RESUMEN

Protein S-sulfhydration involves the regulation of various protein functions, and resolving the S-sulfhydrated proteome (persulfidome) allows for a deeper exploration of various redox regulations. Therefore, we designed a reducible covalent capture method for isolating S-sulfhydrated proteins, which can analyze the persulfidome in biological samples and monitor specific S-sulfhydrated proteins. In this study, we applied this method to reveal the S-sulfhydration levels of proteins, including 3-phosphoglyceraldehyde dehydrogenase, NFκB/p65, and nucleolin. Furthermore, this technique can be used to enrich S-sulfhydrated peptides, aiding in the determination of protein S-sulfhydration modification sites. Finally, we observed that the S-sulfhydration and oxidation of nucleolin on the C543 residue correlate with its nuclear translocation, downstream regulation of p53, Bcl-xL, and Bcl-2 RNA levels and protein expression, as well as the protective function against oxidative stress. Therefore, this method may facilitate the understanding of the regulation of protein function by redox perturbation.

Asunto(s)

Nucleolina , Oxidación-Reducción , Fosfoproteínas , Proteínas de Unión al ARN , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/aislamiento & purificación , Fosfoproteínas/metabolismo , Fosfoproteínas/química , Fosfoproteínas/análisis , Humanos , Proteoma/análisis , Proteoma/química

RESUMEN

Visualization of multiple targets in living cells is important for understanding complex biological processes, but it still faces difficulties, such as complex operation, difficulty in multiplexing, and expensive equipment. Here, we developed a nanoplatform integrating a nucleic acid aptamer and DNA nanotechnology for living cell imaging. Aptamer-based recognition probes (RPs) were synthesized through rolling circle amplification, which were further self-assembled into DNA nanoflowers encapsulated by an aptamer loop. The signal probes (SPs) were obtained by conjugation of multicolor emission carbon quantum dots with oligonucleotides complementary to RPs. Through base pairing, RPs and SPs were hybridized to generate aptamer sgc8-, AS1411-, and Apt-based imaging systems. They were used for individual/simultaneous imaging of cellular membrane protein PTK7, nucleolin, and adenosine triphosphate (ATP) molecules. Fluorescence imaging and intensity analysis showed that the living cell imaging system can not only specifically recognize and efficiently bind their respective targets but also provide a 5-10-fold signal amplification. Cell-cycle-dependent distribution of nucleolin and concentration-dependent fluorescence intensity of ATP demonstrated the utility of the system for tracking changes in cellular status. Overall, this system shows the potential to be a simple, low-cost, highly selective, and sensitive living cell imaging platform.

Asunto(s)

Adenosina Trifosfato , Aptámeros de Nucleótidos , Carbono , Nucleolina , Puntos Cuánticos , Puntos Cuánticos/química , Aptámeros de Nucleótidos/química , Humanos , Carbono/química , Adenosina Trifosfato/química , Adenosina Trifosfato/análisis , Colorantes Fluorescentes/química , Fosfoproteínas/química , Fosfoproteínas/metabolismo , ADN/química , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Células HeLa , Imagen Óptica , Proteínas Tirosina Quinasas/química , Proteínas Tirosina Quinasas/metabolismo , Antígenos de Neoplasias/metabolismo , Antígenos de Neoplasias/química , Moléculas de Adhesión Celular , Proteínas Tirosina Quinasas Receptoras

RESUMEN

Parasites have been associated with possible anticancer activity, including Trypanosoma cruzi, which has been linked to inhibiting the growth of solid tumors. To better understand this antitumor effect, we investigated the association of anti-T. cruzi antibodies with B cells of the acute lymphoblastic leukemia (ALL) SUPB15 cell line. The antibodies were generated in rabbits. IgGs were purified by affinity chromatography. Two procedures (flow cytometry (CF) and Western blot(WB)) were employed to recognize anti-T. cruzi antibodies on SUPB15 cells. We also used CF to determine whether the anti-T. cruzi antibodies could suppress SUPB15 cells. The anti-T. cruzi antibodies recognized 35.5% of the surface antigens of SUPB15. The complement-dependent cytotoxicity (CDC) results demonstrate the cross-suppression of anti-T. cruzi antibodies on up to 8.4% of SUPB15 cells. For the WB analysis, a band at 100 kDa with high intensity was sequenced using mass spectrometry, identifying the protein as nucleolin. This protein may play a role in the antitumor effect on T. cruzi. The anti-T. cruzi antibodies represent promising polyclonal antibodies that have the effect of tumor-suppressive cross-linking on cancer cells, which should be further investigated.

