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1.
Antioxidants (Basel) ; 11(12)2022 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-36552550

RESUMO

Parkinson's disease (PD) is due to the oxidation of alpha synuclein (αSyn) contributing to motor impairment. We developed a transgenic mouse model of PD that overexpresses the mutated human αSyn gene (A53T) crossed to a mouse expressing the human MPO gene. This model exhibits increased oxidation and chlorination of αSyn leading to greater motor impairment. In the current study, the hMPO-A53T mice were treated with thiocyanate (SCN-) which is a favored substrate of MPO as compared to chlorine. We show that hMPO-A53T mice treated with SCN- have less chlorination in the brain and show an improvement in motor skills compared to the nontreated hMPO-A53T mice. Interestingly, in the hMPO-A53T mice we found a possible link between MPO-related disease and the glymphatic system which clears waste including αSyn from the brain. The untreated hMPO-A53T mice exhibited an increase in the size of periventricular glymphatic vessels expressing the glymphatic marker LYVE1 and aquaporin 4 (AQP4). These vessels also exhibited an increase in MPO and HOCl-modified epitopes in the glymphatic vessels correlating with loss of ependymal cells lining the ventricles. These findings suggest that MPO may significantly promote the impairment of the glymphatic waste removal system thus contributing to neurodegeneration in PD. Moreover, the inhibition of MPO chlorination/oxidation by SCN- may provide a potential therapeutic approach to this disease.

2.
Free Radic Biol Med ; 141: 115-140, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31175983

RESUMO

α-Synuclein (αSyn) is central to the neuropathology of Parkinson's disease (PD) due to its propensity for misfolding and aggregation into neurotoxic oligomers. Nitration/oxidation of αSyn leads to dityrosine crosslinking and aggregation. Myeloperoxidase (MPO) is an oxidant-generating enzyme implicated in neurodegenerative diseases. In the present work we have examined the impact of MPO in PD through analysis of postmortem PD brain and in a novel animal model in which we crossed a transgenic mouse expressing the human MPO (hMPO) gene to a mouse expressing human αSyn-A53T mutant (A53T) (hMPO-A53T). Surprisingly, our results show that in PD substantia nigra, the hMPO gene is expressed in neurons containing aggregates of nitrated αSyn as well as MPO-generated HOCl-modified epitopes. In our hMPO-A53T mouse model, we also saw hMPO expression in neurons but not mouse MPO. In the mouse model, hMPO was expressed in neurons colocalizing with nitrated αSyn, carbamylated lysine, nitrotyrosine, as well as HOCl-modified epitopes/proteins. RNAscope in situ hybridization confirmed hMPO mRNA expression in neurons. Interestingly, the hMPO protein expressed in hMPO-A53T brain is primarily the precursor proMPO, which enters the secretory pathway potentially resulting in interneuronal transmission of MPO and oxidative species. Importantly, the hMPO-A53T mouse model, when compared to the A53T model, exhibited significant exacerbation of motor impairment on rotating rods, balance beams, and wire hang tests. Further, hMPO expression in the A53T model resulted in earlier onset of end stage paralysis. Interestingly, there was a high concentration of αSyn aggregates in the stratum lacunosum moleculare of hippocampal CA2 region, which has been associated in humans with accumulation of αSyn pathology and neural atrophy in dementia with Lewy bodies. This accumulation of αSyn aggregates in CA2 was associated with markers of endoplasmic reticulum (ER) stress and the unfolded protein response with expression of activating transcription factor 4 (ATF4), C/EBP homologous protein (CHOP), MPO, and cleaved caspase-3. Together these findings suggest that MPO plays an important role in nitrative and oxidative damage that contributes to αSyn pathology in synucleinopathies.


Assuntos
Modelos Animais de Doenças , Transtornos Motores/enzimologia , Neurônios/enzimologia , Doença de Parkinson/enzimologia , Peroxidase/metabolismo , Substância Negra/enzimologia , Animais , Carbono/química , Epitopos/química , Feminino , Humanos , Masculino , Aprendizagem em Labirinto , Camundongos , Camundongos Transgênicos , Transtornos Motores/fisiopatologia , Destreza Motora , Nitrogênio/química , Estresse Oxidativo , Doença de Parkinson/fisiopatologia , Peroxidase/genética , Substância Negra/citologia
3.
J Biol Chem ; 293(17): 6374-6386, 2018 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-29496995

