RESUMO
The retinal pigmented epithelium (RPE) is a monolayer of multifunctional cells located at the back of the eye. High membrane turnover and polarization, including formation of actin-based apical microvilli, are essential for RPE function and retinal health. Herein, we demonstrate an important role for ßA3/A1-crystallin in RPE. ßA3/A1-crystallin deficiency leads to clathrin-mediated epidermal growth factor receptor (EGFR) endocytosis abnormalities and actin network disruption at the apical side that result in RPE polarity disruption and degeneration. We found that ßA3/A1-crystallin binds to phosphatidylinositol transfer protein (PITPß) and that ßA3/A1-crystallin deficiency diminishes phosphatidylinositol 4,5-biphosphate (PI(4,5)P2), thus probably decreasing ezrin phosphorylation, EGFR activation, internalization, and degradation. We propose that ßA3/A1-crystallin acquired its RPE function before evolving as a structural element in the lens, and that in the RPE, it modulates the PI(4,5)P2 pool through PITPß/PLC signaling axis, coordinates EGFR activation, regulates ezrin phosphorylation and ultimately the cell polarity.
Assuntos
Polaridade Celular/fisiologia , Endocitose , Células Epiteliais/metabolismo , Receptores ErbB/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Cadeia A de beta-Cristalina/metabolismo , Animais , Polaridade Celular/genética , Proteínas do Citoesqueleto/metabolismo , Células Epiteliais/ultraestrutura , Humanos , Camundongos Knockout , Fosfatidilinositol 4,5-Difosfato/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Fosforilação , Ligação Proteica , Epitélio Pigmentado da Retina/citologia , Cadeia A de beta-Cristalina/genéticaRESUMO
Persistent fetal vasculature (PFV) is a human disease that results from failure of the fetal vasculature to regress normally. The regulatory mechanisms responsible for fetal vascular regression remain obscure, as does the underlying cause of regression failure. However, there are a few animal models that mimic the clinical manifestations of human PFV, which can be used to study different aspects of the disease. One such model is the Nuc1 rat model that arose from a spontaneous mutation in the Cryba1 (crystallin, beta 1) gene and exhibits complete failure of the hyaloid vasculature to regress. Our studies with the Nuc1 rat indicate that macroautophagy/autophagy, a process in eukaryotic cells for degrading dysfunctional components to ensure cellular homeostasis, is severely impaired in Nuc1 ocular astrocytes. Further, we show that CRYBA1 interacts with EGFR (epidermal growth factor receptor) and that loss of this interaction in Nuc1 astrocytes increases EGFR levels. Moreover, our data also show a reduction in EGFR degradation in Nuc1 astrocytes compared to control cells that leads to over-activation of the mechanistic target of rapamycin kinase complex 1 (MTORC1) pathway. The impaired EGFR-MTORC1-autophagy signaling in Nuc1 astrocytes triggers abnormal proliferation and migration. The abnormally migrating astrocytes ensheath the hyaloid artery, contributing to the pathogenesis of PFV in Nuc1, by adversely affecting the vascular remodeling processes essential to regression of the fetal vasculature. Herein, we demonstrate in vivo that gefitinib (EGFR inhibitor) can rescue the PFV phenotype in Nuc1 and may serve as a novel therapy for PFV disease by modulating the EGFR-MTORC1-autophagy pathway. ABBREVIATIONS: ACTB: actin, beta; CCND3: cyclin 3; CDK6: cyclin-dependent kinase 6; CHQ: chloroquine; COL4A1: collagen, type IV, alpha 1; CRYBA1: crystallin, beta A1; DAPI: 4'6-diamino-2-phenylindole; EGFR: epidermal growth factor receptor; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFAP: glial fibrillary growth factor; KDR: kinase insert domain protein receptor; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MKI67: antigen identified by monoclonal antibody Ki 67; MTORC1: mechanistic target of rapamycin kinase complex 1; PARP: poly (ADP-ribose) polymerase family; PCNA: proliferating cell nuclear antigen; PFV: persistent fetal vasculature; PHPV: persistent hyperplastic primary vitreous; RPE: retinal pigmented epithelium; RPS6: ribosomal protein S6; RPS6KB1: ribosomal protein S6 kinase, polypeptide 1; SQSTM1/p62: sequestome 1; TUBB: tubulin, beta; VCL: vinculin; VEGFA: vascular endothelial growth factor A; WT: wild type.
