Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 24
Filtrar
1.
Cell ; 166(4): 950-962, 2016 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-27518565

RESUMO

Posttranslational modifications (PTMs) of tubulin specify microtubules for specialized cellular functions and comprise what is termed a "tubulin code." PTMs of histones comprise an analogous "histone code," although the "readers, writers, and erasers" of the cytoskeleton and epigenome have heretofore been distinct. We show that methylation is a PTM of dynamic microtubules and that the histone methyltransferase SET-domain-containing 2 (SETD2), which is responsible for H3 lysine 36 trimethylation (H3K36me3) of histones, also methylates α-tubulin at lysine 40, the same lysine that is marked by acetylation on microtubules. Methylation of microtubules occurs during mitosis and cytokinesis and can be ablated by SETD2 deletion, which causes mitotic spindle and cytokinesis defects, micronuclei, and polyploidy. These data now identify SETD2 as a dual-function methyltransferase for both chromatin and the cytoskeleton and show a requirement for methylation in maintenance of genomic stability and the integrity of both the tubulin and histone codes.


Assuntos
Montagem e Desmontagem da Cromatina , Citoesqueleto/metabolismo , Código das Histonas , Histona-Lisina N-Metiltransferase/metabolismo , Linhagem Celular Tumoral , Citocinese , Instabilidade Genômica , Histona-Lisina N-Metiltransferase/genética , Histonas/metabolismo , Humanos , Lisina/metabolismo , Metilação , Microtúbulos/metabolismo , Mitose , Processamento de Proteína Pós-Traducional , Tubulina (Proteína)/metabolismo
2.
Physiol Rev ; 98(1): 89-115, 2018 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-29167332

RESUMO

Peroxisomes are highly dynamic intracellular organelles involved in a variety of metabolic functions essential for the metabolism of long-chain fatty acids, d-amino acids, and many polyamines. A byproduct of peroxisomal metabolism is the generation, and subsequent detoxification, of reactive oxygen and nitrogen species, particularly hydrogen peroxide (H2O2). Because of its relatively low reactivity (as a mild oxidant), H2O2 has a comparatively long intracellular half-life and a high diffusion rate, all of which makes H2O2 an efficient signaling molecule. Peroxisomes also have intricate connections to mitochondria, and both organelles appear to play important roles in regulating redox signaling pathways. Peroxisomal proteins are also subject to oxidative modification and inactivation by the reactive oxygen and nitrogen species they generate, but the peroxisomal LonP2 protease can selectively remove such oxidatively damaged proteins, thus prolonging the useful lifespan of the organelle. Peroxisomal homeostasis must adapt to the metabolic state of the cell, by a combination of peroxisome proliferation, the removal of excess or badly damaged organelles by autophagy (pexophagy), as well as by processes of peroxisome inheritance and motility. More recently the tumor suppressors ataxia telangiectasia mutate (ATM) and tuberous sclerosis complex (TSC), which regulate mTORC1 signaling, have been found to regulate pexophagy in response to variable levels of certain reactive oxygen and nitrogen species. It is now clear that any significant loss of peroxisome homeostasis can have devastating physiological consequences. Peroxisome dysregulation has been implicated in several metabolic diseases, and increasing evidence highlights the important role of diminished peroxisomal functions in aging processes.


Assuntos
Homeostase/fisiologia , Mitocôndrias/metabolismo , Peroxissomos/metabolismo , Proteostase/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Homeostase/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/farmacologia , Peroxissomos/efeitos dos fármacos , Proteostase/efeitos dos fármacos
3.
Brain ; 144(8): 2527-2540, 2021 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-34014281

