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1.
Anticancer Res ; 41(9): 4321-4331, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34475052

RESUMO

BACKGROUND/AIM: Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are key drugs in cancer treatment due to their minor adverse effects and outstanding anticancer effects. However, drugs for overcoming EGFR-TKI resistance are not in clinical use so far. Therefore, to overcome resistance, we focused on lurasidone, a new antipsychotic drug, due to its mild adverse effect profile from the viewpoint of drug repositioning. MATERIALS AND METHODS: We explored the effects of lurasidone alone or in combination with EGFR-TKI on the growth of osimertinib-resistant cancer cells the anti-apoptotic marker expression such as survivin, and autophagy levels by LC-3B expression. RESULTS: Within a non-toxic concentration range in normal cells, lurasidone and osimertinib combination therapy showed a growth-inhibitory effect in osimertinib-resistant cancer cells in vitro and in vivo. Furthermore, lurasidone decreased survivin expression and mildly induced autophagy. CONCLUSION: Lurasidone may increase the sensitivity to osimertinib in osimertinib-resistant cancer cells in drug repurposing.


Assuntos
Acrilamidas/administração & dosagem , Compostos de Anilina/administração & dosagem , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Cloridrato de Lurasidona/administração & dosagem , Survivina/metabolismo , Células A549 , Acrilamidas/farmacologia , Compostos de Anilina/farmacologia , Animais , Autofagia/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/metabolismo , Cloridrato de Lurasidona/farmacologia , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
2.
Int J Mol Sci ; 22(15)2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34360830

RESUMO

Photoreceptors are highly compartmentalized cells with large amounts of proteins synthesized in the inner segment (IS) and transported to the outer segment (OS) and synaptic terminal. Tulp1 is a photoreceptor-specific protein localized to the IS and synapse. In the absence of Tulp1, several OS-specific proteins are mislocalized and synaptic vesicle recycling is impaired. To better understand the involvement of Tulp1 in protein trafficking, our approach in the current study was to physically isolate Tulp1-containing photoreceptor compartments by serial tangential sectioning of retinas and to identify compartment-specific Tulp1 binding partners by immunoprecipitation followed by liquid chromatography tandem mass spectrometry. Our results indicate that Tulp1 has two distinct interactomes. We report the identification of: (1) an IS-specific interaction between Tulp1 and the motor protein Kinesin family member 3a (Kif3a), (2) a synaptic-specific interaction between Tulp1 and the scaffold protein Ribeye, and (3) an interaction between Tulp1 and the cytoskeletal protein microtubule-associated protein 1B (MAP1B) in both compartments. Immunolocalization studies in the wild-type retina indicate that Tulp1 and its binding partners co-localize to their respective compartments. Our observations are compatible with Tulp1 functioning in protein trafficking in multiple photoreceptor compartments, likely as an adapter molecule linking vesicles to molecular motors and the cytoskeletal scaffold.


Assuntos
Oxirredutases do Álcool/metabolismo , Proteínas Correpressoras/metabolismo , Proteínas do Olho/metabolismo , Cinesina/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Transporte Proteico , Animais , Cromatografia Líquida , Cílios , Proteínas do Olho/genética , Imunoprecipitação , Camundongos , Camundongos Knockout , Ligação Proteica , Proteômica , Ratos , Sinapses , Espectrometria de Massas em Tandem
3.
Int J Mol Sci ; 22(16)2021 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-34445628

RESUMO

We investigated the alterations of hippocampal and reticulo-thalamic (RT) GABAergic parvalbumin (PV) interneurons and their synaptic re-organizations underlying the prodromal local sleep disorders in the distinct rat models of Parkinson's disease (PD). We demonstrated for the first time that REM sleep is a predisposing state for the high-voltage sleep spindles (HVS) induction in all experimental models of PD, particularly during hippocampal REM sleep in the hemiparkinsonian models. There were the opposite underlying alterations of the hippocampal and RT GABAergic PV+ interneurons along with the distinct MAP2 and PSD-95 expressions. Whereas the PD cholinopathy enhanced the number of PV+ interneurons and suppressed the MAP2/PSD-95 expression, the hemiparkinsonism with PD cholinopathy reduced the number of PV+ interneurons and enhanced the MAP2/PSD-95 expression in the hippocampus. Whereas the PD cholinopathy did not alter PV+ interneurons but partially enhanced MAP2 and suppressed PSD-95 expression remotely in the RT, the hemiparkinsonism with PD cholinopathy reduced the PV+ interneurons, enhanced MAP2, and did not change PSD-95 expression remotely in the RT. Our study demonstrates for the first time an important regulatory role of the hippocampal and RT GABAergic PV+ interneurons and the synaptic protein dynamic alterations in the distinct rat models of PD neuropathology.


