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1.
Cell ; 177(2): 428-445.e18, 2019 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-30951670

RESUMO

The heterogeneity of small extracellular vesicles and presence of non-vesicular extracellular matter have led to debate about contents and functional properties of exosomes. Here, we employ high-resolution density gradient fractionation and direct immunoaffinity capture to precisely characterize the RNA, DNA, and protein constituents of exosomes and other non-vesicle material. Extracellular RNA, RNA-binding proteins, and other cellular proteins are differentially expressed in exosomes and non-vesicle compartments. Argonaute 1-4, glycolytic enzymes, and cytoskeletal proteins were not detected in exosomes. We identify annexin A1 as a specific marker for microvesicles that are shed directly from the plasma membrane. We further show that small extracellular vesicles are not vehicles of active DNA release. Instead, we propose a new model for active secretion of extracellular DNA through an autophagy- and multivesicular-endosome-dependent but exosome-independent mechanism. This study demonstrates the need for a reassessment of exosome composition and offers a framework for a clearer understanding of extracellular vesicle heterogeneity.


Assuntos
Exossomos/metabolismo , Exossomos/fisiologia , Anexina A1/metabolismo , Proteínas Argonautas/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Micropartículas Derivadas de Células/metabolismo , DNA/metabolismo , Exossomos/química , Vesículas Extracelulares , Feminino , Humanos , Lisossomos/metabolismo , Masculino , Proteínas/metabolismo , RNA/metabolismo
2.
Cell ; 149(1): 146-58, 2012 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-22464327

RESUMO

Lineage mapping has identified both proliferative and quiescent intestinal stem cells, but the molecular circuitry controlling stem cell quiescence is incompletely understood. By lineage mapping, we show Lrig1, a pan-ErbB inhibitor, marks predominately noncycling, long-lived stem cells that are located at the crypt base and that, upon injury, proliferate and divide to replenish damaged crypts. Transcriptome profiling of Lrig1(+) colonic stem cells differs markedly from the profiling of highly proliferative, Lgr5(+) colonic stem cells; genes upregulated in the Lrig1(+) population include those involved in cell cycle repression and response to oxidative damage. Loss of Apc in Lrig1(+) cells leads to intestinal adenomas, and genetic ablation of Lrig1 results in heightened ErbB1-3 expression and duodenal adenomas. These results shed light on the relationship between proliferative and quiescent intestinal stem cells and support a model in which intestinal stem cell quiescence is maintained by calibrated ErbB signaling with loss of a negative regulator predisposing to neoplasia.


Assuntos
Colo/metabolismo , Genes Supressores de Tumor , Intestino Delgado/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Adenoma/patologia , Proteína da Polipose Adenomatosa do Colo/metabolismo , Animais , Colo/citologia , Receptores ErbB/metabolismo , Perfilação da Expressão Gênica , Humanos , Neoplasias Intestinais/patologia , Intestino Delgado/citologia , Camundongos , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Células-Tronco/citologia , Células-Tronco/metabolismo
3.
Cell Mol Life Sci ; 81(1): 28, 2024 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-38212428

RESUMO

Although amplifications and mutations in receptor tyrosine kinases (RTKs) act as bona fide oncogenes, in most cancers, RTKs maintain moderate expression and remain wild-type. Consequently, cognate ligands control many facets of tumorigenesis, including resistance to anti-RTK therapies. Herein, we show that the ligands for the RTKs MET and RON, HGF and HGFL, respectively, are synthesized as inactive precursors that are activated by cellular proteases. Our newly generated HGF/HGFL protease inhibitors could overcome both de novo and acquired cetuximab resistance in colorectal cancer (CRC). Conversely, HGF overexpression was necessary and sufficient to induce cetuximab resistance and loss of polarity. Moreover, HGF-induced cetuximab resistance could be overcome by the downstream MET inhibitor, crizotinib, and upstream protease inhibitors. Additionally, HAI-1, an endogenous inhibitor of HGF proteases, (i) was downregulated in CRC, (ii) exhibited increased genomic methylation that correlated with poor prognosis, (iii) HAI-1 expression correlated with cetuximab response in a panel of cancer cell lines, and (iv) exogenous addition of recombinant HAI-1 overcame cetuximab resistance in CC-HGF cells. Thus, we describe a targetable, autocrine HAI-1/Protease/HGF/MET axis in cetuximab resistance in CRC.


