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1.
Cells ; 13(16)2024 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-39195284

RESUMO

The AurkA serine/threonine kinase is a key regulator of cell division controlling mitotic entry, centrosome maturation, and chromosome segregation. The microtubule-associated protein TPX2 controls spindle assembly and is the main AurkA regulator, contributing to AurkA activation, localisation, and stabilisation. Since their identification, AurkA and TPX2 have been described as being overexpressed in cancer, with a significant correlation with highly proliferative and aneuploid tumours. Despite the frequent occurrence of AurkA/TPX2 co-overexpression in cancer, the investigation of their involvement in tumorigenesis and cancer therapy resistance mostly arises from studies focusing only on one at the time. Here, we review the existing literature and discuss the mitotic phenotypes described under conditions of AurkA, TPX2, or AurkA/TPX2 overexpression, to build a picture that may help clarify their oncogenic potential through the induction of chromosome instability. We highlight the relevance of the AurkA/TPX2 complex as an oncogenic unit, based on which we discuss recent strategies under development that aim at disrupting the complex as a promising therapeutic perspective.


Assuntos
Aurora Quinase A , Proteínas Associadas aos Microtúbulos , Neoplasias , Humanos , Aurora Quinase A/metabolismo , Aurora Quinase A/genética , Neoplasias/genética , Neoplasias/patologia , Neoplasias/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Animais , Mitose/genética , Aberrações Cromossômicas , Instabilidade Cromossômica/genética , Regulação Neoplásica da Expressão Gênica
2.
Biochim Biophys Acta Mol Basis Dis ; 1870(4): 167116, 2024 04.
Artigo em Inglês | MEDLINE | ID: mdl-38447882

RESUMO

The Aurora-A kinase (AurkA) and its major regulator TPX2 (Targeting Protein for Xklp2) are key mitotic players frequently co-overexpressed in human cancers, and the link between deregulation of the AurkA/TPX2 complex and tumourigenesis is actively investigated. Chromosomal instability, one of the hallmarks of cancer related to the development of intra-tumour heterogeneity, metastasis and chemo-resistance, has been frequently associated with TPX2-overexpressing tumours. In this study we aimed to investigate the actual contribution to chromosomal instability of deregulating the AurkA/TPX2 complex, by overexpressing it in nontransformed hTERT RPE-1 cells. Our results show that overexpression of both AurkA and TPX2 results in increased AurkA activation and severe mitotic defects, compared to AurkA overexpression alone. We also show that AurkA/TPX2 co-overexpression yields increased aneuploidy in daughter cells and the generation of micronucleated cells. Interestingly, the p53/p21 axis response is impaired in AurkA/TPX2 overexpressing cells subjected to different stimuli; consistently, cells acquire increased ability to proliferate after independent induction of mitotic errors, i.e. following nocodazole treatment. Based on our observation that increased levels of the AurkA/TPX2 complex affect chromosome segregation fidelity and interfere with the activation of a pivotal surveillance mechanism in response to altered cell division, we propose that co-overexpression of AurkA and TPX2 per se represents a condition promoting the generation of a genetically unstable context in nontransformed human cells.


Assuntos
Aurora Quinase A , Proteínas de Ciclo Celular , Humanos , Aurora Quinase A/genética , Aurora Quinase A/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteína Supressora de Tumor p53/genética , Segregação de Cromossomos/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Instabilidade Genômica , Instabilidade Cromossômica/genética , Cromossomos/metabolismo
3.
Neurochem Int ; 174: 105673, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38185384

