RESUMO
Poly(rC)-binding protein 2 (PCBP2, hnRNP E2) is one of the most abundant RNA-binding proteins in mammalian cells. In humans, it exists in seven isoforms, which are assumed to play similar roles in cells. The protein is shown to bind 3'-untranslated regions (3'-UTRs) of many mRNAs and regulate their translation and/or stability, but nothing is known about the functional consequences of PCBP2 binding to 5'-UTRs. Here we show that the PCBP2 isoform f interacts with the 5'-UTRs of mRNAs encoding eIF4G2 (a translation initiation factor with a yet unknown mechanism of action, also known as DAP5) and Cyclin I, and inhibits their translation in vitro and in cultured cells, while the PCBP2 isoform e only affects Cyclin I translation. Furthermore, eIF4G2 participates in a cap-dependent translation of the PCBP2 mRNA. Thus, PCBP2 and eIF4G2 seem to regulate one another's expression via a novel type of feedback loop formed by the translation initiation factor and the RNA-binding protein.
Assuntos
Regiões 5' não Traduzidas/genética , Fator de Iniciação Eucariótico 4G/genética , Biossíntese de Proteínas , RNA Mensageiro/genética , Proteínas de Ligação a RNA/metabolismo , Células Cultivadas , Fator de Iniciação Eucariótico 4G/metabolismo , Humanos , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genéticaRESUMO
Asialoglycoprotein receptor (ASGP-R) belongs to a wide family of C-type lectins and it is currently regarded as an attractive protein in the field of targeted drug delivery (TDD). It is abundantly expressed in hepatocytes and can be found predominantly on the sinusoidal surface especially of HepG2 cells. Therefore, ASGP-R can be used for the TDD of anticancer therapeutics against HCC and molecular diagnostic tools. To date, a variety of mono- and multivalent selective ASGP-R ligands have been discovered. Although many of these compounds have demonstrated a relatively high binding affinity towards the target, the reported synthetic schemes are not handled, complicated and include many non-trivial steps. In the current study, we describe a convenient and versatile synthetic approach to novel monovalent drug-conjugates containing N-acetyl-2-deoxy-2-aminogalactopyranose fragment as an ASGP-R-recognition "core-head" and well-known nonselective cytostatic - Doxorubicin (Dox). This is the first example of the direct conjugation of a drug molecule to the ASGP-targeted warhead by a really convenient manner via a simple linker sequence. The performed MTS-based biological evaluation in HepG2 cells revealed the novel conjugates as having anticancer activity. Confocal microscopy showed that the molecules readily penetrated HepG2 membrane and were mainly localized within the cytoplasm instead of the nucleus. Per contra, Dox under the same conditions demonstrated good anticancer activity and was predominantly concentrated in the nucleus. Therefore, we speculate that the amide "trigger" that we have used in this study for linker attachment is a sufficiently stable inside the cells to be enzymatically or spontaneously degraded. As a consequence, we did not observe the release of the drug. Ligands containing triggers that are more liable towards endogenous hydrolysis within the tissue of targeting are strongly required.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Receptor de Asialoglicoproteína/antagonistas & inibidores , Doxorrubicina/farmacologia , Galactose/farmacologia , Antibióticos Antineoplásicos/síntese química , Antibióticos Antineoplásicos/química , Receptor de Asialoglicoproteína/metabolismo , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Doxorrubicina/química , Sistemas de Liberação de Medicamentos , Ensaios de Seleção de Medicamentos Antitumorais , Galactose/análogos & derivados , Galactose/química , Células Hep G2 , Humanos , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Asialoglycoprotein receptor (ASGP-R) is a promising biological target for drug delivery into hepatoma cells. Nevertheless, there are only few examples of small-molecule conjugates of ASGP-R selective ligand equipped by a therapeutic agent for the treatment of hepatocellular carcinoma (HCC). In the present work, we describe a convenient and versatile synthetic approach to novel mono- and multivalent drug-conjugates containing N-acetyl-2-deoxy-2-aminogalactopyranose and anticancer drug - paclitaxel (PTX). Several molecules have demonstrated high affinity towards ASGP-R and good stability under physiological conditions, significant in vitro anticancer activity comparable to PTX, as well as good internalization via ASGP-R-mediated endocytosis. Therefore, the conjugates with the highest potency can be regarded as a promising therapeutic option against HCC.