Adenocarcinoma de Pulmão/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Alcaloides Indólicos/farmacologia , Neoplasias Pulmonares/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Fosfatidilinositol 3-Quinases/química , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Adenocarcinoma de Pulmão/tratamento farmacológico , Adenocarcinoma de Pulmão/patologia , Apoptose , Carcinogênese , Proliferação de Células , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Células Tumorais Cultivadas
13.
Biogerontology ; 20(3): 331-335, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30798505

RESUMO

Cellular senescence has recently been established as a key driver of organismal ageing. The state of senescence is controlled by extensive rewiring of signalling pathways, at the heart of which lies the mammalian Target of Rapamycin Complex I (mTORC1). Here we discuss recent publications aiming to establish the mechanisms by which mTORC1 drives the senescence program. In particular, we highlight our data indicating that mTORC1 can be used as a target for senescence cell elimination in vitro. Suppression of mTORC1 is known to extend lifespan of yeast, worms, flies and some mouse models and our proof-of-concept experiments suggest that it can also act by reducing senescent cell load in vivo.


Assuntos
Autofagia , Senescência Celular , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Animais , Masculino , Camundongos , Estudo de Prova de Conceito
14.
Invest New Drugs ; 36(6): 999-1005, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-29594878

RESUMO

Background Acquired resistance (AR) to an epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) is a common event, and several underlying mechanisms, including T790 M, MET amplification and PTEN downregulation, have been reported for the common EGFR mutations. EGFR G719X is an uncommon mutation that has been reported to show sensitivity to EGFR-TKIs. However, no established cell lines harboring the EGFR G719X have been reported in the literature. Materials and Methods G719S-GR cells were established from malignant pleural effusion of a patient whose tumor developed AR from gefitinib treatment. G719S-GR cells were then genotyped and tested for drug sensitivities. Multiplex ligation-dependent probe amplification (MLPA) was used to compare the clinical tumor samples with G719S-GR. Results G719S-GR cells were resistant to EGFR-TKIs with an LC50 of around 10 µM. A genomic analysis showed that G719S-GR cells harbor the EGFR G719S mutation as well as the amplification of EGFR locus. The homozygous deletion of CDKN2A and the loss of PTEN and TSC1 were also detected. On comparing the copy number of tumor suppressor genes using MLPA, G719S-GR cells were found to lack one copy of PTEN, which was not observed in a tumor obtained before gefitinib treatment. Loss of PTEN may result in AKT activation. The mTORC1/2 inhibitor Torin-1 was able to inhibit the downstream signaling when combined with osimertinib. Discussion The newly established G719S-GR cell line may be useful for investigating the mechanism underlying the development of AR in the G719X mutation; the loss of PTEN may be one such mechanism.


Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/genética , Mutação/genética , Inibidores de Proteínas Quinases/farmacologia , Idoso , Sequência de Bases , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Receptores ErbB/metabolismo , Humanos , Masculino
15.
Biochim Biophys Acta Mol Basis Dis ; 1863(1): 33-42, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27760377

RESUMO

Aldehyde dehydrogenase 5a1-deficient (aldh5a1-/-) mice, the murine orthologue of human succinic semialdehyde dehydrogenase deficiency (SSADHD), manifest increased GABA (4-aminobutyric acid) that disrupts autophagy, increases mitochondria number, and induces oxidative stress, all mitigated with the mTOR (mechanistic target of rapamycin) inhibitor rapamycin [1]. Because GABA regulates mTOR, we tested the hypothesis that aldh5a1-/- mice would show altered levels of mRNA for genes associated with mTOR signaling and oxidative stress that could be mitigated by inhibiting mTOR. We observed that multiple metabolites associated with GABA metabolism (γ-hydroxybutyrate, succinic semialdehyde, D-2-hydroxyglutarate, 4,5-dihydrohexanoate) and oxidative stress were significantly increased in multiple tissues derived from aldh5a1-/- mice. These metabolic perturbations were associated with decreased levels of reduced glutathione (GSH) in brain and liver of aldh5a1-/- mice, as well as increased levels of adducts of the lipid peroxidation by-product, 4-hydroxy-2-nonenal (4-HNE). Decreased liver mRNA levels for multiple genes associated with mTOR signaling and oxidative stress parameters were detected in aldh5a1-/- mice, and several were significantly improved with the administration of mTOR inhibitors (Torin 1/Torin 2). Western blot analysis of selected proteins corresponding to oxidative stress transcripts (glutathione transferase, superoxide dismutase, peroxiredoxin 1) confirmed gene expression findings. Our data provide additional preclinical evidence for the potential therapeutic efficacy of mTOR inhibitors in SSADHD.


Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/tratamento farmacológico , Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Deficiências do Desenvolvimento/tratamento farmacológico , Deficiências do Desenvolvimento/metabolismo , Deleção de Genes , Succinato-Semialdeído Desidrogenase/deficiência , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Ácido gama-Aminobutírico/metabolismo , Erros Inatos do Metabolismo dos Aminoácidos/genética , Animais , Deficiências do Desenvolvimento/genética , Modelos Animais de Doenças , Feminino , Glutationa/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Naftiridinas/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Succinato-Semialdeído Desidrogenase/genética , Succinato-Semialdeído Desidrogenase/metabolismo , Transcriptoma/efeitos dos fármacos
16.
Biometals ; 30(6): 975-980, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29063293

RESUMO

It is generally believed that gene-environment interaction may contribute to neurodegeneration. Of particular note is that iron overload may be one of the risk factors for neurodegeneration. However, the mechanisms underlying iron-associated neurotoxicity are not fully understood. Here we explored the effects of mechanistic target of rapamycin (mTOR) inhibition in iron-stressed human neuroblastoma cells. Two mTOR inhibitors, rapamycin and Torin 1, had similar effects in cells exposed to a relatively low concentration of iron. At a higher concentration of iron, Torin 1, instead of rapamycin, could further aggravate iron-induced cytotoxicity, and mitochondrial ROS levels were significantly higher in Torin 1-treated cells. These results suggest that mTOR inhibition may not be able to alleviate iron-induced neurotoxicity.


Assuntos
Ferro/toxicidade , Mitocôndrias/efeitos dos fármacos , Naftiridinas/farmacologia , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Linhagem Celular Tumoral , Humanos , Mitocôndrias/metabolismo , Neuroblastoma/tratamento farmacológico , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Fármacos Neuroprotetores/farmacologia , Espécies Reativas de Oxigênio/metabolismo
17.
Proc Natl Acad Sci U S A ; 111(49): 17450-5, 2014 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-25422441

RESUMO

Anabolic and catabolic signaling oppose one another in adipose tissue to maintain cellular and organismal homeostasis, but these pathways are often dysregulated in metabolic disorders. Although it has long been established that stimulation of the ß-adrenergic receptor inhibits insulin-stimulated glucose uptake in adipocytes, the mechanism has remained unclear. Here we report that ß-adrenergic-mediated inhibition of glucose uptake requires lipolysis. We also show that lipolysis suppresses glucose uptake by inhibiting the mammalian target of rapamycin (mTOR) complexes 1 and 2 through complex dissociation. In addition, we show that products of lipolysis inhibit mTOR through complex dissociation in vitro. These findings reveal a previously unrecognized intracellular signaling mechanism whereby lipolysis blocks the phosphoinositide 3-kinase-Akt-mTOR pathway, resulting in decreased glucose uptake. This previously unidentified mechanism of mTOR regulation likely contributes to the development of insulin resistance.


Assuntos
Adipócitos/citologia , Catecolaminas/química , Glucose/farmacocinética , Lipólise/fisiologia , Serina-Treonina Quinases TOR/metabolismo , Células 3T3-L1 , Animais , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Homeostase , Hiperglicemia/metabolismo , Insulina/metabolismo , Resistência à Insulina , Lipídeos/química , Camundongos , Modelos Biológicos , Naftiridinas/metabolismo , Receptores Adrenérgicos beta/metabolismo , Transdução de Sinais
18.
J Cell Sci ; 127(Pt 6): 1346-56, 2014 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-24424027

RESUMO

The target of rapamycin (TOR) kinase regulates cell growth and division. Rapamycin only inhibits a subset of TOR activities. Here we show that in contrast to the mild impact of rapamycin on cell division, blocking the catalytic site of TOR with the Torin1 inhibitor completely arrests growth without cell death in Schizosaccharomyces pombe. A mutation of the Tor2 glycine residue (G2040D) that lies adjacent to the key Torin-interacting tryptophan provides Torin1 resistance, confirming the specificity of Torin1 for TOR. Using this mutation, we show that Torin1 advanced mitotic onset before inducing growth arrest. In contrast to TOR inhibition with rapamycin, regulation by either Wee1 or Cdc25 was sufficient for this Torin1-induced advanced mitosis. Torin1 promoted a Polo and Cdr2 kinase-controlled drop in Wee1 levels. Experiments in human cell lines recapitulated these yeast observations: mammalian TOR (mTOR) was inhibited by Torin1, Wee1 levels declined and mitotic commitment was advanced in HeLa cells. Thus, the regulation of the mitotic inhibitor Wee1 by TOR signalling is a conserved mechanism that helps to couple cell cycle and growth controls.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Mitose/efeitos dos fármacos , Naftiridinas/farmacologia , Proteínas Nucleares/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/crescimento & desenvolvimento , Sequência de Aminoácidos , Domínio Catalítico , Morte Celular , Resistência a Medicamentos , Pontos de Checagem da Fase G1 do Ciclo Celular , Células HeLa , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 2 de Rapamicina , Dados de Sequência Molecular , Complexos Multiproteicos/antagonistas & inibidores , Complexos Multiproteicos/metabolismo , Mutagênese Sítio-Dirigida , Fosfatidilinositol 3-Quinases/metabolismo , Transporte Proteico , Schizosaccharomyces/efeitos dos fármacos , Schizosaccharomyces/enzimologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo
19.
BMC Musculoskelet Disord ; 17: 150, 2016 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-27052304