Assuntos
Modelos Animais de Doenças , Hipocampo/patologia , Interneurônios/patologia , Doença de Parkinson/complicações , Parvalbuminas/metabolismo , Transtornos do Sono-Vigília/patologia , Sinapses/patologia , Animais , Proteína 4 Homóloga a Disks-Large/genética , Proteína 4 Homóloga a Disks-Large/metabolismo , Hipocampo/metabolismo , Interneurônios/metabolismo , Masculino , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Neuropatologia , Ratos , Ratos Wistar , Formação Reticular/metabolismo , Transtornos do Sono-Vigília/etiologia , Transtornos do Sono-Vigília/metabolismo , Sinapses/metabolismo , Tálamo/metabolismo , Ácido gama-Aminobutírico/metabolismo
4.
Nat Commun ; 12(1): 4722, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34354059

RESUMO

Mutations in the LaminA gene are a common cause of monogenic dilated cardiomyopathy. Here we show that mice with a cardiomyocyte-specific Lmna deletion develop cardiac failure and die within 3-4 weeks after inducing the mutation. When the same Lmna mutations are induced in mice genetically deficient in the LINC complex protein SUN1, life is extended to more than one year. Disruption of SUN1's function is also accomplished by transducing and expressing a dominant-negative SUN1 miniprotein in Lmna deficient cardiomyocytes, using the cardiotrophic Adeno Associated Viral Vector 9. The SUN1 miniprotein disrupts binding between the endogenous LINC complex SUN and KASH domains, displacing the cardiomyocyte KASH complexes from the nuclear periphery, resulting in at least a fivefold extension in lifespan. Cardiomyocyte-specific expression of the SUN1 miniprotein prevents cardiomyopathy progression, potentially avoiding the necessity of developing a specific therapeutic tailored to treating each different LMNA cardiomyopathy-inducing mutation of which there are more than 450.


Assuntos
Cardiomiopatia Dilatada/genética , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Animais , Cardiomiopatia Dilatada/patologia , Cardiomiopatia Dilatada/fisiopatologia , Dependovirus/genética , Feminino , Humanos , Lamina Tipo A/deficiência , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/deficiência , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Transdução Genética
5.
Nat Commun ; 12(1): 4960, 2021 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-34400618

RESUMO

Agonists of glucocorticoid receptor (GR) are frequently given to cancer patients with platinum-containing chemotherapy to reduce inflammation, but how GR influences tumor growth in response to platinum-based chemotherapy such as cisplatin through inflammation-independent signaling remains largely unclear. Combined genomics and transcription factor profiling reveal that MAST1, a critical platinum resistance factor that reprograms the MAPK pathway, is upregulated upon cisplatin exposure through activated transcription factor GR. Mechanistically, cisplatin binds to C622 in GR and recruits GR to the nucleus for its activation, which induces MAST1 expression and consequently reactivates MEK signaling. GR nuclear translocation and MAST1 upregulation coordinately occur in patient tumors collected after platinum treatment, and align with patient treatment resistance. Co-treatment with dexamethasone and cisplatin restores cisplatin-resistant tumor growth, whereas addition of the MAST1 inhibitor lestaurtinib abrogates tumor growth while preserving the inhibitory effect of dexamethasone on inflammation in vivo. These findings not only provide insights into the underlying mechanism of GR in cisplatin resistance but also offer an effective alternative therapeutic strategy to improve the clinical outcome of patients receiving platinum-based chemotherapy with GR agonists.