Assuntos
Astrócitos/metabolismo , Autofagia/genética , Receptores ErbB/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Vítreo Primário Hiperplásico Persistente/metabolismo , Cadeia A de beta-Cristalina/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Células Cultivadas , Modelos Animais de Doenças , Receptores ErbB/antagonistas & inibidores , Olho/metabolismo , Gefitinibe/farmacologia , Lisossomos/efeitos dos fármacos , Lisossomos/genética , Lisossomos/metabolismo , Lisossomos/ultraestrutura , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Microscopia Imunoeletrônica , Morfolinas/farmacologia , Vítreo Primário Hiperplásico Persistente/genética , Vítreo Primário Hiperplásico Persistente/patologia , Vítreo Primário Hiperplásico Persistente/terapia , Ratos , Transdução de Sinais/genética , Sirolimo/farmacologia , Cadeia A de beta-Cristalina/genéticaRESUMO
The high solubility and lifelong stability of crystallins are crucial to the maintenance of lens transparency and optical properties. Numerous crystallin mutations have been linked to congenital cataract, which is one of the leading causes of newborn blindness. Besides cataract, several crystallin mutations have also been linked to syndromes such as congenital microcornea-cataract syndrome (CMCC). However, the molecular mechanism of CMCC caused by crystallin mutations remains elusive. In the present study, we investigated the mechanism of CMCC caused by the X253R mutation in ßB1-crystallin. The exogenously expressed X253R proteins were prone to form p62-negative aggregates in HeLa cells, strongly inhibited cell proliferation and induced cell apoptosis. The intracellular X253R aggregates could be successfully redissolved by lanosterol but not cholesterol. The extra 26 residues at the C-terminus of ßB1-crystallin introduced by the X253R mutation had little impact on ßB1-crystallin structure and stability, but increased ßB1-crystallin hydrophobicity and decreased its solubility. Interestingly, the X253R mutant fully abolished the aggregatory propensity of ßB1- and ßA3/ßB1-crystallins at high temperatures, suggesting that X253R was an aggregation-inhibition mutation of ß-crystallin homomers and heteromers in dilute solutions. Our results suggest that an increase in hydrophobicity and a decrease in solubility might be responsible for cataractogenesis induced by the X253R mutation, while the cytotoxic effect of X253R aggregates might contribute to the defects in ocular development. Our results also highlight that, at least in some cases, the aggregatory propensity in dilute solutions could not fully mimic the behaviours of mutated proteins in the crowded cytoplasm of the cells.
Assuntos
Catarata/genética , Catarata/metabolismo , Doenças da Córnea/genética , Doenças da Córnea/metabolismo , Agregação Patológica de Proteínas/metabolismo , Cadeia B de beta-Cristalina/química , Cadeia B de beta-Cristalina/metabolismo , Dicroísmo Circular , Células HeLa , Humanos , Interações Hidrofóbicas e Hidrofílicas , Mutação/genética , Agregação Patológica de Proteínas/genética , Cadeia A de beta-Cristalina/química , Cadeia A de beta-Cristalina/genética , Cadeia A de beta-Cristalina/metabolismo , Cadeia B de beta-Cristalina/genéticaRESUMO
Alpha beta-crystallin (CRYAB) is a small heat shock protein that can function as a molecular chaperone and has protective effects for cells undergoing a variety of stressors. Surprisingly, CRYAB has been identified as one of the dominant autoantigens in multiple sclerosis. It has been suggested that autoimmune mediated destruction of this small heat shock protein may limit its protective effects, thereby exacerbating inflammation and cellular damage during multiple sclerosis. It is not altogether clear how autoimmunity against CRYAB might develop, or whether there are environmental factors which might facilitate the presentation of this autoantigen to CD4+ T lymphocytes. In the present study, we utilized an animal model of an Epstein Barr Virus (EBV)-like infection, murine gammaherpesvirus 68 (HV-68), to question whether such a virus could modulate the expression of CRYAB by antigen presenting cells. Following exposure to HV-68 and several other stimuli, in vitro secretion of CRYAB and subsequent intracellular accumulation were observed in cultured macrophages and dendritic cells. Following infection of mice with this virus, it was possible to track CRYAB expression in the spleen and in antigen presenting cell subpopulations, as well as its secretion into the blood. Mice immunized with human CRYAB mounted a significant immune response against this heat shock protein. Further, dendritic cells that were exposed to HV-68 could stimulate CD4+ T cells from CRYAB immunized mice to secrete interferon gamma. Taken together these studies are consistent with the notion of a gammaherpesvirus-induced CRYAB response in professional antigen presenting cells in this mouse model.
