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1.
Mol Cell ; 82(1): 75-89.e9, 2022 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-34942120

RESUMO

Circular RNAs (circRNAs) are widely expressed in eukaryotes and are regulated in many biological processes. Although several studies indicate their activity as microRNA (miRNA) and protein sponges, little is known about their ability to directly control mRNA homeostasis. We show that the widely expressed circZNF609 directly interacts with several mRNAs and increases their stability and/or translation by favoring the recruitment of the RNA-binding protein ELAVL1. Particularly, the interaction with CKAP5 mRNA, which interestingly overlaps the back-splicing junction, enhances CKAP5 translation, regulating microtubule function in cancer cells and sustaining cell-cycle progression. Finally, we show that circZNF609 downregulation increases the sensitivity of several cancer cell lines to different microtubule-targeting chemotherapeutic drugs and that locked nucleic acid (LNA) protectors against the pairing region on circZNF609 phenocopy such effects. These data set an example of how the small effects tuned by circZNF609/CKAP5 mRNA interaction might have a potent output in tumor growth and drug response.


Assuntos
Carcinogênese , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Neoplasias/metabolismo , RNA Circular/metabolismo , RNA Mensageiro/metabolismo , Animais , Antineoplásicos/farmacologia , Proteína Semelhante a ELAV 1/genética , Proteína Semelhante a ELAV 1/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Células K562 , Masculino , Camundongos Nus , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/efeitos dos fármacos , Microtúbulos/genética , Microtúbulos/patologia , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , RNA Circular/genética , RNA Mensageiro/genética , Transdução de Sinais , Carga Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto
2.
Nature ; 580(7801): 106-112, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32238932

RESUMO

Radial glial progenitor cells (RGPs) are the major neural progenitor cells that generate neurons and glia in the developing mammalian cerebral cortex1-4. In RGPs, the centrosome is positioned away from the nucleus at the apical surface of the ventricular zone of the cerebral cortex5-8. However, the molecular basis and precise function of this distinctive subcellular organization of the centrosome are largely unknown. Here we show in mice that anchoring of the centrosome to the apical membrane controls the mechanical properties of cortical RGPs, and consequently their mitotic behaviour and the size and formation of the cortex. The mother centriole in RGPs develops distal appendages that anchor it to the apical membrane. Selective removal of centrosomal protein 83 (CEP83) eliminates these distal appendages and disrupts the anchorage of the centrosome to the apical membrane, resulting in the disorganization of microtubules and stretching and stiffening of the apical membrane. The elimination of CEP83 also activates the mechanically sensitive yes-associated protein (YAP) and promotes the excessive proliferation of RGPs, together with a subsequent overproduction of intermediate progenitor cells, which leads to the formation of an enlarged cortex with abnormal folding. Simultaneous elimination of YAP suppresses the cortical enlargement and folding that is induced by the removal of CEP83. Together, these results indicate a previously unknown role of the centrosome in regulating the mechanical features of neural progenitor cells and the size and configuration of the mammalian cerebral cortex.


Assuntos
Centrossomo/metabolismo , Córtex Cerebral/citologia , Córtex Cerebral/embriologia , Células Ependimogliais/citologia , Células-Tronco Neurais/citologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Membrana Celular/metabolismo , Membrana Celular/patologia , Proliferação de Células , Centríolos/metabolismo , Córtex Cerebral/patologia , Feminino , Masculino , Camundongos , Proteínas Associadas aos Microtúbulos/deficiência , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Microtúbulos/patologia , Neurogênese , Proteínas de Sinalização YAP
3.
Prostate ; 84(6): 605-619, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38375594

RESUMO

BACKGROUND: Metastatic castration-resistant prostate cancer (CRPC), the most refractory prostate cancer, inevitably progresses and becomes unresponsive to hormone therapy, revealing a pressing unmet need for this disease. Novel agents targeting HDAC6 and microtubule dynamics can be a potential anti-CRPC strategy. METHODS: Cell proliferation was examined in CRPC PC-3 and DU-145 cells using sulforhodamine B assay and anchorage-dependent colony formation assay. Flow cytometric analysis of propidium iodide staining was used to determine cell-cycle progression. Cell-based tubulin polymerization assay and confocal immunofluorescence microscopic examination determine microtubule assembly/disassembly status. Protein expressions were determined using Western blot analysis. RESULTS: A total of 82 novel derivatives targeting HDAC6 were designed and synthesized, and Compound 25202 stood out, showing the highest efficacy in blocking HDAC6 (IC50, 3.5 nM in enzyme assay; IC50, 1.0 µM in antiproliferative assay in CRPC cells), superior to tubastatin A (IC50, 5.4 µM in antiproliferative assay). The selectivity and superiority of 25202 were validated by examining the acetylation of both α-tubulin and histone H3, detecting cell apoptosis and HDACs enzyme activity assessment. Notably, 25202 but not tubastatin A significantly decreased HDAC6 protein expression. 25202 prolonged mitotic arrest through the detection of cyclin B1 upregulation, Cdk1 activation, mitotic phosphoprotein levels, and Bcl-2 phosphorylation. Compound 25202 did not mimic docetaxel in inducing tubulin polymerization but disrupted microtubule organization. Compound 25202 also increased the phosphorylation of CDC20, BUB1, and BUBR1, indicating the activation of the spindle assembly checkpoint (SAC). Moreover, 25202 profoundly sensitized cisplatin-induced cell death through impairment of cisplatin-evoked DNA damage response and DNA repair in both ATR-Chk1 and ATM-Chk2 pathways. CONCLUSION: The data suggest that 25202 is a novel selective and potent HDAC6 inhibitor. Compound 25202 blocks HDAC6 activity and interferes microtubule dynamics, leading to SAC activation and mitotic arrest prolongation that eventually cause apoptosis of CRPC cells. Furthermore, 25202 sensitizes cisplatin-induced cell apoptosis through impeding DNA damage repair pathways.


