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1.
Int. microbiol ; 22(2): 169-179, jun. 2019. graf, tab
Artigo em Inglês | IBECS | ID: ibc-184824

RESUMO

Oxysterol-binding protein is an important non-vesicular trafficking protein involved in the transportation of lipids in eukaryotic cells. Oxysterol-binding protein is identified as oxysterol-binding protein-related proteins (ORPs) in mammals and oxysterol-binding protein homologue (Osh) in yeast. Research has described the function and structure of oxysterol-binding protein in mammals and yeast, but little information about the protein's structure and function in filamentous fungi has been reported. This article focuses on recent advances in the research of Osh proteins in yeast and filamentous fungi, such as Aspergillus oryzae, Aspergillus nidulans, and Candida albicans. Furthermore, we point out some problems in the field, summarizing the membrane contact sites (MCS) of Osh proteins in yeast, and consider the future of Osh protein development


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Assuntos
Fungos/genética , Receptores de Esteroides/genética , Leveduras/genética , Proteínas de Transporte/genética , Proteínas Fúngicas/genética , Fungos/metabolismo , Receptores de Esteroides/metabolismo , Leveduras/metabolismo , Proteínas de Transporte/metabolismo , Proteínas Fúngicas/metabolismo , Fungos/química , Metabolismo dos Lipídeos , Domínios Proteicos , Receptores de Esteroides/química , Leveduras/química
2.
PLoS One ; 14(3): e0214768, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30925160

RESUMO

The family of oxysterol binding protein (OSBP) and OSBP-related proteins (ORPs) mediate sterol and phospholipid transfer and signaling at membrane contact sites (MCS). The activity of OSBP at MCS is regulated by phosphorylation, but whether this applies to ORPs is unknown. Here we report the functional characterization of a unique proline/serine-rich phosphorylation motif (S762SPSSPSS769) in the lipid binding OSBP-related domain of full-length ORP4L and a truncated variant ORP4S. Phosphorylation was confirmed by mass spectrometry and [32P]PO4 incorporation, and in silico and in vitro assays using purified ORP4L identified putative proline-directed kinases that phosphorylate the site. The functional significance of the phospho-site was assessed by mutating serine 762, S763, S766 and S768 to aspartate or alanine to produce phosphomimetic (S4D) and phosphorylation-deficient (S4A) mutants, respectively. Solution binding of 25-hydroxycholesterol and cholesterol by recombinant ORP4L-S4D and -S4A was similar to wild-type but ORP4L-S4D more effectively extracted cholesterol from liposomes. ORP4L homo-dimerization was unaffected by phosphorylation but gel filtration of ORP4L-S4D indicated that the native conformation was affected. Confocal microscopy revealed that ORP4L-S4D also strongly associated with bundled vimentin filaments, a feature shared with ORP4S which lacks the PH and dimerization domains. We conclude that phosphorylation of a unique serine/proline motif in the ORD induces a conformation change in ORP4L that enhances interaction with vimentin and cholesterol extraction from membranes.


Assuntos
Colesterol/metabolismo , Prolina , Receptores de Esteroides/química , Receptores de Esteroides/metabolismo , Serina , Vimentina/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Linhagem Celular , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Fosforilação , Ligação Proteica , Domínios Proteicos , Receptores de Esteroides/genética
3.
Int Microbiol ; 22(2): 169-179, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30810998

RESUMO

Oxysterol-binding protein is an important non-vesicular trafficking protein involved in the transportation of lipids in eukaryotic cells. Oxysterol-binding protein is identified as oxysterol-binding protein-related proteins (ORPs) in mammals and oxysterol-binding protein homologue (Osh) in yeast. Research has described the function and structure of oxysterol-binding protein in mammals and yeast, but little information about the protein's structure and function in filamentous fungi has been reported. This article focuses on recent advances in the research of Osh proteins in yeast and filamentous fungi, such as Aspergillus oryzae, Aspergillus nidulans, and Candida albicans. Furthermore, we point out some problems in the field, summarizing the membrane contact sites (MCS) of Osh proteins in yeast, and consider the future of Osh protein development.


