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1.
J Cell Sci ; 137(13)2024 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-38841887

RESUMEN

Centrosomal proteins play pivotal roles in orchestrating microtubule dynamics, and their dysregulation leads to disorders, including cancer and ciliopathies. Understanding the multifaceted roles of centrosomal proteins is vital to comprehend their involvement in disease development. Here, we report novel cellular functions of CEP41, a centrosomal and ciliary protein implicated in Joubert syndrome. We show that CEP41 is an essential microtubule-associated protein with microtubule-stabilizing activity. Purified CEP41 binds to preformed microtubules, promotes microtubule nucleation and suppresses microtubule disassembly. When overexpressed in cultured cells, CEP41 localizes to microtubules and promotes microtubule bundling. Conversely, shRNA-mediated knockdown of CEP41 disrupts the interphase microtubule network and delays microtubule reassembly, emphasizing its role in microtubule organization. Further, we demonstrate that the association of CEP41 with microtubules relies on its conserved rhodanese homology domain (RHOD) and the N-terminal region. Interestingly, a disease-causing mutation in the RHOD domain impairs CEP41-microtubule interaction. Moreover, depletion of CEP41 inhibits cell proliferation and disrupts cell cycle progression, suggesting its potential involvement in cell cycle regulation. These insights into the cellular functions of CEP41 hold promise for unraveling the impact of its mutations in ciliopathies.


Asunto(s)
Proliferación Celular , Microtúbulos , Humanos , Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Centrosoma/metabolismo , Retina/metabolismo , Retina/patología , Retina/anomalías , Ciliopatías/metabolismo , Ciliopatías/genética , Ciliopatías/patología , Cerebelo/metabolismo , Cerebelo/anomalías , Cerebelo/patología , Enfermedades Renales Quísticas/metabolismo , Enfermedades Renales Quísticas/genética , Enfermedades Renales Quísticas/patología , Cilios/metabolismo , Cilios/patología , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Animales , Anomalías Múltiples/metabolismo , Anomalías Múltiples/genética , Anomalías Múltiples/patología , Anomalías del Ojo/metabolismo , Anomalías del Ojo/genética , Anomalías del Ojo/patología , Unión Proteica , Ciclo Celular/genética , Células HEK293
2.
IUBMB Life ; 2024 Jul 22.
Artículo en Inglés | MEDLINE | ID: mdl-39038059

RESUMEN

Estramustine (EM), a clinically successful hormone-refractory anti-prostate cancer drug, exhibited potent anti-proliferative activity, depolymerized microtubules, blocked cells at mitosis, and induced cell death in different cancer cells. Altered iron metabolism is a feature of cancer cells. Using EM, we examined the plausible relationship between microtubule depolymerization and induction of ferroptosis in human neuroblastoma (SH-SY5Y and IMR-32) cells. EM reduced glutathione (GSH) levels and induced reactive oxygen species (ROS) generation. The pre-treatment of neuroblastoma cells with ROS scavengers (N-acetyl cysteine and dithiothreitol) reduced the anti-proliferative effects of EM. EM treatment increased labile iron pool (LIP), depleted glutathione peroxidase 4 (GPX4) levels, and lipid peroxidation, hallmark features of ferroptosis, highlighting ferroptosis induction. Ferroptosis inhibitors (deferoxamine mesylate and liproxstatin-1) abrogated the cytotoxic effects of EM, further confirming ferroptosis induction. Vinblastine and nocodazole also increased LIP and induced lipid peroxidation in neuroblastoma cells. This study provides evidence for the coupling of microtubule integrity to ferroptosis. The results also suggest that microtubule-depolymerizing agents may be considered for developing pro-ferroptosis chemotherapeutics.

3.
Mol Cell Biochem ; 478(9): 1961-1971, 2023 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-36585545

RESUMEN

ß-tubulin isotypes regulate the structure and bundling of microtubule (MT) lattice, its dynamics, and resulting functions. They exhibit differential tissue expression, varying due to physical and biochemical cues. In this work, we investigated the effect of transient heat shock at 42 °C on the nuclear and cytoplasmic stiffness of SH-SY5Y neuroblastoma cells through atomic force microscopy. Moreover, the variations in the expression of ß-tubulin isotypes as a heat shock response were also monitored. The heat-exposed cells endured a recovery at 37 °C for 24 h and they manifested an increase of cytoplasmic stiffness by 130 ± 25% with respect to untreated controls. The expression of ß-II tubulin isotype in heat-recovered cells is augmented by 51 ± 5% whereas the levels of total tubulin and ß-III tubulin isotype remain unaltered. Upon depletion of ß-II tubulin isotype using shRNA, the increase in cytoplasmic stiffness was dampened. However, it remained unaffected upon depletion with ß-III tubulin isotype shRNA. This features the role of the ß-II tubulin isotype in regulating cellular stiffness. In addition, neuroblastoma SH-SY5Y cells undergo differentiation by initiating neuritogenesis and prior evidence suggests the indispensable role of ß-II tubulin isotype in this process. The heat-recovered cells which expressed higher levels of ß-II tubulin isotype expedited the differentiation process in 3-day which was around 5-day for control cells, however, upon depletion of ß-II tubulin isotype, the cells almost lost their differentiation potential. Altogether, this work highlights the role of ß-II tubulin isotype as a biomarker for cellular stiffness.


