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1.
Int J Mol Sci ; 23(22)2022 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-36430510

RESUMO

Protein kinase C (PKC) comprises a family of highly related serine/threonine protein kinases involved in multiple signaling pathways, which control cell proliferation, survival, and differentiation. The role of PKCα in cancer has been studied for many years. However, it has been impossible to establish whether PKCα acts as an oncogene or a tumor suppressor. Here, we analyzed the importance of PKCα in cellular processes such as proliferation, migration, or apoptosis by inhibiting its gene expression in a luminal A breast cancer cell line (MCF-7). Differential expression analysis and phospho-kinase arrays of PKCα-KD vs. PKCα-WT MCF-7 cells identified an essential set of proteins and oncogenic kinases of the JAK/STAT and PI3K/AKT pathways that were down-regulated, whereas IGF1R, ERK1/2, and p53 were up-regulated. In addition, unexpected genes related to the interferon pathway appeared down-regulated, while PLC, ERBB4, or PDGFA displayed up-regulated. The integration of this information clearly showed us the usefulness of inhibiting a multifunctional kinase-like PKCα in the first step to control the tumor phenotype. Then allowing us to design a possible selection of specific inhibitors for the unexpected up-regulated pathways to further provide a second step of treatment to inhibit the proliferation and migration of MCF-7 cells. The results of this study suggest that PKCα plays an oncogenic role in this type of breast cancer model. In addition, it reveals the signaling mode of PKCα at both gene expression and kinase activation. In this way, a wide range of proteins can implement a new strategy to fine-tune the control of crucial functions in these cells and pave the way for designing targeted cancer therapies.


Assuntos
Neoplasias , Proteína Quinase C-alfa , Humanos , Proteína Quinase C-alfa/genética , Proteína Quinase C-alfa/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais , Proteína Quinase C/metabolismo , Proliferação de Células
2.
Inorg Chem ; 58(19): 12954-12963, 2019 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-31550148

RESUMO

The luminescent chalcone gold(I) conjugates [Au(PPh3)(AN3E)]PF6(1) and [Au(SIMes)(AN3E)]PF6 (2) (AN3E = (E)-3-(9-anthracenyl)-1-(4-pyridyl)propenone; SIMes = N,N'-dimesitylimidazolidin-2-ylidene; Mes = 2,4,6-trimethylphenyl)) were prepared and characterized; complex 1 was also characterized by X-ray crystallography. In MTT assays against a panel of three human colon, a melanoma and a breast cancer cell lines both complexes were antiproliferative with low micromolar IC50 values. It is noteworthy that HCT116p53-/- colon carcinoma cells lacking functional p53 (a vital tumor suppressor) were more susceptible to them than the wildtype parent cell line. In flow cytometry analyses, the gold conjugates induced a significant arrest in G2/M phase primarily. Complexes 1 and 2 quickly increased the production of reactive oxygen species (ROS) and induced mitochondrial membrane potential depolarization, higher ROS values being obtained after coadministration with enzymatic inhibitors. The free chalcone AN3E and its gold(I) complex conjugates located in the cell mitochondria according to confocal microscopy. In addition, complexes 1 and 2 showed in vivo antivascular effects on the chorioallantoic membrane (CAM) of fertilized specific-pathogen-free (SPF) chicken eggs.


Assuntos
Inibidores da Angiogênese/farmacologia , Antracenos/farmacologia , Antineoplásicos/farmacologia , Chalcona/farmacologia , Neoplasias do Colo/tratamento farmacológico , Compostos Organoáuricos/farmacologia , Inibidores da Angiogênese/química , Animais , Antracenos/química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Chalcona/análogos & derivados , Galinhas , Cristalografia por Raios X , Células HCT116 , Humanos , Modelos Moleculares , Compostos Organoáuricos/química
3.
J Proteome Res ; 17(1): 252-264, 2018 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-29131639

