RESUMO
Neuronal growth regulator 1 (NEGR1) is a newly identified raft-associated protein, which has recently been spotlighted as a new locus related to human obesity. Niemann-Pick disease Type C2 (NPC2) protein functions as a key player in the intracellular cholesterol trafficking, and its defect is linked to a fatal human neurodegenerative disease, NPC. In this study, we identified that NEGR1 interacts with NPC2 and increases its protein stability. Ectopically expressed NEGR1 proteins relieved an abnormal cholesterol accumulation in endosomal compartments. Importantly, NEGR1-defective mouse embryonic fibroblast cells exhibit increased cholesterol levels and triglyceride contents. These findings provide the first insight into the role of NEGR1 in intracellular cholesterol homeostasis, possibly explaining the missing link between NEGR1 with human obesity.
Assuntos
Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Colesterol/química , Doença de Niemann-Pick Tipo C/metabolismo , Animais , Endossomos/metabolismo , Fibroblastos/metabolismo , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Microdomínios da Membrana , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Doenças Neurodegenerativas/metabolismo , Obesidade/metabolismo , Fases de Leitura Aberta , Triglicerídeos/químicaRESUMO
The CENP-T·CENP-W complex is a recently identified inner centromere component that plays crucial roles in the formation of a functional kinetochore involved in cell division during mitosis. Using yeast two-hybrid screening, we identified an interaction between CENP-T and CSN5, the fifth component of the COP9 signalosome and a key modulator of the cell cycle and cancer. Co-immunoprecipitation revealed that CSN5 directly interacts with both CENP-T and CENP-W. Ectopically expressed CSN5 promoted the ubiquitin- and proteasome-dependent degradation of CENP-T·CENP-W. The formation of a CENP-T·CENP-W complex greatly enhanced the stabilities of the respective proteins, possibly by blocking CSN5-mediated degradation. Furthermore, dysregulation of CSN5 induced severe defects in the recruitment of CENP-T·CENP-W to the kinetochore during the prophase stage of mitosis. Thus, our results indicate that CSN5 regulates the stability of the inner kinetochore components CENP-T and CENP-W, providing the first direct link between CSN5 and the mitotic apparatus, highlighting the role of CSN5 as a multifunctional cell cycle regulator.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeo Hidrolases/metabolismo , Prófase/fisiologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Complexo do Signalossomo COP9 , Proteínas Cromossômicas não Histona/genética , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Cinetocoros/metabolismo , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Peptídeo Hidrolases/genética , Complexo de Endopeptidases do Proteassoma/genética , Ubiquitina/genética , Ubiquitina/metabolismoRESUMO
Epigenetic dysregulation characterized by aberrant DNA hypermethylation is a hallmark of cancer, and it can be targeted by hypomethylating agents (HMAs). Recently, we described the superior therapeutic efficacy of a novel HMA, namely, NTX-301, when used as a monotherapy and in combination with venetoclax in the treatment of acute myeloid leukemia. Following a previous study, we further explored the therapeutic properties of NTX-301 based on experimental investigations and integrative data analyses. Comprehensive sensitivity profiling revealed that NTX-301 primarily exerted anticancer effects against blood cancers and exhibited improved potency against a wide range of solid cancers. Subsequent assays showed that the superior efficacy of NTX-301 depended on its strong effects on cell cycle arrest, apoptosis, and differentiation. Due to its superior efficacy, low doses of NTX-301 achieved sufficiently substantial tumor regression in vivo. Multiomics analyses revealed the mechanisms of action (MoAs) of NTX-301 and linked these MoAs to markers of sensitivity to NTX-301 and to the demethylation activity of NTX-301 with high concordance. In conclusion, our findings provide a rationale for currently ongoing clinical trials of NTX-301 and will help guide the development of novel therapeutic options for cancer patients.
RESUMO
CENP-W was originally identified as a putative oncogene, cancer-upregulated gene 2 (CUG2) that was commonly up-regulated in many cancer tissues. Recently, CENP-W has also been identified as a new centromeric component that interacts with CENP-T. As a complex with CENP-T, CENP-W plays crucial roles in assembly of the functional kinetochore complex. In this study, the subnuclear localization of CENP-W was extensively analyzed using various approaches. We found that ectopically expressed CENP-W primarily accumulated in the nucleolus and remained substantially associated with the nucleolus in stable cells. The following fractionation study also showed that CENP-W is associated with RNA as well as DNA. Moreover, a considerable amount of CENP-W was found in the nuclear mesh-like structure, nuclear matrix, possibly indicating that CENP-W participates in diverse subnuclear activities. Finally, biochemical affinity binding analysis revealed that CENP-W specifically interacts with the nucleolar phosphoprotein, nucleophosmin (B23). Depletion of cellular B23 by siRNA treatment induced a dramatic decrease of CENP-W stability and severe mislocalization during prophase. Our data proposed that B23 may function in the assembly of the kinetochore complex by interacting with CENP-W during interphase.
Assuntos
Centrômero/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Associadas à Matriz Nuclear/metabolismo , RNA/química , Linhagem Celular Tumoral , Nucléolo Celular/metabolismo , Núcleo Celular/metabolismo , Células HeLa , Humanos , Cinetocoros/metabolismo , Microscopia de Fluorescência/métodos , Proteínas Nucleares/metabolismo , Nucleofosmina , Estrutura Terciária de Proteína , RNA/metabolismo , RNA Interferente Pequeno/metabolismo , Frações Subcelulares/metabolismoAssuntos
Compostos Bicíclicos Heterocíclicos com Pontes , Leucemia Mieloide Aguda , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêuticoRESUMO
BACKGROUND: Recent studies have shown that heterogeneous nuclear ribonucleoprotein U (hnRNP U), a component of the hnRNP complex, contributes to stabilize the kinetochore-microtubule interaction during mitosis. CENP-W was identified as an inner centromere component that plays crucial roles in the formation of a functional kinetochore complex. RESULTS: We report that hnRNP U interacts with CENP-W, and the interaction between hnRNP U and CENP-W mutually increased each other's protein stability by inhibiting the proteasome-mediated degradation. Further, their co-localization was observed chiefly in the nuclear matrix region and at the microtubule-kinetochore interface during interphase and mitosis, respectively. Both microtubule-stabilizing and microtubule-destabilizing agents significantly decreased the protein stability of CENP-W. Furthermore, loss of microtubules and defects in microtubule organization were observed in CENP-W-depleted cells. CONCLUSION: Our data imply that CENP-W plays an important role in the attachment and interaction between microtubules and kinetochore during mitosis.