Assuntos
Cisplatino , Neoplasias de Próstata Resistentes à Castração , Masculino , Humanos , Cisplatino/farmacologia , Neoplasias de Próstata Resistentes à Castração/patologia , Tubulina (Proteína)/metabolismo , Pontos de Checagem da Fase M do Ciclo Celular , Linhagem Celular Tumoral , Apoptose , Proliferação de Células , Microtúbulos/metabolismo , Microtúbulos/patologia , Desacetilase 6 de Histona/metabolismo
4.
EMBO J ; 39(12): e103499, 2020 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-32368833

RESUMO

Primary cilia are antenna-like organelles on the surface of most mammalian cells that receive sonic hedgehog (Shh) signaling in embryogenesis and carcinogenesis. Cellular cholesterol functions as a direct activator of a seven-transmembrane oncoprotein called Smoothened (Smo) and thereby induces Smo accumulation on the ciliary membrane where it transduces the Shh signal. However, how cholesterol is supplied to the ciliary membrane remains unclear. Here, we report that peroxisomes are essential for the transport of cholesterol into the ciliary membrane. Zellweger syndrome (ZS) is a peroxisome-deficient hereditary disorder with several ciliopathy-related features and cells from these patients showed a reduced cholesterol level in the ciliary membrane. Reverse genetics approaches revealed that the GTP exchange factor Rabin8, the Rab GTPase Rab10, and the microtubule minus-end-directed kinesin KIFC3 form a peroxisome-associated complex to control the movement of peroxisomes along microtubules, enabling communication between peroxisomes and ciliary pocket membranes. Our findings suggest that insufficient ciliary cholesterol levels may underlie ciliopathies.


Assuntos
Colesterol/metabolismo , Cílios/metabolismo , Síndrome de Zellweger/metabolismo , Células Cultivadas , Colesterol/genética , Cílios/genética , Cílios/patologia , Quinases do Centro Germinativo/genética , Quinases do Centro Germinativo/metabolismo , Humanos , Cinesinas/genética , Cinesinas/metabolismo , Microtúbulos/genética , Microtúbulos/metabolismo , Microtúbulos/patologia , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Síndrome de Zellweger/genética , Síndrome de Zellweger/patologia , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo
5.
Ann Neurol ; 93(6): 1158-1172, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-36843330

RESUMO

OBJECTIVE: Identifying cerebrospinal fluid measures of the microtubule binding region of tau (MTBR-tau) species that reflect tau aggregation could provide fluid biomarkers that track Alzheimer's disease related neurofibrillary tau pathological changes. We examined the cerebrospinal fluid (CSF) MTBR-tau species in dominantly inherited Alzheimer's disease (DIAD) mutation carriers to assess the association with Alzheimer's disease (AD) biomarkers and clinical symptoms. METHODS: Cross-sectional and longitudinal CSF from 229 DIAD mutation carriers and 130 mutation non-carriers had sequential characterization of N-terminal/mid-domain phosphorylated tau (p-tau) followed by MTBR-tau species and tau positron emission tomography (tau PET), other soluble tau and amyloid biomarkers, comprehensive clinical and cognitive assessments, and brain magnetic resonance imaging of atrophy. RESULTS: CSF MTBR-tau species located within the putative "border" region and one species corresponding to the "core" region of aggregates in neurofibrillary tangles (NFTs) increased during the presymptomatic stage and decreased during the symptomatic stage. The "border" MTBR-tau species were associated with amyloid pathology and CSF p-tau; whereas the "core" MTBR-tau species were associated stronger with tau PET and CSF measures of neurodegeneration. The ratio of the border to the core species provided a continuous measure of increasing amounts that tracked clinical progression and NFTs. INTERPRETATION: Changes in CSF soluble MTBR-tau species preceded the onset of dementia, tau tangle increase, and atrophy in DIAD. The ratio of 4R-specific MTBR-tau (border) to the NFT (core) MTBR-tau species corresponds to the pathology of NFTs in DIAD and change with disease progression. The dynamics between different MTBR-tau species in the CSF may serve as a marker of tau-related disease progression and target engagement of anti-tau therapeutics. ANN NEUROL 2023;93:1158-1172.