RESUMO

We investigated new transcription and splicing factors associated with the metastatic phenotype in colorectal cancer. A concatenated tandem array of consensus transcription factor (TF)-response elements was used to pull down nuclear extracts in two different pairs of colorectal cancer cells, KM12SM/KM12C and SW620/480, genetically related but differing in metastatic ability. Proteins were analyzed by label-free LC-MS and quantified with MaxLFQ. We found 240 proteins showing a significant dysregulation in highly metastatic KM12SM cells relative to nonmetastatic KM12C cells and 257 proteins in metastatic SW620 versus SW480. In both cell lines there were similar alterations in genuine TFs and components of the splicing machinery like UPF1, TCF7L2/TCF-4, YBX1, or SRSF3. However, a significant number of alterations were cell-line specific. Functional silencing of MAFG, TFE3, TCF7L2/TCF-4, and SRSF3 in KM12 cells caused alterations in adhesion, survival, proliferation, migration, and liver homing, supporting their role in metastasis. Finally, we investigated the prognostic value of the altered TFs and splicing factors in cancer patients. SRSF3 and SFPQ showed significant prognostic value. We observed that SRSF3 displayed a gradual loss of expression associated with cancer progression. Loss of SRSF3 expression was significantly associated with poor survival and shorter disease-free survival, particularly in early stages, in colorectal cancer.


Assuntos
Neoplasias Colorretais/química , Metástase Neoplásica , Proteômica/métodos , Fatores de Processamento de RNA/análise , Fatores de Transcrição/análise , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Humanos , Prognóstico , Fatores de Processamento de Serina-Arginina/análise
4.
J Proteome Res ; 16(12): 4374-4390, 2017 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-28960077

RESUMO

The Human Proteome Project (HPP) aims deciphering the complete map of the human proteome. In the past few years, significant efforts of the HPP teams have been dedicated to the experimental detection of the missing proteins, which lack reliable mass spectrometry evidence of their existence. In this endeavor, an in depth analysis of shotgun experiments might represent a valuable resource to select a biological matrix in design validation experiments. In this work, we used all the proteomic experiments from the NCI60 cell lines and applied an integrative approach based on the results obtained from Comet, Mascot, OMSSA, and X!Tandem. This workflow benefits from the complementarity of these search engines to increase the proteome coverage. Five missing proteins C-HPP guidelines compliant were identified, although further validation is needed. Moreover, 165 missing proteins were detected with only one unique peptide, and their functional analysis supported their participation in cellular pathways as was also proposed in other studies. Finally, we performed a combined analysis of the gene expression levels and the proteomic identifications from the common cell lines between the NCI60 and the CCLE project to suggest alternatives for further validation of missing protein observations.


Assuntos
Proteoma/análise , Proteômica/métodos , Ferramenta de Busca , Linhagem Celular Tumoral , Humanos , Bases de Conhecimento , Proteínas/análise , Software
5.
J Cell Sci ; 128(6): 1097-107, 2015 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-25632158

RESUMO

The exosome acts on different RNA substrates and plays important roles in RNA metabolism. The fact that short non-coding RNAs are involved in the DNA damage response led us to investigate whether the exosome factor RRP6 of Drosophila melanogaster and its human ortholog EXOSC10 play a role in DNA repair. Here, we show that RRP6 and EXOSC10 are recruited to DNA double-strand breaks (DSBs) in S2 cells and HeLa cells, respectively. Depletion of RRP6/EXOSC10 does not interfere with the phosphorylation of the histone variant H2Av (Drosophila) or H2AX (humans), but impairs the recruitment of the homologous recombination factor RAD51 to the damaged sites, without affecting RAD51 levels. The recruitment of RAD51 to DSBs in S2 cells is also inhibited by overexpression of RRP6-Y361A-V5, a catalytically inactive RRP6 mutant. Furthermore, cells depleted of RRP6 or EXOSC10 are more sensitive to radiation, which is consistent with RRP6/EXOSC10 playing a role in DNA repair. RRP6/EXOSC10 can be co-immunoprecipitated with RAD51, which links RRP6/EXOSC10 to the homologous recombination pathway. Taken together, our results suggest that the ribonucleolytic activity of RRP6/EXOSC10 is required for the recruitment of RAD51 to DSBs.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Exorribonucleases/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Recombinação Homóloga/genética , Animais , Western Blotting , Proliferação de Células , Imunoprecipitação da Cromatina , Proteínas de Drosophila/genética , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Exorribonucleases/antagonistas & inibidores , Exorribonucleases/genética , Complexo Multienzimático de Ribonucleases do Exossomo/antagonistas & inibidores , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Células HeLa , Histonas/metabolismo , Humanos , Fosforilação , RNA Interferente Pequeno/genética , Rad51 Recombinase/metabolismo
6.
Methods Mol Biol ; 2817: 33-43, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38907145