Asunto(s)

Anticuerpos Antiprotozoarios , Leucemia-Linfoma Linfoblástico de Células Precursoras , Trypanosoma cruzi , Trypanosoma cruzi/inmunología , Leucemia-Linfoma Linfoblástico de Células Precursoras/inmunología , Humanos , Línea Celular Tumoral , Animales , Conejos , Anticuerpos Antiprotozoarios/inmunología , Proteínas de Unión al ARN/inmunología , Proteínas de Unión al ARN/metabolismo , Nucleolina , Fosfoproteínas/inmunología , Fosfoproteínas/metabolismo

RESUMEN

Pyroptosis of programmed cell death has been recognized as a more effective way to inhibit the occurrence and development of tumors than the better-studied apoptosis. However, it is still challenging to quickly and effectively trigger pyroptosis of cancer cells for high-efficacy cancer treatment. Here, we report on the first use of mild constant-potential electrostimulation (cp-ES) to quickly trigger cancer cell pyroptosis with a probability up to ∼91.4% and significantly shortened time (within 1 h), ∼3-6 times faster than typical drug stimulation to induce pyroptosis. We find that the ES-induced cancer cell pyroptosis is through the activated caspase-3 (pathway) cleavage of gasdermin E (GSDME) to form an N-terminal fragment (GSDME-N) and observe nuclear shrinkage and reduction of the number of nucleoli as well as down-/up-regulated expression of two important nucleoproteins of nucleolin and nucleophosmin (NPM1). The study enriches the basic understanding of pyroptosis and provides a new avenue for potential effective treatment of cancer.

Asunto(s)

Caspasa 3 , Nucleofosmina , Piroptosis , Humanos , Caspasa 3/metabolismo , Nucleolina , Línea Celular Tumoral , Núcleo Celular/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Unión a Fosfato/metabolismo , Proteínas de Unión al ARN/metabolismo , Gasderminas

RESUMEN

A state-of-the-art, ultrasensitive, paper-based SERS sensor has been developed using silver nanostars (AgNSs) in combination with synthetic and natural antibodies. A key component of this innovative sensor is the plastic antibody, which was synthesized using molecularly imprinted polymer (MIP) technology. This ground-breaking combination of paper substrates/MIPs with AgNSs, which is similar to a sandwich immunoassay, is used for the first time with the aim of SERS detection and specifically targets nucleolin (NCL), a cancer biomarker. The sensor device was carefully fabricated by synthesizing a polyacrylamide-based MIP on cellulose paper (Whatman Grade 1 filter) by photopolymerization. The binding of NCL to the MIP was then confirmed by natural antibody binding using a sandwich assay for quantitative SERS analysis. To facilitate the detection of NCL, antibodies were pre-bound to AgNSs with a Raman tag so that the SERS signal could indicate the presence of NCL. The composition of the sensory layers/materials was meticulously optimized. The intensity of the Raman signal at ∼1078 cm-1 showed a linear trend that correlated with increasing concentrations of NCL, ranging from 0.1 to 1000 nmol L-1, with a limit of detection down to 0.068 nmol L-1 in human serum. The selectivity of the sensor was confirmed by testing its analytical response in the presence of cystatin C and lysozyme. The paper-based SERS detection system for NCL is characterized by its simplicity, sustainability, high sensitivity and stability and thus embodies essential properties for point-of-care applications. This approach is promising for expansion to other biomarkers in various fields, depending on the availability of synthetic and natural antibodies.

Asunto(s)

Anticuerpos , Nucleolina , Papel , Fosfoproteínas , Proteínas de Unión al ARN , Plata , Espectrometría Raman , Plata/química , Fosfoproteínas/inmunología , Humanos , Proteínas de Unión al ARN/inmunología , Anticuerpos/química , Anticuerpos/inmunología , Nanopartículas del Metal/química , Límite de Detección , Técnicas Biosensibles/métodos , Polímeros Impresos Molecularmente/química

RESUMEN

Japanese encephalitis virus (JEV) is an arthropod-borne, plus-strand flavivirus causing viral encephalitis in humans with a high case fatality rate. The JEV non-structural protein 5 (NS5) with the RNA-dependent RNA polymerase activity interacts with the viral and host proteins to constitute the replication complex. We have identified the multifunctional protein Nucleolin (NCL) as one of the several NS5-interacting host proteins. We demonstrate the interaction and colocalization of JEV NS5 with NCL in the virus-infected HeLa cells. The siRNA-mediated knockdown of NCL indicated that it was required for efficient viral replication. Importantly, JEV grew to higher titers in cells over-expressing exogenous NCL, demonstrating its pro-viral role. We demonstrated that NS5 interacted with the RRM and GAR domains of NCL. We show that the NCL-binding aptamer AS1411 containing the G-quadruplex (GQ) structure and the GQ ligand BRACO-19 caused significant inhibition of JEV replication. The antiviral effect of AS1411 and BRACO-19 could be overcome in HeLa cells by the overexpression of exogenous NCL. We demonstrated that the synthetic RNAs derived from the 3'-NCR of JEV genomic RNA containing the GQ sequence could bind NCL in vitro. The replication complex binding to the 3'-NCR is required for the viral RNA synthesis. It is likely that NCL present in the replication complex destabilizes the GQ structures in the genomic RNA, thus facilitating the movement of the replication complex resulting in efficient virus replication.IMPORTANCEJapanese encephalitis virus (JEV) is endemic in most parts of South-East Asia and the Western Pacific region, causing epidemics of encephalitis with a high case fatality rate. While a tissue culture-derived JEV vaccine is available, no antiviral therapy exists. The JEV NS5 protein has RNA-dependent RNA polymerase activity. Together with several host and viral proteins, it constitutes the replication complex necessary for virus replication. Understanding the interaction of NS5 with the host proteins could help design novel antivirals. We identified Nucleolin (NCL) as a crucial host protein interactor of JEV NS5 having a pro-viral role in virus replication. The NS5-interacting NCL binds to the G-quadruplex (GQ) structure sequence in the 3'-NCR of JEV RNA. This may smoothen the movement of the replication complex along the genomic RNA, thereby facilitating the virus replication. This study is the first report on how NCL, a host protein, helps in JEV replication through GQ-binding.