Assuntos
Gammaherpesvirinae , Expressão Gênica , Infecções por Herpesviridae/genética , Esclerose Múltipla/genética , Esclerose Múltipla/imunologia , Cadeia A de beta-Cristalina/genética , Animais , Formação de Anticorpos/imunologia , Autoantígenos/genética , Autoantígenos/imunologia , Autoimunidade , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Células da Medula Óssea/virologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Células Dendríticas/virologia , Modelos Animais de Doenças , Feminino , Gammaherpesvirinae/imunologia , Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/metabolismo , Herpesvirus Humano 1/imunologia , Humanos , Interferon gama/biossíntese , Lipopolissacarídeos/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/virologia , Camundongos , Esclerose Múltipla/virologia , Baço/citologia , Baço/imunologia , Baço/metabolismo , Vírus da Estomatite Vesicular Indiana/imunologia , Cadeia A de beta-Cristalina/imunologia , Cadeia A de beta-Cristalina/metabolismoRESUMO
Since crystallins in the human lens do not turnover, they are susceptible to modification by reactive molecules over time. Methylation is a major post-translational lens modification, however the source of the methyl group is not known and the extent of modification across all crystallins has yet to be determined. Sites of methylation in human lens proteins were determined using HPLC/mass spectrometry following digestion with trypsin. The overall extent of protein methylation increased with age, and there was little difference in the extent of modification between soluble and insoluble crystallins. Several different cysteine and histidine residues in crystallins from adult lenses were found to be methylated with one cysteine (Cys 110 in γD crystallin) at a level approaching 70%, however, methylation of crystallins was not detected in fetal or newborn lenses. S-adenosylmethionine (SAM) was quantified at significant (10-50 µM) levels in lenses, and in model experiments SAM reacted readily with N-α-tBoc-cysteine and N-α-tBoc-histidine, as well as ßA3-crystallin. The pattern of lens protein methylation seen in the human lens was consistent with non-enzymatic alkylation. The in vitro data shows that SAM can act directly to methylate lens proteins and SAM was present in significant concentrations in human lens. Thus, non-enzymatic methylation of crystallins by SAM offers a possible explanation for this major human lens modification.
Assuntos
Cristalino/metabolismo , S-Adenosilmetionina/metabolismo , Cadeia A de beta-Cristalina/metabolismo , Adolescente , Adulto , Idoso , Envelhecimento/fisiologia , Cromatografia Líquida de Alta Pressão , Cisteína/metabolismo , Histidina/metabolismo , Humanos , Espectrometria de Massas , Metilação , Pessoa de Meia-Idade , Proteínas Metiltransferases/metabolismo , Processamento de Proteína Pós-Traducional , Proteômica , Doadores de Tecidos , Adulto JovemRESUMO
BACKGROUND: ß-Crystallins are structural proteins maintaining eye lens transparency and opacification. Previous work demonstrated that dimerization of both ßA3 and ßB2 crystallins (ßA3 and ßB2) involves endothermic enthalpy of association (â¼8 kcal/mol) mediated by hydrophobic interactions. METHODOLOGY/PRINCIPAL FINDINGS: Thermodynamic profiles of the associations of dimeric ßA3 and ßB1 and tetrameric ßB1/ßA3 were measured using sedimentation equilibrium. The homo- and heteromolecular associations of ßB1 crystallin are dominated by exothermic enthalpy (-13.3 and -24.5 kcal/mol, respectively). CONCLUSIONS/SIGNIFICANCE: Global thermodynamics of ßB1 interactions suggest a role in the formation of stable protein complexes in the lens via specific van der Waals contacts, hydrogen bonds and salt bridges whereas those ß-crystallins which associate by predominately hydrophobic forces participate in a weaker protein associations.
Assuntos
Multimerização Proteica/fisiologia , Cadeia B de beta-Cristalina/química , Cadeia B de beta-Cristalina/metabolismo , Animais , Metabolismo Energético , Humanos , Camundongos , Modelos Biológicos , Modelos Moleculares , Ligação Proteica/fisiologia , Estrutura Quaternária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Temperatura , Termodinâmica , Cadeia A de beta-Cristalina/química , Cadeia A de beta-Cristalina/metabolismoRESUMO
Congenital cataract-microcornea syndrome (CCMC) is a clinically and genetically heterogeneous condition characterized by lens opacities and microcornea. It appears as a distinct phenotype of heritable congenital cataract. Here we report a large Chinese family with autosomal dominant congenital cataract and microcornea. Evidence for linkage was detected at marker D22S1167 (LOD score [Z]=4.49, recombination fraction [θ]=0.0), which closely flanks the â-crystallin gene cluster locus. Direct sequencing of the candidate âB1-crystallin gene (CRYBB1) revealed a c.387C>A transversion in exon 4, which cosegregated with the disease in the family and resulted in the substitution of serine by arginine at codon 129 (p.Ser129Arg). A comparison of the biophysical properties of the recombinant ß-crystallins revealed that the mutation impaired the structures of both ßB1-crystallin homomer and ßB1/ßA3-crystallin heteromer. More importantly, the mutation significantly decreased the thermal stability of ßB1/ßA3-crystallin but not ßB1-crystallin. These findings highlight the importance of protein-protein interactions among ß-crystallins in maintaining lens transparency, and provide a novel insight into the molecular mechanism underlying the pathogenesis of human CCMC.