RESUMO

Mass spectrometry-based proteomics has traditionally been limited by the amount of input material for analysis. Single-cell proteomics has emerged as a challenging discipline due to the ultra-high sensitivity required. Isobaric labeling-based multiplex strategies with a carrier proteome offer an approach to overcome the sensitivity limitations. Following this as the basic strategy, we show here the general workflow for preparing cells for single-cell mass spectrometry-based proteomics. This protocol can also be applied to manually isolated cells when large cells, such as cardiomyocytes, are difficult to isolate properly with conventional fluorescence-activated cell sorting (FACS) sorter methods.


Assuntos
Proteômica , Análise de Célula Única , Proteômica/métodos , Análise de Célula Única/métodos , Humanos , Espectrometria de Massas/métodos , Citometria de Fluxo/métodos , Proteoma/análise , Animais , Marcação por Isótopo/métodos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/citologia , Coloração e Rotulagem/métodos
7.
J Proteome Res ; 12(4): 1969-79, 2013 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-23477467

RESUMO

The pyrimidine analogue 5-fluorouracil (5FU) is used as a treatment for solid tumors, but its mechanism of action is not fully understood. We have used mass spectrometry to study the mechanism of action of 5FU, and we have measured the effects of this drug on the composition and on the turnover of the proteome of RKO cancer cells. We have identified novel potential targets of 5FU that are affected after very short exposure times. We have also shown that 5FU has a massive effect on the proteins involved in RNA metabolism. After only 1 h of treatment, 5FU causes a post-transcriptional reduction in the abundance of components of the translation machinery (mostly ribosomal proteins), and this reduction is accompanied by a down-regulation of the translational capacity of the cells. Neither rapamycin nor raltitrexed, two drugs that also block cell proliferation, reduce the abundances of ribosomal proteins as 5FU does, which suggests that the down-regulation of ribosomal proteins is coupled to the mechanism of action of 5FU. Some of our observations conflict with previous reports based on RNA quantification. This shows how important it is to complement RNA profiling studies with analyses of drug toxicity at the protein level.


Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Fluoruracila/farmacologia , Proteoma/metabolismo , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Regulação para Baixo/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas Ribossômicas/metabolismo , Espectrometria de Massas em Tandem
8.
Inorg Chem ; 52(2): 974-82, 2013 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-23301676

RESUMO

A series of new organoiridium(III) complexes [Ir(N-C)(2)(N-S)]Cl (HN-C = 2-phenylpyridine (Hppy), N-S = methyl thiosemicarbazide (1), phenyl thiosemicarbazide (2) and naphtyl thiosemicarbazide (3)) have been synthesized and characterized. The crystal structure of (1) has been established by X-ray diffraction, showing the thiosemicarbazide ligand bound to the iridium atom as N,S-chelate. The cytotoxicity studies show that they are more active than cisplatin (about 5-fold) in T47D (breast cancer) at 48 h incubation time. On the other hand, very low resistance factors (RF) of 1-3 in A2780cisR (cisplatin-resistant ovarian carcinoma) at 48 h were observed (RF ≈ 1). Ir accumulation in T47D cell line after 48 h continuous exposure for complexes 1-3 are higher than that corresponding to cisplatin (about 10 times). The complexes 1-3 bind strongly to HSA with binding constants of about 10(4) M(-1) at 296 K, binding occurring at the warfarin site I for 2. Complexes 2 and 3 are also capable of binding in the minor groove of DNA as shown by Hoechst 33258 displacement experiments. Furthermore, complex 2 is also a good cathepsin B inhibitor (an enzyme implicated in a number of cancer related events), being the enzyme reactivated by cysteine.


