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1.
Nature ; 633(8031): 932-940, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-39232161

RESUMO

CDK1 has been known to be the sole cyclin-dependent kinase (CDK) partner of cyclin B1 to drive mitotic progression1. Here we demonstrate that CDK5 is active during mitosis and is necessary for maintaining mitotic fidelity. CDK5 is an atypical CDK owing to its high expression in post-mitotic neurons and activation by non-cyclin proteins p35 and p392. Here, using independent chemical genetic approaches, we specifically abrogated CDK5 activity during mitosis, and observed mitotic defects, nuclear atypia and substantial alterations in the mitotic phosphoproteome. Notably, cyclin B1 is a mitotic co-factor of CDK5. Computational modelling, comparison with experimentally derived structures of CDK-cyclin complexes and validation with mutational analysis indicate that CDK5-cyclin B1 can form a functional complex. Disruption of the CDK5-cyclin B1 complex phenocopies CDK5 abrogation in mitosis. Together, our results demonstrate that cyclin B1 partners with both CDK5 and CDK1, and CDK5-cyclin B1 functions as a canonical CDK-cyclin complex to ensure mitotic fidelity.


Assuntos
Ciclina B1 , Quinase 5 Dependente de Ciclina , Mitose , Complexos Multiproteicos , Humanos , Coenzimas/metabolismo , Ciclina B1/metabolismo , Quinase 5 Dependente de Ciclina/antagonistas & inibidores , Quinase 5 Dependente de Ciclina/deficiência , Quinase 5 Dependente de Ciclina/genética , Quinase 5 Dependente de Ciclina/metabolismo , Células HeLa , Modelos Moleculares , Complexos Multiproteicos/metabolismo , Mutação , Fosfoproteínas/metabolismo , Ligação Proteica , Proteoma/metabolismo , Reprodutibilidade dos Testes
2.
Cell Commun Signal ; 22(1): 101, 2024 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-38326896

RESUMO

BACKGROUND: Our previous studies demonstrated that 1-Pyrroline-5-carboxylate (P5C) released by prostate cancer cells inhibits T cell proliferation and function by increasing SHP1 expression. We designed this study to further explore the influence of P5C on T cell metabolism, and produced an antibody for targeting P5C to restore the functions of T cells. METHOD: We co-immunoprecipated SHP1 from T cells and analyzed the proteins that were bound to it using liquid chromatography mass spectrometry (LC/MS-MS). The influence of P5C on T cells metabolism was also detected by LC/MS-MS. Seahorse XF96 analyzer was further used to identify the effect of P5C on T cells glycolysis. We subsequently designed and produced an antibody for targeting P5C by monoclonal technique and verified its effectiveness to restore the function of T cells in vitro and in vivo. RESULT: PKM2 and LDHB bind SHP1 in T cells, and P5C could increase the levels of p-PKM2 while having no effect on the levels of PKM2 and LDHB. We further found that P5C influences T cell energy metabolism and carbohydrate metabolism. P5C also inhibits the activity of PKM2 and decreases the content of intracellular lactic acid while increasing the activity of LDH. Using seahorse XF96 analyzer, we confirmed that P5C remarkably inhibits glycolysis in T cells. We produced an antibody for targeting P5C by monoclonal technique and verified that the antibody could oppose the influence of P5C to restore the process of glycolysis and function in T cells. Meanwhile, the antibody also inhibits the growth of prostate tumors in an animal model. CONCLUSION: Our study revealed that P5C inhibits the process of glycolysis in T cells by targeting SHP1/PKM2/LDHB complexes. Moreover, it is important that the antibody for targeting P5C could restore the function of T cells and inhibit the growth of prostate tumors.


Assuntos
Neoplasias da Próstata , Pirróis , Linfócitos T , Humanos , Masculino , Animais , Próstata , Microambiente Tumoral , Proliferação de Células , Glicólise , Linhagem Celular Tumoral
3.
Int J Phytoremediation ; 26(11): 1873-1884, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-38845357

RESUMO

In India, ∼30% of total land is degraded due to pollution, salinization, and nutrient loss. Change in soil-quality at urban waste-dumping site prior and after cow-dung amendment was compared with control agriculture soil. The soil at waste-dumping site had elevated pH, EC, temperature and lowered OC and NPK concentrations when compared to control. Polymetallic pollution of Cr, Cd, Pb, and Ni beyond permissible limits was obtained. Cow-dung amendment restored soil physicochemical properties at the waste-dumping site, with increasing soil moisture, CEC and OC; however, a slight change in soil bulk-density and heavy-metal concentration post-amendment was noted. The seven natural colonizers present at the waste-dumping site accumulated more metals in roots than shoots. Datura innoxia had maximum bioaccumulation of Cr, Calotropis procera of Cd and Ni and Parthenium hysterophorus of Pb in roots. All these plants had Bioacccumulation factor (BAfroot )>1 and translocation factor (Tf) <1 for Cd and serve as its phytostabilizer except Calotropis procera which had BAfroot >1 and Tf >1 and is identified as a phytoextractor for Cd. Cow-dung amendment alone appeared to be insufficient and additionally the revegetation of natural colonizers is recommended for effective reduction in heavy metal load and improving overall soil health at wasteland. Such eco-restoration may also minimize risks to biodiversity in India.


