RESUMO
The apolipoprotein B messenger RNA editing enzyme, catalytic polypeptide (APOBEC) family is composed of nucleic acid editors with roles ranging from antibody diversification to RNA editing. APOBEC2, a member of this family with an evolutionarily conserved nucleic acid-binding cytidine deaminase domain, has neither an established substrate nor function. Using a cellular model of muscle differentiation where APOBEC2 is inducibly expressed, we confirmed that APOBEC2 does not have the attributed molecular functions of the APOBEC family, such as RNA editing, DNA demethylation, and DNA mutation. Instead, we found that during muscle differentiation APOBEC2 occupied a specific motif within promoter regions; its removal from those regions resulted in transcriptional changes. Mechanistically, these changes reflect the direct interaction of APOBEC2 with histone deacetylase (HDAC) transcriptional corepressor complexes. We also found that APOBEC2 could bind DNA directly, in a sequence-specific fashion, suggesting that it functions as a recruiter of HDAC to specific genes whose promoters it occupies. These genes are normally suppressed during muscle cell differentiation, and their suppression may contribute to the safeguarding of muscle cell fate. Altogether, our results reveal a unique role for APOBEC2 within the APOBEC family.
Assuntos
Cromatina , Proteínas Musculares , Desaminases APOBEC/genética , Desaminase APOBEC-1/genética , Diferenciação Celular/genética , Cromatina/genética , Citidina Desaminase/metabolismo , DNA , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/metabolismo , Mioblastos/metabolismo , RNA Mensageiro/genética , Animais , CamundongosRESUMO
The ADP-ribosylation factors (ARFs) and ARF-like (ARL) GTPases serve as essential molecular switches governing a wide array of cellular processes. In this study, we used proximity-dependent biotin identification (BioID) to comprehensively map the interactome of 28 out of 29 ARF and ARL proteins in two cellular models. Through this approach, we identified â¼3000 high-confidence proximal interactors, enabling us to assign subcellular localizations to the family members. Notably, we uncovered previously undefined localizations for ARL4D and ARL10. Clustering analyses further exposed the distinctiveness of the interactors identified with these two GTPases. We also reveal that the expression of the understudied member ARL14 is confined to the stomach and intestines. We identified phospholipase D1 (PLD1) and the ESCPE-1 complex, more precisely, SNX1, as proximity interactors. Functional assays demonstrated that ARL14 can activate PLD1 in cellulo and is involved in cargo trafficking via the ESCPE-1 complex. Overall, the BioID data generated in this study provide a valuable resource for dissecting the complexities of ARF and ARL spatial organization and signaling.
Assuntos
Fatores de Ribosilação do ADP , Fosfolipase D , Transdução de Sinais , Fatores de Ribosilação do ADP/metabolismo , Fatores de Ribosilação do ADP/genética , Humanos , Fosfolipase D/metabolismo , Fosfolipase D/genética , Células HEK293 , Animais , Nexinas de Classificação/metabolismo , Nexinas de Classificação/genética , Mapeamento de Interação de ProteínasRESUMO
RNA binding proteins (RBPs) are central regulators of gene expression implicated in all facets of RNA metabolism. As such, they play key roles in cellular physiology and disease etiology. Since different steps of post-transcriptional gene expression tend to occur in specific regions of the cell, including nuclear or cytoplasmic locations, defining the subcellular distribution properties of RBPs is an important step in assessing their potential functions. Here, we present the RBP Image Database, a resource that details the subcellular localization features of 301 RBPs in the human HepG2 and HeLa cell lines, based on the results of systematic immuno-fluorescence studies conducted using a highly validated collection of RBP antibodies and a panel of 12 markers for specific organelles and subcellular structures. The unique features of the RBP Image Database include: (i) hosting of comprehensive representative images for each RBP-marker pair, with â¼250,000 microscopy images; (ii) a manually curated controlled vocabulary of annotation terms detailing the localization features of each factor; and (iii) a user-friendly interface allowing the rapid querying of the data by target or annotation. The RBP Image Database is freely available at https://rnabiology.ircm.qc.ca/RBPImage/.