RESUMO

Glioblastoma (GB) is a very aggressive human brain tumor. The high growth potential and invasiveness make this tumor surgically and pharmacologically untreatable. Our previous work demonstrated that the activation of the M2 muscarinic acetylcholine receptors (M2 mAChRs) inhibited cell proliferation and survival in GB cell lines and in the cancer stem cells derived from human biopsies. The aim of the present study was to investigate the ability of M2 mAChR to modulate cell migration in two different GB cell lines: U87 and U251. By wound healing assay and single cell migration analysis performed by time-lapse microscopy, we demonstrated the ability of M2 mAChRs to negatively modulate cell migration in U251 but not in the U87 cell line. In order to explain the different effects observed in the two cell lines we have evaluated the possible involvement of the intermediate conductance calcium-activated potassium (IKCa) channel. IKCa channel is present in the GB cells, and it has been demonstrated to modulate cell migration. Using the perforated patch-clamp technique we have found that selective activation of M2 mAChR significantly reduced functional density of the IKCa current in U251 but not in U87 cells. To understand whether the M2 mAChR mediated reduction of ion channel density in the U251 cell line was relevant for the cell migration impairment, we tested the effects of TRAM-34, a selective inhibitor of the IKCa channel, in wound healing assay. We found that it was able to markedly reduce U251 cell migration and significantly decrease the number of invadopodia-like structure formations. These results suggest that only in U251 cells the reduced cell migration M2 mAChR-mediated might involve, at least in part, the IKCa channel.


Assuntos
Glioblastoma , Humanos , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Glioblastoma/metabolismo , Receptor Muscarínico M2/metabolismo , Receptores Muscarínicos/metabolismo
4.
Sci Data ; 10(1): 677, 2023 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-37794110

RESUMO

Detecting and tracking multiple moving objects in a video is a challenging task. For living cells, the task becomes even more arduous as cells change their morphology over time, can partially overlap, and mitosis leads to new cells. Differently from fluorescence microscopy, label-free techniques can be easily applied to almost all cell lines, reducing sample preparation complexity and phototoxicity. In this study, we present ALFI, a dataset of images and annotations for label-free microscopy, made publicly available to the scientific community, that notably extends the current panorama of expertly labeled data for detection and tracking of cultured living nontransformed and cancer human cells. It consists of 29 time-lapse image sequences from HeLa, U2OS, and hTERT RPE-1 cells under different experimental conditions, acquired by differential interference contrast microscopy, for a total of 237.9 hours. It contains various annotations (pixel-wise segmentation masks, object-wise bounding boxes, tracking information). The dataset is useful for testing and comparing methods for identifying interphase and mitotic events and reconstructing their lineage, and for discriminating different cellular phenotypes.


Assuntos
Ciclo Celular , Rastreamento de Células , Imagem com Lapso de Tempo , Humanos , Rastreamento de Células/métodos , Células HeLa , Processamento de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/métodos , Imagem com Lapso de Tempo/métodos
5.
Cell Mol Life Sci ; 80(9): 251, 2023 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-37584777

RESUMO

AMBRA1 is a crucial factor for nervous system development, and its function has been mainly associated with autophagy. It has been also linked to cell proliferation control, through its ability to regulate c-Myc and D-type cyclins protein levels, thus regulating G1-S transition. However, it remains still unknown whether AMBRA1 is differentially regulated during the cell cycle, and if this pro-autophagy protein exerts a direct role in controlling mitosis too. Here we show that AMBRA1 is phosphorylated during mitosis on multiple sites by CDK1 and PLK1, two mitotic kinases. Moreover, we demonstrate that AMBRA1 phosphorylation at mitosis is required for a proper spindle function and orientation, driven by NUMA1 protein. Indeed, we show that the localization and/or dynamics of NUMA1 are strictly dependent on AMBRA1 presence, phosphorylation and binding ability. Since spindle orientation is critical for tissue morphogenesis and differentiation, our findings could account for an additional role of AMBRA1 in development and cancer ontogenesis.


Assuntos
Proteínas Serina-Treonina Quinases , Fuso Acromático , Humanos , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Fuso Acromático/metabolismo , Proteínas de Ciclo Celular/metabolismo , Mitose , Ciclo Celular , Células HeLa , Proteína Quinase CDC2/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo
6.
Int J Mol Sci ; 24(5)2023 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-36902175