Assuntos
Neoplasias Colorretais , Transdução de Sinais , Humanos , Cetuximab/farmacologia , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Inibidores de Proteases/farmacologia , Peptídeo Hidrolases/metabolismo , Linhagem Celular Tumoral , Fator de Crescimento de Hepatócito/genética , Fator de Crescimento de Hepatócito/metabolismo , Fator de Crescimento de Hepatócito/farmacologia
4.
Nano Lett ; 23(16): 7500-7507, 2023 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-37552655

RESUMO

This study addresses the challenge of trapping nanoscale biological particles using optical tweezers without the photothermal heating effect and the limitation presented by the diffraction limit. Optical tweezers are effective for trapping microscopic biological objects but not for nanoscale specimens due to the diffraction limit. To overcome this, we present an approach that uses optical anapole states in all-dielectric nanoantenna systems on distributed Bragg reflector substrates to generate strong optical gradient force and potential on nanoscale biological objects with negligible temperature rise below 1 K. The anapole antenna condenses the accessible electromagnetic energy to scales as small as 30 nm. Using this approach, we successfully trapped nanosized extracellular vesicles and supermeres (approximately 25 nm in size) using low laser power of only 10.8 mW. This nanoscale optical trapping platform has great potential for single molecule analysis while precluding photothermal degradation.

5.
J Cell Sci ; 134(18)2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34406412

RESUMO

In polarized MDCK cells, disruption of the tyrosine-based YXXΦ basolateral trafficking motif (Y156A) in the epidermal growth factor receptor (EGFR) ligand epiregulin (EREG), results in its apical mistrafficking and transformation in vivo. However, the mechanisms underlying these dramatic effects are unknown. Using a doxycycline-inducible system in 3D Matrigel cultures, we now show that induction of Y156A EREG in fully formed MDCK cysts results in direct and complete delivery of mutant EREG to the apical cell surface. Within 3 days of induction, ectopic lumens were detected in mutant, but not wild-type, EREG-expressing cysts. Of note, these structures resembled histological features found in subcutaneous xenografts of mutant EREG-expressing MDCK cells. These ectopic lumens formed de novo rather than budding from the central lumen and depended on metalloprotease-mediated cleavage of EREG and subsequent EGFR activity. Moreover, the most frequent EREG mutation in human cancer (R147stop) resulted in its apical mistrafficking in engineered MDCK cells. Thus, induction of EREG apical mistrafficking is sufficient to disrupt selective aspects of polarity of a preformed polarized epithelium. This article has an associated First Person interview with the first author of the paper.


Assuntos
Receptores ErbB , Transdução de Sinais , Epirregulina/genética , Epirregulina/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Fosforilação
6.
Infect Immun ; 87(7)2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31010817

RESUMO

The symbiotic relationship between the gut microbiome and the host provides a nutrient-rich environment for gut microbes and has beneficial effects on host health. Although the composition of the gut microbiome is known to be influenced by both host genetics and environmental factors, host effects on the activities and functions of the gut microbial communities remain poorly understood. Intestinal epithelial cells exert front-line responses to gut microbes and contribute to maintaining a healthy intestinal homeostasis. Here, seeking to elucidate whether intestinal epithelial cells modulate Lactobacillus rhamnosus GG (LGG) functions, we examined the production of p40, an LGG-derived secretory protein that protects intestinal epithelial cells against inflammation. We found that growth medium conditioned with colonic epithelial cell-derived components promotes p40 protein synthesis and secretion by LGG and enhances LGG-stimulated protective responses in intestinal epithelial cells. Furthermore, when LGG was cultured with the colonic luminal contents from healthy mice, p40 production was upregulated but was attenuated with luminal contents from mice with intestinal inflammation. Importantly, the colonic epithelial cell-derived components potentiated LGG-produced p40 levels in a mouse model of colitis and enhanced LGG-mediated amelioration of intestinal inflammation in this model. Notably, we found that colonic epithelial cell-secreted extracellular vesicles participate in communicating with LGG and that heat shock protein 90 (HSP90) in these vesicles might mediate the promotion of p40 production. These results reveal a previously unrecognized mechanism by which the anti-inflammatory effect of LGG is reinforced by intestinal epithelial cells and thereby maintains intestinal health.