Assuntos
Fungos/genética , Fungos/metabolismo , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo , Leveduras/genética , Leveduras/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fungos/química , Metabolismo dos Lipídeos , Domínios Proteicos , Receptores de Esteroides/química , Leveduras/química
4.
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
5.
Nat Commun ; 10(1): 829, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30783101

RESUMO

Phosphatidylinositol phosphates (PIPs) and cholesterol are known to regulate the function of late endosomes and lysosomes (LELs), and ORP1L specifically localizes to LELs. Here, we show in vitro that ORP1 is a PI(4,5)P2- or PI(3,4)P2-dependent cholesterol transporter, but cannot transport any PIPs. In cells, both ORP1L and PI(3,4)P2 are required for the efficient removal of cholesterol from LELs. Structures of the lipid-binding domain of ORP1 (ORP1-ORD) in complex with cholesterol or PI(4,5)P2 display open conformations essential for ORP function. PI(4,5)P2/PI(3,4)P2 can facilitate ORP1-mediated cholesterol transport by promoting membrane targeting and cholesterol extraction. Thus, our work unveils a distinct mechanism by which PIPs may allosterically enhance OSBP/ORPs-mediated transport of major lipid species such as cholesterol.


Assuntos
Colesterol/metabolismo , Fosfatidilinositóis/metabolismo , Receptores de Esteroides/química , Receptores de Esteroides/metabolismo , Regulação Alostérica , Sítios de Ligação , Transporte Biológico , Membrana Celular/metabolismo , Cristalografia por Raios X , Endossomos/metabolismo , Células HEK293 , Humanos , Lisossomos/metabolismo , Mutação , Receptores de Esteroides/genética , Esteróis/metabolismo
6.
J Chem Theory Comput ; 15(2): 1418-1429, 2019 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-30633866

RESUMO

The amphipathic lipid packing sensor (ALPS) motif, originally discovered on the ArfGAP1 membrane-binding protein, binds to pre-existing large packing defects in a membrane (spontaneous or due to membrane curvature), though a more precise relationship between the ALPS peptide and packing defect characteristics of a membrane remains unclear. We developed an image processing technique for identifying packing defects to quantify the relationship between the ALPS peptide of the Osh4 protein in yeast and packing defects on a membrane model using molecular dynamics simulations. We used the highly mobile membrane mimetic (HMMM) model to create very large packing defects and expedite the binding time scale. Most prominently, we show that the probability of the ALPS peptide moving toward the membrane increases when it is near a large packing defect. Deviations from this trend exist for very large packing defects (≳115 Å2), which we propose is due to an overwhelming hydrophobic effect and a reduced electrostatic effect when large portions of the nonpolar core are exposed and the peptide is oriented unfavorably. Furthermore, we compared our HMMM results to similar simulations using all-atom lipid membranes. The binding time scales of the ALPS peptide can be reduced by roughly 1 order of magnitude when HMMM is used, while still maintaining many of the important physical characteristics of the binding process observed when using an all-atom lipid membrane.


Assuntos
Bicamadas Lipídicas/metabolismo , Proteínas de Membrana/metabolismo , Peptídeos/metabolismo , Receptores de Esteroides/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Motivos de Aminoácidos , Bicamadas Lipídicas/química , Proteínas de Membrana/química , Membranas Artificiais , Simulação de Dinâmica Molecular , Peptídeos/química , Ligação Proteica , Conformação Proteica , Receptores de Esteroides/química , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/química
7.
Biomolecules ; 9(1)2019 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-30669684

RESUMO

The FK506-binding protein 51 (FKBP51) has emerged as a key regulator of endocrine stress responses in mammals and as a potential therapeutic target for stress-related disorders (depression, post-traumatic stress disorder), metabolic disorders (obesity and diabetes) and chronic pain. Recently, FKBP51 has been implicated in several cellular pathways and numerous interacting protein partners have been reported. However, no consensus on the underlying molecular mechanisms has yet emerged. Here, we review the protein interaction partners reported for FKBP51, the proposed pathways involved, their relevance to FKBP51's physiological function(s), the interplay with other FKBPs, and implications for the development of FKBP51-directed drugs.


