RESUMO
AIMS: Doxorubicin (DOX) is a widely used anthracycline anticancer agent; however, its irreversible effects on the heart can result in DOX-induced cardiotoxicity (DICT) after cancer treatment. Unfortunately, the pathophysiology of DICT has not yet been fully elucidated, and there are no effective strategies for its prevention or treatment. In this investigation, the novel role of transducin beta-like protein 1 (TBL1) in developing and regulating DICT was explored. METHODS AND RESULTS: We observed a reduction in TBL1 protein expression levels as well as cleavage events in the transplanted cardiac tissues of patients diagnosed with Dilated Cardiomyopathy and DICT. It was revealed that DOX selectively induces TBL1 cleavage at caspase-3 preferred sites-D125, D136, and D215. Interestingly, overexpression of the uncleaved TBL1 mutant (TBL1uclv) variant reduced apoptosis, effectively preventing DOX-induced cell death. We confirmed that cleaved TBL1 cannot form a complex with ß-catenin. As a result, Wnt reporter activity and Wnt target gene expression collectively indicate a decrease in Wnt/ß-catenin signalling, leading to DICT progression. Furthermore, the cleaved TBL1 triggered DOX-induced abnormal electrophysiological features and disrupted calcium homeostasis. However, these effects were improved in TBL1uclv-overexpressing human-induced pluripotent stem cell-derived cardiomyocytes. Finally, in a DICT mouse model, TBL1uclv overexpression inhibited the DICT-induced reduction of cardiac contractility and collagen accumulation, ultimately protecting cardiomyocytes from cell death. CONCLUSION: Our findings reveal that the inhibition of TBL1 cleavage not only mitigates apoptosis but also enhances cardiomyocyte function, even in the context of DOX administration. Consequently, this study's results suggest that inhibiting TBL1 cleavage may be a novel strategy to ameliorate DICT.
Assuntos
Apoptose , Cardiotoxicidade , Doxorrubicina , Miócitos Cardíacos , Via de Sinalização Wnt , beta Catenina , Doxorrubicina/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Miócitos Cardíacos/enzimologia , Via de Sinalização Wnt/efeitos dos fármacos , Humanos , Animais , Apoptose/efeitos dos fármacos , beta Catenina/metabolismo , beta Catenina/genética , Cardiomiopatia Dilatada/metabolismo , Cardiomiopatia Dilatada/patologia , Cardiomiopatia Dilatada/induzido quimicamente , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/enzimologia , Cardiomiopatia Dilatada/fisiopatologia , Masculino , Transducina/metabolismo , Transducina/genética , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/enzimologia , Células-Tronco Pluripotentes Induzidas/patologia , Feminino , Estudos de Casos e Controles , Antibióticos Antineoplásicos/farmacologia , Antibióticos Antineoplásicos/toxicidadeRESUMO
Bitter taste receptors, particularly TAS2R14, play central roles in discerning a wide array of bitter substances, ranging from dietary components to pharmaceutical agents1,2. TAS2R14 is also widely expressed in extragustatory tissues, suggesting its extra roles in diverse physiological processes and potential therapeutic applications3. Here we present cryogenic electron microscopy structures of TAS2R14 in complex with aristolochic acid, flufenamic acid and compound 28.1, coupling with different G-protein subtypes. Uniquely, a cholesterol molecule is observed occupying what is typically an orthosteric site in class A G-protein-coupled receptors. The three potent agonists bind, individually, to the intracellular pockets, suggesting a distinct activation mechanism for this receptor. Comprehensive structural analysis, combined with mutagenesis and molecular dynamic simulation studies, elucidate the broad-spectrum ligand recognition and activation of the receptor by means of intricate multiple ligand-binding sites. Our study also uncovers the specific coupling modes of TAS2R14 with gustducin and Gi1 proteins. These findings should be instrumental in advancing knowledge of bitter taste perception and its broader implications in sensory biology and drug discovery.