Assuntos
Proteínas de Bactérias/metabolismo , Células Epiteliais/microbiologia , Mucosa Intestinal/microbiologia , Lacticaseibacillus rhamnosus/metabolismo , Vesículas Secretórias/microbiologia , Animais , Proteínas de Bactérias/genética , Células Epiteliais/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Mucosa Intestinal/metabolismo , Lacticaseibacillus rhamnosus/genética , Camundongos , Camundongos Endogâmicos C57BL , Vesículas Secretórias/metabolismo
8.
Mol Cell Proteomics ; 12(2): 343-55, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23161513

RESUMO

Activating mutations in KRAS occur in 30% to 40% of colorectal cancers. How mutant KRAS alters cancer cell behavior has been studied intensively, but non-cell autonomous effects of mutant KRAS are less understood. We recently reported that exosomes isolated from mutant KRAS-expressing colon cancer cells enhanced the invasiveness of recipient cells relative to exosomes purified from wild-type KRAS-expressing cells, leading us to hypothesize mutant KRAS might affect neighboring and distant cells by regulating exosome composition and behavior. Herein, we show the results of a comprehensive proteomic analysis of exosomes from parental DLD-1 cells that contain both wild-type and G13D mutant KRAS alleles and isogenically matched derivative cell lines, DKO-1 (mutant KRAS allele only) and DKs-8 (wild-type KRAS allele only). Mutant KRAS status dramatically affects the composition of the exosome proteome. Exosomes from mutant KRAS cells contain many tumor-promoting proteins, including KRAS, EGFR, SRC family kinases, and integrins. DKs-8 cells internalize DKO-1 exosomes, and, notably, DKO-1 exosomes transfer mutant KRAS to DKs-8 cells, leading to enhanced three-dimensional growth of these wild-type KRAS-expressing non-transformed cells. These results have important implications for non-cell autonomous effects of mutant KRAS, such as field effect and tumor progression.


Assuntos
Neoplasias do Colo/química , Neoplasias do Colo/patologia , Exossomos/química , Invasividade Neoplásica , Proteínas de Neoplasias/química , Proteoma/química , Proteínas Proto-Oncogênicas/química , Proteínas ras/química , Alelos , Linhagem Celular Tumoral , Proliferação de Células , Cromatografia Líquida , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Exossomos/metabolismo , Humanos , Mutação , Proteínas de Neoplasias/metabolismo , Transporte Proteico , Proteoma/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas p21(ras) , Transdução de Sinais , Espectrometria de Massas em Tandem , Microambiente Tumoral , Proteínas ras/genética , Proteínas ras/metabolismo
9.
Commun Biol ; 7(1): 677, 2024 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-38830977

RESUMO

We present a quantitative sandwich immunoassay for CD63 Extracellular Vesicles (EVs) and a constituent surface cargo, EGFR and its activity state, that provides a sensitive, selective, fluorophore-free and rapid alternative to current EV-based diagnostic methods. Our sensing design utilizes a charge-gating strategy, with a hydrophilic anion exchange membrane functionalized with capture antibodies and a charged silica nanoparticle reporter functionalized with detection antibodies. With sensitivity and robustness enhancement by the ion-depletion action of the membrane, this hydrophilic design with charged reporters minimizes interference from dispersed proteins, thus enabling direct plasma analysis without the need for EV isolation or sensor blocking. With a LOD of 30 EVs/µL and a high relative sensitivity of 0.01% for targeted proteomic subfractions, our assay enables accurate quantification of the EV marker, CD63, with colocalized EGFR by an operator/sample insensitive universal normalized calibration. We analysed untreated clinical samples of Glioblastoma to demonstrate this new platform. Notably, we target both total and "active" EGFR on EVs; with a monoclonal antibody mAb806 that recognizes a normally hidden epitope on overexpressed or mutant variant III EGFR. Analysis of samples yielded an area-under-the-curve (AUC) value of 0.99 and a low p-value of 0.000033, surpassing the performance of existing assays and markers.