Asunto(s)

Virus de la Encefalitis Japonesa (Especie) , Nucleolina , Fosfoproteínas , Proteínas de Unión al ARN , Proteínas no Estructurales Virales , Replicación Viral , Humanos , Virus de la Encefalitis Japonesa (Especie)/fisiología , Virus de la Encefalitis Japonesa (Especie)/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Proteínas no Estructurales Virales/metabolismo , Proteínas no Estructurales Virales/genética , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Células HeLa , Unión Proteica , Encefalitis Japonesa/virología , Encefalitis Japonesa/metabolismo , Interacciones Huésped-Patógeno , G-Cuádruplex , Animales

RESUMEN

5-Fluorouracil (5-FU) is a conventional nucleotide analogue used for cancer treatment. However, its clinical application faces challenges such as low stability and non-specific toxicity. With the remarkable advancements in DNA nanotechnology, DNA-based self-assembled nanocarriers have emerged as powerful tools for delivering nucleotide drugs. In this study, we have designed a non-linear hybrid chain reaction involving a fuel strand with AS1411 aptamer sequence to construct a dendritic structure capable of carrying 5-FU. This structure specifically targets cancer cells with overexpressed nucleolin on their surface, allowing the 5-FU to exert its anticancer effects and achieve therapeutic outcomes. Furthermore, we have also investigated the mechanistic action of this drug delivery system, aiming to establish a novel therapeutic platform for 5-FU treatment.

Asunto(s)

Aptámeros de Nucleótidos , Fluorouracilo , Fluorouracilo/administración & dosificación , Fluorouracilo/química , Aptámeros de Nucleótidos/administración & dosificación , Aptámeros de Nucleótidos/química , Humanos , Antimetabolitos Antineoplásicos/administración & dosificación , Antimetabolitos Antineoplásicos/química , Animales , Sistemas de Liberación de Medicamentos/métodos , Oligodesoxirribonucleótidos/administración & dosificación , Oligodesoxirribonucleótidos/química , Proteínas de Unión al ARN/metabolismo , Fosfoproteínas/metabolismo , Línea Celular Tumoral , Nucleolina , Neoplasias/tratamiento farmacológico , Portadores de Fármacos/química , Ratones Desnudos , Ratones Endogámicos BALB C

RESUMEN

A cancer-targeted glutathione (GSH)-gated theranostic probe (CGT probe) for intracellular miRNA imaging and combined treatment of self-sufficient starvation therapy (ST) and chemodynamic therapy (CDT) was developed. The CGT probe is constructed using MnO2 nanosheet (MS) as carrier material to adsorb the elaborately designed functional DNAs. It can be internalized by cancer cells via specific recognition between the AS1411 aptamer and nucleolin. After CGT probe entering the cancer cells, the overexpressed GSH, as gate-control, can degrade MS to Mn2+ which can be used for CDT by Fenton-like reaction. Simultaneously, Mn2+-mediated CDT can further cascade with the enzyme-like activities (catalase-like activity and glucose oxidase-like activity) of CGT probe, achieving self-sufficient ST/CDT synergistic therapy. Meanwhile, the anchored DNAs are released, achieving in situ signal amplification via disubstituted-catalytic hairpin assembly (DCHA) and FRET (fluorescence resonance energy transfer) imaging of miR-21. The in vitro and in vivo experiments demonstrated that accurate and sensitive miRNA detection can be achieved using the CGT probe. Overall, the ingenious CGT probe opens a new avenue for the development of early clinical diagnosis and cancer therapy.

