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1.
Dis Model Mech ; 16(7)2023 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-37401371

RESUMO

Oxidative stress has been implicated in the pathogenesis of age-related macular degeneration, the leading cause of blindness in older adults, with retinal pigment epithelium (RPE) cells playing a key role. To better understand the cytotoxic mechanisms underlying oxidative stress, we used cell culture and mouse models of iron overload, as iron can catalyze reactive oxygen species formation in the RPE. Iron-loading of cultured induced pluripotent stem cell-derived RPE cells increased lysosomal abundance, impaired proteolysis and reduced the activity of a subset of lysosomal enzymes, including lysosomal acid lipase (LIPA) and acid sphingomyelinase (SMPD1). In a liver-specific Hepc (Hamp) knockout murine model of systemic iron overload, RPE cells accumulated lipid peroxidation adducts and lysosomes, developed progressive hypertrophy and underwent cell death. Proteomic and lipidomic analyses revealed accumulation of lysosomal proteins, ceramide biosynthetic enzymes and ceramides. The proteolytic enzyme cathepsin D (CTSD) had impaired maturation. A large proportion of lysosomes were galectin-3 (Lgals3) positive, suggesting cytotoxic lysosomal membrane permeabilization. Collectively, these results demonstrate that iron overload induces lysosomal accumulation and impairs lysosomal function, likely due to iron-induced lipid peroxides that can inhibit lysosomal enzymes.


Assuntos
Sobrecarga de Ferro , Proteômica , Camundongos , Animais , Estresse Oxidativo , Lisossomos/metabolismo , Ferro/metabolismo , Sobrecarga de Ferro/metabolismo , Sobrecarga de Ferro/patologia , Células Epiteliais/metabolismo , Pigmentos da Retina/metabolismo , Epitélio Pigmentado da Retina/metabolismo
2.
PLoS One ; 13(10): e0205871, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30335797

RESUMO

The oral bacterium, Aggregatibacter actinomycetemcomitans, which is associated with localized aggressive periodontitis, as well as systemic infections including endocarditis, produces numerous virulence factors, including a repeats-in-toxin (RTX) protein called leukotoxin (LtxA), which kills human immune cells. The strains of A. actinomycetemcomitans most closely associated with disease have been shown to produce the most LtxA, suggesting that LtxA plays a significant role in the virulence of this organism. LtxA, like many of the RTX toxins, can be divided into four functional domains: an N-terminal hydrophobic domain, which contains a significant fraction of hydrophobic residues and has been proposed to play a role in the membrane interaction of the toxin; the central domain, which contains two lysine residues that are the sites of post-translational acylation; the repeat domain that is characteristic of the RTX toxins, and a C-terminal domain thought to be involved in secretion. In its initial interaction with the host cell, LtxA must bind to both cholesterol and an integrin receptor, lymphocyte function-associated antigen-1 (LFA-1). While both interactions are essential for toxicity, the domains of LtxA involved remain unknown. We therefore undertook a series of experiments, including tryptophan quenching and trypsin digestion, to characterize the structure of LtxA upon interaction with membranes of various lipid compositions. Our results demonstrate that LtxA adopts a U-shaped conformation in the membrane, with the N- and C-terminal domains residing outside of the membrane.


Assuntos
Aggregatibacter actinomycetemcomitans/química , Proteínas de Bactérias/química , Colesterol/química , Proteínas Hemolisinas/química , Antígeno-1 Associado à Função Linfocitária/química , Fatores de Virulência/química , Aggregatibacter actinomycetemcomitans/crescimento & desenvolvimento , Aggregatibacter actinomycetemcomitans/patogenicidade , Sequência de Aminoácidos , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Colesterol/metabolismo , Dimiristoilfosfatidilcolina/química , Dimiristoilfosfatidilcolina/metabolismo , Proteínas Hemolisinas/isolamento & purificação , Proteínas Hemolisinas/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Células Jurkat , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Lipossomos/química , Lipossomos/metabolismo , Antígeno-1 Associado à Função Linfocitária/metabolismo , Fosfatidiletanolaminas/química , Fosfatidiletanolaminas/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Proteólise , Tripsina/química , Fatores de Virulência/isolamento & purificação , Fatores de Virulência/metabolismo
3.
Autophagy ; 14(10): 1796-1817, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29979914

