RESUMO
TP53 mutations are prevalent in various cancers, yet the complexity of apoptotic pathway deregulation suggests the involvement of additional factors. HOPS/TMUB1 is known to extend the half-life of p53 under normal and stress conditions, implying a regulatory function. This study investigates, for the first time, the potential modulatory role of the ubiquitin-like-protein HOPS/TMUB1 in p53-mutants. A comprehensive analysis of apoptosis in the most frequent p53-mutants, R175, R248, and R273, in SKBR3, MIA PaCa2, and H1975 cells indicates that the overexpression of HOPS induces apoptosis at least equivalent to that caused by DNA damage. Immunoprecipitation assays confirm HOPS binding to p53-mutant forms. The interaction of HOPS/TMUB1 with p53-mutants strengthens its effect on the apoptotic cascade, showing a context-dependent gain or loss of function. Gene expression analysis of the MYC and TP63 genes shows that H1975 exhibit a gain-of-function profile, while SKBR3 promote apoptosis in a TP63-dependent manner. The TCGA data further corroborate HOPS/TMUB1's positive correlation with apoptotic genes BAX, BBC3, and NOXA1, underscoring its relevance in patient samples. Notably, singular TP53 mutations inadequately explain pathway dysregulation, emphasizing the need to explore additional contributing factors. These findings illuminate the intricate interplay among TP53 mutations, HOPS/TMUB1, and apoptotic pathways, providing valuable insights for targeted cancer interventions.
Assuntos
Apoptose , Mutação , Neoplasias , Proteína Supressora de Tumor p53 , Humanos , Apoptose/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Linhagem Celular Tumoral , Neoplasias/genética , Neoplasias/patologia , Neoplasias/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Fatores de TranscriçãoRESUMO
The physiological importance of the liver is demonstrated by its unique and essential ability to regenerate following extensive injuries affecting its function. By regenerating, the liver reacts to hepatic damage and thus enables homeostasis to be restored. The aim of this review is to add new findings that integrate the regenerative pathway to the current knowledge. An optimal regeneration is achieved through the integration of two main pathways: IL-6/JAK/STAT3, which promotes hepatocyte proliferation, and PI3K/PDK1/Akt, which in turn enhances cell growth. Proliferation and cell growth are events that must be balanced during the three phases of the regenerative process: initiation, proliferation and termination. Achieving the correct liver/body weight ratio is ensured by several pathways as extracellular matrix signalling, apoptosis through caspase-3 activation, and molecules including transforming growth factor-beta, and cyclic adenosine monophosphate. The actors involved in the regenerative process are numerous and many of them are also pivotal players in both the immune and non-immune inflammatory process, that is observed in the early stages of hepatic regeneration. Balance of Th17/Treg is important in liver inflammatory process outcomes. Knowledge of liver regeneration will allow a more detailed characterisation of the molecular mechanisms that are crucial in the interplay between proliferation and inflammation.
Assuntos
Hepatopatias , Regeneração Hepática , Humanos , Regeneração Hepática/fisiologia , Hepatócitos/metabolismo , Fígado/metabolismo , Hepatopatias/metabolismo , Transdução de Sinais , Proliferação de CélulasRESUMO
A cancer outcome is a multifactorial event that comes from both exogenous injuries and an endogenous predisposing background. The healthy state is guaranteed by the fine-tuning of genes controlling cell proliferation, differentiation, and development, whose alteration induces cellular behavioral changes finally leading to cancer. The function of proteins in cells and tissues is controlled at both the transcriptional and translational level, and the mechanism allowing them to carry out their functions is not only a matter of level. A major challenge to the cell is to guarantee that proteins are made, folded, assembled and delivered to function properly, like and even more than other proteins when referring to oncogenes and onco-suppressors products. Over genetic, epigenetic, transcriptional, and translational control, protein synthesis depends on additional steps of regulation. Post-translational modifications are reversible and dynamic processes that allow the cell to rapidly modulate protein amounts and function. Among them, ubiquitination and ubiquitin-like modifications modulate the stability and control the activity of most of the proteins that manage cell cycle, immune responses, apoptosis, and senescence. The crosstalk between ubiquitination and ubiquitin-like modifications and post-translational modifications is a keystone to quickly update the activation state of many proteins responsible for the orchestration of cell metabolism. In this light, the correct activity of post-translational machinery is essential to prevent the development of cancer. Here we summarize the main post-translational modifications engaged in controlling the activity of the principal oncogenes and tumor suppressors genes involved in the development of most human cancers.