Assuntos
Cisplatino/farmacologia , Proteínas Associadas aos Microtúbulos/metabolismo , Platina/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Glucocorticoides/efeitos dos fármacos , Receptores de Glucocorticoides/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Núcleo Celular , Sobrevivência Celular , Citocinas , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/genética , Receptores de Glucocorticoides/genética , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição , Regulação para Cima/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Biophys J ; 120(15): 3192-3210, 2021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-34197801

RESUMO

Proper formation and maintenance of the mitotic spindle is required for faithful cell division. Although much work has been done to understand the roles of the key molecular components of the mitotic spindle, identifying the consequences of force perturbations in the spindle remains a challenge. We develop a computational framework accounting for the minimal force requirements of mitotic progression. To reflect early spindle formation, we model microtubule dynamics and interactions with major force-generating motors, excluding chromosome interactions that dominate later in mitosis. We directly integrate our experimental data to define and validate the model. We then use simulations to analyze individual force components over time and their relationship to spindle dynamics, making it distinct from previously published models. We show through both model predictions and biological manipulation that rather than achieving and maintaining a constant bipolar spindle length, fluctuations in pole-to-pole distance occur that coincide with microtubule binding and force generation by cortical dynein. Our model further predicts that high dynein activity is required for spindle bipolarity when kinesin-14 (HSET) activity is also high. To the best of our knowledge, our results provide novel insight into the role of cortical dynein in the regulation of spindle bipolarity.


Assuntos
Dineínas , Fuso Acromático , Segregação de Cromossomos , Dineínas/metabolismo , Cinesina/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mitose , Fuso Acromático/metabolismo
7.
Nat Commun ; 12(1): 4170, 2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34234130

RESUMO

Genome organization is driven by forces affecting transcriptional state, but the relationship between transcription and genome architecture remains unclear. Here, we identified the Drosophila transcription factor Motif 1 Binding Protein (M1BP) in physical association with the gypsy chromatin insulator core complex, including the universal insulator protein CP190. M1BP is required for enhancer-blocking and barrier activities of the gypsy insulator as well as its proper nuclear localization. Genome-wide, M1BP specifically colocalizes with CP190 at Motif 1-containing promoters, which are enriched at topologically associating domain (TAD) borders. M1BP facilitates CP190 chromatin binding at many shared sites and vice versa. Both factors promote Motif 1-dependent gene expression and transcription near TAD borders genome-wide. Finally, loss of M1BP reduces chromatin accessibility and increases both inter- and intra-TAD local genome compaction. Our results reveal physical and functional interaction between CP190 and M1BP to activate transcription at TAD borders and mediate chromatin insulator-dependent genome organization.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional , Animais , Animais Geneticamente Modificados , Linhagem Celular , Núcleo Celular/metabolismo , Cromatina/genética , Cromatina/metabolismo , Sequenciamento de Cromatina por Imunoprecipitação , Proteínas de Drosophila/genética , Técnicas de Silenciamento de Genes , Genoma de Inseto , Elementos Isolantes/genética , Masculino , Proteínas Associadas aos Microtúbulos/genética , Proteínas Nucleares/genética , Regiões Promotoras Genéticas/genética , RNA-Seq , Proteínas Repressoras/genética , Fatores de Transcrição/genética
8.
Nat Commun ; 12(1): 4194, 2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34234144

RESUMO

Photomorphogenesis, light-mediated development, is an essential feature of all terrestrial plants. While chloroplast development and brassinosteroid (BR) signaling are known players in photomorphogenesis, proteins that regulate both pathways have yet to be identified. Here we report that DE-ETIOLATION IN THE DARK AND YELLOWING IN THE LIGHT (DAY), a membrane protein containing DnaJ-like domain, plays a dual-role in photomorphogenesis by stabilizing the BR receptor, BRI1, as well as a key enzyme in chlorophyll biosynthesis, POR. DAY localizes to both the endomembrane and chloroplasts via its first transmembrane domain and chloroplast transit peptide, respectively, and interacts with BRI1 and POR in their respective subcellular compartments. Using genetic analysis, we show that DAY acts independently on BR signaling and chlorophyll biogenesis. Collectively, this work uncovers DAY as a factor that simultaneously regulates BR signaling and chloroplast development, revealing a key regulator of photomorphogenesis that acts across cell compartments.