Assuntos
Ácidos Aristolóquicos , Colesterol , Ácido Flufenâmico , Receptores Acoplados a Proteínas G , Paladar , Humanos , Ácidos Aristolóquicos/metabolismo , Ácidos Aristolóquicos/química , Ácidos Aristolóquicos/farmacologia , Sítios de Ligação/efeitos dos fármacos , Colesterol/química , Colesterol/metabolismo , Colesterol/farmacologia , Microscopia Crioeletrônica , Ácido Flufenâmico/química , Ácido Flufenâmico/metabolismo , Ácido Flufenâmico/farmacologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Ligantes , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutação , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/ultraestrutura , Paladar/efeitos dos fármacos , Paladar/fisiologia , Transducina/química , Transducina/metabolismoRESUMO
Though rod and cone photoreceptors use similar phototransduction mechanisms, previous model calculations have indicated that the most important differences in their light responses are likely to be differences in amplification of the G-protein cascade, different decay rates of phosphodiesterase (PDE) and pigment phosphorylation, and different rates of turnover of cGMP in darkness. To test this hypothesis, we constructed TrUx;GapOx rods by crossing mice with decreased transduction gain from decreased transducin expression, with mice displaying an increased rate of PDE decay from increased expression of GTPase-activating proteins (GAPs). These two manipulations brought the sensitivity of TrUx;GapOx rods to within a factor of 2 of WT cone sensitivity, after correcting for outer-segment dimensions. These alterations did not, however, change photoreceptor adaptation: rods continued to show increment saturation though at a higher background intensity. These experiments confirm model calculations that rod responses can mimic some (though not all) of the features of cone responses after only a few changes in the properties of transduction proteins.
Assuntos
Células Fotorreceptoras Retinianas Cones , Células Fotorreceptoras Retinianas Bastonetes , Transducina , Animais , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Camundongos , Transducina/metabolismo , Transducina/genética , Retina/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Diester Fosfórico Hidrolases/genéticaRESUMO
Congenital stationary night blindness (CSNB) is an inherited retinal disease that causes a profound loss of rod sensitivity without severe retinal degeneration. One well-studied rhodopsin point mutant, G90D-Rho, is thought to cause CSNB because of its constitutive activity in darkness causing rod desensitization. However, the nature of this constitutive activity and its precise molecular source have not been resolved for almost 30 y. In this study, we made a knock-in (KI) mouse line with a very low expression of G90D-Rho (equal in amount to ~0.1% of normal rhodopsin, WT-Rho, in WT rods), with the remaining WT-Rho replaced by REY-Rho, a mutant with a very low efficiency of activating transducin due to a charge reversal of the highly conserved ERY motif to REY. We observed two kinds of constitutive noise: one being spontaneous isomerization (R*) of G90D-Rho at a molecular rate (R* s-1) 175-fold higher than WT-Rho and the other being G90D-Rho-generated dark continuous noise comprising low-amplitude unitary events occurring at a very high molecular rate equivalent in effect to ~40,000-fold of R* s-1 from WT-Rho. Neither noise type originated from G90D-Opsin because exogenous 11-cis-retinal had no effect. Extrapolating the above observations at low (0.1%) expression of G90D-Rho to normal disease exhibited by a KI mouse model with RhoG90D/WTand RhoG90D/G90D genotypes predicts the disease condition very well quantitatively. Overall, the continuous noise from G90D-Rho therefore predominates, constituting the major equivalent background light causing rod desensitization in CSNB.
Assuntos
Oftalmopatias Hereditárias , Doenças Genéticas Ligadas ao Cromossomo X , Miopia , Cegueira Noturna , Rodopsina , Animais , Cegueira Noturna/genética , Cegueira Noturna/metabolismo , Oftalmopatias Hereditárias/genética , Oftalmopatias Hereditárias/metabolismo , Camundongos , Rodopsina/genética , Rodopsina/metabolismo , Doenças Genéticas Ligadas ao Cromossomo X/genética , Doenças Genéticas Ligadas ao Cromossomo X/metabolismo , Miopia/genética , Miopia/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/patologia , Escuridão , Transducina/genética , Transducina/metabolismo , Técnicas de Introdução de Genes , Modelos Animais de DoençasRESUMO
Bitter taste sensing is mediated by type 2 taste receptors (TAS2Rs (also known as T2Rs)), which represent a distinct class of G-protein-coupled receptors1. Among the 26 members of the TAS2Rs, TAS2R14 is highly expressed in extraoral tissues and mediates the responses to more than 100 structurally diverse tastants2-6, although the molecular mechanisms for recognizing diverse chemicals and initiating cellular signalling are still poorly understood. Here we report two cryo-electron microscopy structures for TAS2R14 complexed with Ggust (also known as gustducin) and Gi1. Both structures have an orthosteric binding pocket occupied by endogenous cholesterol as well as an intracellular allosteric site bound by the bitter tastant cmpd28.1, including a direct interaction with the α5 helix of Ggust and Gi1. Computational and biochemical studies validate both ligand interactions. Our functional analysis identified cholesterol as an orthosteric agonist and the bitter tastant cmpd28.1 as a positive allosteric modulator with direct agonist activity at TAS2R14. Moreover, the orthosteric pocket is connected to the allosteric site via an elongated cavity, which has a hydrophobic core rich in aromatic residues. Our findings provide insights into the ligand recognition of bitter taste receptors and suggest activities of TAS2R14 beyond bitter taste perception via intracellular allosteric tastants.