Assuntos
Doença de Alzheimer , Disfunção Cognitiva , Humanos , Doença de Alzheimer/diagnóstico por imagem , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Estudos Transversais , Proteínas tau/metabolismo , Disfunção Cognitiva/diagnóstico por imagem , Disfunção Cognitiva/genética , Disfunção Cognitiva/líquido cefalorraquidiano , Peptídeos beta-Amiloides/metabolismo , Tomografia por Emissão de Pósitrons/métodos , Atrofia/patologia , Biomarcadores/líquido cefalorraquidiano , Progressão da Doença , Microtúbulos/metabolismo , Microtúbulos/patologia
6.
Eur Heart J ; 44(17): 1560-1570, 2023 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-37122097

RESUMO

BACKGROUND: Amyloid plaques and neurofibrillary tangles, the molecular lesions that characterize Alzheimer's disease (AD) and other forms of dementia, are emerging as determinants of proteinopathies 'beyond the brain'. This study aims to establish tau's putative pathophysiological mechanistic roles and potential future therapeutic targeting of tau in heart failure (HF). METHODS AND RESULTS: A mouse model of tauopathy and human myocardial and brain tissue from patients with HF, AD, and controls was employed in this study. Tau protein expression was examined together with its distribution, and in vitro tau-related pathophysiological mechanisms were identified using a variety of biochemical, imaging, and functional approaches. A novel tau-targeting immunotherapy was tested to explore tau-targeted therapeutic potential in HF. Tau is expressed in normal and diseased human hearts, in contradistinction to the current oft-cited observation that tau is expressed specifically in the brain. Notably, the main cardiac isoform is high-molecular-weight (HMW) tau (also known as big tau), and hyperphosphorylated tau segregates in aggregates in HF and AD hearts. As previously described for amyloid-beta, the tauopathy phenotype in human myocardium is of diastolic dysfunction. Perturbation in the tubulin code, specifically a loss of tyrosinated microtubules, emerged as a potential mechanism of myocardial tauopathy. Monoclonal anti-tau antibody therapy improved myocardial function and clearance of toxic aggregates in mice, supporting tau as a potential target for novel HF immunotherapy. CONCLUSION: The study presents new mechanistic evidence and potential treatment for the brain-heart tauopathy axis in myocardial and brain degenerative diseases and ageing.


Assuntos
Doença de Alzheimer , Tauopatias , Humanos , Camundongos , Animais , Proteínas tau/química , Proteínas tau/genética , Proteínas tau/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Tauopatias/metabolismo , Tauopatias/patologia , Microtúbulos/metabolismo , Microtúbulos/patologia , Miocárdio/patologia
7.
Semin Cell Dev Biol ; 110: 34-42, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-32732132

RESUMO

Neural development requires a series of cellular events starting with cell specification, proliferation, and migration. Subsequently, axons and dendrites project from the cell surface to form connections to other neurons, interneurons and glia. Anomalies in any one of these steps can lead to malformation or malfunction of the nervous system. Here we review the critical role the primary cilium plays in the fundamental steps of neurodevelopment. By highlighting human diseases caused by mutations in cilia-associated proteins, it is clear that cilia are essential to multiple neural processes. Furthermore, we explore whether additional aspects of cilia regulation, most notably post-translational modification of the tubulin scaffold in cilia, play underappreciated roles in neural development. Finally, we discuss whether cilia-associated proteins function outside the cilium in some aspects of neurodevelopment. These data underscore both the importance of cilia in the nervous system and some outstanding questions in the field.


Assuntos
Encéfalo/metabolismo , Cílios/metabolismo , Ciliopatias/genética , Proteínas Hedgehog/genética , Deficiência Intelectual/genética , Células de Purkinje/metabolismo , Animais , Axônios/metabolismo , Axônios/patologia , Encéfalo/anormalidades , Encéfalo/crescimento & desenvolvimento , Cílios/ultraestrutura , Ciliopatias/metabolismo , Ciliopatias/patologia , Embrião de Mamíferos , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Regulação da Expressão Gênica , Proteínas Hedgehog/metabolismo , Humanos , Deficiência Intelectual/metabolismo , Deficiência Intelectual/patologia , Interneurônios/metabolismo , Interneurônios/patologia , Microtúbulos/metabolismo , Microtúbulos/patologia , Neurogênese/genética , Neuroglia/metabolismo , Neuroglia/patologia , Células de Purkinje/patologia , Via de Sinalização Wnt
8.
J Biol Chem ; 298(11): 102526, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36162501