RESUMO

Protein carbamylation by cyanate is a post-translational modification associated with several (patho)physiological conditions, including cardiovascular disorders. However, the biochemical pathways leading to protein carbamylation are incompletely characterized. This work demonstrates that the heme protein myeloperoxidase (MPO), which is secreted at high concentrations at inflammatory sites from stimulated neutrophils and monocytes, is able to catalyze the two-electron oxidation of cyanide to cyanate and promote the carbamylation of taurine, lysine, and low-density lipoproteins. We probed the role of cyanide as both electron donor and low-spin ligand by pre-steady-state and steady-state kinetic analyses and analyzed reaction products by MS. Moreover, we present two further pathways of carbamylation that involve reaction products of MPO, namely oxidation of cyanide by hypochlorous acid and reaction of thiocyanate with chloramines. Finally, using an in vivo approach with mice on a high-fat diet and carrying the human MPO gene, we found that during chronic exposure to cyanide, mimicking exposure to pollution and smoking, MPO promotes protein-bound accumulation of carbamyllysine (homocitrulline) in atheroma plaque, demonstrating a link between cyanide exposure and atheroma. In summary, our findings indicate that cyanide is a substrate for MPO and suggest an additional pathway for in vivo cyanate formation and protein carbamylation that involves MPO either directly or via its reaction products hypochlorous acid or chloramines. They also suggest that chronic cyanide exposure could promote the accumulation of carbamylated proteins in atherosclerotic plaques.


Assuntos
Cianatos , Cianetos , Peroxidase , Placa Aterosclerótica/enzimologia , Carbamilação de Proteínas , Animais , Citrulina/análogos & derivados , Citrulina/química , Citrulina/genética , Citrulina/metabolismo , Cianatos/química , Cianatos/metabolismo , Cianetos/química , Cianetos/metabolismo , Humanos , Camundongos , Camundongos Knockout , Oxirredução , Peroxidase/química , Peroxidase/genética , Peroxidase/metabolismo , Placa Aterosclerótica/genética , Placa Aterosclerótica/patologia
4.
PLoS One ; 11(2): e0149391, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26890638

RESUMO

Among the human heme-peroxidase family, myeloperoxidase (MPO) has a unique disulfide-linked oligomeric structure resulting from multi-step processing of the pro-protein monomer (proMPO) after it exits the endoplasmic reticulum (ER). Related family members undergo some, but not all, of the processing steps involved with formation of mature MPO. Lactoperoxidase has its pro-domain proteolytically removed and is a monomer in its mature form. Eosinophil peroxidase undergoes proteolytic removal of its pro-domain followed by proteolytic separation into heavy and light chains and is a heterodimer. However, only MPO undergoes both these proteolytic modifications and then is further oligomerized into a heterotetramer by a single inter-molecular disulfide bond. The details of how and where the post-ER processing steps of MPO occur are incompletely understood. We report here that T47D breast cancer cells stably transfected with an MPO expression plasmid are able to efficiently replicate all of the processing steps that lead to formation of the mature MPO heterotetramer. MPO also traffics to the lysosome granules of T47D cells where it accumulates, allowing in-depth immunofluorescent microscopy studies of MPO trafficking and storage for the first time. Using this novel cell model we show that formation of MPO's single inter-molecular disulfide bond can occur normally in the absence of the proteolytic events that lead to separation of the MPO heavy and light chains. We further demonstrate that Cys319, which forms MPO's unique inter-molecular disulfide bond, is important for events that precede this step. Mutation of this residue alters the glycosylation and catalytic activity of MPO and blocks its entry into the endocytic pathway where proteolytic processing and disulfide bonding occur. Finally, using the endocytic trafficking of lysosomal hydrolases as a guide, we investigate the role of candidate receptors in the endocytic trafficking of MPO.


Assuntos
Expressão Gênica , Lisossomos/metabolismo , Peroxidase/genética , Peroxidase/metabolismo , Processamento de Proteína Pós-Traducional , Catepsinas/antagonistas & inibidores , Linhagem Celular , Linhagem Celular Tumoral , Cistina/metabolismo , Dissulfetos , Retículo Endoplasmático , Ativação Enzimática , Técnicas de Silenciamento de Genes , Glicosilação , Humanos , Espaço Intracelular , Manosefosfatos/metabolismo , Mutação , Peroxidase/química , Multimerização Proteica , Transporte Proteico , Proteólise , RNA Interferente Pequeno/genética
5.
Tumour Biol ; 35(1): 141-8, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23893381

RESUMO

Myeloperoxidase (MPO) is an oxidant generating enzyme normally restricted to myeloid cells, however aberrant MPO expression has been found to occur in non-myeloid cells in some disease states. The functional -463GA promoter polymorphism alters MPO expression levels. The -463G is within an SP1 binding site and is associated with higher gene expression. The G allele is most frequent with ~62% of European populations being GG homozygotes. The GA polymorphism has been associated with risk or survival in a variety of cancers including lung and breast cancer. In this study we determined the frequency of the -463G/A polymorphism in 230 ovarian cancer patients, 75 patients with borderline ovarian tumors, and 299 healthy controls. The GG genotype was found to be overrepresented in patients with early stage ovarian cancer (83.3% GG, p = 0.008) as compared to healthy controls (62% GG), suggesting that MPO oxidants may increase risk. Immunohistochemical analysis revealed MPO expression in a subset of columnar ovarian epithelial carcinoma cells in early stage carcinomas.