Assuntos
Receptores ErbB , Vesículas Extracelulares , Glioblastoma , Tetraspanina 30 , Humanos , Glioblastoma/sangue , Glioblastoma/diagnóstico , Glioblastoma/metabolismo , Tetraspanina 30/metabolismo , Receptores ErbB/metabolismo , Vesículas Extracelulares/metabolismo , Imunoensaio/métodos , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/sangue , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/diagnóstico
10.
J Extracell Vesicles ; 13(8): e12498, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-39140467

RESUMO

High-sensitivity flow cytometers have been developed for multi-parameter characterization of single extracellular vesicles (EVs), but performance varies among instruments and calibration methods. Here we compare the characterization of identical (split) EV samples derived from human colorectal cancer (DiFi) cells by three high-sensitivity flow cytometers, two commercial instruments, CytoFLEX/CellStream, and a custom single-molecule flow cytometer (SMFC). DiFi EVs were stained with the membrane dye di-8-ANEPPS and with PE-conjugated anti-EGFR or anti-tetraspanin (CD9/CD63/CD81) antibodies for estimation of EV size and surface protein copy numbers. The limits of detection (LODs) for immunofluorescence and vesicle size based on calibration using cross-calibrated, hard-dyed beads were ∼10 PE/∼80 nm EV diameter for CytoFLEX and ∼10 PEs/∼67 nm for CellStream. For the SMFC, the LOD for immunofluorescence was 1 PE and ≤ 35 nm for size. The population of EVs detected by each system (di-8-ANEPPS+/PE+ particles) differed widely depending on the LOD of the system; for example, CellStream/CytoFLEX detected only 5.7% and 1.5% of the tetraspanin-labelled EVs detected by SMFC, respectively, and median EV diameter and antibody copy numbers were much larger for CellStream/CytoFLEX than for SMFC as measured and validated using super-resolution/single-molecule TIRF microscopy. To obtain a dataset representing a common EV population analysed by all three platforms, we filtered out SMFC and CellStream measurements for EVs below the CytoFLEX LODs as determined by bead calibration (10 PE/80 nm). The inter-platform agreement using this filtered dataset was significantly better than for the unfiltered dataset, but even better concordance between results was obtained by applying higher cutoffs (21 PE/120 nm) determined by threshold analysis using the SMFC data. The results demonstrate the impact of specifying LODs to define the EV population analysed on inter-instrument reproducibility in EV flow cytometry studies, and the utility of threshold analysis of SMFC data for providing semi-quantitative LOD values for other flow cytometers.


Assuntos
Vesículas Extracelulares , Citometria de Fluxo , Citometria de Fluxo/métodos , Citometria de Fluxo/instrumentação , Humanos , Vesículas Extracelulares/metabolismo , Neoplasias Colorretais/diagnóstico , Linhagem Celular Tumoral , Imagem Individual de Molécula/métodos , Imagem Individual de Molécula/instrumentação
11.
Traffic ; 12(12): 1793-804, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21917092

RESUMO

Epithelial cells establish apical and basolateral (BL) membranes with distinct protein and lipid compositions. To achieve this spatial asymmetry, the cell utilizes a variety of mechanisms for differential sorting, delivery and retention of cell surface proteins. The EGF receptor (EGFR) and its ligand, amphiregulin (AREG), are transmembrane proteins delivered to the BL membrane in polarized epithelial cells. Herein, we show that the cytoplasmic domain of AREG (ACD) contains dominant BL sorting information; replacement of the cytoplasmic domain of apically targeted nerve growth factor receptor with the ACD redirects the chimera to the BL surface. Using sequential truncations and site-directed mutagenesis of the ACD, we identify a novel BL sorting motif consisting of a single leucine C-terminal to an acidic cluster (EEXXXL). In adaptor protein (AP)-1B-deficient cells, newly synthesized AREG is initially delivered to the BL surface as in AP-1B-expressing cells. However, in these AP-1B-deficient cells, recycling of AREG back to the BL surface is compromised, leading to its appearance at the apical surface. These results show that recycling, but not delivery, of AREG to the BL surface is AP-1B dependent.


