RESUMO
DNA mismatch repair (MMR) is a highly conserved pathway that corrects both base-base mispairs and insertion-deletion loops (IDLs) generated during DNA replication. Defects in MMR have been linked to carcinogenesis and drug resistance. However, the regulation of MMR is poorly understood. Interestingly, CNOT6 is one of four deadenylase subunits in the conserved CCR4-NOT complex and it targets poly(A) tails of mRNAs for degradation. CNOT6 is overexpressed in acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML) and androgen-independent prostate cancer cells, which suggests that an altered expression of CNOT6 may play a role in tumorigenesis. Here, we report that a depletion of CNOT6 sensitizes human U2OS cells to N-methyl-N'nitro-N-nitrosoguanidine (MNNG) and leads to enhanced apoptosis. We also demonstrate that the depletion of CNOT6 upregulates MMR and decreases the mutation frequency in MMR-proficient cells. Furthermore, the depletion of CNOT6 increases the stability of mRNA transcripts from MMR genes, leading to the increased expression of MMR proteins. Our work provides insight into a novel CNOT6-dependent mechanism for regulating MMR.
Assuntos
Reparo de Erro de Pareamento de DNA , Replicação do DNA , Apoptose/genética , Reparo de Erro de Pareamento de DNA/genética , Humanos , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Cellular senescence is an important factor in aging and many age-related diseases, but understanding its role in health is challenging due to the lack of exclusive or universal markers. Using neural networks, we predict senescence from the nuclear morphology of human fibroblasts with up to 95% accuracy, and investigate murine astrocytes, murine neurons, and fibroblasts with premature aging in culture. After generalizing our approach, the predictor recognizes higher rates of senescence in p21-positive and ethynyl-2'-deoxyuridine (EdU)-negative nuclei in tissues and shows an increasing rate of senescent cells with age in H&E-stained murine liver tissue and human dermal biopsies. Evaluating medical records reveals that higher rates of senescent cells correspond to decreased rates of malignant neoplasms and increased rates of osteoporosis, osteoarthritis, hypertension and cerebral infarction. In sum, we show that morphological alterations of the nucleus can serve as a deep learning predictor of senescence that is applicable across tissues and species and is associated with health outcomes in humans.
Assuntos
Senilidade Prematura , Aprendizado Profundo , Humanos , Camundongos , Animais , Senescência Celular/fisiologia , Envelhecimento , BiomarcadoresRESUMO
Human exonuclease 1 (EXO1), a 5'â3' exonuclease, contributes to the regulation of the cell cycle checkpoints, replication fork maintenance, and post replicative DNA repair pathways. These processes are required for the resolution of stalled or blocked DNA replication that can lead to replication stress and potential collapse of the replication fork. Failure to restart the DNA replication process can result in double-strand breaks, cell-cycle arrest, cell death, or cellular transformation. In this review, we summarize the involvement of EXO1 in the replication, DNA repair pathways, cell cycle checkpoints, and the link between EXO1 and cancer.
Assuntos
Enzimas Reparadoras do DNA/metabolismo , Replicação do DNA , Exodesoxirribonucleases/metabolismo , Apoptose , Pontos de Checagem do Ciclo Celular , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Recombinação Homóloga , Humanos , Neoplasias/metabolismo , Neoplasias/patologiaRESUMO
Autophagy is controlled by AMPK and mTOR, both of which associate with ULK1 and control the production of phosphatidylinositol 3-phosphate (PtdIns3P), a prerequisite for autophagosome formation. Here we report that WIPI3 and WIPI4 scaffold the signal control of autophagy upstream of PtdIns3P production and have a role in the PtdIns3P effector function of WIPI1-WIPI2 at nascent autophagosomes. In response to LKB1-mediated AMPK stimulation, WIPI4-ATG2 is released from a WIPI4-ATG2/AMPK-ULK1 complex and translocates to nascent autophagosomes, controlling their size, to which WIPI3, in complex with FIP200, also contributes. Upstream, WIPI3 associates with AMPK-activated TSC complex at lysosomes, regulating mTOR. Our WIPI interactome analysis reveals the scaffold functions of WIPI proteins interconnecting autophagy signal control and autophagosome formation. Our functional kinase screen uncovers a novel regulatory link between LKB1-mediated AMPK stimulation that produces a direct signal via WIPI4, and we show that the AMPK-related kinases NUAK2 and BRSK2 regulate autophagy through WIPI4.