Assuntos
Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Peroxidase/genética , Alelos , Estudos de Casos e Controles , Feminino , Expressão Gênica , Frequência do Gene , Genótipo , Humanos , Linfonodos/patologia , Metástase Neoplásica , Estadiamento de Neoplasias , Neoplasias Ovarianas/metabolismo , Peroxidase/metabolismo , Polimorfismo Genético , Regiões Promotoras Genéticas
6.
Am J Physiol Endocrinol Metab ; 305(1): E67-77, 2013 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-23632635

RESUMO

The human GnRH receptor (GNRHR1) has a specific set of properties with physiological and pharmacological influences not appropriately modeled in laboratory animals or cell-based systems. To address this deficiency, we have generated human GNRHR1 knock-in mice and described their reproductive phenotype. Measurement of pituitary GNRHR1 transcripts from homozygous human GNRHR1 knock-in (ki/ki) mice revealed a severe reduction (7- to 8-fold) compared with the mouse Gnrhr1 in wild-type mice. ¹²5I-GnRH binding assays on pituitary membrane fractions corroborated reduced human GNRHR1 protein expression in ki/ki mice, as occurs with transfection of human GNRHR1 in cell lines. Female homozygous knock-in mice displayed normal pubertal onset, indicating that a large reduction in GNRHR1 expression is sufficient for this process. However, ki/ki females exhibited periods of prolonged estrous and/or metestrous and reduced fertility. No impairment was found in reproductive maturity or adult fertility in male ki/ki mice. Interestingly, the serum LH response to GnRH challenge was reduced in both knock-in males and females, indicating a reduced GNRHR1 signaling capacity. Small molecules targeting human GPCRs usually have poor activities at homologous rodent receptors, thus limiting their use in preclinical development. Therefore, we tested a human-specific GnRH1 antagonist, NBI-42902, in our mouse model and demonstrated abrogation of a GnRH1-induced serum LH rise in ki/ki mice and an absence of effect in littermates expressing the wild-type murine receptor. This novel model provides the opportunity to study the human receptor in vivo and for screening the activity of human-specific GnRH analogs.


Assuntos
Ciclo Estral/fisiologia , Fertilidade/fisiologia , Receptores LHRH/genética , Receptores LHRH/fisiologia , Reprodução/fisiologia , Animais , Retroalimentação Fisiológica/efeitos dos fármacos , Retroalimentação Fisiológica/fisiologia , Feminino , Técnicas de Introdução de Genes , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Modelos Animais , Fenótipo , Hipófise/fisiologia , Gravidez , Receptores LHRH/antagonistas & inibidores , Maturidade Sexual/fisiologia , Testículo/crescimento & desenvolvimento , Testículo/fisiologia , Timina/análogos & derivados , Timina/farmacologia
7.
J Biol Chem ; 284(5): 3158-3169, 2009 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-19059911

RESUMO

Myeloperoxidase (MPO) is expressed in Alzheimer disease (AD) but not normal aged brain. A functional -463G/A MPO promoter polymorphism has been associated with AD risk through as yet unidentified mechanisms. Here we report that human MPO-463G allele, but not MPO-463A or mouse MPO, is strongly expressed in astrocytes and deposited in plaques in huMPO transgenic mice crossed to the APP23 model. MPO is similarly expressed in astrocytes in human AD tissue. In cortical homogenates of the MPOG-APP23 model, MPO expression correlated with increased levels of a lipid peroxidation product, 4-hydroxynonenal. Fluorescence high-performance liquid chromatography and electrospray ionization mass spectroscopy identified selective accumulation of phospholipid hydroperoxides in two classes of anionic phospholipids, phosphatidylserine (PS-OOH) and phosphatidylinositol (PI-OOH). The same molecular species of PS-OOH and PI-OOH were elevated in human AD brains as compared with non-demented controls. Augmented lipid peroxidation in MPOG-APP23 mice correlated with greater memory deficits. We suggest that aberrant huMPO expression in astrocytes leads to a specific pattern of phospholipid peroxidation and neuronal dysfunction contributing to AD.


Assuntos
Doença de Alzheimer/enzimologia , Astrócitos/enzimologia , Modelos Animais de Doenças , Transtornos da Memória/enzimologia , Peroxidase/metabolismo , Fosfolipídeos/metabolismo , Alelos , Animais , Encéfalo/enzimologia , Cromatografia Líquida de Alta Pressão , Ensaio de Imunoadsorção Enzimática , Humanos , Imuno-Histoquímica , Hibridização In Situ , Peroxidação de Lipídeos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Peroxidase/genética , Espectrometria de Fluorescência , Espectrometria de Massas por Ionização por Electrospray
9.
Proc Natl Acad Sci U S A ; 104(30): 12451-6, 2007 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-17640886

RESUMO

Endometriosis affects 10-20% of women of reproductive age and is associated with pelvic pain and infertility, and its pathogenesis is not well understood. We used genomewide transcriptional profiling to characterize endometriosis and found that it exhibits a gene expression signature consistent with an underlying autoimmune mechanism. Endometriosis lesions are characterized by the presence of abundant plasma cells, many of which produce IgM, and macrophages that produce BLyS/BAFF/TNFSF13B, a member of the TNF superfamily implicated in other autoimmune diseases. B lymphocyte stimulator (BLyS) protein was found elevated in the serum of endometriosis patients. These observations suggest a model for the pathology of endometriosis where BLyS-responsive plasma cells interact with retrograde menstrual tissues to give rise to endometriosis lesions.