Assuntos
Proteínas de Arabidopsis/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Membrana/metabolismo , Morfogênese/fisiologia , Proteínas Quinases/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Brassinosteroides/metabolismo , Clorofila/biossíntese , Cloroplastos/metabolismo , Estiolamento/fisiologia , Regulação da Expressão Gênica de Plantas/fisiologia , Técnicas de Silenciamento de Genes , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP40/isolamento & purificação , Luz , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Morfogênese/efeitos da radiação , Mutação , Plantas Geneticamente Modificadas , Proteínas Quinases/genética , RNA-Seq , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Plântula/crescimento & desenvolvimento , Transdução de Sinais/fisiologia
9.
Chem Biol Interact ; 346: 109578, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34265256

RESUMO

Current organophosphate (OP) toxicity research now considers potential non-cholinergic mechanisms for these compounds, since the inhibition of acetylcholinesterase (AChE) cannot completely explain all the adverse biological effects of OP. Thanks to the development of new strategies for OP detection, some potential molecular targets have been identified. Among these molecules are several cytoskeletal proteins, including actin, tubulin, intermediate filament proteins, and associated proteins, such as motor proteins, microtubule-associated proteins (MAPs), and cofilin. in vitro, ex vivo, and some in vivo reports have identified alterations in the cytoskeleton following OP exposure, including cell morphology defects, cells detachments, intracellular transport disruption, aberrant mitotic spindle formation, modification of cell motility, and reduced phagocytic capability, which implicate the cytoskeleton in OP toxicity. Here, we reviewed the evidence indicating the cytoskeletal targets of OP compounds, including their strategies, the potential effects of their alterations, and their possible participation in neurotoxicity, embryonic development, cell division, and immunotoxicity related to OP compounds exposure.


Assuntos
Citoesqueleto/metabolismo , Compostos Organofosforados/metabolismo , Actinas/metabolismo , Animais , Carcinogênese , Divisão Celular/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Compostos Organofosforados/química , Compostos Organofosforados/toxicidade , Tubulina (Proteína)/metabolismo
10.
Int J Mol Sci ; 22(13)2021 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-34281286

RESUMO

Norepinephrine (NE) neurons and extracellular NE exert some protective effects against a variety of insults, including methamphetamine (Meth)-induced cell damage. The intimate mechanism of protection remains difficult to be analyzed in vivo. In fact, this may occur directly on target neurons or as the indirect consequence of NE-induced alterations in the activity of trans-synaptic loops. Therefore, to elude neuronal networks, which may contribute to these effects in vivo, the present study investigates whether NE still protects when directly applied to Meth-treated PC12 cells. Meth was selected based on its detrimental effects along various specific brain areas. The study shows that NE directly protects in vitro against Meth-induced cell damage. The present study indicates that such an effect fully depends on the activation of plasma membrane ß2-adrenergic receptors (ARs). Evidence indicates that ß2-ARs activation restores autophagy, which is impaired by Meth administration. This occurs via restoration of the autophagy flux and, as assessed by ultrastructural morphometry, by preventing the dissipation of microtubule-associated protein 1 light chain 3 (LC3) from autophagy vacuoles to the cytosol, which is produced instead during Meth toxicity. These findings may have an impact in a variety of degenerative conditions characterized by NE deficiency along with autophagy impairment.


Assuntos
Metanfetamina/antagonistas & inibidores , Metanfetamina/toxicidade , Proteínas Associadas aos Microtúbulos/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Norepinefrina/farmacologia , Receptores Adrenérgicos beta 2/metabolismo , Adrenérgicos/farmacologia , Animais , Autofagia/efeitos dos fármacos , Compartimento Celular/efeitos dos fármacos , Estimulantes do Sistema Nervoso Central/administração & dosagem , Estimulantes do Sistema Nervoso Central/antagonistas & inibidores , Estimulantes do Sistema Nervoso Central/toxicidade , Desipramina/farmacologia , Relação Dose-Resposta a Droga , Metanfetamina/administração & dosagem , Microscopia Eletrônica de Transmissão , Modelos Neurológicos , Neurônios/ultraestrutura , Fármacos Neuroprotetores/farmacologia , Norepinefrina/metabolismo , Células PC12 , Ratos , Vacúolos/efeitos dos fármacos , Vacúolos/metabolismo , Vacúolos/ultraestrutura
11.
Nat Commun ; 12(1): 4096, 2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34215746