Assuntos
Bases de Dados Factuais , Imagem Óptica , Proteínas de Ligação a RNA , Humanos , Anticorpos/metabolismo , Células HeLa , RNA/química , Proteínas de Ligação a RNA/metabolismo , Células Hep G2RESUMO
Homologous recombination (HR) plays an essential role in the maintenance of genome stability by promoting the repair of cytotoxic DNA double strand breaks (DSBs). More recently, the HR pathway has emerged as a core component of the response to replication stress, in part by protecting stalled replication forks from nucleolytic degradation. In that regard, the mammalian RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3) have been involved in both HR-mediated DNA repair and collapsed replication fork resolution. Still, it remains largely obscure how they participate in both processes, thereby maintaining genome stability and preventing cancer development. To gain better insight into their contribution in cellulo, we mapped the proximal interactome of the classical RAD51 paralogs using the BioID approach. Aside from identifying the well-established BCDX2 and CX3 sub-complexes, the spliceosome machinery emerged as an integral component of our proximal mapping, suggesting a crosstalk between this pathway and the RAD51 paralogs. Furthermore, we noticed that factors involved RNA metabolic pathways are significantly modulated within the BioID of the classical RAD51 paralogs upon exposure to hydroxyurea (HU), pointing towards a direct contribution of RNA processing during replication stress. Importantly, several members of these pathways have prognostic potential in breast cancer (BC), where their RNA expression correlates with poorer patient outcome. Collectively, this study uncovers novel functionally relevant partners of the different RAD51 paralogs in the maintenance of genome stability that could be used as biomarkers for the prognosis of BC.
Assuntos
Instabilidade Genômica , Rad51 Recombinase , Animais , Humanos , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Instabilidade Genômica/genética , Recombinação Homóloga/genética , Quebras de DNA de Cadeia Dupla , RNA , Reparo do DNA/genética , Mamíferos/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismoRESUMO
Distinct functions mediated by members of the monopolar spindle-one-binder (MOB) family of proteins remain elusive beyond the evolutionarily conserved and well-established roles of MOB1 (MOB1A/B) in regulating tissue homeostasis within the Hippo pathway. Since MOB proteins are adaptors, understanding how they engage in protein-protein interactions and help assemble complexes is essential to define the full scope of their biological functions. To address this, we undertook a proximity-dependent biotin identification approach to define the interactomes of all seven human MOB proteins in HeLa and human embryonic kidney 293 cell lines. We uncovered >200 interactions, of which at least 70% are unreported on BioGrid. The generated dataset reliably recalled the bona fide interactors of the well-studied MOBs. We further defined the common and differential interactome between different MOBs on a subfamily and an individual level. We discovered a unique association between MOB3C and 7 of 10 protein subunits of the RNase P complex, an endonuclease that catalyzes tRNA 5' maturation. As a proof of principle for the robustness of the generated dataset, we validated the specific interaction of MOB3C with catalytically active RNase P by using affinity purification-mass spectrometry and pre-tRNA cleavage assays of MOB3C pulldowns. In summary, our data provide novel insights into the biology of MOB proteins and reveal the first interactors of MOB3C, components of the RNase P complex, and hence an exciting nexus with RNA biology.