Assuntos
Antineoplásicos/síntese química , Catepsina B/antagonistas & inibidores , DNA/química , Irídio/química , Semicarbazidas/química , Antineoplásicos/química , Antineoplásicos/farmacologia , Ligação Competitiva , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cristalografia por Raios X , DNA/metabolismo , Eletroforese em Gel Bidimensional , Ativação Enzimática/efeitos dos fármacos , Feminino , Humanos , Concentração Inibidora 50 , Compostos Organometálicos/síntese química , Compostos Organometálicos/química , Neoplasias Ovarianas/tratamento farmacológico , Semicarbazidas/síntese química , Semicarbazidas/farmacologia , Albumina Sérica/química , Albumina Sérica/metabolismo
9.
Proc Natl Acad Sci U S A ; 106(16): 6603-7, 2009 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-19346474

RESUMO

C2 domains are widely-spread protein signaling motifs that in classical PKCs act as Ca(2+)-binding modules. However, the molecular mechanisms of their targeting process at the plasma membrane remain poorly understood. Here, the crystal structure of PKCalpha-C2 domain in complex with Ca(2+), 1,2-dihexanoyl-sn-glycero-3-[phospho-L-serine] (PtdSer), and 1,2-diayl-sn-glycero-3-[phosphoinositol-4,5-bisphosphate] [PtdIns(4,5)P(2)] shows that PtdSer binds specifically to the calcium-binding region, whereas PtdIns(4,5)P(2) occupies the concave surface of strands beta3 and beta4. Strikingly, the structure reveals a PtdIns(4,5)P(2)-C2 domain-binding mode in which the aromatic residues Tyr-195 and Trp-245 establish direct interactions with the phosphate moieties of the inositol ring. Mutations that abrogate Tyr-195 and Trp-245 recognition of PtdIns(4,5)P(2) severely impaired the ability of PKCalpha to localize to the plasma membrane. Notably, these residues are highly conserved among C2 domains of topology I, and a general mechanism of C2 domain-membrane docking mediated by PtdIns(4,5)P(2) is presented.


Assuntos
Fosfatidilinositol 4,5-Difosfato/metabolismo , Proteína Quinase C-alfa/química , Proteína Quinase C-alfa/metabolismo , Aminoácidos/metabolismo , Animais , Cátions/metabolismo , Membrana Celular/enzimologia , Sequência Conservada , Genes Dominantes , Modelos Moleculares , Proteínas Mutantes/metabolismo , Células PC12 , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transporte Proteico , Ratos , Relação Estrutura-Atividade
10.
J Proteome Res ; 10(2): 529-40, 2011 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-20973479

RESUMO

PKCα is a key mediator of the neuronal differentiation controlled by NGF and ATP. However, its downstream signaling pathways remain to be elucidated. To identify the signaling partners of PKCα, we analyzed proteins coimmunoprecipitated with this enzyme in PC12 cells differentiated with NGF and ATP and compared them with those obtained with NGF alone or growing media. Mass spectrometry analysis (LC-MS/MS) identified plectin, peripherin, filamin A, fascin, and ß-actin as potential interacting proteins. The colocalization of PKCα and its interacting proteins increased when PC12 cells were differentiated with NGF and ATP. Peripherin and plectin organization and the cortical remodeling of ß-actin were dramatically affected when PKCα was down-regulated, suggesting that all three proteins might be functional targets of ATP-dependent PKCα signaling. Taken together, these data demonstrate that PKCα is essential for controlling the neuronal development induced by NGF and ATP and interacts with the cytoskeletal components at two levels: assembly of the intermediate filament peripherin and organization of cortical actin.


Assuntos
Trifosfato de Adenosina/metabolismo , Diferenciação Celular/fisiologia , Proteínas do Citoesqueleto/metabolismo , Neurônios/metabolismo , Proteína Quinase C-alfa/metabolismo , Animais , Western Blotting , Regulação para Baixo , Proteínas de Filamentos Intermediários/metabolismo , Filamentos Intermediários/metabolismo , Espaço Intracelular/metabolismo , Glicoproteínas de Membrana/metabolismo , Microscopia de Fluorescência , Modelos Biológicos , Fator de Crescimento Neural/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neuritos/metabolismo , Neurônios/citologia , Neurônios/enzimologia , Células PC12 , Periferinas , Plectina/metabolismo , Mapeamento de Interação de Proteínas , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Ratos , Reprodutibilidade dos Testes , Transdução de Sinais/fisiologia , Espectrometria de Massas em Tandem
11.
Inorg Chem ; 50(6): 2151-8, 2011 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-21314142