Asunto(s)
Neuroblastoma , Tubulina (Proteína) , Humanos , Tubulina (Proteína)/metabolismo , Neuroblastoma/metabolismo , Microtúbulos/metabolismo , Diferenciación Celular , ARN Interferente Pequeño/metabolismo
4.
Bioorg Med Chem ; 95: 117489, 2023 11 15.
Artículo en Inglés | MEDLINE | ID: mdl-37816266

RESUMEN

In this study of creating new molecules from clinical trial agents, an approach of Combretastatin structural modulation with the installation of NP-privileged motifs was considered, and a series of trimethoxyphenyl-2-aminoimidazole with functionalized quinolines and isoquinolines was investigated. An exciting method of quinoline C3-H iodination coupled with imidazopyridine-C3-H arylation and hydrazine-mediated fused-ring cleavage enabled synthesizing a class of compounds with two specific unsymmetric aryl substitutions. Interestingly, three compounds (6, 11, and 13) strongly inhibited HeLa cell proliferation with a half-maximal inhibitory concentration (10-46 nM). Among the compounds, compound 6 (QTMP) showed stronger antiproliferative ability than CA-4 (a clinical trial agent) in various cancer cell lines, including cervical, lung, breast, highly metastatic breast, and melanoma cells. QTMP inhibited the assembly of purified tubulin, depolymerized microtubules of A549 lung carcinoma cells, produced defective spindles, and arrested the cells in the G2/M phase. Further, QTMP binds to the colchicine site in tubulin with a dissociation constant of 5.0 ± 0.6 µM. QTMP displayed higher aqueous stability than CA-4 at 37 °C. Further, in silico analysis of QTMP indicated excellent drug-like properties, including good aqueous solubility, balanced hydrophilicity-lipophilicity, and high GI-absorption ability. The results together suggest that QTMP has anticancer potential.


Asunto(s)
Antineoplásicos , Tubulina (Proteína) , Humanos , Tubulina (Proteína)/metabolismo , Estructura Molecular , Relación Estructura-Actividad , Moduladores de Tubulina/farmacología , Células HeLa , Línea Celular Tumoral , Antineoplásicos/farmacología , Antineoplásicos/química , Proliferación Celular , Ensayos de Selección de Medicamentos Antitumorales
5.
Biochem J ; 479(14): 1543-1558, 2022 07 29.
Artículo en Inglés | MEDLINE | ID: mdl-35789252

RESUMEN

The respiratory pathogen, Streptococcus pneumoniae has acquired multiple-drug resistance over the years. An attractive strategy to combat pneumococcal infection is to target cell division to inhibit the proliferation of S. pneumoniae. This work presents Vitamin K3 as a potential anti-pneumococcal drug that targets FtsZ, the master coordinator of bacterial cell division. Vitamin K3 strongly inhibited S. pneumoniae proliferation with a minimum inhibitory concentration (MIC) and a minimum bactericidal concentration (MBC) of 6 µg/ml. Vitamin K3 disrupted the Z-ring localization in both S. pneumoniae and Bacillus subtilis within 30 min of treatment, while the membrane integrity and nucleoid segregation remain unchanged. Several complementary experiments showed that Vitamin K3 inhibits the assembly of purified S. pneumoniae FtsZ (SpnFtsZ) and induces conformational changes in the protein. Interestingly, Vitamin K3 interfered with GTP binding onto FtsZ and increased the GTPase activity of FtsZ polymers. The intrinsic tryptophan fluorescence of SpnFtsZ revealed that Vitamin K3 delays the nucleation of FtsZ polymers and reduces the rate of polymerization. In the presence of a non-hydrolyzable analog of GTP, Vitamin K3 did not show inhibition of FtsZ polymerization. These results indicated that Vitamin K3 induces conformational changes in FtsZ that increase GTP hydrolysis and thereby, destabilize the FtsZ polymers. Together, our data provide evidence that Vitamin K3 derives its potent anti-pneumococcal activity by inhibiting FtsZ assembly.