Asunto(s)

Transferencia Resonante de Energía de Fluorescencia , Glutatión , Compuestos de Manganeso , MicroARNs , Óxidos , Humanos , Glutatión/química , Glutatión/metabolismo , Animales , Compuestos de Manganeso/química , Óxidos/química , Aptámeros de Nucleótidos/química , Ratones , Ratones Desnudos , Nanomedicina Teranóstica/métodos , Nucleolina , Neoplasias/diagnóstico por imagen , Nanoestructuras/química , Oligodesoxirribonucleótidos/química , Ratones Endogámicos BALB C , Colorantes Fluorescentes/química

RESUMEN

BACKGROUND: Triple-negative breast cancer (TNBC) is a recurrent, heterogeneous, and invasive form of breast cancer. The treatment of TNBC patients with paclitaxel and fluorouracil in a sequential manner has shown promising outcomes. However, it is challenging to deliver these chemotherapeutic agents sequentially to TNBC tumors. We aim to explore a precision therapy strategy for TNBC through the sequential delivery of paclitaxel and fluorouracil. METHODS: We developed a dual chemo-loaded aptamer with redox-sensitive caged paclitaxel for rapid release and non-cleavable caged fluorouracil for slow release. The binding affinity to the target protein was validated using Enzyme-linked oligonucleotide assays and Surface plasmon resonance assays. The targeting and internalization abilities into tumors were confirmed using Flow cytometry assays and Confocal microscopy assays. The inhibitory effects on TNBC progression were evaluated by pharmacological studies in vitro and in vivo. RESULTS: Various redox-responsive aptamer-paclitaxel conjugates were synthesized. Among them, AS1411-paclitaxel conjugate with a thioether linker (ASP) exhibited high anti-proliferation ability against TNBC cells, and its targeting ability was further improved through fluorouracil modification. The fluorouracil modified AS1411-paclitaxel conjugate with a thioether linker (FASP) exhibited effective targeting of TNBC cells and significantly improved the inhibitory effects on TNBC progression in vitro and in vivo. CONCLUSIONS: This study successfully developed fluorouracil-modified AS1411-paclitaxel conjugates with a thioether linker for targeted combination chemotherapy in TNBC. These conjugates demonstrated efficient recognition of TNBC cells, enabling targeted delivery and controlled release of paclitaxel and fluorouracil. This approach resulted in synergistic antitumor effects and reduced toxicity in vivo. However, challenges related to stability, immunogenicity, and scalability need to be further investigated for future translational applications.

Asunto(s)

Aptámeros de Nucleótidos , Preparaciones de Acción Retardada , Liberación de Fármacos , Fluorouracilo , Nucleolina , Paclitaxel , Fosfoproteínas , Proteínas de Unión al ARN , Neoplasias de la Mama Triple Negativas , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/patología , Aptámeros de Nucleótidos/farmacología , Aptámeros de Nucleótidos/química , Humanos , Paclitaxel/uso terapéutico , Paclitaxel/farmacología , Línea Celular Tumoral , Animales , Femenino , Fluorouracilo/farmacología , Fluorouracilo/uso terapéutico , Proteínas de Unión al ARN/metabolismo , Fosfoproteínas/metabolismo , Oligodesoxirribonucleótidos/farmacología , Oligodesoxirribonucleótidos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Ratones Desnudos , Ensayos Antitumor por Modelo de Xenoinjerto , Proliferación Celular/efectos de los fármacos , Oxidación-Reducción/efectos de los fármacos , Ratones Endogámicos BALB C

RESUMEN

Rationale: Myocardial infarction (MI) is a severe global clinical condition with widespread prevalence. The adult mammalian heart's limited capacity to generate new cardiomyocytes (CMs) in response to injury remains a primary obstacle in developing effective therapies. Current approaches focus on inducing the proliferation of existing CMs through cell-cycle reentry. However, this method primarily elevates cyclin dependent kinase 6 (CDK6) and DNA content, lacking proper cytokinesis and resulting in the formation of dysfunctional binucleated CMs. Cytokinesis is dependent on ribosome biogenesis (Ribo-bio), a crucial process modulated by nucleolin (Ncl). Our objective was to identify a novel approach that promotes both DNA synthesis and cytokinesis. Methods: Various techniques, including RNA/protein-sequencing analysis, Ribo-Halo, Ribo-disome, flow cytometry, and cardiac-specific tumor-suppressor retinoblastoma-1 (Rb1) knockout mice, were employed to assess the series signaling of proliferation/cell-cycle reentry and Ribo-bio/cytokinesis. Echocardiography, confocal imaging, and histology were utilized to evaluate cardiac function. Results: Analysis revealed significantly elevated levels of Rb1, bur decreased levels of circASXL1 in the hearts of MI mice compared to control mice. Deletion of Rb1 induces solely cell-cycle reentry, while augmenting the Ribo-bio modulator Ncl leads to cytokinesis. Mechanically, bioinformatics and the loss/gain studies uncovered that circASXL1/CDK6/Rb1 regulates cell-cycle reentry. Moreover, Ribo-Halo, Ribo-disome and circRNA pull-down assays demonstrated that circASXL1 promotes cytokinesis through Ncl/Ribo-bio. Importantly, exosomes derived from umbilical cord mesenchymal stem cells (UMSC-Exo) had the ability to enhance cardiac function by facilitating the coordinated signaling of cell-cycle reentry and Ribo-bio/cytokinesis. These effects were attenuated by silencing circASXL1 in UMSC-Exo. Conclusion: The series signaling of circASXL1/CDK6/Rb1/cell-cycle reentry and circASXL1/Ncl/Ribo-bio/cytokinesis plays a crucial role in cardiac repair. UMSC-Exo effectively repairs infarcted myocardium by stimulating CM cell-cycle reentry and cytokinesis in a circASXL1-dependent manner. This study provides innovative therapeutic strategies targeting the circASXL1 signaling network for MI and offering potential avenues for enhanced cardiac repair.

