Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 13 de 13
Filtrar
Mais filtros

Base de dados
Tipo de documento
Intervalo de ano de publicação
1.
Mol Cell ; 70(6): 1025-1037.e5, 2018 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-29861160

RESUMO

When faced with proteotoxic stress, cells mount adaptive responses to eliminate aberrant proteins. Adaptive responses increase the expression of protein folding and degradation factors to enhance the cellular quality control machinery. However, it is unclear whether and how this augmented machinery acquires new activities during stress. Here, we uncover a regulatory cascade in budding yeast that consists of the hydrophilin protein Roq1/Yjl144w, the HtrA-type protease Ynm3/Nma111, and the ubiquitin ligase Ubr1. Various stresses stimulate ROQ1 transcription. The Roq1 protein is cleaved by Ynm3. Cleaved Roq1 interacts with Ubr1, transforming its substrate specificity. Altered substrate recognition by Ubr1 accelerates proteasomal degradation of misfolded as well as native proteins at the endoplasmic reticulum membrane and in the cytosol. We term this pathway stress-induced homeostatically regulated protein degradation (SHRED) and propose that it promotes physiological adaptation by reprogramming a key component of the quality control machinery.


Assuntos
Adaptação Fisiológica/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Dobramento de Proteína , Proteólise , Saccharomyces cerevisiae/enzimologia , Serina Endopeptidases/metabolismo , Estresse Fisiológico/fisiologia , Especificidade por Substrato , Ubiquitina/metabolismo
2.
PLoS Biol ; 16(1): e2004411, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29381698

RESUMO

The AP-5 adaptor protein complex is presumed to function in membrane traffic, but so far nothing is known about its pathway or its cargo. We have used CRISPR-Cas9 to knock out the AP-5 ζ subunit gene, AP5Z1, in HeLa cells, and then analysed the phenotype by subcellular fractionation profiling and quantitative mass spectrometry. The retromer complex had an altered steady-state distribution in the knockout cells, and several Golgi proteins, including GOLIM4 and GOLM1, were depleted from vesicle-enriched fractions. Immunolocalisation showed that loss of AP-5 led to impaired retrieval of the cation-independent mannose 6-phosphate receptor (CIMPR), GOLIM4, and GOLM1 from endosomes back to the Golgi region. Knocking down the retromer complex exacerbated this phenotype. Both the CIMPR and sortilin interacted with the AP-5-associated protein SPG15 in pull-down assays, and we propose that sortilin may act as a link between Golgi proteins and the AP-5/SPG11/SPG15 complex. Together, our findings suggest that AP-5 functions in a novel sorting step out of late endosomes, acting as a backup pathway for retromer. This provides a mechanistic explanation for why mutations in AP-5/SPG11/SPG15 cause cells to accumulate aberrant endolysosomes, and highlights the role of endosome/lysosome dysfunction in the pathology of hereditary spastic paraplegia and other neurodegenerative disorders.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/fisiologia , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Sistemas CRISPR-Cas , Endossomos/fisiologia , Complexo de Golgi/fisiologia , Células HeLa , Humanos , Lisossomos/genética , Lisossomos/fisiologia , Espectrometria de Massas , Proteínas de Membrana/metabolismo , Fenótipo , Transporte Proteico , Paraplegia Espástica Hereditária/genética , Proteínas de Transporte Vesicular/metabolismo
3.
Science ; 375(6585): eabi6983, 2022 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-35271311

RESUMO

Elucidating the wiring diagram of the human cell is a central goal of the postgenomic era. We combined genome engineering, confocal live-cell imaging, mass spectrometry, and data science to systematically map the localization and interactions of human proteins. Our approach provides a data-driven description of the molecular and spatial networks that organize the proteome. Unsupervised clustering of these networks delineates functional communities that facilitate biological discovery. We found that remarkably precise functional information can be derived from protein localization patterns, which often contain enough information to identify molecular interactions, and that RNA binding proteins form a specific subgroup defined by unique interaction and localization properties. Paired with a fully interactive website (opencell.czbiohub.org), our work constitutes a resource for the quantitative cartography of human cellular organization.