Assuntos
Citoplasma/metabolismo , Glicoproteínas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Leucina/metabolismo , Proteínas de Membrana/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Anfirregulina , Animais , Linhagem Celular , Membrana Celular/metabolismo , Polaridade Celular/fisiologia , Cães , Família de Proteínas EGF , Células Epiteliais/metabolismo , Receptores ErbB/metabolismo , Humanos , Células LLC-PK1 , Dados de Sequência Molecular , Estrutura Terciária de Proteína/fisiologia , Transporte Proteico , Receptores de Fator de Crescimento Neural/metabolismo , Suínos
12.
Nat Protoc ; 18(5): 1462-1487, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-36914899

RESUMO

There is an increasing appreciation for the heterogeneous nature of extracellular vesicles (EVs). In addition, two nonvesicular extracellular nanoparticles (NVEPs), exomeres and supermeres, have been discovered recently that are enriched in many cargo previously ascribed to EVs. The EV field has largely focused on EV isolation and characterization, while studies on NVEPs are limited. At this juncture, it is critically important to have robust and reliable methods to separate distinct populations of EVs and NVEPs to assign cargo to their correct carrier. Here, we provide a comprehensive step-by-step protocol for sequential isolation of large and small EVs, nonvesicular fractions, exomeres and supermeres from the same starting material. We describe in detail the use of differential ultracentrifugation, filtration, concentration and high-resolution density-gradient fractionation to obtain purified fractions of distinct populations of EVs and NVEPs. This protocol allows assignment and enrichment of a biomolecule of interest to its specific extracellular compartment. Compared to other isolation methods, our protocol has unique advantages, including high purity and reproducibility, with minimal expertise required. The protocol can be applied to purification of EVs and NVEPs from cell culture medium and human plasma and requires ~72 h to complete. Adoption of this protocol will help translational investigators identify potential circulating biomarkers and therapeutic targets for a host of human diseases and allow basic scientists to better understand EV and NVEP biogenesis and function. Overall, this protocol will allow those interested in isolating EVs and extracellular particles to advance scientific inquiry to answer outstanding questions in the field.


Assuntos
Vesículas Extracelulares , Nanopartículas , Humanos , Reprodutibilidade dos Testes , Ultracentrifugação , Filtração
13.
bioRxiv ; 2023 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-37905113

RESUMO

We present a novel quantitative immunoassay for CD63 EVs (extracellular vesicles) and a constituent surface cargo, EGFR and its activity state, that provides a sensitive, selective, fluorophore-free and rapid alternative to current EV-based diagnostic methods. Our sensing design utilizes a charge-gating strategy, with a hydrophilic anion exchange membrane and a charged silica nanoparticle reporter. With sensitivity and robustness enhancement by the ion-depletion action of the membrane, this hydrophilic design with charged reporters minimizes interference from dispersed proteins and fluorophore degradation, thus enabling direct plasma analysis. With a limit of detection of 30 EVs/µL and a high relative sensitivity of 0.01% for targeted proteomic subfractions, our assay enables accurate quantification of the EV marker, CD63, with colocalized EGFR by an operator/sample insensitive universal normalized calibration. Glioblastoma necessitates improved non-invasive diagnostic approaches for early detection and monitoring. Notably, we target both total and "active" EGFR on EVs; with a monoclonal antibody mAb806 that recognizes a normally hidden epitope on overexpressed or mutant variant III EGFR. This approach offers direct glioblastoma detection from untreated human patient samples. Analysis of glioblastoma clinical samples yielded an area-under-the-curve (AUC) value of 0.99 and low p-value of 0.000033, significantly surpassing the performance of existing assays and markers.