RESUMO

The synthesis of the novel anthraquinone platinum derivate [Pt(ppy)Cl(1C3)] (2) [Hppy = N,C-chelating 2-phenylpyridine; 1C3 = 1-[(3-aminopropyl)amino]-anthracene-9,10-dione] and its values of IC(50) against a panel of human tumor cell lines representative of ovarian (A2780 and A2780cisR) and breast cancers (T47D) are reported. At 24 h incubation time, complex 2 was more active than cisplatin (about 9-fold) and the free ligand 1C3 (about 2-fold) in T47-D. The observation that the cisplatin IC(50) falls by about 10-fold from 24 to 72 h, whereas that for 2 changes little, suggests substantial differences in the mode of action. Complex 2 also showed high cytotoxicity against A2780 (about 3-fold greater than cisplatin at 24 h). On the other hand, very low resistance factors (RF) of 2 in A2780cisR at 24-72 h (RF = 1.3) were observed. The interaction of 2 with DNA was followed by electrophoretic mobility and UV-visible spectroscopy, and its reaction with the model nucleobase 9-EtG was studied by (1)H NMR and ESI-MS. Theoretical calculations at the B3LYP/def2-TZVPP//BP86/def2-TZVP level of theory on complex 2 show a labile Pt-Cl bond that allows easy replacement of Cl by N-nucleophiles such as 9-EtG, which forms a stronger Pt-N bond.


Assuntos
Antraquinonas/química , Antineoplásicos/farmacologia , Compostos Organoplatínicos/farmacologia , Piridinas/química , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Modelos Moleculares , Estrutura Molecular , Compostos Organoplatínicos/síntese química , Compostos Organoplatínicos/química , Teoria Quântica , Relação Estrutura-Atividade , Células Tumorais Cultivadas
12.
Phys Chem Chem Phys ; 12(41): 13372-4, 2010 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-20856953

RESUMO

Detailed analysis of >18 400 high-mass accuracy tandem mass spectra resulting from higher energy collisional dissociation yields further evidence of the cyclic nature of a(2)-ions.


Assuntos
Íons/química , Dicetopiperazinas/química , Oxazolona/química , Espectrometria de Massas em Tandem
13.
Proteomics Clin Appl ; 14(1): e1900052, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31502404

RESUMO

PURPOSE: Successful prevention of colorectal cancer (CRC) would benefit from a rapid serum screening for early detection. Here, a novel strategy for CRC biomarker discovery and validation exclusively based on MS procedures is reported. EXPERIMENTAL DESIGN: Identification of CRC serum biomarkers is initially made using label-free quantification on pooled serum samples from different CRC stages followed by two consecutive steps of targeted parallel reaction monitoring assays in different serum cohorts. Relevance of different protein depletion and peptide fractionation extent is investigated. Absolute quantification of a selected peptide is performed as a proof-of-concept. RESULTS: A total of 945 proteins showed differential abundance in the discovery phase. Based on their statistical significance and relative expression in disease stages, 123 potential biomarkers are selected for a training step. In the final validation step, five peptides belonging to four proteins are consistently quantified in individual CRC serum samples and controls. Different statistical analyses indicate that peptides GWVTDGFSSLK (APOC3) and LCNNPTPQFGGK (THBS1) are candidate biomarkers. Absolute quantification of LCNNPTPQFGGK shows statistical significance for the diagnosis of early respect to late CRC stages. CONCLUSIONS AND CLINICAL RELEVANCE: Two peptides from APOC3 and THBS1 are validated by PRM as potential biomarkers for non-invasive diagnosis of colorectal cancer.


Assuntos
Apolipoproteína C-III/sangue , Biomarcadores Tumorais/sangue , Neoplasias Colorretais/sangue , Peptídeos/sangue , Trombospondinas/sangue , Neoplasias Colorretais/patologia , Detecção Precoce de Câncer , Feminino , Humanos , Masculino , Proteínas de Neoplasias/sangue , Proteoma/genética
14.
J Mol Biol ; 371(3): 608-21, 2007 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-17586528