The novelty of the work lies in revegetation of natural colonizers at polluted wasteland to reduce heavy metal load and improve overall soil health. Calotropis procera, Datura innoxia, Parthenium hysterophorus, and S. nigrum showed maximum bioaccumulation of Cr, Cd, Pb, and Ni. The work confirms C. procera as non-edible, fast growing natural colonizer as potential phytoextractor for Cd and taken into consideration to effectively restore heavy metals polluted wasteland.


Assuntos
Biodegradação Ambiental , Metais Pesados , Poluentes do Solo , Metais Pesados/metabolismo , Poluentes do Solo/metabolismo , Índia , Esterco , Bovinos , Animais , Solo/química , Plantas/metabolismo
4.
Mol Cancer ; 22(1): 186, 2023 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-37993880

RESUMO

Recent studies have uncovered various physiological functions of CDK5 in many nonneuronal tissues. Upregulation of CDK5 and/or its activator p35 in neurons promotes healthy neuronal functions, but their overexpression in nonneuronal tissues is causally linked to cancer of many origins. This review focuses on the molecular mechanisms by which CDK5 recruits diverse tissue-specific substrates to elicit distinct phenotypes in sixteen different human cancers. The emerging theme suggests that CDK5's role as an oncogene or anti-oncogene depends upon its subcellular localization. CDK5 mostly acts as an oncogene, but in gastric cancer, it is a tumor suppressor due to its unique nuclear localization. This indicates that CDK5's access to certain nuclear substrates converts it into an anti-oncogenic kinase. While acting as a bonafide oncogene, CDK5 also activates a few cancer-suppressive pathways in some cancers, presumably due to the mislocalization of nuclear substrates in the cytoplasm. Therefore, directing CDK5 to the nucleus or exporting tumor-suppressive nuclear substrates to the cytoplasm may be promising approaches to combat CDK5-induced oncogenicity, analogous to neurotoxicity triggered by nuclear CDK5. Furthermore, while p35 overexpression is oncogenic, hyperactivation of CDK5 by inducing p25 formation results in apoptosis, which could be exploited to selectively kill cancer cells by dialing up CDK5 activity, instead of inhibiting it. CDK5 thus acts as a molecular rheostat, with different activity levels eliciting distinct functional outcomes. Finally, as CDK5's role is defined by its substrates, targeting them individually or in conjunction with CDK5 should create potentially valuable new clinical opportunities.


Assuntos
Apoptose , Proteínas do Tecido Nervoso , Humanos , Proteínas do Tecido Nervoso/genética , Oncogenes , Citoplasma/metabolismo , Genes Supressores de Tumor , Quinase 5 Dependente de Ciclina/genética , Quinase 5 Dependente de Ciclina/metabolismo
5.
Bioorg Med Chem ; 93: 117459, 2023 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-37659217

RESUMO

A facile and efficient approach utilizing copper-mediated cross-coupling reaction of N-boc-3-indolylsulfoximines with aryl iodides was developed to synthesize a diverse range of N-arylated indolylsulfoximines 11a-m in excellent yields (up to 91%). The key precursors, free NH sulfoximines 9 were readily prepared by the treatment of N-boc-3-methylthioindoles 8 with a combination of IBD and ammonium carbamate. Under similar conditions NH-free indolylsulfoximine 9a was successfully prepared in gram-scale quantities. The reaction is highly chemoselective and tolerant of a wide range of functional groups. The process is environmentally friendly and is amenable to scale-up. Among the prepared N-arylated indolylsulfoximines 11a-m, compounds 11i-j (2.68-2.76 µM), 11f-g (1.9-3.7 µM) and 11k (1.28 µM) showed potent and selective cytotoxicity against 22Rv1, C4-2 and MCF7 cells, respectively. Indolylsulfoximine derivative 11l displayed a broad spectrum of activity (1.7-8.2 µM) against the tested cancer cell lines. These compounds were found to be non-cytotoxic to normal HEK293 cells, indicating their potential selectivity for cancer cells. We analysed the impact of 11l on various cellular assays to uncover its mechanism of action. Cellular assay shows that 11l increases the endogenous level of ROS, leading to the increased level of p-53 and c-jun inducing apoptosis. 11l also induced mitochondrial dysfunction, further promoting apoptotic pathways. Besides, 11l also restricts cell invasiveness, indicating that it could serve as an effective anti-metastatic agent. As oxidative stress severe F actin causing tubulin depolymerization, we examined the impact of 11l on tubulin dynamics. Accordingly, 11l treatment decreased the levels of polymerized tubulin in 22Rv1 and C4-2 cells. Although future studies are needed to determine their exact molecular target(s), our data shows that N-aryl indolylsulfoximines could serve as effective anti-cancer agents.