RESUMO

Myc transcription factors are key regulators of many cellular processes, with Myc target genes crucially implicated in the management of cell proliferation and stem pluripotency, energy metabolism, protein synthesis, angiogenesis, DNA damage response, and apoptosis. Given the wide involvement of Myc in cellular dynamics, it is not surprising that its overexpression is frequently associated with cancer. Noteworthy, in cancer cells where high Myc levels are maintained, the overexpression of Myc-associated kinases is often observed and required to foster tumour cells' proliferation. A mutual interplay exists between Myc and kinases: the latter, which are Myc transcriptional targets, phosphorylate Myc, allowing its transcriptional activity, highlighting a clear regulatory loop. At the protein level, Myc activity and turnover is also tightly regulated by kinases, with a finely tuned balance between translation and rapid protein degradation. In this perspective, we focus on the cross-regulation of Myc and its associated protein kinases underlying similar and redundant mechanisms of regulation at different levels, from transcriptional to post-translational events. Furthermore, a review of the indirect effects of known kinase inhibitors on Myc provides an opportunity to identify alternative and combined therapeutic approaches for cancer treatment.


Assuntos
Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas c-myc , Aurora Quinase B/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Fosfatos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo
7.
Life Sci Alliance ; 6(5)2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-36797043

RESUMO

The AurkA kinase is a well-known mitotic regulator, frequently overexpressed in tumors. The microtubule-binding protein TPX2 controls AurkA activity, localization, and stability in mitosis. Non-mitotic roles of AurkA are emerging, and increased nuclear localization in interphase has been correlated with AurkA oncogenic potential. Still, the mechanisms leading to AurkA nuclear accumulation are poorly explored. Here, we investigated these mechanisms under physiological or overexpression conditions. We observed that AurkA nuclear localization is influenced by the cell cycle phase and nuclear export, but not by its kinase activity. Importantly, AURKA overexpression is not sufficient to determine its accumulation in interphase nuclei, which is instead obtained when AURKA and TPX2 are co-overexpressed or, to a higher extent, when proteasome activity is impaired. Expression analyses show that AURKA, TPX2, and the import regulator CSE1L are co-overexpressed in tumors. Finally, using MCF10A mammospheres we show that TPX2 co-overexpression drives protumorigenic processes downstream of nuclear AurkA. We propose that AURKA/TPX2 co-overexpression in cancer represents a key determinant of AurkA nuclear oncogenic functions.


Assuntos
Aurora Quinase A , Neoplasias , Humanos , Aurora Quinase A/genética , Aurora Quinase A/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteólise , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo
8.
Life Sci Alliance ; 6(2)2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36450448

RESUMO

Mitotic kinase Aurora A (AURKA) diverges from other kinases in its multiple active conformations that may explain its interphase roles and the limited efficacy of drugs targeting the kinase pocket. Regulation of AURKA activity by the cell is critically dependent on destruction mediated by the anaphase-promoting complex (APC/CFZR1) during mitotic exit and G1 phase and requires an atypical N-terminal degron in AURKA called the "A-box" in addition to a reported canonical D-box degron in the C-terminus. Here, we find that the reported C-terminal D-box of AURKA does not act as a degron and instead mediates essential structural features of the protein. In living cells, the N-terminal intrinsically disordered region of AURKA containing the A-box is sufficient to confer FZR1-dependent mitotic degradation. Both in silico and in cellulo assays predict the QRVL short linear interacting motif of the A-box to be a phospho-regulated D-box. We propose that degradation of full-length AURKA also depends on an intact C-terminal domain because of critical conformational parameters permissive for both activity and mitotic degradation of AURKA.


Assuntos
Aurora Quinase A , Bioensaio , Humanos , Aurora Quinase A/genética , Núcleo Celular , Proteínas Cdh1
9.
Int J Mol Sci ; 22(23)2021 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-34884931

