Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 7 de 7
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Nat Chem Biol ; 18(11): 1253-1262, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36229681

RESUMO

Fungal transcription factor Upc2 senses ergosterol levels and regulates sterol biosynthesis and uptake. Constitutive activation of Upc2 causes azole resistance in Candida species. We determined the structure of ergosterol-bound Upc2, revealing the ligand specificity and transcriptional regulation. Ergosterol binding involves conformational changes of the ligand-binding domain, creating a shape-complementary hydrophobic pocket. The conserved helix α12 and glycine-rich loop are critical for sterol recognition by forming the pocket wall. The mutations of the glycine-rich loop inhibit ligand binding by steric clashes and constitutively activate Upc2. The translocation of Upc2 is regulated by Hsp90 chaperone in a sterol-dependent manner. Ergosterol-bound Upc2 associates with Hsp90 using the C-terminal tail, which retains the inactive Upc2 in the cytosol. Ergosterol dissociation induces a conformational change of the C-terminal tail, releasing Upc2 from Hsp90 for nuclear transport by importin α. The understanding of the regulatory mechanism provides an antifungal target for the treatment of azole-resistant Candida infections.


Assuntos
Antifúngicos , Azóis , Azóis/farmacologia , Antifúngicos/farmacologia , Farmacorresistência Fúngica/genética , Esteróis , Ligantes , alfa Carioferinas/genética , alfa Carioferinas/metabolismo , Ergosterol/genética , Ergosterol/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Glicina/metabolismo , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica
2.
Acta Crystallogr D Struct Biol ; 78(Pt 7): 853-864, 2022 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-35775985

RESUMO

Sec14-like phosphatidylinositol transfer proteins (PITPs) are involved in lipid metabolism and phosphatidylinositol 4-phosphate signaling by transporting phosphatidylinositol (PI) and a secondary ligand between the organellar membranes in eukaryotes. Yeast Sfh2 is a PITP that transfers PI and squalene without phosphatidylcholine transfer activity. To investigate the structural determinants for ligand specificity and transport in Sfh2, crystal structures of Sfh2 in complex with PI and squalene were determined at 1.5 and 2.4 Šresolution, respectively. The inositol head group of PI is recognized by highly conserved residues around the pocket entrance. The acyl chains of PI bind into a large hydrophobic cavity. Squalene is accommodated in the bottom of the cavity entirely by hydrophobic interactions. The binding of PI and squalene are mutually exclusive due to their overlapping binding sites, correlating with the role in lipid exchange. The binding mode of PI is well conserved in Sfh family proteins. However, squalene binding is unique to the Sfh2 homolog due to the specific hydrophobic residues forming a shape-complementary binding pocket. Recombinant apo Sfh2 forms a homodimer in vitro by the hydrophobic interaction of the gating α10-α11 helices in an open conformation. Ligand binding closes the lid and dissociates the dimer into monomers. This study reveals the structural determinants for the recognition of the conserved PI and a secondary ligand, squalene, and provides implications for the lipid-transfer function of Sfh2.


Assuntos
Fosfatidilinositóis , Proteínas de Transferência de Fosfolipídeos , Ligantes , Fosfatidilinositóis/química , Fosfatidilinositóis/metabolismo , Proteínas de Transferência de Fosfolipídeos/química , Proteínas de Transferência de Fosfolipídeos/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Esqualeno/metabolismo
3.
Sci Rep ; 11(1): 18859, 2021 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-34552186

RESUMO

The membrane contact sites (MCSs) between the ER and late endosomes (LEs) are essential for the regulation of endosomal protein sorting, dynamics, and motility. PDZD8 is an ER transmembrane protein containing a Synaptotagmin-like Mitochondrial lipid-binding Proteins (SMP) domain. PDZD8 tethers the ER to late endosomes and lysosomes by associating its C-terminal coiled-coil (CC) with the LE Rab7. To identify the structural determinants for the PDZD8-Rab7 interaction, we determined the crystal structure of the human PDZD8 CC domain in complex with the GTP-bound form of Rab7. The PDZD8 CC contains one short helix and the two helices forming an antiparallel coiled-coil. Two Rab7 molecules bind to the opposite sides of the PDZD8 CC in a 2:1 ratio. The switch I/II and interswitch regions of the GTP-loaded Rab7 form the binding interfaces, which correlates with the GTP-dependent interaction of PDZD8 and Rab7. Analysis of the protein interaction by isothermal titration calorimetry confirms that two Rab7 molecules bind the PDZD8 CC in a GTP-dependent manner. The structural model of the PDZD8 CC-Rab7 complex correlates with the recruitment of PDZD8 at the LE-ER interface and its role in lipid transport and regulation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Cristalografia por Raios X , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Lisossomos , Domínios Proteicos , Proteínas rab de Ligação ao GTP/química
4.
PLoS One ; 16(4): e0248781, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33857182

RESUMO

Human ORP3 belongs to the oxysterol-binding protein (OSBP) family of lipid transfer proteins and is involved in lipid trafficking and cell signaling. ORP3 localizes to the ER-PM interfaces and is implicated in lipid transport and focal adhesion dynamics. Here, we report the 2.6-2.7 Å structures of the ORD (OSBP-related domain) of human ORP3 in apo-form and in complex with phosphatidylinositol 4-phosphate. The ORP3 ORD displays a helix grip ß-barrel fold with a deep hydrophobic pocket which is conserved in the OSBP gene family. ORP3 binds PI(4)P by the residues around tunnel entrance and in the hydrophobic pocket, whereas it lacks sterol binding due to the narrow hydrophobic tunnel. The heterologous expression of the ORDs of human ORP3 or OSBP1 rescued the lethality of seven ORP (yeast OSH1-OSH7) knockout in yeast. In contrast, the PI(4)P-binding site mutant of ORP3 did not complement the OSH knockout cells. The N-terminal PH domain and FFAT motif of ORP3 are involved in protein targeting but are not essential in yeast complementation. This observation suggests that the essential function conserved in the ORPs of yeast and human is mediated by PI(4)P-binding of the ORD domain. This study suggests that the non-vesicular PI(4)P transport is a conserved function of all ORPs in eukaryotes.