RESUMO

Non-centrosomal microtubule arrays serve crucial functions in cells, yet the mechanisms of their generation are poorly understood. During budding of the epithelial tubes of the salivary glands in the Drosophila embryo, we previously demonstrated that the activity of pulsatile apical-medial actomyosin depends on a longitudinal non-centrosomal microtubule array. Here we uncover that the exit from the last embryonic division cycle of the epidermal cells of the salivary gland placode leads to one centrosome in the cells losing all microtubule-nucleation capacity. This restriction of nucleation activity to the second, Centrobin-enriched, centrosome is key for proper morphogenesis. Furthermore, the microtubule-severing protein Katanin and the minus-end-binding protein Patronin accumulate in an apical-medial position only in placodal cells. Loss of either in the placode prevents formation of the longitudinal microtubule array and leads to loss of apical-medial actomyosin and impaired apical constriction. We thus propose a mechanism whereby Katanin-severing at the single active centrosome releases microtubule minus-ends that are then anchored by apical-medial Patronin to promote formation of the longitudinal microtubule array crucial for apical constriction and tube formation.


Assuntos
Divisão Celular/fisiologia , Centrossomo/metabolismo , Microtúbulos/metabolismo , Actinas , Actomiosina/metabolismo , Animais , Centrossomo/ultraestrutura , Proteínas do Citoesqueleto/metabolismo , Drosophila , Katanina , Masculino , Proteínas dos Microfilamentos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Morfogênese , Glândulas Salivares , Tubulina (Proteína)/metabolismo
12.
J Cell Sci ; 134(14)2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34297125

RESUMO

As one of four filament types, microtubules are a core component of the cytoskeleton and are essential for cell function. Yet how microtubules are nucleated from their building blocks, the αß-tubulin heterodimer, has remained a fundamental open question since the discovery of tubulin 50 years ago. Recent structural studies have shed light on how γ-tubulin and the γ-tubulin complex proteins (GCPs) GCP2 to GCP6 form the γ-tubulin ring complex (γ-TuRC). In parallel, functional and single-molecule studies have informed on how the γ-TuRC nucleates microtubules in real time, how this process is regulated in the cell and how it compares to other modes of nucleation. Another recent surprise has been the identification of a second essential nucleation factor, which turns out to be the well-characterized microtubule polymerase XMAP215 (also known as CKAP5, a homolog of chTOG, Stu2 and Alp14). This discovery helps to explain why the observed nucleation activity of the γ-TuRC in vitro is relatively low. Taken together, research in recent years has afforded important insight into how microtubules are made in the cell and provides a basis for an exciting era in the cytoskeleton field.


Assuntos
Proteínas Associadas aos Microtúbulos , Microtúbulos , Proteínas Associadas aos Microtúbulos/metabolismo , Centro Organizador dos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Estrutura Secundária de Proteína , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo
13.
Int J Mol Sci ; 22(13)2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-34281194

RESUMO

Cockayne syndrome group A (CS-A) is a rare recessive progeroid disorder characterized by sun sensitivity and neurodevelopmental abnormalities. Cells derived from CS-A patients present as pathological hallmarks excessive oxidative stress, mitochondrial fragmentation and apoptosis associated with hyperactivation of the mitochondrial fission dynamin related protein 1 (DRP1). In this study, by using human cell models we further investigated the interplay between DRP1 and CSA and we determined whether pharmacological or genetic inhibition of DRP1 affects disease progression. Both reactive oxygen and nitrogen species are in excess in CS-A cells and when the mitochondrial translocation of DRP1 is inhibited a reduction of these species is observed together with a recovery of mitochondrial integrity and a significant decrease of apoptosis. This study indicates that the CSA-driven modulation of DRP1 pathway is key to control mitochondrial homeostasis and apoptosis and suggests DRP1 as a potential target in the treatment of CS patients.