RESUMO

Regulation of the neuronal microtubule cytoskeleton is achieved through the coordination of microtubule-associated proteins (MAPs). MAP-Tau, the most abundant MAP in the axon, functions to modulate motor motility, participate in signaling cascades, as well as directly mediate microtubule dynamics. Tau misregulation is associated with a class of neurodegenerative diseases, known as tauopathies, including progressive supranuclear palsy, Pick's disease, and Alzheimer's disease. Many disease-associated mutations in Tau are found in the C-terminal microtubule-binding domain. These mutations decrease microtubule-binding affinity and are proposed to reduce microtubule stability, leading to disease. N-terminal disease-associated mutations also exist, but the mechanistic details of their downstream effects are not as clear. Here, we investigate the effect of the progressive supranuclear palsy-associated N-terminal R5L mutation on Tau-mediated microtubule dynamics using an in vitro reconstituted system. We show that the R5L mutation does not alter Tau interactions with tubulin by fluorescence correlation spectroscopy. Using total internal reflection fluorescence microscopy, we determined that the R5L mutation has no effect on microtubule growth rate, catastrophe frequency, or rescue frequency. Rather, the R5L mutation increases microtubule shrinkage rate. We determine this is due to disruption of Tau patches, larger order Tau complexes known to form on the GDP-microtubule lattice. Altogether, these results provide insight into the role of Tau patches in mediating microtubule dynamics and suggesting a novel mechanism by which mutations in the N-terminal projection domain reduce microtubule stability.


Assuntos
Paralisia Supranuclear Progressiva , Tauopatias , Proteínas tau , Humanos , Microtúbulos/metabolismo , Microtúbulos/patologia , Mutação , Paralisia Supranuclear Progressiva/genética , Paralisia Supranuclear Progressiva/metabolismo , Proteínas tau/genética , Proteínas tau/metabolismo , Tauopatias/genética , Tauopatias/metabolismo
9.
Hum Mol Genet ; 29(24): 3935-3944, 2021 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-33410474

RESUMO

Spinal muscular atrophy (SMA) is a devastating childhood disease primarily affecting lower motoneurons in the spinal cord. SMA is caused by the loss of functional survival of motoneuron (SMN) protein, leading to structural and functional alterations of the cytoskeleton in motoneurons and other cells. Loss of SMN results in impairments of microtubule architecture, but the underlying mechanisms are not completely understood. In this study, we mechanistically analyzed the effects of SMN deficiency on microtubules, demonstrating a reduced stability together with a reduction in alpha tubulin detyrosination. This was caused by increased levels of microtubule-associated protein 1B and tubulin tyrosine ligase, resulting in mitochondrial mislocalization in SMA. Our findings suggest that altered tubulin post-translational modifications and microtubule-associated proteins are involved in the pathomechanisms of SMA, such as an impaired axonal transport of mitochondria.


Assuntos
Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/patologia , Neurônios Motores/patologia , Atrofia Muscular Espinal/patologia , Mutação , Peptídeo Sintases/metabolismo , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Animais , Transporte Axonal , Transporte Biológico , Células Cultivadas , Humanos , Camundongos , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/metabolismo , Mitocôndrias , Neurônios Motores/metabolismo , Atrofia Muscular Espinal/etiologia , Atrofia Muscular Espinal/metabolismo , Peptídeo Sintases/genética , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Tubulina (Proteína)/metabolismo , Tirosina/metabolismo
10.
Cancer ; 129(15): 2348-2359, 2023 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-37080942

RESUMO

BACKGROUND: E7130 is a novel anticancer agent created from a total synthetic study of norhalichondrin B. The authors report the E7130 dose-escalation part of a first-in-human study of patients with advanced solid tumors (NCT03444701). METHODS: Japanese patients ≥20 years of age were enrolled. E7130 was administered intravenously in two cycles: day 1 of a 21-day cycle (Q3W) or days 1 and 15 of a 28-day cycle (Q2W). Doses were escalated from 270 to 550 µg/m2 for the Q3W group or 25-400 µg/m2 for the Q2W group. The primary end point of the dose-escalation phase was safety and tolerability as assessed by the incidence of dose-limiting toxicities (DLTs) and adverse events. Other end points included determination of the maximum tolerated dose (MTD), pharmacokinetics, and pharmacodynamics. RESULTS: Forty-four patients were enrolled: 15 in the E7130 Q3W group and 29 in the Q2W group. Treatment-emergent adverse events (TEAEs) occurred in all patients; the most common TEAE overall was leukopenia (78.6%). Grade 3-4 TEAEs occurred in 93.3% of patients in the Q3W group and 86.2% of patients in the Q2W group. None had a TEAE resulting in study drug discontinuation, and no treatment-related deaths were reported. Per the DLT evaluation, the MTDs were determined as 480 µg/m2 Q3W and 300 µg/m2 Q2W. Significant changes in multiple plasma biomarkers, including vascular endothelial growth factor 3 and matrix metallopeptidase 9, were dose-dependent after initial doses of 350-480 µg/m2 . CONCLUSIONS: E7130 480 µg/m2 Q3W was chosen for the dose-expansion part over 300 µg/m2 Q2W primarily per dose-dependent biomarker results.