Assuntos
Fator Ativador de Células B/metabolismo , Endometriose/metabolismo , Endometriose/patologia , Regulação da Expressão Gênica , Plasmócitos/metabolismo , Fator Ativador de Células B/genética , Fator Ativador de Células B/imunologia , Antígeno de Maturação de Linfócitos B/imunologia , Antígeno de Maturação de Linfócitos B/metabolismo , Endometriose/genética , Endometriose/imunologia , Feminino , Humanos , Macrófagos/citologia , Macrófagos/metabolismo , Plasmócitos/citologia , Transdução de Sinais/imunologia
10.
Endocrinology ; 148(2): 857-67, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17095587

RESUMO

Suppression of the hypothalamic-pituitary-gonadal axis by peptides that act at the GnRH receptor has found widespread use in clinical practice for the management of sex-steroid-dependent diseases (such as prostate cancer and endometriosis) and reproductive disorders. Efforts to develop orally available GnRH receptor antagonists have led to the discovery of a novel, potent nonpeptide antagonist, NBI-42902, that suppresses serum LH concentrations in postmenopausal women after oral administration. Here we report the in vitro and in vivo pharmacological characterization of this compound. NBI-42902 is a potent inhibitor of peptide radioligand binding to the human GnRH receptor (K(i) = 0.56 nm). Tritiated NBI-42902 binds with high affinity (K(d) = 0.19 nm) to a single class of binding sites and can be displaced by a range of peptide and nonpeptide GnRH receptor ligands. In vitro experiments demonstrate that NBI-42902 is a potent functional, competitive antagonist of GnRH stimulated IP accumulation, Ca(2+) flux, and ERK1/2 activation. It did not stimulate histamine release from rat peritoneal mast cells. Finally, it is effective in lowering serum LH in castrated male macaques after oral administration. Overall, these data provide a benchmark of pharmacological characteristics required for a nonpeptide GnRH antagonist to effectively suppress gonadotropins in humans and suggest that NBI-42902 may have clinical utility as an oral agent for suppression of the hypothalamic-pituitary-gonadal axis.


Assuntos
Receptores LHRH/antagonistas & inibidores , Timina/análogos & derivados , Administração Oral , Animais , Sítios de Ligação , Ligação Competitiva , Bloqueadores dos Canais de Cálcio/farmacologia , Linhagem Celular , Ativação Enzimática/efeitos dos fármacos , Hormônio Liberador de Gonadotropina/farmacologia , Liberação de Histamina/efeitos dos fármacos , Humanos , Fosfatos de Inositol/antagonistas & inibidores , Fosfatos de Inositol/metabolismo , Ligantes , Hormônio Luteinizante/sangue , Macaca , Masculino , Mastócitos/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Orquiectomia , Receptores LHRH/metabolismo , Timina/administração & dosagem , Timina/metabolismo , Timina/farmacologia
11.
J Biol Chem ; 282(9): 6338-46, 2007 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-17192263

RESUMO

Natural peptide agonists of corticotrophin-releasing factor (CRF) receptors bind to the receptor by a two-site mechanism as follows: the carboxyl end of the ligand binds the N-terminal extracellular domain (ECD) of the receptor and the amino portion of the ligand binds the extracellular face of the seven transmembrane region. Recently, peptide antagonists homologous to the 12 C-terminal residues of CRF have been derived, which bind the CRF(1) receptor through an interaction with the ECD. Here we characterized the binding of a minimal 12-residue peptide antagonist while bound to the isolated ECD of the CRF(1) receptor. We have expressed and purified soluble and properly folded ECD independent from the seven-transmembrane region as a thioredoxin fusion protein in Escherichia coli. A model of the peptide antagonist, cyclic corticotrophin-releasing factor residues 30-41 (cCRF(30-41)), was calculated while bound to the recombinant ECD using transferred nuclear Overhauser effect spectroscopy. Although the peptide is unstructured in solution, it adopts an alpha-helical conformation when bound to the ECD. Residues of cCRF(30-41) comprising the binding interface with the ECD were mapped using saturation transfer difference NMR. Two hydrophobic residues (Met(38) and Ile(41)) as well as two amide groups (Asn(34) and the C-terminal amide) on one face of the helix defined the binding epitope of the antagonist. This epitope may be used as a starting point for development of non-peptide antagonists targeting the ECD of this receptor.