14.
Commun Biol ; 5(1): 1358, 2022 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-36496485

RESUMO

Superparamagnetic nanobeads offer several advantages over microbeads for immunocapture of nanocarriers (extracellular vesicles, lipoproteins, and viruses) in a bioassay: high-yield capture, reduction in incubation time, and higher capture capacity. However, nanobeads are difficult to "pull-down" because their superparamagnetic feature requires high nanoscale magnetic field gradients. Here, an electrodeposited track-etched membrane is shown to produce a unique superparamagnetic nano-edge ring with multiple edges around nanopores. With a uniform external magnetic field, the induced monopole and dipole of this nano edge junction combine to produce a 10× higher nanobead trapping force. A dense nanobead suspension can be filtered through the magnetic nanoporous membrane (MNM) at high throughput with a 99% bead capture rate. The yield of specific nanocarriers in heterogeneous media by nanobeads/MNM exceeds 80%. Reproducibility, low loss, and concentration-independent capture rates are also demonstrated. This MNM material hence expands the application of nanobead immunocapture to physiological samples.


Assuntos
Vesículas Extracelulares , Reprodutibilidade dos Testes , Campos Magnéticos , Membranas
15.
iScience ; 25(8): 104653, 2022 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-35958027

RESUMO

The extracellular RNA communication consortium (ERCC) is an NIH-funded program aiming to promote the development of new technologies, resources, and knowledge about exRNAs and their carriers. After Phase 1 (2013-2018), Phase 2 of the program (ERCC2, 2019-2023) aims to fill critical gaps in knowledge and technology to enable rigorous and reproducible methods for separation and characterization of both bulk populations of exRNA carriers and single EVs. ERCC2 investigators are also developing new bioinformatic pipelines to promote data integration through the exRNA atlas database. ERCC2 has established several Working Groups (Resource Sharing, Reagent Development, Data Analysis and Coordination, Technology Development, nomenclature, and Scientific Outreach) to promote collaboration between ERCC2 members and the broader scientific community. We expect that ERCC2's current and future achievements will significantly improve our understanding of exRNA biology and the development of accurate and efficient exRNA-based diagnostic, prognostic, and theranostic biomarker assays.

16.
Nat Cell Biol ; 23(12): 1240-1254, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34887515

RESUMO

Extracellular vesicles and exomere nanoparticles are under intense investigation as sources of clinically relevant cargo. Here we report the discovery of a distinct extracellular nanoparticle, termed supermere. Supermeres are morphologically distinct from exomeres and display a markedly greater uptake in vivo compared with small extracellular vesicles and exomeres. The protein and RNA composition of supermeres differs from small extracellular vesicles and exomeres. Supermeres are highly enriched with cargo involved in multiple cancers (glycolytic enzymes, TGFBI, miR-1246, MET, GPC1 and AGO2), Alzheimer's disease (APP) and cardiovascular disease (ACE2, ACE and PCSK9). The majority of extracellular RNA is associated with supermeres rather than small extracellular vesicles and exomeres. Cancer-derived supermeres increase lactate secretion, transfer cetuximab resistance and decrease hepatic lipids and glycogen in vivo. This study identifies a distinct functional nanoparticle replete with potential circulating biomarkers and therapeutic targets for a host of human diseases.


Assuntos
Vesículas Extracelulares/metabolismo , MicroRNAs/metabolismo , Nanopartículas/metabolismo , Doença de Alzheimer/patologia , Enzima de Conversão de Angiotensina 2/metabolismo , Transporte Biológico/fisiologia , Biomarcadores/metabolismo , COVID-19/patologia , Doenças Cardiovasculares/patologia , Comunicação Celular/fisiologia , Linhagem Celular Tumoral , Células HeLa , Humanos , Ácido Láctico/metabolismo , MicroRNAs/genética , Nanopartículas/classificação , Neoplasias/patologia , Microambiente Tumoral
17.
Mol Cell Proteomics ; 7(9): 1651-67, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18504258