Assuntos
Antineoplásicos , Neoplasias , Humanos , Fator A de Crescimento do Endotélio Vascular , Microambiente Tumoral , Neoplasias/patologia , Antineoplásicos/efeitos adversos , Biomarcadores , Microtúbulos/metabolismo , Microtúbulos/patologia , Dose Máxima Tolerável
11.
Liver Int ; 43(12): 2794-2807, 2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-37833852

RESUMO

BACKGROUND AND AIMS: Hepatocellular carcinoma (HCC) is a typically fatal malignancy with limited treatment options and poor survival rates, despite recent FDA approvals of newer treatment options. We aim to address this unmet need by using a proprietary computational drug discovery platform that identifies drug candidates with the potential to advance rapidly and successfully through preclinical studies. METHODS: We generated an in silico model of HCC biology to identify the top 10 small molecules with predicted efficacy. The most promising candidate, CYT997, was tested for its in vitro effects on cell viability and cell death, colony formation, cell cycle changes, and cell migration/invasion in HCC cells. We used an HCC patient-derived xenograft (PDX) mouse model to assess its in vivo efficacy. RESULTS: CYT997 was significantly more cytotoxic against HCC cells than against primary human hepatocytes, and sensitized HCC cells to sorafenib. It arrested cell cycle at the G2/M phase with associated up-regulations of p21, p-MEK1/2, p-ERK, and down-regulation of cyclin B1. Cell apoptosis and senescence-like morphology were also observed. CYT997 inhibited HCC cell migration and invasion, and down-regulated the expressions of acetylated tubulins, ß-tubulin, glypican-3 (GPC3), ß-catenin, and c-Myc. In vivo, CYT997 (20 mg/kg, three times weekly by oral gavage) significantly inhibited PDX growth, while being non-toxic to mice. Immunohistochemistry confirmed the down-regulation of GPC3, c-Myc, and Ki-67, supporting its anti-proliferative effect. CONCLUSION: CYT997 is a potentially efficacious and non-toxic drug candidate for HCC therapy. Its ability to down-regulate GPC3, ß-catenin, and c-Myc highlights a novel mechanism of action.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Camundongos , Animais , Carcinoma Hepatocelular/patologia , beta Catenina/metabolismo , Neoplasias Hepáticas/patologia , Apoptose , Microtúbulos/metabolismo , Microtúbulos/patologia , Linhagem Celular Tumoral , Proliferação de Células , Glipicanas
12.
Basic Res Cardiol ; 117(1): 41, 2022 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-36006489

RESUMO

The mechanical environment of the myocardium has a potent effect on cardiomyocyte form and function, yet an understanding of the cardiomyocyte responses to extracellular stiffening remains incomplete. We therefore employed a cell culture substrate with tunable stiffness to define the cardiomyocyte responses to clinically relevant stiffness increments in the absence of cell-cell interactions. When cultured on substrates magnetically actuated to mimic the stiffness of diseased myocardium, isolated rat adult cardiomyocytes exhibited a time-dependent reduction of sarcomere shortening, characterized by slowed contraction and relaxation velocity, and alterations of the calcium transient. Cardiomyocytes cultured on stiff substrates developed increases in viscoelasticity and microtubule detyrosination in association with early increases in the α-tubulin detyrosinating enzyme vasohibin-2 (Vash2). We found that knockdown of Vash2 was sufficient to preserve contractile performance as well as calcium transient properties in the presence of extracellular substrate stiffening. Orthogonal prevention of detyrosination by overexpression of tubulin tyrosine ligase (TTL) was also able to preserve contractility and calcium homeostasis. These data demonstrate that a pathologic increment of extracellular stiffness induces early, cell-autonomous remodeling of adult cardiomyocytes that is dependent on detyrosination of α-tubulin.


Assuntos
Microtúbulos , Miócitos Cardíacos , Animais , Cálcio , Microtúbulos/patologia , Microtúbulos/fisiologia , Miocárdio , Miócitos Cardíacos/patologia , Miócitos Cardíacos/fisiologia , Ratos , Tubulina (Proteína)/química
13.
Reprod Biol Endocrinol ; 20(1): 154, 2022 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-36329464

RESUMO

The importance of actin and microtubule (MT) cytoskeletons in testis function in rodents is known to some extent, but its role in the etiology of azoospermia in humans remains unexplored. Here, we examined if MT cytoskeleton was defective in NOA (non-obstructive azoospermia) testes versus normal human testes based on histopathological, immunofluorescence (IF), and scRNA-Seq transcriptome profiling. Testis biopsy samples from n = 6 normal men versus n = 3 Sertoli cell only (SCO) and n = 3 MA (meiotic arrest) of NOA patients were used for histopathological analysis. IF analysis was also used to examine MT organization across the seminiferous epithelium, investigating the likely involvement of microtubule-associated proteins (MAPs). scRNA-Seq transcriptome profiling datasets from testes of 3 SCO patients versus 3 normal men in public domain in Gene Expression Omnibus (GEO) Sample (GSM) with identifiers were analyzed to examine relevant genes that regulate MT dynamics. NOA testes of MA and SCO patients displayed notable defects in MT organization across the epithelium with extensive truncation, mis-alignments and appeared as collapsed structures near the base of the tubules. These changes are in contrast to MTs in testes of normal men. scRNA-Seq analyses revealed considerable loss of spermatogenesis capacity in SCO testes of NOA patients versus normal men. An array of genes that support MT dynamics displayed considerable changes in expression and in spatial distribution. In summary, defects in MT cytoskeleton were noted in testes of NOA (SCO) patients, possibly mediated by defective spatial expression and/or distribution of MAPs. These changes, in turn, may impede spermatogenesis in SCO testes of NOA patients.


