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1.
Immunity ; 57(9): 2216-2231.e11, 2024 Sep 10.
Artículo en Inglés | MEDLINE | ID: mdl-39151426

RESUMEN

Microglia are the resident macrophages of the central nervous system (CNS). Their phagocytic activity is central during brain development and homeostasis-and in a plethora of brain pathologies. However, little is known about the composition, dynamics, and function of human microglial phagosomes under homeostatic and pathological conditions. Here, we developed a method for rapid isolation of pure and intact phagosomes from human pluripotent stem cell-derived microglia under various in vitro conditions, and from human brain biopsies, for unbiased multiomic analysis. Phagosome profiling revealed that microglial phagosomes were equipped to sense minute changes in their environment and were highly dynamic. We detected proteins involved in synapse homeostasis, or implicated in brain pathologies, and identified the phagosome as the site where quinolinic acid was stored and metabolized for de novo nicotinamide adenine dinucleotide (NAD+) generation in the cytoplasm. Our findings highlight the central role of phagosomes in microglial functioning in the healthy and diseased brain.


Asunto(s)
Microglía , Fagocitosis , Fagosomas , Humanos , Microglía/metabolismo , Fagosomas/metabolismo , Encéfalo/metabolismo , Encéfalo/citología , Células Cultivadas , Células Madre Pluripotentes/metabolismo , Proteómica/métodos
2.
Nat Commun ; 15(1): 584, 2024 Jan 17.
Artículo en Inglés | MEDLINE | ID: mdl-38233389

RESUMEN

Unbiased phenotypic screens in patient-relevant disease models offer the potential to detect therapeutic targets for rare diseases. In this study, we developed a high-throughput screening assay to identify molecules that correct aberrant protein trafficking in adapter protein complex 4 (AP-4) deficiency, a rare but prototypical form of childhood-onset hereditary spastic paraplegia characterized by mislocalization of the autophagy protein ATG9A. Using high-content microscopy and an automated image analysis pipeline, we screened a diversity library of 28,864 small molecules and identified a lead compound, BCH-HSP-C01, that restored ATG9A pathology in multiple disease models, including patient-derived fibroblasts and induced pluripotent stem cell-derived neurons. We used multiparametric orthogonal strategies and integrated transcriptomic and proteomic approaches to delineate potential mechanisms of action of BCH-HSP-C01. Our results define molecular regulators of intracellular ATG9A trafficking and characterize a lead compound for the treatment of AP-4 deficiency, providing important proof-of-concept data for future studies.


Asunto(s)
Paraplejía Espástica Hereditaria , Humanos , Paraplejía Espástica Hereditaria/tratamiento farmacológico , Paraplejía Espástica Hereditaria/genética , Paraplejía Espástica Hereditaria/metabolismo , Proteómica , Neuronas/metabolismo , Transporte de Proteínas , Proteínas/metabolismo , Mutación
3.
Nat Commun ; 14(1): 5252, 2023 08 29.
Artículo en Inglés | MEDLINE | ID: mdl-37644046

RESUMEN

The Dynamic Organellar Maps (DOMs) approach combines cell fractionation and shotgun-proteomics for global profiling analysis of protein subcellular localization. Here, we enhance the performance of DOMs through data-independent acquisition (DIA) mass spectrometry. DIA-DOMs achieve twice the depth of our previous workflow in the same mass spectrometry runtime, and substantially improve profiling precision and reproducibility. We leverage this gain to establish flexible map formats scaling from high-throughput analyses to extra-deep coverage. Furthermore, we introduce DOM-ABC, a powerful and user-friendly open-source software tool for analyzing profiling data. We apply DIA-DOMs to capture subcellular localization changes in response to starvation and disruption of lysosomal pH in HeLa cells, which identifies a subset of Golgi proteins that cycle through endosomes. An imaging time-course reveals different cycling patterns and confirms the quantitative predictive power of our translocation analysis. DIA-DOMs offer a superior workflow for label-free spatial proteomics as a systematic phenotype discovery tool.