Assuntos
Colesterol , Espaço Intracelular , Receptores Acoplados a Proteínas G , Paladar , Humanos , Regulação Alostérica/efeitos dos fármacos , Sítio Alostérico , Colesterol/química , Colesterol/metabolismo , Colesterol/farmacologia , Microscopia Crioeletrônica , Interações Hidrofóbicas e Hidrofílicas , Espaço Intracelular/química , Espaço Intracelular/metabolismo , Ligantes , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/ultraestrutura , Reprodutibilidade dos Testes , Paladar/efeitos dos fármacos , Paladar/fisiologia , Transducina/química , Transducina/metabolismo , Transducina/ultraestruturaRESUMO
Humans with the mutation Y509C in transducin beta like 1 X-linked (TBL1X HGNC ID HGNC:11585) have been reported to present with the combination of central congenital hypothyroidism and impaired hearing. TBL1X belongs to the WD40 repeat-containing protein family, is part of NCoR and SMRT corepressor complexes, and thereby involved in thyroid hormone signaling. In order to investigate the effects of the Y509C mutation in TBL1X on cellular thyroid hormone action, we aimed to generate a hemizygous male mouse cohort carrying the Tbl1x Y459C mutation which is equivalent to the human TBL1X Y509C mutation using CRISPR/Cas9 technology. Hemizygous male mice were small at birth and inactive. Their life span (median life span 93 days) was very short compared with heterozygous female mice (survived to >200 days with no welfare issues). About 52% of mice did not survive to weaning (133 mice). Of the remaining 118 mice, only 8 were hemizygous males who were unable to mate whereby it was impossible to generate homozygous female mice. In conclusion, the Tbl1x Y459C mutation in male mice has a marked negative effect on birth weight, survival, and fertility of male mice. The present findings are unexpected as they are in contrast to the mild phenotype in human males carrying the equivalent TBL1X Y509C mutation.
Assuntos
Longevidade , Mutação , Transducina , Animais , Feminino , Humanos , Masculino , Camundongos , Hemizigoto , Longevidade/genética , Mutação/genética , Fenótipo , Transducina/genética , Transducina/metabolismoRESUMO
Except the addition of TBL1Y in human, transducing beta like 1 (TBL1) family mainly consists of two members TBL1X and TBL1XR1, taking part in multiple intracellular signaling pathways such as Wnt/ß-catenin and NF-κB in cancer progression. However, the gene expression patterns of this family during embryonic development remain largely unknown. Here we took advantage of zebrafish model to characterize the spatial and temporal expression patterns of TBL1 family genes including tbl1x, tbl1xr1a and tbl1xr1b. The in situ hybridization studies of gene expression showed robust expressions of tbl1x and tbl1xr1b as maternal transcripts except tbl1xr1a. As the embryo develops, zygotic expressions of all TBL1 family members occur and have a redundant and broad pattern including in brain, neural retina, pharyngeal arches, otic vesicles, and pectoral fins. Ubiquitous expression of all family members were ranked from the strongest to the weakest: tbl1xr1a, tbl1x, and tbl1xr1b. In addition, one tbl1xr1a transcript tbl1xr1a202 showed unique and rich expression in the developing heart and lateral line neuromasts. Overall, all members of zebrafish TBL1 family shared numerous similarities and exhibited certain distinctions in the expression patterns, indicating that they might have redundant and exclusive functions to be further explored.