Assuntos
Síndrome de Cockayne/metabolismo , Dinaminas/metabolismo , Mitocôndrias/metabolismo , Animais , Apoptose/genética , Linhagem Celular , Síndrome de Cockayne/fisiopatologia , Progressão da Doença , Dinaminas/genética , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/fisiologia , Doenças Mitocondriais/genética , Doenças Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Modelos Biológicos , Estresse Oxidativo , Quinazolinonas/metabolismo , Quinazolinonas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais
14.
Int J Mol Sci ; 22(14)2021 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-34299022

RESUMO

Neuronal cell adhesion molecule 2 (NCAM2) is a membrane protein with an important role in the morphological development of neurons. In the cortex and the hippocampus, NCAM2 is essential for proper neuronal differentiation, dendritic and axonal outgrowth and synapse formation. However, little is known about NCAM2 functional mechanisms and its interactive partners during brain development. Here we used mass spectrometry to study the molecular interactome of NCAM2 in the second postnatal week of the mouse cerebral cortex. We found that NCAM2 interacts with >100 proteins involved in numerous processes, including neuronal morphogenesis and synaptogenesis. We validated the most relevant interactors, including Neurofilaments (NEFs), Microtubule-associated protein 2 (MAP2), Calcium/calmodulin kinase II alpha (CaMKIIα), Actin and Nogo. An in silico analysis of the cytosolic tail of the NCAM2.1 isoform revealed specific phosphorylation site motifs with a putative affinity for some of these interactors. Our results expand the knowledge of NCAM2 interactome and confirm the key role of NCAM2 in cytoskeleton organization, neuronal morphogenesis and synaptogenesis. These findings are of interest in explaining the phenotypes observed in different pathologies with alterations in the NCAM2 gene.


Assuntos
Córtex Cerebral/metabolismo , Citoesqueleto/metabolismo , Espectrometria de Massas , Moléculas de Adesão de Célula Nervosa/metabolismo , Neurogênese , Neurônios/metabolismo , Actinas/metabolismo , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Córtex Cerebral/crescimento & desenvolvimento , Biologia Computacional , Citoplasma/genética , Citoplasma/metabolismo , Bases de Dados de Compostos Químicos , Ontologia Genética , Técnicas In Vitro , Filamentos Intermediários/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Neurogênese/genética , Proteínas Nogo , Fosforilação , Domínios Proteicos , Mapas de Interação de Proteínas , Proteoma/genética , Proteoma/metabolismo , Transcriptoma/genética
15.
Nat Chem Biol ; 17(9): 964-974, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34083810

RESUMO

A remarkable feature of the microtubule cytoskeleton is the coexistence of subpopulations having different dynamic properties. A prominent example is the anaphase spindle, where stable antiparallel bundles exist alongside dynamic microtubules and provide spatial cues for cytokinesis. How are the dynamics of spatially proximal arrays differentially regulated? We reconstitute a minimal system of three midzone proteins: microtubule-crosslinker PRC1 and its interactors CLASP1 and Kif4A, proteins that promote and suppress microtubule elongation, respectively. We find that their collective activity promotes elongation of single microtubules while simultaneously stalling polymerization of crosslinked bundles. This differentiation arises from (1) strong rescue activity of CLASP1, which overcomes the weaker effects of Kif4A on single microtubules, and (2) lower microtubule- and PRC1-binding affinity of CLASP1, which permits the dominance of Kif4A at overlaps. In addition to canonical mechanisms where antagonistic regulators set microtubule length, our findings illuminate design principles by which collective regulator activity creates microenvironments of arrays with distinct dynamic properties.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Cinesina/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/isolamento & purificação , Humanos , Cinesina/genética , Cinesina/isolamento & purificação , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/isolamento & purificação
16.
J Photochem Photobiol B ; 221: 112245, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34182186