Asunto(s)
Streptococcus pneumoniae , Vitamina K 3 , Bacillus subtilis , Proteínas Bacterianas/química , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/genética , Guanosina Trifosfato/metabolismo , Polímeros/metabolismo , Streptococcus pneumoniae/metabolismo , Vitamina K 3/metabolismo
6.
FASEB J ; 35(4): e21539, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33742719

RESUMEN

An aberrant accumulation of nuclear ß-catenin is closely associated with the augmentation of cancer malignancy. In this work, we report that several microtubule-targeting agents (MTAs) such as vinblastine, taxol, and C12 (combretastatin-2-aminoimidazole analog) inhibit Wnt/ß-catenin signaling in oral squamous cell carcinoma (OSCC). We showed that the inhibition of microtubule dynamics by MTAs decreased the level of ß-catenin by increasing Axin and adenomatous polyposis coli levels and reducing the level of dishevelled. Furthermore, MTAs strongly reduced the localization of ß-catenin in the nucleus. The reduction in the level of nuclear ß-catenin was neither due to the degradation of ß-catenin in the nucleus nor due to an increase in the export of nuclear ß-catenin from the nucleus. A motor protein kinesin-2 was found to assist the nuclear transportation of ß-catenin. Interestingly, Wnt/ß-catenin signaling antagonist treatment synergized with MTAs and the activators of Wnt/ß-catenin signaling antagonized with the MTAs. C12 potently suppressed the growth of 4-Nitroquinoline 1-oxide-induced OSCC in the tongue of C57 black 6 mice and also abrogated Wnt/ß-catenin signaling pathway in the tumor. Our results provide evidence that the decrease in Wnt/ß-catenin signaling is an important antitumor effect of MTAs and the combined use of MTAs with Wnt/ß-catenin signaling antagonists could be a promising strategy for cancer chemotherapy.


Asunto(s)
Carcinoma de Células Escamosas/metabolismo , Microtúbulos/metabolismo , Neoplasias de la Boca/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismo , beta Catenina/metabolismo , Transporte Activo de Núcleo Celular/fisiología , Animales , Línea Celular Tumoral , Humanos , Ratones Endogámicos C57BL , Vía de Señalización Wnt/fisiología
7.
Phys Chem Chem Phys ; 24(27): 16694-16700, 2022 Jul 13.
Artículo en Inglés | MEDLINE | ID: mdl-35766982

RESUMEN

Microtubules (MTs) are widely targeted for the treatment of various types of cancer due to their essential role in cell division. MTs are polymers made of αß-tubulin heterodimers. These α- and ß-tubulins have 8 and 10 different isotypes, respectively. It is known that a few tubulin isotypes have anti-cancer drug resistance properties, especially ßIII, which shows poor sensitivity to many potent anti-cancer drugs such as eribulin. However, the molecular-level understanding of drug-resistance due to tubulin isotype variation is poorly understood. This paper presents the study of differential binding affinities of different tubulin isotypes with the potent anti-cancer drug eribulin. Eribulin (MT destabilizer) binds at the inter-dimer interface of MTs near the vinca site and induces a lattice deformation, which results in catastrophic events in MT dynamics. In this study, sequence analysis has been done throughway and the binding sites and based on that 2α-tubulin isotypes (αI and αVIII) and 7ß tubulin isotypes (ßI, ßIIa, ßIII, ßIVa, ßVI, ßVII and ßVIII) were selected. In total, 14 combinations were prepared after building homology models of these selected isotypes. Molecular docking and molecular dynamics simulations were performed to deeply understand the binding mode of eribulin at different MT compositions. RMSD, RMSF, radius of gyration, SASA, ligand-protein interactions, and calculations of binding free energy were performed to investigate the eribulin binding variations to tubulin isotypes and it was found that αIßII showed the maximum binding affinity among all 14 systems to eribulin. The ßIII-tubulin isotype, which shows low sensitivity to eribulin in experimental results, had the least binding affinity in the system αVIIIßIII complex and the average binding affinity in the system αIßIII among all 14 systems. Additionally, we performed steered MD simulations and DynDom analysis of the systems with the lowest binding energy (αIßII) and the highest binding energy (αVIIIßIII) and extracted force, displacement, and H-bonding profiles during the pulling simulations to get a better insight.


Asunto(s)
Antineoplásicos , Tubulina (Proteína) , Antineoplásicos/metabolismo , Furanos , Humanos , Cetonas , Microtúbulos , Simulación del Acoplamiento Molecular , Unión Proteica , Isoformas de Proteínas/metabolismo , Tubulina (Proteína)/química
8.
Biochem J ; 478(1): 179-196, 2021 01 15.
Artículo en Inglés | MEDLINE | ID: mdl-33346336