Assuntos
Proteínas de Ligação a Tacrolimo/metabolismo , Glucocorticoides/química , Glucocorticoides/metabolismo , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Doenças Metabólicas/metabolismo , Doenças Metabólicas/patologia , NF-kappa B/química , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-akt/química , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Esteroides/química , Receptores de Esteroides/metabolismo , Transtornos de Estresse Pós-Traumáticos/metabolismo , Transtornos de Estresse Pós-Traumáticos/patologia , Proteínas de Ligação a Tacrolimo/química , Proteínas de Ligação a Tacrolimo/genética
8.
J Steroid Biochem Mol Biol ; 186: 42-55, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30243841

RESUMO

The dominant vector of dengue and Zika diseases is a female Aedes aegypti mosquito. Its reproduction is controlled by the formation of an active heterodimer complex of the 20-hydroxyecdysone receptor (EcR) and Ultraspiracle protein (Usp). Although EcR exhibits a structural and functional organization typical of nuclear receptors (NRs), the EcR C-terminus has an additional F domain (AaFEcR) that is rarely present in the NRs superfamily. The presence of F domains is evolutionarily not well conserved in the NRs. The structure-function relationship of EcR F domains in arthropods is unclear and enigmatic. To date, there have been no data concerning the structure and function of AaFEcR. Our results showed that AaFEcR belongs to a family of intrinsically disordered proteins (IDPs) and possesses putative pre-molten globule (PMG) characteristics. Unexpectedly, additional amino acid composition in silico analyses revealed the presence of short unique repeated Pro-His clusters forming an HGPHPHPHG motif, which is similar to those responsible for Zn2+ and Cu2+ binding in histidine-proline-rich glycoproteins (HPRGs). Using SEC, SV-AUC and ESI-TOF MS, we showed that the intrinsically disordered AaFEcR is able to bind metal ions and form complexes with these ions. Our studies provide new insight into the structural organization and activities of the F domains of NRs. This unique for the F domains of NRs ion-binding propensity demonstrated by the AaFEcR domain may be a part of the ecdysteroid receptor's mechanism for regulating the expression of genes encoding oxidative stress-protecting proteins.


Assuntos
Aedes/metabolismo , Proteínas de Insetos/metabolismo , Proteínas Intrinsicamente Desordenadas/metabolismo , Metais/metabolismo , Receptores de Esteroides/metabolismo , Aedes/química , Animais , Proteínas de Insetos/química , Proteínas Intrinsicamente Desordenadas/química , Metais/química , Ligação Proteica , Domínios Proteicos , Receptores de Esteroides/química
9.
Cell Mol Neurobiol ; 39(4): 539-549, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30291573

RESUMO

Corticosteroid hormones act in the brain to support adaptation to stress via binding to mineralocorticoid and glucocorticoid receptors (MR and GR). These receptors act in large measure as transcription factors. Corticosteroid effects can be highly divergent, depending on the receptor type, but also on brain region, cell type, and physiological context. These differences ultimately depend on differential interactions of MR and GR with other proteins, which determine ligand binding, nuclear translocation, and transcriptional activities. In this review, we discuss established and potential mechanisms that confer receptor and cell type-specific effects of the MR and GR-mediated transcriptional effects in the brain.


Assuntos
Encéfalo/metabolismo , Receptores de Esteroides/metabolismo , Transcrição Genética , Animais , Genoma , Humanos , Modelos Biológicos , Receptores de Esteroides/química , Receptores de Esteroides/genética , Ativação Transcricional/genética
10.
Mol Cell ; 73(3): 458-473.e7, 2019 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-30581148

RESUMO

Cholesterol is highly enriched at the plasma membrane (PM), and lipid transfer proteins may deliver cholesterol to the PM in a nonvesicular manner. Here, through a mini-screen, we identified the oxysterol binding protein (OSBP)-related protein 2 (ORP2) as a novel mediator of selective cholesterol delivery to the PM. Interestingly, ORP2-mediated enrichment of PM cholesterol was coupled with the removal of phosphatidylinositol 4, 5-bisphosphate (PI(4,5)P2) from the PM. ORP2 overexpression or deficiency impacted the levels of PM cholesterol and PI(4,5)P2, and ORP2 efficiently transferred both cholesterol and PI(4,5)P2in vitro. We determined the structure of ORP2 in complex with PI(4,5)P2 at 2.7 Å resolution. ORP2 formed a stable tetramer in the presence of PI(4,5)P2, and tetramerization was required for ORP2 to transfer PI(4,5)P2. Our results identify a novel pathway for cholesterol delivery to the PM and establish ORP2 as a key regulator of both cholesterol and PI(4,5)P2 of the PM.