RESUMO

Treatment of rats with the cholesterol pathway inhibitor AY9944 produces an animal model of Smith-Lemli-Opitz syndrome (SLOS), an autosomal recessive disease caused by defective cholesterol synthesis. This SLOS rat model undergoes progressive and irreversible degeneration of the neural retina, with associated pathological features of the retinal pigmented epithelium (RPE). Here, we provide further insights into the mechanism involved in the RPE pathology. In the SLOS rat model, markedly increased RPE apical autofluorescence is observed, compared to untreated animals, which correlates with increased levels of A2E and other bisretinoids. Utilizing cultured human induced pluripotent stem cell (iPSC)- derived SLOS RPE cells, we found significantly elevated steady-state levels of 7-dehydrocholesterol (7DHC) and decreased cholesterol levels (key biochemical hallmarks of SLOS). Western blot analysis revealed altered levels of the macroautophagy/autophagy markers MAP1LC3B-II and SQSTM1/p62, and build-up of ubiquitinated proteins. Accumulation of immature autophagosomes was accompanied by inefficient degradation of phagocytized, exogenously supplied retinal rod outer segments (as evidenced by persistence of the C-terminal 1D4 epitope of RHO [rhodopsin]) in SLOS RPE compared to iPSC-derived normal human control. SLOS RPE cells exhibited lysosomal pH levels and CTSD activity within normal physiological limits, thus discounting the involvement of perturbed lysosomal function. Furthermore, 1D4-positive phagosomes that accumulated in the RPE in both pharmacological and genetic rodent models of SLOS failed to fuse with lysosomes. Taken together, these observations suggest that defective phagosome maturation underlies the observed RPE pathology. The potential relevance of these findings to SLOS and the requirement of cholesterol for phagosome maturation are discussed.


Assuntos
Fagossomos/metabolismo , Epitélio Pigmentado da Retina/patologia , Síndrome de Smith-Lemli-Opitz/patologia , Animais , Biomarcadores/metabolismo , Catepsina D/metabolismo , Bovinos , Técnicas de Cultura de Células , Desidrocolesteróis/metabolismo , Modelos Animais de Doenças , Humanos , Lisossomos/metabolismo , Fusão de Membrana , Fagocitose , Biossíntese de Proteínas , Ratos , Epitélio Pigmentado da Retina/metabolismo , Retinoides/metabolismo , Segmento Externo da Célula Bastonete/metabolismo , Síndrome de Smith-Lemli-Opitz/genética , Transcrição Gênica , Proteínas Ubiquitinadas/metabolismo , Dicloridrato de trans-1,4-Bis(2-clorobenzaminometil)ciclo-hexano
4.
Cell Signal ; 26(5): 968-78, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24407175

RESUMO

Phagocytosis of shed photoreceptor outer segments by the retinal pigment epithelium (RPE) is critical for maintenance of visual function. Because changes in intracellular Ca(2+) regulate phagocytosis, we studied in vitro the impact of different ion channels in addition to mice deficient for Cav1.3 L-type Ca(2+) channels (Ca1.3(-/-)) and maxiK Ca(2+)-dependent K(+) channels (BK(-/-)). The knockdown of Bestrophin-1 protein, a regulator of intracellular Ca(2+) homeostasis, affected phagocytosis in porcine RPE cultures. Blockage of voltage-gated L-type channels by (+)BayK8644 inhibitor reduced phagocytosis in vitro, in contrast L-type activation by (-)BayK8644 had no impact. The expression rate of Cav1.3, the predominant L-type Ca(2+) channel in RPE cells, varied at different times of day. CaV1.3(-/-) RPE lacked peak phagocytic activity following morning photoreceptor shedding in wild-type RPE and retained a higher number of phagosomes at a later time of day. The BK-channel blocker paxilline lowered phagocytosis in RPE cultures in a concentration-dependent manner. BK(-/-) RPE in vivo retained phagocytic capability but this activity, which is normally well synchronized with circadian photoreceptor shedding, shifted out of phase. Retinae of older BK(-/-) mice showed shortened photoreceptor outer segments and diminished rhodopsin content. Store-operated Ca(2+) channels Orai-1 did not affect phagocytosis in cultured RPE. TRPV channel inhibition by ruthenium-red reduced phagocytosis, whereas activation at high concentrations of 2-APB increased phagocytosis. Our data demonstrate essential roles for bestrophin-1, BK, TRPV and L-type channels in regulating retinal phagocytosis. These data indicate further the importance of BK and CaV1.3 for rhythmic phagocytic activity synchronized with photoreceptor shedding.


Assuntos
Canais de Cálcio Tipo L/metabolismo , Canais de Cloreto/metabolismo , Éster Metílico do Ácido 3-Piridinacarboxílico, 1,4-Di-Hidro-2,6-Dimetil-5-Nitro-4-(2-(Trifluormetil)fenil)/farmacologia , Animais , Agonistas dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/química , Canais de Cálcio Tipo L/genética , Células Cultivadas , Canais de Cloreto/antagonistas & inibidores , Canais de Cloreto/genética , Canais de Potássio Ativados por Cálcio de Condutância Alta/deficiência , Canais de Potássio Ativados por Cálcio de Condutância Alta/genética , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fagocitose/efeitos dos fármacos , Interferência de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/metabolismo , Rodopsina/metabolismo , Suínos , Canais de Cátion TRPV/metabolismo
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