RESUMO
Integration and down-regulation of cell growth and differentiation signals rely on plasma membrane receptor endocytosis and sorting towards either recycling vesicles or degradative lysosomes via multivesicular bodies (MVB). In this process, the endosomal sorting complex-III required for transport (ESCRT-III) controls membrane deformation and scission triggering intraluminal vesicle (ILV) formation at early endosomes. Here, we show that the ESCRT-III member CHMP1B can be ubiquitinated within a flexible loop known to undergo conformational changes during polymerization. We demonstrate further that CHMP1B is deubiquitinated by the ubiquitin specific protease USP8 (syn. UBPY) and found fully devoid of ubiquitin in a ~500 kDa large complex that also contains its ESCRT-III partner IST1. Moreover, EGF stimulation induces the rapid and transient accumulation of ubiquitinated forms of CHMP1B on cell membranes. Accordingly, CHMP1B ubiquitination is necessary for CHMP1B function in both EGF receptor trafficking in human cells and wing development in Drosophila. Based on these observations, we propose that CHMP1B is dynamically regulated by ubiquitination in response to EGF and that USP8 triggers CHMP1B deubiquitination possibly favoring its subsequent assembly into a membrane-associated ESCRT-III polymer.
Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Ubiquitina Tiolesterase/metabolismo , Animais , Membrana Celular/metabolismo , Drosophila , Proteínas de Drosophila/metabolismo , Endocitose/fisiologia , Endopeptidases/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Endossomos/metabolismo , Receptores ErbB/metabolismo , Feminino , Células HEK293 , Células HeLa , Humanos , Ligação Proteica , Transporte Proteico , Ubiquitina/metabolismo , UbiquitinaçãoRESUMO
Arrestins are key adaptor proteins that control the fate of cell-surface membrane proteins and modulate downstream signaling cascades. The Dictyostelium discoideum genome encodes six arrestin-related proteins, harboring additional modules besides the arrestin domain. Here, we studied AdcB and AdcC, two homologs that contain C2 and SAM domains. We showed that AdcC - in contrast to AdcB - responds to various stimuli (such as the chemoattractants cAMP and folate) known to induce an increase in cytosolic calcium by transiently translocating to the plasma membrane, and that calcium is a direct regulator of AdcC localization. This response requires the calcium-dependent membrane-targeting C2 domain and the double SAM domain involved in AdcC oligomerization, revealing a mode of membrane targeting and regulation unique among members of the arrestin clan. AdcB shares several biochemical properties with AdcC, including in vitro binding to anionic lipids in a calcium-dependent manner and auto-assembly as large homo-oligomers. AdcB can interact with AdcC; however, its intracellular localization is insensitive to calcium. Therefore, despite their high degree of homology and common characteristics, AdcB and AdcC are likely to fulfill distinct functions in amoebae.
Assuntos
Arrestinas/metabolismo , Cálcio/metabolismo , Fatores Quimiotáticos/farmacologia , Dictyostelium/metabolismo , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Animais , Arrestinas/química , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , AMP Cíclico/farmacologia , Dictyostelium/efeitos dos fármacos , Ácido Fólico/farmacologia , Proteínas de Fluorescência Verde/metabolismo , Espaço Intracelular/metabolismo , Lipossomos , Fosfolipídeos/metabolismo , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , Multimerização Proteica , Transporte Proteico/efeitos dos fármacos , Proteínas de Protozoários/química , Proteínas Recombinantes/metabolismoRESUMO
Lysosomes are membrane-bound endocytic organelles that play a major role in degrading cell macromolecules and recycling their building blocks. A comprehensive knowledge of the lysosome function requires an extensive description of its content, an issue partially addressed by previous proteomic analyses. However, the proteins underlying many lysosomal membrane functions, including numerous membrane transporters, remain unidentified. We performed a comparative, semi-quantitative proteomic analysis of rat liver lysosome-enriched and lysosome-nonenriched membranes and used spectral counts to evaluate the relative abundance of proteins. Among a total of 2,385 identified proteins, 734 proteins were significantly enriched in the lysosomal fraction, including 207 proteins already known or predicted as endo-lysosomal and 94 proteins without any known or predicted subcellular localization. The remaining 433 proteins had been previously assigned to other subcellular compartments but may in fact reside on lysosomes either predominantly or as a secondary location. Many membrane-associated complexes implicated in diverse processes such as degradation, membrane trafficking, lysosome biogenesis, lysosome acidification, signaling, and nutrient sensing were enriched in the lysosomal fraction. They were identified to an unprecedented extent as most, if not all, of their subunits were found and retained by our screen. Numerous transporters were also identified, including 46 novel potentially lysosomal proteins. We expressed 12 candidates in HeLa cells and observed that most of them colocalized with the lysosomal marker LAMP1, thus confirming their lysosomal residency. This list of candidate lysosomal proteins substantially increases our knowledge of the lysosomal membrane and provides a basis for further characterization of lysosomal functions.
