RESUMEN
BACKGROUND: TBK1 positively regulates the growth factor-mediated mTOR signaling pathway by phosphorylating mTOR. However, it remains unclear how the TBK1-mTOR signaling pathway is regulated. Considering that STING not only interacts with TBK1 but also with MARCH1, we speculated that MARCH1 might regulate the mTOR signaling pathway by targeting TBK1. The aim of this study was to determine whether MARCH1 regulates the mTOR signaling pathway by targeting TBK1. METHODS: The co-immunoprecipitation (Co-IP) assay was used to verify the interaction between MARCH1 with STING or TBK1. The ubiquitination of STING or TBK1 was analyzed using denatured co-immunoprecipitation. The level of proteins detected in the co-immunoprecipitation or denatured co-immunoprecipitation samples were determined by Western blotting. Stable knocked-down cells were constructed by infecting lentivirus bearing the related shRNA sequences. Scratch wound healing and clonogenic cell survival assays were used to detect the migration and proliferation of breast cancer cells. RESULTS: We showed that MARCH1 played an important role in growth factor-induced the TBK1- mTOR signaling pathway. MARCH1 overexpression attenuated the growth factor-induced activation of mTOR signaling pathway, whereas its deficiency resulted in the opposite effect. Mechanistically, MARCH1 interacted with and promoted the K63-linked ubiquitination of TBK1. This ubiquitination of TBK1 then attenuated its interaction with mTOR, thereby inhibiting the growth factor-induced mTOR signaling pathway. Importantly, faster proliferation induced by MARCH1 deficiency was weakened by mTOR, STING, or TBK1 inhibition. CONCLUSION: MARCH1 suppressed growth factors mediated the mTOR signaling pathway by targeting the STING-TBK1-mTOR axis.
Asunto(s)
Proliferación Celular , Proteínas Serina-Treonina Quinasas , Transducción de Señal , Serina-Treonina Quinasas TOR , Ubiquitina-Proteína Ligasas , Ubiquitinación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Humanos , Serina-Treonina Quinasas TOR/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Línea Celular Tumoral , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Femenino , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Neoplasias de la Mama/genética , Movimiento CelularRESUMEN
Metal-organic frameworks (MOFs), a distinctive funtionalmaterials which is constructed by various metal ions and organic molecules, have gradually attracted researchers' attention from they were founded. In the last decade, MOFs emerge as a biomedical material with potential applications due to their unique properties. However, the MOFs performed as nanocarriers for functional nucleic acid delivery in biomedical applications rarely summarized. In this review, we introduce recent developments of MOFs for nucleic acid delivery in various biologically relevant applications, with special emphasis on cancer therapy (including siRNA, ASO, DNAzyme, miRNA and CpG oligodeoxynucleotides), bioimaging, biosensors and separation of biomolecules. We expect the accomplishment of this review could benefit certain researchers in biomedical field to develop novel sophisticated nanocarriers for functional nucleic acid delivery based on the promising material of MOFs.
Asunto(s)
Estructuras Metalorgánicas , Ácidos Nucleicos , Sistemas de Liberación de Medicamentos , Portadores de Fármacos , MetalesRESUMEN
DNA damage and apoptosis lead to the release of free nucleosomes-the basic structural repeating units of chromatin-into the blood circulation system. We recently reported that free nucleosomes that enter the cytoplasm of mammalian cells trigger immune responses by activating cGMP-AMP synthase (cGAS). In the present study, we designed experiments to reveal the mechanism of nucleosome uptake by human cells. We showed that nucleosomes are first absorbed on the cell membrane through nonspecific electrostatic interactions between positively charged histone N-terminal tails and ligands on the cell surface, followed by internalization via clathrin- or caveolae-dependent endocytosis. After cellular internalization, endosomal escape occurs rapidly, and nucleosomes are released into the cytosol, maintaining structural integrity for an extended period. The efficient endocytosis of extracellular nucleosomes suggests that circulating nucleosomes may lead to cellular disorders as well as immunostimulation, and thus, the biological effects exerted by endocytic nucleosomes should be addressed in the future.