Asunto(s)

Ciclo Celular , Citocinesis , Ratones Noqueados , Infarto del Miocardio , Miocitos Cardíacos , Ribosomas , Animales , Ratones , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Miocitos Cardíacos/metabolismo , Ribosomas/metabolismo , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Nucleolina , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Proteína de Retinoblastoma/metabolismo , Proteína de Retinoblastoma/genética , Proliferación Celular , Masculino , Humanos

RESUMEN

Despite the demonstrated importance of DNA G-quadruplexes (G4s) in health and disease, technologies to readily manipulate specific G4 folding for functional analysis and therapeutic purposes are lacking. Here we employ G4-stabilizing protein/ligand in conjunction with CRISPR to selectively facilitate single or multiple targeted G4 folding within specific genomic loci. We demonstrate that fusion of nucleolin with a catalytically inactive Cas9 can specifically stabilize G4s in the promoter of oncogene MYC and muscle-associated gene Itga7 as well as telomere G4s, leading to cell proliferation arrest, inhibition of myoblast differentiation and cell senescence, respectively. Furthermore, CRISPR can confer intra-G4 selectivity to G4-binding compounds pyridodicarboxamide and pyridostatin. Compared with traditional G4 ligands, CRISPR-guided biotin-conjugated pyridodicarboxamide enables a more precise investigation into the biological functionality of de novo G4s. Our study provides insights that will enhance understanding of G4 functions and therapeutic interventions.

Asunto(s)

Sistemas CRISPR-Cas , G-Cuádruplex , Nucleolina , Proteínas de Unión al ARN , Humanos , Ligandos , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Ácidos Picolínicos/farmacología , Ácidos Picolínicos/química , Proliferación Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Animales , Senescencia Celular/efectos de los fármacos , Senescencia Celular/genética , Proteína 9 Asociada a CRISPR/metabolismo , Proteína 9 Asociada a CRISPR/genética , Regiones Promotoras Genéticas , Telómero/metabolismo , Telómero/genética , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Piridinas/farmacología , Piridinas/química , ADN/metabolismo , ADN/genética , Ratones , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Células HEK293 , Mioblastos/metabolismo , Mioblastos/citología , Aminoquinolinas

RESUMEN

O6-methylguanine DNA methyltransferase (MGMT) is a crucial determinant of temozolomide (TMZ) sensitivity in patients with glioblastoma (GBM). The therapeutic potential of small interfering RNA (siRNA) targeting MGMT to enhance TMZ sensitivity has been hampered by serum nuclease degradation, off-target effects, poor accumulation at tumor sites, and low circulation in blood stream. In this study, we developed a framework nucleic acid-based nanoparticles (FNN), which is constructed from a six-helix DNA bundle, to encapsulate and protect siMGMT for improving TMZ sensitivity in GBM treatment. For better blood-brain barrier (BBB) penetration and GBM targeting, we conjugated Angiopep-2 (ANG) targeting modules to each end of the FNN. Nucleolin (NCL)-responsive locks were engineered along the sides of the six-helix DNA bundle, which safeguard siMGMT before tumor entry. Upon interaction with tumor-overexpressed NCL, these locks unlock, exposing siMGMT, this allows for effective suppression of MGMT, resulting in a significant improvement of TMZ therapeutic efficacy in GBM. This innovative strategy has the potential to transform the current treatment landscape for GBM.

Asunto(s)

Antineoplásicos Alquilantes , Barrera Hematoencefálica , Neoplasias Encefálicas , Glioblastoma , Nanopartículas , Temozolomida , Glioblastoma/tratamiento farmacológico , Glioblastoma/patología , Temozolomida/farmacología , Temozolomida/administración & dosificación , Temozolomida/uso terapéutico , Humanos , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/genética , Antineoplásicos Alquilantes/farmacología , Antineoplásicos Alquilantes/uso terapéutico , Nanopartículas/química , Animales , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Línea Celular Tumoral , Proteínas de Unión al ARN/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Enzimas Reparadoras del ADN/genética , Metilasas de Modificación del ADN/metabolismo , Nucleolina , Fosfoproteínas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas Supresoras de Tumor/genética , ARN Interferente Pequeño/administración & dosificación , Ácidos Nucleicos , Péptidos