Assuntos
Antineoplásicos , Tubulina (Proteína) , Humanos , Células HEK293 , Antineoplásicos/farmacologia , Estresse Oxidativo , Citoesqueleto de Actina
6.
Br J Cancer ; 124(5): 995-1008, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33311589

RESUMO

BACKGROUND: SPOP, an E3 ubiquitin ligase adaptor, can act either as a tumour suppressor or a tumour promoter. In prostate cancer (PCa), it inhibits tumorigenesis by degrading several oncogenic substrates. SPOP is the most altered gene in PCa (~15%), which renders it ineffective, promoting cancer. The remaining PCa tumours, which retain WT-SPOP, still progress to castration-resistant (CRPC) stage, indicating that other critical mechanisms exist for downregulating SPOP. SPOP is reduced in ~94% of WT-SPOP-bearing prostate tumours; however, no molecular mechanism is known for its downregulation. METHODS: SPOP was identified as a direct target of LIMK2 using an innovative technique. The reciprocal relationship between SPOP and LIMK2 and its consequences on oncogenicity were analysed using a variety of biochemical assays. To probe this relationship in vivo, xenograft studies were conducted. RESULTS: LIMK2 degrades SPOP by direct phosphorylation at three sites. SPOP promotes LIMK2's ubiquitylation, creating a feedback loop. SPOP's degradation stabilises AR, ARv7 and c-Myc promoting oncogenicity. Phospho-resistant SPOP completely suppresses tumorigenesis in vivo, indicating that LIMK2-mediated SPOP degradation is a key event in PCa progression. CONCLUSIONS: While genomically altered SPOP-bearing tumours require gene therapy, uncovering LIMK2-SPOP relationship provides a powerful opportunity to retain WT-SPOP by inhibiting LIMK2, thereby halting disease progression.


Assuntos
Biomarcadores Tumorais/metabolismo , Regulação Neoplásica da Expressão Gênica , Quinases Lim/metabolismo , Proteínas Nucleares/metabolismo , Neoplasias de Próstata Resistentes à Castração/patologia , Receptores Androgênicos/metabolismo , Proteínas Repressoras/metabolismo , Animais , Apoptose , Biomarcadores Tumorais/genética , Movimento Celular , Proliferação de Células , Humanos , Quinases Lim/genética , Masculino , Camundongos , Camundongos Nus , Mutação , Proteínas Nucleares/genética , Fosforilação , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/metabolismo , Receptores Androgênicos/genética , Proteínas Repressoras/genética , Células Tumorais Cultivadas , Ubiquitinação , Ensaios Antitumorais Modelo de Xenoenxerto
7.
J Biomed Sci ; 28(1): 68, 2021 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-34625072

RESUMO

BACKGROUND: NKX3.1, a prostate-specific tumor suppressor, is either genomically lost or its protein levels are severely downregulated, which are invariably associated with poor prognosis in prostate cancer (PCa). Nevertheless, a clear disconnect exists between its mRNA and protein levels, indicating that its post-translational regulation may be critical in maintaining its protein levels. Similarly, AURKA is vastly overexpressed in all stages of prostate cancer (PCa), including castration-resistant PCa (CRPC) and neuroendocrine PCa (NEPC), although its transcripts are only increased in ~ 15% of cases, hinting at additional mechanisms of deregulation. Thus, identifying the upstream regulators that control AURKA and NKX3.1's levels and/or their downstream effectors offer an alternative route to inhibit AURKA and upregulate NKX3.1 in highly fatal CRPC and NEPC. AURKA and NKX3.1 have not linked to each other in any study to date. METHODS: A chemical genetic screen revealed NKX3.1 as a direct target of AURKA. AURKA-NKX3.1 cross-talk was analyzed using several biochemical techniques in CRPC and NEPC cells. RESULTS: We uncovered a reciprocal loop between AURKA and NKX3.1 in CRPC and NEPC cells. We observed that AURKA-mediated NKX3.1 downregulation is a major mechanism that drives CRPC pathogenesis and NEPC differentiation. AURKA phosphorylates NKX3.1 at three sites, which degrades it, but AURKA does not regulate NKX3.1 mRNA levels. NKX3.1 degradation drives highly aggressive oncogenic phenotypes in cells. NKX3.1 also degrades AURKA in a feedback loop. NKX3.1-AURKA loop thus upregulates AKT, ARv7 and Androgen Receptor (AR)-signaling in tandem promoting highly malignant phenotypes. Just as importantly, we observed that NKX3.1 overexpression fully abolished synaptophysin and enolase expression in NEPC cells, uncovering a strong negative relationship between NKX3.1 and neuroendocrine phenotypes, which was further confirmed be measuring neurite outgrowth. While WT-NKX3.1 inhibited neuronal differentiation, 3A-NKX3.1 expression obliterated it. CONCLUSIONS: NKX3.1 loss could be a major mechanism causing AURKA upregulation in CRPC and NEPC and vice versa. NKX3.1 genomic loss requires gene therapy, nonetheless, targeting AURKA provides a powerful tool to maintain NKX3.1 levels. Conversely, when NKX3.1 upregulation strategy using small molecules comes to fruition, AURKA inhibition should work synergistically due to the reciprocal loop in these highly aggressive incurable diseases.


