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1.
Physiol Rev ; 98(2): 697-725, 2018 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-29442594

RESUMEN

After synthesis, proteins are folded into their native conformations aided by molecular chaperones. Dysfunction in folding caused by genetic mutations in numerous genes causes protein conformational diseases. Membrane proteins are more prone to misfolding due to their more intricate folding than soluble proteins. Misfolded proteins are detected by the cellular quality control systems, especially in the endoplasmic reticulum, and proteins may be retained there for eventual degradation by the ubiquitin-proteasome system or through autophagy. Some misfolded proteins aggregate, leading to pathologies in numerous neurological diseases. In vitro, modulating mutant protein folding by altering molecular chaperone expression can ameliorate some misfolding. Some small molecules known as chemical chaperones also correct mutant protein misfolding in vitro and in vivo. However, due to their lack of specificity, their potential as therapeutics is limited. Another class of compounds, known as pharmacological chaperones (pharmacoperones), binds with high specificity to misfolded proteins, either as enzyme substrates or receptor ligands, leading to decreased folding energy barriers and correction of the misfolding. Because many of the misfolded proteins are misrouted but do not have defects in function per se, pharmacoperones have promising potential in advancing to the clinic as therapeutics, since correcting routing may ameliorate the underlying mechanism of disease. This review will comprehensively summarize this exciting area of research, surveying the literature from in vitro studies in cell lines to transgenic animal models and clinical trials in several protein misfolding diseases.


Asunto(s)
Retículo Endoplásmico/metabolismo , Chaperonas Moleculares/metabolismo , Transporte de Proteínas/fisiología , Deficiencias en la Proteostasis/metabolismo , Animales , Humanos , Chaperonas Moleculares/genética , Conformación Proteica , Pliegue de Proteína , Transporte de Proteínas/genética , Deficiencias en la Proteostasis/terapia
2.
Mol Cell ; 54(1): 166-179, 2014 04 10.
Artículo en Inglés | MEDLINE | ID: mdl-24685158

RESUMEN

Molecular chaperones triage misfolded proteins via action as substrate selectors for quality control (QC) machines that fold or degrade clients. Herein, the endoplasmic reticulum (ER)-associated Hsp40 JB12 is reported to participate in partitioning mutant conformers of gonadotropin-releasing hormone receptor (GnRHR), a G protein-coupled receptor, between ER-associated degradation (ERAD) and an ERQC autophagy pathway. ERQC autophagy degrades E90K-GnRHR because pools of its partially folded and detergent-soluble degradation intermediates are resistant to ERAD. S168R-GnRHR is globally misfolded and disposed of via ERAD, but inhibition of p97, the protein retrotranslocation motor, shunts S168R-GnRHR from ERAD to ERQC autophagy. Partially folded and grossly misfolded forms of GnRHR associate with JB12 and Hsp70. Elevation of JB12 promotes ERAD of S168R-GnRHR, with E90K-GnRHR being resistant. E90K-GnRHR elicits association of the Vps34 autophagy initiation complex with JB12. Interaction between ER-associated Hsp40s and the Vps34 complex permits the selective degradation of ERAD-resistant membrane proteins via ERQC autophagy.


Asunto(s)
Autofagia , Degradación Asociada con el Retículo Endoplásmico , Pliegue de Proteína , Receptores LHRH/metabolismo , Animales , Autofagia/efectos de los fármacos , Células COS , Chlorocebus aethiops , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Degradación Asociada con el Retículo Endoplásmico/efectos de los fármacos , Proteínas del Choque Térmico HSP40/metabolismo , Humanos , Cinética , Modelos Moleculares , Mutación , Inhibidores de Proteasoma/farmacología , Conformación Proteica , Pliegue de Proteína/efectos de los fármacos , Transporte de Proteínas , Proteolisis , Interferencia de ARN , Receptores LHRH/química , Receptores LHRH/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Transfección
3.
Proc Natl Acad Sci U S A ; 111(15): 5735-40, 2014 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-24706813