RESUMO

BACKGROUND: Recent studies have shown that autophagy was associated with the development of osteoarthritis (OA), the purpose of this research was to determine the exact role of autophagy in OA and investigate effective therapeutic drugs to inhibit the pathological progression of OA. METHODS: In this study, a cellular OA model was generated by stimulating SW1353 cells with IL-1ß and a rabbit OA model was established by intra-articular injection of collagenase, followed by treatment with Torin 1 or 3-Methyladenine (3-MA). The mRNA expression levels of VEGF, MMP-13 and TIMP-1 were determined by quantitative real-time PCR. The caitilage degeneration was examined by histological evaluation, chondrocytes degeneration and autophagosomes were observed by transmission electron microscopy. Expression levels of Beclin-1 and LC3 were evaluated by western blotting and immunofluorescence. RESULTS: The degeneration of SW 1353 cells, cartilage and chondrocytes was related to the loss of autophagy in experimental OA. 3-MA increased the severity of degeneration of cells and cartilage by autophagy inhibition, while Torin 1 reduced that by autophagy activation. CONCLUSIONS: The loss of autophagy is linked with the experimental OA and autophagy may play a protective role in the pathogenesis of OA. Treatment of Torin 1 can inhibit the degenerative changes of experimental OA by activating autophagy and it may be a useful therapeutic drug for OA.


Assuntos
Artrite Experimental/tratamento farmacológico , Autofagia/efeitos dos fármacos , Cartilagem/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Naftiridinas/farmacologia , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Artrite Experimental/metabolismo , Artrite Experimental/patologia , Proteína Beclina-1 , Cartilagem/metabolismo , Cartilagem/ultraestrutura , Linhagem Celular Tumoral , Condrócitos/metabolismo , Condrócitos/ultraestrutura , Citoproteção , Humanos , Masculino , Metaloproteinase 13 da Matriz/genética , Metaloproteinase 13 da Matriz/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Coelhos , Índice de Gravidade de Doença , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo
20.
Drug Discov Ther ; 18(2): 134-139, 2024 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-38569833

RESUMO

Both PAK1 (RAC/CDC42-activating kinase 1) and TOR (Target of Rapamycin) are among the major oncogenic/ageing kinases. However, they play the opposite role in our immune system, namely immune system is suppressed by PAK1, while it requires TOR. Thus, PAK1-blockers, would be more effective for therapy of cancers, than TOR-blockers. Since 2015 when we discovered genetically that PDGF-induced melanogenesis depends on "PAK1", we are able to screening a series of PAK1-blockers as melanogenesis-inhibitors which could eventually promote longevity. Interestingly, rapamycin, the first TOR-inhibitor, promotes melanogenesis, clearly indicating that TOR suppresses melanogenesis. However, a new TOR-inhibitor called TORin-1 no longer suppresses immune system, and blocks melanogenesis in cell culture. These observations strongly indicate that TORin-1 acts as PAK1-blockers, instead of TOR-blockers, in vivo. Thus, it is most likely that melanogenesis in cell culture could enable us to discriminate PAK1-blockers from TORblockers.


Assuntos
Mesilato de Imatinib , Pirimidinas , Sirolimo , Serina-Treonina Quinases TOR , Quinases Ativadas por p21 , Quinases Ativadas por p21/metabolismo , Quinases Ativadas por p21/genética , Humanos , Serina-Treonina Quinases TOR/metabolismo , Pirimidinas/farmacologia , Sirolimo/farmacologia , Sirolimo/uso terapêutico , Mesilato de Imatinib/farmacologia , Mesilato de Imatinib/uso terapêutico , Animais , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Melaninas/biossíntese , Melaninas/metabolismo , Inibidores de MTOR/farmacologia , Inibidores de MTOR/uso terapêutico , Naftiridinas
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