RESUMO
PURPOSE: To assess the efficacy, safety, and tolerability of a topical water-free cyclosporine A formulation (CyclASol 0.1% ophthalmic solution) in comparison with vehicle for the treatment of dry eye disease (DED). METHODS: Three hundred twenty-eight patients were enrolled in this prospective, 12-week, multicenter, randomized, double-masked, confirmatory, vehicle-controlled clinical study. After a 2-week run-in period, eligible DED patients were randomized 1:1 to either CyclASol 0.1% or vehicle twice daily. The primary efficacy endpoint was change from baseline in total corneal fluorescein staining (National Eye Institute scale), and the second hierarchical primary efficacy endpoint was change from baseline in the Ocular Surface Disease Index score, both at 4 weeks. Secondary efficacy and safety assessments included conjunctival lissamine green staining (Oxford scale), visual analog scales for dry eye symptoms, and adverse event. RESULTS: Treatment with CyclASol 0.1% was superior to vehicle in the primary endpoint: total corneal fluorescein staining at week 4 (Δ -0.8; 95% confidence interval, -1.3 to -0.4; P = 0.0002, analysis of covariance). This difference had already reached statistical significance after 2 weeks and was maintained throughout the study. The study did not statistically meet its second hierarchically tested primary endpoint: Ocular Surface Disease Index score (P = 0.2634). However, CyclASol 0.1% treatment showed statistically significant improvement compared with that of vehicle in the eye dryness score at week 4 (Δ -4.783; 95% confidence interval, -9.129 to -0.438; P = 0.0311). CONCLUSIONS: CyclASol 0.1% was effective in treating signs and symptoms of DED. It significantly reduced corneal and conjunctival staining and improved ocular dryness compared with vehicle. CyclASol 0.1% was safe and showed excellent tolerability.
Assuntos
Ciclosporina/administração & dosagem , Síndromes do Olho Seco/tratamento farmacológico , Imunossupressores/administração & dosagem , Administração Oftálmica , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Túnica Conjuntiva/metabolismo , Córnea/metabolismo , Ciclosporina/efeitos adversos , Método Duplo-Cego , Síndromes do Olho Seco/metabolismo , Síndromes do Olho Seco/fisiopatologia , Feminino , Fluoresceína/metabolismo , Corantes Fluorescentes/metabolismo , Humanos , Imunossupressores/efeitos adversos , Masculino , Pessoa de Meia-Idade , Soluções Oftálmicas , Estudos Prospectivos , Coloração e Rotulagem/métodos , Resultado do Tratamento , ÁguaRESUMO
The nuclear receptor retinoic acid receptor-related orphan receptor gamma t (RORγt) is a transcription factor that drives Th17 cell differentiation and IL-17 production in both innate and adaptive immune cells. The IL-23/IL-17 pathway is implicated in major autoimmune and inflammatory diseases. RORγt lies at the core of this pathway and represents an attractive opportunity for intervention with small molecule therapeutics. Despite diverse chemical series having been reported, combining high potency and nuclear receptor selectivity with good physicochemical properties remains a challenging endeavor in the field of RORγt drug discovery. We recently described the discovery and evaluation of a new class of potent and selective RORγt inverse agonists based on a thiazole scaffold. Herein we describe the successful optimization of this class by incorporation of an additional amide moiety at the 4-position of the thiazole core. In several optimization cycles, we have reduced human PXR activation, improved solubility, and increased potency while maintaining nuclear receptor selectivity. X-ray crystallographic analysis of compound 1g bound in the sterol binding site of the ligand binding domain of RORγt was largely consistent with an earlier structure, guiding further insight into the molecular mechanism for RORγt inhibition with this series. Compound 1g is orally bioavailable, potent in a human whole blood assay and proved to be efficacious in an ex-vivo IL-17A assay, and was selected for preclinical evaluation.
