RESUMO
Transparency in the lens is accomplished by the dense packing and short-range order interactions of the crystallin proteins in fiber cells lacking organelles. These features are accompanied by a lack of protein turnover, leaving lens proteins susceptible to a number of damaging modifications and aggregation. The loss of lens transparency is attributed in part to such aggregation during aging. Among the damaging post-translational modifications that accumulate in long-lived proteins, isomerization at aspartate residues has been shown to be extensive throughout the crystallins. In this study of the human lens, we localize the accumulation of l-isoaspartate within water-soluble protein extracts primarily to crystallin peptides in high-molecular weight aggregates and show with MS that these peptides are from a variety of crystallins. To investigate the consequences of aspartate isomerization, we investigated two αA crystallin peptides 52LFRTVLDSGISEVR65 and 89VQDDFVEIH98, identified within this study, with the l-isoaspartate modification introduced at Asp58 and Asp91, respectively. Importantly, whereas both peptides modestly increase protein precipitation, the native 52LFRTVLDSGISEVR65 peptide shows higher aggregation propensity. In contrast, the introduction of l-isoaspartate within a previously identified anti-chaperone peptide from water-insoluble aggregates, αA crystallin 66SDRDKFVIFL(isoAsp)VKHF80, results in enhanced amyloid formation in vitro The modification of this peptide also increases aggregation of the lens chaperone αB crystallin. These findings may represent multiple pathways within the lens wherein the isomerization of aspartate residues in crystallin peptides differentially results in peptides associating with water-soluble or water-insoluble aggregates. Here the eye lens serves as a model for the cleavage and modification of long-lived proteins within other aging tissues.
Assuntos
Cristalinas/química , Ácido Isoaspártico/química , Cristalino/metabolismo , Agregados Proteicos , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Cristalinas/metabolismo , Humanos , Isomerismo , Espectrometria de Massas , Peptídeos/análise , Peptídeos/química , Peptídeos/isolamento & purificação , Proteína D-Aspartato-L-Isoaspartato Metiltransferase/genética , Proteína D-Aspartato-L-Isoaspartato Metiltransferase/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Cadeia A de alfa-Cristalina/química , Cadeia A de alfa-Cristalina/genética , Cadeia A de alfa-Cristalina/metabolismo , Cadeia B de alfa-Cristalina/química , Cadeia B de alfa-Cristalina/genética , Cadeia B de alfa-Cristalina/metabolismoRESUMO
Application of hydrostatic pressure shifts protein conformational equilibria in a direction to reduce the volume of the system. A current view is that the volume reduction is dominated by elimination of voids or cavities in the protein interior via cavity hydration, although an alternative mechanism wherein cavities are filled with protein side chains resulting from a structure relaxation has been suggested [López CJ, Yang Z, Altenbach C, Hubbell WL (2013) Proc Natl Acad Sci USA 110(46):E4306-E4315]. In the present study, mechanisms for elimination of cavities under high pressure are investigated in the L99A cavity mutant of T4 lysozyme and derivatives thereof using site-directed spin labeling, pressure-resolved double electron-electron resonance, and high-pressure circular dichroism spectroscopy. In the L99A mutant, the ground state is in equilibrium with an excited state of only â¼ 3% of the population in which the cavity is filled by a protein side chain [Bouvignies et al. (2011) Nature 477(7362):111-114]. The results of the present study show that in L99A the native ground state is the dominant conformation to pressures of 3 kbar, with cavity hydration apparently taking place in the range of 2-3 kbar. However, in the presence of additional mutations that lower the free energy of the excited state, pressure strongly populates the excited state, thereby eliminating the cavity with a native side chain rather than solvent. Thus, both cavity hydration and structure relaxation are mechanisms for cavity elimination under pressure, and which is dominant is determined by details of the energy landscape.