Assuntos
Processamento de Proteína Pós-Traducional , Ubiquitina , Humanos , Ubiquitinação , Ubiquitina/metabolismo , Proteínas/metabolismo , HomeostaseRESUMO
Autophagy selectively degrades aggregation-prone misfolded proteins caused by defective cellular proteostasis. However, the complexity of autophagy may prevent the full appreciation of how its modulation could be used as a therapeutic strategy in disease management. Here, we define a molecular pathway through which recombinant IL-1 receptor antagonist (IL-1Ra, anakinra) affects cellular proteostasis independently from the IL-1 receptor (IL-1R1). Anakinra promoted H2O2-driven autophagy through a xenobiotic sensing pathway involving the aryl hydrocarbon receptor that, activated through the indoleamine 2,3-dioxygenase 1-kynurenine pathway, transcriptionally activated NADPH oxidase 4 independent of the IL-1R1. By coupling the mitochondrial redox balance to autophagy, anakinra improved the dysregulated proteostasis network in murine and human cystic fibrosis. We anticipate that anakinra may represent a therapeutic option in addition to its IL-1R1-dependent antiinflammatory properties by acting at the intersection of mitochondrial oxidative stress and autophagy with the capacity to restore conditions in which defective proteostasis leads to human disease.
Assuntos
Autofagia/efeitos dos fármacos , Proteína Antagonista do Receptor de Interleucina 1/farmacologia , Mitocôndrias/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Proteostase/efeitos dos fármacos , Animais , Feminino , Masculino , Camundongos , Camundongos Knockout , Oxirredução/efeitos dos fármacosRESUMO
HOPS is a ubiquitin-like protein implicated in many aspects of cellular function including the regulation of mitotic activity, proliferation, and cellular stress responses. In this study, we focused on the complex relationship between HOPS and the tumor suppressor p53, investigating both transcriptional and non-transcriptional p53 responses. Here, we demonstrated that Hops heterozygous mice and mouse embryonic fibroblasts exhibit an impaired DNA-damage response to etoposide-induced double-strand breaks when compared to wild-type genes. Specifically, alterations in HOPS levels caused significant defects in the induction of apoptosis, including a reduction in p53 protein level and percentage of apoptotic cells. We also analyzed the effect of reduced HOPS levels on the DNA-damage response by examining the transcript profiles of p53-dependent genes, showing a suggestive deregulation of the mRNA levels for a number of p53-dependent genes. Taken together, these results show an interesting haploinsufficiency effect mediated by Hops monoallelic deletion, which appears to be enough to destabilize the p53 protein and its functions. Finally, these data indicate a novel role for Hops as a tumor-suppressor gene in DNA damage repair in mammalian cells.
Assuntos
Apoptose , Reparo do DNA , Haploinsuficiência , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas de Membrana/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Animais , Dano ao DNA , Feminino , Heterozigoto , Masculino , CamundongosRESUMO
Non-small-cell-lung cancer accounts for 80-85% of all forms of lung cancer as leading cause of cancer-related death in human. Despite remarkable advances in the diagnosis and therapy of lung cancer, no significant improvements have thus far been achieved in terms of patients' prognosis. Here, we investigated the role of INSL4 - a member of the relaxin-family - in NSCLC. We overexpressed INSL4 in NSCLC cells to analyse in vitro the growth rate and the tumourigenic features. We investigated the signalling pathways engaged in INSL4 overexpressing cells and the tumour growth ability by studying the tumour development in a patient derived tumour xenograft mouse model. We found an INSL4 cell growth promoting effect in vitro in H1299 cells and in vivo in NOD/SCID mice. Surprisingly, in NSCLC-A549 cells, INSL4 overexpression has not similar effect, despite huge basal INSL4-mRNA expression respect to H1299. The INSL4-mRNA analysis of eight different NSCLC-derived cell lines, revealed highly difference in the INSL4-mRNA amount. Transfection of NSCLC lines with INSL4-Myc showed huge level of INSL4-mRNA with a very low amount of protein expressed. Notably, similar discrepancy has been observed in NSCLC patients. However, in a cohort of NSCLC patients analysing a database, we found a significant inverse correlation between INSL4 expression and Overall Survival. By combining the in vitro and in vivo results, suggest that in patients whose NSCLC adenocarcinoma spontaneously expressed high levels of INSL4 post-transcriptional modifications affecting INSL4 do not allow to assess precision therapy in selected patients without consider protein INSL4 amount.