RESUMEN

Human body temperature limits below 40°C during heat stroke or fever. The implications of prolonged exposure to the physiologically relevant temperature (40°C) on cellular mechanobiology is poorly understood. Here, we have examined the effects of heat stress (40°C for 72 h incubation) in human lung adenocarcinoma (A549), mouse melanoma (B16F10), and non-cancerous mouse origin adipose tissue cells (L929). Hyperthermia increased the level of ROS, γ-H2AX and HSP70 and decreased mitochondrial membrane potential in the cells. Heat stress impaired cell division, caused G1 arrest, induced cellular senescence, and apoptosis in all the tested cell lines. The cells incubated at 40°C for 72 h displayed a significant decrease in the f-actin level and cellular traction as compared with cells incubated at 37°C. Also, the cells showed a larger focal adhesion area and stronger adhesion at 40°C than at 37°C. The mitotic cells at 40°C were unable to round up properly and displayed retracting actin stress fibers. Hyperthermia down-regulated HDAC6, increased the acetylation level of microtubules, and perturbed the chromosome alignment in the mitotic cells at 40°C. Overexpression of HDAC6 rescued the cells from the G1 arrest and reduced the delay in cell rounding at 40°C suggesting a crucial role of HDAC6 in hyperthermia mediated responses. This study elucidates the significant role of cellular traction, focal adhesions, and cytoskeletal networks in mitotic cell rounding and chromosomal misalignment. It also highlights the significance of HDAC6 in heat-evoked senile cellular responses.


Asunto(s)
Senescencia Celular/fisiología , Citoesqueleto/metabolismo , Puntos de Control de la Fase G1 del Ciclo Celular , Histona Desacetilasa 6/metabolismo , Hipertermia/metabolismo , Fibras de Estrés/metabolismo , Acetilación , Actinas/metabolismo , Apoptosis/fisiología , Adhesión Celular/fisiología , División Celular/fisiología , Línea Celular Tumoral , Proliferación Celular/fisiología , Forma de la Célula , Tamaño de la Célula , Puntos de Control de la Fase G1 del Ciclo Celular/genética , Puntos de Control de la Fase G1 del Ciclo Celular/fisiología , Proteínas HSP70 de Choque Térmico/metabolismo , Histona Desacetilasa 6/genética , Humanos , Hipertermia/genética , Potencial de la Membrana Mitocondrial/fisiología , Microtúbulos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Regulación hacia Arriba
9.
Nanomedicine ; 41: 102529, 2022 04.
Artículo en Inglés | MEDLINE | ID: mdl-35104671

RESUMEN

Hepatocellular carcinoma (HCC) is the most aggressive form of cancer with poor drug responses. Developing an effective drug treatment remains a major unmet clinical need for HCC. We report a comprehensive study of combinatorial Cetuximab (Cet) targeted polymeric poly(D, L-lactide-co-glycolide)-b-poly(ethylene glycol) nanocomplexes delivery of Combretastatin A4 (CA4) and 2-Methoxyestradiol (2ME) (Cet-PLGA-b-PEG-CA4 NP + Cet-PLGA-b-PEG-2ME NP) against metastatic HCC in SCID mice. 125I-Cet-PLGA-b-PEG NP showed potent accumulation and retention in HCC tumors with longer circulation time up to 48 h (18 ±â€¯1.0% ID/g, P < .0001). Combinatorial treatment with targeted polymeric nanocomplexes presented significant tumor growth inhibition (85%, P < .0001) than the free drug combinatorial counterpart, effectively inhibited orthotopic HCC and prevented lung metastasis. Combinatorial nanocomplexes treatment significantly blocked PRC1, a novel target of therapeutic response against HCC. Thus, the combinatorial cetuximab-targeted polymeric nanocomplexes possess superior antitumor activity against metastatic HCC and provide supports for the clinical translation ahead.


Asunto(s)
Antineoplásicos , Carcinoma Hepatocelular , Neoplasias Hepáticas , Nanopartículas , Animales , Antineoplásicos/farmacología , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Cetuximab/farmacología , Cetuximab/uso terapéutico , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/patología , Ratones , Ratones SCID , Polietilenglicoles/uso terapéutico , Radioisótopos
10.
Molecules ; 27(20)2022 Oct 18.
Artículo en Inglés | MEDLINE | ID: mdl-36296585

RESUMEN

Multi-drug resistance is increasing in the pathogenic bacterium S. pneumoniae, which is mainly responsible for meningitis and community-acquired pneumonia (CAP), highlighting the need for new anti-pneumococcal agents. We have identified a potential anti-pneumococcal agent, enol 3, which acts by hindering the cell division process by perturbing Z-ring dynamics inside the cell. Enol 3 was also shown to inhibit FtsZ polymerization and induce its aggregation in vitro but does not affect the activity of tubulin and alkaline phosphatase. Docking studies show that 3 binds near the T7 loop, which is the catalytic site of FtsZ. Similar effects on Z-ring and FtsZ assembly were observed in B. subtilis, indicating that 3 could be a broad-spectrum anti-bacterial agent useful in targeting Gram-positive bacteria. In conclusion, compound 3 shows strong anti-pneumococcal activity, prompting further pre-clinical studies to explore its potential.