Assuntos
Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Transdução de Sinais , Transducina/genética , Transducina/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
We previously reported that 2-arachidonoylglycerol (2-AG) synthesis by diacylglycerol lipase (DAGL) and lysophosphatidate phosphohydrolase (LPAP) and hydrolysis by monoacylglycerol lipase (MAGL) in rod outer segments (ROS) from bovine retina were differently modified by light applied to the retina. Based on these findings, the aim of the present research was to evaluate whether 2-AG metabolism could be modulated by proteins involved in the visual process. To this end, ROS kept in darkness (DROS) or obtained in darkness and then subjected to light (BROS) were treated with GTPγS and GDPßS, or with low and moderate ionic strength buffers for detaching soluble and peripheral proteins, or soluble proteins, respectively. Only DAGL activity was stimulated by the application of light to the ROS. GTPγS-stimulated DAGL activity in DROS reached similar values to that observed in BROS. The studies using different ionic strength show that (1) the highest decrease in DROS DAGL activity was observed when both phosphodiesterase (PDE) and transducin α (Tα) are totally membrane-associated; (2) the decrease in BROS DAGL activity does not depend on PDE association to membrane, and that (3) MAGL activity decreases, both in DROS and BROS, when PDE is not associated to the membrane. Our results indicate that the bioavailability of 2-AG under light conditions is favored by G protein-stimulated increase in DAGL activity and hindered principally by Tα/PDE association with the ROS membrane, which decreases DAGL activity.
Assuntos
Ácidos Araquidônicos , Endocanabinoides , Glicerídeos , Segmento Externo da Célula Bastonete , Animais , Endocanabinoides/metabolismo , Ácidos Araquidônicos/metabolismo , Segmento Externo da Célula Bastonete/metabolismo , Bovinos , Glicerídeos/metabolismo , Transdução de Sinal Luminoso , Transducina/metabolismo , Luz , Lipase Lipoproteica/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Visão Ocular/fisiologia , Visão Ocular/efeitos dos fármacosRESUMO
BACKGROUND: Bitter taste receptors (Tas2rs) are generally considered to sense various bitter compounds to escape the intake of toxic substances. Bitter taste receptors have been found to widely express in extraoral tissues and have important physiological functions outside the gustatory system in vivo. METHODS: To investigate the physiological functions of the bitter taste receptor cluster Tas2r106/Tas2r104/Tas2r105/Tas2r114 in lingual and extraoral tissues, multiple Tas2rs mutant mice and Gnat3 were produced using CRISPR/Cas9 gene-editing technique. A mixture containing Cas9 and sgRNA mRNAs for Tas2rs and Gnat3 gene was microinjected into the cytoplasm of the zygotes. Then, T7EN1 assays and sequencing were used to screen genetic mutation at the target sites in founder mice. Quantitative real-time polymerase chain reaction (qRT-PCR) and immunostaining were used to study the expression level of taste signaling cascade and bitter taste receptor in taste buds. Perception to taste substance was also studied using two-bottle preference tests. RESULTS: We successfully produced several Tas2rs and Gnat3 mutant mice using the CRISPR/Cas9 technique. Immunostaining results showed that the expression of GNAT3 and PLCB2 was not altered in Tas2rs mutant mice. But qRT-PCR results revealed the changed expression profile of mTas2rs gene in taste buds of these mutant mice. With two-bottle preference tests, these mutant mice eliminate responses to cycloheximide due to genetic mutation of Tas2r105. In addition, these mutant mice showed a loss of taste perception to quinine dihydrochloride, denatonium benzoate, and cucurbitacin B (CuB). Gnat3-mediated taste receptor and its signal pathway contribute to CuB perception. CONCLUSIONS: These findings implied that these mutant mice would be a valuable means to understand the biological functions of TAS2Rs in extraoral tissues and investigate bitter compound-induced responses mediated by these TAS2Rs in many extraoral tissues.