RESUMO

By interacting with the cytoplasmic tail of a Golgi-processed form of transforming growth factor-alpha (TGFalpha), Naked2 coats TGFalpha-containing exocytic vesicles and directs them to the basolateral corner of polarized epithelial cells where the vesicles dock and fuse in a Naked2 myristoylation-dependent manner. These TGFalpha-containing Naked2-associated vesicles are not directed to the subapical Sec6/8 exocyst complex as has been reported for other basolateral cargo, and thus they appear to represent a distinct set of basolaterally targeted vesicles. To identify constituents of these vesicles, we exploited our finding that myristoylation-deficient Naked2 G2A vesicles are unable to fuse at the plasma membrane. Isolation of a population of myristoylation-deficient, green fluorescent protein-tagged G2A Naked2-associated vesicles was achieved by biochemical enrichment followed by flow cytometric fluorescence-activated vesicle sorting. The protein content of these plasma membrane de-enriched, flow-sorted fluorescent G2A Naked2 vesicles was determined by LC/LC-MS/MS analysis. Three independent isolations were performed, and 389 proteins were found in all three sets of G2A Naked2 vesicles. Rab10 and myosin IIA were identified as core machinery, and Na(+)/K(+)-ATPase alpha1 was identified as an additional cargo within these vesicles. As an initial validation step, we confirmed their presence and that of three additional proteins tested (annexin A1, annexin A2, and IQGAP1) in wild-type Naked2 vesicles. To our knowledge, this is the first large scale protein characterization of a population of basolaterally targeted exocytic vesicles and supports the use of fluorescence-activated vesicle sorting as a useful tool for isolation of cellular organelles for comprehensive proteomics analysis.


Assuntos
Proteínas de Transporte/metabolismo , Exocitose , Proteínas/análise , Proteômica/métodos , Vesículas Transportadoras/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Western Blotting , Proteínas de Ligação ao Cálcio , Proteínas de Transporte/genética , Linhagem Celular , Membrana Celular/metabolismo , Cromatografia Líquida/métodos , Cães , Fluorescência , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/metabolismo , Humanos , Espectrometria de Massas , Transporte Proteico/efeitos da radiação , Proteínas/metabolismo , Fator de Crescimento Transformador alfa/metabolismo , Vesículas Transportadoras/efeitos da radiação , Ácidos Tri-Iodobenzoicos/metabolismo
18.
Oncotarget ; 10(13): 1320-1333, 2019 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-30863492

RESUMO

It is increasingly appreciated that 3D cultures are more predictive of in vivo therapeutic efficacy than 2D cultures. Using in vitro 3D type I collagen cultures of human colorectal cancer (CRC) cell line HCA-7 derivatives CC, SC, and CC-CR, we previously identified that activation of receptor tyrosine kinases (RTKs) MET and RON contributed to resistance to the EGF receptor (EGFR)-directed therapeutic antibody cetuximab. The de novo mode of cetuximab resistance in SC cells could be overcome by crizotinib, a multi-RTK inhibitor that also targets MET and RON. We now show that crizotinib also overcomes acquired cetuximab resistance in CC-CR cells. Phospho-RTK array analysis showed increased phosphorylation of several RTKs, including MET and RON, in SC and CC-CR cells compared to cetuximab-sensitive CC counterparts. Furthermore, other multi-RTK inhibitors cabozantinib and BMS-777607 helped overcome cetuximab resistance, as measured by 3D colony growth and activation state of key signaling molecules. Conversely, addition of RTK ligands HGF and NRG1 induced cetuximab resistance in CC cells, which could be blocked by addition of crizotinib. We further determined the mechanism of the cooperative effect of cetuximab and crizotinib by FACS analysis and observed increased cell cycle arrest in G1 phase in cetuximab-resistant CRC 3D cultures. Finally, we show that crizotinib overcomes cetuximab resistance in vivo in SC nude mice xenografts. Thus, our work shows that multi-RTK inhibition strategy is a potent, broadly applicable strategy to overcome resistance to EGFR-targeted therapeutics in CRC and highlights the relevance of 3D cultures in these studies. Statement of implication: Using in vitro 3D CRC cultures and in vivo CRC xenografts, we show that parallel inhibition of multiple RTKs with small molecule inhibitors overcomes de novo and acquired resistance to EGFR-directed therapies in CRC.