Assuntos
Catarata/congênito , Catarata/genética , Doenças da Córnea/congênito , Doenças da Córnea/genética , Mutação de Sentido Incorreto , Cadeia A de beta-Cristalina/química , Cadeia B de beta-Cristalina/química , Cadeia B de beta-Cristalina/genética , Alelos , Substituição de Aminoácidos , Sequência de Bases , Catarata/patologia , Doenças da Córnea/patologia , Análise Mutacional de DNA , Frequência do Gene , Ligação Genética , Humanos , Cristalino/patologia , Multimerização Proteica , Estabilidade Proteica , Análise de Sequência de DNA , Cadeia A de beta-Cristalina/genética , Cadeia A de beta-Cristalina/metabolismo , Cadeia B de beta-Cristalina/metabolismoRESUMO
ß-Crystallins are the major structural proteins in mammalian lens, and their stability is critical in maintaining the transparency and refraction index of the lens. Among the seven ß-crystallins, ßA3-crystallin and ßB1-crystallin, an acidic and a basic ß-crystallin, respectively, can form heteromers in vivo. However, the physiological roles of the heteromer have not been fully elucidated. In this research, we studied whether the basic ß-crystallin facilitates the folding of acidic ß-crystallin. Equilibrium folding studies revealed that the ßA3-crystallin and ßB1-crystallin homomers and the ßA3/ßB1-crystallin heteromer all undergo similar five-state folding pathways which include one dimeric and two monomeric intermediates. ßA3-Crystallin was found to be the most unstable among the three proteins, and the transition curve of ßA3/ßB1-crystallin was close to that of ßB1-crystallin. The dimeric intermediate may be a critical determinant in the aggregation process and thus is crucial to the lifelong stability of the ß-crystallins. A comparison of the Gibbs free energy of the equilibrium folding suggested that the formation of heteromer contributed to the stabilization of the dimer interface. On the other hand, ßA3-crystallin, the only protein whose refolding is challenged by serious aggregation, can be protected by ßB1-crystallin in a dose-dependent manner during the kinetic co-refolding. However, the protection is not observed in the presence of the pre-existed well-folded ßB1-crystallin. These findings suggested that the formation of ß-crystallin heteromers not only stabilizes the unstable acidic ß-crystallin but also protects them against aggregation during refolding from the stress-denatured states.
Assuntos
Multimerização Proteica , Redobramento de Proteína , Cadeia A de beta-Cristalina/química , Cadeia A de beta-Cristalina/metabolismo , Cadeia B de beta-Cristalina/química , Cadeia B de beta-Cristalina/fisiologia , Dimerização , HumanosRESUMO
PURPOSE: The purpose of the study was to determine whether the autodegradation of human ßA3-crystallin is due to its intrinsic protease activity. METHODS: Recombinant His-tagged human ßA3-crystallin was expressed in E. coli and purified by a Ni+2-affinity column chromatographic method. To determine protease activity, the purified crystallin was incubated for 24 h with either sodium deoxycholate, Triton X-100, or CHAPS {3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate} and with benzoyl DL-arginine p-nitroanilide (BAPNA), a colorimetric protease substrate. The autodegradation of the crystallin at 0 h and 24 h on incubation at 37 °C with and without detergents (CHAPS/Triton X-100) was also determined by sodium dodecylsulfate-PAGE (SDS-PAGE) method. To examine whether the autodegradation of the crystallin was due to its protease activity, the crystallin was incubated with inhibitors of serine-, metallo- and cysteine-proteases. The binding of the intact ßA3-crystallin and its autodegradation products to FFCK [5-carboxyfluorescenyl-1-phenylalaninyl-chloromethyl ketone], an analog of TPCK [1-Chloro-3-tosylamido-4-phenyl-2-butanone, a chymotrypsin-type serine protease inhibitor] was also determined by their incubation followed by SDS-PAGE and scanning for fluorescence using a Typhoon 9400 scanner. RESULTS: ßA3-crystallin protease activity showed activation in the presence of CHAPS but not in presence of Triton X-100. Upon incubation of ßA3-crystallin for 24 h with CHAPS or sodium deoxycholate and BAPNA as a substrate, a time-dependent increase in the Arg-bond hydrolyzing activity was observed. SDS-PAGE analysis exhibited autodegradation products with Mr of 22, 27 and 30 kDa, which on partial NH2-terminal sequencing showed cleavage of Lys17-Met18, Gln4-Ala5 and Thr-Gly (in the NH2-terminal His-tag region) bonds, respectively. Almost no autodegradation of the ßA3-crystallin occurred during its incubation alone or with CHAPS plus serine protease inhibitors (phenylmethylsulfonyl fluoride [PMSF], approtinin, and chymostatin). In contrast, the autodegradation occurred in the presence of metallo-protease inhibitors (EDTA and EGTA) and cysteine protease inhibitors (E-64, N-methylmaleimide and iodoacetamide). The ßA3-crystallin also exhibited binding to FFCK, suggesting existence of a chymotrypsin-type active site in the ßA3-crystallin protease. CONCLUSIONS: The results suggested that a serine-type protease activity of ßA3-crystalllin was responsible for its autodegradation. The specific bonds cleaved during autodegradation (Gln4-Ala5 and Lys17-Met18), were localized in the NH2-terminal arm of ßA3-crystallin.