Assuntos
Via de Sinalização Hippo , Mapeamento de Interação de Proteínas , Proteínas Serina-Treonina Quinases , Ribonuclease P , Humanos , Células HeLa , Via de Sinalização Hippo/fisiologia , Ribonuclease P/metabolismo , Células HEK293 , Subunidades Proteicas/metabolismoRESUMO
HIV-1 encodes several accessory proteins-Nef, Vif, Vpr, and Vpu-whose functions are to modulate the cellular environment to favor immune evasion and viral replication. While Vpr was shown to mediate a G2/M cell cycle arrest and provide a replicative advantage during infection of myeloid cells, the mechanisms underlying these functions remain unclear. In this study, we defined HIV-1 Vpr proximity interaction network using the BioID proximity labeling approach and identified 352 potential Vpr partners/targets, including several complexes, such as the cell cycle-regulatory anaphase-promoting complex/cyclosome (APC/C). Herein, we demonstrate that both the wild type and cell cycle-defective mutants of Vpr induce the degradation of APC1, an essential APC/C scaffolding protein, and show that this activity relies on the recruitment of DCAF1 by Vpr and the presence of a functional proteasome. Vpr forms a complex with APC1, and the APC/C coactivators Cdh1 and Cdc20 are associated with these complexes. Interestingly, we found that Vpr encoded by the prototypic HIV-1 NL4.3 does not interact efficiently with APC1 and is unable to mediate its degradation as a result of a N28S-G41N amino acid substitution. In contrast, we show that APC1 degradation is a conserved feature of several primary Vpr variants from transmitted/founder virus. Functionally, Vpr-mediated APC1 degradation did not impact the ability of the protein to induce a G2 cell cycle arrest during infection of CD4+ T cells or enhance HIV-1 replication in macrophages, suggesting that this conserved activity may be important for other aspects of HIV-1 pathogenesis. IMPORTANCE The function of the Vpr accessory protein during HIV-1 infection remains poorly defined. Several cellular targets of Vpr were previously identified, but their individual degradation does not fully explain the ability of Vpr to impair the cell cycle or promote HIV-1 replication in macrophages. Here, we used the unbiased proximity labeling approach, called BioID, to further define the Vpr proximity interaction network and identified several potentially new Vpr partners/targets. We validated our approach by focusing on a cell cycle master regulator, the APC/C complex, and demonstrated that Vpr mediated the degradation of a critical scaffolding component of APC/C called APC1. Furthermore, we showed that targeting of APC/C by Vpr did not impact the known activity of Vpr. Since degradation of APC1 is a conserved feature of several primary variants of Vpr, it is likely that the interplay between Vpr and APC/C governs other aspects of HIV-1 pathogenesis.
Assuntos
Subunidade Apc1 do Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Infecções por HIV/patologia , HIV-1/crescimento & desenvolvimento , Proteínas Serina-Treonina Quinases/genética , Ubiquitina-Proteína Ligases/genética , Replicação Viral/genética , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/metabolismo , Linfócitos T CD4-Positivos/virologia , Linhagem Celular Tumoral , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Células HEK293 , HIV-1/metabolismo , Células HeLa , Humanos , Macrófagos/virologia , Interferência de RNA , RNA Interferente Pequeno/genética , Espectrometria de Massas em Tandem , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/genéticaRESUMO
Cyclin M (CNNM1-4) proteins maintain cellular and body magnesium (Mg2+) homeostasis. Using various biochemical approaches, we have identified members of the CNNM family as direct interacting partners of ADP-ribosylation factor-like GTPase 15 (ARL15), a small GTP-binding protein. ARL15 interacts with CNNMs at their carboxyl-terminal conserved cystathionine-ß-synthase (CBS) domains. In silico modeling of the interaction between CNNM2 and ARL15 supports that the small GTPase specifically binds the CBS1 and CNBH domains. Immunocytochemical experiments demonstrate that CNNM2 and ARL15 co-localize in the kidney, with both proteins showing subcellular localization in the endoplasmic reticulum, Golgi apparatus and the plasma membrane. Most importantly, we found that ARL15 is required for forming complex N-glycosylation of CNNMs. Overexpression of ARL15 promotes complex N-glycosylation of CNNM3. Mg2+ uptake experiments with a stable isotope demonstrate that there is a significant increase of 25Mg2+ uptake upon knockdown of ARL15 in multiple kidney cancer cell lines. Altogether, our results establish ARL15 as a novel negative regulator of Mg2+ transport by promoting the complex N-glycosylation of CNNMs.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Ciclinas/metabolismo , Homeostase , Magnésio/metabolismo , Fatores de Ribosilação do ADP/genética , Transporte Biológico , Ciclinas/genética , Glicosilação , Células HEK293 , Humanos , Modelos Moleculares , Ligação ProteicaRESUMO