RESUMO

There is currently no clear understanding on the pathways involved in the process of cell inhibition by photobiomodulation (PBM). The present study evaluated the influence of PBM on the expression of autophagy markers in vitro in an in situ model of oral carcinoma. Oral squamous cell carcinoma (Cal27) and stromal fibroblasts (FG) cultures were used. The independent variables were 'cell type' (FG and CAL27) 'culture condition' (monocultures or co-cultures) and PBM (placebo and 36 J/cm2). The cultures were irradiated from a red LED source for mRNA expression and protein expression analyses. The autophagy markers evaluated were Beclin-1, LC3B and p62 as well as adjuvant markers (BAX Bcl-2, VEGF, CD105, CD34, PRDX1, PRDX4 and GRP78). The Cal27 cells upregulated the autophagy markers upon exposure to PBM both at the mRNA and protein expression levels, providing evidence to explain malignant cell inhibition by PBM.


Assuntos
Autofagia/genética , Luz , Regulação para Cima/efeitos da radiação , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular , Técnicas de Cocultura , Humanos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Neoplasias Bucais/metabolismo , Neoplasias Bucais/patologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/metabolismo
17.
J Biol Chem ; 297(1): 100873, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34126070

RESUMO

Macroautophagy dysregulation is implicated in multiple neurological disorders, such as Parkinson's disease. While autophagy pathways are heavily researched in heterologous cells and neurons, regulation of autophagy in the astrocyte, the most abundant cell type in the mammalian brain, is less well understood. Missense mutations in the Synj1 gene encoding Synaptojanin1 (Synj1), a neuron-enriched lipid phosphatase, have been linked to Parkinsonism with seizures. Our previous study showed that the Synj1 haploinsufficient (Synj1+/-) mouse exhibits age-dependent autophagy impairment in multiple brain regions. Here, we used cultured astrocytes from Synj1-deficient mice to investigate its role in astrocyte autophagy. We report that Synj1 is expressed in low levels in astrocytes and represses basal autophagosome formation. We demonstrate using cellular imaging that Synj1-deficient astrocytes exhibit hyperactive autophagosome formation, represented by an increase in the size and number of GFP-microtubule-associated protein 1A/1B-light chain 3 structures. Interestingly, Synj1 deficiency is also associated with an impairment in stress-induced autophagy clearance. We show, for the first time, that the Parkinsonism-associated R839C mutation impacts autophagy in astrocytes. The impact of this mutation on the phosphatase function of Synj1 resulted in elevated basal autophagosome formation that mimics Synj1 deletion. We found that the membrane expression of the astrocyte-specific glucose transporter GluT-1 was reduced in Synj1-deficient astrocytes. Consistently, AMP-activated protein kinase activity was elevated, suggesting altered glucose sensing in Synj1-deficient astrocytes. Expressing exogenous GluT-1 in Synj1-deficient astrocytes reversed the autophagy impairment, supporting a role for Synj1 in regulating astrocyte autophagy via disrupting glucose-sensing pathways. Thus, our work suggests a novel mechanism for Synj1-related Parkinsonism involving astrocyte dysfunction.


Assuntos
Astrócitos/metabolismo , Autofagossomos/metabolismo , Proteínas do Tecido Nervoso/genética , Doença de Parkinson/genética , Monoéster Fosfórico Hidrolases/genética , Animais , Autofagia , Células Cultivadas , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação de Sentido Incorreto , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/metabolismo , Monoéster Fosfórico Hidrolases/deficiência , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas Quinases/metabolismo , Regulação para Cima
18.
J Biol Chem ; 297(1): 100877, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34139237