Assuntos
Hepatócitos/metabolismo , Membranas Intracelulares/química , Fígado/metabolismo , Lisossomos/química , Proteínas de Membrana Transportadoras/isolamento & purificação , Proteoma/isolamento & purificação , Animais , Biomarcadores/metabolismo , Expressão Gênica , Células HeLa , Hepatócitos/química , Humanos , Fígado/química , Proteínas de Membrana Lisossomal/genética , Proteínas de Membrana Lisossomal/metabolismo , Masculino , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Microscopia de Fluorescência , Anotação de Sequência Molecular , Proteoma/genética , Proteoma/metabolismo , Ratos , Ratos WistarRESUMO
Cystinosin, the lysosomal cystine exporter defective in cystinosis, is the founding member of a family of heptahelical membrane proteins related to bacteriorhodopsin and characterized by a duplicated motif termed the PQ loop. PQ-loop proteins are more frequent in eukaryotes than in prokaryotes; except for cystinosin, their molecular function remains elusive. In this study, we report that three yeast PQ-loop proteins of unknown function, Ypq1, Ypq2, and Ypq3, localize to the vacuolar membrane and are involved in homeostasis of cationic amino acids (CAAs). We also show that PQLC2, a mammalian PQ-loop protein closely related to yeast Ypq proteins, localizes to lysosomes and catalyzes a robust, electrogenic transport that is selective for CAAs and strongly activated at low extracytosolic pH. Heterologous expression of PQLC2 at the yeast vacuole rescues the resistance phenotype of an ypq2 mutant to canavanine, a toxic analog of arginine efficiently transported by PQLC2. Finally, PQLC2 transports a lysine-like mixed disulfide that serves as a chemical intermediate in cysteamine therapy of cystinosis, and PQLC2 gene silencing trapped this intermediate in cystinotic cells. We conclude that PQLC2 and Ypq1-3 proteins are lysosomal/vacuolar exporters of CAAs and suggest that small-molecule transport is a conserved feature of the PQ-loop protein family, in agreement with its distant similarity to SWEET sugar transporters and to the mitochondrial pyruvate carrier. The elucidation of PQLC2 function may help improve cysteamine therapy. It may also clarify the origin of CAA abnormalities in Batten disease.
Assuntos
Sistemas de Transporte de Aminoácidos Básicos/química , Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Cisteamina/uso terapêutico , Cistinose/tratamento farmacológico , Cistinose/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Sistemas de Transporte de Aminoácidos Básicos/genética , Animais , Sequência de Bases , Proteínas de Caenorhabditis elegans/genética , Canavanina/metabolismo , RNA Helicases DEAD-box , DNA Complementar/genética , Proteínas de Drosophila , Fenômenos Eletrofisiológicos , Feminino , Genes Fúngicos , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Oócitos/metabolismo , Estrutura Secundária de Proteína , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade da Espécie , Vacúolos/metabolismo , Xenopus laevisRESUMO
The cellular slime mold Dictyostelium discoideum is a soil-living eukaryote, which feeds on microorganisms engulfed by phagocytosis. Axenic laboratory strains have been produced that are able to use liquid growth medium internalized by macropinocytosis as the source of food. To better define the macropinocytosis process, we established the inventory of proteins associated with this pathway using mass spectrometry-based proteomics. Using a magnetic purification procedure and high-performance LC-MS/MS proteome analysis, a list of 2108 non-redundant proteins was established, of which 24% featured membrane-spanning domains. Bioinformatics analyses indicated that the most abundant proteins were linked to signaling, vesicular trafficking and the cytoskeleton. The present repertoire validates our purification method and paves the way for a future proteomics approach to study the dynamics of macropinocytosis.