RESUMO

Gene discovery efforts in autism spectrum disorder have identified heterozygous defects in chromatin remodeller genes, the 'readers, writers and erasers' of methyl marks on chromatin, as major contributors to this disease. Despite this advance, a convergent aetiology between these defects and aberrant chromatin architecture or gene expression has remained elusive. Recently, data have begun to emerge that chromatin remodellers also function directly on the cytoskeleton. Strongly associated with autism spectrum disorder, the SETD2 histone methyltransferase for example, has now been shown to directly methylate microtubules of the mitotic spindle. However, whether microtubule methylation occurs in post-mitotic cells, for example on the neuronal cytoskeleton, is not known. We found the SETD2 α-tubulin lysine 40 trimethyl mark occurs on microtubules in the brain and in primary neurons in culture, and that the SETD2 C-terminal SRI domain is required for binding and methylation of α-tubulin. A CRISPR knock-in of a pathogenic SRI domain mutation (Setd2SRI) that disables microtubule methylation revealed at least one wild-type allele was required in mice for survival, and while viable, heterozygous Setd2SRI/wtmice exhibited an anxiety-like phenotype. Finally, whereas RNA-sequencing (RNA-seq) and chromatin immunoprecipitation-sequencing (ChIP-seq) showed no concomitant changes in chromatin methylation or gene expression in Setd2SRI/wtmice, primary neurons exhibited structural deficits in axon length and dendritic arborization. These data provide the first demonstration that microtubules of neurons are methylated, and reveals a heterozygous chromatin remodeller defect that specifically disables microtubule methylation is sufficient to drive an autism-associated phenotype.


Assuntos
Ansiedade/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Microtúbulos/metabolismo , Neurônios/metabolismo , Animais , Encéfalo/metabolismo , Histonas/metabolismo , Metilação , Camundongos , Fenótipo
4.
Biochem Biophys Res Commun ; 558: 202-208, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-33036756

RESUMO

The process of autophagy is dysregulated in many cancers including clear cell renal cell carcinoma (ccRCC). Autophagy involves the coordination of numerous autophagy-related (ATG) genes, as well as processes involving the actin cytoskeleton. The histone methyltransferase SETD2, frequently inactivated in ccRCC, has recently been shown to also methylate cytoskeletal proteins, which in the case of actin lysine 68 trimethylation (ActK68me3) regulates actin polymerization dynamics. Here we show that cells lacking SETD2 exhibit autophagy defects, as well as decreased interaction of the actin nucleation promoting factor WHAMM with its target actin, which is required for initiation of autophagy. Interestingly, the WHAMM actin binding deficit could be rescued with pharmacologic induction of actin polymerization in SETD2-null cells using Jasplakinolide. These data indicate that the decreased interaction between WHAMM and its target actin in SETD2-null cells was secondary to altered actin dynamics rather than loss of the SETD2 ActK68me3 mark itself, and underscores the importance of the functional defect in actin polymerization in SETD2-null cells exhibiting autophagy defects.


Assuntos
Actinas/metabolismo , Carcinoma de Células Renais/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Neoplasias Renais/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Autofagia/genética , Autofagia/fisiologia , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Linhagem Celular , Linhagem Celular Tumoral , Regulação para Baixo , Técnicas de Inativação de Genes , Histona-Lisina N-Metiltransferase/deficiência , Histona-Lisina N-Metiltransferase/genética , Humanos , Neoplasias Renais/genética , Neoplasias Renais/patologia
5.
J Cell Sci ; 131(24)2018 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-30518623

RESUMO

Loss of the gene von Hippel-Lindau (VHL) is associated with loss of primary cilia and is causally linked to elevated levels of Aurora kinase A (AURKA). We developed an image-based high-throughput screening (HTS) assay using a dual-labeling image analysis strategy that identifies both the cilium and the basal body. By using this strategy, we screened small-molecule compounds for the targeted rescue of cilia defects associated with VHL deficiency with high accuracy and reproducibility. Bexarotene was identified and validated as a positive regulator of the primary cilium. Importantly, the inability of an alternative retinoid X receptor (RXR) agonist to rescue ciliogenesis, in contrast to bexarotene, suggested that multiple bexarotene-driven mechanisms were responsible for the rescue. We found that bexarotene decreased AURKA expression in VHL-deficient cells, thereby restoring the ability of these cells to ciliate in the absence of VHL Finally, bexarotene treatment reduced the propensity of subcutaneous lesions to develop into tumors in a mouse xenograft model of renal cell carcinoma (RCC), with a concomitant decrease in activated AURKA, highlighting the potential of bexarotene treatment as an intervention strategy in the clinic to manage renal cystogenesis associated with VHL deficiency and elevated AURKA expression.