RESUMO

C2 domains are conserved protein modules in many eukaryotic signaling proteins, including the protein kinase (PKCs). The C2 domains of classical PKCs bind to membranes in a Ca(2+)-dependent manner and thereby act as cellular Ca(2+) effectors. Recent findings suggest that the C2 domain of PKCalpha interacts specifically with phosphatidylinositols 4,5-bisphosphate (PtdIns(4,5)P(2)) through its lysine rich cluster, for which it shows higher affinity than for POPS. In this work, we compared the three C2 domains of classical PKCs. Isothermal titration calorimetry revealed that the C2 domains of PKCalpha and beta display a greater capacity to bind to PtdIns(4,5)P(2)-containing vesicles than the C2 domain of PKCgamma. Comparative studies using lipid vesicles containing both POPS and PtdIns(4,5)P(2) as ligands revealed that the domains behave as PtdIns(4,5)P(2)-binding modules rather than as POPS-binding modules, suggesting that the presence of the phosphoinositide in membranes increases the affinity of each domain. When the magnitude of PtdIns(4,5)P(2) binding was compared with that of other polyphosphate phosphatidylinositols, it was seen to be greater in both PKCbeta- and PKCgamma-C2 domains. The concentration of Ca(2+) required to bind to membranes was seen to be lower in the presence of PtdIns(4,5)P(2) for all C2 domains, especially PKCalpha. In vivo experiments using differentiated PC12 cells transfected with each C2 domain fused to ECFP and stimulated with ATP demonstrated that, at limiting intracellular concentration of Ca(2+), the three C2 domains translocate to the plasma membrane at very similar rates. However, the plasma membrane dissociation event differed in each case, PKCalpha persisting for the longest time in the plasma membrane, followed by PKCgamma and, finally, PKCbeta, which probably reflects the different levels of Ca(2+) needed by each domain and their different affinities for PtdIns(4,5)P(2).


Assuntos
Membrana Celular/enzimologia , Fosfatidilinositol 4,5-Difosfato/metabolismo , Proteína Quinase C/química , Proteína Quinase C/metabolismo , Animais , Cálcio/farmacologia , Membrana Celular/efeitos dos fármacos , Cinética , Modelos Biológicos , Modelos Moleculares , Células PC12 , Ligação Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , Ratos , Receptores de Superfície Celular/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato/efeitos dos fármacos , Termodinâmica
15.
Inorg Chem ; 47(15): 6990-7001, 2008 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-18593114

RESUMO

The new complexes [Pd(dmba)( N10-9AA)(PPh 3)]ClO 4 ( 1), [Pt(dmba)( N9-9AA)(PPh 3)]ClO 4 ( 2), [Pd(dmba)( N10-9AA)Cl] ( 3), and [Pd(C 6F 5)( N10-9AA)(PPh 3)Cl] ( 4) (9-AA = 9-aminoacridine; dmba = N,C-chelating 2-(dimethylaminomethyl)phenyl) have been prepared. The crystal structures have been established by X-ray diffraction. In complex 2, an anagostic C-H...Pt interaction is observed. All complexes are luminescent in the solid state at room temperature, showing important differences between the palladium and platinum complexes. Complex 2 shows two structured emission bands at high and low energies in the solid state, and the lifetimes are in agreement with excited states of triplet parentage. Density functional theory and time-dependent density functional theory calculations for complex 2 have been done. Values of IC 50 were also calculated for the new complexes 1- 4 against the tumor cell line HL-60. All of the new complexes were more active than cisplatin (up to 30-fold in some cases). The DNA adduct formation of the new complexes synthesized was followed by circular dichroism and electrophoretic mobility. Atomic force microscopy images of the modifications caused by the complexes on plasmid DNA pB R322 were also obtained.


Assuntos
Aminacrina/química , Antineoplásicos/farmacologia , Luminescência , Compostos Organometálicos/síntese química , Compostos Organometálicos/farmacologia , Paládio/química , Platina/química , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quelantes , Dicroísmo Circular , Eletroforese em Gel de Ágar , Humanos , Microscopia de Força Atômica , Compostos Organometálicos/química , Teoria Quântica , Fatores de Tempo
16.
Mol Biol Cell ; 16(6): 2848-61, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15814842