Assuntos
Dictyostelium/metabolismo , Espectrometria de Massas/métodos , Pinocitose , Proteoma/análise , Proteômica/métodos , Western Blotting , Cromatografia Líquida/métodos , Biologia Computacional , Eletroforese em Gel de Poliacrilamida , Lisossomos/metabolismo , Imãs , Transporte Proteico , Proteoma/isolamento & purificação , Proteoma/metabolismo , Proteínas de Protozoários/análise , Proteínas de Protozoários/metabolismoRESUMO
The ubiquitin-specific protease USP8 plays a major role in controlling the stability and intracellular trafficking of numerous cell surface proteins among which the EGF receptor that regulates cell growth and proliferation in many physio-pathological processes. The function of USP8 at the endocytic pathway level partly relies on binding to and deubiquitination of the Endosomal Sorting Complex Required for Transport (ESCRT) protein CHMP1B. In the aim of finding chemical inhibitors of the USP8::CHMP1B interaction, we performed a high-throughput screening campaign using an HTRF® assay to monitor the interaction directly in lysates of cells co-expressing both partners. The assay was carried out in an automated format to screen the academic Fr-PPIChem library (Bosc N et al., 2020), which includes 10,314 compounds dedicated to the targeting of protein-protein interactions (PPIs). Eleven confirmed hits inhibited the USP8::CHMP1B interaction within a range of 30% to 70% inhibition at 50 µM, while they were inactive on a set of other PPI interfaces demonstrating the feasibility of specifically disrupting this particular interface. In parallel, we adapted this HTRF® assay to compare the USP8 interacting capacity of CHMP1B variants. As anticipated from earlier studies, a deletion of the MIM (Microtubule Interacting and Trafficking domain Interacting Motif) domain or mutation of two conserved leucine residues, L192 and L195, in this domain respectively abolished or strongly impeded the USP8::CHMP1B interaction. By contrast, a CHMP1B mutant that displays a highly decreased ubiquitination level following mutation of four lysine residues in arginine interacted at a similar level as the wild-type form with USP8. Therefore, conserved leucine residues within the MIT domain rather than its ubiquitinated status triggers CHMP1B substrate recognition by USP8.
Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte , Ubiquitina Tiolesterase , Arginina , Endopeptidases/genética , Endopeptidases/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/química , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Receptores ErbB/genética , Leucina , Lisina , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , Proteases Específicas de UbiquitinaRESUMO
Gap junctions (GJs) are dynamic structures composed of hexamers of connexins (Cxs), a class of transmembrane proteins enabling channel-mediated direct intercellular communication through cell-cell diffusion of ions and small metabolites. In defined conditions, Cxs also work as hemichannels allowing exchanges between the cytoplasm and the extracellular medium. The most common GJ channel is formed by connexin 43 (Cx43) and plays an important role in physiological and pathological processes in excitable tissues, such as heart and brain. Hence, Cx43 has been largely envisioned as a new therapeutic target in cancer, neurological and psychiatric indications, or cardiovascular diseases. Identifying new pharmacological inhibitors of Cx43 GJs with different mechanisms of action and from diverse chemical classes is thus highly challenging. We present here a high-content screening method, based on the evaluation of fluorescent dye transfer rates between adjacent cells to monitor the function of GJs in U251 glioblastoma cells expressing high levels of Cx43. This assay was validated using well-described pharmacological GJ inhibitors such as mefloquine. The method was adapted to screen a library of 1,280 Food and Drug Administration- and European Medicines Agency-approved drugs that led to the selection of both known and new inhibitors of GJ channel function. We further focused on a specific class of microtubule-targeting agents, confirming that a proper tubulin network is required for functional Cx43 GJ channels.