Assuntos
Aurora Quinase A/genética , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/genética , Neoplasias da Próstata/genética , Fatores de Transcrição/genética , Aurora Quinase A/metabolismo , Castração , Proteínas de Homeodomínio/metabolismo , Humanos , Masculino , Fatores de Transcrição/metabolismo
8.
Physiol Mol Biol Plants ; 27(3): 535-541, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33854282

RESUMO

In salt-prone areas, plant growth and productivity is adversely affected. In the present study, the ZT1-ZT6 transgenic tomato lines having BcZAT12 gene under the regulatory control of the stress inducible Bclea1 promoter were exposed to three salinity levels (50, 100 and 200 mM) at the four leaf stage for 10 days. The transgenic lines showed improved growth in stem height, leaf area, root length and shoot length under saline conditions, as compared to control. Moreover, ZT1 and ZT5 lines showed lower electrolyte leakage and decreased hydrogen peroxide formation, in combination with elevated relative water content, proline and chlorophyll levels. The enzyme activity of catalase was also enhanced in ZT1 and ZT5. These results poses the present lines as an attractive alternative for tomato cultivation in salinity-affected areas.

9.
J Cell Sci ; 130(18): 3023-3039, 2017 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-28751497

RESUMO

Cdk5 deregulation is highly neurotoxic in Alzheimer's disease (AD). We identified Mcl-1 as a direct Cdk5 substrate using an innovative chemical screen in mouse brain lysates. Our data demonstrate that Mcl-1 levels determine the threshold for cellular damage in response to neurotoxic insults. Mcl-1 is a disease-specific target of Cdk5, which associates with Cdk5 under basal conditions, but is not regulated by it. Neurotoxic insults hyperactivate Cdk5 causing Mcl-1 phosphorylation at T92. This phosphorylation event triggers Mcl-1 ubiquitylation, which directly correlates with mitochondrial dysfunction. Consequently, ectopic expression of phosphorylation-dead T92A-Mcl-1 fully prevents mitochondrial damage and subsequent cell death triggered by neurotoxic treatments in neuronal cells and primary cortical neurons. Notably, enhancing Mcl-1 levels offers comparable neuroprotection to that observed upon Cdk5 depletion, suggesting that Mcl-1 degradation by direct phosphorylation is a key mechanism by which Cdk5 promotes neurotoxicity in AD. The clinical significance of the Mcl-1-Cdk5 axis was investigated in human AD clinical specimens, revealing an inverse correlation between Mcl-1 levels and disease severity. These results emphasize the potential of Mcl-1 upregulation as an attractive therapeutic strategy for delaying or preventing neurodegeneration in AD.


Assuntos
Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Quinase 5 Dependente de Ciclina/metabolismo , Mitocôndrias/metabolismo , Modelos Biológicos , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Animais , Núcleo Celular/metabolismo , Ácido Glutâmico/farmacologia , Células HEK293 , Humanos , Camundongos , Mitocôndrias/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurotoxinas/toxicidade , Fosforilação/efeitos dos fármacos , Fosfotreonina/metabolismo , Transporte Proteico/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Índice de Gravidade de Doença , Transdução de Sinais/efeitos dos fármacos , Frações Subcelulares/metabolismo , Especificidade por Substrato/efeitos dos fármacos , Ubiquitinação/efeitos dos fármacos
10.
J Cell Sci ; 130(6): 1078-1093, 2017 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-28167680

RESUMO

We uncovered a crucial role for the Aurora kinase A (AURKA)-Twist1 axis in promoting epithelial-to-mesenchymal transition (EMT) and chemoresistance in pancreatic cancer. Twist1 is the first EMT-specific target of AURKA that was identified using an innovative screen. AURKA phosphorylates Twist1 at three sites, which results in its multifaceted regulation - AURKA inhibits its ubiquitylation, increases its transcriptional activity and favors its homodimerization. Twist1 reciprocates and prevents AURKA degradation, thereby triggering a feedback loop. Ablation of either AURKA or Twist1 completely inhibits EMT, highlighting both proteins as central players in EMT progression. Phosphorylation-dead Twist1 serves as a dominant-negative and fully reverses the EMT phenotype induced by Twist1, underscoring the crucial role of AURKA-mediated phosphorylation in mediating Twist1-induced malignancy. Likewise, Twist1-overexpressing BxPC3 cells formed large tumors in vivo, whereas expression of phosphorylation-dead Twist1 fully abrogated this effect. Furthermore, immunohistochemical analysis of pancreatic cancer specimens revealed a 3-fold higher level of Twist1 compared to that seen in healthy normal tissues. This is the first study that links Twist1 in a feedback loop with its activating kinase, which indicates that concurrent inhibition of AURKA and Twist1 will be synergistic in inhibiting pancreatic tumorigenesis and metastasis.


