RESUMO
N-Linked glycosylation is one of the most essential post-translational modifications of proteins. However, N-glycan structural determination remains challenging because of the small differences in structures between isomers. In this study, we constructed a database containing collision-induced dissociation MSn mass spectra and chromatograms of high-performance liquid chromatography for the rapid identification of high-mannose and paucimannose N-glycan isomers. These N-glycans include isomers by breaking of arbitrary numbers of glycosidic bonds at arbitrary positions of canonical Man9GlcNAc2 N-glycans. In addition, some GlcMannGlcNAc2 N-glycan isomers were included in the database. This database is particularly useful for the identification of the N-glycans not in conventional N-glycan standards. This study demonstrated the application of the database to structural assignment for high-mannose N-glycans extracted from bovine whey proteins, soybean proteins, human mammary epithelial cells, and human breast carcinoma cells. We found many N-glycans that are not expected to be generated by conventional biosynthetic pathways of multicellular eukaryotes.
Assuntos
Mama , Manose , Humanos , Animais , Bovinos , Cromatografia Líquida de Alta Pressão , Bases de Dados Factuais , PolissacarídeosRESUMO
N-linked glycosylation is one of the most important post-translational modifications of proteins. Current knowledge of multicellular eukaryote N-glycan biosynthesis suggests high mannose N-glycans are generated in the endoplasmic reticulum and Golgi apparatus through conserved biosynthetic pathways. According to conventional biosynthetic pathways, four Man7GlcNAc2 isomers, three Man6GlcNAc2 isomers, and one Man5GlcNAc2 isomer are generated during this process. In this study, we applied our latest mass spectrometry method, logically derived sequence tandem mass spectrometry (LODES/MSn), to re-examine high mannose N-glycans extracted from various multicellular eukaryotes which are not glycosylation mutants. LODES/MSn identified many high mannose N-glycan isomers previously unreported in plantae, animalia, cancer cells, and fungi. A database consisting of retention time and CID MSn mass spectra was constructed for all possible MannGlcNAc2 (n = 5, 6, 7) isomers that include the isomers by removing arbitrary numbers and positions of mannose from canonical N-glycan, Man9GlcNAc2. Many N-glycans in this database are not found in current N-glycan mass spectrum libraries. The database is useful for rapid high mannose N-glycan isomeric identification.
Assuntos
Eucariotos , Manose , Humanos , Manose/química , Eucariotos/metabolismo , Vias Biossintéticas , Polissacarídeos/química , Espectrometria de Massas em Tandem/métodosRESUMO
Tracking DNA double strand break (DSB) repair is paramount for the understanding and therapeutic development of various diseases including cancers. Herein, we describe a multiplexed bioluminescent repair reporter (BLRR) for non-invasive monitoring of DSB repair pathways in living cells and animals. The BLRR approach employs secreted Gaussia and Vargula luciferases to simultaneously detect homology-directed repair (HDR) and non-homologous end joining (NHEJ), respectively. BLRR data are consistent with next-generation sequencing results for reporting HDR (R2 = 0.9722) and NHEJ (R2 = 0.919) events. Moreover, BLRR analysis allows longitudinal tracking of HDR and NHEJ activities in cells, and enables detection of DSB repairs in xenografted tumours in vivo. Using the BLRR system, we observed a significant difference in the efficiency of CRISPR/Cas9-mediated editing with guide RNAs only 1-10 bp apart. Moreover, BLRR analysis detected altered dynamics for DSB repair induced by small-molecule modulators. Finally, we discovered HDR-suppressing functions of anticancer cardiac glycosides in human glioblastomas and glioma cancer stem-like cells via inhibition of DNA repair protein RAD51 homolog 1 (RAD51). The BLRR method provides a highly sensitive platform to simultaneously and longitudinally track HDR and NHEJ dynamics that is sufficiently versatile for elucidating the physiology and therapeutic development of DSB repair.
Assuntos
Genes Reporter , Luciferases/genética , Reparo de DNA por Recombinação , Animais , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Copépodes/enzimologia , Reparo do DNA por Junção de Extremidades , Feminino , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Luciferases/metabolismo , Camundongos , Camundongos Nus , Reação em Cadeia da Polimerase Multiplex/métodos , Imagem Óptica/métodos , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Análise de Sequência de DNA/métodosRESUMO
Extracellular vesicles (EVs) including exosomes and microvesicles are lipid bilayer-encapsulated nanoparticles released by cells, ranging from 40 nm to several microns in diameter. Biological cargoes including proteins, RNAs, and DNAs can be ferried by EVs to neighboring and distant cells via biofluids, serving as a means of cell-to-cell communication under normal and pathological conditions, especially cancers. On the other hand, EVs have been investigated as a novel "information capsule" for early disease detection and monitoring via liquid biopsy. This review summarizes current advancements in EV subtype characterization, cancer EV capture, proteomic analysis technologies, as well as possible EV-based multiomics for cancer diagnostics.