Asunto(s)
Proteínas Bacterianas , Proteínas del Citoesqueleto , Proteínas del Citoesqueleto/metabolismo , Proteínas Bacterianas/metabolismo , Tubulina (Proteína)/metabolismo , Fosfatasa Alcalina/metabolismo , Antibacterianos/farmacología , Antibacterianos/metabolismo , Bacillus subtilis
11.
Bioorg Chem ; 107: 104538, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33349456

RESUMEN

Tuberculosis is the leading cause of death globally among infectious diseases. Due to the development of resistance of Mycobacterium tuberculosis to currently used anti-TB medicines and the TB-HIV synergism the urgent need to develop novel anti-mycobacterial agents has been realized. The drug-to-target path has been the successful strategy for new anti-TB drug development. All the six drug candidates that have shown promise during the clinical trials and some of these being approved for treatment against MDR TB are the results of phenotype screening of small molecule compound libraries. In search of compounds belonging to novel pharmacophoric class that could be subjected to whole cell assay to generate new anti-TB leads the benzo[d]imidazole-2-carboxamide moiety has been designed as a novel anti-TB scaffold. The design was based on the identification of the benzimidazole ring as a prominent substructure of the FDA approved drugs, the structural analysis of reported anti-TB benzimidazoles, and the presence of the C-2 carboxamido functionality in novel bioisoteric anti-TB benzothiazoles. Twenty seven final compounds have been prepared via NH4Cl-catalyzed amidation of ethyl benzo[d]imidazole-2-carboxylates, as the required intermediates, obtained through a green "all water" one-pot synthetic route following a tandem N-arylation-reduction-cyclocondensation procedure. All of the synthesised target compounds were assessed for anti-TB potential using H37Rv ATCC27294 strain. Thirteen compounds were found with better MIC (0.78-6.25 µg/mL) than the standard drugs and being non-cytotoxic nature (<50% inhibition against RAW 264.7 cell lines at 50 µg/mL). The compound 8e exhibited best anti-TB activity (MIC: 2.15 µM and selectivity index: > 60) and a few others e.g., 8a, 8f, 8k and 8o are the next best anti-TB hits (MIC: 1.56 µg/mL). The determination and analysis of various physiochemical parameters revealed favorable druglike properties of the active compounds. The compounds 8a-l and 8o, with MIC values of ≤ 6.25 µg/mL, have high LipE values (10.66-11.77) that are higher than that of the suggested value of > 6 derived from empirical evidence for quality drug candidates and highlight their therapeutic potential. The highest LipE value of 11.77 of the best active compound 8e with the MIC of 0.78 µg/mL indicates its better absorption and clearance as a probable clinical candidate for anti-TB drug discovery. These findings highlight the discovery of benzimidazole-2-carboxamides for further development as new anti-TB agents.


Asunto(s)
Amidas/química , Antituberculosos/síntesis química , Diseño de Fármacos , Imidazoles/química , Amidas/farmacología , Amidas/uso terapéutico , Cloruro de Amonio/química , Animales , Antituberculosos/farmacología , Antituberculosos/uso terapéutico , Catálisis , Supervivencia Celular/efectos de los fármacos , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Ratones , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/efectos de los fármacos , Células RAW 264.7 , Relación Estructura-Actividad , Tuberculosis/tratamiento farmacológico , Tuberculosis/microbiología
12.
Mol Pharmacol ; 97(6): 409-422, 2020 06.
Artículo en Inglés | MEDLINE | ID: mdl-32241960

RESUMEN

The microtubule-binding taxanes, docetaxel and cabazitaxel, are administered intravenously for the treatment of castration-resistant prostate cancer (CRPC) as the oral administration of these drugs is largely hampered by their low and highly variable bioavailabilities. Using a simple, rapid, and environmentally friendly microwave-assisted protocol, we have synthesized a number of 3,5-bis(styryl)pyrazoles 2a-l, thus allowing for their screening for antiproliferative activity in the androgen-independent PC3 prostate cancer cell line. Surprisingly, two of these structurally simple 3,5-bis(styryl)pyrazoles (2a and 2l) had concentrations which gave 50% of the maximal inhibition of cell proliferation (GI50) in the low micromolar range in the PC3 cell line and were thus selected for extensive further biologic evaluation (apoptosis and cell cycle analysis, and effects on tubulin and microtubules). Our findings from these studies show that 3,5-bis[(1E)-2(2,6-dichlorophenyl)ethenyl]-1H-pyrazole 2l 1) caused significant effects on the cell cycle in PC3 cells, with the vast majority of treated cells in the G2/M phase (89%); 2) induces cell death in PC3 cells even after the removal of the compound; 3) binds to tubulin [dissociation constant (Kd) 0.4 ± 0.1 µM] and inhibits tubulin polymerization in vitro; 4) had no effect upon the polymerization of the bacterial cell division protein FtsZ (a homolog of tubulin); 5) is competitive with paclitaxel for binding to tubulin but not with vinblastine, crocin, or colchicine; and 6) leads to microtubule depolymerization in PC3 cells. Taken together, these results suggest that 3,5-bis(styryl)pyrazoles warrant further investigation as lead compounds for the treatment of CRPC. SIGNIFICANCE STATEMENT: The taxanes are important components of prostate cancer chemotherapy regimens, but their oral administration is hampered by very low and highly variable oral bioavailabilities resulting from their poor absorption, poor solubility, high first-pass metabolism, and efficient efflux by P-glycoprotein. New chemical entities for the treatment of prostate cancer are thus required, and we report here the synthesis and investigation of the mechanism of action of some bis(styryl)pyrazoles, demonstrating their potential as lead compounds for the treatment of prostate cancer.