RESUMO

Neuroblastoma is a severe childhood disease, accounting for ~10% of all infant cancers. The amplification of the MYCN gene, coding for the N-Myc transcription factor, is an essential marker correlated with tumor progression and poor prognosis. In neuroblastoma cells, the mitotic kinase Aurora-A (AURKA), also frequently overexpressed in cancer, prevents N-Myc degradation by directly binding to a highly conserved N-Myc region. As a result, elevated levels of N-Myc are observed. During recent years, it has been demonstrated that some ATP competitive inhibitors of AURKA also cause essential conformational changes in the structure of the activation loop of the kinase that prevents N-Myc binding, thus impairing the formation of the AURKA/N-Myc complex. In this study, starting from a screening of crystal structures of AURKA in complexes with known inhibitors, we identified additional compounds affecting the conformation of the kinase activation loop. We assessed the ability of such compounds to disrupt the interaction between AURKA and N-Myc in vitro, using Surface Plasmon Resonance competition assays, and in tumor cell lines overexpressing MYCN, by performing Proximity Ligation Assays. Finally, their effects on N-Myc cellular levels and cell viability were investigated. Our results identify PHA-680626 as an amphosteric inhibitor both in vitro and in MYCN overexpressing cell lines, thus expanding the repertoire of known conformational disrupting inhibitors of the AURKA/N-Myc complex and confirming that altering the conformation of the activation loop of AURKA with a small molecule is an effective strategy to destabilize the AURKA/N-Myc interaction in neuroblastoma cancer cells.


Assuntos
Aurora Quinase A/metabolismo , Proteína Proto-Oncogênica N-Myc/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Pirróis/farmacologia , Trifosfato de Adenosina/metabolismo , Antineoplásicos/farmacologia , Aurora Quinase A/antagonistas & inibidores , Aurora Quinase A/química , Azepinas/metabolismo , Azepinas/farmacologia , Benzazepinas/metabolismo , Benzazepinas/farmacologia , Sítios de Ligação , Ligação Competitiva , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Proteína Proto-Oncogênica N-Myc/química , Neuroblastoma/tratamento farmacológico , Neuroblastoma/metabolismo , Conformação Proteica , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Pirazóis/metabolismo , Pirimidinas/metabolismo , Pirimidinas/farmacologia , Pirróis/metabolismo , Ressonância de Plasmônio de Superfície
10.
Cells ; 10(7)2021 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-34359896

RESUMO

BACKGROUND: Glioblastoma multiforme (GBM) is characterized by several genetic abnormalities, leading to cell cycle deregulation and abnormal mitosis caused by a defective checkpoint. We previously demonstrated that arecaidine propargyl ester (APE), an orthosteric agonist of M2 muscarinic acetylcholine receptors (mAChRs), arrests the cell cycle of glioblastoma (GB) cells, reducing their survival. The aim of this work was to better characterize the molecular mechanisms responsible for this cell cycle arrest. METHODS: The arrest of cell proliferation was evaluated by flow cytometry analysis. Using immunocytochemistry and time-lapse analysis, the percentage of abnormal mitosis and aberrant mitotic spindles were assessed in both cell lines. Western blot analysis was used to evaluate the modulation of Sirtuin2 and acetylated tubulin-factors involved in the control of cell cycle progression. RESULTS: APE treatment caused arrest in the M phase, as indicated by the increase in p-HH3 (ser10)-positive cells. By immunocytochemistry, we found a significant increase in abnormal mitoses and multipolar mitotic spindle formation after APE treatment. Time-lapse analysis confirmed that the APE-treated GB cells were unable to correctly complete the mitosis. The modulated expression of SIRT2 and acetylated tubulin in APE-treated cells provides new insights into the mechanisms of altered mitotic progression in both GB cell lines. CONCLUSIONS: Our data show that the M2 agonist increases aberrant mitosis in GB cell lines. These results strengthen the idea of considering M2 acetylcholine receptors a novel promising therapeutic target for the glioblastoma treatment.


Assuntos
Glioblastoma/metabolismo , Glioblastoma/patologia , Mitose , Receptor Muscarínico M2/metabolismo , Fuso Acromático/metabolismo , Acetilação/efeitos dos fármacos , Arecolina/análogos & derivados , Arecolina/farmacologia , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Humanos , Metáfase/efeitos dos fármacos , Sirtuína 2/metabolismo , Imagem com Lapso de Tempo , Tubulina (Proteína)/metabolismo
11.
Oncogene ; 40(23): 3917-3928, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33981003

RESUMO

The Aurora-A kinase regulates cell division, by controlling centrosome biology and spindle assembly. Cancer cells often display elevated levels of the kinase, due to amplification of the gene locus, increased transcription or post-translational modifications. Several inhibitors of Aurora-A activity have been developed as anti-cancer agents and are under evaluation in clinical trials. Although the well-known mitotic roles of Aurora-A point at chromosomal instability, a hallmark of cancer, as a major link between Aurora-A overexpression and disease, recent evidence highlights the existence of non-mitotic functions of potential relevance. Here we focus on a nuclear-localised fraction of Aurora-A with oncogenic roles. Interestingly, this pool would identify not only non-mitotic, but also kinase-independent functions of the kinase. We review existing data in the literature and databases, examining potential links between Aurora-A stabilisation and localisation, and discuss them in the perspective of a more effective targeting of Aurora-A in cancer therapy.