Assuntos
Aurora Quinase A/metabolismo , Bexaroteno/farmacologia , Carcinoma de Células Renais/tratamento farmacológico , Aurora Quinase A/genética , Linhagem Celular Tumoral , Cílios/efeitos dos fármacos , Cílios/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Mutação/efeitos dos fármacos , Mutação/genética , Proteína Supressora de Tumor Von Hippel-Lindau/efeitos dos fármacos , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo
6.
Sci Rep ; 11(1): 10461, 2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-34002003

RESUMO

Loss of primary cilia in cells deficient for the tumor suppressor von Hippel Lindau (VHL) arise from elevated Aurora Kinase A (AURKA) levels. VHL in its role as an E3 ubiquitin ligase targets AURKA for degradation and in the absence of VHL, high levels of AURKA result in destabilization of the primary cilium. We identified NVP-BEZ235, a dual PI3K/AKT and mTOR inhibitor, in an image-based high throughput screen, as a small molecule that restored primary cilia in VHL-deficient cells. We identified the ability of AKT to modulate AURKA expression at the transcript and protein level. Independent modulation of AKT and mTOR signaling decreased AURKA expression in cells confirming AURKA as a new signaling node downstream of the PI3K cascade. Corroborating these data, a genetic knockdown of AKT in cells deficient for VHL rescued the ability of these cells to ciliate. Finally, inhibition of AKT/mTOR using NVP-BEZ235 was efficacious in reducing tumor burden in a 786-0 xenograft model of renal cell carcinoma. These data highlight a previously unappreciated signaling node downstream of the AKT/mTOR pathway via AURKA that can be targeted in VHL-null cells to restore ciliogenesis.


Assuntos
Aurora Quinase A/metabolismo , Carcinoma de Células Renais/tratamento farmacológico , Cílios/efeitos dos fármacos , Imidazóis/farmacologia , Neoplasias Renais/tratamento farmacológico , Quinolinas/farmacologia , Doença de von Hippel-Lindau/tratamento farmacológico , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Cílios/patologia , Técnicas de Silenciamento de Genes , Humanos , Imidazóis/uso terapêutico , Neoplasias Renais/genética , Neoplasias Renais/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Quinolinas/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Carga Tumoral/efeitos dos fármacos , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Doença de von Hippel-Lindau/complicações , Doença de von Hippel-Lindau/genética , Doença de von Hippel-Lindau/patologia
7.
Sci Adv ; 6(40)2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33008892

RESUMO

The methyltransferase SET domain-containing 2 (SETD2) was originally identified as Huntingtin (HTT) yeast partner B. However, a SETD2 function associated with the HTT scaffolding protein has not been elucidated, and no linkage between HTT and methylation has yet been uncovered. Here, we show that SETD2 is an actin methyltransferase that trimethylates lysine-68 (ActK68me3) in cells via its interaction with HTT and the actin-binding adapter HIP1R. ActK68me3 localizes primarily to the insoluble F-actin cytoskeleton in cells and regulates actin polymerization/depolymerization dynamics. Disruption of the SETD2-HTT-HIP1R axis inhibits actin methylation, causes defects in actin polymerization, and impairs cell migration. Together, these data identify SETD2 as a previously unknown HTT effector regulating methylation and polymerization of actin filaments and provide new avenues for understanding how defects in SETD2 and HTT drive disease via aberrant cytoskeletal methylation.


Assuntos
Actinas , Proteínas de Ligação ao GTP/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Lisina , Actinas/metabolismo , Citoesqueleto/metabolismo , Lisina/metabolismo , Metilação , Processamento de Proteína Pós-Traducional
8.
In Vivo ; 23(2): 303-7, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19414419

RESUMO

BACKGROUND: Recent reports regarding acetylsalicylic acid (ASA) and its metabolites suggest suppressive effects against mitomycin C (MMC)-induced genotoxicity in a mice chromosomal aberration assay. Keeping this in mind, the potential anti-genotoxic effect of the thio-analogue of salicylic acid namely thio-salicylic acid (TSA) was speculated upon. The present study investigated and compared the anti-genotoxic potential of ASA and TSA. MATERIALS AND METHODS: The study was performed in male swiss mice (20+/-2 g) using single-cell gel electrophoresis and a peripheral blood micronucleus assay. ASA and TSA (5, 10 or 20 mg/kg) were administered 15 minutes after MMC (1 mg/kg) once daily for 3 or 7 days. RESULTS: Both ASA and TSA significantly decreased the DNA damage induced by MMC as indicated by a decrease in the comet parameters in bone marrow cells and decreased frequencies of micronucleated reticulocytes in peripheral blood. CONCLUSION: The results clearly demonstrate the anti-genotoxic potential of ASA and TSA.