RESUMEN

FSH and luteinizing hormone (LH) are secreted constitutively or in pulses, respectively, from pituitary gonadotropes in many vertebrates, and regulate ovarian function. The molecular basis for this evolutionarily conserved gonadotropin-specific secretion pattern is not understood. Here, we show that the carboxyterminal heptapeptide in LH is a gonadotropin-sorting determinant in vivo that directs pulsatile secretion. FSH containing this heptapeptide enters the regulated pathway in gonadotropes of transgenic mice, and is released in response to gonadotropin-releasing hormone, similar to LH. FSH released from the LH secretory pathway rescued ovarian defects in Fshb-null mice as efficiently as constitutively secreted FSH. Interestingly, the rerouted FSH enhanced ovarian follicle survival, caused a dramatic increase in number of ovulations, and prolonged female reproductive lifespan. Furthermore, the rerouted FSH vastly improved the in vivo fertilization competency of eggs, their subsequent development in vitro and when transplanted, the ability to produce offspring. Our study demonstrates the feasibility to fine-tune the target tissue responses by modifying the intracellular trafficking and secretory fate of a pituitary trophic hormone. The approach to interconvert the secretory fate of proteins in vivo has pathophysiological significance, and could explain the etiology of several hormone hyperstimulation and resistance syndromes.


Asunto(s)
Evolución Biológica , Hormona Folículo Estimulante/metabolismo , Gonadotrofos/metabolismo , Hormona Luteinizante/metabolismo , Ovario/fisiología , Transducción de Señal/fisiología , Análisis de Varianza , Animales , Western Blotting , Femenino , Fertilidad/fisiología , Ratones , Ratones Transgénicos , Microscopía Confocal , Microscopía Inmunoelectrónica , Folículo Ovárico/metabolismo , Ovario/metabolismo , Ovulación/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa
4.
J Biol Chem ; 290(5): 2699-714, 2015 Jan 30.
Artículo en Inglés | MEDLINE | ID: mdl-25525274

RESUMEN

Pituitary gonadotropins follicle-stimulating hormone and luteinizing hormone are heterodimeric glycoproteins expressed in gonadotropes. They act on gonads and promote their development and functions including steroidogenesis and gametogenesis. Although transcriptional regulation of gonadotropin subunits has been well studied, the post-transcriptional regulation of gonadotropin subunits is not well understood. To test if microRNAs regulate the hormone-specific gonadotropin ß subunits in vivo, we deleted Dicer in gonadotropes by a Cre-lox genetic approach. We found that many of the DICER-dependent microRNAs, predicted in silico to bind gonadotropin ß subunit mRNAs, were suppressed in purified gonadotropes of mutant mice. Loss of DICER-dependent microRNAs in gonadotropes resulted in profound suppression of gonadotropin-ß subunit proteins and, consequently, the heterodimeric hormone secretion. In addition to suppression of basal levels, interestingly, the post-gonadectomy-induced rise in pituitary gonadotropin synthesis and secretion were both abolished in mutants, indicating a defective gonadal negative feedback control. Furthermore, mutants lacking Dicer in gonadotropes displayed severely reduced fertility and were rescued with exogenous hormones confirming that the fertility defects were secondary to suppressed gonadotropins. Our studies reveal that DICER-dependent microRNAs are essential for gonadotropin homeostasis and fertility in mice. Our studies also implicate microRNAs in gonadal feedback control of gonadotropin synthesis and secretion. Thus, DICER-dependent microRNAs confer a new layer of transcriptional and post-transcriptional regulation in gonadotropes to orchestrate the hypothalamus-pituitary-gonadal axis physiology.


Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Gonadotrofos/metabolismo , Gonadotropinas/metabolismo , Ribonucleasa III/metabolismo , Animales , ARN Helicasas DEAD-box/genética , Femenino , Fertilidad/genética , Fertilidad/fisiología , Gonadotropinas/genética , Masculino , Ratones , Ratones Noqueados , MicroARNs/genética , Ratas , Reacción en Cadena en Tiempo Real de la Polimerasa , Ribonucleasa III/genética
5.
Proc Natl Acad Sci U S A ; 110(52): 21030-5, 2013 Dec 24.
Artículo en Inglés | MEDLINE | ID: mdl-24324164