Asunto(s)
Caveolinas/metabolismo , Membrana Celular/metabolismo , Clatrina/metabolismo , Endocitosis , Nucleosomas/metabolismo , Animales , Línea Celular , Toxina del Cólera/metabolismo , Retículo Endoplásmico/metabolismo , Endosomas/metabolismo , Aparato de Golgi/metabolismo , Células HeLa , Células Hep G2 , Humanos , Lisosomas/metabolismo , Ratones , Microscopía Confocal , Nucleosomas/genética , Células THP-1 , Transferrina/metabolismoRESUMEN
8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) is a biomarker of oxidative DNA damage and can be repaired by hOGG1 and APE1 via the base excision repair (BER) pathway. In this work, we studied coordinated BER of 8-oxodGuo by hOGG1 and APE1 in nucleosome core particles and found that histones transiently formed DNA-protein cross-links (DPCs) with active repair intermediates such as 3'-phospho-α,ß-unsaturated aldehyde (PUA) and 5'-deoxyribosephosphate (dRP). The effects of histone participation could be beneficial or deleterious to the BER process, depending on the circumstances. In the absence of APE1, histones enhanced the AP lyase activity of hOGG1 by cross-linking with 3'-PUA. However, the formed histone-PUA DPCs hampered the subsequent repair process. In the presence of APE1, both the AP lyase activity of hOGG1 and the formation of histone-PUA DPCs were suppressed. In this case, histones could catalyse removal of the 5'-dRP by transiently cross-linking with the active intermediate. That is, histones promoted the repair by acting as 5'-dRP lyases. Our findings demonstrate that histones participate in multiple steps of 8-oxodGuo repair in nucleosome core particles, highlighting the diverse roles that histones may play during DNA repair in eukaryotic cells.
Asunto(s)
8-Hidroxi-2'-Desoxicoguanosina/metabolismo , Reparación del ADN/fisiología , Histonas/fisiología , Nucleosomas/metabolismo , Liasas de Fósforo-Oxígeno/metabolismo , ADN Glicosilasas/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Humanos , Modelos Moleculares , Conformación de Ácido Nucleico , Nucleosomas/ultraestructura , Conformación Proteica , Ribosamonofosfatos/metabolismoRESUMEN
Nitrogen mustards have long been used in cancer chemotherapy, and their cytotoxicity has traditionally been attributed to the formation of DNA interstrand cross-links and DNA monoalkylation. Recent studies have shown that exposure to nitrogen mustards also induces the formation of DNA-protein cross-links (DPCs) via bridging between N7 of a deoxyguanosine residue in the DNA and the side chain of a Cys residue in the protein. However, the formation of nitrogen mustard-induced DNA-histone cross-links has never been observed. Herein, we demonstrate that treating reconstituted nucleosome core particles (NCPs) with the nitrogen mustard mechlorethamine results in the formation of DNA-histone cross-links in addition to DNA monoalkylation and interstrand cross-link formation. The yields of these three types of DNA lesions in the NCPs decreased in the following order: DNA monoalkylation â« DNA interstrand cross-links > DNA-histone cross-links. Mechanistic studies involving tailless histones and competitive inhibition by a polyamine demonstrated that Lys residues in the N- and C-terminal tails of the histones were the predominant sites involved in DNA-histone cross-link formation. Given that NCPs are the fundamental repeating units of chromatin in eukaryotes, our findings suggest that nitrogen mustard-induced formation of DNA-histone cross-links may occur in living cells and that DPC formation may contribute to the cytotoxicity of nitrogen mustards.
Asunto(s)
Alquilantes/química , Reactivos de Enlaces Cruzados/química , ADN/efectos de los fármacos , Histonas/efectos de los fármacos , Mecloretamina/química , Nucleosomas/efectos de los fármacos , Secuencia de Aminoácidos , Animales , ADN/química , Histonas/química , Masculino , Modelos Químicos , Nucleosomas/química , Salmón , Espermatozoides/químicaRESUMEN
The mammalian target of rapamycin complex1 (mTORC1) can response to amino acid to regulate metabolism and cell growth. GATOR2 act as important role in amino acid mediated mTORC1 signaling pathway by repressing GTPase activity (GAP) of GATOR1. However, it is still unclear how GATOR2 regulates mTORC1 signaling pathway. Here, we found that K63-ubiquitination of Sce13, one component of GATOR2, suppresses the mTORC1 activity by lessening the inter-interaction of GATOR2. Mechanistically, the ubiquitination of Sec13 was mediated by SPOP. Subsequently, the ubiquitination of Sec13 attenuated its interaction with the other component of GATOR2, thus suppressing the activity of mTORC1. Importantly, the deficiency of SPOP promoted the faster proliferation and migration of breast cancer cells, which was attenuated by knocking down of Sec13. Therefore, SPOP can act as a tumor suppressor gene by negatively regulating mTORC1 signaling pathway.
Asunto(s)
Aminoácidos , Serina-Treonina Quinasas TOR , Ciclo Celular , Proliferación Celular , Diana Mecanicista del Complejo 1 de la RapamicinaRESUMEN
Triple negative breast cancer (TNBC) is a greatly aggressive subtype of breast cancer with high recurrence and mortality rates. Chemotherapy as a primary treatment for cancer is limited due to toxic side effects and drug resistance. Therefore, low toxicity and more effective breast cancer therapeutic approaches are greatly desired. In this study, a strategy which using ZIF-90 nanoparticles co-deliver Ce6-anti-miR-21 and Ce6-anti-miR-155 into the tumor cells was developed. Due to the pH responsive drug release of ZIF-90, antisense oligonucleotides (anti-miRNAs) and photosensitizers are able to be efficiently released inside tumor microenvironment. The nano delivery system captures overexpressed oncogenic miRNAs while the photosensitizer Ce6 generates ROS under light irradiation to effectively induce the apoptosis of tumor cell. This combinatorial effect was verified by results showing that the purposed therapic method could effectively inhibit tumor cell proliferation and metastasis. The concept of antisense oligonucleotide combined with photodynamic therapy has great potential in cancer treatment or adjuvant therapy.
Asunto(s)
MicroARNs , Oligonucleótidos Antisentido , Fotoquimioterapia , Fármacos Fotosensibilizantes , Neoplasias de la Mama Triple Negativas , Humanos , Línea Celular , Proliferación Celular/efectos de los fármacos , Liberación de Fármacos , Concentración de Iones de Hidrógeno , MicroARNs/efectos de los fármacos , Sistema de Administración de Fármacos con Nanopartículas , Metástasis de la Neoplasia , Oligonucleótidos Antisentido/farmacología , Tamaño de la Partícula , Fotoquimioterapia/métodos , Fármacos Fotosensibilizantes/farmacología , Propiedades de Superficie , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
The cGAS-STING pathway discovered ten years ago is an important component of the innate immune system. Activation of cGAS-STING triggers downstream signalling, such as TBK1-IRF3, NF-κB and autophagy, which in turn leads to antipathogen responses, durable antitumour immunity or autoimmune diseases. 2',3'-Cyclic GMP-AMP dinucleotides (2',3'-cGAMP), the key second messengers produced by cGAS, play a pivotal role in cGAS-STING signalling by binding and activating STING. Thus, 2',3'-cGAMP has immunotherapeutic potential, which in turn has stimulated research on the design and synthesis of 2',3'-cGAMP analogues for clinical applications over the past ten years. This review presents the discovery, metabolism, and function of 2',3'-cGAMP in the cGAS-STING innate immune signalling axis. The enzymatic and chemical syntheses of 2',3'-cGAMP analogues as STING-targeting therapeutics are also summarized.