Assuntos
Bacteriófago T4/enzimologia , Muramidase/química , Mutação , Dicroísmo Circular , Espectroscopia de Ressonância de Spin Eletrônica , Concentração de Íons de Hidrogênio , Pressão Hidrostática , Ligantes , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Muramidase/genética , Mutagênese Sítio-Dirigida , Desnaturação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Solventes , Relação Estrutura-Atividade , Temperatura , TermodinâmicaRESUMO
Excited states of proteins may play important roles in function, yet are difficult to study spectroscopically because of their sparse population. High hydrostatic pressure increases the equilibrium population of excited states, enabling their characterization [Akasaka K (2003) Biochemistry 42:10875-85]. High-pressure site-directed spin-labeling EPR (SDSL-EPR) was developed recently to map the site-specific structure and dynamics of excited states populated by pressure. To monitor global secondary structure content by circular dichroism (CD) at high pressure, a modified optical cell using a custom MgF2 window with a reduced aperture is introduced. Here, a combination of SDSL-EPR and CD is used to map reversible structural transitions in holomyoglobin and apomyoglobin (apoMb) as a function of applied pressure up to 2 kbar. CD shows that the high-pressure excited state of apoMb at pH 6 has helical content identical to that of native apoMb, but reversible changes reflecting the appearance of a conformational ensemble are observed by SDSL-EPR, suggesting a helical topology that fluctuates slowly on the EPR time scale. Although the high-pressure state of apoMb at pH 6 has been referred to as a molten globule, the data presented here reveal significant differences from the well-characterized pH 4.1 molten globule of apoMb. Pressure-populated states of both holomyoglobin and apoMb at pH 4.1 have significantly less helical structure, and for the latter, that may correspond to a transient folding intermediate.
Assuntos
Modelos Moleculares , Mioglobina/química , Pressão , Conformação Proteica , Dicroísmo Circular/métodos , Clonagem Molecular , Espectroscopia de Ressonância de Spin Eletrônica , Concentração de Íons de Hidrogênio , Espectrofotometria Ultravioleta , Marcadores de Spin , TermodinâmicaRESUMO
αA-Crystallin (αA) and αB-crystallin (αB), the two prominent members of the small heat shock family of proteins are considered to be two subunits of one multimeric protein, α-crystallin, within the ocular lens. Outside of the ocular lens, however, αA and αB are known to be two independent proteins, with mutually exclusive expression in many tissues. This dichotomous view is buoyed by the high expression of αA and αB in the lens and their co-fractionation from lens extracts as one multimeric entity, α-crystallin. To understand the biological function(s) of each of these two proteins, it is important to investigate the biological basis of this perceived dichotomy; in this report, we address the question whether αA and αB exist as independent proteins in the ocular lens. Discontinuous sucrose density gradient fractionation and immunoconfocal localization reveal that in early developing rat lens αA is a membrane-associated small heat shock protein similar to αB but with remarkable differences. Employing an established protocol, we demonstrate that αB predominantly sediments with rough endoplasmic reticulum, whereas αA fractionates with smooth membranes. These biochemical observations were corroborated with immunogold labeling and transmission electron microscopy. Importantly, in the rat heart also, which does not contain αA, αB fractionates with rough endoplasmic reticulum, suggesting that αA has no influence on the distribution of αB. These data demonstrate presence of αA and αB in two separate subcellular membrane compartments, pointing to their independent existence in the developing ocular lens.
Assuntos
Retículo Endoplasmático/metabolismo , Cristalino/embriologia , Cristalino/ultraestrutura , Cadeia A de alfa-Cristalina/metabolismo , Cadeia B de alfa-Cristalina/metabolismo , Animais , Retículo Endoplasmático/ultraestrutura , Ratos , Ratos Sprague-Dawley , Ultracentrifugação , Cadeia A de alfa-Cristalina/química , Cadeia B de alfa-Cristalina/químicaRESUMO
PURPOSE: The roles that crystallin proteins play during lens development are not well understood. Similarities in the adult crystallin composition of mammalian and zebrafish lenses have made the latter a valuable model for examining lens function. In this study, we describe the changing zebrafish lens proteome during development to identify ontogenetic shifts in crystallin expression that may provide insights into age-specific functions. METHODS: Two-dimensional gel electrophoresis and size exclusion chromatography were used to characterize the lens crystallin content of 4.5-day to 27-month-old zebrafish. Protein spots were identified with mass spectrometry and comparisons with previously published proteomic maps, and quantified with densitometry. Constituents of size exclusion chromatography elution peaks were identified with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. RESULTS: Zebrafish lens crystallins were expressed in three ontogenetic patterns, with some crystallins produced at relatively constant levels throughout development, others expressed primarily before 10 weeks of age (ßB1-, ßA1-, and γN2-crystallins), and a third group primarily after 10 weeks (α-, ßB3-, and γS-crystallins). Alpha-crystallins comprised less than 1% of total lens protein in 4.5-day lenses and increased to less than 7% in adult lenses. The developmental period between 6 weeks and 4 months contained the most dramatic shifts in lens crystallin expression. CONCLUSIONS: These data provide the first two-dimensional gel electrophoresis maps of the developing zebrafish lens, with quantification of changing crystallin abundance and visualization of post-translational modification. Results suggest that some crystallins may play stage specific roles during lens development. The low levels of zebrafish lens α-crystallin relative to mammals may be due to the high concentrations of γ-crystallins in this aquatic lens. Similarities with mammalian crystallin expression continue to support the use of the zebrafish as a model for lens crystallin function.