Asunto(s)
Endosomas , Humanos , Células HeLa , Reproducibilidad de los Resultados , Fraccionamiento Celular , Espectrometría de Masas
4.
Science ; 380(6651): 1258-1265, 2023 06 23.
Artículo en Inglés | MEDLINE | ID: mdl-37347855

RESUMEN

During initiation of antiviral and antitumor T cell-mediated immune responses, dendritic cells (DCs) cross-present exogenous antigens on major histocompatibility complex (MHC) class I molecules. Cross-presentation relies on the unusual "leakiness" of endocytic compartments in DCs, whereby internalized proteins escape into the cytosol for proteasome-mediated generation of MHC I-binding peptides. Given that type 1 conventional DCs excel at cross-presentation, we searched for cell type-specific effectors of endocytic escape. We devised an assay suitable for genetic screening and identified a pore-forming protein, perforin-2 (Mpeg1), as a dedicated effector exclusive to cross-presenting cells. Perforin-2 was recruited to antigen-containing compartments, where it underwent maturation, releasing its pore-forming domain. Mpeg1-/- mice failed to efficiently prime CD8+ T cells to cell-associated antigens, revealing an important role for perforin-2 in cytosolic entry of antigens during cross-presentation.


Asunto(s)
Presentación de Antígeno , Linfocitos T CD8-positivos , Endocitosis , Proteínas Citotóxicas Formadoras de Poros , Animales , Ratones , Antígenos/inmunología , Linfocitos T CD8-positivos/inmunología , Reactividad Cruzada/genética , Reactividad Cruzada/inmunología , Células Dendríticas/inmunología , Endocitosis/genética , Endocitosis/inmunología , Pruebas Genéticas , Antígenos de Histocompatibilidad Clase I , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Proteolisis
5.
Science ; 376(6599): eabf9088, 2022 06 17.
Artículo en Inglés | MEDLINE | ID: mdl-35709258

RESUMEN

The centrosome provides an intracellular anchor for the cytoskeleton, regulating cell division, cell migration, and cilia formation. We used spatial proteomics to elucidate protein interaction networks at the centrosome of human induced pluripotent stem cell-derived neural stem cells (NSCs) and neurons. Centrosome-associated proteins were largely cell type-specific, with protein hubs involved in RNA dynamics. Analysis of neurodevelopmental disease cohorts identified a significant overrepresentation of NSC centrosome proteins with variants in patients with periventricular heterotopia (PH). Expressing the PH-associated mutant pre-mRNA-processing factor 6 (PRPF6) reproduced the periventricular misplacement in the developing mouse brain, highlighting missplicing of transcripts of a microtubule-associated kinase with centrosomal location as essential for the phenotype. Collectively, cell type-specific centrosome interactomes explain how genetic variants in ubiquitous proteins may convey brain-specific phenotypes.


Asunto(s)
Centrosoma , Células-Madre Neurales , Neurogénesis , Neuronas , Heterotopia Nodular Periventricular , Mapas de Interacción de Proteínas , Empalme Alternativo , Animales , Encéfalo/anomalías , Centrosoma/metabolismo , Humanos , Células Madre Pluripotentes Inducidas , Ratones , Microtúbulos/metabolismo , Neuronas/metabolismo , Heterotopia Nodular Periventricular/metabolismo , Proteoma/metabolismo , Factores de Empalme de ARN/metabolismo , Factores de Transcripción/metabolismo
6.
Nat Commun ; 13(1): 1058, 2022 02 25.
Artículo en Inglés | MEDLINE | ID: mdl-35217685

RESUMEN

The adaptor protein complex AP-4 mediates anterograde axonal transport and is essential for axon health. AP-4-deficient patients suffer from a severe neurodevelopmental and neurodegenerative disorder. Here we identify DAGLB (diacylglycerol lipase-beta), a key enzyme for generation of the endocannabinoid 2-AG (2-arachidonoylglycerol), as a cargo of AP-4 vesicles. During normal development, DAGLB is targeted to the axon, where 2-AG signalling drives axonal growth. We show that DAGLB accumulates at the trans-Golgi network of AP-4-deficient cells, that axonal DAGLB levels are reduced in neurons from a patient with AP-4 deficiency, and that 2-AG levels are reduced in the brains of AP-4 knockout mice. Importantly, we demonstrate that neurite growth defects of AP-4-deficient neurons are rescued by inhibition of MGLL (monoacylglycerol lipase), the enzyme responsible for 2-AG hydrolysis. Our study supports a new model for AP-4 deficiency syndrome in which axon growth defects arise through spatial dysregulation of endocannabinoid signalling.