Assuntos
Azoospermia , Humanos , Masculino , Azoospermia/genética , Azoospermia/patologia , Testículo/metabolismo , Espermatogênese/genética , Microtúbulos/metabolismo , Microtúbulos/patologia , Citoesqueleto/genética , Citoesqueleto/metabolismo
14.
Mov Disord ; 37(3): 598-607, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34927746

RESUMO

BACKGROUND: Haploinsufficiency is widely accepted as the pathogenic mechanism of spastic paraplegia type 4 (SPG4). However, there are some cases that cannot be explained by reduced function of the spastin protein encoded by SPAST. OBJECTIVES: To identify the causative gene of autosomal dominant hereditary spastic paraplegia in three large Chinese families and explore the pathological mechanism of a spastin variant. METHODS: Three large Chinese hereditary spastic paraplegia families with a total of 247 individuals (67 patients) were investigated, of whom 59 members were recruited to the study. Genetic testing was performed to identify the causative gene. Western blotting and immunofluorescence were used to analyze the effects of the mutant proteins in vitro. RESULTS: In the three hereditary spastic paraplegia families, of whom three index cases were misdiagnosed as other types of neurological diseases, a novel c.985dupA (p.Met329Asnfs*3) variant in SPAST was identified and was shown to cosegregate with the phenotype in the three families. The c.985dupA mutation produced two truncated mutants (mutant M1 and M87 isoforms) that accumulated to a higher level than their wild-type counterparts. Furthermore, the mutant M1 isoform heavily decorated the microtubules and rendered them resistant to depolymerization. In contrast, the mutant M87 isoform was diffusely localized in both the nucleus and the cytoplasm, could not decorate microtubules, and was not able to promote microtubule disassembly. CONCLUSIONS: SPAST mutations leading to premature stop codons do not always act through haploinsufficiency. The truncated spastin may damage the corticospinal tracts through an isoform-specific toxic effect.


Assuntos
Paraplegia Espástica Hereditária , Humanos , Microtúbulos/genética , Microtúbulos/metabolismo , Microtúbulos/patologia , Mutação/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Paraplegia Espástica Hereditária/genética , Espastina/genética , Espastina/metabolismo
15.
J Pharmacol Sci ; 150(4): 259-266, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36344048

RESUMO

Uterine leiomyosarcoma is an aggressive soft tissue tumor. Stathmin, a phosphoprotein that modulates microtubule dynamics, is highly expressed in many malignancies including leiomyosarcoma. The microtubule-depolymerizing agent eribulin has been recently approved for treating malignant soft tissue tumors. Although eribulin inhibits microtubule polymerization, little is known about the relationship between eribulin treatment and stathmin dynamics. In this study, we explored the role of stathmin expression in the action of eribulin in leiomyosarcoma cells. Eribulin induced phosphorylation of stathmin and reduced expression of subunits A and C of protein phosphatase 2A (PP2A) in a leiomyosarcoma cell line. The PP2A activator FTY720 reduced levels of phosphorylated stathmin. Eribulin decreased stathmin protein levels without affecting stathmin mRNA expression. Furthermore, stathmin knockdown attenuated the inhibitory effects of eribulin on cell viability, whereas stathmin overexpression enhanced the anti-proliferative effect of eribulin. Eribulin-resistant leiomyosarcoma cell lines had enhanced expression of the class Ⅰ ß-tubulin TUBB1, multi-drug resistance 1 protein MDR1 and breast cancer-resistance protein BCRP, and decreased expression of stathmin. Taken together, these results suggest that stathmin expression modulates the pharmacological efficacy of eribulin in uterine leiomyosarcoma cells.


Assuntos
Leiomiossarcoma , Estatmina , Humanos , Estatmina/genética , Estatmina/metabolismo , Estatmina/farmacologia , Leiomiossarcoma/tratamento farmacológico , Leiomiossarcoma/genética , Leiomiossarcoma/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Neoplasias/metabolismo , Microtúbulos/metabolismo , Microtúbulos/patologia
16.
Clin Exp Pharmacol Physiol ; 49(4): 445-452, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35066919

RESUMO

Breast cancer is the most common malignant neoplasm among women, responsible for 30% of all malignant tumours, and the second most significant reason of cancer fatality in women. Treatment failure and tumour recurrence are common outcomes of chemotherapy when patients develop multidrug resistance (MDR). New therapeutic methods like molecularly targeted therapeutic interventions need a thorough understanding of malignant tumour's molecular processes. Numerous studies published in the last few years indicate that stathmin and tubulin-associated units (tau) are upregulated in a range of human malignant tumours, suggesting that they may enhance the incidence and progression of malignancies. By promoting cancer cell reproduction, infiltration and generating drug resistance, these proteins aid in the disease's development. Existing information on the expression of tau protein and stathmin in breast cancer, as well as their involvement in treatment methods, is summarized in this literature review.