RESUMO

The human ZC3H14 gene, which encodes a ubiquitously expressed polyadenosine zinc finger RNA-binding protein, is mutated in an inherited form of autosomal recessive, nonsyndromic intellectual disability. To gain insight into neurological functions of ZC3H14, we previously developed a Drosophila melanogaster model of ZC3H14 loss by deleting the fly ortholog, Nab2. Studies in this invertebrate model revealed that Nab2 controls final patterns of neuron projection within fully developed adult brains, but the role of Nab2 during development of the Drosophila brain is not known. Here, we identify roles for Nab2 in controlling the dynamic growth of axons in the developing brain mushroom bodies, which support olfactory learning and memory, and regulating abundance of a small fraction of the total brain proteome. The group of Nab2-regulated brain proteins, identified by quantitative proteomic analysis, includes the microtubule-binding protein Futsch, the neuronal Ig-family transmembrane protein turtle, the glial:neuron adhesion protein contactin, the Rac GTPase-activating protein tumbleweed, and the planar cell polarity factor Van Gogh, which collectively link Nab2 to the processes of brain morphogenesis, neuroblast proliferation, circadian sleep/wake cycles, and synaptic development. Overall, these data indicate that Nab2 controls the abundance of a subset of brain proteins during the active process of wiring the pupal brain mushroom body and thus provide a window into potentially conserved functions of the Nab2/ZC3H14 RNA-binding proteins in neurodevelopment.


Assuntos
Encéfalo/metabolismo , Proteínas de Drosophila/metabolismo , Neurogênese , Proteoma/genética , Proteínas de Ligação a RNA/metabolismo , Animais , Encéfalo/crescimento & desenvolvimento , Contactinas/genética , Contactinas/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Imunoglobulinas/genética , Imunoglobulinas/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Memória , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteoma/metabolismo , Proteínas de Ligação a RNA/genética
19.
Genes (Basel) ; 12(5)2021 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-34068194

RESUMO

Congenital microcephaly is the clinical presentation of significantly reduced head circumference at birth. It manifests as both non-syndromic-microcephaly primary hereditary (MCPH)-and syndromic forms and shows considerable inter- and intrafamilial variability. It has been hypothesized that additional genetic variants may be responsible for this variability, but data are sparse. We have conducted deep phenotyping and genotyping of five Pakistani multiplex families with either MCPH (n = 3) or Seckel syndrome (n = 2). In addition to homozygous causal variants in ASPM or CENPJ, we discovered additional heterozygous modifier variants in WDR62, CEP63, RAD50 and PCNT-genes already known to be associated with neurological disorders. MCPH patients carrying an additional heterozygous modifier variant showed more severe phenotypic features. Likewise, the phenotype of Seckel syndrome caused by a novel CENPJ variant was aggravated to microcephalic osteodysplastic primordial dwarfism type II (MOPDII) in conjunction with an additional PCNT variant. We show that the CENPJ missense variant impairs splicing and decreases protein expression. We also observed centrosome amplification errors in patient cells, which were twofold higher in MOPDII as compared to Seckel cells. Taken together, these observations advocate for consideration of additional variants in related genes for their role in modifying the expressivity of the phenotype and need to be considered in genetic counseling and risk assessment.


Assuntos
Genes Modificadores , Microcefalia/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas do Tecido Nervoso/genética , Hidrolases Anidrido Ácido/genética , Adulto , Antígenos/genética , Proteínas de Ciclo Celular/genética , Criança , Proteínas de Ligação a DNA/genética , Feminino , Heterozigoto , Humanos , Masculino , Microcefalia/patologia , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação , Linhagem , Fenótipo
20.
FEBS Lett ; 595(15): 1987-1996, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34107052

RESUMO

Microtubule nucleation is mainly mediated by the γ-tubulin ring complex (γTuRC), whose core components are γ-tubulin and γ-tubulin complex proteins GCP2-6. A substantial fraction of γ-tubulin also exists with GCP2 and GCP3 in a tetramer called the γ-tubulin small complex (γTuSC). To date, the mechanisms underlying the turnover of γ-tubulin and GCPs have remained unclear. Here, we show that γ-tubulin, GCP2, and GCP3 are proteolyzed by the ubiquitin-proteasome system, and we identify cullin 1, cullin 4A, and cullin 4B as the E3 ligases that mediate the ubiquitination and, consequently, the degradation of γ-tubulin. Notably, we found that γTuSC disassembly promotes the degradation of γ-tubulin, GCP2, and GCP3, which indicates a role for γTuSCs in the stabilization of its components.


Assuntos
Complexo de Endopeptidases do Proteassoma/metabolismo , Tubulina (Proteína)/metabolismo , Ubiquitina/metabolismo , Linhagem Celular Tumoral , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Estabilidade Proteica , Proteólise , Ubiquitinação
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