Asunto(s)
Antineoplásicos/uso terapéutico , Plomo/uso terapéutico , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Pirazoles/uso terapéutico , Tubulina (Proteína)/metabolismo , Antineoplásicos/síntesis química , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Sitios de Unión , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Estabilidad de Medicamentos , Humanos , Plomo/química , Masculino , Microtúbulos/efectos de los fármacos , Modelos Moleculares , Células PC-3 , Pirazoles/síntesis química , Pirazoles/química
13.
IUBMB Life ; 72(5): 978-990, 2020 05.
Artículo en Inglés | MEDLINE | ID: mdl-31984664

RESUMEN

The assembly and disassembly of FtsZ play an essential role in bacterial cell division. Using single-cell imaging, we report that short exposure to BT-benzo-29 inhibits Z-ring formation in live Bacillus subtilis cells. Fluorescence recovery after photobleaching of the Z-ring in live bacteria demonstrated that BT-benzo-29 strongly suppressed the assembly dynamics of FtsZ in the Z-ring. Furthermore, B. subtilis cells expressing V275A-FtsZ resisted the antibacterial activity of BT-benzo-29 providing evidence that BT-benzo-29 inhibits bacterial proliferation by targeting FtsZ. In addition, a brief (8 min) exposure of BT-benzo-29 destroyed the Z-ring without perturbing the localization of a late cell division protein, DivIVA, the nucleoid segregation, and membrane permeability. BT-benzo-29, when used in combination with vancomycin and polymyxin B (PMB), produced a much stronger inhibitory effect on Bacillus subtilis and Escherichia coli cells, respectively. The combination index of BT-benzo-29 with vancomycin and PMB was determined to be <1, suggesting that BT-benzo-29 exhibits synergistic inhibitory effects on bacterial proliferation when used along with these antibiotics.


Asunto(s)
Antibacterianos/farmacología , Bacillus subtilis/efectos de los fármacos , Proteínas Bacterianas/antagonistas & inhibidores , Bencimidazoles/farmacología , División Celular/efectos de los fármacos , Proteínas del Citoesqueleto/antagonistas & inhibidores , Farmacorresistencia Bacteriana/genética , Tiofenos/farmacología , Sustitución de Aminoácidos , Bacillus subtilis/genética , Bacillus subtilis/crecimiento & desarrollo , Bacillus subtilis/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Permeabilidad de la Membrana Celular/efectos de los fármacos , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Combinación de Medicamentos , Sinergismo Farmacológico , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Recuperación de Fluorescencia tras Fotoblanqueo , Expresión Génica , Pruebas de Sensibilidad Microbiana , Mutación , Polimixina B/farmacología , Análisis de la Célula Individual , Vancomicina/farmacología
14.
Mol Cell Biochem ; 465(1-2): 27-36, 2020 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-31782084

RESUMEN

Polo-like kinase 1 (Plk1) is a mitotic serine/threonine kinase implicated in spindle formation and cytokinesis in mammalian cells. Here, purified Plk1 was found to bind to reconstituted microtubules in vitro. Further, Plk1 was found to co-localize with interphase microtubules in MCF-7 cells and to co-immunoprecipitate with polymerized tubulin. The binding of Plk1 to interphase microtubules appeared to increase with an increase in the level of tubulin acetylation in MCF-7 cells. Interestingly, Plk1 inhibitor III, an inhibitor of Plk1 kinase activity, treatment increased the association of Plk1 with the interphase microtubules in MCF-7 cells. Therefore, the effect of inhibition of Plk1 kinase activity on the dynamic instability of microtubules was determined by time-lapse imaging in MCF-7 cells. Plk1 inhibitor III dampened the dynamic instability of microtubules. For example, Plk1 inhibitor III (3 µM) reduced the rate and extent of the growing phase by 28 and 48%, respectively, and inhibited the dynamicity of microtubules by 53% as compared to the microtubules in control MCF-7 cells. Plk1 inhibitor III treatment also increased the level of acetylated microtubules, indicating that it stabilizes microtubules. The findings indicated that Plk1 interacts with microtubules and Plk1 may have a role in the regulation of microtubule dynamics.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Femenino , Humanos , Células MCF-7 , Microtúbulos/genética , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/genética , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Quinasa Tipo Polo 1
15.
Biochem J ; 476(9): 1359-1376, 2019 05 12.
Artículo en Inglés | MEDLINE | ID: mdl-31085712