Assuntos
Cristalino/metabolismo , Processamento de Proteína Pós-Traducional , Serina Proteases/metabolismo , Cadeia A de beta-Cristalina/metabolismo , Ácidos Cólicos/farmacologia , Cromatografia de Afinidade , Ácido Desoxicólico/farmacologia , Detergentes/farmacologia , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática/efeitos dos fármacos , Fluorescência , Humanos , Cristalino/efeitos dos fármacos , Inibidores de Proteases/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Cadeia A de beta-Cristalina/isolamento & purificaçãoRESUMO
The human lens proteins beta-crystallins are subdivided into acidic (betaA1-betaA4) and basic (betaB1-betaB3) subunit groups. These structural proteins exist at extremely high concentrations and associate into oligomers under physiological conditions. Crystallin acidic-basic pairs tend to form strong heteromolecular associations. The long N-terminal extensions of beta-crystallins may influence both homo- and heteromolecular interactions. However, identification of the critical regions of the extensions mediating protein associations has not been previously addressed. This was studied by comparing the self-association and heteromolecular associations of wild-type recombinant betaA3- and betaB1-crystallins and their N-terminally truncated counterparts (betaA3DeltaN30 and betaB1DeltaN56) using several biophysical techniques, including analytical ultracentrifugation and fluorescence spectroscopy. Removal of the N-terminal extension of betaA3 had no effect on dimerization or heteromolecular tetramer formation with betaB1. In contrast, the level of self-association of betaB1DeltaN56 increased, resulting in homotetramer formation, and heteromolecular association with betaA3 was blocked. Limited proteolysis of betaB1 produced betaB1DeltaN47, which is similar to intact protein formed dimers but in contrast showed enhanced heteromolecular tetramer formation with betaA3. The tryptic digestion was physiologically significant, corresponding to protease processing sites observed in vivo. Molecular modeling of the N-terminal betaB1 extension indicates structural features that position a mobile loop in the vicinity of these processing sites. The loop is derived from residues 48-56 which appear to be critical for mediating protein interactions with betaA3-crystallin.
Assuntos
Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/fisiologia , Cadeia A de beta-Cristalina/química , Cadeia A de beta-Cristalina/metabolismo , Cadeia B de beta-Cristalina/química , Cadeia B de beta-Cristalina/fisiologia , Animais , Dimerização , Humanos , Camundongos , Fragmentos de Peptídeos/genética , Peptídeo Hidrolases/metabolismo , Mapeamento de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Deleção de Sequência/genética , Cadeia A de beta-Cristalina/genética , Cadeia B de beta-Cristalina/antagonistas & inibidores , Cadeia B de beta-Cristalina/genéticaRESUMO
The purpose of the study was to determine the effects of truncation of various regions of betaB1-crystallin on its structural properties and stability of heterooligomers formed by wild-type (WT) betaB1 or its deletion mutants with WT betaA3-crystallin. For these analyses, seven deletion mutants of betaB1-crystallin were generated with the following sequential deletions of either N-terminal arm [betaB1(59-252)], N-terminal arm + motif I [betaB1(99-252)], N-terminal arm + motif I + motif II [betaB1(144-252)], N-terminal arm + motif I + motif II + connecting peptide [betaB1(149-252)], C-terminal extension [betaB1(1-234)], C-terminal extension plus motif IV [betaB1(1-190)], or C-terminal extension + motif III + motif IV [betaB1(1-148)]. The betaB1-crystallin became water insoluble on the deletion of C-terminal extension and subsequent deletions of the C-terminal domain (C-terminal extension plus motifs III and IV) while it remained partially soluble on the deletion of the N-terminal domain (N-terminal arm plus motifs I and II). However, circular dichroism spectral analysis showed that the deletion of the N-terminal domain but not the C-terminal domain exhibited relatively greater structural changes in the crystallin. The deletion of the C-terminal domain resulted in a greater exposure and disturbance in the microenvironment of Trp-100, Trp-123, and Trp-126 (localized in the motif II), suggesting a relatively greater role of the C-terminal domain than the N-terminal domain in the structural stability of the crystallin. The deletion of the N-terminal extension in betaB1 resulted in maximum exposure of hydrophobic patches and compact structure and in a maximum loss of subunit exchange with WT betaA3-crystallin compared to deletion of either the C-terminal extension, the N-terminal domain, or the C-terminal domain. The thermal stability results of the heterooligomer of betaB1- plus betaA3-crystallins suggested that oligomers lose their stability on deletion of the C-terminal domain. Together, the results suggested that the N-terminal arm of betaB1-crystallin plays a major role in interaction with betaA3-crystallin during heterooligomer formation, and the solubility of betaB1-crystallin per se and that of the heterooligomer with betaA3-crystallin are dependent on the intact C-terminal domain of betaB1-crystallin.