Assuntos
Mutação , Receptores Acoplados a Proteínas G , Percepção Gustatória , Animais , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Percepção Gustatória/genética , Percepção Gustatória/efeitos dos fármacos , Camundongos , Compostos de Amônio Quaternário/farmacologia , Papilas Gustativas/efeitos dos fármacos , Papilas Gustativas/metabolismo , Sistemas CRISPR-Cas , Paladar/efeitos dos fármacos , Paladar/genética , Transducina/genética , Transducina/metabolismo , Edição de Genes , Triterpenos , Proteínas Heterotriméricas de Ligação ao GTP , Fosfolipase C betaRESUMO
Phototransduction in retinal rods occurs when the G protein-coupled photoreceptor rhodopsin triggers the activation of phosphodiesterase 6 (PDE6) by GTP-bound alpha subunits of the G protein transducin (GαT). Recently, we presented a cryo-EM structure for a complex between two GTP-bound recombinant GαT subunits and native PDE6, that included a bivalent antibody bound to the C-terminal ends of GαT and the inhibitor vardenafil occupying the active sites on the PDEα and PDEß subunits. We proposed GαT-activated PDE6 by inducing a striking reorientation of the PDEγ subunits away from the catalytic sites. However, questions remained including whether in the absence of the antibody GαT binds to PDE6 in a similar manner as observed when the antibody is present, does GαT activate PDE6 by enabling the substrate cGMP to access the catalytic sites, and how does the lipid membrane enhance PDE6 activation? Here, we demonstrate that 2:1 GαT-PDE6 complexes form with either recombinant or retinal GαT in the absence of the GαT antibody. We show that GαT binding is not necessary for cGMP nor competitive inhibitors to access the active sites; instead, occupancy of the substrate binding sites enables GαT to bind and reposition the PDE6γ subunits to promote catalytic activity. Moreover, we demonstrate by reconstituting GαT-stimulated PDE6 activity in lipid bilayer nanodiscs that the membrane-induced enhancement results from an increase in the apparent binding affinity of GαT for PDE6. These findings provide new insights into how the retinal G protein stimulates rapid catalytic turnover by PDE6 required for dim light vision.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 6 , Modelos Moleculares , Transducina , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/química , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/metabolismo , Guanosina Trifosfato/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/enzimologia , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Transducina/química , Transducina/genética , Transducina/metabolismo , Animais , Bovinos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Estrutura Quaternária de Proteína , Ligação Proteica/efeitos dos fármacos , Domínio Catalítico , 1-Metil-3-Isobutilxantina/farmacologia , Bicamadas Lipídicas/metabolismo , Ativação EnzimáticaRESUMO
T-box transcription factor 20 (Tbx20) plays a multifaceted role in cardiac morphogenesis and controls a broad gene regulatory network. However, the mechanism by which Tbx20 activates and represses target genes in a tissue-specific and temporal manner remains unclear. Studies show that Tbx20 directly interacts with the Transducin-like Enhancer of Split (TLE) family of proteins to mediate transcriptional repression. However, a function for the Tbx20-TLE transcriptional repression complex during heart development has yet to be established. We created a mouse model with a two amino acid substitution in the Tbx20 EH1 domain, thereby disrupting the Tbx20-TLE interaction. Disruption of this interaction impaired crucial morphogenic events, including cardiac looping and chamber formation. Transcriptional profiling of Tbx20EH1Mut hearts and analysis of putative direct targets revealed misexpression of the retinoic acid pathway and cardiac progenitor genes. Further, we show that altered cardiac progenitor development and function contribute to the severe cardiac defects in our model. Our studies indicate that TLE-mediated repression is a primary mechanism by which Tbx20 controls gene expression.
Assuntos
Proteínas com Domínio T , Transducina , Animais , Camundongos , Transducina/genética , Transducina/metabolismo , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Coração , Sequências Reguladoras de Ácido NucleicoRESUMO
BACKGROUND: Endoplasmic reticulum (ER) stress has a close relation with cancer progression. Blocking the adaptive pathway of ER stress could be an anticancer strategy. Here, we identified an ER stress-related gene, Transducin beta-like 2 (TBL2), an ER-localized type I transmembrane protein, on increased chromosome 7q as a candidate driver gene of lung adenocarcinoma (LUAD). METHODS: The association between TBL2 mRNA expression and prognostic outcomes and clinicopathological factors was analyzed using The Cancer Genome Atlas (TCGA) datasets of LUAD and lung squamous cell carcinoma (LUSC). Localization of TBL2 in tumor tissues was observed by immunohistochemical staining. Gene set enrichment analysis (GSEA) was conducted using TCGA dataset. In vitro cell proliferation assays were performed using TBL2 knockdown LUAD cells, LUSC cells, and LUAD cells overexpressing TBL2. Apoptosis and ATF4 expression (ER stress marker) were evaluated by western blotting. RESULTS: TBL2 was overexpressed in LUAD and LUSC cells. Multivariate analysis indicated high TBL2 mRNA expression was an independent poor prognostic factor of LUAD. GSEA revealed high TBL2 expression was positively correlated to the ER stress response in LUAD. TBL2 knockdown attenuated LUAD cell proliferation under ER stress. TBL2 inhibited apoptosis in LUAD cells under ER stress. TBL2 knockdown reduced ATF4 expression under ER stress. CONCLUSIONS: TBL2 may be a novel driver gene that facilitates cell proliferation, possibly by upregulating ATF4 expression followed by adaptation to ER stress, and it is a poor prognostic biomarker of LUAD.
Assuntos
Adenocarcinoma de Pulmão , Carcinoma Pulmonar de Células não Pequenas , Carcinoma de Células Escamosas , Proteínas de Ligação ao GTP , Neoplasias Pulmonares , Humanos , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/patologia , Carcinoma de Células Escamosas/patologia , Estresse do Retículo Endoplasmático , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transducina/metabolismo , Proteínas de Ligação ao GTP/genéticaRESUMO
Photoreceptors are highly polarized sensory neurons. Precise localization of signaling molecules within the ciliary outer segment is critical for photoreceptor function and viability. The small GTPase Arl3 plays a particularly important role in photoreceptors as it regulates outer segment enrichment of lipidated proteins essential for the visual response: transducin-α, transducin-γ, PDEα, PDE ß, and Grk1. Recently, mutations in Arl3 have been identified in human patients with nonsyndromic autosomal recessive and dominant inherited retinal degenerations as well as syndromic Joubert syndrome including retinal dystrophy.
Assuntos
Proteínas Monoméricas de Ligação ao GTP , Distrofias Retinianas , Humanos , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Transducina/metabolismo , Cílios/genética , Cílios/metabolismo , Fatores de Ribosilação do ADP/genética , Distrofias Retinianas/genética , MutaçãoRESUMO
Despite recent advances in cancer immunotherapy, their efficacy for treating patients with prostate adenocarcinoma (PRAD) is low due to complex immune evasion mechanisms. However, the function of long non-coding RNA (lncRNAs) in immune evasion has not been fully clarified. This study aimed to expound the role of myocardial infarction-associated transcript (MIAT), a lncRNA significantly upregulated in three PRAD-associated datasets, in immune evasion and try to reveal the potential mechanism. MIAT was highly expressed in PRAD tissues and predicted poor prognosis, and suppression of MIAT inhibited the malignant biological behavior of PRAD cells. Moreover, the depletion of MIAT promoted the immune response of CD8+ T cells and hampered the immune evasion of PRAD cells. In addition, MIAT downregulated TP53 protein expression by recruiting transducin beta-like protein 1X (TBL1X) for ubiquitination modification. Silencing of TP53 or overexpression of TBL1X was enough to abate the tumor suppressive effects of MIAT knockdown in vitro and in vivo. Our results provide evidence for a novel regulation mechanism of CD8+ T cells in PRAD and MIAT may serve as a potential therapeutic target in PRAD.
Assuntos
Adenocarcinoma , MicroRNAs , RNA Longo não Codificante , Humanos , Masculino , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , MicroRNAs/genética , Próstata/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Transducina/genética , Transducina/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , UbiquitinaçãoRESUMO
Mammalian UNC119 is a ciliary trafficking chaperone highly expressed in the inner segment of retinal photoreceptors. Previous research has shown that UNC119 can bind to transducin, the synaptic ribbon protein RIBEYE, and the calcium-binding protein CaBP4, suggesting that UNC119 may have a role in synaptic transmission. We made patch-clamp recordings from retinal slices in mice with the UNC119 gene deleted and showed that removal of even one gene of UNC119 has no effect on the rod outer segment photocurrent, but acted on bipolar cells much like background light: it depolarized membrane potential, decreased sensitivity, accelerated response decay, and decreased the Hill coefficient of the response-intensity relationship. Similar effects were seen on rod bipolar-cell current and voltage responses, and after exposure to bright light to translocate transducin into the rod inner segment. These findings indicate that UNC119 deletion reduces the steady-state glutamate release rate at rod synapses, though no change in the voltage dependence of the synaptic Ca current was detected. We conclude that UNC119, either by itself or together with transducin, can facilitate the release of glutamate at rod synapses, probably by some interaction with RIBEYE or other synaptic proteins rather than by binding to CaBP4 or calcium channels.
Assuntos
Transmissão Sináptica , Transducina , Animais , Camundongos , Glutamatos/metabolismo , Mamíferos/metabolismo , Retina/metabolismo , Sinapses/metabolismo , Transmissão Sináptica/fisiologia , Transducina/metabolismoRESUMO
BACKGROUND: G protein-coupled receptors (GPCRs) transduce external stimuli into the cell by G proteins via an allosteric mechanism. Agonist binding to the receptor stimulates GDP/GTP exchange within the heterotrimeric G protein complex, whereas recent structures of GPCR-G protein complexes revealed that the H5, S1 and S2 domains of Gα are involved in binding the active receptor, earlier studies showed that a short peptide analog derived from the C-terminus (H5) of the G protein transducin (Gt) is sufficient to stabilize rhodopsin in an active form. METHODS: We have used Molecular Dynamics simulations along with biological evaluation by means of radio-ligand binding assay to study the interactions between Gαi-derived peptide (G-peptide) and the µ-opioid receptor (µOR). RESULTS: Here, we show that a Gαi-derived peptide of 12 amino acids binds the µ-opioid receptor and acts as an allosteric modulator. The Gαi-derived peptide increases µOR affinity for its agonist morphine in a dose-dependent way. CONCLUSIONS: These results indicate that the GPCR-Gα peptide interaction observed so far for only rhodopsin can be extrapolated to µOR. In addition, we show that the C-terminal peptide of the Gαi subunit is sufficient to stabilize the active conformation of the receptor. Our approach opens the possibility to investigate the GPCR-G protein interface with peptide modification.
Assuntos
Receptores Opioides , Rodopsina , Rodopsina/química , Rodopsina/metabolismo , Receptores Opioides/metabolismo , Peptídeos , Receptores Acoplados a Proteínas G/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Transducina/química , Transducina/metabolismo , Ligação ProteicaRESUMO
The light sensor of vertebrate scotopic (low-light) vision, rhodopsin, is a G-protein-coupled receptor comprising a polypeptide chain with bound chromophore, 11-cis-retinal, that exhibits remarkable physicochemical properties. This photopigment is extremely stable in the dark, yet its chromophore isomerises upon photon absorption with 70% efficiency, enabling the activation of its G-protein, transducin, with high efficiency. Rhodopsin's photochemical and biochemical activities occur over very different time-scales: the energy of retinaldehyde's excited state is stored in <1 ps in retinal-protein interactions, but it takes milliseconds for the catalytically active state to form, and many tens of minutes for the resting state to be restored. In this review, we describe the properties of rhodopsin and its role in rod phototransduction. We first introduce rhodopsin's gross structural features, its evolution, and the basic mechanisms of its activation. We then discuss light absorption and spectral sensitivity, photoreceptor electrical responses that result from the activity of individual rhodopsin molecules, and recovery of rhodopsin and the visual system from intense bleaching exposures. We then provide a detailed examination of rhodopsin's molecular structure and function, first in its dark state, and then in the active Meta states that govern its interactions with transducin, rhodopsin kinase and arrestin. While it is clear that rhodopsin's molecular properties are exquisitely honed for phototransduction, from starlight to dawn/dusk intensity levels, our understanding of how its molecular interactions determine the properties of scotopic vision remains incomplete. We describe potential future directions of research, and outline several major problems that remain to be solved.
Assuntos
Rodopsina , Transducina , Células Fotorreceptoras/metabolismo , Retina/metabolismo , Rodopsina/metabolismo , Transducina/metabolismo , Visão Ocular , AnimaisRESUMO
Retinal photoreceptor cells, rods and cones, convert photons of light into chemical and electrical signals as the first step of the visual transduction cascade. Although the chemical processes in the phototransduction system are very similar to each other in these photoreceptors, the light sensitivity and time resolution of the photoresponse in rods are functionally different than those in the photoresponses of cones. To systematically investigate how photoresponses are divergently regulated in rods and cones, we have developed a detailed mathematical model on the basis of the Hamer model. The current model successfully reconstructed light intensity-, ATP- and GTP-dependent changes in concentrations of phosphorylated visual pigments (VPs), activated transducins (Tr*s) and phosphodiesterases (PDEs) in rods and cones. In comparison to rods, the lower light sensitivity of cones was attributed not only to the lower affinity of activated VPs for Trs but also to the faster desensitization of the VPs. The assumption of an intermediate inactive state, MIIi, in the thermal decay of activated VPs was essential for inducing faster inactivation of VPs in rods, and possibly also in cones.
Assuntos
Fotofobia , Células Fotorreceptoras Retinianas Bastonetes , Humanos , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Transdução de Sinal Luminoso/fisiologia , Transducina/metabolismoRESUMO
Understanding the physiology of the retina, and especially of the highly polarized photoreceptors, is essential not only to broaden our knowledge of the processes required for normal vision, but also to develop effective therapies to prevent or slow retinal degenerative diseases. However, the molecular analysis of photoreceptors is a challenge due to the heterogeneity of the retinal tissue and the lack of easy and reliable methods for cell separation. Here we present the ReLayS method-a simple technique for the separation of photoreceptor segments (PS) containing both inner and outer segments, outer nuclear layer (ONL), and inner retina (InR) that contains the remaining retinal layers. The layer-specific material isolated from a mouse half-retina with the ReLayS method was sufficient for protein isolation and Western blotting or RNA isolation and real-time PCR studies. The separation of PS, ONL, and InR was successfully validated by Western blotting and real-time PCR using proteins and genes with known expression profiles within the retina. Furthermore, the separation of the PS from the ONL enabled the detection of light-driven translocation of transducin from the PS to the soma. ReLayS is a simple and useful method to address protein and possibly metabolites distribution in photoreceptor compartments in various situations including development, ageing, and degenerative diseases.
Assuntos
Corpo Celular , Retina , Camundongos , Animais , Corpo Celular/metabolismo , Retina/metabolismo , Células Fotorreceptoras/metabolismo , Transducina/metabolismoRESUMO
Over the past 2 decades, the adaptor protein transducin ß-like 1 (TBL1X) and its homolog TBL1XR1 have been shown to be upregulated in solid tumors and hematologic malignancies, and their overexpression is associated with poor clinical outcomes. Moreover, dysregulation of the TBL1 family of proteins has been implicated as a key component of oncogenic prosurvival signaling, cancer progression, and metastasis. Herein, we discuss how TBL1X and TBL1XR1 are required for the regulation of major transcriptional programs through the silencing mediator for tetanoid and thyroid hormone receptor (SMRT)/nuclear receptor corepressor (NCOR)/ B cell lymphoma 6 (BCL6) complex, Wnt/ß catenin, and NF-κB signaling. We outline the utilization of tegavivint (Iterion Therapeutics), a first-in-class small molecule targeting the N-terminus domain of TBL1, as a novel therapeutic strategy in preclinical models of cancer and clinically. Although most published work has focused on the transcriptional role of TBL1X, we recently showed that in diffuse large B-cell lymphoma (DLBCL), the most common lymphoma subtype, genetic knockdown of TBL1X and treatment with tegavivint resulted in decreased expression of critical (onco)-proteins in a posttranscriptional/ß-catenin-independent manner by promoting their proteasomal degradation through a Skp1/Cul1/F-box (SCF)/TBL1X supercomplex and potentially through the regulation of protein synthesis. However, given that TBL1X controls multiple oncogenic signaling pathways in cancer, treatment with tegavivint may ultimately result in drug resistance, providing the rationale for combination strategies. Although many questions related to TBL1X function remain to be answered in lymphoma and other diseases, these data provide a growing body of evidence that TBL1X is a promising therapeutic target in oncology.