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1.
Biochem J ; 2022 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-35015072

RESUMO

The ability of the cellular immune system to discriminate self from foreign antigens depends on the appropriate calibration of the T-cell receptor (TCR) signalling threshold. The lymphocyte homeostatic cytokine interleukin 7 (IL-7) is known to affect TCR thresholding, but the molecular mechanism is not fully elucidated. A better understanding of this process is highly relevant in the context of autoimmune disease therapy and cancer immunotherapy. We sought to characterise the early signalling events attributable to IL-7 priming; in particular, the altered phosphorylation of signal transduction proteins and their molecular localisation to the TCR. By integrating high-resolution proximity- phospho-proteomic and imaging approaches using primary T cells, rather than engineered cell lines or an in vitro expanded T cell population, we uncovered transduction events previously not linked to IL-7. We show that IL-7 leads to dephosphorylation of cytohesin interacting protein (CYTIP) at a hitherto undescribed phosphorylation site (pThr280) and alters the co-localisation of cytohesin 1 with the TCR and LFA-1 integrin. These results show that IL-7, acting via CYTIP and cytohesin-1, may impact TCR activation thresholds by enhancing the co-clustering of TCR and LFA-1 integrin.

2.
Drug Discov Today Technol ; 39: 57-67, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34906326

RESUMO

Spatial proteomics has provided important insights into the relationship between protein function and subcellular location. Localization of Organelle Proteins by Isotope Tagging (LOPIT) and its variants are proteome-wide techniques, not matched in scale by microscopy-based or proximity tagging-based techniques, allowing holistic mapping of protein subcellular location and re-localization events downstream of cellular perturbations. LOPIT can be a powerful and versatile tool in drug discovery for unlocking important information on disease pathophysiology, drug mechanism of action, and off-target toxicity screenings. Here, we discuss technical concepts of LOPIT with its potential applications in drug discovery and development research.


Assuntos
Proteoma , Proteômica , Descoberta de Drogas , Isótopos , Organelas
3.
Mol Cell Proteomics ; : 100186, 2021 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-34922010

RESUMO

The internal environment of cells is molecularly crowded, which requires spatial organization via subcellular compartmentalization. These compartments harbor specific conditions for molecules to perform their biological functions, such as coordination of the cell cycle, cell survival, and growth. This compartmentalization is also not static, with molecules trafficking between these subcellular neighborhoods to carry out their functions. For example, some biomolecules are multifunctional, requiring an environment with differing conditions or interacting partners, and others traffic to export such molecules. Aberrant localization of proteins or RNA species have been linked to many pathological conditions, such as neurological, cancer and pulmonary diseases. Differential expression studies in transcriptomics and proteomics are relatively common, but the majority have overlooked the importance of subcellular information. Additionally, subcellular transcriptomics and proteomics data do not always co-locate due to the biochemical processes that occur during and after translation, highlighting the complementary nature of these fields. In this review, we discuss and directly compare the current methods in spatial proteomics and transcriptomics, which include sequencing- and imaging-based strategies, to give the reader an overview of the current tools available. We also discuss current limitations of these strategies, as well as future developments in the field of spatial -omics.

4.
Nat Commun ; 12(1): 5773, 2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34599159

RESUMO

Protein localisation and translocation between intracellular compartments underlie almost all physiological processes. The hyperLOPIT proteomics platform combines mass spectrometry with state-of-the-art machine learning to map the subcellular location of thousands of proteins simultaneously. We combine global proteome analysis with hyperLOPIT in a fully Bayesian framework to elucidate spatiotemporal proteomic changes during a lipopolysaccharide (LPS)-induced inflammatory response. We report a highly dynamic proteome in terms of both protein abundance and subcellular localisation, with alterations in the interferon response, endo-lysosomal system, plasma membrane reorganisation and cell migration. Proteins not previously associated with an LPS response were found to relocalise upon stimulation, the functional consequences of which are still unclear. By quantifying proteome-wide uncertainty through Bayesian modelling, a necessary role for protein relocalisation and the importance of taking a holistic overview of the LPS-driven immune response has been revealed. The data are showcased as an interactive application freely available for the scientific community.