Assuntos
Proteínas de Ligação a Ácido Graxo/fisiologia , Proteínas de Ligação a Ácido Graxo/ultraestrutura , Sítios de Ligação , Transporte Biológico , Proteínas de Transporte , Proteínas de Ligação a Ácido Graxo/genética , Humanos , Fosfatos de Fosfatidilinositol/metabolismo , Ligação Proteica , Domínios Proteicos , Receptores de Esteroides
5.
Biochem Biophys Res Commun ; 520(2): 466-472, 2019 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-31607485

RESUMO

The steroidogenic acute regulatory protein (StAR)-related lipid transfer domain-4 (STARD4) is a sterol-binding protein that is involved in cholesterol homeostasis by intracellular sterol transport. In this work, we determined the crystal structures of human STARD4 and its Ω1-loop mutant in apo forms at 1.95 and 1.7 Šresolutions, respectively. The structure of human STARD4 displays a conserved α-helix/ß-grip fold containing a deep hydrophobic pocket. The Ω1-loop which serves as a lid for the hydrophobic pocket has a closed conformation. The shape of the sterol-binding cavity in the closed form is not complementary to accommodate cholesterol, suggesting that a conformational change of the Ω1-loop is essential for sterol binding. The human STARD4 displayed sterol transfer activity between liposomes, and the mutations in the Ω1-loop and the hydrophobic wall abolished the transfer activity. This study confirms the structural conservation of the STARD4 subfamily proteins and the flexibility of the Ω1-loop and helix α4 required for sterol transport.


Assuntos
Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Humanos , Interações Hidrofóbicas e Hidrofílicas , Lipossomos/metabolismo , Proteínas de Membrana Transportadoras/genética , Modelos Moleculares , Conformação Proteica , Dobramento de Proteína , Esteróis/metabolismo
6.
PLoS One ; 14(2): e0211724, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30721249

RESUMO

Oxysterol-binding protein (OSBP) and OSBP-related proteins (ORPs) constitute a family of lipid transfer proteins conserved in eukaryotes. ORP1 transports cholesterol at the interface between the late endosomes/lysosomes (LELs) and the endoplasmic reticulum (ER). ORP1 is targeted to the endosomal membranes by forming a tripartite complex with the LE GTPase Rab7 and its effector RILP (Rab7-interacting lysosomal protein). Here, we determined the crystal structure of human ORP1 ANK domain in complex with the GTP-bound form of Rab7. ORP1 ANK binds to the helix α3 of Rab7 located away from the switching regions, which makes the interaction independent of the nucleotide-binding state of Rab7. Thus, the effector-interacting switch regions of Rab7 are accessible for RILP binding, allowing formation of the ORP1-Rab7-RILP complex. ORP1 ANK binds to Rab7 and the Rab7-RILP complex with similar micro-molar affinities, which is consistent with the independence binding of ORP1 and RILP to Rab7. The structural model of the ORP1-Rab7-RILP complex correlates with the recruitment of ORP1 at the LEL-ER interface and the role in lipid transport and regulation.


Assuntos
Endossomos/metabolismo , Lisossomos/metabolismo , Receptores de Esteroides/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Sítios de Ligação , Calorimetria , Clonagem Molecular , Cristalografia por Raios X , Retículo Endoplasmático/metabolismo , Humanos , Ligação Proteica , Conformação Proteica , Receptores de Esteroides/química , Proteínas rab de Ligação ao GTP/química , proteínas de unión al GTP Rab7
7.
Sci Rep ; 7(1): 16837, 2017 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-29203903

RESUMO

Peroxisome proliferator-activator receptor (PPAR) γ is a nuclear hormone receptor that regulates glucose homeostasis, lipid metabolism, and adipocyte function. PPARγ is a target for thiazolidinedione (TZD) class of drugs which are widely used for the treatment of type 2 diabetes. Recently, lobeglitazone was developed as a highly effective TZD with reduced side effects by Chong Kun Dang Pharmaceuticals. To identify the structural determinants for the high potency of lobeglitazone as a PPARγ agonist, we determined the crystal structures of the PPARγ ligand binding domain (LBD) in complex with lobeglitazone and pioglitazone at 1.7 and 1.8 Å resolutions, respectively. Comparison of ligand-bound PPARγ structures revealed that the binding modes of TZDs are well conserved. The TZD head group forms hydrogen bonds with the polar residues in the AF-2 pocket and helix 12, stabilizing the active conformation of the LBD. The unique p-methoxyphenoxy group of lobeglitazone makes additional hydrophobic contacts with the Ω-pocket. Docking analysis using the structures of TZD-bound PPARγ suggested that lobeglitazone displays 12 times higher affinity to PPARγ compared to rosiglitazone and pioglitazone. This structural difference correlates with the enhanced affinity and the low effective dose of lobeglitazone compared to the other TZDs.


Assuntos
Hipoglicemiantes/metabolismo , PPAR gama/metabolismo , Pioglitazona/metabolismo , Pirimidinas/metabolismo , Tiazolidinedionas/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Humanos , Hipoglicemiantes/química , Simulação de Acoplamento Molecular , PPAR gama/química , PPAR gama/genética , Pioglitazona/química , Estrutura Terciária de Proteína , Pirimidinas/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Tiazolidinedionas/química
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...