Assuntos
Espectroscopia de Ressonância Magnética , Peptídeos/farmacologia , Receptores de Hormônio Liberador da Corticotropina/antagonistas & inibidores , Receptores de Hormônio Liberador da Corticotropina/química , Aminoácidos , Sítios de Ligação , Humanos , Fragmentos de Peptídeos/farmacologia , Peptídeos/química , Ligação Proteica , Conformação Proteica
12.
Mol Cell Endocrinol ; 259(1-2): 1-9, 2006 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-16987592

RESUMO

The capacity of novel benzopyridazinone-based antagonists to inhibit MCH-R1 function, relative to their affinity for the receptor, has been investigated. Three compounds that differ by the addition of either a chlorine atom, or trifluoromethyl group, have nearly identical receptor affinities; however their abilities to inhibit receptor elicited signaling events, measured as a function of time, are dramatically altered. Both the chlorinated and trifluoromethyl modified compounds have a very slow on-rate to maximal functional inhibition relative to the unmodified base compound. A similar impact on inhibitory capacity can be achieved by modifying the side-chain composition at position 2.53 of the receptor; replacement of the native phenylalanine with alanine significantly reduces the amount of time required by the chlorinated compound to attain maximal functional inhibition. The primary attribute responsible for this alteration in inhibitory capacity appears to be the overall bulk of the amino acid at this position-substitution of the similarly sized amino acids leucine and tyrosine results in phenotypes that are indistinguishable from the wild type receptor. Finally, the impact of these differential inhibitory kinetics has been examined in cultured rat neurons by measuring the ability of the compounds to reverse MCH mediated inhibition of calcium currents. As observed using the cell expression models, the chlorinated compound has a diminished capacity to interfere with receptor function. Collectively, these data suggest that differential inhibitory on rates between a small-molecule antagonist and its target receptor can impact the ability of the compound to modify the biological response(s) elicited by the receptor.


Assuntos
Piridazinas/química , Piridazinas/farmacocinética , Receptores de Somatostatina/antagonistas & inibidores , Receptores de Somatostatina/metabolismo , Somatostatina/antagonistas & inibidores , Aminoácidos/química , Animais , Cálcio/metabolismo , Agonistas dos Canais de Cálcio/química , Agonistas dos Canais de Cálcio/farmacocinética , Canais de Cálcio/metabolismo , Células Cultivadas , Desenho de Fármacos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Biológicos , Neurônios/efeitos dos fármacos , Ratos , Receptores de Somatostatina/química
13.
Mol Endocrinol ; 19(10): 2579-90, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15928315

RESUMO

G protein-coupled receptors (GPCRs) are heptahelical integral membrane proteins that require cell surface expression to elicit their effects. The lack of appropriate expression of GPCRs may be the underlying cause of a number of inherited disorders. There is evidence that newly synthesized GPCRs must attain a specific conformation for their correct trafficking to the cell surface. In this study, we show that a single point mutation in human melanin-concentrating hormone receptor (hMCHR1) at position 255 (T255A), which is located at the junction of intracellular loop 3 and transmembrane domain 6, reduces the hMCHR1 cell surface expression level to 20% of that observed for the wild-type receptor. Most of these mutant receptors are located intracellularly, as opposed to the wild-type receptor, which is located primarily on the cell surface. Immunoprecipitation experiments show that hMCHR1-T255A has reduced glycosylation compared with the wild-type receptor and is associated with the chaperone protein, calnexin, and it colocalizes in the endoplasmic reticulum with KDEL-containing proteins. We also demonstrate that a cell-permeable small molecule antagonist of hMCHR1 can function as a pharmacological chaperone to restore cell surface expression of this and other MCHR1 mutants to wild-type levels. Once rescued, the T255A mutant couples to Gq proteins as efficiently as the wild-type receptor. These data suggest that this single mutation produces an hMCHR1 that folds incorrectly, resulting in its retention in the endoplasmic reticulum, but once rescued to the cell surface can still function normally.


Assuntos
Hormônios Hipotalâmicos/metabolismo , Melaninas/metabolismo , Hormônios Hipofisários/metabolismo , Mutação Puntual , Receptores de Somatostatina/química , Receptores de Somatostatina/genética , Sequência de Aminoácidos , Transporte Biológico Ativo , Linhagem Celular , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Expressão Gênica , Glicosilação , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Dobramento de Proteína , Estrutura Terciária de Proteína , Receptores de Somatostatina/antagonistas & inibidores , Receptores de Somatostatina/metabolismo , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfecção
14.
J Biol Chem ; 280(12): 11560-8, 2005 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-15653688

RESUMO

The corticotropin releasing factor (CRF) type 1 receptor (CRF1) is a class B family G protein-coupled receptor that regulates the hypothalamic-pituitary-adrenal stress axis. Astressin is an amino-terminal truncated analog of CRF that retains high affinity binding to the extracellular domain of the receptor and is believed to act as a neutral competitive antagonist of receptor activation. Here we show that despite being unable to activate the CRF1 receptor, astressin binding results in the internalization of the receptor. Furthermore, entirely different pathways of internalization of CRF1 receptors are utilized following CRF and astressin binding. CRF causes the receptor to be phosphorylated, recruit beta-arrestin2, and to be internalized rapidly, likely through clathrin-coated pits. Astressin, however, fails to induce receptor phosphorylation or beta-arrestin2 recruitment, and internalization is slow and occurs through a pathway that is insensitive to inhibitors of clathrin-coated pits and caveolae. The fate of the internalized receptors also differs because only CRF-induced internalization results in receptor down-regulation. Furthermore, we present evidence that for astressin to induce internalization it must interact with both the extracellular amino terminus and the juxtamembrane domain of the receptor. Astressin binds with 6-fold higher affinity to full-length CRF1 receptors than to a chimeric protein containing only the extracellular domain attached to the transmembrane domain of the activin IIB receptor, yet two 12-residue analogs of astressin have similar affinities for both proteins but are unable to induce receptor internalization. These data demonstrate that agonists and antagonists for CRF1 receptors promote distinct conformations, which are then differentially regulated.


Assuntos
Hormônio Liberador da Corticotropina/farmacologia , Fragmentos de Peptídeos/farmacologia , Receptores de Hormônio Liberador da Corticotropina/química , Animais , Células CHO , Hormônio Liberador da Corticotropina/metabolismo , Cricetinae , Endocitose , Humanos , Camundongos , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Receptores de Hormônio Liberador da Corticotropina/antagonistas & inibidores , Receptores de Hormônio Liberador da Corticotropina/metabolismo
15.
Ann Thorac Surg ; 78(2): 436-43; discussion 436-43, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15276492

RESUMO

BACKGROUND: Malignant pleural mesothelioma (MPM) is an uncommon but highly fatal neoplasm for which only limited treatment is available. METHODS: Immunohistochemical analysis was used to determine the expression of interleukin-4 receptors (IL-4R) on mesothelioma cell lines and resected mesothelioma tumors. Radioreceptor binding assays were used to show that these IL-4R were high-affinity receptors. Previously, we had shown that a chimeric protein composed of a circularly permuted IL-4 molecule fused to a truncated form of Pseudomonas exotoxin A, IL-4(38-37)-PE38KDEL, could be used to kill IL-4R-bearing tumor cells in vitro. The toxicity of this molecule to mesothelioma cell lines was tested using a protein synthesis inhibition assay. A human mesothelioma xenograft model was then developed to assess the efficacy of this molecule in vivo. RESULTS: All MPM cell lines tested were found to express high-affinity cell-surface IL-4R. Immunohistochemical analysis of resected mesothelioma tumor specimens from 13 patients revealed that all tumors expressed moderate-to-high levels of IL-4R. Coculture of malignant mesothelioma cell lines with IL-4(38-37)-PE38KDEL resulted in a dose-dependent inhibition of tumor cell protein synthesis through an interaction with cell-surface IL-4R. In a nude mouse xenograft model of human MPM, intratumoral administration of IL-4(38-37)-PE38KDEL mediated a dose-dependent decrease in tumor volume and a dose-dependent increase in survival. CONCLUSIONS: The chimeric protein, IL-4(38-37)-PE38KDEL, has potent antitumor effects against MPM both in vitro and in vivo.


Assuntos
ADP Ribose Transferases/uso terapêutico , Toxinas Bacterianas/uso terapêutico , Exotoxinas/uso terapêutico , Imunotoxinas/uso terapêutico , Mesotelioma/tratamento farmacológico , Proteínas de Neoplasias/efeitos dos fármacos , Neoplasias Pleurais/tratamento farmacológico , Receptores de Interleucina-4/efeitos dos fármacos , Fatores de Virulência/uso terapêutico , ADP Ribose Transferases/administração & dosagem , ADP Ribose Transferases/química , Idoso , Animais , Toxinas Bacterianas/administração & dosagem , Toxinas Bacterianas/química , Linhagem Celular Tumoral/efeitos dos fármacos , Exotoxinas/administração & dosagem , Exotoxinas/química , Feminino , Humanos , Interleucina-4/administração & dosagem , Interleucina-4/química , Masculino , Mesotelioma/química , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Proteínas de Neoplasias/análise , Neoplasias Pleurais/química , Receptores de Interleucina-4/análise , Organismos Livres de Patógenos Específicos , Fatores de Virulência/administração & dosagem , Fatores de Virulência/química , Ensaios Antitumorais Modelo de Xenoenxerto , Exotoxina A de Pseudomonas aeruginosa
16.
J Immunol ; 171(7): 3333-7, 2003 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-14500624

RESUMO

APCs of the nonobese diabetic (NOD) mouse have a genetically programmed capacity to overexpress IL-12p40, a cytokine critical for development of pathogenic autoreactive Th1 cells. To determine whether a diabetes-associated NOD chromosomal locus (i.e., Idd) was responsible for this defect, LPS-stimulated macrophages from several recombinant congenic inbred mice with Idd loci on a C57BL/6 background or with different combinations of NOD and CBA genomic segments were screened for IL-12p40 production. Only macrophages from the congenic strains containing the Idd4 locus showed IL-12p40 overproduction/expression. Moreover, analysis of IL-12p40 sequence polymorphisms demonstrated that the Idd4 intervals in these strains contained the IL-12p40 allele of the NOD, although further analysis is required to determine whether the IL-12p40 allele itself is responsible for its overexpression. Thus, the non-MHC-associated Idd4 locus appears responsible for IL-12p40 overexpression, which may be a predisposing factor for type 1 diabetes in NOD mice.


Assuntos
Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/imunologia , Regulação da Expressão Gênica/imunologia , Interleucina-12/biossíntese , Interleucina-12/genética , Camundongos Endogâmicos NOD/genética , Camundongos Endogâmicos NOD/imunologia , Subunidades Proteicas/biossíntese , Subunidades Proteicas/genética , Locos de Características Quantitativas/imunologia , Alelos , Animais , Células Cultivadas , Mapeamento Cromossômico , Marcadores Genéticos , Predisposição Genética para Doença , Genoma , Interleucina-12/deficiência , Interleucina-12/fisiologia , Subunidade p40 da Interleucina-12 , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/metabolismo , Masculino , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Subunidades Proteicas/deficiência , Subunidades Proteicas/fisiologia , RNA Mensageiro/biossíntese
17.
Neurosci Lett ; 346(3): 182-6, 2003 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-12853114

RESUMO

Different potencies for hypocretin-1 and -2 in sleep-wake regulation and feeding after intracerebroventricular (ICV) administration have been reported. These differences were often explained by the selectivity of the two hypocretins for hypocretin receptor-1 and -2, but little attention has been paid to kinetics of hypocretin peptides. We investigated the kinetics of the ICV hypocretin-1 and -2 in rats. ICV hypocretin-1 (10 nmol) increased hypocretin-1 peptide level in the CSF by 800-fold from baseline with the elevation lasting over 4 h. In contrast, after ICV hypocretin-2 (10 nmol), no significant rise in the CSF was found. CSF hypocretin levels were significantly correlated with the biological activities of CSF hypocretin-1 and -2 using the Ca(2+) mobilization assay. Difference in the kinetics of hypocretins should be considered for interpreting ICV effects of hypocretins.


Assuntos
Proteínas de Transporte/líquido cefalorraquidiano , Peptídeos e Proteínas de Sinalização Intracelular , Neuropeptídeos/líquido cefalorraquidiano , Neurotransmissores/líquido cefalorraquidiano , Animais , Proteínas de Transporte/administração & dosagem , Proteínas de Transporte/farmacocinética , Linhagem Celular , Humanos , Injeções Intraventriculares , Masculino , Neuropeptídeos/administração & dosagem , Neuropeptídeos/farmacocinética , Orexinas , Radioimunoensaio , Ratos , Ratos Wistar
18.
J Pharmacol Exp Ther ; 304(3): 1217-27, 2003 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-12604699

RESUMO

The molecular basis of ligand recognition by the melanocortin 4 receptor (MC4R) has not been fully defined. In this study, we investigated the molecular determinants of MC4R ligand binding, employing a large array of ligands, using three approaches. First, molecular modeling of the receptor was used to identify Phe284, in transmembrane (TM) 7, as a potential site of ligand interaction. Mutation of Phe284 to alanine reduced binding affinity and potency of peptides containing L-Phe by up to 71-fold but did not appreciably affect binding of linear peptides containing D-Phe, consistent with a hydrophobic interaction between the Phe7 of alpha-melanocyte-stimulating hormone and Phe284. Second, we examined the effect of a naturally occurring mutation in TM3 (I137T) that is linked to obesity. This mutation decreased affinity and potency of cyclic, rigid peptides but not more flexible peptides, consistent with an indirect effect of the mutation on the tertiary structure of the receptor. Third, we examined the residues that support ligand selectivity for the MC4R over the MC3R. Mutation of Ile125 (TM3) of the MC4R to the equivalent residue of the MC3R (phenylalanine) selectively decreased affinity and potency of MC4R-selective ligands. This effect was mirrored by the reciprocal MC3R mutation F157I. The magnitude of this effect indicates that this locus is not of major importance. However, it is considered that an isoleucine/phenylalanine mutation may affect the orientation of Asp122, which has been identified as a major determinant of ligand binding affinity. Thus, this study provides further characterization of the MC4R binding pocket.