Assuntos
Inflamação/metabolismo , Leucemia/metabolismo , Leucemia/patologia , Lipopolissacarídeos/farmacologia , Proteômica , Algoritmos , Anti-Infecciosos/metabolismo , Anti-Inflamatórios/metabolismo , Apresentação do Antígeno , Autofagossomos/metabolismo , Teorema de Bayes , Pontos de Checagem do Ciclo Celular , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Forma Celular , Humanos , Imunidade , Inflamação/patologia , Leucemia/imunologia , Ativação Linfocitária/imunologia , Lisossomos/metabolismo , Proteínas de Neoplasias/metabolismo , Transporte Proteico , Proteoma/metabolismo , Transdução de Sinais , Linfócitos T/imunologia , Células THP-1 , Fatores de Tempo , Vesículas Transportadoras/metabolismo , Regulação para Cima , Proteínas rho de Ligação ao GTP/metabolismo
5.
Artigo em Inglês | MEDLINE | ID: mdl-34549195

RESUMO

The eukaryotic cell is compartmentalized into subcellular niches, including membrane-bound and membrane-less organelles. Proteins localize to these niches to fulfil their function, enabling discreet biological processes to occur in synchrony. Dynamic movement of proteins between niches is essential for cellular processes such as signalling, growth, proliferation, motility and programmed cell death, and mutations causing aberrant protein localization are associated with a wide range of diseases. Determining the location of proteins in different cell states and cell types and how proteins relocalize following perturbation is important for understanding their functions, related cellular processes and pathologies associated with their mislocalization. In this Primer, we cover the major spatial proteomics methods for determining the location, distribution and abundance of proteins within subcellular structures. These technologies include fluorescent imaging, protein proximity labelling, organelle purification and cell-wide biochemical fractionation. We describe their workflows, data outputs and applications in exploring different cell biological scenarios, and discuss their main limitations. Finally, we describe emerging technologies and identify areas that require technological innovation to allow better characterization of the spatial proteome.

6.
Mol Cell ; 81(13): 2851-2867.e7, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34118193

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19). SARS-CoV-2 relies on cellular RNA-binding proteins (RBPs) to replicate and spread, although which RBPs control its life cycle remains largely unknown. Here, we employ a multi-omic approach to identify systematically and comprehensively the cellular and viral RBPs that are involved in SARS-CoV-2 infection. We reveal that SARS-CoV-2 infection profoundly remodels the cellular RNA-bound proteome, which includes wide-ranging effects on RNA metabolic pathways, non-canonical RBPs, and antiviral factors. Moreover, we apply a new method to identify the proteins that directly interact with viral RNA, uncovering dozens of cellular RBPs and six viral proteins. Among them are several components of the tRNA ligase complex, which we show regulate SARS-CoV-2 infection. Furthermore, we discover that available drugs targeting host RBPs that interact with SARS-CoV-2 RNA inhibit infection. Collectively, our results uncover a new universe of host-virus interactions with potential for new antiviral therapies against COVID-19.


Assuntos
COVID-19/metabolismo , Proteoma/metabolismo , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , SARS-CoV-2/fisiologia , Proteínas Virais/metabolismo , Replicação Viral/fisiologia , Células A549 , COVID-19/genética , Humanos , Proteoma/genética , RNA Viral/genética , Proteínas de Ligação a RNA/genética , Proteínas Virais/genética
7.
Commun Biol ; 4(1): 810, 2021 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-34188175

RESUMO

The thermal stability of proteins can be altered when they interact with small molecules, other biomolecules or are subject to post-translation modifications. Thus monitoring the thermal stability of proteins under various cellular perturbations can provide insights into protein function, as well as potentially determine drug targets and off-targets. Thermal proteome profiling is a highly multiplexed mass-spectrommetry method for monitoring the melting behaviour of thousands of proteins in a single experiment. In essence, thermal proteome profiling assumes that proteins denature upon heating and hence become insoluble. Thus, by tracking the relative solubility of proteins at sequentially increasing temperatures, one can report on the thermal stability of a protein. Standard thermodynamics predicts a sigmoidal relationship between temperature and relative solubility and this is the basis of current robust statistical procedures. However, current methods do not model deviations from this behaviour and they do not quantify uncertainty in the melting profiles. To overcome these challenges, we propose the application of Bayesian functional data analysis tools which allow complex temperature-solubility behaviours. Our methods have improved sensitivity over the state-of-the art, identify new drug-protein associations and have less restrictive assumptions than current approaches. Our methods allows for comprehensive analysis of proteins that deviate from the predicted sigmoid behaviour and we uncover potentially biphasic phenomena with a series of published datasets.