Assuntos
Aurora Quinase A/metabolismo , Proteínas Nucleares/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Proteína 1 Relacionada a Twist/metabolismo , Animais , Biomarcadores Tumorais/metabolismo , Carcinogênese/metabolismo , Carcinogênese/patologia , Linhagem Celular Tumoral , Movimento Celular , Sobrevivência Celular , Resistencia a Medicamentos Antineoplásicos , Transição Epitelial-Mesenquimal , Retroalimentação Fisiológica , Feminino , Humanos , Camundongos Nus , Invasividade Neoplásica , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fenótipo , Fosforilação , Multimerização Proteica , Estabilidade Proteica , Transporte Proteico , Proteólise , Frações Subcelulares/metabolismo , Especificidade por Substrato , Ensaios Antitumorais Modelo de Xenoenxerto , Neoplasias Pancreáticas
11.
IUBMB Life ; 71(6): 721-737, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30801966

RESUMO

Protein kinases function as key signaling hubs in the intricate network of biochemical signaling processes in the living cell. More than two-thirds of the human proteome is estimated to be phosphorylated at ~960,000 phosphosites, which makes it challenging to identify the direct contribution of any desired kinase in generating this phosphoproteome. In this review, we discuss some of the methods that have been developed over the years for global identification of kinase substrates. The methods are essentially categorized into two classes, namely, (i) direct tagging of kinase substrates and (ii) indirect phosphoproteomics-based approaches. We discuss the advantages and limitations entailed to each of the method introduced, with a special emphasis on the analog-sensitive (as) kinase approach method. © 2019 IUBMB Life, 71(6):721-737, 2019.


Assuntos
Fosfoproteínas/genética , Proteínas Quinases/genética , Proteoma/genética , Humanos , Fosfoproteínas/química , Fosforilação/genética , Proteínas Quinases/química , Transdução de Sinais/genética , Especificidade por Substrato
12.
Arch Virol ; 164(4): 949-960, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30680529

RESUMO

HIV is one of the most lethal viral diseases in the human population. Patients often suffer from drug resistance, which hampers HIV therapy. Eleven different structural analogues of saquinavir (SQV), designed using ChemSketch™ and named S1 through S11, were compared with SQV with respect to their pharmacodynamic and pharmacokinetic properties. Pharmacokinetic predictions were carried out using AutoDock, and molecular docking between macromolecule HIV protease (PDB ID: 3IXO) and analogues S1 - S11 as ligands was performed. Analogues S1, S3, S4, S9 and S11 had lower binding scores when compared with saquinavir, whereas that of analogue S5 was similar. Pharmacokinetic predictions made using ACDilab2, including the Lipinski profile, general physical features, absorption, distribution, metabolism and excretion parameters, and toxicity values, for the eleven analogues and SQV suggested that S1 and S5 are pharmacodynamically and pharmacokinetically robust molecules that could be developed and established as lead molecules after in vitro and in vivo studies.


Assuntos
Infecções por HIV/tratamento farmacológico , Inibidores da Protease de HIV/química , Inibidores da Protease de HIV/farmacocinética , HIV-1/enzimologia , Saquinavir/análogos & derivados , Saquinavir/farmacocinética , Animais , Infecções por HIV/virologia , Protease de HIV/química , Protease de HIV/genética , Protease de HIV/metabolismo , Inibidores da Protease de HIV/administração & dosagem , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , Humanos , Camundongos , Simulação de Acoplamento Molecular , Saquinavir/administração & dosagem
13.
J Cell Sci ; 129(9): 1815-1830, 2016 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-28157684

