RESUMO
Many eukaryotic cells ingest extracellular particles in a process termed phagocytosis which entails the generation of a new intracellular compartment, the phagosome. Phagosomes change their composition over time and this maturation process culminates in their fusion with acidic, hydrolase-rich lysosomes. During the maturation process, degradation and, when applicable, killing of the cargo may ensue. Many of the events that are pathologically relevant depend on strong acidification of phagosomes by the 'vacuolar' ATPase (V-ATPase). This protein complex acidifies the lumen of some intracellular compartments at the expense of ATP hydrolysis. We discuss here the roles and importance of V-ATPase in intracellular trafficking, its distribution, inhibition and activities, its role in the defense against microorganisms and the counteractivities of pathogens.
Assuntos
Lisossomos/metabolismo , Fagossomos/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , Anti-Infecciosos , Autofagia , Bactérias/crescimento & desenvolvimento , Bactérias/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Lisossomos/química , Lisossomos/microbiologia , Fusão de Membrana , Fagossomos/química , Fagossomos/microbiologia , Transporte Proteico , ATPases Vacuolares Próton-Translocadoras/antagonistas & inibidoresRESUMO
The vacuolar H(+)-ATPase (v-ATPase) complex is instrumental in establishing and maintaining acidification of some cellular compartments, thereby ensuring their functionality. Recently it has been proposed that the transmembrane V0 sector of v-ATPase and its a-subunits promote membrane fusion in the endocytic and exocytic pathways independent of their acidification functions. Here, we tested if such a proton-pumping independent role of v-ATPase also applies to phagosome-lysosome fusion. Surprisingly, endo(lyso)somes in mouse embryonic fibroblasts lacking the V0 a3 subunit of the v-ATPase acidified normally, and endosome and lysosome marker proteins were recruited to phagosomes with similar kinetics in the presence or absence of the a3 subunit. Further experiments used macrophages with a knockdown of v-ATPase accessory protein 2 (ATP6AP2) expression, resulting in a strongly reduced level of the V0 sector of the v-ATPase. However, acidification appeared undisturbed, and fusion between latex bead-containing phagosomes and lysosomes, as analyzed by electron microscopy, was even slightly enhanced, as was killing of non-pathogenic bacteria by V0 mutant macrophages. Pharmacologically neutralized lysosome pH did not affect maturation of phagosomes in mouse embryonic cells or macrophages. Finally, locking the two large parts of the v-ATPase complex together by the drug saliphenylhalamide A did not inhibit in vitro and in cellulo fusion of phagosomes with lysosomes. Hence, our data do not suggest a fusion-promoting role of the v-ATPase in the formation of phagolysosomes.
Assuntos
Lisossomos/metabolismo , Fagossomos/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Receptores de Superfície Celular/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , Animais , Escherichia coli/metabolismo , Feminino , Fibroblastos/metabolismo , Concentração de Íons de Hidrogênio , Macrófagos/citologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Masculino , Fusão de Membrana , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Microscopia Eletrônica , Microscopia de Fluorescência , Frações Subcelulares/metabolismoRESUMO
Mast cell (MC) activation through the high-affinity IgE receptor FcεRI leads to the release of mediators involved in immediate-type allergic reactions. Although Abs against the tetraspanins CD63 and CD81 inhibit FcεRI-induced MC degranulation, the intrinsic role of these molecules in FcεRI-induced MC activation is unknown. In MCs, CD63 is expressed at the cell surface and in lysosomes (particularly secretory lysosomes that contain allergic mediators). In this study, we investigated the role of CD63 in MC using a CD63 knockout mouse model. CD63-deficiency did not affect in vivo MC numbers and tissue distribution. Bone marrow-derived MC developed normally in the absence of CD63 protein. However, CD63-deficient bone marrow-derived MC showed a significant decrease in FcεRI-mediated degranulation, but not PMA/ionomycin-induced degranulation, as shown by ß-hexosaminidase release assays. The secretion of TNF-α, which is both released from granules and synthesized de novo upon MC activation, was also decreased. IL-6 secretion and production of the lipid mediator leukotriene C4 were unaffected. There were no ultrastructural differences in granule content and morphology, late endosomal/lysosomal marker expression, FcεRI-induced global tyrosine phosphorylation, and Akt phosphorylation. Finally, local reconstitution in genetically MC-deficient Kit(w/w-v) mice was unaffected by the absence of CD63. However, the sites reconstituted with CD63-deficient MC developed significantly attenuated cutaneous anaphylactic reactions. These findings demonstrate that the absence of CD63 results in a significant decrease of MC degranulation, which translates into a reduction of acute allergic reactions in vivo, thus identifying CD63 as an important component of allergic inflammation.