Assuntos
Membrana Celular/metabolismo , Colesterol/metabolismo , Hepatócitos/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Receptores de Esteroides/metabolismo , Transporte Biológico , Linhagem Celular Tumoral , Células HEK293 , Humanos , Modelos Moleculares , Multimerização Proteica , Estrutura Quaternária de Proteína , Receptores de Esteroides/química , Receptores de Esteroides/genética , Relação Estrutura-Atividade
11.
ACS Chem Biol ; 14(2): 276-287, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30576108

RESUMO

Oxysterol-binding protein (OSBP) is a lipid transport and regulatory protein required for the replication of Enterovirus genus viruses, which includes many significant human pathogens. Short-term exposure (i.e., 1-6 h) to a low dose (i.e., 1 nM) of the natural product compound OSW-1 induces a reduction of cellular OSBP levels by ∼90% in multiple different cell lines with no measurable cytotoxicity, defect in cellular proliferation, or global proteome reduction. Interestingly, the reduction of OSBP levels persists multiple days after the low-dose, transient OSW-1 compound treatment is ended and the intracellular OSW-1 compound levels drop to undetectable levels. The reduction in OSBP levels is inherited in multiple generations of cells that are propagated after the OSW-1 compound treatment is stopped. The enduring multiday, multigenerational reduction of OSBP levels triggered by the OSW-1 compound is not due to proteasome degradation of OSBP or due to a reduction in OSBP mRNA levels. OSW-1 compound treatment induces transient autophagy in cells, but blocking autophagy does not rescue OSBP levels. Although the specific cellular mechanism of long-term OSBP repression is not yet identified, these results clearly show the existence of an OSBP specific cellular regulation process that is triggered upon treatment with an OSBP-binding compound. The stable reduction of OSBP levels upon short-term, transient OSW-1 compound treatment will be a powerful tool to understand OSBP regulation and cellular function. Additionally, the persistent reduction in OSBP levels triggered by the transient OSW-1 compound treatment substantially reduces viral replication in treated cells. Therefore, the long-term, compound-induced reduction of OSBP in cells presents a new route to broad spectrum anti- Enterovirus activity, including as a novel route to antiviral prophylactic treatment through small molecule targeting a human host protein.


Assuntos
Antivirais/farmacologia , Enterovirus/efeitos dos fármacos , Receptores de Esteroides/química , Replicação Viral/efeitos dos fármacos , Antivirais/administração & dosagem , Relação Dose-Resposta a Droga , Enterovirus/metabolismo , Enterovirus/fisiologia , Humanos , Proteínas Virais/metabolismo
12.
Biomolecules ; 8(4)2018 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-30360548

RESUMO

An increasing occurrence of resistance in insect pests and high mammal toxicity exhibited by common pesticides increase the need for new alternative molecules. Among these alternatives, bioinsecticides are considered to be environmentally friendly and safer than synthetic insecticides. Particularly, plant extracts have shown great potential in laboratory conditions. However, the lack of studies that confirm their mechanisms of action diminishes their potential applications on a large scale. Previously, we have reported the insect growth regulator and insecticidal activities of secondary metabolites isolated from plants of the Calceolaria genus. Herein, we report an in silico study of compounds isolated from Calceolaria against acetylcholinesterase, prophenoloxidase, and ecdysone receptor. The molecular docking results are consistent with the previously reported experimental results, which were obtained during the bioevaluation of Calceolaria extracts. Among the compounds, phenylethanoid glycosides, such as verbascoside, exhibited good theoretical affinity to all the analyzed targets. In light of these results, we developed an index to evaluate potential multitarget insecticides based on docking scores.