Assuntos
Vesículas Extracelulares/metabolismo , Neoplasias/diagnóstico , Neoplasias/metabolismo , Proteômica/métodos , Micropartículas Derivadas de Células/metabolismo , Exossomos/metabolismo , HumanosRESUMO
Extracellular vesicles (EVs) are lipid bilayer-enclosed nanoparticles released by cells. They range from 30 nm to several micrometers in diameter, and ferry biological cargos such as proteins, lipids, RNAs and DNAs for local and distant intercellular communications. EVs have since been found to play a role in development, as well as in diseases including cancers. To elucidate the roles of EVs, researchers have established different methods to visualize and study their spatiotemporal properties. However, since EV are nanometer-sized, imaging them demands a full understanding of each labeling strategy to ensure accurate monitoring. This review covers current and emerging strategies for EV imaging for prospective studies.
Assuntos
Diagnóstico por Imagem/métodos , Vesículas Extracelulares/fisiologia , Microscopia Eletrônica/métodos , Diagnóstico por Imagem/instrumentação , Vesículas Extracelulares/ultraestrutura , Microscopia Eletrônica/instrumentaçãoRESUMO
Extracellular vesicles (EVs) are released by cells to mediate intercellular communication under pathological and physiological conditions. While small EVs (sEVs; <100-200 nm, exosomes) are intensely investigated, the properties and functions of medium and large EVs (big EVs (bEVs); >200 nm, microvesicles) are less well explored. Here, bEVs and sEVs are identified as distinct EV populations, and it is determined that bEVs are released in a greater bEV:sEV ratio in the aggressive human triple-negative breast cancer (TNBC) subtype. PalmGRET, bioluminescence-resonance-energy-transfer (BRET)-based EV reporter, reveals dose-dependent EV biodistribution at nonlethal and physiological EV dosages, as compared to lipophilic fluorescent dyes. Remarkably, the bEVs and sEVs exhibit unique biodistribution profiles, yet individually promote in vivo tumor growth in a syngeneic immunocompetent TNBC breast tumor murine model. The bEVs and sEVs share mass-spectrometry-identified tumor-progression-associated EV surface membrane proteins (tpEVSurfMEMs), which include solute carrier family 29 member 1, Cd9, and Cd44. tpEVSurfMEM depletion attenuates EV lung organotropism, alters biodistribution, and reduces protumorigenic potential. This study identifies distinct in vivo property and function of bEVs and sEVs in breast cancer, which suggest the significant role of bEVs in diseases, diagnostic and therapeutic applications.
Assuntos
Exossomos , Vesículas Extracelulares , Neoplasias de Mama Triplo Negativas , Camundongos , Humanos , Animais , Distribuição Tecidual , Proteínas de Membrana/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Vesículas Extracelulares/metabolismo , Exossomos/metabolismo , Carcinogênese/metabolismoRESUMO
The dynamics of DNA double-strand break (DSB) repairs including homology-directed repair and nonhomologous end joining play an important role in diseases and therapies. However, investigating DSB repair is typically a low-throughput and cross-sectional process, requiring disruption of cells and organisms for subsequent nuclease-, sequencing- or reporter-based assays. In this protocol, we provide instructions for establishing a bioluminescent repair reporter system using engineered Gaussia and Vargula luciferases for noninvasive tracking of homology-directed repair and nonhomologous end joining, respectively, induced by SceI meganuclease, SpCas9 or SpCas9 D10A nickase-mediated editing. We also describe complementation with orthogonal DSB repair assays and omics analyses to validate the reporter readouts. The bioluminescent repair reporter system provides longitudinal and rapid readout (~seconds per sample) to accurately and efficiently measure the efficacy of genome-editing tools and small-molecule modulators on DSB repair. This protocol takes ~2-4 weeks to establish, and as little as 2 h to complete the assay. The entire bioluminescent repair reporter procedure can be performed by one person with standard molecular biology expertise and equipment. However, orthogonal DNA repair assays would require a specialized facility that performs Sanger sequencing or next-generation sequencing.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA/fisiologia , Medições Luminescentes/métodos , Animais , Sistemas CRISPR-Cas , Linhagem Celular , Humanos , Camundongos , Camundongos Transgênicos , Neoplasias ExperimentaisRESUMO