Assuntos
Mitomicina/farmacologia , Ácido Salicílico/farmacologia , Animais , Células da Medula Óssea , Ensaio Cometa , Dano ao DNA , Masculino , Camundongos , Testes para Micronúcleos , Testes de Mutagenicidade , Mutagênicos/toxicidade , Reticulócitos/metabolismo , Ácido Salicílico/química , Fatores de Tempo
9.
Cancer Res ; 78(12): 3135-3146, 2018 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-29724720

RESUMO

Loss of the short arm of chromosome 3 (3p) occurs early in >95% of clear cell renal cell carcinoma (ccRCC). Nearly ubiquitous 3p loss in ccRCC suggests haploinsufficiency for 3p tumor suppressors as early drivers of tumorigenesis. We previously reported methyltransferase SETD2, which trimethylates H3 histones on lysine 36 (H3K36me3) and is located in the 3p deletion, to also trimethylate microtubules on lysine 40 (αTubK40me3) during mitosis, with αTubK40me3 required for genomic stability. We now show that monoallelic, Setd2-deficient cells retaining H3K36me3, but not αTubK40me3, exhibit a dramatic increase in mitotic defects and micronuclei count, with increased viability compared with biallelic loss. In SETD2-inactivated human kidney cells, rescue with a pathogenic SETD2 mutant deficient for microtubule (αTubK40me3), but not histone (H3K36me3) methylation, replicated this phenotype. Genomic instability (micronuclei) was also a hallmark of patient-derived cells from ccRCC. These data show that the SETD2 tumor suppressor displays a haploinsufficiency phenotype disproportionately impacting microtubule methylation and serves as an early driver of genomic instability.Significance: Loss of a single allele of a chromatin modifier plays a role in promoting oncogenesis, underscoring the growing relevance of tumor suppressor haploinsufficiency in tumorigenesis. Cancer Res; 78(12); 3135-46. ©2018 AACR.


Assuntos
Carcinoma de Células Renais/genética , Cromossomos Humanos Par 3/genética , Histona-Lisina N-Metiltransferase/genética , Neoplasias Renais/genética , Microtúbulos/metabolismo , Animais , Carcinogênese/genética , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Fibroblastos , Técnicas de Silenciamento de Genes , Instabilidade Genômica , Haploinsuficiência , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Humanos , Neoplasias Renais/patologia , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/patologia , Lisina/metabolismo , Metilação , Camundongos , Micronúcleos com Defeito Cromossômico
10.
FEBS Lett ; 581(5): 1071-8, 2007 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-17316625

RESUMO

Diabetic nephropathy (DN) is one of the main causes of end stage renal disease (ESRD) and a leading cause of diabetes mellitus related morbidity and mortality. Recently, sirtuin are reported to have emerging pathogenetic roles in cancer, muscle differentiation, heart failure, neurodegeneration, diabetes and aging. The aim of the present study was to study the role of intermittent fasting (IF) on DN and studying the expression of Sir2 and p53. At biochemical level, we found that IF causes significant improvement in blood urea nitrogen (BUN), creatinine, albumin and HDL cholesterol, parameters that are associated with the development of DN. Diabetic rats on IF also show significant improvement in onset of hypertension. Interestingly, the expression of Sir2, a NAD dependent histone deacetylase, decreases in diabetic rat kidney and this decrease is overcome by IF. Moreover, we provide evidence for involvement of mitogen activated protein kinases (MAPK) cascade in mediating the effects of IF as there is reduction in the expression of p38 which gets induced under diabetic condition. This was further accompanied by the concomitant decrease in cleavage of caspase3 and p53 expression. These findings suggest that IF significantly improves biochemical parameters associated with development of DN and changes the expression of Sir2 and p53.