Assuntos
Calceolariaceae/química , Simulação por Computador , Inibidores Enzimáticos/toxicidade , Glicosídeos/toxicidade , Praguicidas/toxicidade , Acetilcolinesterase/química , Animais , Drosophila melanogaster/química , Inibidores Enzimáticos/química , Glucosídeos/química , Glicosídeos/química , Humanos , Simulação de Acoplamento Molecular , Praguicidas/química , Fenóis/química , Receptores de Esteroides/química
13.
J Phys Chem B ; 122(42): 9713-9723, 2018 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-30281310

RESUMO

Amphipathic helices are key domains of peripheral membrane proteins, targeting specific membranes to enable proper protein function as well as changing the local topology and lipid dynamics of the membranes they bind. Here, we use extended all-atom molecular dynamics to study, in detail, the binding mechanism and conformation of the N-terminus of the lipid-transport protein Osh4 in yeast, that is, the amphipathic lipid-packing sensor (ALPS) motif. We identified two binding conformations: (i) a vertical one with the N-terminus of the peptide embedded into the hydrophobic core and (ii) a horizontal, and energetically favored, conformation in which the hydrophobic side chains of ALPS are fully embedded into the membrane hydrophobic core. From extensive analysis on 21 trajectories of 2 µs each, we describe peptide binding in terms of the structural changes that both the peptide and the membrane undergo upon binding as well as energetics of this interaction. The membrane models in this study include a simple binary lipid mixture, with a neutral and a charged lipid (1,2-dioleoyl- sn-glycero-3-phosphocholine-1,2-dioleoyl- sn-glycero-3-phospho-l-serine) and complex mixtures with lipid compositions characteristic of two organelles in yeast (each with more than six lipid types and an accurate sterol content). Our conclusions are in agreement with available literature, showing that the ALPS peptide is more likely to bind membrane surfaces with packing defects and higher anionic character. In addition, we show that there is an interplay between ALPS binding an existing packing defect and creating or enhancing one as the peptide binds to the membrane, which was previously suggested in the literature.


Assuntos
Bicamadas Lipídicas/metabolismo , Proteínas de Membrana/metabolismo , Fragmentos de Peptídeos/metabolismo , Receptores de Esteroides/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Motivos de Aminoácidos , Interações Hidrofóbicas e Hidrofílicas , Bicamadas Lipídicas/química , Proteínas de Membrana/química , Simulação de Dinâmica Molecular , Fragmentos de Peptídeos/química , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Fosfatidilserinas/química , Fosfatidilserinas/metabolismo , Ligação Proteica , Conformação Proteica , Receptores de Esteroides/química , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/química , Termodinâmica
14.
Biochim Biophys Acta Biomembr ; 1860(11): 2356-2365, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-29879417

RESUMO

OSBP binds, extracts and transfers sterols and phosphatidylinositol-4-phosphate (PI(4)P between liposomes, but the sequence of steps at the membrane surface leading to ligand removal is poorly characterized. In this study, we used dual polarization interferometry (DPI), a label-free surface analytical technique, to characterize the interaction of recombinant, purified OSBP as it flows over immobilized dioleoyl-phosphatidylcholine (DOPC) bilayers containing PI(4)P, cholesterol or 25-hydroxycholesterol. Kinetics of membrane interaction were analyzed for PI(4)P-binding and phosphorylation mutants of OSBP. Wild-type OSBP demonstrated a distinctive association with immobilized DOPC bilayers containing 1-8 mol% PI(4)P that was characterized by initial saturable binding followed by desorption, indicative of PI(4)P extraction. In support of this conclusion, an OSBP mutant with impaired binding and extraction of PI(4)P was stably absorbed to PI(4)P-containing membranes, while a pleckstrin homology domain mutant did not associate with PI(4)P-containing membranes. The inclusion of >2 mol% cholesterol, but not 25-hydroxycholesterol, in membranes, enhanced the absorption of the wild-type OSBP. A phosphomimetic of OSBP with enhanced in vitro sterol binding activity displayed membrane interaction properties similar to wild-type. These real-time flow studies allow us to dissect the association of OSBP with PI(4)P into discrete components; initial recruitment to PI(4)P membranes by the PH domain, detection and extraction of PI(4)P, and desorption due to ligand depletion.


