RESUMEN
Amyotrophic Lateral Sclerosis (ALS) is a rare neurological disorder that causes degeneration of upper and lower motor neurons and their axons. ALS is mostly sporadic, but there are familial forms. In more than half of the familial forms, a pathogenic variant is found in one of the following genes: C9ORF72, SOD1, TDP-43, FUS, and VCP. SOD1 is the 2nd most common gene involved in genetic forms of ALS. Genotype-phenotype relationships are occasionally established in genetic forms of ALS associated with SOD1 mutations pathogenic variants. The c.281G > T (p.[G93V]) variant in SOD1 is associated with a rarely described and unexplained anticipation phenomenon. We report a large family from Martinique in whom ALS is associated with a c.281G > T (p.[G93V]) pathogenic variant in SOD1 and a statistically suggested anticipation. A whole-exome study and detection of CNVs (CoDESeq) from 3 affected members of this family revealed the presence of variants of uncertain signification (VUS) in other ALS genes. VUS in DCTN1 and NEFH were present in patients of the 2nd generation, and CNVs involving UBQLN2 and C21orf2 were found in the youngest case of the family.
Asunto(s)
Esclerosis Amiotrófica Lateral , Esclerosis Amiotrófica Lateral/genética , Proteínas de Unión al ADN/genética , Humanos , Martinica , Mutación/genética , Superóxido Dismutasa-1/genéticaRESUMEN
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.
Asunto(s)
Evolución Molecular , Lisosomas/metabolismo , Microdominios de Membrana/metabolismo , Fagosomas/metabolismo , Proteómica/métodos , Animales , Línea Celular , Detergentes/farmacología , Lisosomas/efectos de los fármacos , Microdominios de Membrana/efectos de los fármacos , Ratones , Péptidos/metabolismo , Fagosomas/efectos de los fármacos , Proteoma/metabolismo , Reproducibilidad de los Resultados , Homología de Secuencia de Aminoácido , Factores de TiempoRESUMEN
Phagosomes, by killing and degrading pathogens for antigen presentation, are organelles implicated in key aspects of innate and adaptive immunity. Although it has been well established that phagosomes consist of membranes from the plasma membrane, endosomes, and lysosomes, the notion that the endoplasmic reticulum (ER) membrane could play an important role in the formation of the phagosome is debated. However, a method to accurately estimate the contribution of potential source organelles and contaminants to the phagosome proteome has been lacking. Herein, we have developed a proteomic approach for objectively quantifying the contribution of various organelles to the early and late phagosomes by comparing these fractions to their total membrane and postnuclear supernatant of origin in the J774A.1 murine macrophage cell line. Using quantitative label-free mass spectrometry, the abundance of peptides corresponding to hundreds of proteins was estimated and attributed to one of five organelles (e.g. plasma membrane, endosomes/lysosomes, ER, Golgi, and mitochondria). These data in combination with a stable isotope labeling in cell culture method designed to detect potential contaminant sources revealed that the ER is part of the phagosomal membrane and contributes ≈ 20% of the early phagosome proteome. In addition, only a subset of ER proteins is recruited to the phagosome, suggesting that a specific subdomain(s) of the ER might be involved in phagocytosis. Western blotting and immunofluorescence substantially validated this conclusion; we were able to demonstrate that the fraction of the ER in which the ER marker GFP-KDEL accumulates is excluded from the phagosomes, whereas that containing the mVenus-Syntaxin 18 is recruited. These results highlight promising new avenues for the description of the pathogenic mechanisms used by Leishmania, Brucella, and Legionella spp., which thrive in ER-rich phagosomes.
Asunto(s)
Retículo Endoplásmico/química , Macrófagos/metabolismo , Fagosomas/química , Proteómica/métodos , Animales , Biomarcadores/análisis , Western Blotting , Línea Celular , Membrana Celular/química , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/ultraestructura , Endosomas/química , Endosomas/metabolismo , Endosomas/ultraestructura , Técnica del Anticuerpo Fluorescente , Aparato de Golgi/química , Aparato de Golgi/metabolismo , Aparato de Golgi/ultraestructura , Marcaje Isotópico , Lisosomas/química , Lisosomas/metabolismo , Lisosomas/ultraestructura , Macrófagos/citología , Macrófagos/ultraestructura , Espectrometría de Masas , Ratones , Oligopéptidos , Fagocitosis , Fagosomas/metabolismo , Fagosomas/ultraestructura , Plásmidos , Señales de Clasificación de Proteína , Proteínas Qa-SNARE , TransfecciónRESUMEN
Proteomics has significantly contributed to improve our understanding of cell structures and functions in the last decade. The possibility to identify large sets of proteins from minute amount of material, linked with the isolation of cellular organelles using various cell fractionation methods, has provided unique insights into the molecular mechanisms governing cell functions in health and disease. The success of this approach relies on the isolation of highly enriched cell fractions enabling the separation of organelles with minimal contamination by other cellular structures.