RESUMEN

Hepatocyte growth factor receptor (c-Met) is a suitable molecular target for the targeted therapy of cancer. Novel c-Met-targeting drugs need to be developed because conventional small-molecule inhibitors and antibodies of c-Met have some limitations. To synthesize such drugs, we developed a bispecific DNA nanoconnector (STPA) to inhibit c-Met function. STPA was constructed by using DNA triangular prism as a scaffold and aptamers as binding molecules. After c-Met-specific SL1 and nucleolin-specific AS1411 aptamers were integrated with STPA, STPA could bind to c-Met and nucleolin on the cell membrane. This led to the formation of the c-Met/STPA/nucleolin complex, which in turn blocked c-Met activation. In vitro experiments showed that STPA could not only inhibit the c-Met signaling pathways but also facilitate c-Met degradation through lysosomes. STPA also inhibited c-Met-promoted cell migration, invasion, and proliferation. The results of in vivo experiments showed that STPA could specifically target to tumor site in xenograft mouse model, and inhibit tumor growth with low toxicity by downregulating c-Met pathways. This study provided a novel and simple strategy to develop c-Met-targeting drugs for the targeted therapy of cancer.

Asunto(s)

Aptámeros de Nucleótidos , Proliferación Celular , Neoplasias , Proteínas Proto-Oncogénicas c-met , Transducción de Señal , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-met/metabolismo , Humanos , Animales , Transducción de Señal/efectos de los fármacos , Ratones , Proliferación Celular/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Neoplasias/patología , Línea Celular Tumoral , Aptámeros de Nucleótidos/farmacología , Aptámeros de Nucleótidos/química , Nucleolina , Movimiento Celular/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas de Unión al ARN/metabolismo , Fosfoproteínas/metabolismo , Terapia Molecular Dirigida , ADN/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/química , Oligodesoxirribonucleótidos

RESUMEN

De-differentiation and subsequent increased proliferation and inflammation of vascular smooth muscle cells (VSMCs) is one of the mechanisms of atherogenesis. Maintaining VSMCs in a contractile differentiated state is therefore a promising therapeutic strategy for atherosclerosis. We have reported the 18-base myogenetic oligodeoxynucleotide, iSN04, which serves as an anti-nucleolin aptamer and promotes skeletal and myocardial differentiation. The present study investigated the effect of iSN04 on VSMCs because nucleolin has been reported to contribute to VSMC de-differentiation under pathophysiological conditions. Nucleolin is localized in the nucleoplasm and nucleoli of both rat and human VSMCs. iSN04 without a carrier was spontaneously incorporated into VSMCs, indicating that iSN04 would serve as an anti-nucleolin aptamer. iSN04 treatment decreased the ratio of 5-ethynyl-2'-deoxyuridine (EdU)-positive proliferating VSMCs and increased the expression of α-smooth muscle actin, a contractile marker of VSMCs. iSN04 also suppressed angiogenesis of mouse aortic rings ex vivo, which is a model of pathological angiogenesis involved in plaque formation, growth, and rupture. These results demonstrate that antagonizing nucleolin with iSN04 preserves VSMC differentiation, providing a nucleic acid drug candidate for the treatment of vascular disease.

Asunto(s)

Aptámeros de Nucleótidos , Diferenciación Celular , Proliferación Celular , Músculo Liso Vascular , Miocitos del Músculo Liso , Nucleolina , Fosfoproteínas , Proteínas de Unión al ARN , Animales , Proteínas de Unión al ARN/metabolismo , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Aptámeros de Nucleótidos/farmacología , Proliferación Celular/efectos de los fármacos , Fosfoproteínas/metabolismo , Diferenciación Celular/efectos de los fármacos , Humanos , Ratas , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/citología , Ratones , Células Cultivadas , Oligodesoxirribonucleótidos/farmacología , Masculino , Ratas Sprague-Dawley , Ratones Endogámicos C57BL

RESUMEN

The interaction between glioma cells and astrocytes promotes the proliferation of gliomas. Micro-RNAs (miRNAs) carried by astrocyte exosomes (exos) may be involved in this process, but the mechanism remains unclear. The oligonucleotide AS1411, which consists of 26 bases and has a G-quadruplex structure, is an aptamer that targets nucleolin. In this study, we demonstrate exosome-miRNA-27a-mediated cross-activation between astrocytes and glioblastoma and show that AS1411 reduces astrocytes' pro-glioma activity. The enhanced affinity of AS1411 toward nucleolin is attributed to its G-quadruplex structure. After binding to nucleolin, AS1411 inhibits the entry of the NF-κB pathway transcription factor P65 into the nucleus, then downregulates the expression of miRNA-27a in astrocytes surrounding gliomas. Then, AS1411 downregulates astrocyte exosome-miRNA-27a and upregulates the expression of INPP4B, the target gene of miRNA-27a in gliomas, thereby inhibiting the PI3K/AKT pathway and inhibiting glioma proliferation. These results were verified in mouse orthotopic glioma xenografts and human glioma samples. In conclusion, the parallel structure of AS1411 allows it to bind to nucleolin and disrupt the exosome-miRNA-27a-mediated reciprocal activation loop between glioma cells and astrocytes. Our results may help in the development of a novel approach to therapeutic modulation of the glioma microenvironment.