RESUMO

Deregulated Cdk5 causes neurotoxic amyloid beta peptide (Aß) processing and cell death, two hallmarks of Alzheimer's disease, through the Foxo3 transcriptional factor in hippocampal cells, primary neurons and an Alzheimer's disease mouse model. Using an innovative chemical genetic screen, we identified Foxo3 as a direct substrate of Cdk5 in brain lysates. Cdk5 directly phosphorylates Foxo3, which increased its levels and nuclear translocation. Nuclear Foxo3 initially rescued cells from ensuing oxidative stress by upregulating MnSOD (also known as SOD2). However, following prolonged exposure, Foxo3 upregulated Bim (also known as BCL2L11) and FasL (also known as FASLG) causing cell death. Active Foxo3 also increased Aß(1-42) levels in a phosphorylation-dependent manner. These events were completely inhibited either by expressing phosphorylation-resistant Foxo3 or by depleting Cdk5 or Foxo3, highlighting a key role for Cdk5 in regulating Foxo3. These results were confirmed in an Alzheimer's disease mouse model, which exhibited increased levels and nuclear localization of Foxo3 in hippocampal neurons, which preceded neurodegeneration and Aß plaque formation, indicating this phenomenon is an early event in Alzheimer's disease pathogenesis. Collectively, these results show that Cdk5-mediated phospho-regulation of Foxo3 can activate several genes that promote neuronal death and aberrant Aß processing, thereby contributing to the progression of neurodegenerative pathologies.


Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Quinase 5 Dependente de Ciclina/metabolismo , Proteína Forkhead Box O3/metabolismo , Hipocampo/metabolismo , Neurônios/metabolismo , Fragmentos de Peptídeos/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/genética , Animais , Proteína 11 Semelhante a Bcl-2/genética , Proteína 11 Semelhante a Bcl-2/metabolismo , Quinase 5 Dependente de Ciclina/genética , Modelos Animais de Doenças , Proteína Forkhead Box O3/genética , Células HEK293 , Hipocampo/patologia , Humanos , Camundongos , Neurônios/patologia , Estresse Oxidativo/genética , Fragmentos de Peptídeos/genética , Fosforilação/genética , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo
14.
J Mol Recognit ; 31(7): e2709, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29630761

RESUMO

Epitope imprinting is a promising technique for fabrication of novel diagnostic tools. In this study, an epitope imprinted methodology for recognition of target epitope sequence as well as targeted protein infused by bacterial infection in blood samples of patients suffering from brain fever is developed. Template sequence chosen is a ferric iron binding fbp A protein present in Neisseria meningitidis bacteria. To orient the imprinting template peptide sequence on gold surface of electrochemical quartz crystal microbalance (EQCM), thiol chemistry was utilized to form the self-assembled monolayer on EQCM electrode. Here, synergistic effects induced by various noncovalent interactions extended by multiple monomers (3-sulfopropyl methacrylate potassium-salt and benzyl methacrylate) were used in fabricating the imprinting polymeric matrix with additional firmness provided by N,N-methylene-bis-acrylamide as cross-linker and azo-isobutyronitrile as initiator. Extraction of template molecule was carried out with phosphate buffer solution. After extraction of epitope molecules from the polymeric film, epitope molecularly imprinted polymeric films were fabricated on EQCM electrode surface. Nonimprinted polymers were also synthesized in the similar manner without epitope molecule. Detection limit of epitope molecularly imprinted polymers and imprinting factor (epitope molecularly imprinted polymers/nonimprinted polymers) was calculated 1.39 ng mL-1 and 12.27 respectively showing high binding capacity and specific recognition behavior toward template molecule. Simplicity of present method would put forward a fast, facile, cost-effective diagnostic tool for mass health care.


Assuntos
Proteínas de Bactérias/sangue , Técnicas Biossensoriais/métodos , Epitopos/análise , Proteínas de Ligação ao Ferro/sangue , Impressão Molecular/métodos , Neisseria meningitidis/química , Peptídeos/química , Acrilamidas/química , Sequência de Aminoácidos , Reagentes de Ligações Cruzadas/química , Eletrodos , Epitopos/química , Humanos , Proteínas de Ligação ao Ferro/análise , Limite de Detecção , Meningite Meningocócica/sangue , Meningite Meningocócica/diagnóstico , Meningite Meningocócica/microbiologia , Metacrilatos/química , Técnicas de Microbalança de Cristal de Quartzo
15.
Bioorg Med Chem Lett ; 28(17): 2842-2845, 2018 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-30072225

RESUMO

A new library of thirteen indolylisoxazolines 6a-m has been synthesized by the treatment of indolylchalcones with hydroxylamine hydrochloride. Evaluation of anticancer activity of indolylisoxazolines 6a-m led to the identification of potent compounds 6c-d, 6i and 6l, with IC50 ranging 2.5-5.0 µM against the tested cancer cell lines. Using a number of complementary techniques such as acridine orange/ethidium bromide staining, PARP1 cleavage and DNA strand breaks assay, we show that the compounds 6c and 6i induce apoptosis in highly aggressive C4-2 cells. Our data further revealed that 6c and 6i inhibited C4-2 cells proliferation without inducing reactive oxygen species (ROS). Finally, we show that compounds 6c and 6i also potently inhibit cell migration, indicating these compounds have the potential to serve as effective anti-cancer agents.