Asunto(s)
Orgánulos/química , Proteómica/métodos , Animales , Biomarcadores/análisis , Fraccionamiento Celular/métodos , Línea Celular , Interferón gamma/farmacología , Ratones , Orgánulos/efectos de los fármacos , Proteoma/análisisRESUMEN
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.
Asunto(s)
Células Dendríticas/citología , Endosomas/metabolismo , Macrófagos/citología , Proteínas/metabolismo , Proteoma/metabolismo , Animales , Médula Ósea/inmunología , Médula Ósea/metabolismo , Fraccionamiento Celular/métodos , Línea Celular , Centrifugación por Gradiente de Densidad , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Endocitosis/efectos de los fármacos , Endocitosis/inmunología , Endosomas/química , Endosomas/inmunología , Lipopolisacáridos/farmacología , Lisosomas/química , Lisosomas/inmunología , Lisosomas/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Ratones , Microesferas , Fagosomas/inmunología , Fagosomas/metabolismo , Proteínas/análisis , Proteínas/clasificación , Proteínas/inmunología , Proteoma/análisis , Proteoma/clasificación , Proteoma/inmunología , Sacarosa/químicaRESUMEN
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.
Asunto(s)
Interferón gamma/farmacología , Macrófagos/inmunología , Fagosomas/inmunología , Proteoma/análisis , Proteómica/métodos , Animales , Línea Celular , Cromatografía Liquida , Reactividad Cruzada/efectos de los fármacos , Electroforesis en Gel Bidimensional , Antígenos de Histocompatibilidad Clase I/inmunología , Espectrometría de Masas , Ratones , Fagosomas/química , Fagosomas/efectos de los fármacosRESUMEN
The small GTPase rab5 has been shown to play key roles in the function of both endocytic and phagocytic organelles. Although these organelles share several additional common features, different processes have been proposed to explain their biogenesis. In the present study, we provide evidence that lysosome biogenesis involves mechanisms similar to those previously described for the formation of phagolysosomes. Transient interactions ('kiss and run') between endocytic organelles are shown to occur during lysosome biogenesis. These interactions are regulated initially by the GTPase activity of rab5, as demonstrated by the loss of size-selective fusion between endosomes in cells expressing a GTPase-deficient mutant of rab5. Endocytic compartments in these cells sequentially display properties of early and late endosomes. However, the formation of lysosomes and the sorting of endocytic solute materials to small electron dense vacuoles are not affected by the rab5 mutation. Together, our results indicate that endosome maturation occurs during the early part of lysosome biogenesis. This process involves transient fusion events regulated, in part, by the small GTPase rab5.
Asunto(s)
Endosomas/metabolismo , Lisosomas/metabolismo , Macrólidos , Fusión de Membrana , Fagosomas/metabolismo , Proteínas de Unión al GTP rab5/metabolismo , Animales , Antibacterianos/farmacología , Línea Celular , Endosomas/efectos de los fármacos , Endosomas/ultraestructura , Macrófagos/fisiología , Macrófagos/ultraestructura , Glicoproteínas de Membrana/metabolismo , Microscopía Fluorescente , Microscopía Inmunoelectrónica , Mutación , Fagosomas/ultraestructura , Factores de Tiempo , Proteínas de Unión al GTP rab5/genéticaRESUMEN
Phagocytosis is a key aspect of our innate ability to fight infectious diseases. In this study, we have found that fusion of the endoplasmic reticulum (ER) with the macrophage plasmalemma, underneath phagocytic cups, is a source of membrane for phagosome formation in macrophages. Successive waves of ER become associated with maturing phagosomes during phagolysosome biogenesis. Thus, the ER appears to possess unexpectedly pluripotent fusion properties. ER-mediated phagocytosis is regulated in part by phosphatidylinositol 3-kinase and used for the internalization of inert particles and intracellular pathogens, regardless of their final trafficking in the host. In neutrophils, where pathogens are rapidly killed, the ER is not used as a major source of membrane for phagocytosis. We propose that intracellular pathogens have evolved to adapt and exploit ER-mediated phagocytosis to avoid destruction in host cells.