Asunto(s)

Aptámeros de Nucleótidos , Astrocitos , Exosomas , Glioma , MicroARNs , Nucleolina , Oligodesoxirribonucleótidos , Fosfoproteínas , Proteínas de Unión al ARN , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Humanos , Astrocitos/metabolismo , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , MicroARNs/genética , MicroARNs/metabolismo , Animales , Glioma/metabolismo , Glioma/patología , Glioma/genética , Ratones , Oligodesoxirribonucleótidos/genética , Oligodesoxirribonucleótidos/metabolismo , Oligodesoxirribonucleótidos/farmacología , Aptámeros de Nucleótidos/metabolismo , Aptámeros de Nucleótidos/genética , Exosomas/metabolismo , Exosomas/genética , Línea Celular Tumoral , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Ratones Desnudos , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/genética , Transducción de Señal

RESUMEN

Post-translational modifications of proteins (PTMs) introduce an extra layer of complexity to cellular regulation. Although phosphorylation of serine, threonine, and tyrosine residues is well-known as PTMs, lysine is, in fact, the most heavily modified amino acid, with over 30 types of PTMs on lysine having been characterized. One of the most recently discovered PTMs on lysine residues is polyphosphorylation, which sees linear chains of inorganic polyphosphates (polyP) attached to lysine residues. The labile nature of phosphoramidate bonds raises the question of whether this modification is covalent in nature. Here, we used buffers with very high ionic strength, which would disrupt any non-covalent interactions, and confirmed that lysine polyphosphorylation occurs covalently on proteins containing PASK domains (polyacidic, serine-, and lysine-rich), such as the budding yeast protein nuclear signal recognition 1 (Nsr1) and the mammalian protein nucleolin. This Matters Arising Response paper addresses the Neville et al. (2024) Matters Arising paper, published concurrently in Molecular Cell.

Asunto(s)

Lisina , Fosfoproteínas , Procesamiento Proteico-Postraduccional , Proteínas de Unión al ARN , Fosforilación , Lisina/metabolismo , Fosfoproteínas/metabolismo , Fosfoproteínas/química , Fosfoproteínas/genética , Humanos , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/química , Nucleolina , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Animales , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Polifosfatos/metabolismo , Polifosfatos/química , Concentración Osmolar

RESUMEN

Nucleolin is overexpressed on the surface of pancreatic cancer cells and are regarded as the remarkable therapeutic target. Aptamers are capable of binding the external domain of nucleolin on the cell surface with high affinity and specificity. But nucleolin has not been localized on pancreatic cancer cells at very high spatial resolution, and the interactions between nucleolin and aptamers have not been investigated at very high force resolution level. In this work, nucleolin was localized on pancreatic cancer and normal cells by aptamers (9FU-AS1411-NH2, AS1411-NH2 and CRONH2) in Single Molecule Recognition Imaging mode of Atomic Force Microscopy. There are plenty of nucleolin on the surfaces of pancreatic cancer cells (area percentage about 5 %), while there are little nucleolin on the surfaces of normal cells. The interactions between three types of aptamers and nucleolins on the surfaces of pancreatic cancer cells were investigated by Single Molecule Force Spectroscopy. The unbinding forces of nucleolins-(9FU-AS1411-NH2) are larger than nucleolins-(AS1411-NH2). The dissociation activation energy on nucleolin-(9FU-AS1411-NH2) is higher than nucleolin-(AS1411-NH2), which indicates that the former complex is more stable and harder to dissociate than the later complex. There are no unbinding forces between nucleolin and CRONH2. All these demonstrate that nucleolin was localized on pancreatic cancer and normal cells at single molecule level quantitatively, and the interactions (unbinding forces and kinetics) between nucleolin and aptamers were studied at picoNewton level. The approaches and results of this work will pave new ways in the investigations of nucleolin and aptamers, and will also be useful in the studies on other proteins and their corresponding aptamers.