Assuntos
Antineoplásicos/farmacologia , Indóis/farmacologia , Isoxazóis/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Indóis/síntese química , Indóis/química , Isoxazóis/síntese química , Isoxazóis/química , Estrutura Molecular , Espécies Reativas de Oxigênio/metabolismo , Relação Estrutura-Atividade
16.
Bioorg Med Chem Lett ; 28(8): 1278-1282, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29573910

RESUMO

A series of sixteen ß-carbolines, bearing chalcone moiety at C-1 position, were prepared from easily accessible 1-acetyl-ß-carboline and various aldehydes under basic conditions followed by N2-alkylation using different alkyl bromides. The prepared compounds were evaluated for in vitro cytotoxicity against a panel of human tumor cell lines. N2-Alkylated-ß-carboline chalcones 13a-i represented the interesting anticancer activities compared to N2-unsubstituted ß-carboline chalcones 12a-g. Off the prepared ß-carbolines, 13g exhibited broad spectrum of activity with IC50 values lower than 22.5 µM against all the tested cancer cell lines. Further, the N2-alkylated-ß-carboline chalcone 13g markedly induced cell death in MDA-MB-231 cells by AO/EB staining assay. The most cytotoxic compound 13g possessed a relatively high drug score of 0.48. Additionally, the prepared ß-carboline chalcones displayed moderate antibacterial activities against tested bacterial strains.


Assuntos
Antibacterianos/farmacologia , Antineoplásicos/farmacologia , Brometos/farmacologia , Carbolinas/farmacologia , Chalconas/farmacologia , Sais/farmacologia , Antibacterianos/síntese química , Antineoplásicos/síntese química , Apoptose/efeitos dos fármacos , Bacillus subtilis/efeitos dos fármacos , Brometos/síntese química , Carbolinas/síntese química , Linhagem Celular Tumoral , Chalconas/síntese química , Cloranfenicol/farmacologia , Doxorrubicina/farmacologia , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Enterobacter cloacae/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/efeitos dos fármacos , Sais/síntese química , Staphylococcus aureus/efeitos dos fármacos
17.
BMC Biol ; 15(1): 10, 2017 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-28193222

RESUMO

BACKGROUND: Epithelial-to-mesenchymal transition (EMT) and cancer stem cell (CSC) formation are key underlying causes that promote extensive metastasis, drug resistance, and tumor recurrence in highly lethal pancreatic cancer. The mechanisms leading to EMT and CSC phenotypes are not fully understood, which has hindered the development of effective targeted therapies capable of improving treatment outcomes in patients with pancreatic cancer. RESULTS: We show a central role of Aurora kinase A (AURKA) in promoting EMT and CSC phenotypes via ALDH1A1, which was discovered as its direct substrate using an innovative chemical genetic screen. AURKA phosphorylates ALDH1A1 at three critical residues which exert a multifaceted regulation over its level, enzymatic activity, and quaternary structure. While all three phosphorylation sites contribute to its increased stability, T267 phosphorylation primarily regulates ALDH1A1 activity. AURKA-mediated phosphorylation rapidly dissociates tetrameric ALDH1A1 into a highly active monomeric species. ALDH1A1 also reciprocates and prevents AURKA degradation, thereby triggering a positive feedback activation loop which drives highly aggressive phenotypes in cancer. Phospho-resistant ALDH1A1 fully reverses EMT and CSC phenotypes, thus serving as dominant negative, which underscores the clinical significance of the AURKA-ALDH1A1 signaling axis in pancreatic cancer. CONCLUSIONS: While increased levels and activity of ALDH1A1 are hallmarks of CSCs, the underlying molecular mechanism remains unclear. We show the first phosphorylation-dependent regulation of ALDH1A1, which increases its levels and activity via AURKA. Recent global phospho-proteomic screens have revealed increased phosphorylation of ALDH1A1 at the T267 site in human cancers and healthy liver tissues where ALDH1A1 is highly expressed and active, indicating that this regulation is likely crucial both in normal and diseased states. This is also the first study to demonstrate oligomer-dependent activity of ALDH1A1, signifying that targeting its oligomerization state may be an effective therapeutic approach for counteracting its protective functions in cancer. Finally, while AURKA inhibition provides a potent tool to reduce ALDH1A1 levels and activity, the reciprocal loop between them ensures that their concurrent inhibition will be highly synergistic when inhibiting tumorigenesis, chemoresistance, and metastasis in highly aggressive pancreatic cancer and beyond.