BACKGROUND: Vertical transmission is the major cause of pediatric hepatitis C virus (HCV) infection. The objective of this study was to better understand HCV pathogenesis in pregnant women and provide insights into risk factors and mechanisms involved in vertical transmission. METHODS: Evolutionary dynamics of HCV variant spectra and HCV-specific neutralizing antibody responses were examined using high-throughput sequencing and pseudoparticle-based assays in pregnant women monoinfected with HCV (n = 17) or coinfected with HCV and human immunodeficiency virus (HIV)-1 (n = 15). RESULTS: Overall, statistically significant associations were found between HCV quasispecies diversity, selective pressure exerted on the HCV E2 envelope protein, and neutralizing activity of maternal immunoglobulins. Women with low quasispecies diversity displayed significantly higher mean aspartate aminotransferase and alanine aminotransferase levels throughout pregnancy, but this difference was restricted to monoinfected participants. Low quasispecies diversity and inefficient neutralizing activity were also significantly associated with vertical transmission, but only in the monoinfected group. CONCLUSIONS: These results indicate that maternal neutralizing antibody responses play a role in the prevention of vertical HCV transmission, but not in presence of HIV-1 coinfection, and suggest that the mechanism of vertical transmission may be different between monoinfected and coinfected women. These findings could inform management strategies for the prevention of vertical HCV transmission.
Assuntos
Variação Genética , Hepacivirus/classificação , Hepatite C/transmissão , Hepatite C/virologia , Transmissão Vertical de Doenças Infecciosas , Complicações Infecciosas na Gravidez/virologia , Quase-Espécies , Adulto , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Feminino , Infecções por HIV/complicações , Hepacivirus/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Recém-Nascido , Masculino , Gravidez , Fatores de Risco , Adulto JovemRESUMO
Hepatitis C virus (HCV) can be transmitted from mother to child during pregnancy and childbirth. However, the timing and precise biological mechanisms that are involved in this process are incompletely understood, as are the determinants that influence transmission of particular HCV variants. Here we report results of a longitudinal assessment of HCV quasispecies diversity and composition in 5 cases of vertical HCV transmission, including 3 women coinfected with human immunodeficiency virus type 1 (HIV-1). The population structure of HCV variant spectra based on E2 envelope gene sequences (nucleotide positions 1491 to 1787), including hypervariable regions 1 and 2, was characterized using next-generation sequencing and median-joining network analysis. Compatible with a loose transmission bottleneck, larger numbers of shared HCV variants were observed in the presence of maternal coinfection. Coalescent Bayesian Markov chain Monte Carlo simulations revealed median times of transmission between 24.9 weeks and 36.1 weeks of gestation, with some confidence intervals ranging into the 1st trimester, considerably earlier than previously thought. Using recombinant autologous HCV pseudoparticles, differences were uncovered in HCV-specific antibody responses between coinfected mothers and mothers infected with HCV alone, in whom generalized absence of neutralization was observed. Finally, shifts in HCV quasispecies composition were seen in children around 1 year of age, compatible with the disappearance of passively transferred maternal immunoglobulins and/or the development of HCV-specific humoral immunity. Taken together, these results provide insights into the timing, dynamics, and biologic mechanisms involved in vertical HCV transmission and inform preventative strategies.IMPORTANCE Although it is well established that hepatitis C virus (HCV) can be transmitted from mother to child, the manner and the moment at which transmission operates have been the subject of conjecture. By carrying out a detailed examination of viral sequences, we showed that transmission could take place comparatively early in pregnancy. In addition, we showed that when the mother also carried human immunodeficiency virus type 1 (HIV-1), many more HCV variants were shared between her and her child, suggesting that the mechanism and/or the route of transmission of HCV differed in the presence of coinfection with HIV-1. These results could explain why cesarean section is ineffective in preventing vertical HCV transmission and guide the development of interventions to avert pediatric HCV infection.