Assuntos
Nefropatias Diabéticas/prevenção & controle , Jejum/fisiologia , Sirtuínas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Glicemia/metabolismo , Pressão Sanguínea , Nitrogênio da Ureia Sanguínea , Peso Corporal , HDL-Colesterol/sangue , Creatinina/sangue , Nefropatias Diabéticas/metabolismo , Ingestão de Alimentos , Histonas/química , Histonas/metabolismo , Peroxidação de Lipídeos , Sistema de Sinalização das MAP Quinases , Masculino , Modelos Biológicos , Fosforilação , Ratos , Ratos Sprague-Dawley , Albumina Sérica/metabolismo , Superóxido Dismutase/metabolismo
11.
Curr Opin Cell Biol ; 39: 109-12, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26967755

RESUMO

Peroxisomes participate in lipid metabolism, and are a major source of ROS in the cell. Their importance in cellular energy balance and redox homeostasis is well-established, as is the need to maintain peroxisome homeostasis to prevent pathologies associated with too few, or too many, of these organelles. How cells regulate peroxisome number has remained somewhat elusive. Recently, the tumor suppressors ATM and TSC, which regulate mTORC1 signaling, have been localized to peroxisomes. When activated by peroxisomal ROS, ATM signals to TSC to repress mTORC1 signaling and increase autophagic flux in cells, and also phosphorylates the peroxisomal protein PEX 5 to target peroxisomes for selective autophagy (pexophagy), providing a mechanism for regulation of peroxisomal homeostasis using ROS as a rheostat.


Assuntos
Organelas/metabolismo , Peroxissomos/metabolismo , Transdução de Sinais , Animais , Autofagia , Homeostase , Humanos
12.
Autophagy ; 12(4): 711-2, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27050462

RESUMO

Peroxisomes are autonomously replicating and highly metabolic organelles necessary for ß-oxidation of fatty acids, a process that generates large amounts of reactive oxygen species (ROS). Maintaining a balance between biogenesis and degradation of peroxisomes is essential to maintain cellular redox balance, but how cells do this has remained somewhat of a mystery. While it is known that peroxisomes can be degraded via selective autophagy (pexophagy), little is known about how mammalian cells regulate pexophagy to maintain peroxisome homeostasis. We have uncovered a mechanism for regulating pexophagy in mammalian cells that defines a new role for ATM (ATM serine/threonine kinase) kinase as a "first responder" to peroxisomal ROS. ATM is delivered to the peroxisome by the PEX5 import receptor, which recognizes an SRL sequence located at the C terminus of ATM to localize this kinase to peroxisomes. In response to ROS, the ATM kinase is activated and performs 2 functions: i) it signals to AMPK, which activates TSC2 to suppresses MTORC1 and phosphorylates ULK1 to induce autophagy, and ii) targets specific peroxisomes for pexophagy by phosphorylating PEX5 at Ser141, which triggers ubiquitination of PEX5 at Lys209 and binding of the autophagy receptor protein SQSTM1/p62 to induce pexophagy.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Autofagia , Peroxissomos/metabolismo , Animais , Humanos , Mamíferos/metabolismo , Modelos Biológicos , Espécies Reativas de Oxigênio/metabolismo
13.
Curr Neuropharmacol ; 14(6): 627-40, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26725888

RESUMO

The mechanisms underlying neurodegenerative disorders are complex and multifactorial; however, accumulating evidences suggest few common shared pathways. These common pathways include mitochondrial dysfunction, intracellular Ca2+ overload, oxidative stress and inflammation. Often multiple pathways co-exist, and therefore limit the benefits of therapeutic interventions. Nutraceuticals have recently gained importance owing to their multifaceted effects. These food-based approaches are believed to target multiple pathways in a slow but more physiological manner without causing severe adverse effects. Available information strongly supports the notion that apart from preventing the onset of neuronal damage, nutraceuticals can potentially attenuate the continued progression of neuronal destruction. In this article, we i) review the common pathways involved in the pathogenesis of the toxicants-induced neurotoxicity and neurodegenerative disorders with special emphasis on Alzheimer`s disease (AD), Parkinson`s disease (PD), Huntington`s disease (HD), Multiple sclerosis (MS) and Amyotrophic lateral sclerosis (ALS), and ii) summarize current research advancements on the effects of nutraceuticals against these detrimental pathways.