Assuntos
Conexina 43/antagonistas & inibidores , Junções Comunicantes/efeitos dos fármacos , Ensaios de Triagem em Larga Escala , Mefloquina/farmacologia , Linhagem Celular Tumoral , Conexina 43/metabolismo , Relação Dose-Resposta a Droga , Junções Comunicantes/metabolismo , HumanosRESUMO
Most soluble lysosomal proteins carry Man6P (mannose 6-phosphate), a specific carbohydrate marker that enables their binding to cellular MPRs (Man6P receptors) and their subsequent targeting towards the lysosome. This characteristic was exploited to identify novel soluble lysosomal proteins by proteomic analysis of Man6P proteins purified from a human cell line. Among the proteins identified during the course of the latter study [Journet, Chapel, Kieffer, Roux and Garin (2002) Proteomics, 2, 1026-1040], some had not been previously described as lysosomal proteins. We focused on a protein detected at 76 kDa by SDS/PAGE. We named this protein 'p76' and it appeared later in the NCBI protein database as the 'hypothetical protein LOC196463'. In the present paper, we describe the identification of p76 by MS and we analyse several of its biochemical characteristics. The presence of Man6P sugars was confirmed by an MPR overlay experiment, which showed the direct and Man6P-dependent interaction between p76 and the MPR. The presence of six N-glycosylation sites was validated by progressive peptide-N-glycosidase F deglycosylation. Experiments using N- and C-termini directed anti-p76 antibodies provided insights into p76 maturation. Most importantly, we were able to demonstrate the lysosomal localization of this protein, which was initially suggested by its Man6P tags, by both immunofluorescence and sub-cellular fractionation of mouse liver homogenates.
Assuntos
Glicoproteínas/metabolismo , Lisossomos/metabolismo , Animais , Biomarcadores , Linhagem Celular , Glicosilação , Humanos , Fígado/metabolismo , Camundongos , Processamento de Proteína Pós-Traducional , RatosRESUMO
Dictyostelium discoideum has been used largely as a model organism to study the organization and function of the endocytic pathway. Here we describe dense structures present in D. discoideum endocytic compartments, which we named pycnosomes. Pycnosomes are constitutively secreted in the extracellular medium, from which they can be recovered by differential centrifugation. We identified the most abundant protein present in secreted pycnosomes, that we designated SctA. SctA defines a new family of proteins with four members in D. discoideum, and homologous proteins in other protists and eumetazoa. We developed a monoclonal antibody specific for SctA and used it to further characterize secreted and intracellular pycnosomes. Within cells, immunofluorescence as well as electron microscopy identified pycnosomes as SctA-enriched dense structures in the lumen of endocytic compartments. Pycnosomes are occasionally seen in continuity with intra-endosomal membranes, particularly in U18666A-treated cells where intraluminal budding is highly enhanced. While the exact nature, origin and cellular function of pycnosomes remain to be established, this study provides a first description of these structures as well as a characterization of reagents that can be used for further studies.
Assuntos
Dictyostelium/metabolismo , Endossomos/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Transporte Biológico , Dictyostelium/imunologia , Dictyostelium/ultraestrutura , Endocitose , Endossomos/imunologia , Endossomos/ultraestrutura , Membranas Intracelulares/metabolismo , Camundongos , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/metabolismoRESUMO
S-palmitoylation is a reversible post-translational modification important for controlling the membrane targeting and function of numerous membrane proteins with diverse roles in signalling, scaffolding, and trafficking. We sought to identify novel palmitoylated proteins in B lymphocytes using acyl-biotin exchange chemistry, coupled with differential analysis by liquid-chromatography tandem mass spectrometry. In total, we identified 57 novel palmitoylated protein candidates from human EBV-transformed lymphoid cells. Two of them, namely CD20 and CD23 (low affinity immunoglobulin epsilon Fc receptor), are immune regulators that are effective/potential therapeutic targets for haematological malignancies, autoimmune diseases and allergic disorders. Palmitoylation of CD20 and CD23 was confirmed by heterologous expression of alanine mutants coupled with bioorthogonal metabolic labeling. This study demonstrates a new subset of palmitoylated proteins in B cells, illustrating the ubiquitous role of protein palmitoylation in immune regulation.