Assuntos
Mapeamento de Interação de Proteínas , Proteínas/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Sistemas CRISPR-Cas , Análise por Conglomerados , Conjuntos de Dados como Assunto , Corantes Fluorescentes , Células HEK293 , Humanos , Imunoprecipitação , Aprendizado de Máquina , Espectrometria de Massas , Microscopia Confocal , Proteínas de Ligação a RNA/metabolismo , Análise Espacial
4.
Science ; 367(6482): 1140-1146, 2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-32139545

RESUMO

Ribosome profiling has revealed pervasive but largely uncharacterized translation outside of canonical coding sequences (CDSs). In this work, we exploit a systematic CRISPR-based screening strategy to identify hundreds of noncanonical CDSs that are essential for cellular growth and whose disruption elicits specific, robust transcriptomic and phenotypic changes in human cells. Functional characterization of the encoded microproteins reveals distinct cellular localizations, specific protein binding partners, and hundreds of microproteins that are presented by the human leukocyte antigen system. We find multiple microproteins encoded in upstream open reading frames, which form stable complexes with the main, canonical protein encoded on the same messenger RNA, thereby revealing the use of functional bicistronic operons in mammals. Together, our results point to a family of functional human microproteins that play critical and diverse cellular roles.


Assuntos
Fases de Leitura Aberta , Peptídeos/genética , Biossíntese de Proteínas/genética , RNA Mensageiro , Sistemas CRISPR-Cas , Humanos , Óperon , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Transcriptoma
5.
Cell Rep ; 32(2): 107905, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32668257

RESUMO

Cross-presentation of antigens by dendritic cells (DCs) is critical for initiation of anti-tumor immune responses. Yet, key steps involved in trafficking of antigens taken up by DCs remain incompletely understood. Here, we screen 700 US Food and Drug Administration (FDA)-approved drugs and identify 37 enhancers of antigen import from endolysosomes into the cytosol. To reveal their mechanism of action, we generate proteomic organellar maps of control and drug-treated DCs (focusing on two compounds, prazosin and tamoxifen). By combining organellar mapping, quantitative proteomics, and microscopy, we conclude that import enhancers undergo lysosomal trapping leading to membrane permeation and antigen release. Enhancing antigen import facilitates cross-presentation of soluble and cell-associated antigens. Systemic administration of prazosin leads to reduced growth of MC38 tumors and to a synergistic effect with checkpoint immunotherapy in a melanoma model. Thus, inefficient antigen import into the cytosol limits antigen cross-presentation, restraining the potency of anti-tumor immune responses and efficacy of checkpoint blockers.


Assuntos
Antineoplásicos/farmacologia , Citosol/metabolismo , Endossomos/metabolismo , Imunidade , Neoplasias/imunologia , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Antígenos/metabolismo , Transporte Biológico/efeitos dos fármacos , Apresentação Cruzada/efeitos dos fármacos , Citosol/efeitos dos fármacos , Células Dendríticas/metabolismo , Degradação Associada com o Retículo Endoplasmático/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Imunidade/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neoplasias/tratamento farmacológico , Permeabilidade , Prazosina/farmacologia , Quinazolinas/farmacologia , Tamoxifeno/farmacologia , beta-Lactamases/metabolismo
6.
Curr Protoc Cell Biol ; 83(1): e81, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30489039

RESUMO

Eukaryotic cells are highly compartmentalized and protein subcellular localization critically influences protein function. Identification of the subcellular localizations of proteins and their translocation events upon perturbation has mostly been confined to targeted studies or laborious microscopy-based methods. Here we describe a systematic mass spectrometry-based method for spatial proteomics. The approach uses simple fractionation profiling and has two applications: Firstly it can be used to infer subcellular protein localization on a proteome-wide scale, resulting in a protein map of the cell. Secondly, the method permits identification of changes in protein localization, by comparing maps made under different conditions, as a tool for unbiased systems cell biology. © 2018 by John Wiley & Sons, Inc.


Assuntos
Organelas/metabolismo , Proteômica/métodos , Espaço Intracelular/metabolismo , Espectrometria de Massas/métodos , Organelas/ultraestrutura , Proteínas/análise , Proteínas/metabolismo , Frações Subcelulares/química
7.
Dis Model Mech ; 12(11)2019 11 11.
Artigo em Inglês | MEDLINE | ID: mdl-31628211

RESUMO

The unfolded protein response (UPR) involves extensive proteome remodeling in many cellular compartments. To date, a comprehensive analysis of the UPR has not been possible because of technological limitations. Here, we employ stable isotope labeling with amino acids in cell culture (SILAC)-based proteomics to quantify the response of over 6200 proteins to increasing concentrations of tunicamycin in HeLa cells. We further compare the effects of tunicamycin (5 µg/ml) to those of thapsigargin (1 µM) and DTT (2 mM), both activating the UPR through different mechanisms. This systematic quantification of the proteome-wide expression changes that follow proteostatic stress is a resource for the scientific community, enabling the discovery of novel players involved in the pathophysiology of the broad range of disorders linked to proteostasis. We identified increased expression in 38 proteins not previously linked to the UPR, of which 15 likely remediate ER stress, and the remainder may contribute to pathological outcomes. Unexpectedly, there are few strongly downregulated proteins, despite expression of the pro-apoptotic transcription factor CHOP, suggesting that IRE1-dependent mRNA decay (RIDD) has a limited contribution to ER stress-mediated cell death in our system.