Assuntos
Amidas/química , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/agonistas , Bibliotecas de Moléculas Pequenas/química , Tiazóis/química , Doenças Autoimunes/tratamento farmacológico , Sítios de Ligação , Cristalografia por Raios X , Avaliação Pré-Clínica de Medicamentos , Humanos , Inflamação/tratamento farmacológico , Interleucina-17/química , Modelos Moleculares , Estrutura Molecular , Ligação Proteica , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-Atividade , Tiazóis/farmacologiaRESUMO
Starting from previously identified thiazole-2-carboxamides exemplified by compound 1/6, two new series of RORγt inverse agonists with significantly improved aqueous solubility, ADME parameters and oral PK properties were discovered. These scaffolds were identified from a bioisosteric amide replacement approach. Amongst the variety of heterocycles explored, a 1,3,4-oxadiazole led to compounds with the best overall profile for SAR development and in vivo exploration. In an ex vivo mouse PD model, concentration dependent efficacy was demonstrated and compounds 3/5 and 6/3 were profiled in a 5-day rat tolerability study.
Assuntos
Amidas/farmacologia , Descoberta de Drogas , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/agonistas , Oxidiazóis/farmacologia , Tiazóis/farmacologia , Administração Oral , Amidas/administração & dosagem , Amidas/química , Animais , Relação Dose-Resposta a Droga , Microssomos Hepáticos/química , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Oxidiazóis/administração & dosagem , Oxidiazóis/química , Ratos , Relação Estrutura-Atividade , Tiazóis/administração & dosagem , Tiazóis/químicaRESUMO
The pleiotropic cytokine IL-1 mediates its biological functions via association with the signaling receptor IL-1R1. Despite an apparent simplicity in IL-1 signaling activation, multiple negative regulators have been identified. The decoy receptor IL-1R2 (also known as CD121b) can suppress IL-1 maturation, sequester its active forms or hinder the signaling complex assembly. IL-1R2 is differentially expressed among numerous cell types and displays cis- and trans- modes of action. In this review, we link different forms of IL-1R2 (membrane-bound (mIL-1R2), secreted (sIL-1R2), shedded (shIL-1R2), cytoplasmic, and intracellular domain (IL-1R2ICD) restricted) with their ability to interfere with IL-1, thereby regulating immune responses. We also discuss the intriguing possible function of IL-1R2 as a transcriptional regulator. Finally, we summarize the known impact of IL-1R2 in disease pathogenesis and discuss its potential role in treatment of inflammatory conditions.
Assuntos
Interleucina-1/metabolismo , Receptores Tipo II de Interleucina-1/metabolismo , Animais , Humanos , Transdução de SinaisRESUMO
The nuclear receptor retinoic acid receptor-related orphan receptor gamma t (RORγt) is a transcription factor that drives Th17 cell differentiation and IL-17 production in both innate and adaptive immune cells. The IL-23/IL-17 pathway is implicated in major autoimmune and inflammatory diseases. RORγt lies at the core of this pathway and represents an attractive opportunity for intervention with a small molecule. Despite diverse chemical series having been reported, combining high potency and nuclear receptor selectivity with good physicochemical properties remains a challenging endeavor in the field of RORγt drug discovery. We describe the discovery and evaluation of a new class of potent and selective RORγt inverse agonists based on a thiazole core. Acid analog 1j demonstrated oral bioavailability in rats and was potent in a human whole blood assay, suggesting potential utility in treating autoimmune and inflammatory diseases such as psoriasis. X-ray crystallographic data helped to elucidate the molecular mechanism for RORγt inhibition with this series.
Assuntos
Receptores do Ácido Retinoico/agonistas , Tiazóis/farmacologia , Animais , Cristalografia por Raios X , Humanos , Modelos Moleculares , Estrutura Molecular , Ratos , Relação Estrutura-Atividade , Tiazóis/síntese química , Tiazóis/químicaRESUMO
Cervical cancer is the fourth common cancer in women resulting worldwide in 266,000 deaths per year. Belonging to the carcinomas, new insights into cervical cancer biology may also have great implications for finding new treatment strategies for other kinds of epithelial cancers. Although the transcription factor NF-κB is known as a key player in tumor formation, the relevance of its particular subunits is still underestimated. Here, we applied CRISPR/Cas9n-mediated genome editing to successfully knockout the NF-κB subunit c-REL in HeLa Kyoto cells as a model system for cervical cancers. We successfully generated a homozygous deletion in the c-REL gene, which we validated using sequencing, qPCR, immunocytochemistry, western blot analysis, EMSA and analysis of off-target effects. On the functional level, we observed the deletion of c-REL to result in a significantly decreased cell proliferation in comparison to wildtype (wt) without affecting apoptosis. The impaired proliferative behavior of c-REL-/- cells was accompanied by a strongly decreased amount of the H2B protein as well as a significant delay in the prometaphase of mitosis compared to c-REL+/+ HeLa Kyoto cells. c-REL-/- cells further showed significantly decreased expression levels of c-REL target genes in comparison to wt. In accordance to our proliferation data, we observed the c-REL knockout to result in a significantly increased resistance against the chemotherapeutic agents 5-Fluoro-2'-deoxyuridine (5-FUDR) and cisplatin. In summary, our findings emphasize the importance of c-REL signaling in a cellular model of cervical cancer with direct clinical implications for the development of new treatment strategies.
Assuntos
Técnicas de Inativação de Genes , Histonas/metabolismo , Fator de Transcrição RelA/genética , Neoplasias do Colo do Útero/genética , Sistemas CRISPR-Cas , Ciclo Celular , Proliferação de Células , Feminino , Células HeLa , Humanos , Modelos Biológicos , Deleção de SequênciaRESUMO
Several isoxazole-containing series of FXR agonists have been published over the last 15years, subsequent to the prototypical amphiphilic 'hammerhead'-type structure that was originally laid out by GW4064, the first potent synthetic FXR agonist. A set of novel compounds where the hammerhead is connected to the terminal carboxylic acid-bearing aryl or heteroaryl moiety by either a cyclopropyl, a hydroxycyclobutyl or a hydroxyazetidinyl linker was synthesized in order to improve upon the ADME properties of such isoxazoles. The resulting compounds all demonstrated high potencies at the target receptor FXR but with considerable differences in their physicochemical and in vivo profiles. The structure-activity relationships for key chemical features that have a major impact on the in vivo pharmacology of this series are discussed.
Assuntos
Isoxazóis/farmacologia , Receptores Citoplasmáticos e Nucleares/agonistas , Relação Dose-Resposta a Droga , Humanos , Isoxazóis/síntese química , Isoxazóis/química , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Retinoic acid receptor-related orphan nuclear receptor gamma t (RORγt) is a key transcription factor for the development of Th17 cells. Inhibiting RORγt activity is thought to be beneficial in targeting a variety of inflammatory and autoimmune disorders. Recently N-(5-(arylcarbonyl)thiazol-2-yl)amides were described as RORγt antagonists with in vivo efficacy in experimental autoimmune encephalomyelitis (EAE) and collagen-induced arthritis (CIA) via oral administration. So far no selective small molecule ligands have been revealed for RORß. We show, that one compound of this class, namely N-[5-(2-chloro-benzoyl)-4-(3-chlorophenyl)-thiazol-2-yl]-2-(4-ethanesulfonyl-phenyl)-acetamide (4) is a potent dual inverse agonist towards RORγt and RORß devoid of activity to 18 other human nuclear receptors and thus can serve as chemical probe to deepen our understanding about RORß and its biology.
Assuntos
Benzenoacetamidas/química , Membro 2 do Grupo F da Subfamília 1 de Receptores Nucleares/agonistas , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/agonistas , Tiazóis/química , Tretinoína/química , Benzenoacetamidas/metabolismo , Agonismo Inverso de Drogas , Transferência Ressonante de Energia de Fluorescência , Humanos , Ligantes , Membro 2 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Ligação Proteica , Células Th17/imunologia , Células Th17/metabolismo , Tiazóis/metabolismo , Tretinoína/metabolismoRESUMO
Thymocytes mature in a series of stages by migrating through specific areas of the thymus and interacting with other cells to receive the necessary developmental signals; however, little is known about the molecular mechanisms governing this migration. We report that murine thymocytes with a knockout mutation in α-PAK (p21-activated kinase)-interacting exchange factor (PIX; Arhgef6), an activator of Rho GTPases, showed greatly increased motility and altered morphology in two-dimensional migration on ICAM-1. αPIX was also required for efficient positive selection, but not negative selection, of thymocytes. TCR signaling was normal in αPix(-) thymocytes, indicating that the effects of αPIX on positive selection are largely independent of TCR signaling. αPix(-) thymocytes also paused less during migration in the thymic cortex, interacted less with ICAM-1 coated beads, and could overcome TCR stop signals, consistent with defective scanning behavior. These results identify αPIX as a regulator of thymocyte migration and subsequent arrest that is linked to positive selection.
Assuntos
Movimento Celular/imunologia , Fatores de Troca de Nucleotídeo Guanina Rho/imunologia , Timócitos/imunologia , Timo/imunologia , Animais , Movimento Celular/genética , Células Cultivadas , Citometria de Fluxo , Molécula 1 de Adesão Intercelular/imunologia , Molécula 1 de Adesão Intercelular/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia de Fluorescência por Excitação Multifotônica , Ligação Proteica/imunologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Timócitos/citologia , Timócitos/metabolismo , Timo/citologia , Timo/metabolismoRESUMO
The farnesoid X receptor (FXR) is expressed predominantly in tissues exposed to high levels of bile acids and controls bile acid and lipid homeostasis. FXR(-/-) mice develop hepatocellular carcinoma (HCC) and show an increased prevalence for intestinal malignancies, suggesting a role of FXR as a tumor suppressor in enterohepatic tissues. The N-myc downstream-regulated gene 2 (NDRG2) has been recognized as a tumor suppressor gene, which is downregulated in human hepatocellular carcinoma, colorectal carcinoma and many other malignancies.We show reduced NDRG2 mRNA in livers of FXR(-/-) mice compared to wild type mice and both, FXR and NDRG2 mRNAs, are reduced in human HCC compared to normal liver. Gene reporter assays and Chromatin Immunoprecipitation data support that FXR directly controls NDRG2 transcription via IR1-type element(s) identified in the first introns of the human, mouse and rat NDRG2 genes. NDRG2 mRNA was induced by non-steroidal FXR agonists in livers of mice and the magnitude of induction of NDRG2 mRNA in three different human hepatoma cell lines was increased when ectopically expressing human FXR. Growth and metastasis of SK-Hep-1 cells was strongly reduced by non-steroidal FXR agonists in an orthotopic liver xenograft tumor model. Ectopic expression of FXR in SK-Hep1 cells reduced tumor growth and metastasis potential of corresponding cells and increased the anti-tumor efficacy of FXR agonists, which may be partly mediated via increased NDRG2 expression. FXR agonists may show a potential in the prevention and/or treatment of human hepatocellular carcinoma, a devastating malignancy with increasing prevalence and limited therapeutic options.
Assuntos
Isoxazóis/farmacologia , Neoplasias Hepáticas/prevenção & controle , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Sítios de Ligação/genética , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Células Hep G2 , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Nus , Metástase Neoplásica , Ligação Proteica , Interferência de RNA , Receptores Citoplasmáticos e Nucleares/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Carga Tumoral/efeitos dos fármacos , Proteínas Supressoras de Tumor/genéticaRESUMO
Farnesoid X receptor (FXR), a bile acid-activated nuclear hormone receptor, plays an important role in the regulation of cholesterol and more specifically high-density lipoprotein (HDL) homeostasis. Activation of FXR is reported to lead to both pro- and anti-atherosclerotic effects. In the present study we analyzed the impact of different FXR agonists on cholesterol homeostasis, plasma lipoprotein profiles, and transhepatic cholesterol efflux in C57BL/6J mice and cynomolgus monkeys and atherosclerosis development in cholesteryl ester transfer protein transgenic (CETPtg) low-density lipoprotein receptor (LDLR) (-/-) mice. In C57BL/6J mice on a high-fat diet the synthetic FXR agonists isopropyl 3-(3,4-difluorobenzoyl)-1,1-dimethyl-1,2,3,6-tetrahydroazepino[4,5-b]indole-5-carboxylate (FXR-450) and 4-[2-[2-chloro-4-[[5-cyclopropyl-3-(2,6-dichlorophenyl)-4-isoxazolyl]methoxy]phenyl]cyclopropyl]benzoic acid (PX20606) demonstrated potent plasma cholesterol-lowering activity that affected all lipoprotein species, whereas 3-[2-[2-chloro-4-[[3-(2,6-dichlorophenyl)-5-(1-methylethyl)-4-isoxazolyl]methoxy]phenyl]ethenyl]benzoic acid (GW4064) and 6-ethyl chenodeoxycholic acid (6-ECDCA) showed only limited effects. In FXR wild-type mice, but not FXR(-/-) mice, the more efficacious FXR agonists increased fecal cholesterol excretion and reduced intestinal cholesterol (re)uptake. In CETPtg-LDLR(-/-) mice PX20606 potently lowered total cholesterol and, despite the observed HDL cholesterol (HDLc) reduction, caused a highly significant decrease in atherosclerotic plaque size. In normolipidemic cynomolgus monkeys PX20606 and 6-ECDCA both reduced total cholesterol, and PX20606 specifically lowered HDL(2c) but not HDL(3c) or apolipoprotein A1. That pharmacological FXR activation specifically affects this cholesterol-rich HDL(2) subclass is a new and highly interesting finding and sheds new light on FXR-dependent HDLc lowering, which has been perceived as a major limitation for the clinical development of FXR agonists.
Assuntos
Anticolesterolemiantes/farmacologia , Aterosclerose/prevenção & controle , Benzoatos/farmacologia , Proteínas de Transferência de Ésteres de Colesterol/metabolismo , Colesterol/sangue , Isoxazóis/farmacologia , Lipoproteínas HDL/sangue , Fígado/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores de LDL/metabolismo , Animais , Anticolesterolemiantes/química , Anticolesterolemiantes/uso terapêutico , Aorta/efeitos dos fármacos , Aorta/metabolismo , Aorta/patologia , Aterosclerose/sangue , Aterosclerose/metabolismo , Benzoatos/química , Benzoatos/uso terapêutico , Transporte Biológico , Colesterol/administração & dosagem , Colesterol/metabolismo , Proteínas de Transferência de Ésteres de Colesterol/genética , Dieta Hiperlipídica , Modelos Animais de Doenças , Fezes/química , Feminino , Humanos , Isoxazóis/química , Isoxazóis/uso terapêutico , Fígado/metabolismo , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Estrutura Molecular , Ratos , Ratos Sprague-Dawley , Receptores de LDL/genética , Especificidade da Espécie , Relação Estrutura-AtividadeRESUMO
There is an intense discussion about the subcellular origin of the generation of reactive oxygen species (ROS) under hypoxia. Since this fundamental question can be addressed only in a cellular system, the O(2) -sensing rat pheochromocytoma (PC12) cells were used. Severe hypoxia is known to elevate non-esterified fatty acids. Therefore, the site(s) of ROS generation were studied in cells which we simultaneously exposed to hypoxia (1% oxygen) and free fatty acids (FFA). We obtained the following results: (i) at hypoxia, ROS generation increases in PC12 cells but not in mitochondria isolated therefrom. (ii) Non-esterified polyunsaturated fatty acids (PUFA) enhance the ROS release from PC12 cells as well as from mitochondria, both in normoxia and in hypoxia. (iii) PUFA-induced ROS generation by PC12 cells is not decreased either by inhibitors of the cell membrane NAD(P)H oxidase or inhibitors impairing the PUFA metabolism. (iv) PUFA-induced ROS generation of mitochondria is paralleled by a decline of the NADH-cytochrome c reductase activity (reflecting combined enzymatic activity of complex I plus III). (v) Mitochondrial superoxide indicator (MitoSOXred)-loaded cells exposed to PUFA exhibit increased fluorescence indicating mitochondrial ROS generation. In conclusion, elevated PUFA levels enhance cellular ROS level in hypoxia, most likely by impairing the electron flux within the respiratory chain. Thus, we propose that PUFAs are likely to act as important extrinsic factor to enhance the mitochondria-associated intracellular ROS signaling in hypoxia.
Assuntos
Ácidos Graxos Insaturados/farmacologia , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Ácidos Graxos , Peróxido de Hidrogênio/metabolismo , Hipóxia/metabolismo , Mitocôndrias/efeitos dos fármacos , NADH Desidrogenase/metabolismo , Oxigênio/metabolismo , Células PC12/efeitos dos fármacos , Células PC12/metabolismo , RatosRESUMO
To overcome the known liabilities of GW4064 a series of analogs were synthesized where the stilbene double bond is replaced by an oxymethylene or amino-methylene linker connecting a terminal benzoic acid with a substituted heteroaryl in the middle ring position. As a result we discovered compounds with increased potency in vitro that cause dose-dependent reduction of plasma triglycerides and cholesterol in db/db mice down to 2 x 1 mg/kg/day upon oral administration.
Assuntos
Fármacos Antiobesidade/síntese química , Isoxazóis/química , Receptores Citoplasmáticos e Nucleares/agonistas , Administração Oral , Animais , Fármacos Antiobesidade/química , Fármacos Antiobesidade/farmacologia , Sítios de Ligação , Colesterol/sangue , Simulação por Computador , Isoxazóis/síntese química , Isoxazóis/farmacologia , Camundongos , Camundongos Obesos , Receptores Citoplasmáticos e Nucleares/metabolismo , Triglicerídeos/sangueRESUMO
The endocytic protein Numb3 was found to bind to the cytosolic tail of the leukocyte adhesion receptor P-selectin. The N-terminal phosphotyrosine-binding (PTB) domain of Numb3 is responsible for this activity. An alanine scan revealed the FTNAAFD sequence as recognition region in P-selectin. Structural modeling of the interaction between the Numb PTB domain and the P-selectin tail suggests that both phenylalanines within the recognition sequence fit into hydrophobic cavities of the PTB surface. Their exchange for alanine gave Numb-negative mutants detaining the inhibition of P-selectin endocytosis by Numb PTB overexpression. Cells stable expressing P-selectins internalized the negative mutants markedly slower than the wild type. Consistent with other reports on the phosphorylation of Numb, we found that only the dephospho-Numb is able to bind P-selectin. Our observations demonstrate that Numb3 is an endocytic receptor for P-selectin and may be responsible for the rapid internalization of P-selectin when endothelial activation ends.
Assuntos
Endocitose , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Selectina-P/metabolismo , Processamento Alternativo , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Selectina-P/química , Selectina-P/genética , Fenilalanina/química , Fenilalanina/genética , Fenilalanina/metabolismo , Proteínas Quinases/metabolismo , Estrutura Terciária de Proteína , Técnicas do Sistema de Duplo-HíbridoRESUMO
The mammalian sorting nexin (SNX) proteins are involved in the endocytosis and the sorting machinery of transmembrane proteins. Additionally to the family defining phox homology (PX) domain, SNX17 is the only member with a truncated FERM (4.1, ezrin, radixin, and moesin) domain and a unique C-terminal region (together designated as FC unit). By gel filtration and lipid overlay assays we show that SNX17 is a non-self-assembling and a PtdIns(3)P high class affinity protein. A SNX17 affinity to any other phosphoinositides was not detected. By yeast two-hybrid- and GST-trapping assays we identified KRIT1 (krev1 interaction trapped 1) as a new specific interaction partner of the FC unit of SNX17. KRIT1 binds SNX17 by its N-terminal region like the known interaction partner ICAP1alpha (integrin cytoplasmic domain-associated protein-1). The interaction was also detected in HEK 293 cells transiently expressing GFP-tagged KRIT1 and Xpress-tagged SNX17. KRIT1 mutations cause cerebral cavernous malformation (CCM1). Our finding suggests a SNX17 involvement in the indicated KRIT1 function in cell adhesion processes by integrin signaling.
Assuntos
Proteínas de Transporte/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Fosfatidilinositol 3-Quinases/química , Proteínas Proto-Oncogênicas/metabolismo , Proteínas de Transporte/química , Comunicação Celular , Cromatografia em Gel , Células Endoteliais/citologia , Glutationa/metabolismo , Hemangioma Cavernoso do Sistema Nervoso Central/metabolismo , Humanos , Integrinas/metabolismo , Proteína KRIT1 , Cinética , Fosfatidilinositóis/química , Estrutura Terciária de Proteína , Nexinas de Classificação , Técnicas do Sistema de Duplo-Híbrido , Proteínas de Transporte VesicularRESUMO
SNX17 is a member of the sorting nexin family (SNX), a group of hydrophilic proteins whose common characteristic property is a phox homology (PX) domain. The PX domain directs SNXs to phosphatidylinositides containing membranes of the endosomal compartment, where the SNXs are involved in the sorting of transmembrane proteins. SNX17 is known to interact with P-selectin and the LDL receptor family. Here, we report that the PX domain of SNX17 specifically binds to phosphatidylinositol 3-phosphate-containing membranes. The functional part of SNX17 that binds P-selectin or Patched (PTCH) consists of a truncated FERM domain and a unique C terminus together (FC-unit). In a yeast two-hybrid analysis a putative recognition motif for the FC-unit was revealed within P-selectin as FxNaa(F/Y). When HepG2 cells overexpress P-selectin together with SNX17, SNX17 changes its distribution from early endosomes to lysobisphosphatidic acid-containing late endosomes. Furthermore, overexpressed SNX17 restrains P-selectin in the outer membrane of the late endosomal compartment, thus preventing the normal lysosomal accumulation of P-selectin. These results suggest that the PX domain is necessary for the intracellular localisation, while the FC-unit is required for cargo recognition. We hypothesise that the expression level of SNX17 may regulate the lysosomal degradation, at least for P-selectin, by suppressing its entry into the inner vesicles of the multi-vesicular bodies (MVBs).
Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Selectina-P/metabolismo , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Proteínas de Transporte/genética , Linhagem Celular , Cricetinae , Endossomos/metabolismo , Expressão Gênica , Humanos , Dados de Sequência Molecular , Mutação/genética , Selectina-P/química , Selectina-P/genética , Fosfatos de Fosfatidilinositol/farmacologia , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Especificidade por Substrato , Proteínas de Transporte Vesicular/genéticaRESUMO
The transient appearance of P-selectin on the surface of endothelial cells helps recruit leukocytes into sites of inflammation. The tight control of cell surface P-selectin on these cells depends on regulated exocytosis of Weibel-Palade bodies where the protein is stored and on its rapid endocytosis. After endocytosis, P-selectin is either sorted via endosomes and the Golgi apparatus for storage in Weibel-Palade bodies or targeted to lysosomes for degradation. A potential player in this complex endocytic itinerary is SNX17, a member of the sorting nexin family, which has been shown in a yeast two-hybrid assay to bind P-selectin. Here, we show that overexpression of SNX17 in mammalian cells can influence two key steps in the endocytic trafficking of P-selectin. First, it promotes the endocytosis of P-selectin from the plasma membrane. Second, it inhibits the movement of P-selectin into lysosomes, thereby reducing its degradation.
Assuntos
Proteínas de Transporte/metabolismo , Endocitose , Endossomos/fisiologia , Selectina-P/metabolismo , Androstadienos/farmacologia , Animais , Proteínas de Transporte/análise , Proteínas de Transporte/genética , Membrana Celular/química , Membrana Celular/metabolismo , Células Cultivadas , Endocitose/fisiologia , Endossomos/imunologia , Expressão Gênica , Humanos , Lisossomos/fisiologia , Selectina-P/análise , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Estrutura Terciária de Proteína/genética , Transporte Proteico/fisiologia , Nexinas de Classificação , Proteínas de Transporte Vesicular/metabolismo , WortmaninaRESUMO
In a prospective naturalistic design 31 patients with long-term behavior therapy (average 63 treatment hours) and 31 patients with psychoanalytically-oriented long-term therapy (average 185 treatment hours) were compared. All patients were examined by extern interviewers with the Structured Clinical Interview for Diagnosis (SCID) before they were included in the study. Only patients that showed DSM-III-R criteria of a depressive or anxiety disorder were included. At four times the patient goals in therapy and the actual state was examined: At the beginning of treatment, after 1, after 2,5 and after 3,5 years. At all times the patients could formulate new goals and release old ones. The symptoms were registered by SCL-90-R and the interpersonal problems were recorded by IIP-D. After 3,5 years a follow-up interview was conducted. Although all patients were comparable in their diagnoses the patients in behavior therapy and those in psychoanalytically-oriented therapy differed in a number of characteristics, for example by the way they gained access to therapy (doctors' subscription versus personal initiative for an appointment), the education, the consuming of psychotropic medication and the strain of symptoms. The patients did not differ remarkably in their goals in therapy. The symptoms as a goal did not have the highest priority in both groups, but the category "self-worth-problems" was found in both groups with a high priority. About one third of therapy goals were redefined by the patients in both groups within one year. After 2,5 years the number of goals in the category "interpersonal conflicts" increases remarkably in both treatment groups. For both groups we found the extent of reaching aimed goals in therapy (recorded by GAS) was significant over time. The results seem to prove that psychotherapy under naturalistic conditions aims more at improving the general level of functioning than at reducing the symptoms.