Assuntos
Teorema de Bayes , Estabilidade Proteica , Proteoma , Solubilidade , Temperatura , Termodinâmica
8.
Nat Biotechnol ; 39(7): 846-854, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33767396

RESUMO

Accurate quantification of the proteome remains challenging for large sample series and longitudinal experiments. We report a data-independent acquisition method, Scanning SWATH, that accelerates mass spectrometric (MS) duty cycles, yielding quantitative proteomes in combination with short gradients and high-flow (800 µl min-1) chromatography. Exploiting a continuous movement of the precursor isolation window to assign precursor masses to tandem mass spectrometry (MS/MS) fragment traces, Scanning SWATH increases precursor identifications by ~70% compared to conventional data-independent acquisition (DIA) methods on 0.5-5-min chromatographic gradients. We demonstrate the application of ultra-fast proteomics in drug mode-of-action screening and plasma proteomics. Scanning SWATH proteomes capture the mode of action of fungistatic azoles and statins. Moreover, we confirm 43 and identify 11 new plasma proteome biomarkers of COVID-19 severity, advancing patient classification and biomarker discovery. Thus, our results demonstrate a substantial acceleration and increased depth in fast proteomic experiments that facilitate proteomic drug screens and clinical studies.


Assuntos
Proteômica/métodos , Espectrometria de Massas em Tandem , Arabidopsis/metabolismo , Biomarcadores/metabolismo , COVID-19/sangue , COVID-19/diagnóstico , Linhagem Celular , Humanos , Peptídeos/análise , Proteoma/análise , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Índice de Gravidade de Doença
9.
NPJ Genom Med ; 6(1): 4, 2021 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-33495453

RESUMO

Uncharacterized and unannotated open-reading frames, which we refer to as novel open reading frames (nORFs), may sometimes encode peptides that remain unexplored for novel therapeutic opportunities. To our knowledge, no systematic identification and characterization of transcripts encoding nORFs or their translation products in cancer, or in any other physiological process has been performed. We use our curated nORFs database (nORFs.org), together with RNA-Seq data from The Cancer Genome Atlas (TCGA) and Genotype-Expression (GTEx) consortiums, to identify transcripts containing nORFs that are expressed frequently in cancer or matched normal tissue across 22 cancer types. We show nORFs are subject to extensive dysregulation at the transcript level in cancer tissue and that a small subset of nORFs are associated with overall patient survival, suggesting that nORFs may have prognostic value. We also show that nORF products can form protein-like structures with post-translational modifications. Finally, we perform in silico screening for inhibitors against nORF-encoded proteins that are disrupted in stomach and esophageal cancer, showing that they can potentially be targeted by inhibitors. We hope this work will guide and motivate future studies that perform in-depth characterization of nORF functions in cancer and other diseases.

10.
PLoS Comput Biol ; 16(11): e1008288, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33166281

RESUMO

The cell is compartmentalised into complex micro-environments allowing an array of specialised biological processes to be carried out in synchrony. Determining a protein's sub-cellular localisation to one or more of these compartments can therefore be a first step in determining its function. High-throughput and high-accuracy mass spectrometry-based sub-cellular proteomic methods can now shed light on the localisation of thousands of proteins at once. Machine learning algorithms are then typically employed to make protein-organelle assignments. However, these algorithms are limited by insufficient and incomplete annotation. We propose a semi-supervised Bayesian approach to novelty detection, allowing the discovery of additional, previously unannotated sub-cellular niches. Inference in our model is performed in a Bayesian framework, allowing us to quantify uncertainty in the allocation of proteins to new sub-cellular niches, as well as in the number of newly discovered compartments. We apply our approach across 10 mass spectrometry based spatial proteomic datasets, representing a diverse range of experimental protocols. Application of our approach to hyperLOPIT datasets validates its utility by recovering enrichment with chromatin-associated proteins without annotation and uncovers sub-nuclear compartmentalisation which was not identified in the original analysis. Moreover, using sub-cellular proteomics data from Saccharomyces cerevisiae, we uncover a novel group of proteins trafficking from the ER to the early Golgi apparatus. Overall, we demonstrate the potential for novelty detection to yield biologically relevant niches that are missed by current approaches.