Assuntos
Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Cadeia A de beta-Cristalina , Cadeia B de beta-Cristalina , Naftalenossulfonato de Anilina/química , Dicroísmo Circular , Transferência Ressonante de Energia de Fluorescência , Humanos , Mutagênese Sítio-Dirigida , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Espectrometria de Fluorescência , Cadeia A de beta-Cristalina/química , Cadeia A de beta-Cristalina/genética , Cadeia A de beta-Cristalina/metabolismo , Cadeia B de beta-Cristalina/química , Cadeia B de beta-Cristalina/genética , Cadeia B de beta-Cristalina/metabolismoRESUMO
PURPOSE: Cataracts are a major cause of blindness worldwide. A potential mechanism for loss of visual acuity may be due to light scattering from disruption of normal protein-protein interactions. During aging, the lens accumulates extensively deamidated crystallins. We have previously reported that deamidation in the betaA3-crystallin (betaA3) dimer decreased the stability of the dimer in vitro. The purpose of the present study was to investigate if deamidation altered the interaction of betaA3 with other beta-crystallin subunits. METHODS: Deamidation was mimicked by replacing glutamines, Q85 and Q180, at the predicted interacting interface between betaA3 domains with glutamic acids by site-directed mutagenesis. Human recombinant wild type betaA3 or the doubly deamidated mutant betaA3 Q85E/Q180E (DM betaA3) were mixed with either betaB1- or betaB2-crystallin (betaB1 or betaB2) subunits. After incubation at increasing temperatures, hetero-oligomers were resolved from individual subunits and their molar masses determined by size exclusion chromatography with in line multiangle laser light scattering. Structural changes of hetero-oligomers were analyzed with fluorescence spectroscopy and blue-native PAGE. RESULTS: Molar masses of the hetero-oligomer complexes indicated betaA3 formed a polydispersed hetero-tetramer with betaB1 and a mondispersed hetero-dimer with betaB2. Deamidation at the interface in the betaA3 dimer decreased formation of the hetero-oligomer with betaB1 and further decreased formation of the hetero-dimer with betaB2. During thermal-induced denaturation of the deamidated betaA3 dimer, betaB1 but not betaB2 was able to prevent precipitation of betaA3. CONCLUSIONS: Deamidation decreased formation of hetero-oligomers between beta-crystallin subunits. An excess accumulation of deamidated beta-crystallins in vivo may disrupt normal protein-protein interactions and diminish the stabilizing effects between them, thus, contributing to the accumulation of insoluble beta-crystallins during aging and cataracts.
Assuntos
Cadeia A de beta-Cristalina/química , Cadeia B de beta-Cristalina/química , Substituição de Aminoácidos , Cromatografia em Gel , Dicroísmo Circular , Ácido Glutâmico/química , Glutamina/química , Humanos , Multimerização Proteica , Estrutura Quaternária de Proteína , Espalhamento de Radiação , Cadeia A de beta-Cristalina/genética , Cadeia A de beta-Cristalina/metabolismo , Cadeia B de beta-Cristalina/genética , Cadeia B de beta-Cristalina/metabolismoRESUMO
Protein aggregation is a hallmark of several neurodegenerative diseases and also of cataracts. The major proteins in the lens of the eye are crystallins, which accumulate throughout life and are extensively modified. Deamidation is the major modification in the lens during aging and cataracts. Among the crystallins, the betaA3-subunit has been found to have multiple sites of deamidation associated with the insoluble proteins in vivo. Several sites were predicted to be exposed on the surface of betaA3 and were investigated in this study. Deamidation was mimicked by site-directed mutagenesis at Q42 and N54 on the N-terminal domain, N133 and N155 on the C-terminal domain, and N120 in the peptide connecting the domains. Deamidation altered the tertiary structure without disrupting the secondary structure or the dimer formation of betaA3. Deamidations in the C-terminal domain and in the connecting peptide decreased stability to a greater extent than deamidations in the N-terminal domain. Deamidation at N54 and N155 also disrupted the association with the betaB1-subunit. Sedimentation velocity experiments integrated with high-resolution analysis detected soluble aggregates at 15%-20% in all deamidated proteins, but not in wild-type betaA3. These aggregates had elevated frictional ratios, suggesting that they were elongated. The detection of aggregates in vitro strongly suggests that deamidation may contribute to protein aggregation in the lens. A potential mechanism may include decreased stability and/or altered interactions with other beta-subunits. Understanding the role of deamidation in the long-lived crystallins has important implications in other aggregation diseases.