Assuntos
Autofagia , Proteínas de Transporte/química , Proteínas Serina-Treonina Quinases/química , Transdução de Sinais , Quinases Proteína-Quinases Ativadas por AMP , Proteínas Quinases Ativadas por AMP/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/química , Proteínas Relacionadas à Autofagia/química , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Lisossomos/metabolismo , Fagossomos/metabolismo , Fosfatos de Fosfatidilinositol/química , Ligação Proteica , Conformação Proteica , Proteínas de Transporte Vesicular/químicaRESUMO
Autophagy is a lysosomal bulk degradation pathway for cytoplasmic cargo, such as long-lived proteins, lipids, and organelles. Induced upon nutrient starvation, autophagic degradation is accomplished by the concerted actions of autophagy-related (ATG) proteins. Here we demonstrate that two ATGs, human Atg2A and Atg14L, colocalize at cytoplasmic lipid droplets (LDs) and are functionally involved in controlling the number and size of LDs in human tumor cell lines. We show that Atg2A is targeted to cytoplasmic ADRP-positive LDs that migrate bidirectionally along microtubules. The LD localization of Atg2A was found to be independent of the autophagic status. Further, Atg2A colocalized with Atg14L under nutrient-rich conditions when autophagy was not induced. Upon nutrient starvation and dependent on phosphatidylinositol 3-phosphate [PtdIns(3)P] generation, both Atg2A and Atg14L were also specifically targeted to endoplasmic reticulum-associated early autophagosomal membranes, marked by the PtdIns(3)P effectors double-FYVE containing protein 1 (DFCP1) and WD-repeat protein interacting with phosphoinositides 1 (WIPI-1), both of which function at the onset of autophagy. These data provide evidence for additional roles of Atg2A and Atg14L in the formation of early autophagosomal membranes and also in lipid metabolism.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Autofagossomos/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Membrana Celular/metabolismo , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Relacionadas à Autofagia/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Membrana Celular/genética , Células HeLa , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Fosfatos de Fosfatidilinositol/genética , Fosfatos de Fosfatidilinositol/metabolismoRESUMO
Autophagy is a catabolic pathway in which the cell sequesters cytoplasmic material, including long-lived proteins, lipids and organelles, in specialized double-membrane vesicles, called autophagosomes. Subsequently, autophagosomes communicate with the lysosomal compartment and acquire acidic hydrolases for final cargo degradation. This process of partial self-eating secures the survival of eukaryotic cells during starvation periods and is critically regulated by mTORC1 (mammalian target of rapamycin complex 1). Under nutrient-poor conditions, inhibited mTORC1 permits localized PtdIns(3)P production at particular membranes that contribute to autophagosome formation. Members of the human WIPI (WD-repeat protein interacting with phosphoinositides) family fulfil an essential role as PtdIns(3)P effectors at the initiation step of autophagosome formation. In the present article, we discuss the role of human WIPIs in autophagy, and the identification of evolutionarily conserved amino acids of WIPI-1 that confer PtdIns(3)P binding downstream of mTORC1 inhibition. We also discuss the PtdIns(3)P effector function of WIPIs in the context of longevity and autophagy-related human diseases, such as cancer and neurodegeneration.
Assuntos
Autofagia , Longevidade , Proteínas de Membrana/fisiologia , Sequência de Aminoácidos , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Complexos Multiproteicos/fisiologia , Fosfatidilinositóis/metabolismo , Ligação Proteica , Homologia de Sequência de Aminoácidos , Serina-Treonina Quinases TOR/fisiologiaRESUMO
Autophagy is a conserved degradative transport pathway. It is characterized by the formation of double-membrane autophagosomes at the phagophore assembly site (PAS). Atg18 is essential for autophagy but also for vacuole homeostasis and probably endosomal functions. This protein is basically a ß-propeller, formed by seven WD40 repeats, that contains a conserved FRRG motif that binds to phosphoinositides and promotes Atg18 recruitment to the PAS, endosomes and vacuoles. However, it is unknown how Atg18 association with these organelles is regulated, as the phosphoinositides bound by this protein are present on the surface of all of them. We have investigated Atg18 recruitment to the PAS and found that Atg18 binds to Atg2 through a specific stretch of amino acids in the ß-propeller on the opposite surface to the FRRG motif. As in the absence of the FRRG sequence, the inability of Atg18 to interact with Atg2 impairs its association with the PAS, causing an autophagy block. Our data provide a model whereby the Atg18 ß-propeller provides organelle specificity by binding to two determinants on the target membrane.
Assuntos
Autofagia/fisiologia , Proteínas de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Proteínas Relacionadas à Autofagia , Sítios de Ligação , Proteínas de Membrana/genética , Modelos Moleculares , Dados de Sequência Molecular , Fagossomos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Relação Estrutura-AtividadeRESUMO
Gossypol, a natural Bcl-2 homology domain 3 mimetic compound isolated from cottonseeds, is currently being evaluated in clinical trials. Here, we provide evidence that gossypol induces autophagy followed by apoptotic cell death in both the MCF-7 human breast adenocarcinoma and HeLa cell lines. We first show that knockdown of the Bcl-2 homology domain 3-only protein Beclin 1 reduces gossypol-induced autophagy in MCF-7 cells, but not in HeLa cells. Gossypol inhibits the interaction between Beclin 1 and Bcl-2 (B-cell leukemia/lymphoma 2), antagonizes the inhibition of autophagy by Bcl-2, and hence stimulates autophagy. We then show that knockdown of Vps34 reduces gossypol-induced autophagy in both cell lines, and consistent with this, the phosphatidylinositol 3-phosphate-binding protein WIPI-1 is recruited to autophagosomal membranes. Further, Atg5 knockdown also reduces gossypol-mediated autophagy. We conclude that gossypol induces autophagy in both a canonical and a noncanonical manner. Notably, we found that gossypol-mediated apoptotic cell death was potentiated by treatment with the autophagy inhibitor wortmannin or with small interfering RNA against essential autophagy genes (Vps34, Beclin 1, and Atg5). Our findings support the notion that gossypol-induced autophagy is cytoprotective and not part of the cell death process induced by this compound.