Assuntos
Estresse do Retículo Endoplasmático/fisiologia , Espectrometria de Massas/métodos , Proteômica/métodos , Aminoácidos/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Células HeLa , Humanos , Marcação por Isótopo , Mapas de Interação de Proteínas , Tapsigargina/farmacologia , Tunicamicina/farmacologia , Resposta a Proteínas não Dobradas/efeitos dos fármacos
8.
Nat Commun ; 9(1): 3958, 2018 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-30262884

RESUMO

Adaptor protein 4 (AP-4) is an ancient membrane trafficking complex, whose function has largely remained elusive. In humans, AP-4 deficiency causes a severe neurological disorder of unknown aetiology. We apply unbiased proteomic methods, including 'Dynamic Organellar Maps', to find proteins whose subcellular localisation depends on AP-4. We identify three transmembrane cargo proteins, ATG9A, SERINC1 and SERINC3, and two AP-4 accessory proteins, RUSC1 and RUSC2. We demonstrate that AP-4 deficiency causes missorting of ATG9A in diverse cell types, including patient-derived cells, as well as dysregulation of autophagy. RUSC2 facilitates the transport of AP-4-derived, ATG9A-positive vesicles from the trans-Golgi network to the cell periphery. These vesicles cluster in close association with autophagosomes, suggesting they are the "ATG9A reservoir" required for autophagosome biogenesis. Our study uncovers ATG9A trafficking as a ubiquitous function of the AP-4 pathway. Furthermore, it provides a potential molecular pathomechanism of AP-4 deficiency, through dysregulated spatial control of autophagy.


Assuntos
Complexo 4 de Proteínas Adaptadoras/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Autofagia , Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Vesículas Transportadoras/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Células HeLa , Humanos , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Modelos Biológicos , Fagossomos/metabolismo , Fagossomos/ultraestrutura , Fenótipo , Ligação Proteica , Proteômica , Vesículas Transportadoras/ultraestrutura , Rede trans-Golgi/metabolismo , Rede trans-Golgi/ultraestrutura
9.
Elife ; 72018 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-29809151

RESUMO

The endoplasmic reticulum (ER) supports biosynthesis of proteins with diverse transmembrane domain (TMD) lengths and hydrophobicity. Features in transmembrane domains such as charged residues in ion channels are often functionally important, but could pose a challenge during cotranslational membrane insertion and folding. Our systematic proteomic approaches in both yeast and human cells revealed that the ER membrane protein complex (EMC) binds to and promotes the biogenesis of a range of multipass transmembrane proteins, with a particular enrichment for transporters. Proximity-specific ribosome profiling demonstrates that the EMC engages clients cotranslationally and immediately following clusters of TMDs enriched for charged residues. The EMC can remain associated after completion of translation, which both protects clients from premature degradation and allows recruitment of substrate-specific and general chaperones. Thus, the EMC broadly enables the biogenesis of multipass transmembrane proteins containing destabilizing features, thereby mitigating the trade-off between function and stability.


Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Biossíntese de Proteínas , Saccharomyces cerevisiae/metabolismo , Humanos , Chaperonas Moleculares/metabolismo , Complexos Multiproteicos/metabolismo , Transporte Proteico , Proteômica , Ribossomos/metabolismo
10.
Cell Rep ; 20(11): 2706-2718, 2017 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-28903049

RESUMO

We previously developed a mass spectrometry-based method, dynamic organellar maps, for the determination of protein subcellular localization and identification of translocation events in comparative experiments. The use of metabolic labeling for quantification (stable isotope labeling by amino acids in cell culture [SILAC]) renders the method best suited to cells grown in culture. Here, we have adapted the workflow to both label-free quantification (LFQ) and chemical labeling/multiplexing strategies (tandem mass tagging [TMT]). Both methods are highly effective for the generation of organellar maps and capture of protein translocations. Furthermore, application of label-free organellar mapping to acutely isolated mouse primary neurons provided subcellular localization and copy-number information for over 8,000 proteins, allowing a detailed analysis of organellar organization. Our study extends the scope of dynamic organellar maps to any cell type or tissue and also to high-throughput screening.