RESUMO
The circadian clock driven by the daily light-dark and temperature cycles of the environment regulates fundamental physiological processes and perturbations of these sophisticated mechanisms may result in pathological conditions, including cancer. While experimental evidence is building up to unravel the link between circadian rhythms and tumorigenesis, it is becoming increasingly apparent that the response to antitumor agents is similarly dependent on the circadian clock, given the dependence of each drug on the circadian regulation of cell cycle, DNA repair and apoptosis. However, the molecular mechanisms that link the circadian machinery to the action of anticancer treatments is still poorly understood, thus limiting the application of circadian rhythms-driven pharmacological therapy, or chronotherapy, in the clinical practice. Herein, we demonstrate the circadian protein period 1 (PER1) and the tumor suppressor p53 negatively cross-regulate each other's expression and activity to modulate the sensitivity of cancer cells to anticancer treatments. Specifically, PER1 physically interacts with p53 to reduce its stability and impair its transcriptional activity, while p53 represses the transcription of PER1. Functionally, we could show that PER1 reduced the sensitivity of cancer cells to drug-induced apoptosis, both in vitro and in vivo in NOD scid gamma (NSG) mice xenotransplanted with a lung cancer cell line. Therefore, our results emphasize the importance of understanding the relationship between the circadian clock and tumor regulatory proteins as the basis for the future development of cancer chronotherapy.
Assuntos
Carcinogênese/genética , Neoplasias/genética , Proteínas Circadianas Period/genética , Proteína Supressora de Tumor p53/genética , Células A549 , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Ritmo Circadiano/efeitos dos fármacos , Cisplatino/farmacologia , Docetaxel/farmacologia , Cronofarmacoterapia , Etoposídeo/farmacologia , Humanos , Camundongos , Neoplasias/patologia , Neoplasias/terapia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Liver regeneration represents an outstanding tool to study not only proliferation, but also other important processes such as inflammation, regenerative response or stem cell biology. Several novel genes have been identified as being involved in the proliferation of residual hepatocytes. One of them, HOPS/TMUB1, is proving to be a significant player in the control of proliferation, both contributing to genomic stability and as a partner of essential molecules. HOPS is an ubiquitin-like protein, shuttling from nucleus to cytoplasm, and it is engaged in a number of biological and physiopathological functions. HOPS overexpression in tumour cell lines strongly reduces proliferation, arresting cell cycle in G0 /G1 . HOPS is involved in centrosome assembly and maintenance, and its knockdown causes genomic instability. Moreover, a direct interaction of HOPS with nucleophosmin (NPM) and p19Arf has been established, resulting in proper control of p19Arf stability and localization. These data indicate that HOPS acts as a functional bridge in the interaction between NPM and p19Arf , providing new mechanistic insight into how NPM and p19Arf will oppose cell proliferation. HOPS exerts a control in p53 stability, directing p53 mitochondrial apoptosis and cytoplasmic localization. HOPS plays a direct role as novel post-translational modifier of p53, much like SUMO or NEDD. HOPS is overexpressed in a high number of human tumours in patients affected by large intestinal, CNS, liver and oesophageal tumours. This review highlights HOPS involvement in distinct cellular functions, establishing its role as a key player in cell biology and pathology in a broader context.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Proteínas Nucleares/genética , Estatmina/genética , Proteína Supressora de Tumor p53/genética , Animais , Núcleo Celular/genética , Regulação da Expressão Gênica/genética , Humanos , Inflamação/genética , Inflamação/patologia , Fígado/metabolismo , Fígado/patologia , Regeneração Hepática/genética , Camundongos , NucleofosminaRESUMO