Assuntos
Teorema de Bayes , Proteínas de Saccharomyces cerevisiae/metabolismo , Frações Subcelulares/metabolismo , Algoritmos , Animais , Conjuntos de Dados como Assunto , Humanos , Aprendizado de Máquina , Espectrometria de Massas , Camundongos , Proteômica
11.
Nat Commun ; 11(1): 5987, 2020 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-33239640

RESUMO

Intracellular traffic between compartments of the secretory and endocytic pathways is mediated by vesicle-based carriers. The proteomes of carriers destined for many organelles are ill-defined because the vesicular intermediates are transient, low-abundance and difficult to purify. Here, we combine vesicle relocalisation with organelle proteomics and Bayesian analysis to define the content of different endosome-derived vesicles destined for the trans-Golgi network (TGN). The golgin coiled-coil proteins golgin-97 and GCC88, shown previously to capture endosome-derived vesicles at the TGN, were individually relocalised to mitochondria and the content of the subsequently re-routed vesicles was determined by organelle proteomics. Our findings reveal 45 integral and 51 peripheral membrane proteins re-routed by golgin-97, evidence for a distinct class of vesicles shared by golgin-97 and GCC88, and various cargoes specific to individual golgins. These results illustrate a general strategy for analysing intracellular sub-proteomes by combining acute cellular re-wiring with high-resolution spatial proteomics.


Assuntos
Autoantígenos/metabolismo , Proteínas da Matriz do Complexo de Golgi/metabolismo , Proteínas de Membrana/metabolismo , Rede trans-Golgi/metabolismo , Autoantígenos/genética , Endossomos/metabolismo , Técnicas de Silenciamento de Genes , Proteínas da Matriz do Complexo de Golgi/genética , Células HEK293 , Células HeLa , Humanos , Mitocôndrias/metabolismo , Proteômica/métodos , Análise Espacial
12.
Cell Host Microbe ; 28(5): 752-766.e9, 2020 11 11.
Artigo em Inglês | MEDLINE | ID: mdl-33053376

RESUMO

Apicomplexan parasites cause major human disease and food insecurity. They owe their considerable success to highly specialized cell compartments and structures. These adaptations drive their recognition, nondestructive penetration, and elaborate reengineering of the host's cells to promote their growth, dissemination, and the countering of host defenses. The evolution of unique apicomplexan cellular compartments is concomitant with vast proteomic novelty. Consequently, half of apicomplexan proteins are unique and uncharacterized. Here, we determine the steady-state subcellular location of thousands of proteins simultaneously within the globally prevalent apicomplexan parasite Toxoplasma gondii. This provides unprecedented comprehensive molecular definition of these unicellular eukaryotes and their specialized compartments, and these data reveal the spatial organizations of protein expression and function, adaptation to hosts, and the underlying evolutionary trajectories of these pathogens.


Assuntos
Proteoma , Proteínas de Protozoários/metabolismo , Toxoplasma/metabolismo , Apicomplexa , Evolução Biológica , Epitopos , Interações Hospedeiro-Patógeno , Humanos , Proteômica , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Toxoplasma/genética
13.
PLoS One ; 15(9): e0239641, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32941543

RESUMO

[This corrects the article DOI: 10.1371/journal.pone.0233048.].

14.
Cell Syst ; 11(1): 11-24.e4, 2020 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-32619549

RESUMO

The COVID-19 pandemic is an unprecedented global challenge, and point-of-care diagnostic classifiers are urgently required. Here, we present a platform for ultra-high-throughput serum and plasma proteomics that builds on ISO13485 standardization to facilitate simple implementation in regulated clinical laboratories. Our low-cost workflow handles up to 180 samples per day, enables high precision quantification, and reduces batch effects for large-scale and longitudinal studies. We use our platform on samples collected from a cohort of early hospitalized cases of the SARS-CoV-2 pandemic and identify 27 potential biomarkers that are differentially expressed depending on the WHO severity grade of COVID-19. They include complement factors, the coagulation system, inflammation modulators, and pro-inflammatory factors upstream and downstream of interleukin 6. All protocols and software for implementing our approach are freely available. In total, this work supports the development of routine proteomic assays to aid clinical decision making and generate hypotheses about potential COVID-19 therapeutic targets.