Assuntos
Neoplasias da Mama , Estatmina , Neoplasias da Mama/patologia , Feminino , Humanos , Masculino , Microtúbulos/metabolismo , Microtúbulos/patologia , Recidiva Local de Neoplasia/metabolismo , Estatmina/metabolismo
17.
Hum Mol Genet ; 28(24): 4043-4052, 2019 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-29893868

RESUMO

Mutations in the lamin A/C gene (LMNA) cause an autosomal dominant inherited form of dilated cardiomyopathy associated with cardiac conduction disease (hereafter referred to as LMNA cardiomyopathy). Compared with other forms of dilated cardiomyopathy, mutations in LMNA are responsible for a more aggressive clinical course owing to a high rate of malignant ventricular arrhythmias. Gap junctions are intercellular channels that allow direct communication between neighboring cells, which are involved in electrical impulse propagation and coordinated contraction of the heart. For gap junctions to properly control electrical synchronization in the heart, connexin-based hemichannels must be correctly targeted to intercalated discs, Cx43 being the major connexin in the working myocytes. We here showed an altered distribution of Cx43 in a mouse model of LMNA cardiomyopathy. However, little is known on the molecular mechanisms of Cx43 remodeling in pathological context. We now show that microtubule cytoskeleton alteration and decreased acetylation of α-tubulin lead to remodeling of Cx43 in LMNA cardiomyopathy, which alters the correct communication between cardiomyocytes, ultimately leading to electrical conduction disturbances. Preventing or reversing this process could offer a strategy to repair damaged heart. Stabilization of microtubule cytoskeleton using Paclitaxel improved intraventricular conduction defects. These results indicate that microtubule cytoskeleton contributes to the pathogenesis of LMNA cardiomyopathy and that drugs stabilizing the microtubule may be beneficial for patients.


Assuntos
Cardiomiopatias/genética , Cardiomiopatias/metabolismo , Conexina 43/metabolismo , Lamina Tipo A/genética , Paclitaxel/farmacologia , Acetilação/efeitos dos fármacos , Animais , Doença do Sistema de Condução Cardíaco/genética , Cardiomiopatias/patologia , Conexina 43/genética , Citoesqueleto/metabolismo , Citoesqueleto/patologia , Junções Comunicantes/efeitos dos fármacos , Junções Comunicantes/metabolismo , Junções Comunicantes/patologia , Lamina Tipo A/metabolismo , Masculino , Camundongos , Camundongos Knockout , Microtúbulos/metabolismo , Microtúbulos/patologia , Mutação , Miocárdio/patologia , Miócitos Cardíacos/patologia
18.
J Cell Sci ; 132(3)2019 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-30635446

RESUMO

Sperm cells are highly specialized mammalian cells, and their biogenesis requires unique intracellular structures. Perturbation of spermatogenesis often leads to male infertility. Here, we assess the role of a post-translational modification of tubulin, glutamylation, in spermatogenesis. We show that mice lacking the tubulin deglutamylase CCP5 (also known as AGBL5) do not form functional sperm. In these mice, spermatids accumulate polyglutamylated tubulin, accompanied by the occurrence of disorganized microtubule arrays, in particular in the sperm manchette. Spermatids further fail to re-arrange their intracellular space and accumulate organelles and cytosol, while nuclei condense normally. Strikingly, spermatids lacking CCP5 show supernumerary centrioles, suggesting that glutamylation could control centriole duplication. We show that most of these observed defects are also present in mice in which CCP5 is deleted only in the male germ line, strongly suggesting that they are germ-cell autonomous. Our findings reveal that polyglutamylation is, beyond its known importance for sperm flagella, an essential regulator of several microtubule-based functions during spermatogenesis. This makes enzymes involved in glutamylation prime candidates for being genes involved in male sterility.


Assuntos
Carboxipeptidases/genética , Infertilidade Masculina/genética , Microtúbulos/metabolismo , Processamento de Proteína Pós-Traducional , Espermátides/metabolismo , Espermatogênese/genética , Tubulina (Proteína)/metabolismo , Animais , Carboxipeptidases/deficiência , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Centríolos/metabolismo , Centríolos/patologia , Centríolos/ultraestrutura , Citosol/metabolismo , Citosol/ultraestrutura , Ácido Glutâmico/metabolismo , Humanos , Infertilidade Masculina/metabolismo , Infertilidade Masculina/patologia , Masculino , Camundongos , Camundongos Knockout , Microtúbulos/patologia , Microtúbulos/ultraestrutura , Cauda do Espermatozoide/metabolismo , Cauda do Espermatozoide/patologia , Cauda do Espermatozoide/ultraestrutura , Espermátides/patologia , Espermátides/ultraestrutura , Tubulina (Proteína)/genética
19.
Toxicol Appl Pharmacol ; 421: 115534, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-33852878