Assuntos
Anafilaxia/imunologia , Degranulação Celular/imunologia , Mastócitos/imunologia , Tetraspanina 30/imunologia , Transferência Adotiva , Anafilaxia/metabolismo , Animais , Citocinas/biossíntese , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Técnica Indireta de Fluorescência para Anticorpo , Immunoblotting , Imunoglobulina E/imunologia , Mastócitos/metabolismo , Camundongos , Camundongos Knockout , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Tetraspanina 30/metabolismoRESUMO
The vacuolar-type H+-translocating ATPase (v-H+-ATPase) has been implicated in the amino acid-dependent activation of the mechanistic target of rapamycin complex 1 (MTORC1), an important regulator of macroautophagy. To reveal the mechanistic links between the v-H+-ATPase and MTORC1, we destablilized v-H+-ATPase complexes in mouse liver cells by induced deletion of the essential chaperone ATP6AP2. ATP6AP2-mutants are characterized by massive accumulation of endocytic and autophagic vacuoles in hepatocytes. This cellular phenotype was not caused by a block in endocytic maturation or an impaired acidification. However, the degradation of LC3-II in the knockout hepatocytes appeared to be reduced. When v-H+-ATPase levels were decreased, we observed lysosome association of MTOR and normal signaling of MTORC1 despite an increase in autophagic marker proteins. To better understand why MTORC1 can be active when v-H+-ATPase is depleted, the activation of MTORC1 was analyzed in ATP6AP2-deficient fibroblasts. In these cells, very little amino acid-elicited activation of MTORC1 was observed. In contrast, insulin did induce MTORC1 activation, which still required intracellular amino acid stores. These results suggest that in vivo the regulation of macroautophagy depends not only on v-H+-ATPase-mediated regulation of MTORC1.
Assuntos
Autofagia , Fígado/enzimologia , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Receptores de Superfície Celular/metabolismo , Vacúolos/enzimologia , Aminoácidos/farmacologia , Animais , Autofagia/efeitos dos fármacos , Células Cultivadas , Embrião de Mamíferos/citologia , Endocitose/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/enzimologia , Insulina/farmacologia , Fígado/efeitos dos fármacos , Fígado/ultraestrutura , Lisossomos/efeitos dos fármacos , Camundongos Knockout , ATPases Translocadoras de Prótons/deficiência , Receptores de Superfície Celular/deficiência , Vacúolos/efeitos dos fármacosRESUMO
Endolysosomal organelles play a key role in trafficking, breakdown and receptor-mediated recycling of different macromolecules such as low-density lipoprotein (LDL)-cholesterol, epithelial growth factor (EGF) or transferrin. Here we examine the role of two-pore channel (TPC) 2, an endolysosomal cation channel, in these processes. Embryonic mouse fibroblasts and hepatocytes lacking TPC2 display a profound impairment of LDL-cholesterol and EGF/EGF-receptor trafficking. Mechanistically, both defects can be attributed to a dysfunction of the endolysosomal degradation pathway most likely on the level of late endosome to lysosome fusion. Importantly, endolysosomal acidification or lysosomal enzyme function are normal in TPC2-deficient cells. TPC2-deficient mice are highly susceptible to hepatic cholesterol overload and liver damage consistent with non-alcoholic fatty liver hepatitis. These findings indicate reduced metabolic reserve of hepatic cholesterol handling. Our results suggest that TPC2 plays a crucial role in trafficking in the endolysosomal degradation pathway and, thus, is potentially involved in the homoeostatic control of many macromolecules and cell metabolites.
Assuntos
Canais de Cálcio/genética , Fígado Gorduroso/genética , Fígado Gorduroso/fisiopatologia , Animais , Transporte Biológico/genética , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Colesterol/metabolismo , LDL-Colesterol/metabolismo , Endossomos/metabolismo , Receptores ErbB/metabolismo , Fígado Gorduroso/etiologia , Predisposição Genética para Doença , Lisossomos/metabolismo , Masculino , Camundongos KnockoutRESUMO
The aryl-hydrocarbon receptor-interacting protein (AIP) interacts with several protein binding partners and has been associated with pituitary tumor development. Here, we report nearly complete (1)H, (13)C and (15)N chemical shift assignments for the N-terminal AIP(2-166) segment, which has been predicted to represent a FKBP-type PPIase domain. Sequence alignment with the prototypic FKBP12, however, reveals disagreements between the AIP chemical shift index consensus and the corresponding FKBP12 secondary structure elements.