Assuntos
Bicamadas Lipídicas/metabolismo , Receptores de Esteroides/metabolismo , Animais , Colesterol/química , Hidroxicolesteróis/química , Interferometria , Bicamadas Lipídicas/química , Fosfatidilcolinas/química , Fosfatos de Fosfatidilinositol/química , Receptores de Esteroides/química , Receptores de Esteroides/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Sf9 , Spodoptera
15.
Methods Mol Biol ; 1786: 117-130, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29786790

RESUMO

Nuclear receptors play an important role in prostate cancer and the androgen receptor is a key transcription factor in regulation of cellular events. Androgen receptor-associated coregulators may be upregulated or downregulated in prostate cancer. Altered expression of regulators may potentiate androgen-induced proliferation, migration, and invasion. Therapies aimed to modulate the function of coregulators in prostate cancer may be based on the use of small molecule inhibitors. Expression and function of AR-associated proteins could be investigated after overexpression and gene silencing followed by hormonal treatment, real-time RT-PCR and ChIP.


Assuntos
Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo , Progressão da Doença , Humanos , Masculino , Complexos Multiproteicos/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Ligação Proteica , Reação em Cadeia da Polimerase em Tempo Real , Receptores Androgênicos/metabolismo , Receptores de Esteroides/química , Fatores de Transcrição/metabolismo , Transcrição Genética
16.
Expert Opin Drug Metab Toxicol ; 14(6): 635-647, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29757018

RESUMO

INTRODUCTION: Pregnane X receptor (PXR) and the constitutive androstane receptor (CAR) are two members of the nuclear receptor superfamily that play major roles in the expression of various drug metabolism enzymes and are known for their ligand promiscuity. As with other nuclear receptors, PXR and CAR are each composed of a ligand-binding domain (LBD) and a DNA-binding domain (DBD) connected by a hinge region. Areas covered: This review focuses on the information obtained over the last 15+ years from X-ray crystallography studies of the structure of PXR and CAR. Areas of focus include the mobility of each structure, based on temperature factors (B factors); multimeric interactions; the binding of coregulators and ligands; and how the crystal structures were obtained. The first use of hydrogen-deuterium exchange coupled with mass spectroscopy (HDX-MS) to study compound-protein interactions in the PXR-LBD is also addressed. Expert opinion: X-ray crystallography studies have provided us with an excellent understanding of how the LBDs of each receptor function; however, many questions remain concerning the structure of these receptors. Future research should focus on determining the co-crystal structure of an antagonist bound to PXR and on studying the structural aspects of the full-length CAR and PXR proteins.


Assuntos
Preparações Farmacêuticas/metabolismo , Receptores Citoplasmáticos e Nucleares/química , Receptores de Esteroides/química , Animais , Cristalografia por Raios X , Medição da Troca de Deutério/métodos , Humanos , Ligantes , Espectrometria de Massas/métodos , Receptor de Pregnano X , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Esteroides/metabolismo
17.
Angew Chem Int Ed Engl ; 57(27): 8048-8052, 2018 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-29697176

RESUMO

The bioactivity of vancomycin is enabled by three aromatic crosslinks, the biosynthesis of which has been an active area of investigation for two decades. Two cytochrome P450 enzymes, OxyB and OxyA, have been shown to introduce bisaryl ether linkages with the help of a so-called X-domain. The final crosslink, however, a biaryl bond thought to be installed by OxyC, has remained elusive. We report the in vitro reconstitution of the OxyC reaction and formation of the first carbon-carbon crosslink in any glycopeptide antibiotic. Using a cascade sequence, in which the peptide substrate was incubated with the Oxy enzymes in turn, we completed the chemoenzymatic synthesis of a vancomycin aglycone variant. This approach was also used to generate a new analogue carrying a thioamide linkage at residue 4, a precursor to the amidine derivative, which is effective against vancomycin-resistant pathogens. Our results set the stage for creating therapeutic vancomycin derivatives by using the native metalloenzymes.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Vancomicina/biossíntese , Biocatálise , Ciclização , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Isoquinolinas/química , Isoquinolinas/metabolismo , Receptores de Esteroides/química , Receptores de Esteroides/metabolismo , Especificidade por Substrato , Vancomicina/análogos & derivados
18.
Biochem Biophys Res Commun ; 499(4): 836-842, 2018 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-29621549

RESUMO

In this study, we investigated the role of OSH6, which encodes a homolog of the oxysterol-binding protein, in the assimilation of n-alkanes in the yeast Yarrowia lipolytica. The deletion mutant of OSH6 showed growth defects on n-alkanes of 10-16 carbons. In the deletion mutant, production of the functional cytochrome P450 was not observed. However, transcription of ALK1, encoding a major P450 belonging to the CYP52 family that plays a critical role in n-alkane hydroxylation, and further translation of its transcript were noted in the deletion mutant as well as in the wild-type strain. The phospholipid composition was altered and, the ratio of phosphatidylserine (PS) was reduced by the deletion of OSH6. Residues involved in the transport of PS and phosphatidylinositol-4-phosphate in Osh6 of Saccharomyces cerevisiae are conserved in Y. lipolytica Osh6p and substitutions of these residues resulted in a defect in the n-alkane assimilation by Y. lipolytica. From these results, we propose a hypothesis that Osh6p provides an ideal endoplasmic reticulum membrane environment for Alk proteins to have a functional conformation via lipid transport activity in Y. lipolytica.


Assuntos
Alcanos/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Receptores de Esteroides/química , Receptores de Esteroides/metabolismo , Homologia de Sequência de Aminoácidos , Yarrowia/metabolismo , Sequência de Aminoácidos , Transporte Biológico , Proteínas Fúngicas/metabolismo , Deleção de Genes , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilserinas/metabolismo , Saccharomyces cerevisiae/metabolismo , Yarrowia/crescimento & desenvolvimento
19.
J Mol Graph Model ; 81: 77-85, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29529496

RESUMO

Insect growth is regulated by the steroid hormone 20-hydroxyecdysone (20E), which works via the ecdysone receptor (EcR). To identify biologically active and novel ecdysone agonists/antagonists, ligand/structure-based virtual screening combined with pharmacophore modeling and molecular docking was performed to identify novel nonsteroidal lead compounds. Nine molecules were screened and selected for an in vitro cell-based reporter bioassay. The results showed that VS-006 and VS-009 exhibited antagonistic activity in S2 cells, whereas only VS-006 exhibited antagonistic activity in Bm5 cells. Molecular dynamic simulation of VS-006 complexed with the ligand binding domain of EcR validated the binding stability of VS-006 and highlighted the key residues for further lead optimization.


Assuntos
Descoberta de Drogas , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Receptores de Esteroides/química , Descoberta de Drogas/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Ligantes , Conformação Molecular , Estrutura Molecular , Ligação Proteica , Receptores de Esteroides/agonistas , Receptores de Esteroides/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas , Relação Estrutura-Atividade
20.
Annu Rev Biochem ; 87: 809-837, 2018 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-29596003

RESUMO

To maintain an asymmetric distribution of ions across membranes, protein pumps displace ions against their concentration gradient by using chemical energy. Here, we describe a functionally analogous but topologically opposite process that applies to the lipid transfer protein (LTP) oxysterol-binding protein (OSBP). This multidomain protein exchanges cholesterol for the phosphoinositide phosphatidylinositol 4-phosphate [PI(4)P] between two apposed membranes. Because of the subsequent hydrolysis of PI(4)P, this counterexchange is irreversible and contributes to the establishment of a cholesterol gradient along organelles of the secretory pathway. The facts that some natural anti-cancer molecules block OSBP and that many viruses hijack the OSBP cycle for the formation of intracellular replication organelles highlight the importance and potency of OSBP-mediated lipid exchange. The architecture of some LTPs is similar to that of OSBP, suggesting that the principles of the OSBP cycle-burning PI(4)P for the vectorial transfer of another lipid-might be general.


Assuntos
Colesterol/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Receptores de Esteroides/metabolismo , Transporte Biológico Ativo , Proteínas de Transporte/metabolismo , Complexo de Golgi/metabolismo , Humanos , Ligantes , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Modelos Biológicos , Modelos Moleculares , Oxisteróis/metabolismo , Domínios e Motivos de Interação entre Proteínas , Receptores de Esteroides/química , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Replicação Viral/fisiologia
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