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Receptor de Asialoglicoproteína/antagonistas & inibidores , Carcinoma Hepatocelular/tratamento farmacológico , Galactose/farmacologia , Neoplasias Hepáticas/tratamento farmacológico , Paclitaxel/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Antineoplásicos Fitogênicos/síntese química , Antineoplásicos Fitogênicos/química , Receptor de Asialoglicoproteína/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Sistemas de Liberação de Medicamentos , Ensaios de Seleção de Medicamentos Antitumorais , Galactose/análogos & derivados , Galactose/química , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Estrutura Molecular , Paclitaxel/síntese química , Paclitaxel/química , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-AtividadeRESUMO
Glioblastoma (GBM) is the most frequent and aggressive primary brain cancer in adult patients. A variety of long non-coding RNAs play an important role in the pathogenesis of GBM, however the molecular functions of most of them still remain elusive. Here, we investigated linc-RoR (long intergenic non-protein coding RNA, regulator of reprogramming) using GBM neurospheres obtained from 12 different patients. We demonstrated that the highest level of this transcript is detected in cells with increased EGFR expression. According to our data, linc-RoR knockdown decreases cell proliferation, increases sensitivity to DNA damage, and downregulates the level of cancer stem cell (CSC) markers. On the other hand, linc-RoR overexpression promote cell growth and increases the proportion of CSCs. Analysis of RNA sequencing data revealed that linc-RoR affects expression of genes involved in the regulation of mitosis. In agreement with this observation, we have showen that the highest level of linc-RoR is detected in the G2/M phase of the cell cycle, when linc-RoR is localized on the chromosomes of dividing cells. Based on our results, we can propose that linc-RoR performs pro-oncogenic functions in human gliobalstoma cells, which may be associated with the regulation of mitotic progression and GBM stemness.
Assuntos
Glioblastoma , RNA Longo não Codificante , Carcinogênese , Linhagem Celular Tumoral , Proliferação de Células/genética , Glioblastoma/genética , Humanos , Células-Tronco Neoplásicas/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismoRESUMO
Human protein Ku usually functions in the cell as a complex of two subunits, Ku70 and Ku80. The Ku heterodimer plays a key role in the non-homologous end joining DNA repair pathway by specifically recognizing the DNA ends at the site of the lesion. The binding of the Ku heterodimer to DNA has been well-studied, and its interactions with RNA have been also described. However, Ku70 subunit is known to have independent DNA binding capability, which is less characterized. RNA binding properties of Ku70 have not been yet specially studied. We have prepared recombinant full-length Ku70 and a set of its truncated mutants in E. coli, and studied their interactions with nucleic acids of various structures: linear single- and double-stranded DNA and RNA, as well as closed circular DNA and hairpin RNA. Ku70 has demonstrated a high affinity binding to double stranded DNA and hairpin RNA with a certain structure only. Interestingly, in contrast to the Ku heterodimer, Ku70 is found to interact with closed circular DNA. We also show for the first time that Ku70 employs two different sites for DNA and RNA binding. The double-stranded DNA is recognized by the C-terminal part of Ku70 including SAP domain as it has been earlier demonstrated, whereas hairpin RNA binding is provided by amino acids 251-438.