Assuntos
Hepacivirus/genética , Hepatite C/transmissão , Transmissão Vertical de Doenças Infecciosas , Adulto , Teorema de Bayes , Coinfecção/virologia , Biologia Computacional , Feminino , Variação Genética , Infecções por HIV/complicações , Infecções por HIV/virologia , Soropositividade para HIV , HIV-1/genética , HIV-1/imunologia , Hepacivirus/fisiologia , Hepatite C/complicações , Hepatite C/imunologia , Hepatite C/virologia , Anticorpos Anti-Hepatite C/sangue , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imunidade Humoral , Lactente , Gravidez , Complicações Infecciosas na Gravidez/virologia , Quase-Espécies , Fatores de Risco , Proteínas do Envelope Viral/genéticaRESUMO
Macrophages play an important role in innate and adaptive immunity as professional phagocytes capable of internalizing and degrading pathogens to derive antigens for presentation to T cells. They also produce pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) that mediate local and systemic responses and direct the development of adaptive immunity. The present work describes the use of label-free quantitative proteomics to profile the dynamic changes of proteins from resting and TNF-α-activated mouse macrophages. These analyses revealed that TNF-α activation of macrophages led to the down-regulation of mitochondrial proteins and the differential regulation of several proteins involved in vesicle trafficking and immune response. Importantly, we found that the down-regulation of mitochondria proteins occurred through mitophagy and was specific to TNF-α, as other cytokines such as IL-1ß and IFN-γ had no effect on mitochondria degradation. Furthermore, using a novel antigen presentation system, we observed that the induction of mitophagy by TNF-α enabled the processing and presentation of mitochondrial antigens at the cell surface by MHC class I molecules. These findings highlight an unsuspected role of TNF-α in mitophagy and expanded our understanding of the mechanisms responsible for MHC presentation of self-antigens.
Assuntos
Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/citologia , Macrófagos/metabolismo , Mitofagia/efeitos dos fármacos , Proteômica/métodos , Fator de Necrose Tumoral alfa/farmacologia , Animais , Apresentação de Antígeno/efeitos dos fármacos , Antígenos/metabolismo , Autofagia/efeitos dos fármacos , Linhagem Celular , Biologia Computacional , Regulação para Baixo/efeitos dos fármacos , Antígenos de Histocompatibilidade Classe I/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/ultraestrutura , Proteínas de Membrana/metabolismo , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Fosfolipases A2 Citosólicas/metabolismo , Mapas de Interação de Proteínas/efeitos dos fármacos , Proteoma/metabolismoRESUMO
After their formation at the cell surface, phagosomes become fully functional through a complex maturation process involving sequential interactions with various intracellular organelles. In the last decade, series of data indicated that some of the phagosome functional properties occur in specialized membrane microdomains. The molecules associated with membrane microdomains, as well as the organization of these structures during phagolysosome biogenesis are largely unknown. In this study, we combined proteomics and bioinformatics analyses to characterize the dynamic association of proteins to maturing phagosomes. Our data indicate that groups of proteins shuffle from detergent-soluble to detergent-resistant membrane microdomains during maturation, supporting a model in which the modulation of the phagosome functional properties involves an important reorganization of the phagosome proteome by the coordinated spatial segregation of proteins.