RESUMEN

Microtubules, composed of αß-tubulin heterodimers, exhibit diverse structural and functional properties in different cell types. The diversity in the microtubule structure originates from tubulin heterogeneities, namely tubulin isotypes and their post-translational modifications (PTMs). These heterogeneities confer differential stability to microtubules and provide spatial cues for the functioning of the cell. Furthermore, the altered expressions of tubulin isotypes and PTMs are prominent factors for the development of resistance against some cancer drugs. In this review, we summarize our current knowledge of the tubulin isotypes and PTMs and how, together, they control the cellular functions of the microtubules. We also describe how cancer cells use this tubulin heterogeneity to acquire resistance against clinical agents and discuss existing attempts to counter the developed resistance.


Asunto(s)
Resistencia a Antineoplásicos , Regulación Neoplásica de la Expresión Génica , Microtúbulos/metabolismo , Proteínas de Neoplasias/biosíntesis , Neoplasias/metabolismo , Tubulina (Proteína)/biosíntesis , Animales , Humanos , Microtúbulos/patología , Neoplasias/patología , Isoformas de Proteínas/biosíntesis
16.
Biochem J ; 476(10): 1401-1417, 2019 05 21.
Artículo en Inglés | MEDLINE | ID: mdl-31036717

RESUMEN

In Alzheimer's disease, tau is predominantly acetylated at K174, K274, K280, and K281 residues. The acetylation of K274-tau is linked with memory loss and dementia. In this study, we have examined the molecular mechanism of the toxicity of acetylated K274-tau. We incorporated an acetylation mimicking mutation at K274 (K→Q) residue of tau. The mutation (K274Q) strongly reduced the ability of tau to bind to tubulin and also to polymerize tubulin while K274R mutation did not reduce the ability of tau either to bind or polymerize tubulin. In addition, K274Q-tau displayed a higher aggregation propensity than wild-type tau as evident from thioflavin S fluorescence, tryptophan fluorescence, and electron microscopic images. Furthermore, dynamic light scattering, atomic force microscopy, and dot blot analysis using an oligomer-specific antibody suggested that K274Q mutation enhanced the oligomerization of tau. The K274Q mutation also strongly decreased the critical concentration for the liquid-liquid phase separation of tau. The oligomeric forms of K274Q-tau were found to be more toxic than wild tau to neuroblastoma cells. Using circular dichroism and fluorescence spectroscopy, we provide evidence indicating that the acetylation mimicking mutation (K274Q) induced conformational changes in tau. The results suggested that the acetylation of tau at 274 residues can increase tau aggregation and enhance the cytotoxicity of tau oligomers.


Asunto(s)
Mutación Missense , Agregación Patológica de Proteínas , Tubulina (Proteína) , Proteínas tau , Acetilación , Sustitución de Aminoácidos , Línea Celular Tumoral , Humanos , Agregación Patológica de Proteínas/genética , Agregación Patológica de Proteínas/metabolismo , Agregación Patológica de Proteínas/patología , Tubulina (Proteína)/química , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Proteínas tau/química , Proteínas tau/genética , Proteínas tau/metabolismo
17.
Traffic ; 18(2): 123-133, 2017 02.
Artículo en Inglés | MEDLINE | ID: mdl-27976831

RESUMEN

The assembly of microtubule-based cytoskeleton propels the cilia and flagella growth. Previous studies have indicated that the kinesin-2 family motors transport tubulin into the cilia through intraflagellar transport. Here, we report a direct interaction between the C-terminal tail fragments of heterotrimeric kinesin-2 and α-tubulin1 isoforms in vitro. Blot overlay screen, affinity purification from tissue extracts, cosedimentation with subtilisin-treated microtubule and LC-ESI-MS/MS characterization of the tail-fragment-associated tubulin identified an association between the tail domains and α-tubulin1A/D isotype. The interaction was confirmed by Forster's resonance energy transfer assay in tissue-cultured cells. The overexpression of the recombinant tails in NIH3T3 cells affected the primary cilia growth, which was rescued by coexpression of a α-tubulin1 transgene. Furthermore, fluorescent recovery after photobleach analysis in the olfactory cilia of Drosophila indicated that tubulin is transported in a non-particulate form requiring kinesin-2. These results provide additional new insight into the mechanisms underlying selective tubulin isoform enrichment in the cilia.