RESUMO

Monomethyl auristatin E (MMAE) is a potent anti-cancer microtubule-targeting agent (MTA) used as a payload in three approved MMAE-containing antibody drug conjugates (ADCs) and multiple ADCs in clinical development to treat different types of cancers. Unfortunately, MMAE-ADCs can induce peripheral neuropathy, a frequent adverse event leading to treatment dose reduction or discontinuation and subsequent clinical termination of many MMAE-ADCs. MMAE-ADC-induced peripheral neuropathy is attributed to non-specific uptake of the ADC in peripheral nerves and release of MMAE, disrupting microtubules (MTs) and causing neurodegeneration. However, molecular mechanisms underlying MMAE and MMAE-ADC effects on MTs remain unclear. Here, we characterized MMAE-tubulin/MT interactions in reconstituted in vitro soluble tubulin or MT systems and evaluated MMAE and vcMMAE-ADCs in cultured human MCF7 cells. MMAE bound to soluble tubulin heterodimers with a maximum stoichiometry of ~1:1, bound abundantly along the length of pre-assembled MTs and with high affinity at MT ends, introduced structural defects, suppressed MT dynamics, and reduced the kinetics and extent of MT assembly while promoting tubulin ring formation. In cells, MMAE and MMAE-ADC (via nonspecific uptake) suppressed proliferation, mitosis and MT dynamics, and disrupted the MT network. Comparing MMAE action to other MTAs supports the hypothesis that peripheral neuropathy severity is determined by the precise mechanism(s) of each individual drug-MT interaction (location of binding, affinity, effects on morphology and dynamics). This work demonstrates that MMAE binds extensively to tubulin and MTs and causes severe MT dysregulation, providing convincing evidence that MMAE-mediated inhibition of MT-dependent axonal transport leads to severe peripheral neuropathy.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Microtúbulos/efeitos dos fármacos , Oligopeptídeos/toxicidade , Doenças do Sistema Nervoso Periférico/induzido quimicamente , Sistema Nervoso Periférico/efeitos dos fármacos , Moduladores de Tubulina/toxicidade , Tubulina (Proteína)/metabolismo , Transporte Axonal/efeitos dos fármacos , Sítios de Ligação , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Células MCF-7 , Microtúbulos/metabolismo , Microtúbulos/patologia , Mitose/efeitos dos fármacos , Oligopeptídeos/metabolismo , Sistema Nervoso Periférico/metabolismo , Sistema Nervoso Periférico/patologia , Doenças do Sistema Nervoso Periférico/metabolismo , Doenças do Sistema Nervoso Periférico/patologia , Ligação Proteica , Medição de Risco , Fuso Acromático/efeitos dos fármacos , Fuso Acromático/metabolismo , Fuso Acromático/patologia , Moduladores de Tubulina/metabolismo
20.
BMC Cancer ; 21(1): 981, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34470602

RESUMO

BACKGROUND: Paclitaxel (Taxol) is a microtubule-stabilizing drug used to treat several solid tumors, including ovarian, breast, non-small cell lung, and pancreatic cancers. The current treatment of ovarian cancer is chemotherapy using paclitaxel in combination with carboplatin as a frontline agent, and paclitaxel is also used in salvage treatment as a second line drug with a dose intensive regimen following recurrence. More recently, a dose dense approach for paclitaxel has been used to treat metastatic breast cancer with success. Paclitaxel binds to beta tubulin with high affinity and stabilizes microtubule bundles. As a consequence of targeting microtubules, paclitaxel kills cancer cells through inhibition of mitosis, causing mitotic catastrophes, and by additional, not yet well defined non-mitotic mechanism(s). RESULTS: In exploring methods to modulate activity of paclitaxel in causing cancer cell death, we unexpectedly found that a brief exposure of paclitaxel-treated cells in culture to low intensity ultrasound waves prevented the paclitaxel-induced cytotoxicity and death of the cancer cells. The treatment with ultrasound shock waves was found to transiently disrupt the microtubule cytoskeleton and to eliminate paclitaxel-induced rigid microtubule bundles. When cellular microtubules were labelled with a fluorescent paclitaxel analog, exposure to ultrasound waves led to the disassembly of the labeled microtubules and localization of the signals to perinuclear compartments, which were determined to be lysosomes. CONCLUSIONS: We suggest that ultrasound disrupts the paclitaxel-induced rigid microtubule cytoskeleton, generating paclitaxel bound fragments that undergo degradation. A new microtubule network forms from tubulins that are not bound by paclitaxel. Hence, ultrasound shock waves are able to abolish paclitaxel impact on microtubules. Thus, our results demonstrate that a brief exposure to low intensity ultrasound can reduce and/or eliminate cytotoxicity associated with paclitaxel treatment of cancer cells in cultures.


Assuntos
Neoplasias da Mama/patologia , Microtúbulos/patologia , Mitose , Neoplasias Ovarianas/patologia , Paclitaxel/farmacologia , Ondas Ultrassônicas , Antineoplásicos Fitogênicos/farmacologia , Apoptose , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/radioterapia , Proliferação de Células , Citoesqueleto/metabolismo , Feminino , Humanos , Microtúbulos/efeitos dos fármacos , Microtúbulos/efeitos da radiação , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/radioterapia , Tubulina (Proteína)/metabolismo , Células Tumorais Cultivadas
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