Assuntos
Cristalinas/metabolismo , Cristalino/crescimento & desenvolvimento , Cristalino/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismo , Animais , Cristalinas/genética , Eletroforese em Gel Bidimensional , Proteoma/genética , Proteoma/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Proteínas de Peixe-Zebra/genética , alfa-Cristalinas/genética , alfa-Cristalinas/metabolismo , beta-Cristalinas/genética , beta-Cristalinas/metabolismo , gama-Cristalinas/genética , gama-Cristalinas/metabolismoRESUMO
Despite the enormous number of studies demonstrating changes in the chaperone-like activity of α-crystallins in vitro, little is known about how these changes influence life-long lens transparency in vivo. Using the γB-crystallin I4F mutant protein as a target for αA-crystallins, we examined how cataract phenotypes are modulated by interactions between α-crystallins with altered chaperone-like activities and γB-I4F proteins in vivo. Double heterozygous α-crystallin knock-out αA(+/-) αB(+/-) mice with a decreased amount of α-crystallins were used to simulate reduced total α-crystallin chaperone-like activity in vivo. We found that triple heterozygous αA(+/-) αB(+/-) γB(I4F/+) mice developed more severe whole cataracts than heterozygous γB(I4F/+) mice. Thus, total chaperone-like activity of α-crystallins is important for maintaining lens transparency. We further tested whether mutant αA-crystallin Y118D proteins with increased chaperone-like activity influenced the whole cataract caused by the γB-I4F mutation. Unexpectedly, compound αA(Y118D/+) γB(I4F/+) mutant lenses displayed severe nuclear cataracts, whereas the lens cortex remained unaffected. Thus, the synergistic effect of αA-Y118D and γB-I4F mutant proteins is detrimental to the transparency only in the lens core. α-Crystallins with different chaperone-like activities are likely required in the lens cortex and nucleus for maintaining transparency.
Assuntos
Catarata , Córtex do Cristalino , Mutação , Cadeia A de alfa-Cristalina , beta-Cristalinas , Substituição de Aminoácidos , Animais , Catarata/genética , Catarata/metabolismo , Catarata/patologia , Linhagem Celular Transformada , Heterozigoto , Córtex do Cristalino/metabolismo , Córtex do Cristalino/patologia , Camundongos , Camundongos Knockout , Cadeia A de alfa-Cristalina/genética , Cadeia A de alfa-Cristalina/metabolismo , beta-Cristalinas/genética , beta-Cristalinas/metabolismoRESUMO
The generation of stable clones for biomolecule production is a common but lengthy and labor-intensive process. For complex molecules, such as viruses or virus-like particles (VLPs), the timeline becomes even more cumbersome. Thus, in the early stages of development, transient production methods serve as a reasonable alternative to stable clone construction. In this work, an investigation of a polyethylenimine- (PEI-) based transfection method for the transient production of Chikungunya (Chik) VLPs, a vaccine candidate molecule, was undertaken. This effort focuses on tracking cell population responses during transfection, understanding how process changes affect these responses, and monitoring patterns in cell performance over the culture duration. Plasmid labeling and VLP staining were employed to comprehensively track cells via flow cytometry and to draw correlations between plasmid DNA (pDNA) uptake and the resulting VLP expression. The method detected high transfection efficiency (≥97%) in all samples tested and demonstrated the capability to track kinetics of plasmid-cell binding. With varied transfection cell concentrations, the pDNA binding kinetics are altered and saturation binding is observed in the lowest cell concentration sample tested in less than 3 hours of incubation. Interestingly, in all samples, the flow cytometry analysis of relative pDNA amount versus VLP expression staining showed that cells which contained fewer pDNA complexes resulted in the highest levels of VLP stain. Finally, to determine the potential breadth of our observations, we compared daily expression patterns of ChikVLP with a reporter, monomeric GFP molecule. The similarities detected suggest the interpretations presented here to likely be more broadly informative and applicable to PEI-based transient production of additional biological products as well.