RESUMEN

Mutations in receptors, ion channels, and enzymes are frequently recognized by the cellular quality control system as misfolded and retained in the endoplasmic reticulum (ER) or otherwise misrouted. Retention results in loss of function at the normal site of biological activity and disease. Pharmacoperones are target-specific small molecules that diffuse into cells and serve as folding templates that enable mutant proteins to pass the criteria of the quality control system and route to their physiologic site of action. Pharmacoperones of the gonadotropin releasing hormone receptor (GnRHR) have efficacy in cell culture systems, and their cellular and biochemical mechanisms of action are known. Here, we show the efficacy of a pharmacoperone drug in a small animal model, a knock-in mouse, expressing a mutant GnRHR. This recessive mutation (GnRHR E(90)K) causes hypogonadotropic hypogonadism (failed puberty associated with low or apulsatile luteinizing hormone) in both humans and in the mouse model described. We find that pulsatile pharmacoperone therapy restores E(90)K from ER retention to the plasma membrane, concurrently with responsiveness to the endogenous natural ligand, gonadotropin releasing hormone, and an agonist that is specific for the mutant. Spermatogenesis, proteins associated with steroid transport and steroidogenesis, and androgen levels were restored in mutant male mice following pharmacoperone therapy. These results show the efficacy of pharmacoperone therapy in vivo by using physiological, molecular, genetic, endocrine and biochemical markers and optimization of pulsatile administration. We expect that this newly appreciated approach of protein rescue will benefit other disorders sharing pathologies based on misrouting of misfolded protein mutants.


Asunto(s)
Hipogonadismo/tratamiento farmacológico , Chaperonas Moleculares/farmacología , Pliegue de Proteína/efectos de los fármacos , Deficiencias en la Proteostasis/genética , Receptores LHRH/genética , Testículo/fisiología , Animales , Biomarcadores/metabolismo , Retículo Endoplásmico/metabolismo , Técnicas de Sustitución del Gen , Hipogonadismo/genética , Masculino , Ratones , Chaperonas Moleculares/uso terapéutico , Mutación/genética , Testículo/efectos de los fármacos
6.
Rev Invest Clin ; 67(1): 15-9, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25857579

RESUMEN

Pharmacoperones are hydrophobic molecule drugs that enter cells and serve as a molecular framework to cause misfolded mutant proteins to fold properly and adopt a stable conformation compatible with proper intracellular trafficking. Pharmacoperones have successfully been used experimentally to rescue function of some misfolded proteins (enzymes, receptors, channels) that lead to disease. Identification of pharmacoperones by high-throughput screens of drug libraries will likely provide new molecules that may be potentially useful to treat diseases caused by protein misfolding.


Asunto(s)
Chaperonas Moleculares/metabolismo , Proteínas/metabolismo , Deficiencias en la Proteostasis/tratamiento farmacológico , Animales , Diseño de Fármacos , Ensayos Analíticos de Alto Rendimiento , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Chaperonas Moleculares/química , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Pliegue de Proteína , Proteínas/química , Deficiencias en la Proteostasis/patología
7.
Pharmacol Res ; 83: 38-51, 2014 May.
Artículo en Inglés | MEDLINE | ID: mdl-24373832

RESUMEN

A pharmacoperone (from "pharmacological chaperone") is a small molecule that enters cells and serves as molecular scaffolding in order to cause otherwise-misfolded mutant proteins to fold and route correctly within the cell. Pharmacoperones have broad therapeutic applicability since a large number of diseases have their genesis in the misfolding of proteins and resultant misrouting within the cell. Misrouting may result in loss-of-function and, potentially, the accumulation of defective mutants in cellular compartments. Most known pharmacoperones were initially derived from receptor antagonist screens and, for this reason, present a complex pharmacology, although these are highly target specific. In this summary, we describe efforts to produce high throughput screens that identify these molecules from chemical libraries as well as a mouse model which provides proof-of-principle for in vivo protein rescue using existing pharmacoperones.