Assuntos
Evolução Molecular , Lisossomos/metabolismo , Microdomínios da Membrana/metabolismo , Fagossomos/metabolismo , Proteômica/métodos , Animais , Linhagem Celular , Detergentes/farmacologia , Lisossomos/efeitos dos fármacos , Microdomínios da Membrana/efeitos dos fármacos , Camundongos , Peptídeos/metabolismo , Fagossomos/efeitos dos fármacos , Proteoma/metabolismo , Reprodutibilidade dos Testes , Homologia de Sequência de Aminoácidos , Fatores de TempoRESUMO
Growth-arrest specific 6 (GAS6) is a secreted protein that acts as a ligand for TAM receptors (TYRO3, AXL and MERTK). In humans, GAS6 circulating levels and genetic variations in GAS6 are associated with hyperglycemia and increased risk of type 2 diabetes. However, the mechanisms by which GAS6 influences glucose metabolism are not understood. Here, we show that Gas6 deficiency in mice increases insulin sensitivity and protects from diet-induced insulin resistance. Conversely, increasing GAS6 circulating levels is sufficient to reduce insulin sensitivity in vivo. GAS6 inhibits the activation of the insulin receptor (IR) and reduces insulin response in muscle cells in vitro and in vivo. Mechanistically, AXL and IR form a complex, while GAS6 reprograms signaling pathways downstream of IR. This results in increased IR endocytosis following insulin treatment. This study contributes to a better understanding of the cellular and molecular mechanisms by which GAS6 and AXL influence insulin sensitivity.
RESUMO
The ADP-ribosylation factors (ARFs) and ARF-like (ARLs) GTPases serve as essential molecular switches governing a wide array of cellular processes. In this study, we utilized proximity-dependent biotin identification (BioID) to comprehensively map the interactome of 28 out of 29 ARF and ARL proteins in two cellular models. Through this approach, we identified ~3000 high-confidence proximal interactors, enabling us to assign subcellular localizations to the family members. Notably, we uncovered previously undefined localizations for ARL4D and ARL10. Clustering analyses further exposed the distinctiveness of the interactors identified with these two GTPases. We also reveal that the expression of the understudied member ARL14 is confined to the stomach and intestines. We identified phospholipase D1 (PLD1) and the ESCPE-1 complex, more precisely SNX1, as proximity interactors. Functional assays demonstrated that ARL14 can activate PLD1 in cellulo and is involved in cargo trafficking via the ESCPE-1 complex. Overall, the BioID data generated in this study provide a valuable resource for dissecting the complexities of ARF and ARL spatial organization and signaling.
RESUMO
The ubiquitin proteasome system performs the covalent attachment of lysine 48-linked polyubiquitin chains to substrate proteins, thereby targeting them for degradation, while deubiquitylating enzymes (DUBs) reverse this process. This posttranslational modification regulates key features both of innate and adaptative immunity, including antigen presentation, protein homeostasis and signal transduction. Here we show that loss of one of the most highly expressed DUBs, Otub1, results in changes in murine splenic B cell subsets, leading to a significant increase in marginal zone and transitional B cells and a concomitant decrease in follicular B cells. We demonstrate that Otub1 interacts with the γ-subunit of the heterotrimeric G protein, Gng2, and modulates its ubiquitylation status, thereby controlling Gng2 stability. Proximal mapping of Gng2 revealed an enrichment in partners associated with chemokine signaling, actin cytoskeleton and cell migration. In line with these findings, we show that Otub1-deficient B cells exhibit greater Ca2+ mobilization, F-actin polymerization and chemotactic responsiveness to Cxcl12, Cxcl13 and S1P in vitro, which manifests in vivo as altered localization of B cells within the spleen. Together, our data establishes Otub1 as a novel regulator of G-protein coupled receptor signaling in B cells, regulating their differentiation and positioning in the spleen.
Assuntos
Quimiotaxia de Leucócito , Enzimas Desubiquitinantes , Baço , Ubiquitina , Animais , Camundongos , Enzimas Desubiquitinantes/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Transdução de Sinais , Baço/metabolismo , Ubiquitina/metabolismo , Ubiquitinação , Cisteína Endopeptidases/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Linfócitos B/metabolismo , Quimiotaxia de Leucócito/genéticaRESUMO
Interstrand DNA cross-links (ICLs) represent complex lesions that compromise genomic stability. Several pathways have been involved in ICL repair, but the extent of factors involved in the resolution of ICL-induced DNA double-strand breaks (DSBs) remains poorly defined. Using CRISPR-based genomics, we identified FIGNL1 interacting regulator of recombination and mitosis (FIRRM) as a sensitizer of the ICL-inducing agent mafosfamide. Mechanistically, we showed that FIRRM, like its interactor Fidgetin like 1 (FIGNL1), contributes to the resolution of RAD51 foci at ICL-induced DSBs. While the stability of FIGNL1 and FIRRM is interdependent, expression of a mutant of FIRRM (∆WCF), which stabilizes the protein in the absence of FIGNL1, allows the resolution of RAD51 foci and cell survival, suggesting that FIRRM has FIGNL1-independent function during DNA repair. In line with this model, FIRRM binds preferentially single-stranded DNA in vitro, raising the possibility that it directly contributes to RAD51 disassembly by interacting with DNA. Together, our findings establish FIRRM as a promoting factor of ICL repair.