Asunto(s)
Complejo 4 de Proteína Adaptadora , Endocannabinoides , Neuronas , Complejo 4 de Proteína Adaptadora/metabolismo , Animales , Transporte Axonal , Axones/metabolismo , Endocannabinoides/metabolismo , Humanos , Ratones , Monoacilglicerol Lipasas/genética , Monoacilglicerol Lipasas/metabolismo , Neuronas/metabolismo
7.
EMBO J ; 40(8): e105492, 2021 04 15.
Artículo en Inglés | MEDLINE | ID: mdl-33709510

RESUMEN

Cells release diverse types of extracellular vesicles (EVs), which transfer complex signals to surrounding cells. Specific markers to distinguish different EVs (e.g. exosomes, ectosomes, enveloped viruses like HIV) are still lacking. We have developed a proteomic profiling approach for characterizing EV subtype composition and applied it to human Jurkat T cells. We generated an interactive database to define groups of proteins with similar profiles, suggesting release in similar EVs. Biochemical validation confirmed the presence of preferred partners of commonly used exosome markers in EVs: CD81/ADAM10/ITGB1, and CD63/syntenin. We then compared EVs from control and HIV-1-infected cells. HIV infection altered EV profiles of several cellular proteins, including MOV10 and SPN, which became incorporated into HIV virions, and SERINC3, which was re-routed to non-viral EVs in a Nef-dependent manner. Furthermore, we found that SERINC3 controls the surface composition of EVs. Our workflow provides an unbiased approach for identifying candidate markers and potential regulators of EV subtypes. It can be widely applied to in vitro experimental systems for investigating physiological or pathological modifications of EV release.


Asunto(s)
Vesículas Extracelulares/metabolismo , Infecciones por VIH/metabolismo , Proteoma/metabolismo , Células Cultivadas , Células HEK293 , VIH-1 , Humanos , Células Jurkat , Leucosialina/metabolismo , Glicoproteínas de Membrana/metabolismo , ARN Helicasas/metabolismo
8.
Curr Protoc Bioinformatics ; 71(1): e105, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32931150

RESUMEN

The Perseus software provides a comprehensive framework for the statistical analysis of large-scale quantitative proteomics data, also in combination with other omics dimensions. Rapid developments in proteomics technology and the ever-growing diversity of biological studies increasingly require the flexibility to incorporate computational methods designed by the user. Here, we present the new functionality of Perseus to integrate self-made plugins written in C#, R, or Python. The user-written codes will be fully integrated into the Perseus data analysis workflow as custom activities. This also makes language-specific R and Python libraries from CRAN (cran.r-project.org), Bioconductor (bioconductor.org), PyPI (pypi.org), and Anaconda (anaconda.org) accessible in Perseus. The different available approaches are explained in detail in this article. To facilitate the distribution of user-developed plugins among users, we have created a plugin repository for community sharing and filled it with the examples provided in this article and a collection of already existing and more extensive plugins. © 2020 The Authors. Basic Protocol 1: Basic steps for R plugins Support Protocol 1: R plugins with additional arguments Basic Protocol 2: Basic steps for python plugins Support Protocol 2: Python plugins with additional arguments Basic Protocol 3: Basic steps and construction of C# plugins Basic Protocol 4: Basic steps of construction and connection for R plugins with C# interface Support Protocol 4: Advanced example of R Plugin with C# interface: UMAP Basic Protocol 5: Basic steps of construction and connection for python plugins with C# interface Support Protocol 5: Advanced example of python plugin with C# interface: UMAP Support Protocol 6: A basic workflow for the analysis of label-free quantification proteomics data using perseus.


Asunto(s)
Biología Computacional , Proteómica , Programas Informáticos , Lenguajes de Programación
9.
Cell Rep ; 32(2): 107905, 2020 07 14.
Artículo en Inglés | MEDLINE | ID: mdl-32668257

RESUMEN

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.