Asunto(s)
Inmunoterapia , Proteínas de la Membrana/antagonistas & inhibidores , Neoplasias/terapia , Nucleótidos Cíclicos/farmacología , Nucleótidos/farmacología , Nucleotidiltransferasas/antagonistas & inhibidores , Humanos , Proteínas de la Membrana/inmunología , Modelos Moleculares , Conformación Molecular , Neoplasias/inmunología , Nucleótidos/síntesis química , Nucleótidos/química , Nucleótidos Cíclicos/síntesis química , Nucleótidos Cíclicos/química , Nucleotidiltransferasas/inmunología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunologíaRESUMEN
The nucleosome is the basic structural repeating unit of chromatin. DNA damage and cell apoptosis release nucleosomes into the blood circulatory system, and increased levels of circulating nucleosomes have been observed to be related to inflammation and autoimmune diseases. However, how circulating nucleosomes trigger immune responses has not been fully elucidated. cGAS (cGMP-AMP synthase) is a recently discovered pattern recognition receptor that senses cytoplasmic double-stranded DNA (dsDNA). In this study, we employed in vitro reconstituted nucleosomes to examine whether extracellular nucleosomes can gain access to the cytoplasm of mammalian cells to induce immune responses by activating cGAS. We showed that nucleosomes can be taken up by various mammalian cells. Additionally, we found that in vitro reconstituted mononucleosomes and oligonucleosomes can be recognized by cGAS. Compared to dsDNA, nucleosomes exhibit higher binding affinities to cGAS but considerably lower potency in cGAS activation. Incubation of monocytic cells with reconstituted nucleosomes leads to limited production of type I interferons and proinflammatory cytokines via a cGAS-dependent mechanism. This proof-of-concept study reveals the cGAS-dependent immunogenicity of nucleosomes and highlights the potential roles of circulating nucleosomes in autoimmune diseases, inflammation, and antitumour immunity.
Asunto(s)
Inmunidad Innata/inmunología , Nucleosomas/inmunología , Nucleotidiltransferasas/metabolismo , Adenosina Monofosfato/metabolismo , Animales , Apoptosis , Línea Celular , Cromatina/metabolismo , GMP Cíclico/metabolismo , Citocinas/metabolismo , Citosol/metabolismo , ADN/metabolismo , Daño del ADN , Vesículas Extracelulares/inmunología , Células HeLa , Células Hep G2 , Humanos , Inflamación/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Nucleosomas/metabolismo , Nucleotidiltransferasas/inmunología , Transducción de Señal/genética , Células THP-1RESUMEN
Many reagents have been developed for cysteine-specific protein modification. However, few of them allow for multi-functionalization of a single Cys residue and disulfide bridging bioconjugation. Herein, we report 3-bromo-5-methylene pyrrolones (3Br-5MPs) as a simple, robust, and versatile class of reagents for cysteine-specific protein modification. These compounds can be facilely synthesized via a one-pot mild reaction and they show comparable tagging efficiency but higher cysteine specificity than the maleimide counterparts. The addition of cysteine to 3Br-5MPs generates conjugates that are amenable to secondary addition by another thiol or cysteine, making 3Br-5MPs valuable for multi-functionalization of a single cysteine and disulfide bridging bioconjugation. The labeling reaction and subsequent treatments are mild enough to produce stable and active protein conjugates for biological applications.
Asunto(s)
Cisteína/química , Proteínas/química , Técnicas de Química Sintética/métodos , Disulfuros/química , Indicadores y Reactivos/química , Fenómenos Químicos Orgánicos , Pirroles/química , Somatostatina/químicaRESUMEN
When designing prodrugs, choosing an appropriate linker is the key to achieving efficient, controlled drug delivery. Herein, we report the use of a photocaged C4'-oxidized abasic site (PC4AP) as a light-responsive, self-immolative linker. Any amine- or hydroxyl-bearing drug can be loaded onto the linker via a carbamate or carbonate bond, and the linker is then conjugated to a carrier peptide or protein via an alkyl chain. The PC4AP linker is stable under physiologically relevant conditions. However, photodecaging of the linker generates an active intermediate that reacts intramolecularly with a primary amine (the ε-amine of a lysine residue and the N-terminal amine) on the carrier, leading to rapid and efficient release of the drug via an addition-elimination cascade, without generating any toxic side products. We demonstrated that the use of this self-immolative linker to conjugate the anticancer drug doxorubicin to a cell-penetrating peptide or an antibody enabled targeted, controlled delivery of the drug to cells. Our results suggest that the linker can be used with a broad range of carriers, such as cell-penetrating peptides, proteins, antibodies, and amine-functionalized polymers, and thus will find a wide range of practical applications.