Assuntos
Aurora Quinase A/antagonistas & inibidores , Aurora Quinase A/metabolismo , Centrossomo/metabolismo , Neoplasias/metabolismo , Antineoplásicos/farmacologia , Núcleo Celular/metabolismo , Humanos , Mitose/fisiologia , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Inibidores de Proteínas Quinases/farmacologia
12.
Biomolecules ; 11(4)2021 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-33921540

RESUMO

The conformational state of the activation loop (A-loop) is pivotal for the activity of most protein kinases. Hence, the characterization of the conformational dynamics of the A-loop is important to increase our understanding of the molecular processes related to diseases and to support the discovery of small molecule kinase inhibitors. Here, we carry out a combination of molecular dynamics (MD) and essential dynamics (ED) analyses to fully map the effects of phosphorylation, ADP, and conformation disrupting (CD) inhibitors (i.e., CD532 and MLN8054) on the dynamics of the A-loop of Aurora-A. MD revealed that the stability of the A-loop in an open conformation is enhanced by single phospho-Thr-288, while paradoxically, the presence of a second phosphorylation at Thr-287 decreases such stability and renders the A-loop more fluctuant in time and space. Moreover, we found that this post-translational modification has a significant effect on the direction of the A-loop motions. ED analysis suggests that the presence of the phosphate moiety induces the dynamics of Aurora-A to sample two distinct energy minima, instead of a single large minimum, as in unphosphorylated Aurora-A states. This observation indicates that the conformational distributions of Aurora-A with both single and double phospho-threonine modifications are remarkably different from the unphosphorylated state. In the closed states, binding of CD532 and MLN8054 inhibitors has the effect of increasing the distance of the N- and C-lobes of the kinase domain of Aurora-A, and the angle analysis between those two lobes during MD simulations showed that the N- and C-lobes are kept more open in presence of CD532, compared to MLN8054. As the A-loop is a common feature of Aurora protein kinases, our studies provide a general description of the conformational dynamics of this structure upon phosphorylation and different ligands binding.


Assuntos
Difosfato de Adenosina/metabolismo , Aurora Quinase A/química , Simulação de Dinâmica Molecular , Inibidores de Proteínas Quinases/farmacologia , Difosfato de Adenosina/química , Aurora Quinase A/antagonistas & inibidores , Aurora Quinase A/metabolismo , Benzazepinas/química , Benzazepinas/farmacologia , Domínio Catalítico , Humanos , Compostos de Fenilureia/química , Compostos de Fenilureia/farmacologia , Fosforilação , Ligação Proteica , Inibidores de Proteínas Quinases/química , Pirimidinas/química , Pirimidinas/farmacologia
13.
Curr Biol ; 31(3): 658-667.e5, 2021 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-33275894

RESUMO

Mitotic spindle orientation is a crucial process that defines the axis of cell division, contributing to daughter cell positioning and fate, and hence to tissue morphogenesis and homeostasis.1,2 The trimeric NuMA/LGN/Gαi complex, the major determinant of spindle orientation, exerts pulling forces on the spindle poles by anchoring astral microtubules (MTs) and dynein motors to the cell cortex.3,4 Mitotic kinases contribute to correct spindle orientation by regulating nuclear mitotic apparatus protein (NuMA) localization,5-7 among which the Aurora-A centrosomal kinase regulates NuMA targeting to the cell cortex in metaphase.8,9 Aurora-A and its activator targeting protein for Xklp2 (TPX2) are frequently overexpressed in cancer,10-12 raising the question as to whether spindle orientation is among the processes downstream the Aurora-A/TPX2 signaling axis altered under pathological conditions. Here, we investigated the role of TPX2 in the Aurora-A- and NuMA-dependent spindle orientation. We show that, in cultured adherent human cells, the interaction with TPX2 is required for Aurora-A to exert this function. We also show that Aurora-A, TPX2, and NuMA are part of a complex at spindle MTs, where TPX2 acts as a platform for Aurora-A regulation of NuMA. Interestingly, excess TPX2 does not influence NuMA localization but induces a "super-alignment" of the spindle axis with respect to the substrate, although an excess of Aurora-A induces spindle misorientation. These opposite effects are both linked to altered MT stability. Overall, our results highlight the importance of TPX2 for spindle orientation and suggest that spindle orientation is differentially sensitive to unbalanced levels of Aurora-A, TPX2, or the Aurora-A/TPX2 complex.