RESUMO
Alpha A and alpha B crystallins are key members of the small heat-shock protein family. In addition to being a major structural protein of the lens, they are constitutively found in many other cells, where their function is not completely understood. Alpha B crystallin is also known to be over-expressed in many neurological diseases. To date, all efforts to crystallize alpha A or alpha B have failed. Thus, high-resolution data on the tertiary and quaternary structures of alpha crystallin is not available. The main reason for this failure seems to be the polydisperse nature of alpha crystallin. This review deals mainly with the polydisperse properties of alpha crystallin and the influence of post-translational modification, chemical modifications, truncations and mutation on its quaternary structure.
Assuntos
Núcleo do Cristalino/metabolismo , Estrutura Quaternária de Proteína , alfa-Cristalinas/química , Animais , Dicroísmo Circular , Humanos , Peso Molecular , Processamento de Proteína Pós-Traducional , Espalhamento de Radiação , Relação Estrutura-AtividadeRESUMO
AlphaB-crystallin is a member of the sHsp (small heat-shock protein) family that prevents misfolded target proteins from aggregating and precipitating. Phosphorylation at three serine residues (Ser19, Ser45 and Ser59) is a major post-translational modification that occurs to alphaB-crystallin. In the present study, we produced recombinant proteins designed to mimic phosphorylation of alphaB-crystallin by incorporating a negative charge at these sites. We employed these mimics to undertake a mechanistic and structural investigation of the effect of phosphorylation on the chaperone activity of alphaB-crystallin to protect against two types of protein misfolding, i.e. amorphous aggregation and amyloid fibril assembly. We show that mimicking phosphorylation of alphaB-crystallin results in more efficient chaperone activity against both heat-induced and reduction-induced amorphous aggregation of target proteins. Mimick-ing phosphorylation increased the chaperone activity of alphaB-crystallin against one amyloid-forming target protein (kappa-casein), but decreased it against another (ccbeta-Trp peptide). We observed that both target protein identity and solution (buffer) conditions are critical factors in determining the relative chaperone ability of wild-type and phosphorylated alphaB-crystallins. The present study provides evidence for the regulation of the chaperone activity of alphaB-crystallin by phosphorylation and indicates that this may play an important role in alleviating the pathogenic effects associated with protein conformational diseases.