RESUMO

Signal transduction through protein kinase Cs (PKCs) strongly depends on their subcellular localization. Here, we investigate the molecular determinants of PKCalpha localization by using a model system of neural growth factor (NGF)-differentiated pheochromocytoma (PC12) cells and extracellular stimulation with ATP. Strikingly, the Ca2+ influx, initiated by the ATP stimulation of P2X receptors, rather than the Ca2+ released from the intracellular stores, was the driving force behind the translocation of PKCalpha to the plasma membrane. Furthermore, the localization process depended on two regions of the C2 domain: the Ca2+-binding region and the lysine-rich cluster, which bind Ca2+ and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], respectively. It was demonstrated that diacylglycerol was not involved in the localization of PKCalpha through its C1 domain, and in lieu, the presence of PtdIns(4,5)P2 increased the permanence of PKCalpha in the plasma membrane. Finally, it also was shown that ATP cooperated with NGF during the differentiation process of PC12 cells by increasing the length of the neurites, an effect that was inhibited when the cells were incubated in the presence of a specific inhibitor of PKCalpha, suggesting a possible role for this isoenzyme in the neural differentiation process. Overall, these results show a novel mechanism of PKCalpha activation in differentiated PC12 cells, where Ca2+ influx, together with the endogenous PtdIns(4,5)P2, anchor PKCalpha to the plasma membrane through two distinct motifs of its C2 domain, leading to enzyme activation.


Assuntos
Trifosfato de Adenosina/farmacologia , Cálcio/metabolismo , Diferenciação Celular , Membrana Celular/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Animais , Cálcio/análise , Ativação Enzimática/efeitos dos fármacos , Fura-2/farmacologia , Cinética , Lisina/metabolismo , Microscopia Confocal , Modelos Biológicos , Fatores de Crescimento Neural/farmacologia , Neuritos/efeitos dos fármacos , Neuritos/metabolismo , Células PC12 , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Transporte Proteico , Ratos
17.
J Mol Biol ; 362(5): 901-14, 2006 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-16949603

RESUMO

The C2 domain is a targeting domain that responds to intracellular Ca2+ signals in classical protein kinases (PKCs) and mediates the translocation of its host protein to membranes. Recent studies have revealed a new motif in the C2 domain, named the lysine-rich cluster, that interacts with acidic phospholipids. The purpose of this work was to characterize the molecular mechanism by which PtdIns(4,5)P2 specifically interacts with this motif. Using a combination of isothermal titration calorimetry, fluorescence resonance energy transfer and time-lapse confocal microscopy, we show here that Ca2+ specifically binds to the Ca2+ -binding region, facilitating PtdIns(4,5)P2 access to the lysine-rich cluster. The magnitude of PtdIns(4,5)P2 binding is greater than in the case of other polyphosphate phosphatidylinositols. Very importantly, the residues involved in PtdIns(4,5)P2 binding are essential for the plasma membrane localization of PKCalpha when RBL-2H3 cells are stimulated through their IgE receptors. Additionally, CFP-PH and CFP-C1 domains were used as bioprobes to demonstrate the co-existence of PtdIns(4,5)P2 and diacylglycerol in the plasma membrane, and it was shown that although a fraction of PtdIns(4,5)P2 is hydrolyzed to generate diacylglycerol and IP3, an important amount still remains in the membrane where it is available to activate PKCalpha. These findings entail revision of the currently accepted model of PKCalpha recruitment to the membrane and its activation.


Assuntos
Cálcio/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Proteína Quinase C-alfa/química , Proteína Quinase C-alfa/metabolismo , Animais , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Cinética , Lisina/metabolismo , Modelos Moleculares , Mutação , Ligação Proteica , Proteína Quinase C-alfa/genética , Estrutura Terciária de Proteína , Ratos , Termodinâmica
18.
J Mol Biol ; 357(4): 1105-20, 2006 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-16476439

RESUMO

Arachidonic acid, one of the major unsaturated fatty acids released during cell stimulation, participates in the signaling necessary for activation of different enzymes, including protein kinase C (PKC). Here, we demonstrate that arachidonic acid is a direct activator of PKCalpha, but needs the cooperation of Ca(2+) to exert its function. By using several mutants of the C2 and C1 domains, we were able to determine the molecular mechanism of this activation. More specifically, site-directed mutagenesis in key residues found in the C2 domain showed that the Ca(2+)-binding region was essential for the arachidonic acid-dependent localization and activation of PKCalpha. However, the lysine-rich cluster, also located in the C2 domain, played no relevant role in either the membrane localization or activation of the enzyme. Moreover, site-directed mutagenesis in key residues placed in the C1A and C1B subdomains, which are responsible for the diacylglycerol/phorbil ester interaction, demonstrated that the C1A subdomain was involved in the membrane localization and activation mechanism. Taken together, these data suggest a very precise mechanism for PKCalpha activation by arachidonic acid, involving a sequential model of activation in which an increase in intracytosolic Ca(2+) leads to the interaction of arachidonic acid with the Ca(2+)-binding region; only after this step, does the C1A subdomain interact with arachidonic acid, leading to full activation of the enzyme.