Cell-released, membrane-encapsulated extracellular vesicles (EVs) serve as a means of intercellular communication by delivering bioactive cargos including proteins, nucleic acids and lipids. EVs have been widely used for a variety of biomedical applications such as biomarkers for disease diagnosis and drug delivery vehicles for therapy. Herein, this study reports a novel method for label-free, contact-free isolation and recovery of EVs via optically-induced dielectrophoresis (ODEP) on a pneumatically-driven microfluidic platform with minimal human intervention. At an optimal driving frequency of 20 kHz and a voltage of 20 Vpp, an ODEP force from a 75 µm moving light beam was characterized to be 23.5-97.7 fN in 0.2 M sucrose solution. Furthermore, rapid enrichment of EVs with a small volume of only 27 pL in 32 s achieved an increase of 272-fold by dynamically shrinking circular light patterns. Moreover, EVs could be automatically isolated and recovered within 25 min, while achieving a releasing efficiency of 99.8% and a recovery rate of 52.2% by using an integrated microfluidics-based optically-induced EV isolation (OIEV) platform. Given the capacity of label-free, contact-free EV isolation, and automatic, easy-releasing EV recovery, this integrated OIEV platform provides a unique approach for EV-based disease diagnosis and drug delivery applications.
Assuntos
Vesículas Extracelulares , Ácidos Nucleicos , Comunicação Celular , Humanos , Lipídeos , MicrofluídicaRESUMO
Extracellular particles (EPs) including extracellular vesicles (EVs) and exomeres play significant roles in diseases and therapeutic applications. However, their spatiotemporal dynamics in vivo have remained largely unresolved in detail due to the lack of a suitable method. Therefore, a bioluminescence resonance energy transfer (BRET)-based reporter, PalmGRET, is created to enable pan-EP labeling ranging from exomeres (<50 nm) to small (<200 nm) and medium and large (>200 nm) EVs. PalmGRET emits robust, sustained signals and allows the visualization, tracking, and quantification of the EPs from whole animal to nanoscopic resolutions under different imaging modalities, including bioluminescence, BRET, and fluorescence. Using PalmGRET, it is shown that EPs released by lung metastatic hepatocellular carcinoma (HCC) exhibit lung tropism with varying distributions to other major organs in immunocompetent mice. It is further demonstrated that gene knockdown of lung-tropic membrane proteins, solute carrier organic anion transporter family member 2A1, alanine aminopeptidase/Cd13, and chloride intracellular channel 1 decreases HCC-EP distribution to the lungs and yields distinct biodistribution profiles. It is anticipated that EP-specific imaging, quantitative assays, and detailed in vivo characterization are a starting point for more accurate and comprehensive in vivo models of EP biology and therapeutic design.
RESUMO
Extracellular vesicles (EVs) released by cells play a role in intercellular communication. Reporter and targeting proteins can be modified and exposed on the surface of EVs to investigate their half-life and biodistribution. A characterization of membrane-bound Gaussia luciferase (mbGluc) revealed that its signal was detected also in a form smaller than common EVs (<70 nm). We demonstrated that mbGluc initially exposed on the surface of EVs, likely undergoes proteolytic cleavage and processed fragments of the protein are released into the extracellular space in active form. Based on this observation, we developed a new assay to quantitatively track shedding of membrane proteins from the surface of EVs. We used this assay to show that ectodomain shedding in EVs is continuous and is mediated by specific proteases, e.g. metalloproteinases. Here, we present a novel tool to study membrane protein cleavage and release using both in vitro and in vivo models.