Assuntos
Microtúbulos , Fuso Acromático , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Divisão Celular , Dineínas/metabolismo , Células HeLa , Humanos , Metáfase , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mitose , Fuso Acromático/metabolismo
14.
J Cell Sci ; 133(12)2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32393600

RESUMO

Activity of AURKA is controlled through multiple mechanisms including phosphorylation, ubiquitin-mediated degradation and allosteric interaction with TPX2. Activity peaks at mitosis, before AURKA is degraded during and after mitotic exit in a process strictly dependent on the APC/C coactivator FZR1. We used FZR1 knockout cells (FZR1KO) and a novel FRET-based AURKA biosensor to investigate how AURKA activity is regulated in the absence of destruction. We found that AURKA activity in FZR1KO cells dropped at mitotic exit as rapidly as in parental cells, despite absence of AURKA destruction. Unexpectedly, TPX2 was degraded normally in FZR1KO cells. Overexpression of an N-terminal TPX2 fragment sufficient for AURKA binding, but that is not degraded at mitotic exit, caused delay in AURKA inactivation. We conclude that inactivation of AURKA at mitotic exit is determined not by AURKA degradation but by degradation of TPX2 and therefore is dependent on CDC20 rather than FZR1. The biosensor revealed that FZR1 instead suppresses AURKA activity in interphase and is critically required for assembly of the interphase mitochondrial network after mitosis.This article has an associated First Person interview with the first authors of the paper.


Assuntos
Aurora Quinase A , Proteínas de Ciclo Celular , Ciclossomo-Complexo Promotor de Anáfase , Aurora Quinase A/genética , Proteínas de Ciclo Celular/genética , Interfase , Mitose/genética , Complexos Ubiquitina-Proteína Ligase
15.
Sci Rep ; 10(1): 6562, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32300147

RESUMO

Exposure of the developing or adult brain to ionizing radiation (IR) can cause cognitive impairment and/or brain cancer, by targeting neural stem/progenitor cells (NSPCs). IR effects on NSPCs include transient cell cycle arrest, permanent cell cycle exit/differentiation, or cell death, depending on the experimental conditions. In vivo studies suggest that brain age influences NSPC response to IR, but whether this is due to intrinsic NSPC changes or to niche environment modifications remains unclear. Here, we describe the dose-dependent, time-dependent effects of X-ray IR in NSPC cultures derived from the mouse foetal cerebral cortex. We show that, although cortical NSPCs are resistant to low/moderate IR doses, high level IR exposure causes cell death, accumulation of DNA double-strand breaks, activation of p53-related molecular pathways and cell cycle alterations. Irradiated NSPC cultures transiently upregulate differentiation markers, but recover control levels of proliferation, viability and gene expression in the second week post-irradiation. These results are consistent with previously described in vivo effects of IR in the developing mouse cortex, and distinct from those observed in adult NSPC niches or in vitro adult NSPC cultures, suggesting that intrinsic differences in NSPCs of different origins might determine, at least in part, their response to IR.