Assuntos
Proteínas Sanguíneas/metabolismo , Infecções por Coronavirus/sangue , Pneumonia Viral/sangue , Proteômica/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Betacoronavirus/isolamento & purificação , Biomarcadores/sangue , Proteínas Sanguíneas/análise , COVID-19 , Infecções por Coronavirus/classificação , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pandemias/classificação , Pneumonia Viral/classificação , Pneumonia Viral/patologia , Pneumonia Viral/virologia , SARS-CoV-2 , Adulto Jovem
15.
Nat Protoc ; 15(8): 2568-2588, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32651564

RESUMO

RNA-protein interactions play a pivotal role in cell homeostasis and disease, but current approaches to study them require a considerable amount of starting material, favor the recovery of only a subset of RNA species or are complex and time-consuming. We recently developed orthogonal organic phase separation (OOPS): a quick, efficient and reproducible method to purify cross-linked RNA-protein adducts in an unbiased way. OOPS avoids molecular tagging or the capture of polyadenylated RNA. Instead, it is based on sampling the interface of a standard TRIzol extraction to enrich RNA-binding proteins (RBPs) and their cognate bound RNA. OOPS specificity is achieved by digesting the enriched interfaces with RNases or proteases to release the RBPs or protein-bound RNA, respectively. Here we present a step-by-step protocol to purify protein-RNA adducts, free protein and free RNA from the same sample. We further describe how OOPS can be applied in human cell lines, Arabidopsis thaliana, Schizosaccharomyces pombe and Escherichia coli and how it can be used to study RBP dynamics.


Assuntos
Fracionamento Químico/métodos , Proteoma/isolamento & purificação , Proteínas de Ligação a RNA/isolamento & purificação , RNA/isolamento & purificação , Transcriptoma , Linhagem Celular , Humanos , Proteoma/metabolismo , RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fluxo de Trabalho
16.
Proteomics ; 20(23): e1900392, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32558233

RESUMO

The spatial subcellular proteome is a dynamic environment; one that can be perturbed by molecular cues and regulated by post-translational modifications. Compartmentalization of this environment and management of these biomolecular dynamics allows for an array of ancillary protein functions. Profiling spatial proteomics has proved to be a powerful technique in identifying the primary subcellular localization of proteins. The approach has also been refashioned to study multi-localization and localization dynamics. Here, the analytical approaches that have been applied to spatial proteomics thus far are critiqued, and challenges particularly associated with multi-localization and dynamic relocalization is identified. To meet some of the current limitations in analytical processing, it is suggested that Bayesian modeling has clear benefits over the methods applied to date and should be favored whenever possible. Careful consideration of the limitations and challenges, and development of robust statistical frameworks, will ensure that profiling spatial proteomics remains a valuable technique as its utility is expanded.


Assuntos
Proteoma , Proteômica , Teorema de Bayes , Processamento de Proteína Pós-Traducional , Proteoma/metabolismo
17.
PLoS One ; 15(5): e0233048, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32453791

RESUMO

Panagrolaimus sp. DAW1, a nematode cultured from the Antarctic, has the extraordinary physiological ability to survive total intracellular freezing throughout all of its compartments. While a few other organisms, all nematodes, have subsequently also been found to survive freezing in this manner, P. sp. DAW1 has so far shown the highest survival rates. In addition, P. sp. DAW1 is also, depending on the rate or extent of freezing, able to undergo cryoprotective dehydration. In this study, the proteome of P. sp DAW1 is explored, highlighting a number of differentially expressed proteins and pathways that occur when the nematodes undergo intracellular freezing. Among the strongest signals after being frozen is an upregulation of proteases and the downregulation of cytoskeletal and antioxidant activity, the latter possibly accumulated before freezing much in the way the sugar trehalose has been shown to be stored during acclimation.