Assuntos
Ácido Araquidônico/metabolismo , Proteína Quinase C-alfa/química , Proteína Quinase C-alfa/metabolismo , Animais , Cálcio/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Ativação Enzimática , Humanos , Modelos Moleculares , Proteína Quinase C-alfa/genética , Estrutura Terciária de Proteína , Ratos
19.
J Chem Theory Comput ; 13(8): 3898-3910, 2017 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-28641006

RESUMO

In this work, we use DFT-based methods to simulate the chemical structures, optical properties, and interaction with DNA of a recently synthesized chelated C^N 9-aminoacridine arene Ru(II) anticancer agent and two new closely related Rh(III) and Ir(III) complexes using DFT-based methods. Four chemical models and a number of theoretical approaches, which representatively include the PBE0, B97D, ωB97X, ωB97X-D, M06, and M06-L density functionals and the LANL2DZ, def2-SVP, and def2-TZVP basis sets, are tested. The best overall accuracy/cost performance for the optimization process is reached at the ωB97X-D/def2-SVP and M06/def2-SVP levels of theory. Inclusion of explicit solvent molecules (CHCl3) further refines the geometry, while taking into account the crystal network gives no significant improvements of the computed bond distances and angles. The analysis of the excited states reveals that the M06 level matches better the experimental absorption spectra, compared to ωB97X-D. The use of the M06/def2-SVP approach is therefore a well-balanced method to study theoretically the bioactivity of this type of antitumoral complexes, so we couple this TD-DFT approach to molecular dynamics simulations in order to assess their reactivity with DNA. The reported results demonstrate that these drugs could be used to inject electrons into DNA, which might broaden their applications in photoactivated chemotherapy and as new materials for DNA-based electrochemical nanodevices.


Assuntos
Aminacrina/análogos & derivados , Antineoplásicos/química , Complexos de Coordenação/química , DNA/química , Substâncias Intercalantes/química , Elétrons , Irídio/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Teoria Quântica , Ródio/química , Rutênio/química
20.
ACS Chem Biol ; 12(6): 1524-1537, 2017 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-28388047

RESUMO

Two new 1-acridin-9-yl-3-methylthiourea Au(I) DNA intercalators [Au(ACRTU)2]Cl (2) and [Au(ACRTU) (PPh3)]PF6 (3) have been prepared. Both complexes were highly active in the human ovarian carcinoma cisplatin-sensitive A2780 cell line, exhibiting IC50 values in the submicromolar range. Compounds 2 and 3 are also cytotoxic toward different phenotypes of breast cancer cell lines MDA-MB-231 (triple negative), SK-BR-3 (HER2+, ERα-, and ERß-), and MCF-7 (ER+). Both complexes induce apoptosis through activation of caspase-3 in vitro. While inhibition of some proteins (thiol-containing enzymes) seems to be the main mechanism of action for cytotoxic gold complexes, 2 and 3 present a DNA-dependent mechanism of action. They locate in the cell nucleus according to confocal microscopy and transmission electronic microscopy. The binding to DNA resulted to be via intercalation as shown by spectroscopic methods and viscometry, exhibiting a dose-dependent response on topoisomerase I mediated DNA unwinding. In addition, 2 and 3 exhibit potent antiangiogenic effects and are also able to inhibit vasculogenic mimicry of highly invasive MDA-MB-231 cells.


Assuntos
Antineoplásicos/química , Substâncias Intercalantes/química , Acridinas/química , Inibidores da Angiogênese/química , Inibidores da Angiogênese/farmacologia , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Ouro/química , Humanos , Substâncias Intercalantes/farmacologia , Mimetismo Molecular , Invasividade Neoplásica/prevenção & controle , Neovascularização Patológica/tratamento farmacológico , Tioureia/química
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