Assuntos
Suplementos Nutricionais , Doenças Neurodegenerativas/dietoterapia , Doenças Neurodegenerativas/metabolismo , Animais , Humanos
14.
MAbs ; 8(8): 1590-1597, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27594515

RESUMO

Posttranslational modifications (PTMs) on microtubules differentiate these cytoskeletal elements for a variety of cellular functions. We recently identified SETD2 as a dual-function histone and microtubule methyltransferase, and methylation as a new microtubule PTM that occurs on lysine 40 of α-tubulin, which is trimethylated (α-TubK40me3) by SETD2. In the course of these studies, we generated polyclonal (α-TubK40me3 pAb) and monoclonal (α-TubK40me3 mAb) antibodies to a methylated α-tubulin peptide (GQMPSD-Kme3-TIGGGDC). Here, we characterize these antibodies, and the specific mono-, di- or tri-methylated lysine residues they recognize. While both the pAb and mAb antibodies recognized lysines methylated by SETD2 on microtubules and histones, the clone 18 mAb was more specific for methylated microtubules, with little cross-reactivity for methylated histones. The clone 18 mAb recognized specific subsets of microtubules during mitosis and cytokinesis, and lacked the chromatin staining seen by immunocytochemistry with the pAb. Western blot analysis using these antibodies revealed that methylated α-tubulin migrated faster than unmethylated α-tubulin, suggesting methylation may be a signal for additional processing of α-tubulin and/or microtubules. As the first reagents that specifically recognize methylated α-tubulin, these antibodies are a valuable tool for studying this new modification of the cytoskeleton, and the function of methylated microtubules.


Assuntos
Anticorpos Monoclonais/imunologia , Lisina/imunologia , Tubulina (Proteína)/química , Tubulina (Proteína)/imunologia , Anticorpos/imunologia , Humanos , Lisina/química , Lisina/metabolismo , Metilação , Microtúbulos/química , Microtúbulos/imunologia , Microtúbulos/metabolismo , Mitose/fisiologia , Processamento de Proteína Pós-Traducional , Tubulina (Proteína)/metabolismo
16.
PLoS One ; 10(10): e0139254, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26440941

RESUMO

MDM2 (mouse double minute 2) inhibitors that activate p53 and induce apoptosis in a non-genotoxic manner are in clinical development for treatment of leukemias. P53 can modulate other programmed cell death pathways including autophagy both transcriptionally and non-transcriptionally. We investigated autophagy induction in acute leukemia by Nutlin 3a, a first-in-class MDM2 inhibitor. Nutlin 3a induced autophagy in a p53 dependent manner and transcriptional activation of AMP kinase (AMPK) is critical, as this effect is abrogated in AMPK -/- mouse embryonic fibroblasts. Nutlin 3a induced autophagy appears to be pro-apoptotic as pharmacological (bafilomycin) or genetic inhibition (BECLIN1 knockdown) of autophagy impairs apoptosis induced by Nutlin 3a.


Assuntos
Adenilato Quinase/metabolismo , Autofagia/efeitos dos fármacos , Imidazóis/farmacologia , Leucemia/metabolismo , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismo , Western Blotting , Linhagem Celular Tumoral , Ativação Enzimática/efeitos dos fármacos , Citometria de Fluxo , Humanos , Lentivirus/genética , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Proteína Supressora de Tumor p53/genética
17.
Nat Cell Biol ; 17(10): 1259-1269, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26344566

RESUMO

Peroxisomes are highly metabolic, autonomously replicating organelles that generate reactive oxygen species (ROS) as a by-product of fatty acid ß-oxidation. Consequently, cells must maintain peroxisome homeostasis, or risk pathologies associated with too few peroxisomes, such as peroxisome biogenesis disorders, or too many peroxisomes, inducing oxidative damage and promoting diseases such as cancer. We report that the PEX5 peroxisome import receptor binds ataxia-telangiectasia mutated (ATM) and localizes this kinase to the peroxisome. In response to ROS, ATM signalling activates ULK1 and inhibits mTORC1 to induce autophagy. Specificity for autophagy of peroxisomes (pexophagy) is provided by ATM phosphorylation of PEX5 at Ser 141, which promotes PEX5 monoubiquitylation at Lys 209, and recognition of ubiquitylated PEX5 by the autophagy adaptor protein p62, directing the autophagosome to peroxisomes to induce pexophagy. These data reveal an important new role for ATM in metabolism as a sensor of ROS that regulates pexophagy.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Autofagia , Peroxissomos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Western Blotting , Células Cultivadas , Células HEK293 , Células Hep G2 , Humanos , Peróxido de Hidrogênio/farmacologia , Células MCF-7 , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos Knockout , Microscopia Eletrônica , Microscopia de Fluorescência , Complexos Multiproteicos/metabolismo , Mutação , Receptor 1 de Sinal de Orientação para Peroxissomos , Peroxissomos/ultraestrutura , Fagossomos/metabolismo , Fagossomos/ultraestrutura , Fosforilação/efeitos dos fármacos , Ligação Proteica , Interferência de RNA , Receptores Citoplasmáticos e Nucleares/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Sequestossoma-1 , Serina/genética , Serina/metabolismo , Serina-Treonina Quinases TOR/metabolismo
18.
Nat Cell Biol ; 15(10): 1186-96, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23955302