Asunto(s)
Cilios/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Subunidades de Proteína/metabolismo , Tubulina (Proteína)/metabolismo , Secuencia de Aminoácidos , Animales , Transporte Biológico/fisiología , Drosophila , Flagelos/metabolismo , Cinesinas , Ratones , Microtúbulos/metabolismo , Células 3T3 NIH , Unión Proteica/fisiología , Dominios Proteicos/fisiología
18.
Biochem J ; 475(15): 2473-2489, 2018 08 14.
Artículo en Inglés | MEDLINE | ID: mdl-30006469

RESUMEN

The role of FtsZ-associated proteins in the regulation of the assembly dynamics of Mycobacterium smegmatis FtsZ is not clear. In this work, we examined the effect of M. smegmatis SepF on the assembly and stability of M. smegmatis FtsZ polymers. We discovered a single dominant point mutation in SepF (G51D or G51R) that renders the protein inactive. SepF promoted the polymerization of FtsZ, induced the bundling of FtsZ filaments, stabilized FtsZ filaments and reduced the GTPase activity of FtsZ. Surprisingly, both G51D-SepF and G51R-SepF neither stabilized FtsZ filaments nor showed a discernable effect on the GTPase activity of FtsZ. The binding affinity of SepF to FtsZ was found to be stronger than the binding affinity of G51R/D-SepF to FtsZ. Interestingly, the binding affinity of SepF to G51R-SepF was determined to be 45 times stronger than FtsZ. In addition, the interaction of SepF with G51R-SepF was found to be 2.6 times stronger than SepF-SepF interaction. Furthermore, G51R-SepF impaired the ability of SepF to promote the assembly of FtsZ. In addition, the overexpression of G51R-SepF in M. smegmatis mc2 155 cells retarded the proliferation of these cells and increased the average length of the cells. The results indicated that SepF positively regulates the assembly of M. smegmatis FtsZ and the G51 residue has an important role in the functioning of SepF.


Asunto(s)
Proteínas Bacterianas/metabolismo , División Celular , Proteínas del Citoesqueleto/metabolismo , Mutación Missense , Mycobacterium smegmatis/metabolismo , Sustitución de Aminoácidos , Proteínas Bacterianas/genética , Proteínas del Citoesqueleto/genética , Mycobacterium smegmatis/genética
19.
IUBMB Life ; 70(7): 602-611, 2018 07.
Artículo en Inglés | MEDLINE | ID: mdl-29734495

RESUMEN

The centrosome executes diverse functions including the nucleation and organization of microtubules. A subset of centrosomal proteins is found to be involved in regulating the nucleation, stability, and dynamics of microtubules. Literature is flooded with reports of centrosomal proteins regulating microtubule nucleation. However, the centrosomal proteins that regulate microtubule stability are underexplored. Here, we review the centrosomal proteins, which either enhance or reduce the stability of microtubules and thereby regulate microtubule dynamics. We also discuss unexplored aspects of the centrosomal proteins that influence microtubule stability. © 2018 IUBMB Life, 70(7):602-611, 2018.


Asunto(s)
Centrosoma/metabolismo , Microtúbulos/metabolismo , Proteínas/metabolismo , Animales , Humanos
20.
Biochem J ; 474(16): 2841-2859, 2017 08 10.
Artículo en Inglés | MEDLINE | ID: mdl-28694353

RESUMEN

Here, we report that a centrosomal protein FOR20 [FOP (FGFR1 (fibroblast growth factor receptor 1) oncogene protein)-like protein of molecular mass of 20 kDa; also named as C16orf63, FLJ31153 or PHSECRG2] can regulate the assembly and stability of microtubules. Both FOR20 IgG antibody and GST (glutathione S-transferase)-tagged FOR20 could precipitate tubulin from the HeLa cell extract, indicating a possible interaction between FOR20 and tubulin. FOR20 was also detected in goat brain tissue extract and it cycled with microtubule-associated proteins. Furthermore, FOR20 bound to purified tubulin and inhibited the assembly of tubulin in vitro. The overexpression of FOR20 depolymerized interphase microtubules and the depletion of FOR20 prevented nocodazole-induced depolymerization of microtubules in HeLa cells. In addition, the depletion of FOR20 suppressed the dynamics of individual microtubules in live HeLa cells. FOR20-depleted MDA-MB-231 cells displayed zigzag motion and migrated at a slower rate than the control cells, indicating that FOR20 plays a role in directed cell migration. The results suggested that the centrosomal protein FOR20 is a new member of the microtubule-associated protein family and that it regulates the assembly and dynamics of microtubules.


Asunto(s)
Cilios/metabolismo , Microtúbulos/metabolismo , Proteínas Nucleares/genética , Proteínas/genética , Factores de Transcripción/genética , Tubulina (Proteína)/genética , Animales , Química Encefálica , Línea Celular Tumoral , Movimiento Celular , Cilios/ultraestructura , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/ultraestructura , Adhesiones Focales/efectos de los fármacos , Adhesiones Focales/metabolismo , Adhesiones Focales/ultraestructura , Expresión Génica , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Cabras , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Ratones , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Células 3T3 NIH , Nocodazol/farmacología , Proteínas Nucleares/metabolismo , Proteínas/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Imagen de Lapso de Tiempo , Factores de Transcripción/metabolismo , Tubulina (Proteína)/metabolismo
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