Asunto(s)
Evaluación Preclínica de Medicamentos , Ensayos Analíticos de Alto Rendimiento , Proteínas/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Evaluación Preclínica de Medicamentos/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Transporte de Proteínas/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química
8.
Subcell Biochem ; 63: 263-89, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-23161143

RESUMEN

G-protein-coupled receptors (GPCRs) are a large superfamily of plasma membrane proteins that play central roles in transducing endocrine, neural and -sensory signals. In humans, more than 30 disorders are associated with mutations in GPCRs and these proteins are common drug development targets, with 30-50% of drugs targeting them. GPCR mutants are frequently misfolded, recognized as defective by the cellular quality control system, retained in the endoplasmic reticulum and do not traffic to the plasma membrane. The use of small molecules chaperones (pharmacological chaperones or "pharmacoperones") to rescue misfolded GPCRs has provided a new approach for treatment of human diseases caused by misfolding and misrouting. This chapter provides an overview of the molecular basis of this approach using the human gonadotropin-releasing hormone receptor (hGnRHR) as model for treatment of conformational diseases provoked by -misfolded GPCRs.


Asunto(s)
Preparaciones Farmacéuticas/metabolismo , Pliegue de Proteína/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Receptores Acoplados a Proteínas G/metabolismo , Receptores LHRH/metabolismo , Animales , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Humanos , Transducción de Señal/efectos de los fármacos
9.
Proc Natl Acad Sci U S A ; 107(9): 4454-8, 2010 Mar 02.
Artículo en Inglés | MEDLINE | ID: mdl-20160100

RESUMEN

G protein-coupled receptors (GPCRs) play central roles in almost all physiological functions; mutations in GPCRs are responsible for more than 30 disorders. There is a great deal of information about GPCR structure but little information that directly relates structure to protein trafficking or to activation. The gonadotropin releasing hormone receptor, because of its small size among GPCRs, is amenable to preparation of mutants and was used in this study to establish the relation among a salt bridge, protein trafficking, and receptor activation. This bridge, between residues E(90) [located in transmembrane segment (TM) 2] and K(121) (TM3), is associated with correct trafficking to the plasma membrane. Agonists, but not antagonists, interact with residue K(121), and destabilize the TM2-TM3 association of the receptor in the plasma membrane. The hGnRHR mutant E(90)K has a broken salt bridge, which also destabilizes the TM2-TM3 association and is typically retained in the endoplasmic reticulum. We show that this mutant, if rescued to the plasma membrane by either of two different means, has constitutive activity and shows modified ligand specificity, revealing a role for the salt bridge in receptor activation, ligand specificity, trafficking, and structure. The data indicate that destabilizing the TM2-TM3 relation for receptor activation, while requiring an intact salt bridge for correct trafficking, provides a mechanism that protects the cell from plasma membrane expression of constitutive activity.


Asunto(s)
Transporte de Proteínas , Receptores Acoplados a Proteínas G/metabolismo , Sales (Química)/metabolismo , Humanos , Receptores Acoplados a Proteínas G/química
10.
BMC Cancer ; 12: 550, 2012 Nov 23.
Artículo en Inglés | MEDLINE | ID: mdl-23176180

RESUMEN

BACKGROUND: Gonadotropin-releasing hormone (GnRH) and its receptor (GnRHR) are both expressed by a number of malignant tumors, including those of the breast. In the latter, both behave as potent inhibitors of invasion. Nevertheless, the signaling pathways whereby the activated GnRH/GnRHR system exerts this effect have not been clearly established. In this study, we provide experimental evidence that describes components of the mechanism(s) whereby GnRH inhibits breast cancer cell invasion. METHODS: Actin polymerization and substrate adhesion was measured in the highly invasive cell line, MDA-MB-231 transiently expressing the wild-type or mutant DesK191 GnRHR by fluorometry, flow cytometric analysis, and confocal microscopy, in the absence or presence of GnRH agonist. The effect of RhoA-GTP on stress fiber formation and focal adhesion assembly was measured in MDA-MB-231 cells co-expressing the GnRHRs and the GAP domain of human p190Rho GAP-A or the dominant negative mutant GAP-Y1284D. Cell invasion was determined by the transwell migration assay. RESULTS: Agonist-stimulated activation of the wild-type GnRHR and the highly plasma membrane expressed mutant GnRHR-DesK191 transiently transfected to MDA-MB-231 cells, favored F-actin polymerization and substrate adhesion. Confocal imaging allowed detection of an association between F-actin levels and the increase in stress fibers promoted by exposure to GnRH. Pull-down assays showed that the effects observed on actin cytoskeleton resulted from GnRH-stimulated activation of RhoA GTPase. Activation of this small G protein favored the marked increase in both cell adhesion to Collagen-I and number of focal adhesion complexes leading to inhibition of the invasion capacity of MDA-MB-231 cells as disclosed by assays in Transwell Chambers. CONCLUSIONS: We here show that GnRH inhibits invasion of highly invasive breast cancer-derived MDA-MB-231 cells. This effect is mediated through an increase in substrate adhesion promoted by activation of RhoA GTPase and formation of stress fibers and focal adhesions. These observations offer new insights into the molecular mechanisms whereby activation of overexpressed GnRHRs affects cell invasion potential of this malignant cell line, and provide opportunities for designing mechanism-based adjuvant therapies for breast cancer.


Asunto(s)
Actinas/metabolismo , Movimiento Celular , Receptores LHRH/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Buserelina/metabolismo , Buserelina/farmacología , Línea Celular Tumoral , Activación Enzimática/efectos de los fármacos , Femenino , Citometría de Flujo , Fluorometría , Adhesiones Focales/efectos de los fármacos , Hormona Liberadora de Gonadotropina/farmacología , Humanos , Immunoblotting , Células MCF-7 , Microscopía Confocal , Mutación , Invasividad Neoplásica , Polimerizacion/efectos de los fármacos , Receptores LHRH/agonistas , Receptores LHRH/genética , Fibras de Estrés/metabolismo , Transfección , Proteína de Unión al GTP rhoA/genética
11.
J Pharmacol Exp Ther ; 338(2): 430-42, 2011 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-21527534

RESUMEN

G protein-coupled receptors (GPCRs) play central roles in most physiological functions, and mutations in them cause heritable diseases. Whereas crystal structures provide details about the structure of GPCRs, there is little information that identifies structural features that permit receptors to pass the cellular quality control system or are involved in transition from the ground state to the ligand-activated state. The gonadotropin-releasing hormone receptor (GnRHR), because of its small size among GPCRs, is amenable to molecular biological approaches and to computer modeling. These techniques and interspecies comparisons are used to identify structural features that are important for both intracellular trafficking and GnRHR activation yet distinguish between these processes. Our model features two salt (Arg(38)-Asp(98) and Glu(90)-Lys(121)) and two disulfide (Cys(14)-Cys(200) and Cys(114)-Cys(196)) bridges, all of which are required for the human GnRHR to traffic to the plasma membrane. This study reveals that both constitutive and ligand-induced activation are associated with a "coincidence detector" that occurs when an agonist binds. The observed constitutive activation of receptors lacking Glu(90)-Lys(121), but not Arg(38)-Asp(98) ionic bridge, suggests that the role of the former connection is holding the receptor in the inactive conformation. Both the aromatic ring and hydroxyl group of Tyr(284) and the hydrogen bonding of Ser(217) are important for efficient receptor activation. Our modeling results, supported by the observed influence of Lys(191) from extracellular loop 2 (EL2) and a four-residue motif surrounding this loop on ligand binding and receptor activation, suggest that the positioning of EL2 within the seven-α-helical bundle regulates receptor stability, proper trafficking, and function.


Asunto(s)
Aminoácidos Básicos/química , Aminoácidos Básicos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores LHRH/química , Receptores LHRH/fisiología , Animales , Sitios de Unión/fisiología , Células COS , Bovinos , Chlorocebus aethiops , Cristalografía por Rayos X , Líquido Extracelular/metabolismo , Humanos , Ratones , Mutación/fisiología , Unión Proteica/fisiología , Estabilidad Proteica , Transporte de Proteínas , Ratas , Receptores Acoplados a Proteínas G/química , Sales (Química)/química , Sales (Química)/metabolismo
12.
Trends Pharmacol Sci ; 30(5): 228-33, 2009 May.
Artículo en Inglés | MEDLINE | ID: mdl-19307028

RESUMEN

Proteins serve in cellular roles that necessitate structural precision, a requirement overseen by the cellular quality control system (QCS). By rejecting misfolded proteins, the QCS protects against aberrant activity. Misfolding and subsequent retention by the QCS results in proteins that might maintain function but become misrouted and cause disease. Correcting the misrouting of misfolded mutant proteins often restores activity and addresses the underlying disease. Because of its small size, the gonadotropin-releasing hormone receptor has been an excellent model for G-protein-coupled receptor trafficking and has recently enabled elucidation of both the requirements to pass the QCS and the biochemical mechanism of rescue by pharmacological chaperones; this information will now enable rational design of these therapeutic agents. Here, we summarize what is known about the relation between receptor structure and interactions with the QCS with a view toward therapeutic development based on the rescue of misfolded and, consequently, misrouted mutants with drugs.


Asunto(s)
Retículo Endoplásmico/metabolismo , Chaperonas Moleculares , Pliegue de Proteína/efectos de los fármacos , Receptores LHRH/química , Animales , Fenómenos Fisiológicos Celulares , Cisteína/química , Descubrimiento de Drogas , Humanos , Modelos Moleculares , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/farmacología , Proteínas Mutantes/efectos de los fármacos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Receptores LHRH/genética , Receptores LHRH/metabolismo
13.
Mol Endocrinol ; 23(2): 157-68, 2009 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-19095769

RESUMEN

The human GnRH receptor (hGnRHR), a G protein-coupled receptor, is a useful model for studying pharmacological chaperones (pharmacoperones), drugs that rescue misfolded and misrouted protein mutants and restore them to function. This technique forms the basis of a therapeutic approach of rescuing mutants associated with human disease and restoring them to function. The present study relies on computational modeling, followed by site-directed mutagenesis, assessment of ligand binding, effector activation, and confocal microscopy. Our results show that two different chemical classes of pharmacoperones act to stabilize hGnRHR mutants by bridging residues D(98) and K(121). This ligand-mediated bridge serves as a surrogate for a naturally occurring and highly conserved salt bridge (E(90)-K(121)) that stabilizes the relation between transmembranes 2 and 3, which is required for passage of the receptor through the cellular quality control system and to the plasma membrane. Our model was used to reveal important pharmacophoric features, and then identify a novel chemical ligand, which was able to rescue a D(98) mutant of the hGnRHR that could not be rescued as effectively by previously known pharmacoperones.


Asunto(s)
Modelos Moleculares , Chaperonas Moleculares/metabolismo , Receptores Acoplados a Proteínas G , Receptores LHRH , Animales , Bovinos , Membrana Celular/metabolismo , Simulación por Computador , Humanos , Ligandos , Estructura Molecular , Mutagénesis Sitio-Dirigida , Mutación , Unión Proteica , Conformación Proteica , Transporte de Proteínas/fisiología , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores LHRH/genética , Receptores LHRH/metabolismo
14.
Cell Biochem Funct ; 28(1): 66-73, 2010 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-20029959

RESUMEN

Retention of misfolded proteins by the endoplasmic reticulum (ER) is a quality control mechanism involving the participation of endogenous chaperones such as calnexin (CANX). CANX interacts with and restricts plasma membrane expression (PME) of the gonadotropin releasing hormone receptor (GnRHR), a G protein-coupled receptor. CANX also interacts with ERP-57 a thiol oxidoreductase chaperone present in the ER. CANX along with ERP-57 promotes the formation of disulfide bond bridges in nascent proteins. The human GnRH receptor (hGnRHR) is stabilized by two disulfide bond bridges (C(14)-C(200) and C(114)-C(196)), that, when broken, lead to a decrease in receptor expression at the plasma membrane. To determine if the presence of chaperones CANX and ERP-57 exerts an influence over membrane routing and second messenger activation, we assessed the effect of various mutants including those with broken disulfide bridges (Cys --> Ala) along with the hGnRHR. The effect of chaperones on mutants was insignificant, whereas the over expression of ERP-57 led to an hGnRHR retention. This effect was further enhanced by cotransfection with cDNA for CANX showing receptor retention by ERP-57 augmented by CANX, suggesting utilization of these chaperones for quality control of the GnRHR.


Asunto(s)
Calnexina/metabolismo , Membrana Celular/metabolismo , Chaperonas Moleculares/metabolismo , Proteína Disulfuro Isomerasas/metabolismo , Receptores LHRH/metabolismo , Sustitución de Aminoácidos , Animales , Células COS , Chlorocebus aethiops , Retículo Endoplásmico/metabolismo , Humanos , Mutagénesis Sitio-Dirigida , Pliegue de Proteína
15.
Endocr Rev ; 26(4): 479-503, 2005 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-15536207

RESUMEN

Receptors, hormones, enzymes, ion channels, and structural components of the cell are created by the act of protein synthesis. Synthesis alone is insufficient for proper function, of course; for a cell to operate effectively, its components must be correctly compartmentalized. The mechanism by which proteins maintain the fidelity of localization warrants attention in light of the large number of different molecules that must be routed to distinct subcellular loci, the potential for error, and resultant disease. This review summarizes diseases known to have etiologies based on defective protein folding or failure of the cell's quality control apparatus and presents approaches for therapeutic intervention.


Asunto(s)
Enfermedad/etiología , Señales de Clasificación de Proteína/fisiología , Proteínas/metabolismo , Animales , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Humanos , Mitocondrias/metabolismo , Chaperonas Moleculares/fisiología , Mutación , Enfermedades del Sistema Nervioso/etiología , Conformación Proteica , Proteínas/química , Proteínas/genética , Receptores Acoplados a Proteínas G
16.
Mol Cell Endocrinol ; 299(2): 137-45, 2009 Feb 27.
Artículo en Inglés | MEDLINE | ID: mdl-19059461

RESUMEN

In order to serve as enzymes, receptors and ion channels, proteins require structural precision. This is monitored by a cellular quality control system (QCS) that rejects misfolded proteins and thereby protects the cell against aberrant activity. Misfolding can result in protein molecules that retain intrinsic function, yet become misrouted within the cell; these cease to perform normally and result in disease. A therapeutic opportunity exists to correct misrouting and rescue mutants using "pharmacoperones" (small molecular folding templates, often peptidomimetics, which promote correct folding and rescue) thereby restoring function and potentially curing the underlying disease. Because of its small size, the GnRH (gonadotropin-releasing hormone) receptor (GnRHR) is an excellent model for GPCR (G protein-coupled receptor) and has allowed elucidation of the precise biochemical mechanism of pharmacoperone action necessary for rational design of new therapeutic agents. This review summarizes what has been learned about the structural requirements of the GnRHR that govern its interaction with the QCS and now presents the potential for the rational design of pharmacoperones. Because of the role of protein processing, this approach is likely to be applicable to other GCPCs and other proteins in general.


Asunto(s)
Enfermedad , Salud , Receptores LHRH/metabolismo , Animales , Retículo Endoplásmico/metabolismo , Humanos , Pliegue de Proteína , Transporte de Proteínas , Receptores LHRH/química
17.
Mol Cell Endocrinol ; 298(1-2): 84-8, 2009 Jan 27.
Artículo en Inglés | MEDLINE | ID: mdl-18848862

RESUMEN

A thienopyr(im)idine (Org41841) activates the luteinizing hormone (LH) receptor but does not compete with the natural ligand binding site and does not show agonistic action on the follicle-stimulating hormone receptor (hFSHR) at sub-millimolar concentrations. When this drug is preincubated at sub-micromolar concentrations with host cells expressing the hFSHR, and then washed out, binding analysis and assessment of receptor-effector coupling show that it increases plasma membrane expression of the hFSHR. Real-time PCR shows that this effect did not result from increased hFSHR mRNA accumulation. It is possible that Org41841 behaves as a pharmacoperone, a drug which increases the percentage of newly synthesized receptor routing to the membrane. Like pharmacoperones for other receptors, this drug was able to rescue a particular mutant hFSHR (A(189)V) associated with misrouting and endoplasmic reticulum retention, although other mutants could not be rescued. This is potentially the first member of the pharmacoperone drug class which binds at a site that is distinctive from the ligand binding site.


Asunto(s)
Membrana Celular/efectos de los fármacos , Piridinas/farmacología , Receptores de HFE/metabolismo , Animales , Células COS , Membrana Celular/metabolismo , Chlorocebus aethiops , Relación Dosis-Respuesta a Droga , Humanos , Modelos Biológicos , Unión Proteica , Transporte de Proteínas/efectos de los fármacos , Piridinas/metabolismo , Pirimidinas/metabolismo , Pirimidinas/farmacología , Receptores de HFE/genética , Receptores de HL/agonistas , Tiofenos/metabolismo , Tiofenos/farmacología , Transfección
19.
Arch Biochem Biophys ; 477(1): 92-7, 2008 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-18541137

RESUMEN

The GnRH receptor is coupled to G proteins of the families G(q) and G(11). G(q) and G(11) coupling leads to intracellular signaling through the phospholipase C pathway. GnRHR coupling to other G proteins is controversial. This study provides evidence that G protein families G(s), G(i), G(q) and G(11) complete for binding with the GnRHR. We quantified interactions of over-expressed G proteins with GnRHR by a competitive binding approach, using measurements of second messengers, IP and cAMP. Transient co-transfection of HEK293 cells with human WT GnRHR and with stimulatory and inhibitory G proteins (G(q), G(11) and G(s), G(i)) led to either production or inhibition of total inositol phosphate (IP) production, depending on the G protein that was over-expressed. Studies were conducted in different human (COS7, HeLa) and rodent-derived (CHO-K1, GH(3)) cell lines in order to confirm that G protein promiscuity observed with the GnRHR was not limited to a particular cell type.


Asunto(s)
Proteínas de Unión al GTP/metabolismo , Receptores LHRH/metabolismo , Animales , Unión Competitiva , Bucladesina/farmacología , Células CHO , Células COS , Chlorocebus aethiops , Toxina del Cólera/farmacología , Colforsina/farmacología , Cricetinae , Cricetulus , Inhibidores Enzimáticos/farmacología , Estrenos/farmacología , Células HeLa , Humanos , Toxina del Pertussis/farmacología , Pirrolidinonas/farmacología , Fosfolipasas de Tipo C/antagonistas & inhibidores
20.
FASEB J ; 21(2): 384-92, 2007 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-17172315

RESUMEN

The primate GnRH receptor (GnRHR) is a GPCR (G-protein-coupled receptor) that transduces both amplitude- and frequency-modulated signals; each modality conveys information that regulates primate reproduction. Slower GnRH pulses favor release (and higher circulating levels) of pituitary FSH, while faster pulses favor LH release. We used radioligand binding and inositol phosphate production (a measure of G-protein coupling) in association with mutational analysis to identify the impact of evolved sequence specializations that regulate receptor concentration at the plasma membrane and Kd in primate GnRHRs. Our results show that mutations appear to provide a mechanism that allows independent adjustment of response sensitivity and squelching (suppression) of low-level signals (noise), both desirable features for recognition of frequency-modulated signals. We identify specific amino acid residues that appear to be involved in these processes. This investigation occurred in light of recent observations that restriction of GnRHR plasma membrane expression developed under strong convergent pressure and concurrently with the complex pattern of cyclicity associated with primate reproduction. The findings present an evolved means for increased effectiveness of detection of a frequency-modulated signal and provide a strategy to identify similar mechanisms in other receptors.


Asunto(s)
Proteínas Mutantes/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores LHRH/metabolismo , Secuencia de Aminoácidos , Animales , Buserelina/farmacología , Células COS , Chlorocebus aethiops , Secuencia Conservada , Cisteína/química , Cisteína/genética , Cisteína/metabolismo , Perros , Humanos , Fosfatos de Inositol/metabolismo , Lisina/química , Lisina/genética , Lisina/metabolismo , Ratones , Modelos Biológicos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis , Proteínas Mutantes/química , Proteínas Mutantes/genética , Primates , Unión Proteica/efectos de los fármacos , Ensayo de Unión Radioligante , Ratas , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Receptores LHRH/química , Receptores LHRH/genética , Especificidad de la Especie , Transfección
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