Assuntos
Cristalino/química , Cadeia A de beta-Cristalina/análise , Cadeia A de beta-Cristalina/química , Cadeia A de beta-Cristalina/metabolismo , Substituição de Aminoácidos , Ácido Aspártico/metabolismo , Dicroísmo Circular , Dimerização , Relação Dose-Resposta a Droga , Escherichia coli/genética , Ácido Glutâmico/metabolismo , Temperatura Alta , Humanos , Modelos Moleculares , Peso Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Conformação Proteica , Desnaturação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Termodinâmica , Ureia/farmacologia , Cadeia A de beta-Cristalina/genética , Cadeia B de beta-Cristalina/metabolismoRESUMO
Gene-expression profiling has revealed several molecular subtypes of breast cancer, which differ in their pathobiology and clinical outcomes. Basal-like tumors are a newly recognized subtype of breast cancer, which express genes that are characteristic of basal epithelial cells, such as the basal cytokeratins, and are associated with poor relapse-free and overall survival. However, the genetic and epigenetic alterations that are responsible for the biologically aggressive phenotype of these estrogen receptor-negative and HER2/ErbB2-negative tumors are not well understood, thereby hindering efforts to develop targeted therapies. Here, we focus on new insights into the molecular pathogenesis of basal-like breast cancer and explore how these discoveries might impact the treatment of these poor-prognosis tumors.
Assuntos
Proteína BRCA1/genética , Biomarcadores Tumorais/análise , Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica , Neoplasia de Células Basais/genética , Receptor ErbB-2/genética , Neoplasias da Mama/etiologia , Neoplasias da Mama/patologia , Neoplasias da Mama/terapia , Feminino , Predisposição Genética para Doença , Humanos , Modelos Biológicos , Neoplasia de Células Basais/etiologia , Neoplasia de Células Basais/patologia , Neoplasia de Células Basais/terapia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/fisiologia , Análise Serial de Proteínas , Transdução de Sinais , Cadeia A de beta-Cristalina/metabolismoRESUMO
The beta-crystallins are a family of long-lived, abundant structural proteins that are coexpressed in the vertebrate lens. As beta-crystallins form heteromers, a process that involves transient exposure of hydrophobic interfaces, we have examined whether in vivobeta-crystallin assembly is enhanced by protein chaperones, either small heat shock proteins, Hsp27 or alphaB-crystallin, or Hsp70. We show here that betaA4-crystallin is abundantly expressed in HeLa cells, but rapidly degraded, irrespective of the presence of Hsp27, alphaB-crystallin or Hsp70. Degradation is even enhanced by Hsp70. Coexpression of betaA4-crystallin with betaB2-crystallin yielded abundant soluble betaA4-betaB2-crystallin heteromers; betaB1-crystallin was much less effective in solubilizing betaA4-crystallin. As betaB2-crystallin competed for betaA4-crystallin with Hsp70 and the proteasomal degradation pathway, betaB2-crystallin probably captures an unstable betaA4-crystallin intermediate. We suggest that the proper folding of betaA4-crystallin is not mediated by general chaperones but requires a heteromeric partner, which then also acts as a dedicated chaperone towards betaA4-crystallin.
Assuntos
Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Cadeia A de beta-Cristalina/metabolismo , Animais , Cricetinae , Inibidores Enzimáticos/farmacologia , Genes Reporter , Células HeLa , Humanos , Focalização Isoelétrica , Leupeptinas/farmacologia , Luciferases/metabolismo , Mutação , Dobramento de Proteína , Solubilidade , Cadeia A de beta-Cristalina/genética , Cadeia B de beta-Cristalina/genética , Cadeia B de beta-Cristalina/metabolismo , beta-Cristalinas/genética , beta-Cristalinas/metabolismoRESUMO
Beta-crystallins are major protein constituents of the mammalian lens, where their stability and association into higher order complexes are critical for lens clarity and refraction. Dimerization is an initial step in formation of beta-crystallin complexes. Beta-crystallin association into dimers is energetically highly favoured, but rapidly reversible under physiological conditions. Beta-crystallin dimers can exchange monomers, probably through a transient and energetically unfavoured monomer intermediate state. As predicted by molecular modelling, the fraction of beta-crystallin present as dimers increases with increasing temperature, implying that beta-crystallin association is entropically driven.