Assuntos
Aclimatação/fisiologia , Redes Reguladoras de Genes , Proteômica/métodos , Rabditídios/fisiologia , Animais , Antioxidantes/metabolismo , Temperatura Baixa , Regulação da Expressão Gênica , Proteínas de Helminto/metabolismo , Peptídeo Hidrolases/metabolismo , Mapas de Interação de Proteínas
18.
Mol Cell Proteomics ; 19(8): 1281-1302, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32434922

RESUMO

Following central nervous system (CNS) demyelination, adult oligodendrocyte progenitor cells (OPCs) can differentiate into new myelin-forming oligodendrocytes in a regenerative process called remyelination. Although remyelination is very efficient in young adults, its efficiency declines progressively with ageing. Here we performed proteomic analysis of OPCs freshly isolated from the brains of neonate, young and aged female rats. Approximately 50% of the proteins are expressed at different levels in OPCs from neonates compared with their adult counterparts. The amount of myelin-associated proteins, and proteins associated with oxidative phosphorylation, inflammatory responses and actin cytoskeletal organization increased with age, whereas cholesterol-biosynthesis, transcription factors and cell cycle proteins decreased. Our experiments provide the first ageing OPC proteome, revealing the distinct features of OPCs at different ages. These studies provide new insights into why remyelination efficiency declines with ageing and potential roles for aged OPCs in other neurodegenerative diseases.


Assuntos
Envelhecimento/metabolismo , Células Precursoras de Oligodendrócitos/metabolismo , Proteoma/metabolismo , Animais , Animais Recém-Nascidos , Biomarcadores/metabolismo , Separação Celular , Colesterol/metabolismo , Bainha de Mielina/metabolismo , Doenças Neurodegenerativas/patologia , Células Precursoras de Oligodendrócitos/citologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Dobramento de Proteína , Proteômica , Proteostase , Ratos Sprague-Dawley , Reprodutibilidade dos Testes
19.
J Cell Biol ; 219(4)2020 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-32328641

RESUMO

Filopodia are finger-like actin-rich protrusions that extend from the cell surface and are important for cell-cell communication and pathogen internalization. The small size and transient nature of filopodia combined with shared usage of actin regulators within cells confounds attempts to identify filopodial proteins. Here, we used phage display phenotypic screening to isolate antibodies that alter the actin morphology of filopodia-like structures (FLS) in vitro. We found that all of the antibodies that cause shorter FLS interact with SNX9, an actin regulator that binds phosphoinositides during endocytosis and at invadopodia. In cells, we discover SNX9 at specialized filopodia in Xenopus development and that SNX9 is an endogenous component of filopodia that are hijacked by Chlamydia entry. We show the use of antibody technology to identify proteins used in filopodia-like structures, and a role for SNX9 in filopodia.


Assuntos
Pseudópodes/metabolismo , Nexinas de Classificação/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Feminino , Células HeLa , Humanos , Masculino , Nexinas de Classificação/genética , Proteínas de Xenopus/genética , Xenopus laevis
20.
Int J Oncol ; 57(1): 87-99, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32319587

RESUMO

The immune checkpoint protein B7­H4 plays an important role in the positive as well as the negative regulation of immune T­cell responses. When expressed on cancer cells, B7­H4 inhibits T­cell activity, and numerous types of cancer cells use upregulation of B7­H4 as a survival strategy. Thus, B7­H4 is a potential target for anticancer drug therapy. Unfortunately, the cell biology of this molecule has yet to be fully elucidated. Even basic properties, such as the nature of B7­H4 interactors, are controversial. In particular, the cis­interactors of B7­H4 on cancer cell plasma membranes have not been investigated to date. The present study used a proteomic proximity­labelling assay to investigate the molecular neighbours of B7­H4 on the surface of the human breast cancer cells SK­BR­3. By comparison to a comprehensive proteome analysis of SK­BR­3 cells, the proximity method detected a relatively small number of low abundance plasma membrane proteins highly enriched for proteins known to modulate cell adhesion and immune recognition. It may be inferred that these molecules contribute to the immunosuppressive behaviour that is characteristic of B7­H4 on cancer cells.


Assuntos
Neoplasias da Mama/imunologia , Mapeamento de Interação de Proteínas , Inibidor 1 da Ativação de Células T com Domínio V-Set/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Adesão Celular/efeitos dos fármacos , Adesão Celular/imunologia , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/imunologia , Membrana Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/imunologia , Feminino , Regulação Neoplásica da Expressão Gênica/imunologia , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Ativação Linfocitária/efeitos dos fármacos , Mapas de Interação de Proteínas/efeitos dos fármacos , Mapas de Interação de Proteínas/imunologia , Proteômica/métodos , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/imunologia , Inibidor 1 da Ativação de Células T com Domínio V-Set/antagonistas & inibidores , Inibidor 1 da Ativação de Células T com Domínio V-Set/imunologia
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