Assuntos
Receptores da Corticotropina/metabolismo , Sequência de Aminoácidos , Células Cultivadas , AMP Cíclico/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Mutagênese , Receptor Tipo 3 de Melanocortina , Receptor Tipo 4 de Melanocortina , Receptores da Corticotropina/genética , Transfecção
19.
Clin Cancer Res ; 8(11): 3503-11, 2002 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-12429641

RESUMO

Previous studies have demonstrated that human lung tumor cell lines express interleukin 4 (IL-4) receptors, and IL-4 can mediate modest to moderate antiproliferative activity in vitro and in vivo in animal models of human lung tumors. On the basis of these studies, IL-4 was tested in clinical trials; however, it showed little antitumor activity in lung cancer patients. In the present study, we examined the expression of IL-4 receptors (IL-4Rs) in lung tumor samples and normal lung tissues and tested whether an IL-4R targeted agent will have better antitumor activity in vitro and in vivo compared with IL-4. IL-4R expression was tested by immunohistochemistry in 54 lung tumor samples and normal lung tissues in a tissue array, by reverse-transcription PCR and Northern blot analyses in lung tumor cell lines. Cytotoxic activity of IL-4 cytotoxin [IL-4(38-37)-PE38KDEL], composed of a circular permuted IL-4 and a mutated form of Pseudomonas exotoxin (PE38KDEL) was tested by protein synthesis inhibition and clonogenic assays in seven lung tumor cell lines. Antitumor activity of IL-4 cytotoxin was tested in vitro and in immunodeficient animal models of human lung tumors. We observed that IL-4Rs are expressed at higher levels in situ in lung tumor samples compared with normal lung tissues and IL-4 cytotoxin is highly and specifically cytotoxic to lung tumor cell lines in vitro. Intratumoral and i.p. administration of IL-4 cytotoxin to immunodeficient mice with s.c. established human lung H358 non-small cell lung cancer tumors mediated considerable antitumor activity in a dose-dependent manner with the higher dose producing durable complete responses. On the other hand, H460 non-small cell lung cancer tumors expressing low levels of IL-4R did not respond to IL-4 cytotoxin therapy. Because IL-4 cytotoxin mediates its antitumor activity through IL-4R, and a variety of lung tumors expressed high levels of IL-4R, we propose testing the safety of this agent in patients with lung cancer.


Assuntos
Neoplasias Pulmonares/metabolismo , Receptores de Interleucina-4/metabolismo , Animais , Northern Blotting , Peso Corporal , Carcinoma Pulmonar de Células não Pequenas/patologia , Citotoxinas/farmacologia , Relação Dose-Resposta a Droga , Exotoxinas/farmacologia , Humanos , Imuno-Histoquímica , Interleucina-4/metabolismo , Cinética , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Nus , Mutação , Transplante de Neoplasias , Reação em Cadeia da Polimerase , Ligação Proteica , Inibidores da Síntese de Proteínas/farmacologia , Pseudomonas/metabolismo , RNA Mensageiro/metabolismo , Ensaio Radioligante , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Células Tumorais Cultivadas
20.
Int Immunol ; 14(7): 723-32, 2002 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12096031

RESUMO

The requirement of the transcription factor PU.1 for macrophage development has been well documented. However, the target genes regulated by PU.1 controlling macrophage maturation are not known. A granulocyte macrophage colony stimulating factor (GM-CSF)-dependent PU.1 null monocytic precursor cell was stably transduced with a PU.1-expressing retrovirus. The expression of PU.1 altered the surface expression of a few proteins expressed on monocytes; these cells, however, remained GM-CSF dependent and maintained an immature phenotype. In contrast to the PU.1 null cells, the cells expressing PU.1 responded to macrophage colony stimulating factor (M-CSF) with subsequent development into mature macrophages. Using suppressive subtractive hybridization between the PU.1 null and immature PU.1 rescued cells, three genes, MRP-14, Dap12 and CD53, were found expressed in the rescued cells, but not in the PU.1 null cells. In addition, these genes were modulated during M-CSF-induced maturation of the PU.1 rescued cells. The PU.1 null and rescued early monocytic cells provide a useful model to study the role of PU.1 in macrophage development.


Assuntos
Antígenos CD/genética , Antígenos de Diferenciação de Linfócitos T/genética , Calgranulina B/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Monócitos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Imunológicos/genética , Transativadores/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Northern Blotting , Diferenciação Celular/genética , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Vetores Genéticos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Fator Estimulador de Colônias de Macrófagos/farmacologia , Proteínas de Membrana , Hibridização de Ácido Nucleico , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , RNA/genética , RNA Mensageiro/análise , Receptores Imunológicos/metabolismo , Retroviridae/genética , Tetraspanina 25 , Transativadores/biossíntese , Transativadores/genética , Transdução Genética , Regulação para Cima
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