Asunto(s)
Antineoplásicos/farmacología , Citosol/metabolismo , Endosomas/metabolismo , Inmunidad , Neoplasias/inmunología , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Antígenos/metabolismo , Transporte Biológico/efectos de los fármacos , Reactividad Cruzada/efectos de los fármacos , Citosol/efectos de los fármacos , Células Dendríticas/metabolismo , Degradación Asociada con el Retículo Endoplásmico/efectos de los fármacos , Endosomas/efectos de los fármacos , Inmunidad/efectos de los fármacos , Ratones Endogámicos C57BL , Ratones Transgénicos , Neoplasias/tratamiento farmacológico , Permeabilidad , Prazosina/farmacología , Quinazolinas/farmacología , Tamoxifeno/farmacología , beta-Lactamasas/metabolismo
10.
Mol Cell Proteomics ; 19(7): 1076-1087, 2020 07.
Artículo en Inglés | MEDLINE | ID: mdl-32345598

RESUMEN

Protein subcellular localization is an essential and highly regulated determinant of protein function. Major advances in mass spectrometry and imaging have allowed the development of powerful spatial proteomics approaches for determining protein localization at the whole cell scale. Here, a brief overview of current methods is presented, followed by a detailed discussion of organellar mapping through proteomic profiling. This relatively simple yet flexible approach is rapidly gaining popularity, because of its ability to capture the localizations of thousands of proteins in a single experiment. It can be used to generate high-resolution cell maps, and as a tool for monitoring protein localization dynamics. This review highlights the strengths and limitations of the approach and provides guidance to designing and interpreting profiling experiments.


Asunto(s)
Fraccionamiento Celular/métodos , Orgánulos/metabolismo , Mapeo de Interacción de Proteínas/métodos , Proteoma/metabolismo , Proteómica/métodos , Fracciones Subcelulares/metabolismo , Animales , Humanos , Espectrometría de Masas/métodos , Transporte de Proteínas , Análisis Espacio-Temporal
11.
Hum Mol Genet ; 29(2): 320-334, 2020 01 15.
Artículo en Inglés | MEDLINE | ID: mdl-31915823

RESUMEN

Deficiency of the adaptor protein complex 4 (AP-4) leads to childhood-onset hereditary spastic paraplegia (AP-4-HSP): SPG47 (AP4B1), SPG50 (AP4M1), SPG51 (AP4E1) and SPG52 (AP4S1). This study aims to evaluate the impact of loss-of-function variants in AP-4 subunits on intracellular protein trafficking using patient-derived cells. We investigated 15 patient-derived fibroblast lines and generated six lines of induced pluripotent stem cell (iPSC)-derived neurons covering a wide range of AP-4 variants. All patient-derived fibroblasts showed reduced levels of the AP4E1 subunit, a surrogate for levels of the AP-4 complex. The autophagy protein ATG9A accumulated in the trans-Golgi network and was depleted from peripheral compartments. Western blot analysis demonstrated a 3-5-fold increase in ATG9A expression in patient lines. ATG9A was redistributed upon re-expression of AP4B1 arguing that mistrafficking of ATG9A is AP-4-dependent. Examining the downstream effects of ATG9A mislocalization, we found that autophagic flux was intact in patient-derived fibroblasts both under nutrient-rich conditions and when autophagy is stimulated. Mitochondrial metabolism and intracellular iron content remained unchanged. In iPSC-derived cortical neurons from patients with AP4B1-associated SPG47, AP-4 subunit levels were reduced while ATG9A accumulated in the trans-Golgi network. Levels of the autophagy marker LC3-II were reduced, suggesting a neuron-specific alteration in autophagosome turnover. Neurite outgrowth and branching were reduced in AP-4-HSP neurons pointing to a role of AP-4-mediated protein trafficking in neuronal development. Collectively, our results establish ATG9A mislocalization as a key marker of AP-4 deficiency in patient-derived cells, including the first human neuron model of AP-4-HSP, which will aid diagnostic and therapeutic studies.