Assuntos
Córtex Cerebral/citologia , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos da radiação , Animais , Biomarcadores/metabolismo , Ciclo Celular/efeitos da radiação , Morte Celular/efeitos da radiação , Diferenciação Celular/efeitos da radiação , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Células Cultivadas , Relação Dose-Resposta à Radiação , Histonas/metabolismo , Cinética , Camundongos , Modelos Biológicos , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima/efeitos da radiação , Raios X
16.
Cells ; 9(2)2020 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-32041138

RESUMO

The microtubule-associated protein TPX2 is a key mitotic regulator that contributes through distinct pathways to spindle assembly. A well-characterised function of TPX2 is the activation, stabilisation and spindle localisation of the Aurora-A kinase. High levels of TPX2 are reported in tumours and the effects of its overexpression have been investigated in cancer cell lines, while little is known in non-transformed cells. Here we studied TPX2 overexpression in hTERT RPE-1 cells, using either the full length TPX2 or a truncated form unable to bind Aurora-A, to identify effects that are dependent-or independent-on its interaction with the kinase. We observe significant defects in mitotic spindle assembly and progression through mitosis that are more severe when overexpressed TPX2 is able to interact with Aurora-A. Furthermore, we describe a peculiar, and Aurora-A-interaction-independent, phenotype in telophase cells, with aberrantly stable microtubules interfering with nuclear reconstitution and the assembly of a continuous lamin B1 network, resulting in daughter cells displaying doughnut-shaped nuclei. Our results using non-transformed cells thus reveal a previously uncharacterised consequence of abnormally high TPX2 levels on the correct microtubule cytoskeleton remodelling and G1 nuclei reformation, at the mitosis-to-interphase transition.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mitose , Aurora Quinase A/metabolismo , Linhagem Celular , Cromatina/metabolismo , Citoesqueleto/metabolismo , Complexo de Golgi/metabolismo , Humanos , Lamina Tipo B/metabolismo , Metáfase , Ligação Proteica , Telófase
17.
PLoS Comput Biol ; 15(12): e1007219, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31846452

RESUMO

The most frequently used approach for protein structure prediction is currently homology modeling. The 3D model building phase of this methodology is critical for obtaining an accurate and biologically useful prediction. The most widely employed tool to perform this task is MODELLER. This program implements the "modeling by satisfaction of spatial restraints" strategy and its core algorithm has not been altered significantly since the early 1990s. In this work, we have explored the idea of modifying MODELLER with two effective, yet computationally light strategies to improve its 3D modeling performance. Firstly, we have investigated how the level of accuracy in the estimation of structural variability between a target protein and its templates in the form of σ values profoundly influences 3D modeling. We show that the σ values produced by MODELLER are on average weakly correlated to the true level of structural divergence between target-template pairs and that increasing this correlation greatly improves the program's predictions, especially in multiple-template modeling. Secondly, we have inquired into how the incorporation of statistical potential terms (such as the DOPE potential) in the MODELLER's objective function impacts positively 3D modeling quality by providing a small but consistent improvement in metrics such as GDT-HA and lDDT and a large increase in stereochemical quality. Python modules to harness this second strategy are freely available at https://github.com/pymodproject/altmod. In summary, we show that there is a large room for improving MODELLER in terms of 3D modeling quality and we propose strategies that could be pursued in order to further increase its performance.


Assuntos
Modelos Moleculares , Software , Homologia Estrutural de Proteína , Algoritmos , Biologia Computacional , Simulação de Dinâmica Molecular/estatística & dados numéricos , Proteínas/química , Alinhamento de Sequência/estatística & dados numéricos
18.
Mol Biol Cell ; 29(13): 1611-1627, 2018 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-29742020

RESUMO

A primary cilium is found on most mammalian cells, where it acts as a cellular antenna for the reception of both mechanical and chemical signals. A variety of diseases are associated with defective ciliogenesis, reflecting the ubiquity of the function of cilia and the number of proteins required for their assembly. Proper cilia length is necessary for cilia signaling and is regulated through a poorly understood balance of assembly and disassembly rates. FHDC1 is a unique member of the formin family of cytoskeletal regulatory proteins. Overexpression of FHDC1 induces F-actin accumulation and microtubule stabilization and acetylation. We find that overexpression of FHDC1 also has profound effects on ciliogenesis; in most cells FHDC1 overexpression blocks cilia assembly, but the cilia that are present are immensely elongated. FHDC1-induced cilia growth requires the FHDC1 FH2 and microtubule-binding domain and results from F-actin-dependent inhibition of cilia disassembly. FHDC1 depletion, or treatment with a pan-formin inhibitor, inhibits cilia assembly and induces cilia resorption. Endogenous FHDC1 protein localizes to cytoplasmic microtubules converging on the base of the cilia, and we identify the subdistal appendage protein Cep170 as an FHDC1 interacting protein. Our results suggest that FHDC1 plays a role in coordinating cytoskeletal dynamics during normal cilia assembly.