Assuntos
Aldeído Desidrogenase/metabolismo , Aurora Quinase A/metabolismo , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/patologia , Família Aldeído Desidrogenase 1 , Linhagem Celular Tumoral , Movimento Celular , Resistencia a Medicamentos Antineoplásicos , Estabilidade Enzimática , Transição Epitelial-Mesenquimal , Retroalimentação Fisiológica , Humanos , Invasividade Neoplásica , Fenótipo , Fosforilação , Fosfotreonina/metabolismo , Multimerização Proteica , Transporte Proteico , Proteólise , Retinal Desidrogenase , Frações Subcelulares/enzimologia
18.
Bioorg Med Chem Lett ; 27(6): 1379-1384, 2017 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-28254167

RESUMO

A series of novel ß-carbolinium bromides has been synthesized from easily accessible ß-carbolines and 1-aryl-2-bromoethanones. The newly synthesized compounds were evaluated for their in vitro anticancer activity. Among the synthesized derivatives, compounds 16l, 16o and 16s exhibited potent anticancer activity with IC50 values of <10µM against tested cancer cell lines. The most potent analogue 16l was broadly active against all the tested cancer cell lines (IC50=3.16-7.93µM). In order to test the mechanism of cell death, we exposed castration resistant prostate cancer cell line (C4-2) to compounds 16l and 16s, which resulted in increased levels of cleaved PARP1 and AO/EB staining, indicating that ß-carbolinium salts induce apoptosis in these cells. Additionally, the most potent ß-carbolines 16l and 16s were found to inhibit tubulin polymerization.


Assuntos
Brometos/síntese química , Brometos/farmacologia , Carbolinas/síntese química , Carbolinas/farmacologia , Brometos/química , Carbolinas/química , Linhagem Celular Tumoral , Desenho de Fármacos , Humanos , Masculino , Neoplasias da Próstata/patologia
19.
J Cell Sci ; 127(Pt 11): 2391-400, 2014 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-24879856

RESUMO

Cyclin dependent kinase-5 (Cdk5), a family member of the cyclin-dependent kinases, plays a pivotal role in the central nervous system. During embryogenesis, Cdk5 is indispensable for brain development and, in the adult brain, it is essential for numerous neuronal processes, including higher cognitive functions such as learning and memory formation. However, Cdk5 activity becomes deregulated in several neurological disorders, such as Alzheimer's disease, Parkinson's disease and Huntington's disease, which leads to neurotoxicity. Therefore, precise control over Cdk5 activity is essential for its physiological functions. This Commentary covers the various mechanisms of Cdk5 regulation, including several recently identified protein activators and inhibitors of Cdk5 that control its activity in normal and diseased brains. We also discuss the autoregulatory activity of Cdk5 and its regulation at the transcriptional, post-transcriptional and post-translational levels. We finally highlight physiological and pathological roles of Cdk5 in the brain. Specific modulation of these protein regulators is expected to provide alternative strategies for the development of effective therapeutic interventions that are triggered by deregulation of Cdk5.


Assuntos
Quinase 5 Dependente de Ciclina/metabolismo , Regulação da Expressão Gênica , Doenças Neurodegenerativas/metabolismo , Processamento de Proteína Pós-Traducional , Interferência de RNA , Animais , Cognição , Quinase 5 Dependente de Ciclina/genética , Desenvolvimento Fetal , Homeostase , Humanos , Memória , Terapia de Alvo Molecular , Doenças Neurodegenerativas/tratamento farmacológico
20.
J Mol Recognit ; 29(12): 572-579, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27488811

RESUMO

Neisseria meningitidis, a human-specific bacterial pathogen causes bacterial meningitis by invading the meninges (outer lining) of central nervous system. It is the polysaccharide present on the bacterial capsid that distinguishes various serogroups of N. meningitidis and can be utilized as antigens to elicit immune response. A computational approach identified candidate T-cell epitopes from outer membrane proteins Por B of N. meningitidis (MC58): (273 KGLVDDADI282 in loop VII and 170 GRHNSESYH179 in loop IV) present on the exposed surface of immunogenic loops of class 3 outer membrane proteins allele of N. meningitidis. One of them, KGLVDDADI is used here for designing a diagnostic tool via molecularly imprinted piezoelectric sensor (molecularly imprinted polymer-quartz crystal microbalance) for N. meningitidis strain MC58. Methacrylic acid, ethylene glycol dimethacrylate and azoisobutyronitrile were used as functional monomer, cross-linker and initiator, respectively. The epitope can be simultaneously bound to methacrylic acid and fitted into the shape-selective cavities. On extraction of epitope sequence from thus grafted polymeric film, shape-selective and sensitive sites were generated on electrochemical quartz crystal microbalance crystal, ie, known as epitope imprinted polymers. Imprinting was characterized by atomic force microscopy images. The epitope-imprinted sensor was able to selectively bind N. meningitidis proteins present in blood serum of patients suffering from brain fever. Thus, fabricated sensor can be used as a diagnostic tool for meningitis disease.


Assuntos
Epitopos de Linfócito T/metabolismo , Meningite Meningocócica/diagnóstico , Impressão Molecular/métodos , Neisseria meningitidis/isolamento & purificação , Biologia Computacional , Epitopos de Linfócito T/química , Humanos , Metacrilatos , Microscopia de Força Atômica , Modelos Moleculares , Neisseria meningitidis/imunologia , Técnicas de Microbalança de Cristal de Quartzo
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