Asunto(s)
Complejo 4 de Proteína Adaptadora/genética , Complejo 4 de Proteína Adaptadora/metabolismo , Proteínas Relacionadas con la Autofagia/metabolismo , Proteínas de la Membrana/metabolismo , Transporte de Proteínas/genética , Paraplejía Espástica Hereditaria/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Red trans-Golgi/metabolismo , Complejo 4 de Proteína Adaptadora/deficiencia , Subunidades beta de Complejo de Proteína Adaptadora/metabolismo , Adolescente , Autofagosomas/metabolismo , Autofagia/genética , Línea Celular , Niño , Preescolar , Femenino , Fibroblastos/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Hierro/metabolismo , Mutación con Pérdida de Función , Masculino , Proteínas Asociadas a Microtúbulos/metabolismo , Mitocondrias/metabolismo , Neurogénesis/genética , Neuronas/metabolismo , Paraplejía Espástica Hereditaria/genética , Red trans-Golgi/genética
12.
Proteomics ; 20(23): e1900328, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-33615682

Asunto(s)
Proteómica
13.
Nat Rev Mol Cell Biol ; 20(5): 285-302, 2019 05.
Artículo en Inglés | MEDLINE | ID: mdl-30659282

RESUMEN

Protein subcellular localization is tightly controlled and intimately linked to protein function in health and disease. Capturing the spatial proteome - that is, the localizations of proteins and their dynamics at the subcellular level - is therefore essential for a complete understanding of cell biology. Owing to substantial advances in microscopy, mass spectrometry and machine learning applications for data analysis, the field is now mature for proteome-wide investigations of spatial cellular regulation. Studies of the human proteome have begun to reveal a complex architecture, including single-cell variations, dynamic protein translocations, changing interaction networks and proteins localizing to multiple compartments. Furthermore, several studies have successfully harnessed the power of comparative spatial proteomics as a discovery tool to unravel disease mechanisms. We are at the beginning of an era in which spatial proteomics finally integrates with cell biology and medical research, thereby paving the way for unbiased systems-level insights into cellular processes. Here, we discuss current methods for spatial proteomics using imaging or mass spectrometry and specifically highlight global comparative applications. The aim of this Review is to survey the state of the field and also to encourage more cell biologists to apply spatial proteomics approaches.


Asunto(s)
Espectrometría de Masas , Proteoma/metabolismo , Proteómica , Animales , Humanos , Transporte de Proteínas/fisiología , Proteoma/genética
14.
Curr Protoc Cell Biol ; 83(1): e81, 2019 06.
Artículo en Inglés | MEDLINE | ID: mdl-30489039

RESUMEN

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.


Asunto(s)
Orgánulos/metabolismo , Proteómica/métodos , Espacio Intracelular/metabolismo , Espectrometría de Masas/métodos , Orgánulos/ultraestructura , Proteínas/análisis , Proteínas/metabolismo , Fracciones Subcelulares/química
15.
Nat Commun ; 9(1): 3958, 2018 09 27.
Artículo en Inglés | MEDLINE | ID: mdl-30262884

RESUMEN

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.


Asunto(s)
Complejo 4 de Proteína Adaptadora/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Relacionadas con la Autofagia/metabolismo , Autofagia , Proteínas Portadoras/metabolismo , Proteínas de la Membrana/metabolismo , Vesículas Transportadoras/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Células HeLa , Humanos , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Modelos Biológicos , Fagosomas/metabolismo , Fagosomas/ultraestructura , Fenotipo , Unión Proteica , Proteómica , Vesículas Transportadoras/ultraestructura , Red trans-Golgi/metabolismo , Red trans-Golgi/ultraestructura
16.
Mol Cell ; 70(6): 1025-1037.e5, 2018 06 21.
Artículo en Inglés | MEDLINE | ID: mdl-29861160

RESUMEN

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.


Asunto(s)
Adaptación Fisiológica/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Citosol/metabolismo , Retículo Endoplásmico/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Pliegue de Proteína , Proteolisis , Saccharomyces cerevisiae/enzimología , Serina Endopeptidasas/metabolismo , Estrés Fisiológico/fisiología , Especificidad por Sustrato , Ubiquitina/metabolismo
17.
Sci Rep ; 8(1): 2340, 2018 02 05.
Artículo en Inglés | MEDLINE | ID: mdl-29402896