Assuntos
Actinas/metabolismo , Cílios/metabolismo , Proteínas Fetais/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Nucleares/metabolismo , Animais , Centríolos/metabolismo , Forminas , Complexo de Golgi/metabolismo , Camundongos , Células NIH 3T3 , Ligação Proteica
19.
Sci Rep ; 8(1): 1850, 2018 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-29382863

RESUMO

Karyopherin beta-1/Importin beta-1 is a conserved nuclear transport receptor, acting in protein nuclear import in interphase and as a global regulator of mitosis. These pleiotropic functions reflect its ability to interact with, and regulate, different pathways during the cell cycle, operating as a major effector of the GTPase RAN. Importin beta-1 is overexpressed in cancers characterized by high genetic instability, an observation that highlights the importance of identifying its partners in mitosis. Here we present the first comprehensive profile of importin beta-1 interactors from human mitotic cells. By combining co-immunoprecipitation and proteome-wide mass spectrometry analysis of synchronized cell extracts, we identified expected (e.g., RAN and SUMO pathway factors) and novel mitotic interactors of importin beta-1, many with RNA-binding ability, that had not been previously associated with importin beta-1. These data complement interactomic studies of interphase transport pathways. We further developed automated proximity ligation assay (PLA) protocols to validate selected interactors. We succeeded in obtaining spatial and temporal resolution of genuine importin beta-1 interactions, which were visualized and localized in situ in intact mitotic cells. Further developments of PLA protocols will be helpful to dissect importin beta-1-orchestrated pathways during mitosis.


Assuntos
Processamento de Imagem Assistida por Computador/métodos , Imunoprecipitação/métodos , Mitose , beta Carioferinas/metabolismo , Bioensaio , Células HeLa , Humanos , Reação em Cadeia da Polimerase , Domínios e Motivos de Interação entre Proteínas
20.
Oncotarget ; 8(19): 32117-32133, 2017 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-28389630

RESUMO

Aurora kinases are a family of cell division regulators that govern the correct assembly of a bipolar mitotic spindle and the fidelity of chromosome segregation. Their overexpression is associated with genomic instability and aneuploidy, and is frequently observed in cancer. Accordingly, competitive inhibitors targeting Aurora kinase activity at the ATP-binding site are being investigated for therapeutic purposes. Despite promising pre-clinical data, these molecules display moderate effects in clinical trials and incomplete selectivity, either against distinct family members, or other kinases. As an alternative approach, protein-protein interaction inhibitors targeting mitotic kinases and their activators can be exploited to achieve increased specificity of action. In this study, a virtual screening of small molecules led to the identification of 25 potential inhibitors of the interaction between Aurora-A and its activator TPX2. In vitro experiments confirmed that 4 hits bind Aurora-A in the low micromolar range and compete for TPX2 binding. Immunofluorescence assays showed that 2 compounds also yield lowered Aurora-A activity and spindle pole defects in cultured osteosarcoma cells. The identified protein-protein interaction inhibitors of the Aurora-A/TPX2 complex might represent lead compounds for further development towards pioneering anti-cancer drugs and provide the proof-of-concept for a new exploitable strategy to target mitotic kinases.


Assuntos
Aurora Quinase A/metabolismo , Proteínas de Ciclo Celular/metabolismo , Descoberta de Drogas , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Antineoplásicos/química , Antineoplásicos/farmacologia , Aurora Quinase A/química , Sítios de Ligação , Proteínas de Ciclo Celular/química , Linhagem Celular Tumoral , Simulação por Computador , Humanos , Proteínas Associadas aos Microtúbulos/química , Modelos Moleculares , Conformação Molecular , Proteínas Nucleares/química , Ligação Proteica/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas , Relação Estrutura-Atividade
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