Assuntos
Linfócitos B/metabolismo , Lipoilação , Antígenos CD20/metabolismo , Linhagem Celular Transformada , Ácidos Graxos Insaturados , Células HEK293 , Herpesvirus Humano 4 , Humanos , Processamento de Proteína Pós-Traducional , Proteômica/métodos , Receptores de IgE/metabolismoRESUMO
Because of the great number of women who are diagnosed with breast cancer each year, and though this disease presents the lowest mortality rate among cancers, breast cancer remains a major public health problem. As for any cancer, the tumorigenic and metastatic processes are still hardly understood, and the biochemical markers that allow either a precise monitoring of the disease or the classification of the numerous forms of breast cancer remain too scarce. Therefore, great hopes are put on the development of high-throughput genomic and proteomic technologies. Such comprehensive techniques should help in understanding the processes and in defining steps of the disease by depicting specific genes or protein profiles. Because techniques dedicated to the current proteomic challenges are continuously improving, the probability of the discovery of new potential protein biomarkers is rapidly increasing. In addition, the identification of such markers should be eased by lowering the sample complexity; e.g., by sample fractionation, either according to specific physico-chemical properties of the proteins, or by focusing on definite subcellular compartments. In particular, proteins of the lysosomal compartment have been shown to be prone to alterations in their localization, expression, or post-translational modifications (PTMs) during the cancer process. Some of them, such as the aspartic protease cathepsin D (CatD), have even been proven as participating actively in the disease progression. The present review aims at giving an overview of the implication of the lysosome in breast cancer, and at showing how subproteomics and the constantly refining MS-based proteomic techniques may help in making breast cancer research progress, and thus, hopefully, in improving disease treatment.
Assuntos
Biomarcadores Tumorais/análise , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/enzimologia , Hidrolases/análise , Lisossomos/enzimologia , Espectrometria de Massas/métodos , Proteômica/métodos , Biomarcadores Tumorais/metabolismo , Humanos , Hidrolases/metabolismo , Proteínas de Neoplasias/análise , Proteínas de Neoplasias/metabolismoRESUMO
To date, about fifty lysosomal hydrolases have been identified, and most of them are targeted towards the lysosomes through a specific mannose-6-phosphate (M-6-P) tag. As more lysosomal hydrolases were expected to be discovered, we performed a proteomic study of soluble lysosomal proteins. Human cells were induced to secrete M-6-P proteins which were affinity purified on immobilized M-6-P receptor. The purified proteins were resolved by two-dimensional electrophoresis and analyzed by mass spectrometry. Twenty-two proteins were identified, among which 16 were well-known lysosomal hydrolases. The remaining species distributed as follows: epididymis-specific alpha-mannosidase is a new mannosidase homolog, cystatin F and CREG (cellular repressor of E1A-stimulated genes) were previously identified as M-6-P proteins (Journet et al., Electrophoresis 2000, 21, 3411-3419), and the last three, which are not hydrolases, were up to now considered as nonlysosomal. This two-dimensional reference map of human U937 M-6-P proteins was afterwards used for comparison with M-6-P proteins purified either from U937 differentiated into macrophage-like cells, or from human breast cancer MCF7 cells. Phorbol ester induced differentiation of U937 cells led to limited proteolytic cleavage or maturation of a discrete number of hydrolases. Five additional lysosomal hydrolases were identified from MCF7 samples. These results prove the usefulness of such a procedure to analyze the lysosomal content of various cell lines, to discover new M-6-P proteins, as well as to point towards unknown biological processes.