RESUMEN

The repertoire of cell types in the human nervous system arises through a highly orchestrated process, the complexity of which is still being discovered. Here, we present evidence that CHC22 has a non-redundant role in an early stage of neural precursor differentiation, providing a potential explanation of why CHC22 deficient patients are unable to feel touch or pain. We show the CHC22 effect on neural differentiation is independent of the more common clathrin heavy chain CHC17, and that CHC22-dependent differentiation is mediated through an autocrine/paracrine mechanism. Using quantitative proteomics, we define the composition of clathrin-coated vesicles in SH-SY5Y cells, and determine proteome changes induced by CHC22 depletion. In the absence of CHC22 a subset of dense core granule (DCG) neuropeptides accumulated, were processed into biologically active 'mature' forms, and secreted in sufficient quantity to trigger neural differentiation. When CHC22 is present, however, these DCG neuropeptides are directed to the lysosome and degraded, thus preventing differentiation. This suggests that the brief reduction seen in CHC22 expression in sensory neural precursors may license a step in neuron precursor neurodevelopment; and that this step is mediated through control of a novel neuropeptide processing pathway.


Asunto(s)
Cadenas Pesadas de Clatrina/metabolismo , Neuropéptidos/metabolismo , Proteolisis , Comunicación Autocrina , Diferenciación Celular , Línea Celular Tumoral , Cadenas Pesadas de Clatrina/genética , Técnicas de Silenciamiento del Gen , Humanos , Lisosomas , Neuronas , Comunicación Paracrina , Transporte de Proteínas
18.
PLoS Biol ; 16(1): e2004411, 2018 01.
Artículo en Inglés | MEDLINE | ID: mdl-29381698

RESUMEN

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.


Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/fisiología , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Sistemas CRISPR-Cas , Endosomas/fisiología , Aparato de Golgi/fisiología , Células HeLa , Humanos , Lisosomas/genética , Lisosomas/fisiología , Espectrometría de Masas , Proteínas de la Membrana/metabolismo , Fenotipo , Transporte de Proteínas , Paraplejía Espástica Hereditaria/genética , Proteínas de Transporte Vesicular/metabolismo
19.
Cell Rep ; 20(11): 2706-2718, 2017 Sep 12.
Artículo en Inglés | MEDLINE | ID: mdl-28903049

RESUMEN

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.


Asunto(s)
Neuronas/metabolismo , Proteoma/metabolismo , Proteómica/métodos , Espectrometría de Masas en Tándem/métodos , Animales , Biomarcadores/metabolismo , Fraccionamiento Celular , Células Cultivadas , Células HeLa , Humanos , Marcaje Isotópico , Ratones , Orgánulos/metabolismo , Transporte de Proteínas , Coloración y Etiquetado , Fracciones Subcelulares/metabolismo
20.
Mol Biol Cell ; 28(20): 2676-2685, 2017 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-28814506

RESUMEN

The dense core vesicles (DCVs) of neuroendocrine cells are a rich source of bioactive molecules such as peptides, hormones, and neurotransmitters, but relatively little is known about how they are formed. Using fractionation profiling, a method that combines subcellular fractionation with mass spectrometry, we identified ∼1200 proteins in PC12 cell vesicle-enriched fractions, with DCV-associated proteins showing distinct profiles from proteins associated with other types of vesicles. To investigate the role of clathrin in DCV biogenesis, we stably transduced PC12 cells with an inducible short hairpin RNA targeting clathrin heavy chain, resulting in ∼85% protein loss. DCVs could still be observed in the cells by electron microscopy, but mature profiles were approximately fourfold less abundant than in mock-treated cells. By quantitative mass spectrometry, DCV-associated proteins were found to be reduced approximately twofold in clathrin-depleted cells as a whole and approximately fivefold in vesicle-enriched fractions. Our combined data sets enabled us to identify new candidate DCV components. Secretion assays revealed that clathrin depletion causes a near-complete block in secretagogue-induced exocytosis. Taken together, our data indicate that clathrin has a function in DCV biogenesis beyond its established role in removing unwanted proteins from the immature vesicle.


Asunto(s)
Clatrina/metabolismo , Vesículas Secretoras/metabolismo , Animales , Proteínas de Unión al Calcio/metabolismo , Células Cultivadas , Cadenas Pesadas de Clatrina/metabolismo , Exocitosis/fisiología , Espectrometría de Masas/métodos , Células Neuroendocrinas/metabolismo , Células PC12 , Ratas , Fracciones Subcelulares
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