Assuntos
Neurônios/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Animais , Biomarcadores/metabolismo , Fracionamento Celular , Células Cultivadas , Células HeLa , Humanos , Marcação por Isótopo , Camundongos , Organelas/metabolismo , Transporte Proteico , Coloração e Rotulagem , Frações Subcelulares/metabolismo
11.
Nat Commun ; 8(1): 1469, 2017 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-29133944

RESUMO

The heart is a central human organ and its diseases are the leading cause of death worldwide, but an in-depth knowledge of the identity and quantity of its constituent proteins is still lacking. Here, we determine the healthy human heart proteome by measuring 16 anatomical regions and three major cardiac cell types by high-resolution mass spectrometry-based proteomics. From low microgram sample amounts, we quantify over 10,700 proteins in this high dynamic range tissue. We combine copy numbers per cell with protein organellar assignments to build a model of the heart proteome at the subcellular level. Analysis of cardiac fibroblasts identifies cellular receptors as potential cell surface markers. Application of our heart map to atrial fibrillation reveals individually distinct mitochondrial dysfunctions. The heart map is available at maxqb.biochem.mpg.de as a resource for future analyses of normal heart function and disease.


Assuntos
Coração/fisiologia , Miocárdio/metabolismo , Proteoma/metabolismo , Células Cultivadas , Vasos Coronários/citologia , Células Endoteliais/metabolismo , Átrios do Coração/citologia , Átrios do Coração/metabolismo , Ventrículos do Coração/citologia , Ventrículos do Coração/metabolismo , Humanos , Masculino , Miócitos Cardíacos/metabolismo , Miócitos de Músculo Liso/metabolismo , Proteômica/métodos
12.
Elife ; 52016 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-27278775

RESUMO

Subcellular localization critically influences protein function, and cells control protein localization to regulate biological processes. We have developed and applied Dynamic Organellar Maps, a proteomic method that allows global mapping of protein translocation events. We initially used maps statically to generate a database with localization and absolute copy number information for over 8700 proteins from HeLa cells, approaching comprehensive coverage. All major organelles were resolved, with exceptional prediction accuracy (estimated at >92%). Combining spatial and abundance information yielded an unprecedented quantitative view of HeLa cell anatomy and organellar composition, at the protein level. We subsequently demonstrated the dynamic capabilities of the approach by capturing translocation events following EGF stimulation, which we integrated into a quantitative model. Dynamic Organellar Maps enable the proteome-wide analysis of physiological protein movements, without requiring any reagents specific to the investigated process, and will thus be widely applicable in cell biology.


Assuntos
Técnicas Citológicas/métodos , Células Epiteliais/química , Proteínas/análise , Proteômica/métodos , Células HeLa , Humanos , Análise Espaço-Temporal
13.
Mol Cell Biol ; 35(12): 2186-202, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25870107

RESUMO

The unfolded protein response (UPR) remediates endoplasmic reticulum (ER) stress. IRE1, a component of the UPR, senses misfolded protein and cleaves XBP1 mRNA, which is ligated to code for the prosurvival transcription factor. IRE1 also cleaves other mRNAs preceding their degradation, termed regulated IRE1-dependent mRNA decay (RIDD). It has been reported that RIDD may be involved in cell viability under stress and therefore may contribute to cancer cell viability. To investigate RIDD targets that may have functional relevance in cell survival, we identified conserved RIDD targets containing stringent IRE1 RNase target sequences. Using a systematic bioinformatics approach with quantitative-PCR (qPCR) validation, we show that only BLOC1S1 is consistently a RIDD target in all systems tested. Using cancer cell lines, we show that BLOC1S1 is specifically cleaved by IRE1 at guanine 444, but only under conditions of IRE1 hyperactivation. BLOC1S1 cleavage is temporally separate from XBP1 splicing, occurring after depletion of unspliced XBP1. Expression of an uncleavable BLOC1S1 mutant or inhibition of RIDD using an IRE1 RNase inhibitor did not affect cellular recovery from acute ER stress. These data demonstrate that although hyperactivated IRE1 specifically cleaves BLOC1S1, this cleavage event and RIDD as a whole are dispensable for cell viability under acute stress.


Assuntos
Proteínas de Ligação a DNA/genética , Estresse do Retículo Endoplasmático , Endorribonucleases/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/química , RNA Mensageiro/genética , Fatores de Transcrição/genética , Animais , Sequência de Bases , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Humanos , Dados de Sequência Molecular , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Splicing de RNA , Estabilidade de RNA , RNA Mensageiro/metabolismo , Fatores de Transcrição de Fator Regulador X , Proteína 1 de Ligação a X-Box
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA