Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 6 de 6
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
2.
Proc Natl Acad Sci U S A ; 119(2)2022 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-34992143

RESUMO

Low-density lipoprotein (LDL) delivers cholesterol to mammalian cells through receptor-mediated endocytosis. The LDL cholesterol is liberated in lysosomes and transported to the plasma membrane (PM) and from there to the endoplasmic reticulum (ER). Excess ER cholesterol is esterified with a fatty acid for storage as cholesteryl esters. Recently, we showed that PM-to-ER transport of LDL cholesterol requires phosphatidylserine (PS). Others showed that PM-to-ER transport of cholesterol derived from other sources requires Asters (also called GRAMD1s), a family of three ER proteins that bridge between the ER and PM by binding to PS. Here, we use a cholesterol esterification assay and other measures of ER cholesterol delivery to demonstrate that Asters participate in PM-to-ER transport of LDL cholesterol in Chinese hamster ovary cells. Knockout of the gene encoding PTDSS1, the major PS-synthesizing enzyme, lowered LDL-stimulated cholesterol esterification by 85%, whereas knockout of all three Aster genes lowered esterification by 65%. The reduction was even greater (94%) when the genes encoding PTDSS1 and the three Asters were knocked out simultaneously. We conclude that Asters participate in LDL cholesterol delivery from PM to ER, and their action depends in large part, but not exclusively, on PS. The data also indicate that PS participates in another delivery pathway, so far undefined, that is independent of Asters.


Assuntos
LDL-Colesterol/metabolismo , Proteínas de Membrana/metabolismo , Fosfatidilserinas/metabolismo , Animais , Transporte Biológico , Células CHO , Membrana Celular/metabolismo , Colesterol/metabolismo , Ésteres do Colesterol/metabolismo , Cricetinae , Cricetulus , Endocitose , Retículo Endoplasmático/metabolismo , Lisossomos/metabolismo
3.
Proc Natl Acad Sci U S A ; 117(31): 18521-18529, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32690708

RESUMO

Animal cells acquire cholesterol from receptor-mediated uptake of low-density lipoprotein (LDL), which releases cholesterol in lysosomes. The cholesterol moves to the endoplasmic reticulum (ER), where it inhibits production of LDL receptors, completing a feedback loop. Here we performed a CRISPR-Cas9 screen in human SV589 cells for genes required for LDL-derived cholesterol to reach the ER. We identified the gene encoding PTDSS1, an enzyme that synthesizes phosphatidylserine (PS), a phospholipid constituent of the inner layer of the plasma membrane (PM). In PTDSS1-deficient cells where PS is low, LDL cholesterol leaves lysosomes but fails to reach the ER, instead accumulating in the PM. The addition of PS restores cholesterol transport to the ER. We conclude that LDL cholesterol normally moves from lysosomes to the PM. When the PM cholesterol exceeds a threshold, excess cholesterol moves to the ER in a process requiring PS. In the ER, excess cholesterol acts to reduce cholesterol uptake, preventing toxic cholesterol accumulation. These studies reveal that one lipid-PS-controls the movement of another lipid-cholesterol-between cell membranes. We relate these findings to recent evidence indicating that PM-to-ER cholesterol transport is mediated by GRAMD1/Aster proteins that bind PS and cholesterol.


Assuntos
Membrana Celular/metabolismo , LDL-Colesterol/metabolismo , Retículo Endoplasmático/metabolismo , Lisossomos/metabolismo , Fosfatidilserinas/metabolismo , Animais , Transporte Biológico , Linhagem Celular , Colesterol/metabolismo , Humanos
4.
Carbohydr Res ; 456: 24-29, 2018 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-29247910

RESUMO

Properties of di(triethylene glycol monomethyl ether) squarate relevant to conjugation of carbohydrates to proteins have been reinvestigated and compared with those of dimethyl squarate. It is concluded that the commercially available, crystalline dimethyl squarate remains the most convenient and efficient reagent for conjugation of amine-containing carbohydrates to proteins by a two-step or one-pot conjugation protocol.