HOPS/Tmub1 is a ubiquitously expressed transmembrane ubiquitin-like protein that shuttles between nucleus and cytoplasm during cell cycle progression. HOPS causes cell cycle arrest in G0/G1 phase, an event associated to stabilization of p19Arf, an important tumor suppressor protein. Moreover, HOPS plays an important role in driving centrosomal assembly and maintenance, mitotic spindle proper organization, and ultimately a correct cell division. Recently, HOPS has been described as an important regulator of p53, which acts as modifier, stabilizing p53 half-life and playing a key role in p53 mediating apoptosis after DNA damage. NF-κB is a transcription factor with a central role in many cellular events, including inflammation and apoptosis. Our experiments demonstrate that the transcriptional activity of the p65/RelA NF-κB subunit is regulated by HOPS. Importantly, Hops-/- cells have remarkable alterations of pro-inflammatory responses. Specifically, we found that HOPS enhances NF-κB activation leading to increase transcription of inflammatory mediators, through the reduction of IκBα stability. Notably, this effect is mediated by a direct HOPS binding to the E3 ubiquitin ligase TRAF6, which lessens TRAF6 stability ultimately leading increased IKK complex activation. These findings uncover a previously unidentified function of HOPS/Tmub1 as a novel modulator of TRAF6, regulating inflammatory responses driven by activation of the NF-κB signaling pathway. The comprehension on how HOPS/Tmub1 takes part to the inflammatory processes in vivo and whether this function is important in the control of proliferation and tumorigenesis could establish the basis for the development of novel pharmacological strategies.
Assuntos
Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , NF-kappa B/metabolismo , Fator de Transcrição RelA/metabolismo , Núcleo Celular/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Ligação Proteica/fisiologia , Transdução de Sinais/fisiologia , Proteínas Supressoras de Tumor/metabolismoRESUMO
The oncosuppressor protein p53 plays a major role in transcriptionally controlling the expression of a number of genes, which in turn regulates many functions in response to DNA damage, oncogene triggering, oxidative, and additional cell stresses. A developing area of interest in p53 is the studies related to its cytoplasmic function(s). Many investigations revealed the significant role of p53 in the cytoplasm, acting in a transcriptional-independent manner in important processes related to cell homeostasis such as; apoptosis, autophagy, metabolism control, drug, and oxidative stress response. The studies on cytoplasmic p53 have shown intricate mechanisms by which posttranslational modifications allow p53 to perform its cytoplasmic functions. A number of ubiquitins, deubiquitins, and small ubiquitin-like proteins, have a pivotal role in controlling cytoplasmic stability and localization. Recently, HOPS/TMUB1 a novel small ubiquitin-like protein has been described as a vital molecule stabilizing p53 half-life, directing it to the mitochondria and favoring p53-mediated apoptosis. Furthermore, HOPS/TMUB1 competing with importin-α lessens p53 nuclear localization, thereby increasing cytoplasmic concentration. HOPS/TMUB1 as p53 modifiers could be attractive candidates to elucidate apoptosis or other important transcriptional-independent functions which are key in cancer research in order to develop new therapeutic approaches.
Assuntos
Proteínas de Transporte/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Nucleares/metabolismo , Transdução de Sinais/genética , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose/genética , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Dano ao DNA/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Neoplasias/metabolismoRESUMO
Apoptotic signalling by p53 occurs at both transcriptional and non-transcriptional levels, as p53 may act as a direct apoptogenic stimulus via activation of the intrinsic mitochondrial pathway. HOPS is a highly conserved, ubiquitously expressed shuttling protein with an ubiquitin-like domain. We generated Hops-/- mice and observed that they are viable with no apparent phenotypic defects. However, when treated with chemotherapeutic agents, Hops-/- mice display a significant reduction in apoptosis, suggesting an impaired ability to respond to genotoxic stressors. We show that HOPS acts as a regulator of cytoplasmic p53 levels and function. By binding p53, HOPS inhibits p53 proteasomal degradation and favours p53 recruitment to mitochondria and apoptosis induction. By interfering with importin α, HOPS further increases p53 cytoplasmic levels. Thus, HOPS promotes the p53-dependent mitochondrial apoptosis pathway by preserving cytoplasmic p53 from both degradation and nuclear uptake.