Asunto(s)

Aptámeros de Nucleótidos , Microscopía de Fuerza Atómica , Nucleolina , Neoplasias Pancreáticas , Fosfoproteínas , Proteínas de Unión al ARN , Proteínas de Unión al ARN/metabolismo , Fosfoproteínas/metabolismo , Humanos , Neoplasias Pancreáticas/metabolismo , Microscopía de Fuerza Atómica/métodos , Línea Celular Tumoral , Unión Proteica , Imagen Individual de Molécula/métodos

RESUMEN

Despite many advances in the use of DNA nanodevices as assembly or disassembly modules to build various complex structures, the simultaneous assembly and disassembly of DNA structures in living cells remains a challenge. In this study, we present a modular engineering approach for assembling and disassembling DNA nanodevices in response to endogenous biomarkers. As a result of pairwise prehybridization of original DNA strands, the DNA nanodevice is initially inert. In an effort to bind one of the paired strands and release its complement, nucleolin competes. Assembly of the DNA nanodevice is initiated when the released complement binds to it, and disassembly is initiated when APE1 shears the assembled binding site of the DNA nanodevice. Spatial-temporal logic control is achieved through our approach during the assembly and disassembly of DNA nanodevices. Furthermore, by means of this assembly and disassembly procedure, the sequential detection and imaging of two tumor markers can be achieved, thereby effectively reducing false-positive signal results and accelerating the detection time. This study emphasizes the simultaneous assembly and disassembly of DNA nanodevices controlled by biomarkers in a simple and versatile manner; it has the potential to expand the application scope of DNA nanotechnology and offers an idea for the implementation of precision medicine testing.

Asunto(s)

ADN , Nanoestructuras , Fosfoproteínas , Humanos , ADN/química , ADN/metabolismo , Fosfoproteínas/metabolismo , Fosfoproteínas/química , Nanoestructuras/química , Nucleolina , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/química , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Biomarcadores de Tumor/metabolismo , Nanotecnología

RESUMEN

Background: Sepsis-induced myocardial injury, as one of the important complications of sepsis, can significantly increase the mortality of septic patients. Our previous study found that nucleolin affected mitochondrial function in energy synthesis and had a protective effect on septic cardiomyopathy in mice. During sepsis, glucose metabolism disorders aggravated myocardial injury and had a negative effect on septic patients. Objectives: We investigated whether nucleolin could regulate glucose metabolism during endotoxemia-induced myocardial injury. Methods: The study tested whether the nucleolin cardiac-specific knockout in the mice could affect glucose metabolism through untargeted metabolomics, and the results of metabolomics were verified experimentally in H9C2 cells. The ATP content, lactate production, and oxygen consumption rate (OCR) were evaluated. Results: The metabolomics results suggested that glycolytic products were increased in endotoxemia-induced myocardial injury, and that nucleolin myocardial-specific knockout altered oxidative phosphorylation-related pathways. The experiment data showed that TNF-α combined with LPS stimulation could increase the lactate content and the OCR values by about 25%, and decrease the ATP content by about 25%. However, interference with nucleolin expression could further decrease ATP content and OCR values by about 10-20% and partially increase the lactate level in the presence of TNF-α and LPS. However, nucleolin overexpression had the opposite protective effect, which partially reversed the decrease in ATP content and the increase in lactate level. Conclusion: Down-regulation of nucleolin can exacerbate glucose metabolism disorders in endotoxemia-induced myocardial injury. Improving glucose metabolism by regulating nucleolin was expected to provide new therapeutic ideas for patients with septic cardiomyopathy.

Asunto(s)

Endotoxemia , Glucosa , Nucleolina , Fosfoproteínas , Proteínas de Unión al ARN , Animales , Ratones , Adenosina Trifosfato/metabolismo , Cardiomiopatías/metabolismo , Cardiomiopatías/genética , Cardiomiopatías/etiología , Línea Celular , Endotoxemia/metabolismo , Glucosa/metabolismo , Lipopolisacáridos , Metabolómica , Ratones Noqueados , Miocardio/metabolismo , Miocardio/patología , Fosforilación Oxidativa , Consumo de Oxígeno , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/deficiencia , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/genética

RESUMEN

We fabricated hybrid nanoparticles consisting of organic semiconducting material with peptide sequence to reflect the target protein interaction. A phosphorescent OLED material, platinum octaethylporphyrin (PtOEP) was self-assembled by reprecipitation with the A17 peptide (YCAYYSPRHKTTF) selected as a probe ligand in order to recognize heat shock protein 70 (HSP70). The phosphorescence intensity of the PtOEP-A17 assembly was enhanced by 125 % after treatment with HSP70. The specificity of the protein interaction was confirmed in both solution and solid states of the PtOEP-A17 assembly against to BSA and nucleolin. We figured out that the phosphorescence lifetime of PtOEP-A17 assembly after exposed to HSP70 increased significantly to 153 ns from initial 115 ns. These simultaneous enhancements in phosphorescence and lifetime triggered by the specific protein interaction would open new applications of PtOEP, a representative material of light-emitting device fields.

Asunto(s)

Péptidos , Péptidos/química , Unión Proteica , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP70 de Choque Térmico/química , Mediciones Luminiscentes , Porfirinas/química , Platino (Metal)/química , Albúmina Sérica Bovina/química , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/química , Nucleolina , Animales