Assuntos
Carboidratos/química , Ciclobutanos/química , Ésteres/química , Glicoconjugados/química , Proteínas/química , Hidrólise
5.
Proc Natl Acad Sci U S A ; 114(34): 9116-9121, 2017 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-28784760

RESUMO

Niemann-Pick C1 (NPC1) and NPC2 proteins are indispensable for the export of LDL-derived cholesterol from late endosomes. Mutations in these proteins result in Niemann-Pick type C disease, a lysosomal storage disease. Despite recent reports of the NPC1 structure depicting its overall architecture, the function of its C-terminal luminal domain (CTD) remains poorly understood even though 45% of NPC disease-causing mutations are in this domain. Here, we report a crystal structure at 3.3 Å resolution of NPC1* (residues 314-1,278), which-in contrast to previous lower resolution structures-features the entire CTD well resolved. Notably, all eight cysteines of the CTD form four disulfide bonds, one of which (C909-C914) enforces a specific loop that in turn mediates an interaction with a loop of the N-terminal domain (NTD). Importantly, this loop and its interaction with the NTD were not observed in any previous structures due to the lower resolution. Our mutagenesis experiments highlight the physiological relevance of the CTD-NTD interaction, which might function to keep the NTD in the proper orientation for receiving cholesterol from NPC2. Additionally, this structure allows us to more precisely map all of the disease-causing mutations, allowing future molecular insights into the pathogenesis of NPC disease.


Assuntos
Proteínas de Transporte/metabolismo , LDL-Colesterol/metabolismo , Endossomos/metabolismo , Glicoproteínas de Membrana/metabolismo , Sítios de Ligação/genética , Transporte Biológico/genética , Proteínas de Transporte/química , Proteínas de Transporte/genética , Cristalografia por Raios X , Cisteína/química , Cisteína/genética , Cisteína/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Modelos Moleculares , Mutação , Proteína C1 de Niemann-Pick , Doença de Niemann-Pick Tipo C/genética , Doença de Niemann-Pick Tipo C/metabolismo , Domínios Proteicos
6.
Proc Natl Acad Sci U S A ; 114(1): 89-94, 2017 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-27994139

RESUMO

Niemann-Pick C1 (NPC1), a membrane protein of lysosomes, is required for the export of cholesterol derived from receptor-mediated endocytosis of LDL. Lysosomal cholesterol export is reportedly inhibited by itraconazole, a triazole that is used as an antifungal drug [Xu et al. (2010) Proc Natl Acad Sci USA 107:4764-4769]. Here we show that posaconazole, another triazole, also blocks cholesterol export from lysosomes. We prepared P-X, a photoactivatable cross-linking derivative of posaconazole. P-X cross-linked to NPC1 when added to intact cells. Cross-linking was inhibited by itraconazole but not by ketoconazole, an imidazole that does not block cholesterol export. Cross-linking of P-X was also blocked by U18666A, a compound that has been shown to bind to NPC1 and inhibit cholesterol export. P-X also cross-linked to purified NPC1 that was incorporated into lipid bilayer nanodiscs. In this in vitro system, cross-linking of P-X was inhibited by itraconazole, but not by U18666A. P-X cross-linking was not prevented by deletion of the N-terminal domain of NPC1, which contains the initial binding site for cholesterol. In contrast, P-X cross-linking was reduced when NPC1 contained a point mutation (P691S) in its putative sterol-sensing domain. We hypothesize that the sterol-sensing domain has a binding site that can accommodate structurally different ligands.


Assuntos
Transporte Biológico/genética , Proteínas de Transporte/metabolismo , Colesterol/metabolismo , Lisossomos/metabolismo , Glicoproteínas de Membrana/metabolismo , Triazóis/farmacologia , Androstenos/farmacologia , Animais , Antifúngicos/farmacologia , Sítios de Ligação/genética , Células CHO , Linhagem Celular , Cricetulus , Endocitose/fisiologia , Itraconazol/farmacologia , Cetoconazol/farmacologia , Ligação Proteica/fisiologia , Domínios Proteicos/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...