Assuntos
Reparo do DNA , Rad51 Recombinase , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Proteínas/genética , DNA/genética , MitoseRESUMO
Proximity-dependent biotinylation (BioID) screens are excellent tools to capture in cellulo interactomes for a large variety of baits, including transient and weak affinity interactions, as well as localization-specific proximity components, which are much harder to detect with conventional approaches. Here, we describe the major starting steps and a detailed protocol on how to perform BioID in mammalian cells. We also describe the mass spectrometry procedure and the bioinformatics pipeline for the data analysis. For complete details on the use and execution of this profile, please refer to Bagci et al. (2020).
Assuntos
Mapeamento de Interação de Proteínas , Proteínas , Animais , Biotinilação , Biologia Computacional , Mamíferos/metabolismo , Espectrometria de Massas/métodos , Mapeamento de Interação de Proteínas/métodos , Proteínas/metabolismoRESUMO
The essential roles of the endovacuolar system in health and disease call for the development of new tools allowing a better understanding of the complex molecular machinery involved in endocytic processes. We took advantage of the floating properties of small latex beads (sLB) on a discontinuous sucrose gradient to isolate highly purified endosomes following internalization of small latex beads in J774 macrophages and bone marrow-derived dendritic cells (DC). We particularly focused on the isolation of macrophages early endosomes and late endosomes/lysosomes (LE/LYS) as well as the isolation of LE/LYS from immature and lipopolysaccharide-activated (mature) DC. We subsequently performed a comparative analysis of their respective protein contents by MS. As expected, proteins already known to localize to the early endosomes were enriched in the earliest fraction of J774 endosomes, while proteins known to accumulate later in the process, such as hydrolases, were significantly enriched in the LE/LYS preparations. We next compared the LE/LYS protein contents of immature DC and mature DC, which are known to undergo massive reorganization leading to potent immune activation. The differences between the protein contents of endocytic organelles from macrophages and DC were underlined by focusing on previously poorly characterized biochemical pathways, which could have an unexpected but important role in the endosomal functions of these highly relevant immune cell types.
Assuntos
Células Dendríticas/citologia , Endossomos/metabolismo , Macrófagos/citologia , Proteínas/metabolismo , Proteoma/metabolismo , Animais , Medula Óssea/imunologia , Medula Óssea/metabolismo , Fracionamento Celular/métodos , Linhagem Celular , Centrifugação com Gradiente de Concentração , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Endocitose/efeitos dos fármacos , Endocitose/imunologia , Endossomos/química , Endossomos/imunologia , Lipopolissacarídeos/farmacologia , Lisossomos/química , Lisossomos/imunologia , Lisossomos/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Microesferas , Fagossomos/imunologia , Fagossomos/metabolismo , Proteínas/análise , Proteínas/classificação , Proteínas/imunologia , Proteoma/análise , Proteoma/classificação , Proteoma/imunologia , Sacarose/químicaRESUMO
Amoeba use phagocytosis to internalize bacteria as a source of nutrients, whereas multicellular organisms utilize this process as a defense mechanism to kill microbes and, in vertebrates, initiate a sustained immune response. By using a large-scale approach to identify and compare the proteome and phosphoproteome of phagosomes isolated from distant organisms, and by comparative analysis over 39 taxa, we identified an 'ancient' core of phagosomal proteins around which the immune functions of this organelle have likely organized. Our data indicate that a larger proportion of the phagosome proteome, compared with the whole cell proteome, has been acquired through gene duplication at a period coinciding with the emergence of innate and adaptive immunity. Our study also characterizes in detail the acquisition of novel proteins and the significant remodeling of the phagosome phosphoproteome that contributed to modify the core constituents of this organelle in evolution. Our work thus provides the first thorough analysis of the changes that enabled the transformation of the phagosome from a phagotrophic compartment into an organelle fully competent for antigen presentation.