Assuntos
Apoptose , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana , Mitocôndrias , Proteína Supressora de Tumor p53 , Animais , Apoptose/genética , Citoplasma/genética , Citoplasma/metabolismo , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
A tight link has been established between inflammation and cancer. Liver regeneration is a widely used model to study the correlation between inflammation and proliferation. IL-6 is essentially involved in liver regeneration and in cancer. Recently, IL-17A has been shown to regulate not only inflammation, but also cell proliferation. Here, we analyze the role played by IL-17A signaling in liver regeneration by comparing cell proliferation in Wild Type and IL-17RA-/- mice. Partial hepatectomy experiments performed in IL-17RA-/- mice showed a delay in expression of early-genes to prime the residual hepatocyte to proliferate, with subsequent delay in G1/S-phase transition. We demonstrated that IL-17RA regulates, by recruitment of non-parenchymal cell, the expression of IL-6, which in turn triggers the proliferation of residual hepatocytes. Our data indicate an important role played by IL-17RA in liver proliferation via IL-6.
Assuntos
Proliferação de Células , Hepatócitos/metabolismo , Interleucina-6/metabolismo , Regeneração Hepática , Fígado/metabolismo , Receptores de Interleucina-17/deficiência , Animais , Comunicação Celular , Proliferação de Células/genética , Pontos de Checagem da Fase G1 do Ciclo Celular , Hepatectomia , Fígado/cirurgia , Regeneração Hepática/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Interleucina-17/genética , Receptores de Interleucina-17/metabolismo , Transdução de SinaisRESUMO
Liver regeneration is a unique means of studying cell proliferation in vivo. Screening of a large cDNA library from regenerating liver has previously allowed us to identify and characterize a cluster of genes encoding proteins with important roles in proliferative processes. Here, by examining different rat and human tissues as well as cell lines, we characterized a highly conserved gene, guanylyl cyclase domain containing 1 (GUCD1), whose modulation occurs in liver regeneration and cell cycle progression in vitro. High-level expression of GUCD1 transcripts was observed in livers from patients with hepatocellular carcinoma. A yeast two-hybrid interaction assay, aimed at identifying any relevant interaction partners of GUCD1, revealed direct interactions with NEDD4-1 (E3 ubiquitin protein ligase neural precursor cell expressed, developmentally downregulated gene 4), resulting in control of GUCD1 stability. Thus, we have characterized expression and function of a ubiquitous protein, GUCD1, which might have a role in regulating normal and abnormal cell growth in the liver.
Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Guanilato Ciclase/metabolismo , Regeneração Hepática/genética , Fígado/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Ciclo Celular , Proliferação de Células , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Guanilato Ciclase/genética , Humanos , Fígado/citologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Camundongos , Ubiquitina-Proteína Ligases Nedd4 , Ratos Sprague-Dawley , Ubiquitina-Proteína Ligases/genética , Regulação para CimaRESUMO
Hepatocyte odd protein shuttling (HOPS) moves between nucleus and cytoplasm. HOPS overexpression leads to cell cycle arrest in G 0/G 1, and HOPS knockdown causes centrosome alterations, with subsequent abnormal cell division. Recently, we demonstrated that HOPS acts as a functional bridge in NPM-p19(Arf) interactions. Here we show that HOPS is present in 3 different isoforms that play distinct intracellular functions. Although HOPS is a transmembrane ubiquitin, an isoform with intermediate molecular weight is cleaved from the membrane and released into the cytosol, to act as the shuttling protein. We identified a signal peptide peptidase structure in N-terminal membrane-bound HOPS that allows the regulated intramembrane proteolysis (RIP) system to control the relative amounts of the released, shuttling isoform capable of binding NPM. These results argue for distinct, isoform-specific functions of HOPS in the nucleolus, nucleus, and cytoplasm and provide insight into the dynamics of HOPS association with NPM, whose mutation and subsequent delocalization is found in 30% of acute myeloid leukemia patients.