Assuntos
Cristalino/metabolismo , beta-Cristalinas/metabolismo , Cromatografia em Gel , Temperatura Alta , Humanos , Focalização Isoelétrica , Modelos Biológicos , Ultracentrifugação , Cadeia A de beta-Cristalina/metabolismo , Cadeia B de beta-Cristalina/metabolismoRESUMO
PURPOSE: The aim of the study was to comparatively analyze crystallin fragments in the water soluble high molecular weight (WS-HMW) and in the water insoluble (WI) protein fractions of human cataractous (with nuclear opacity) and age matched normal lenses to determine the identity of crystallin species that show cataract specific changes such as truncation and post-translational modifications. Because these changes were cataract specific and not aging specific, the results were expected to provide information regarding potential mechanisms of age related cataract development. METHODS: The WS-alpha-crystallin, WS-HMW protein, and WI protein fractions were isolated from normal lenses of different ages and from cataractous lenses. The three fractions were subjected to two dimensional (2D) gel electrophoresis (IEF in the first dimension and SDS-PAGE in the second dimension). Individual spots from 2D gels were trypsin digested and the tryptic fragments were analyzed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. RESULTS: The 2D protein profiles of WS-alpha-crystallin fractions of normal human lenses showed an age related increase in the number of crystallin fragments. In young normal lenses, the WS-alpha-crystallin fragments were mostly C-terminally truncated, but in older lenses these were both N- and C-terminally truncated. The WS-HMW protein fraction from normal lenses contained mainly fragments of alphaA- and alphaB-crystallin, whereas additional fragments of betaB1- and betaA3-crystallin were present in this fraction from cataractous lenses. Similarly, the WI proteins in normal lenses contained fragments of alphaA- and alphaB-crystallin, but cataractous lenses contained additional fragments of betaA3- and betaB1-crystallin. The modifications identified in the WS-HMW and WI crystallin species of cataractous lenses were truncation, oxidation of Trp residues, and deamidation of Asn to Asp residues. CONCLUSIONS: The results show that the components of WS-HMW and WI protein fractions of cataractous lenses differed from normal lenses. Selective insolubilization of fragments of betaA3/A1- and betaB1-crystallin occurred during cataract development compared to normal lenses. Further, the crystallin species of cataractous lenses showed increased truncation, deamidation of Asn to Asp residues, and oxidation of Trp residue.
Assuntos
Envelhecimento/metabolismo , Catarata/metabolismo , Cristalino/metabolismo , Cadeia A de alfa-Cristalina/metabolismo , Cadeia B de alfa-Cristalina/metabolismo , Cadeia A de beta-Cristalina/metabolismo , Cadeia B de beta-Cristalina/metabolismo , Adolescente , Adulto , Idoso , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel Bidimensional , Humanos , Pessoa de Meia-Idade , Peso Molecular , Fragmentos de Peptídeos/metabolismo , Solubilidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , ÁguaRESUMO
Beta-crystallins are major protein constituents of the mammalian lens, where their stability and association into higher order complexes are critical for lens clarity and refraction. Dimerization is an initial step in formation of beta-crystallin complexes. Beta-crystallin association into dimers is energetically highly favoured, but rapidly reversible under physiological conditions. Beta-crystallin dimers can exchange monomers, probably through a transient and energetically unfavoured monomer intermediate state. As predicted by molecular modelling, the fraction of beta-Crystallin present as dimers increases with increasing temperature, implying that beta-crystallin association is entropically driven.
Assuntos
Cristalino/metabolismo , beta-Cristalinas/metabolismo , Cromatografia em Gel , Temperatura Alta , Humanos , Focalização Isoelétrica , Modelos Biológicos , Ultracentrifugação , Cadeia A de beta-Cristalina/metabolismo , Cadeia B de beta-Cristalina/metabolismoRESUMO
Crystallins are bulk structural proteins of the eye lens that have to last a life time. They gradually become modified with age, denature and form light scattering centres. High thermodynamic and kinetic stability of the crystallins enables them to resist unfolding and delay cataract. Here we have made recombinant human betaA1-, betaA3-, and betaA4-crystallins. The betaA3-crystallin formed higher oligomers that lead to precipitation at ambient temperature. Heat-induced precipitation of betaA3-crystallin was compared with human and calf betaB2-crystallins, showing that the human proteins start to precipitate above 50 degrees C while the calf betaB2-crystallin stays in solution even when unfolded. The stabilities of these human acidic beta-crystallin homo-oligomers have been estimated by measuring their unfolding in urea at neutral pH. BetaA3/1/betaB1 and betaA4/betaB1-crystallin hetero-oligomers have been prepared from homo-oligomers by subunit exchange. The resolution of the methodology used was insufficient to detect a stabilization of the betaA4-crystallin subunit in the hetero-oligomer, the betaA1-crystallin subunit was clearly stabilized by its interaction with betaB1-crystallin. Circular dichroism and fluorescence spectroscopies show that homo-dimer surface tryptophans become buried in the betaA3/1/betaB1-crystallin hetero-dimer concomitant with changes in polypeptide chain conformation.