RESUMO

Subcellular localization is emerging as an important mechanism for mTORC1 regulation. We report that the tuberous sclerosis complex (TSC) signalling node, TSC1, TSC2 and Rheb, localizes to peroxisomes, where it regulates mTORC1 in response to reactive oxygen species (ROS). TSC1 and TSC2 were bound by peroxisomal biogenesis factors 19 and 5 (PEX19 and PEX5), respectively, and peroxisome-localized TSC functioned as a Rheb GTPase-activating protein (GAP) to suppress mTORC1 and induce autophagy. Naturally occurring pathogenic mutations in TSC2 decreased PEX5 binding, and abrogated peroxisome localization, Rheb GAP activity and suppression of mTORC1 by ROS. Cells lacking peroxisomes were deficient in mTORC1 repression by ROS, and peroxisome-localization-deficient TSC2 mutants caused polarity defects and formation of multiple axons in neurons. These data identify a role for the TSC in responding to ROS at the peroxisome, and identify the peroxisome as a signalling organelle involved in regulation of mTORC1.


Assuntos
Autofagia , Regulação Enzimológica da Expressão Gênica , Complexos Multiproteicos/genética , Peroxissomos/metabolismo , Espécies Reativas de Oxigênio , Transdução de Sinais , Serina-Treonina Quinases TOR/genética , Animais , Linhagem Celular , Células HEK293 , Humanos , Células MCF-7 , Alvo Mecanístico do Complexo 1 de Rapamicina , Proteínas de Membrana/metabolismo , Camundongos , Complexos Multiproteicos/metabolismo , Ligação Proteica , Ratos , Serina-Treonina Quinases TOR/metabolismo , Proteína 1 do Complexo Esclerose Tuberosa , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/metabolismo
20.
Fundam Clin Pharmacol ; 26(3): 383-92, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21352352

RESUMO

Furosemide (FS) is a potent loop diuretic widely used in the management of fluid retention associated with cardiac, renal, and hepatic failure as well as for the treatment of hypertension. FS is a well-characterized and known hepatotoxin in both human and animal test systems. In this study, an attempt has been made to investigate the in vivo genotoxicity of FS at the hepatotoxic equivalent doses using the chromosomal aberration and the comet assay in the bone marrow cells of mice as the endpoints of evaluation. The animals were treated with FS at the doses of 2.5, 5, 10, 20, 40, and 80 mg/kg/body weight (bw) intraperitoneal (ip) for both single (24 h) and repeated dose (seven consecutive days) toxicity studies. FS toxicity in the hepatocytes was evaluated using the parameters, such as, alanine-/aspartate-aminotransferase (ALT/AST), single cell gel electrophoresis (comet), tissue histology, DNA fragmentation, and TUNEL assay as the endpoints. The results clearly demonstrate that FS produced toxic responses in the hepatocytes as evident from increased ALT/AST level, DNA damage, TUNEL positive cells and increased DNA fragmentation in mice in vivo. However, it is interesting that in bone marrow cells, FS did not induced structural chromosomal abberations, but produced mild DNA strand breaks as observed by the comet assay. So it is considered as weak genotoxic toward the bone marrow cells when compared to the hepatocytes of mice.


Assuntos
Células da Medula Óssea/efeitos dos fármacos , Citotoxinas/toxicidade , Dano ao DNA/efeitos dos fármacos , Furosemida/toxicidade , Hepatócitos/efeitos dos fármacos , Animais , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Dano ao DNA/fisiologia , Hepatócitos/metabolismo , Hepatócitos/patologia , Masculino , Camundongos , Distribuição Aleatória
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA