Los receptores acoplados a proteínas G como blanco terapéutico / G protein-coupled receptors as therapeutic target

Resumen Los receptores son proteínas codificadas por el ADN, algunos de los cuales ya han sido cristalizados, lo que permite conocer los detalles de su estructura a nivel atómico y algunos aspectos de su función. Esta revisión se enfoca en los más diversos y abundantes, los receptores acoplados a la proteína G. Esta familia de receptores reconoce y media la acción de varios ligandos endógenos (hormonas, neurotransmisores, factores de crecimiento y hormonas locales) y también interviene en la patogenia de diversas enfermedades, por lo que son el blanco terapéutico de aproximadamente 30 a 40 % de los medicamentos que se emplean en la práctica clínica cotidiana y de diversas drogas ilegales. La cristalografía de rayos X es una de las herramientas clave que ha permitido observar la estructura de estos receptores en los aminoácidos que participan en esta interacción, lo que posibilita conocer el sitio de unión del ligando endógeno y de moléculas sintéticas que actúan sobre ellos para modular su acción. El modelado molecular es también una herramienta bioinformática computacional que apoya la investigación sobre la unión receptor-ligando, que hace posible el diseño y desarrollo de fármacos cada vez más específicos. A estos desarrollos se suman importantes cambios en los conceptos farmacodinámicos fundamentales.

Abstract Receptors are proteins coded by DNA, some of which have already been crystalized, thus allowing the details of their structure at the atomic level and some aspects of their function to be known. This review focuses on the most diverse and abundant family of receptors, G protein-coupled receptors. This family of receptors recognizes and mediates the action of several endogenous ligands (hormones, neurotransmitters, growth factors and local hormones) and also intervenes in the pathogenesis of various diseases, which is why they are targeted by approximately 30 to 40% of medications that are used in daily clinical practice and of various illegal drugs as well. X-ray crystallography is one of the essential tools that has allowed to observe the structure of these receptors in the amino acids that participate in this interaction, which allows to know the binding site of the endogenous ligand and of synthetic molecules that act on them to modulate their action. Molecular modeling or "docking" is also a computational bioinformatics tool that supports research on receptor-ligand binding, which allows the design and development of increasingly specific drugs. These developments have brought along significant changes in fundamental pharmacodynamic concepts.

The expression level of β-arrestin 1/2 in mice with Parkinson's disease and its relationship with pathogenesis of Parkinson's disease / 中华老年医学杂志

Objective:To observe the expression level of β-arrestin 1/2 in mice with Parkinson's disease(PD)and its relationship with pathogenesis of PD.Methods:PD model was prepared by using 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine hydrochloride(MPTP). The mice were killed at 3 days after the last administration and the brain tissue was taken for observing brain histopathological changes.The colocalization of β-arrestin1/2 with microglia was detected by using immunofluorescence double-labeling of β-arrestin1/2 and microglia.Tyrosine hydroxylase(TH)and Iba-1 were used to label cells, and then the loss of dopaminergic neurons and the activation of microglia were observed by immunohistochemistry.Results:As compared with the blank control group, the relative expression level of β-arrestin1 protein in brain tissue of PD mice was increased significantly, while the relative expression level of β-arrestin2 protein was decreased significantly( t=11.535, 9.948, both P=0.000), and β-arrestin1/2 shared cell localization with microglia.After MPTP induced PD, the number of Th + neurons in SNc area of midbrain was decreased significantly in β-arrestin1 + /+ group and β-arrestin1 -/- group( t=4.098, 3.571, P=0.000, 0.001), while the number of Iba-1 + cells in SNc area of midbrain was increased significantly( t=10.097、6.448, both P=0.000). After MPTP induced PD, the number of Th + neurons in SNc area of midbrain was decreased significantly in β-arrestin2 + /+ group and β-arrestin2 -/- group( t=3.512, 5.237, P=0.001, 0.000), while the number of Iba-1 + cells in SNc area of midbrain was increased significantly( t=5.816、8.402, P=0.000). Compared with β-arrestin1 + /+ group, the expressions of TRAF6, NF-κB and COX-2 in mouse microglia were significantly increased in β-arrestin1 -/- group( t=5.324, 5.837, 9.350, all P=0.0000). Compared with β-arrestin2 + /+ group, the expressions of TRAF6, NF-κB and COX-2 in mouse microglia were significantly down-regulated in β-arrestin2 -/- group( t=5.094, 6.318, 9.466, all P=0.000). Conclusions:The expression of β-arrestin1 is up-regulated and β-arrestin2 is down-regulated in brain tissue of PD mice.β-arrestin1/2 may affect the proliferation and activation of microglia and the loss of dopaminergic neurons through TRAF6/NF-κB/COX-2 pathway, and participate in the pathological process of PD.

β-arrestin2 Affects Cardiac Progenitor Cell Survival through Cell Mobility and Tube Formation in Severe Hypoxia

BACKGROUND AND OBJECTIVES: β-arrestin2 (β-arr2) basically regulates multiple signaling pathways in mammalian cells by desensitization and internalization of G-protein coupled receptors (GPCRs). We investigated impacts of β-arr2 on survival, mobility, and tube formation of cardiac progenitor cells (CPCs) obtained from wild-type (WT) mouse (CPC-WT), and β-arr2 knock-out (KO) mouse (CPC-KO) cultured in presence or absence of serum and oxygen as non-canonical roles in GPCR system. METHODS: CPCs were cultured in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 -based media containing fetal bovine serum and growth factors. Survival of 2 types of CPCs in hypoxia and/or serum deprivation was measured by fluorescence-activated cell sorting. Wound healing ability, and tube formation ability on Matrigel of 2 kinds of CPCs were compared in normoxic and hypoxic cultures. Protein expression related to survival and mobility were measured with the Western blot for each culture conditions. RESULT: CPC-KO showed significantly worse mobility in the wound healing assay and in tube formation on Matrigel especially in hypoxic culture than did the CPC-WT. Also, CPC-KO showed significantly higher apoptosis fraction in both normoxic and hypoxic cultures than did the CPC-WT. Expression of proteins associated with cell survival and mobility, e.g., protein kinase B (Akt), β-catenin, and glycogen synthase kinase-3β (GSK-3β) was significantly worse in CPC-KO. CONCLUSIONS: The CPC-KO had significantly worse cell mobility, tube formation ability, and survival than the CPC-WT, especially in the hypoxic cultures. Apparently, β-arr2 is important on CPC survival by means of mobility and tube formation in myocardial ischemia.

Role of β-arrestin-1 in penehyclidine hydrochloride-induced inhibition of LPS-caused increase in pulmonary microvascular permeability in human pulmonary microvascular endothelial cells / 中华麻醉学杂志

Objective To evaluate the role of β-arrestin-1 in penehyclidine hydrochloride (PHC)-induced inhibition of lipopolysaccharide (LPS)-caused increase in pulmonary microvascular permeability in human pulmonary microvascular endothelial cells (PMVECs).Methods Human PMVECs were seeded in 6-well plates (2 ml/well) or in culture flasks (4 ml/flask) at the density of 1 × 105 cells/ml and divided into 5 groups (n=15 each) using a random number table:empty plasmid transfection group (group C),LPS plus empty plasmid transfection group (LPS group),PHC plus LPS plus empty plasmid transfection group (P+LPS group),LPS plus β-arrestin-1 short hairpin RNA (shRNA) transfection group (LPS+shRNA group) and PHC plus LPS plus β-arrestin-1 shRNA transfection group (P+LPS+shRNA group).In LPS and LPS+shRNA groups,the cells were transfected with empty plasmid 1.5 μg or with plasmid containing 15 nmol/L β-arrestin-1 shRNA,LPS with the final concentration of 0.1 μg/ml was added at 24 h of incubation,and the cells were then incubated for 1 h.In P+LPS and P+LPS+shRNA groups,the cells were transfected with empty plasmid 1.5 μg or with plasmid containing 15 nmol/L β-arrestin-1 shRNA,PHC with the final concentration of 2 μg/ml was added at 24 h of incubation,LPS with the final concentration of 0.1 μg/ml was added at 1 h of incubation,and the cells were then incubated for 1 h.The cell permeability was measured using Transwell chambers.The expression of heat shock protein (HSP27) was detected by immunofluorescence.The expression of β-arrestin-1,p38 mitogen-activated protein kinase (p38MAPK) and phosphorylated p38MAPK (p-p38MAPK) was detected by Western blot.The ratio of pp38MAPK/p38MAPK was calculated.Results Compared with group C,the cell permeability was significantly increased,the expression of HSP27 was up-regulated,p-p38MAPK/p38MAPK ratio was increased,and the expression of β-arrestin-1 was down-regulated in LPS,LPS + shRNA and P + LPS + shRNA groups (P＜0.05),and no significant change was found in the parameters mentioned above in group P+LPS (P＞ 0.05).Compared with group LPS,the cell permeability was significantly decreased,the expression of HSP27 was down-regulated,p-p38MAPK/p38MAPK ratio was decreased,and the expression of β-arrestin1 was up-regulated in group P +LPS,and p-p38MAPK/p38MAPK ratio was significantly increased (P＜0.05),and no significant change was found in the other parameters in group P+LPS+shRNA (P＞0.05).Compared with group P+LPS,the cell permeability was significantly increased,the expression of HSP27 was up-regulated,p-p38MAPK/p38MAPK ratio was increased,and the expression of β-arrestin-1 was down-regulated in group P+LPS+shRNA (P＜0.05).Conclusion The mechanism by which PHC inhibits LPS-induced increase in pulmonary microvascular permeability is totally related to β-arrestin-1 in human PMVECs.

Role of β-arrestin-1 in inhibition of endoxin-induced activation of MAPK signaling pathway in pulmonary microvascular endothelial cells by penehyclidine hydrochloride / 中华麻醉学杂志

Objective To investigate the role of β-arrestin-1 in inhibition of endoxin-induced activation of MAPK signaling pathway in pulmonary microvascular endothelial cells (PMVECs) by penehyclidine hydrochloride (PHC).Methods Human PMVECs were seeded in 6-well plates (2 ml/well) or in culture flasks (4 ml/flask) at the density of 1×105 cells/ml,and randomly divided into 5 groups (n=20 each) using a random number table:empty plasmid transfection group (group C),lipopolysaccharide (LPS) + empty plasmid transfection group (LPS group),PHC + LPS + empty plasmid transfection group (P + LPS group),LPS+β-arrestin-1 short hairpin RNA (shRNA) transfection group (LPS+shRNA group),and PHC + LPS+β-arrestin-1 shRNA transfection group (P+LPS+shRNA group).After the cells were transfected with empty plasmid 1.5 μg or with plasmid containing 15 nmol/L β-arrestin-1 shRNA,the cells were incubated for 24 h.At 24 h of incubation,LPS with the final concentration of 0.1 μg/ml was added,and the cells were then incubated for 1 h in LPS and LPS+ shRNA groups.In P+LPS and P+LPS+shRNA groups,PHC with the final concentration of 2 μg/ml was added,and the cells were incubated for 1 h,and then LPS with the final concentration of 0.1 μg/ml was added,and the cells were incubated for 1 h.The expression of filamentous actin (F-actin) was detected by flow cytometry.The expression of myosin light chain kinase (MLCK) and vascular endothelial-cadherin (VE-cadherin) was detected by immunofluorescence.The expression of phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) and phosphorylated cJun N-terminal kinase (p-JNK) was determined by Western blot.The expression of β-arrestin-1 mRNA was determined by real-time polymerase chain reaction.Results Compared with group C,the expression of Factin,VE-cadherin and β-arrestin-1 mRNA was significantly down-regulated,and the expression of MLCK,p-ERK1/2 and p-JNK was up-regulated in group LPS,and the expression of p-ERK1/2 and p-JNK was significantly up-regulated (P＜0.05),and no significant change was found in the other parameters mentioned above in group P+LPS (P＞0.05).Compared with group LPS,the expression of F-actin,VE-cadherin and β-arrestin-1 mRNA was significantly up-regulated,and the expression of MLCK,p-ERK1/2 and p-JNK was down-regulated in group P+LPS,and the expression of F-actin,VE-cadherin and β-arrestin-1 mRNA was significantly down-regulated,and the expression of MLCK and p-JNK was up-regulated in group LPS+shRNA (P＜0.05).Compared with group P+LPS,the expression of F-actin,VE-cadherin and β-arrestin-1 mRNA was significantly down-regulated,and the expression of MLCK,p-ERK1/2 and p-JNK was up-regulated in group P+LPS+shRNA (P＜0.05).Conclusion The mechanism by which PHC inhibits endoxin-induced activation of MAPK signaling pathway in PMVECs is partially related to up-regulation of β-arrestin-1 expression.

Roles of β-arrestin-1 in inhibition of endotoxin-induced activation of NF-κB in human pulmonary microvascular endothelial cells by penehyclidine bydrochloride / 中华麻醉学杂志

Objective To evaluate the role of β-arrestin-1 in inhibition of endotoxin-induced activation of nuclear factor kappa B (NF-κB) in human pulmonary microvascular endothelial cells (HPM-VECs) by penehyclidine hydrochloride (PHC).Methods HPMVECs were seeded in 6-well plates (2 ml/hole) or in culture flasks (4 ml/flask) at the density of 1 × 105/ml,and were randomly divided into 5 groups (n =20 each) using a random number table:empty plasmid transfection group (group C),lipopolysaccharide (LPS) + empty plasmid transfection group (group LPS),PHC + LPS + empty plasmid transfection group (group P+LPS),LPS + β-arrestin-1 gene-shRNA transfection group (group LPS+shRNA) and PHC + LPS + β-arrestin-1 gene-shRNA transfection group (group P+LPS+shRNA).HPMVECs were transfected with empty plasmid 1.5 μg or with plasmid containing 15 nmol/L β-arrestin-1gene-shRNA.At 24 h of incubation,PHC with the final concentration of 2 μg/ml was added,the cells were incubated for 1 h,LPS with the final concentration of 0.1 μg/ml was then added,and the cells were continuously incubated for another 1 h.The supernatant was collected to measure the activity of lactic dehydrogenase (LDH).The cell suspension was collected for determination of vascular cell adhesion molecule-1 (VCAM-1) expression and NF-κB activities and NF-κB inhibitor I-κB and β-arrestin-1expression.Results Compared with group C,the activities of LDH in supernatant were increased,VCAM-1 expression was up-regulated,NF-κB activity was significantly increased,and I-κB and β-arrestin-1 expression was down-regulated in LPS and LPS+shRNA groups.Compared with group LPS,the activities of LDH in supernatant were decreased,VCAM-1 expression was down-regulated,NF-κB activity was significantly decreased,and I-κB and β-arrestin-l expression was up-regulated in group P+LPS,and no significant change was found in the parameters mentioned above in group P+LPS+shRNA.Compared with group P+LPS,the activities of LDH in supernatant were increased,VCAM-1 expression was up-regulated,NF-κB activity was significantly increased,and I-κB and β-arrestin-1 expression was down-regulated in group P+LPS+shRNA.Conclusion PHC inhibits endotoxin-induced activation of NF-κB in HPMVECs completely through up-regulating β-arrestin-1 expression.

Research progress of β-arrestins in fibrotic diseases / 中国药理学通报

β-arrestins, a kind of important adaptor protein and signal transduction protein found in the purification process ofβ-adrenergic receptor kinase (β-ARK) ,were first identified as pro-teins that have the ability to desensitize G protein-coupled recep-tors ( GPCR) . Fibrosis is defined by the overgrowth, hardening, and scarring of various tissues and is attributed to excess deposi-tion of extracellular matrix ( ECM ) components including colla-gen . A large number of studies have shown thatβ-arrestins play an important role in the process of fibrotic diseases, involved in inflammatory response and excess deposition of ECM. This re-view discusses the research status and development prospects ofβ-arrestins-mediated fibrotic diseases.

Effects of penehyclidine hydrochloride pretreatment on expression of β-arrestin-1 during sepsis-induced acute lung injury in mice / 中华麻醉学杂志

Objective To investigate the effects of penehyclidine hydrochloride (PHCD) pretreatment on β-arrestin-1 expression during sepsis-induced acute lung injury in mice.Methods Thirty female Kunming mice,weighing 18-20 g,were randomly divided into 3 groups (n =10 each):sham operation group (S group),sepsis group (CLP group) and PHCD group.Sepsis was induced by cecal ligation and puncture (CLP).In PHCD group,PHCD 0.45 mg/kg was injected intraperitoneally 1 h before CLP.The equal volume of normal saline was given instead in groups S and CLP.The mice were sacrificed at 12 h after CLP,bronchoalveolar lavage fluid (BALF) was collected for measurement of the total protein concentration,and the lungs were removed for determination of wet/dry lung weight ratio and expression of myosin light chain kinase (MLCK),vascular endothelial cadherin (VE-cad-herin) and β-arrestin-1 in lung tissues.The pathological changes of the lung were scored.Results Compared with group S,the lung injury score,wet/dry lung weight ratio and total protein concentration in BALF were significantly increased,MLCK expression was up-regulated and VE-cadherin expression was down-regulated in groups CLP and PHCD,β-arrestin-1 expression was down-regulated in group CLP and β-arrestin-1 expression was up-regulated in group PHCD (P ＜ 0.05 or 0.01).The lung injury score,wet/dry lung weight ratio,total protein concentration in BALF,and MLCK expression were significantly lower,while the expression of VE-cadherin and β-arrestin-1 was higher in PHCD group than in CLP group (P ＜ 0.05 or 0.01).Conclusion PHCD pretreatment can ameliorate acute lung injury through up-regulating β-arrestin-1 expression and reducing microvascular permeability in septic mice.

β-arrestins in regulating inflammatory diseases: Recent progress / 第二军医大学学报

β-arrestins, important soluble proteins mediating receptor desensitization, have diverse biological functions, such as regulating cell proliferation, cell survival, apoptosis and gene transcription. β-arrestins regulate inflammatory and immune reactions by inhibiting the basal activity of pro-inflammatory transcription factor NF-kB and participating in Toll-like receptors (TLR)/NF-kB signal pathway-mediated NF-kB activation. β-arrestins are involved in the pathogenesis of various inflammatory diseases, such as inflammatory bowel disease, rheumatoid arthritis, ankylosing spondylitis, asthma and so on. The study on β-arrestins will reveal the mechanisms of inflammatory diseases and provide new strategy for clinical treatment. Here we reviewed the recent progress on the role of β-arrestins in regulating the inflammatory diseases.

Progress in the study of β-arrestins in glucose and lipid metabolism / 中华内分泌代谢杂志

β-arrestins are involved in the insulin signaling pathway, affecting peripheral insulin resistance,also affecting insulin secretion and lipid metabolism. To clarify β-arrestins mediated signaling pathways is an approach to the pathogenesis and effective treatment of diabetes.

Effect of ornithine decarboxylase antizyme inhibitor-1 overexpression on the proliferation of B16-F1 mouse melanoma cells / 肿瘤

Objective: To study the effect of ornithine decarboxylase antizyme inhibitor-1 (OAZI-1) overexpression on the proliferation of B16-F1 mouse melanoma cells. Methods: OAZI-1 gene was cloned out from the cDNAs derived from H22 mouse hepatocellular carcinoma cells, and then subcloned into the eukaryotic expression vector pcDNA3.1(+). The recombinant plasmid pcDNA3.1(+)/OAZI-1 was transfected into B16-F1 cells with LipofectAMINE2000 reagents. The positive clone with OAZI-1 overexpression was identified by Western blotting and real-time fluorescent quantitative PCR. The effects of OAZI-1 overexpression on the cell proliferation and the cell cycle distribution were detected by MTT and flow cytometry. The level of polyamine was detected by reversed-phase high performance liquid chromatography. The chemiluminescence analysis was used to determine the activity of spermine oxidase (SMO). Results: The B16-F1 clone with OAZI-1 overexpression was obtained successfully and named as B16/over. The expression level of OAZI-1 mRNA was 3.6-fold higher in the B16/over cells than that in the control B16/3.1 cells transfected with pcDNA3.1(+). The level of ornithine decarboxylase antizyme (OAZ) mRNA was decreased, and the level of ornithine decarboxylase (ODC) mRNA was elevated in B16/ over cells. Overexpression of OAZI-1 in B16-F1 cells could promote the cell proliferation and influence the cell cycle distribution, including decreasing the cell number in G0/G1 phase and increasing the cell number in S and G2/M phases. In B16/over cells, the content of putrescine was increased, but the contents of spermidine and spermine were decreased, and the activity of SMO was elevated. Conclusion: Overexpression of OAZI-1 may promote the proliferation of B16-F1 cells by elevating the content of putrescine, which indicates that OAZI-1 may be a potential target for melanoma therapy. Copyright© 2011 by the Editorial Board of Tumor.

β-arrestin1 mediates the effect of MK-801 on levodopa-induced dyskinesia in Parkinson ', s disease / 中华神经科杂志

ObjectiveTo investigate the effect of MK-801 on levodopa-induced dyskinesia (LID)in Parkinson' s disease (PD). MethodsRat models ( n = 25) of Parkinsonism related motor complications were established and were randomly divided into levodopa-induced dyskinesia (LID) group (n = 10), MK801 treatment group (n = 10) and PD group (n =5). Another 5 rats were served as control group. The behaviors of LID rats treated with MK-801 were observed. Immunohistochemistry and Western blot analysis were used to determine the expression of β-arrestin1 in the striate of rats.Results After MK-801 treatment, abnormalinvoluntarymovementscores and peakturning weredecreasedin LIDrats.Immunohistochemistry showed that β-arrestin1-positive cells of the lesioned side ((2. 95 ± 0. 44) × 104) in LID rats were decreased compared to the contralateral side ( ( 3.78 ± 0. 37 ) × 104, t = 5. 415, P ＜ 0. 05 ).Western blot showed that the levels of β-arrestinl in PD group ( presented as lesioned side/contralateral side) were ( 81.02％ ± 2. 23％ ). The levels of β-arrestin1 (64. 88％ ± 3. 10％ ) were deceased in LID rats compared to PD rats ( t = 9.47, P ＜ 0. 01 ). However, the levels of β-arrestin1 ( 89. 26％ ± 1.90％ )were increased in MK-801-treated rats (t = 14. 82, P ＜0. 01). ConclusionsMK-801 reduces LID in PD rats. The beneficial effect of MK-801 may be mediated through the increased expression of β-arrestinl which in turninhibits the overactivation of glutamate receptors.

Search for enzyme inhibitors from the ocean / 第二军医大学学报

Some enzyme inhibitors derived from the ocean were described briefly in this review.