Assuntos
Copépodes/enzimologia , Vesículas Extracelulares/metabolismo , Luciferases/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Recombinantes/metabolismo , Animais , Linhagem Celular Tumoral , Copépodes/genética , Copépodes/metabolismo , Feminino , Humanos , Luciferases/genética , Proteínas de Membrana/genética , Membranas/metabolismo , Camundongos , Camundongos Nus , Proteínas Recombinantes/genética , Via Secretória/genética , Distribuição TecidualRESUMO
Analysis of cancer-derived extracellular vesicles (EVs) in biofluids potentially provides a source of disease biomarkers. At present there is no procedure to systematically identify which antigens should be targeted to differentiate cancer-derived from normal host cell-derived EVs. Here, we propose a computational framework that integrates information about membrane proteins in tumors and normal tissues from databases: UniProt, The Cancer Genome Atlas, the Genotype-Tissue Expression Project, and the Human Protein Atlas. We developed two methods to assess capture of EVs from specific cell types. (1) We used palmitoylated fluorescent protein (palmtdTomato) to label tumor-derived EVs. Beads displaying antibodies of interest were incubated with conditioned medium from palmtdTomato-expressing cells. Bound EVs were quantified using flow cytometry. (2) We also showed that membrane-bound Gaussia luciferase allows the detection of cancer-derived EVs in blood of tumor-bearing animals. Our analytical and validation platform should be applicable to identify antigens on EVs from any tumor type.
Assuntos
Biomarcadores Tumorais/metabolismo , Vesículas Extracelulares/metabolismo , Citometria de Fluxo/métodos , Proteínas de Membrana/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Linhagem Celular Tumoral , Feminino , Proteínas de Fluorescência Verde/metabolismo , Humanos , Imunoensaio/métodos , Luciferases/metabolismo , Camundongos , Camundongos Nus , Pessoa de Meia-IdadeRESUMO
Abnormal tumour vasculature has a significant impact on tumour progression and response to therapy. Nitric oxide (NO) regulates angiogenesis and maintains vascular homeostasis and, thus, can be delivered to normalize tumour vasculature. However, a NO-delivery system with a prolonged half-life and a sustained release mechanism is currently lacking. Here we report the development of NanoNO, a nanoscale carrier that enables sustained NO release to efficiently deliver NO into hepatocellular carcinoma. Low-dose NanoNO normalizes tumour vessels and improves the delivery and effectiveness of chemotherapeutics and tumour necrosis factor-related, apoptosis-inducing, ligand-based therapy in both primary tumours and metastases. Furthermore, low-dose NanoNO reprogrammes the immunosuppressive tumour microenvironment toward an immunostimulatory phenotype, thereby improving the efficacy of cancer vaccine immunotherapy. Our findings demonstrate the ability of nanoscale NO delivery to efficiently reprogramme tumour vasculature and immune microenvironments to overcome resistance to cancer therapy, resulting in a therapeutic benefit.
Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Preparações de Ação Retardada/química , Neoplasias Hepáticas/tratamento farmacológico , Nanopartículas/química , Neovascularização Patológica/tratamento farmacológico , Óxido Nítrico/administração & dosagem , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/uso terapêutico , Animais , Carcinoma Hepatocelular/irrigação sanguínea , Humanos , Neoplasias Hepáticas/irrigação sanguínea , Masculino , Camundongos , Óxido Nítrico/uso terapêutico , Microambiente Tumoral/efeitos dos fármacosRESUMO
Elucidating extracellular vesicle (EV; e.g., exosomes, microvesicles) delivery and translation of its RNA cargo with an accurate spatiotemporal resolution is critical in helping understand EV's role under normal and pathological conditions. We here describe a multiplexed fluorescent and bioluminescent reporter strategy to simultaneously monitor and quantify EV delivery, as well as EV-RNA translation in EV-recipient cells.
Assuntos
Vesículas Extracelulares/metabolismo , Expressão Gênica , Genes Reporter , Imagem Molecular , Biossíntese de Proteínas , Transporte de RNA , RNA/metabolismo , Micropartículas Derivadas de Células/metabolismo , Exossomos/metabolismo , Células HEK293 , Humanos , Medições Luminescentes , Microscopia de Fluorescência , Imagem Molecular/métodosRESUMO
Extracellular membrane vesicles (EMVs) are nanometer sized vesicles, including exosomes and microvesicles capable of transferring DNAs, mRNAs, microRNAs, non-coding RNAs, proteins, and lipids among cells without direct cell-to-cell contact, thereby representing a novel form of intercellular communication. Many cells in the nervous system have been shown to release EMVs, implicating their active roles in development, function, and pathologies of this system. While substantial progress has been made in understanding the biogenesis, biophysical properties, and involvement of EMVs in diseases, relatively less information is known about their biological function in the normal nervous system. In addition, since EMVs are endogenous vehicles with low immunogenicity, they have also been actively investigated for the delivery of therapeutic genes/molecules in treatment of cancer and neurological diseases. The present review summarizes current knowledge about EMV functions in the nervous system under both physiological and pathological conditions, as well as emerging EMV-based therapies that could be applied to the nervous system in the foreseeable future.