Assuntos
DNA Circular/metabolismo , DNA/metabolismo , Autoantígeno Ku/metabolismo , RNA/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Ligação Competitiva , Western Blotting , DNA/química , DNA/genética , DNA Circular/química , DNA Circular/genética , Humanos , Cinética , Autoantígeno Ku/química , Autoantígeno Ku/genética , Conformação de Ácido Nucleico , Motivos de Nucleotídeos/genética , Ligação Proteica , Domínios Proteicos , RNA/química , RNA/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homologia de Sequência do Ácido NucleicoRESUMO
Human Ku70/Ku80 protein is known to influence HIV-1 replication. One of the possible reasons may be the protection of integrase from proteasomal degradation by Ku70 subunit. We demonstrated that recombinant HIV-1 integrase and Ku70 form a stable complex, while no interaction of Ku70 with integrase from prototype foamy virus was observed. By analyzing protein subdomains we determined two binding sites in the structure of both Ku70 and integrase: the 51-160 a.a. region of integrase interacts with residues 251-438 of Ku70, whereas Ku70 N-terminal domain (1-250 a.a.) contacts an α6-helix in the 200-220 a.a. integrase region. Single substitutions within integrase (E212A or L213A) block the interaction with Ku70 thus indicating that the binding site formed by the 200-220 a.a. integrase region is crucial for complex formation. E212A/L213A substitutions decreased the integrase capacity to bind Ku70 in HEK293T cells. A conjugate of 2'-ÐMe-GGUUUUUGUGU oligonucleotide with eosin is shown by molecular modeling to shield integrase residues E212/L213 and is effective in blocking complex formation of Ku70 with integrase what makes the complex between α6-helix and Ku70(1-250) a possible target for drug development.
Assuntos
Integrase de HIV/química , Integrase de HIV/metabolismo , HIV-1/enzimologia , Autoantígeno Ku/química , Autoantígeno Ku/metabolismo , Substituição de Aminoácidos , Sítios de Ligação , Domínio Catalítico , Sistemas de Liberação de Medicamentos , Células HEK293 , Integrase de HIV/genética , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Domínios Proteicos , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
Recently a covalent peroxidase-mimicking DNAzyme (cPMDNAzyme) with the improved catalytic activity was prepared. Here we demonstrate that hydrogen peroxide, the oxidant substrate of cPMDNAzyme is an inactivating agent of this catalyst. Presence of the reductant substrate, 2,2'-azino-bis(3-ethylbenthothiazoline-6-sulfonic acid (ABTS) prevents the inactivation of cPMDNAzyme. The experimental conditions (pH-optimum, concentrations of ABTS and H2O2) for the determination of cPMDNAzyme activity were optimized that allows a construction of the colorimetric cPMDNAzyme-based biosensors and assays with improved sensitivity.
Assuntos
Benzotiazóis/metabolismo , Materiais Biomiméticos/metabolismo , DNA Catalítico/metabolismo , Peróxido de Hidrogênio/farmacologia , Peroxidase/metabolismo , Substâncias Redutoras/metabolismo , Ácidos Sulfônicos/metabolismo , Benzotiazóis/farmacologia , Biocatálise , Ativação Enzimática/efeitos dos fármacos , Oxirredução , Substâncias Redutoras/farmacologia , Ácidos Sulfônicos/farmacologiaRESUMO
Lesions in the DNA arise under ionizing irradiation conditions or various chemical oxidants as a single damage or as part of a multiply damaged site within 1-2 helical turns (clustered lesion). Here, we explored the repair opportunity of the apurinic/apyrimidinic site (AP site) composed of the clustered lesion with 5-formyluracil (5-foU) by the base excision repair (BER) proteins. We found, that if the AP site is shifted relative to the 5-foU of the opposite strand, it could be repaired primarily via the short-patch BER pathway. In this case, the cleavage efficiency of the AP site-containing DNA strand catalyzed by human apurinic/apyrimidinic endonuclease 1 (hAPE1) decreased under AP site excursion to the 3'-side relative to the lesion in the other DNA strand. DNA synthesis catalyzed by DNA polymerase lambda was more accurate in comparison to the one catalyzed by DNA polymerase beta. If the AP site was located exactly opposite 5-foU it was expected to switch the repair to the long-patch BER pathway. In this situation, human processivity factor hPCNA stimulates the process.