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas Nucleares/metabolismo , Animais , Proteínas de Transporte/genética , Linhagem Celular , Núcleo Celular/metabolismo , Chlorocebus aethiops , Simulação por Computador , Citosol/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana , Camundongos , Proteínas Nucleares/genética , Nucleofosmina , Especificidade de Órgãos , Ligação Proteica , Conformação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteólise , Ratos , Ubiquitina/metabolismoRESUMO
BACKGROUND/AIMS: Transcription factors coupled to cyclic adenosine mono phosphate (cAMP) signalling in the cAMP-responsive elements binding (CREB)/ATF family constitute a family of activators or repressors that bind to cAMP-responsive promoter elements (CREs) in the regulatory regions of cAMP-inducible genes. A role for CREB/ATF family has been advocated in the control of hepatocellular carcinoma progression. CREB appears to be activated by the X protein of hepatitis B virus, which links to the unphosphorylated form of CREB and activates transcription, thus obviating an otherwise indispensable Ser-133 phosphorylation. Identification of factors capable of triggering transcription via cAMP-responsive elements modulator (CREM)/CREB signalling in the absence of Ser phosphorylation will improve our knowledge of the molecular mechanism of liver cell proliferation. METHODS: To isolate and study proteins binding and activating CREB and/or CREM in the liver, we performed the screening of a mouse liver cDNA library using the Two-Hybrid System. RESULTS: We report the identification and characterization of a novel peptide, VTIP-peptide (VTIP-P), which binds and enhances the activation of CREM/CREB, obviating the need for transcription factor phosphorylation. We demonstrated that VTIP-P physically interacts with the activation domain (AD) of the transcription factors CREB/CREM and activates transcription by modifying their phosphorylation pattern in hepatoma cells. The data allowed the conclusion that VTIP-P binds the AD of CREB and CREM by stabilizing their phosphorylation. CONCLUSION: The characterization of molecules capable of interfering in the liver with an important pathway such as CREB could be significant in designing and/or developing new therapeutic approaches to the control of liver cell proliferation.
Assuntos
Proteínas de Transporte/metabolismo , Modulador de Elemento de Resposta do AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Regeneração Hepática/fisiologia , Fígado/metabolismo , Peptídeos/metabolismo , Animais , Proteínas de Transporte/classificação , Linhagem Celular Tumoral , Modulador de Elemento de Resposta do AMP Cíclico/genética , Biblioteca Gênica , Camundongos , Peptídeos/classificação , Fatores de Transcrição/metabolismo , Transfecção , Técnicas do Sistema de Duplo-HíbridoRESUMO
Centrosomes direct microtubule organization during cell division. Aberrant number of centrosomes results from alteration of its components and leads to abnormal mitoses and chromosome instability. HOPS is a newly discovered protein isolated during liver regeneration, implicated in cell proliferation. Here, we provide evidence that HOPS is an integral constituent of centrosomes. HOPS is associated with classical markers of centrosomes and found in cytosolic complexes containing CRM-1, gamma-tubulin, eEF-1A and HSP70. These features suggest that HOPS is involved in centrosome assembly and maintenance. HOPS depletion generates supernumerary centrosomes, multinucleated cells and multipolar spindle formation leading to activation of p53 checkpoint and cell cycle arrest. The presence of HOPS in cytosolic complexes supports that centrosome proteins might be preassembled in the cytoplasm to then be rapidly recruited for centrosome duplication. Altogether these data show HOPS implication in the control of cell division. HOPS contribution appears relevant to understand genomic instability and centrosome amplification in cancer.
