RESUMO
SLC45A2 encodes a putative transporter expressed primarily in pigment cells. SLC45A2 mutations cause oculocutaneous albinism type 4 (OCA4) and polymorphisms are associated with pigmentation variation, but the localization, function, and regulation of SLC45A2 and its variants remain unknown. We show that SLC45A2 localizes to a cohort of mature melanosomes that only partially overlaps with the cohort expressing the chloride channel OCA2. SLC45A2 expressed ectopically in HeLa cells localizes to lysosomes and raises lysosomal pH, suggesting that in melanocytes SLC45A2 expression, like OCA2 expression, results in the deacidification of maturing melanosomes to support melanin synthesis. Interestingly, OCA2 overexpression compensates for loss of SLC45A2 expression in pigmentation. Analyses of SLC45A2- and OCA2-deficient mouse melanocytes show that SLC45A2 likely functions later during melanosome maturation than OCA2. Moreover, the light skin-associated SLC45A2 allelic F374 variant restores only moderate pigmentation to SLC45A2-deficient melanocytes due to rapid proteasome-dependent degradation resulting in lower protein expression levels in melanosomes than the dark skin-associated allelic L374 variant. Our data suggest that SLC45A2 maintains melanosome neutralization that is initially orchestrated by transient OCA2 activity to support melanization at late stages of melanosome maturation, and that a common allelic variant imparts reduced activity due to protein instability.
Assuntos
Antígenos de Neoplasias/metabolismo , Melanócitos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Pigmentação da Pele/fisiologia , Animais , Antígenos de Neoplasias/fisiologia , Proteínas de Transporte/metabolismo , Linhagem Celular , Canais de Cloreto/metabolismo , Células HeLa , Humanos , Lisossomos/metabolismo , Masculino , Melanossomas/metabolismo , Proteínas de Membrana Transportadoras/fisiologia , Camundongos , Pigmentação/fisiologia , Estabilidade Proteica , Pele/metabolismoRESUMO
Bacterial pathogens that compromise phagosomal membranes stimulate inflammasome assembly in the cytosol, but the molecular mechanisms by which membrane dynamics regulate inflammasome activity are poorly characterized. We show that in murine dendritic cells (DCs), the endosomal adaptor protein AP-3 -which optimizes toll-like receptor signaling from phagosomes-sustains inflammasome activation by particulate stimuli. AP-3 independently regulates inflammasome positioning and autophagy induction, together resulting in delayed inflammasome inactivation by autophagy in response to Salmonella Typhimurium (STm) and other particulate stimuli specifically in DCs. AP-3-deficient DCs, but not macrophages, hyposecrete IL-1ß and IL-18 in response to particulate stimuli in vitro, but caspase-1 and IL-1ß levels are restored by silencing autophagy. Concomitantly, AP-3-deficient mice exhibit higher mortality and produce less IL-1ß, IL-18, and IL-17 than controls upon oral STm infection. Our data identify a novel link between phagocytosis, inflammasome activity and autophagy in DCs, potentially explaining impaired antibacterial immunity in AP-3-deficient patients.
Assuntos
Complexo 3 de Proteínas Adaptadoras/deficiência , Células Dendríticas/imunologia , Células Dendríticas/microbiologia , Inflamassomos/imunologia , Imunidade Adaptativa , Complexo 3 de Proteínas Adaptadoras/genética , Complexo 3 de Proteínas Adaptadoras/imunologia , Animais , Autofagia/imunologia , Células Dendríticas/patologia , Feminino , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Interleucina-17/biossíntese , Interleucina-18/biossíntese , Interleucina-1beta/biossíntese , Interleucina-1beta/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas NLR/genética , Proteínas NLR/imunologia , Fagocitose , Salmonelose Animal/imunologia , Salmonelose Animal/patologia , Salmonella typhimurium/imunologia , Salmonella typhimurium/patogenicidade , Ativação TranscricionalRESUMO
Intracellular ion channels are essential regulators of organellar and cellular function, yet the molecular identity and physiological role of many of these channels remains elusive. In particular, no ion channel has been characterized in melanosomes, organelles that produce and store the major mammalian pigment melanin. Defects in melanosome function cause albinism, characterized by vision and pigmentation deficits, impaired retinal development, and increased susceptibility to skin and eye cancers. The most common form of albinism is caused by mutations in oculocutaneous albinism II (OCA2), a melanosome-specific transmembrane protein with unknown function. Here we used direct patch-clamp of skin and eye melanosomes to identify a novel chloride-selective anion conductance mediated by OCA2 and required for melanin production. Expression of OCA2 increases organelle pH, suggesting that the chloride channel might regulate melanin synthesis by modulating melanosome pH. Thus, a melanosomal anion channel that requires OCA2 is essential for skin and eye pigmentation.
Assuntos
Canais de Cloreto/metabolismo , Espaço Intracelular/metabolismo , Pigmentação , Albinismo Oculocutâneo/metabolismo , Animais , Ânions/metabolismo , Anuros , Proteínas de Transporte/metabolismo , Linhagem Celular , Endossomos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Lisossomos/metabolismo , Melanossomas/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Mutação/genéticaRESUMO
The molecular underpinnings of the oocyte-to-embryo transition are poorly understood. Here we show that two protein tyrosine phosphatase-like (PTPL) family proteins, EGG-4 and EGG-5, are required for key events of the oocyte-to-embryo transition in Caenorhabditis elegans. The predicted EGG-4 and EGG-5 amino acid sequences are 99% identical and their functions are redundant. In embryos lacking EGG-4 and EGG-5, we observe defects in meiosis, polar body formation, the block to polyspermy, F-actin dynamics, and eggshell deposition. During oogenesis, EGG-4 and EGG-5 assemble at the oocyte cortex with the previously identified regulators or effectors of the oocyte-to-embryo transition EGG-3, CHS-1, and MBK-2 [1, 2]. All of these molecules share a complex interdependence with regards to their dynamics and subcellular localization. Shortly after fertilization, EGG-4 and EGG-5 are required to properly coordinate a redistribution of CHS-1 and EGG-3 away from the cortex during meiotic anaphase I. Therefore, EGG-4 and EGG-5 are not only required for critical events of the oocyte-to-embryo transition but also link the dynamics of the regulatory machinery with the advancing cell cycle.