Assuntos
Evolução Biológica , Modelos Biológicos , Fagossomos/fisiologia , Proteoma/fisiologia , Proteômica/métodos , Animais , Linhagem Celular , Análise por Conglomerados , Dictyostelium , Drosophila , Camundongos , Fagossomos/genética , Fagossomos/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Fosfoproteínas/fisiologia , Proteoma/química , Proteoma/metabolismo , Transdução de SinaisRESUMO
Macrophages are immune cells that function in the clearance of infectious particles. This process involves the engulfment of microbes into phagosomes where these particles are lysed and degraded. In the current study, we used a large scale quantitative proteomics approach to analyze the changes in protein abundance induced on phagosomes by interferon-gamma (IFN-gamma), an inflammatory cytokine that activates macrophages. Our analysis identified 167 IFN-gamma-modulated proteins on phagosomes of which more than 90% were up-regulated. The list of phagosomal proteins regulated by IFN-gamma includes proteins expected to alter phagosome maturation, enhance microbe degradation, trigger the macrophage immune response, and promote antigen loading on major histocompatibility complex (MHC) class I molecules. A dynamic analysis of IFN-gamma-sensitive proteins by Western blot indicated that newly formed phagosomes display a delayed proteolytic activity coupled to an increased recruitment of the MHC class I peptide-loading complex. These phagosomal conditions may favor antigen presentation by MHC class I molecules on IFN-gamma-activated macrophages.
Assuntos
Interferon gama/farmacologia , Macrófagos/imunologia , Fagossomos/imunologia , Proteoma/análise , Proteômica/métodos , Animais , Linhagem Celular , Cromatografia Líquida , Apresentação Cruzada/efeitos dos fármacos , Eletroforese em Gel Bidimensional , Antígenos de Histocompatibilidade Classe I/imunologia , Espectrometria de Massas , Camundongos , Fagossomos/química , Fagossomos/efeitos dos fármacosRESUMO
Polycomb Group proteins regulate gene expression by modifying chromatin. Polycomb Repressive Complex 1 (PRC1) has two activities: a ubiquitin ligase activity for histone H2A and a chromatin compacting activity. In Drosophila, the Posterior Sex Combs (PSC) subunit of PRC1 is central to both activities. The N-terminal of PSC assembles into PRC1, including partnering with dRING to form the ubiquitin ligase. The intrinsically disordered C-terminal region of PSC compacts chromatin and inhibits chromatin remodeling and transcription in vitro. Both regions of PSC are essential in vivo. To understand how these two activities may be coordinated in PRC1, we used crosslinking mass spectrometry to analyze the conformations of the C-terminal region of PSC in PRC1 and how they change on binding DNA. Crosslinking identifies interactions between the C-terminal region of PSC and the core of PRC1, including between N and C-terminal regions of PSC. New contacts and overall more compacted PSC C-terminal region conformations are induced by DNA binding. Protein footprinting of accessible lysine residues reveals an extended, bipartite candidate DNA/chromatin binding surface in the C-terminal region of PSC. Our data suggest a model in which DNA (or chromatin) follows a long path on the flexible disordered region of PSC. Intramolecular interactions of PSC detected by crosslinking can bring the high-affinity DNA/chromatin binding region close to the core of PRC1 without disrupting the interface between the ubiquitin ligase and the nucleosome. Our approach may be applicable to understanding the global organization of other large intrinsically disordered regions that bind nucleic acids.