Assuntos
Proteínas de Transporte/metabolismo , Divisão Celular/fisiologia , Centrossomo/fisiologia , Instabilidade Genômica/fisiologia , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Animais , Proteínas de Transporte/genética , Centrossomo/metabolismo , Imunofluorescência , Immunoblotting , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana , Camundongos , Células NIH 3T3 , Proteínas Nucleares/genética , OligonucleotídeosRESUMO
3Beta-hydroxysterol Delta(14)-reductase operates during the conversion of lanosterol to cholesterol in mammalian cells. Besides the endoplasmic reticulum 3beta-hydroxysterol Delta(14)-reductase (C14SR) encoded by TM7SF2 gene, the lamin B receptor (LBR) of the inner nuclear membrane possesses 3beta-hydroxysterol Delta(14)-reductase activity, based on its ability to complement C14SR-defective yeast strains. LBR was indicated as the primary 3beta-hydroxysterol Delta(14)-reductase in human cholesterol biosynthesis, since mutations in LBR gene were found in Greenberg skeletal dysplasia, characterized by accumulation of Delta(14)-unsaturated sterols. This study addresses the issue of C14SR and LBR role in cholesterol biosynthesis. Both human C14SR and LBR expressed in COS-1 cells exhibit 3beta-hydroxysterol Delta(14)-reductase activity in vitro. TM7SF2 mRNA and C14SR protein expression in HepG2 cells grown in delipidated serum (LPDS) plus lovastatin (sterol starvation) were 4- and 8-fold higher, respectively, than in LPDS plus 25-hydroxycholesterol (sterol feeding), resulting in 4-fold higher 3beta-hydroxysterol Delta(14)-reductase activity. No variations in LBR mRNA and protein levels were detected in the same conditions. The induction of TM7SF2 gene expression is turned-on by promoter activation in response to low cell sterol levels and is mediated by SREBP-2. The results suggest a primary role of C14SR in human cholesterol biosynthesis, whereas LBR role in the pathway remains unclear.
Assuntos
Colesterol/genética , Regulação da Expressão Gênica , Proteínas de Membrana/genética , Oxirredutases/metabolismo , Doenças do Desenvolvimento Ósseo/enzimologia , Doenças do Desenvolvimento Ósseo/genética , Colesterol/biossíntese , Humanos , Hidroxicolesteróis/farmacologia , Lovastatina/farmacologia , Proteínas de Membrana/metabolismo , Mutação , Oxirredutases/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , Receptor de Lamina BRESUMO
The liver has the ability to autonomously regulate growth and mass. Following partial hepatectomy, hormones, growth factors, cytokines and their coupled signal transduction pathways have been implicated in hepatocyte proliferation. To understand the mechanisms responsible for the proliferative response, we studied liver regeneration by characterization of novel genes that are activated in residual hepatocytes. A regenerating liver cDNA library screening was performed with cDNA-subtracted probes derived from regenerating and normal liver. Here, we describe the biology of Hops (for hepatocyte odd protein shuttling). HOPS is a novel shuttling protein that contains an ubiquitin-like domain, a putative NES and a proline-rich region. HOPS is rapidly exported from the nucleus and is overexpressed during liver regeneration. Evidence shows that cAMP governs HOPS export in hepatocytes of normal and regenerating liver and is mediated via CRM-1. We demonstrate that HOPS binds to elongation factor eEF-1A and interferes in protein synthesis. HOPS overexpression in H-35-hepatoma and 3T3-NIH cells strongly reduces proliferation.
Assuntos
Proteínas de Transporte/metabolismo , AMP Cíclico/metabolismo , Fígado/metabolismo , Proteínas Nucleares/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Proteínas de Transporte/genética , Processos de Crescimento Celular/fisiologia , Chlorocebus aethiops , Clonagem Molecular , DNA Complementar/genética , Hepatócitos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Fígado/citologia , Regeneração Hepática/genética , Regeneração Hepática/fisiologia , Masculino , Proteínas de Membrana , Camundongos , Dados de Sequência Molecular , Células NIH 3T3 , Proteínas Nucleares/genética , Fator 1 de Elongação de Peptídeos/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Especificidade por SubstratoRESUMO
BACKGROUND: During liver regeneration, 95% of the resting hepatocytes enter in G1/S phase of the cell cycle. A number of hormones, growth factors and cytokines were identified that activate signal transduction pathways playing a primary role in hepatocyte proliferation. A wide and representative cDNA library containing 1.5 x 106 independent clones was constructed from regenerating liver in order to identify and characterize gene the products which play a crucial role in the first hours of the proliferative process of liver regeneration. RESULTS: A novel gene expressed in liver regeneration was cloned by subtractive hybridization. The putative protein displays in the N'-terminal a annexin-like domain and an aminopeptidase domain. We named the novel gene Liver Annexin Like-1 (lal-1). The lal-1 gene is modulated during liver regeneration, in hepatoma cells following physiological stimulation and after cAMP induction. CONCLUSION: The results indicate that lal-1 is involved in liver regeneration and that its expression is finely regulated during proliferative process. The isolation of lal-1 paves the way for a further characterization helping to assess lal-1 involvement in cell function and proliferation.