19.
Cell Rep ; 27(3): 940-954.e6, 2019 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-30956133

RESUMO

Exomeres are a recently discovered type of extracellular nanoparticle with no known biological function. Herein, we describe a simple ultracentrifugation-based method for separation of exomeres from exosomes. Exomeres are enriched in Argonaute 1-3 and amyloid precursor protein. We identify distinct functions of exomeres mediated by two of their cargo, the ß-galactoside α2,6-sialyltransferase 1 (ST6Gal-I) that α2,6- sialylates N-glycans, and the EGFR ligand, amphiregulin (AREG). Functional ST6Gal-I in exomeres can be transferred to cells, resulting in hypersialylation of recipient cell-surface proteins including ß1-integrin. AREG-containing exomeres elicit prolonged EGFR and downstream signaling in recipient cells, modulate EGFR trafficking in normal intestinal organoids, and dramatically enhance the growth of colonic tumor organoids. This study provides a simplified method of exomere isolation and demonstrates that exomeres contain and can transfer functional cargo. These findings underscore the heterogeneity of nanoparticles and should accelerate advances in determining the composition and biological functions of exomeres.


Assuntos
Exossomos/metabolismo , Nanopartículas/metabolismo , Anfirregulina/genética , Anfirregulina/metabolismo , Animais , Linhagem Celular Tumoral , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Cães , Receptores ErbB/química , Receptores ErbB/metabolismo , Exossomos/química , Humanos , Lipídeos/análise , Lipídeos/química , Células Madin Darby de Rim Canino , Camundongos , Camundongos Knockout , Nanopartículas/química , Ácidos Nucleicos/análise , Tamanho da Partícula , Análise de Componente Principal , Proteoma/análise , Proteoma/metabolismo , Proteômica/métodos , Sialiltransferases/análise , Sialiltransferases/metabolismo , beta-D-Galactosídeo alfa 2-6-Sialiltransferase
20.
Mol Immunol ; 44(9): 2173-83, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17196657

RESUMO

Memory B cells and the antibodies they encode are important for protective immunity against infectious pathogens. Characterization of naïve and memory B cell antibody repertoires will elucidate the molecular basis for the generation of antibody diversity in human B cells and the optimization of antibody structures that bind microbial antigens. In this study we aimed to investigate the influence of antigenic selection on the antibody genes of the two CD27+ memory B cell subsets, comparing them with the naïve repertoire in CD27- cells. We analyzed and compared the Ig heavy chain gene transcripts in three recently defined circulating naïve and memory B cell subsets (CD19+IgD+CD27- [naïve], CD19+IgD+CD27+ [un-class-switched memory] or CD19+IgD- CD27+ [class-switched memory]) at the single cell level. We found similar biased patterns of variable, diversity and joining heavy chain gene usages in all three groups of cells. CD19+IgD+CD27+ memory B cells harbored as diverse an antibody gene repertoire as CD19+IgD-CD27+ memory B cells. Interestingly, CD19+IgD+CD27+ memory B cells possessed a lower frequency of somatic mutations, a higher incidence of exonuclease activity at the 3' end of D regions, and a lower frequency of N and P nucleotide additions at both VH-D and D-JH junctions of CDR3 regions compared to CD19+IgD-CD27+ memory B cells. These data suggest distinct functional mechanisms underlying selection of this unique subset of un-class-switched memory B cells.


Assuntos
Linfócitos B/enzimologia , Linfócitos B/imunologia , DNA Nucleotidilexotransferase/metabolismo , Exonucleases/metabolismo , Genes de Imunoglobulinas/genética , Memória Imunológica/imunologia , Adulto , Motivos de Aminoácidos , Animais , Antígenos CD19/imunologia , Linfócitos B/metabolismo , Doadores de Sangue , Separação Celular , Células Clonais , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/imunologia , Citometria de Fluxo , Regulação da Expressão Gênica , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Isotipos de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/imunologia , Camundongos , Mutação/genética , Nucleotídeos , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia
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