Assuntos
Cadeia B de alfa-Cristalina/metabolismo , Animais , Caseínas/isolamento & purificação , Catalase/metabolismo , Bovinos , Feminino , Lactalbumina/metabolismo , Fígado/enzimologia , Leite , Chaperonas Moleculares/fisiologia , Nefelometria e Turbidimetria , Fosforilação , Espalhamento de Radiação , Cadeia B de alfa-Cristalina/química , Cadeia B de alfa-Cristalina/isolamento & purificaçãoRESUMO
PURPOSE: To understand the underlying molecular mechanism for a dominant cataract caused by a point mutation in the gammaD-crystallin gene. METHODS: A dominant cataractous mouse line was identified from chemically induced mouse mutations by phenotypic screening with slit lamp examination. Genomewide linkage analysis and DNA sequencing were used to determine the causative gene mutation. Histology, immunohistochemistry, Western blotting, and in vitro transfection studies were used to characterize mutant lenses. RESULTS: Cataracts in mutant mice were caused by a point mutation in the gammaD-crystallin gene (gammaD-V76D). Intranuclear gamma-crystallin aggregates, incomplete denucleation, and decreased connexins were observed in mutant lens fiber cells. Mutant gammaD-V76D proteins became less soluble in the lens, and structural modeling suggested that the substituted aspartic acid residue (D) altered hydrogen bond formation and surface electrostatic potential of the protein. Unexpectedly, the formation of cold cataracts, which occurred in wild-type lenses at low temperature, was abolished in gammaD-V76D mutant lenses. In vitro transfection studies revealed that wild-type gammaD proteins were uniformly distributed in the cytosol and nucleus of transfected cells, whereas gammaD-V76D proteins formed cytosolic and nuclear aggregates. CONCLUSIONS: Mutant gammaD-V76D reduces protein solubility in the lens and forms substantial intranuclear aggregates that disrupt the denucleation process of inner lens fiber cells. Sustained fiber cell nuclei and nuclear remnants scatter light, whereas other downstream events, such as decreased connexins, presumably disrupt gap junction communication and lens homeostasis, further contributing to the cataract phenotype in mutant lenses. This work also suggests that gammaD-crystallin is one of the crucial components for the formation of cold cataracts in vivo.
Assuntos
Catarata/metabolismo , Células Epiteliais/metabolismo , Cristalino/metabolismo , gama-Cristalinas/metabolismo , Substituição de Aminoácidos , Animais , Catarata/etiologia , Catarata/genética , Linhagem Celular Transformada , Núcleo Celular/metabolismo , Temperatura Baixa/efeitos adversos , Conexinas/metabolismo , Citosol/metabolismo , Células Epiteliais/citologia , Feminino , Cristalino/citologia , Masculino , Camundongos , Camundongos Mutantes , Fenótipo , Mutação Puntual , Estrutura Terciária de Proteína , Ruptura , Solubilidade , Água/metabolismo , gama-Cristalinas/química , gama-Cristalinas/genéticaRESUMO
PURPOSE: Calpain-mediated C-terminal cleavage of alpha A-crystallins occurs during aging and cataractogenesis. The objective of the present study was to explore the role of the ubiquitin-proteasome pathway (UPP) in degrading C-terminal truncated alpha A-crystallins. METHODS: Recombinant wild-type (wt) alpha A-crystallin and C-terminal truncated alpha A(1-168)-, alpha A(1-163)-, and alpha A(1-162)-crystallins were expressed in Escherichia coli and purified to homogeneity. The wt and truncated alpha A-crystallins were labeled with (125)I, and proteolytic degradation was determined using both lens fiber lysate and reticulocyte lysate as sources of ubiquitinating and proteolytic enzymes. Far UV circular dichroism, tryptophan fluorescence intensity, and binding to the hydrophobic fluorescence probe Bis-ANS were used to characterize the wt and truncated alpha A-crystallins. Oligomer sizes of these crystallins were determined by multiangle light-scattering. RESULTS: Whereas wt alpha A-crystallin was degraded moderately in both lens fiber and reticulocyte lysates, alpha A(1-168)-crystallin was resistant to degradation. The susceptibility of alpha A(1-163)-crystallin to degradation was comparable to that of wt alpha A-crystallin. However, alpha A(1-162)-crystallin was much more susceptible than wt alpha A-crystallin to degradation in both lens fiber and reticulocyte lysates. The degradation of both wt and C-terminal truncated alpha A(1-162)-crystallins requires adenosine triphosphate (ATP) and was stimulated by addition of a ubiquitin-conjugating enzyme, Ubc4. The degradation was substantially inhibited by the proteasome inhibitor MG132 and a dominant negative mutant of ubiquitin, K6W-Ub, indicating that at least part of the proteolysis was mediated by the UPP. Spectroscopic analyses of wt and C-terminal truncated alpha A-crystallins revealed that C-terminal truncation of alpha A-crystallin resulted in only subtle changes in secondary structures. However, C-terminal truncations resulted in significant changes in surface hydrophobicity and thermal stability. Thus, these conformational changes may reveal or mask the signals for the ubiquitin-dependent degradation. CONCLUSIONS: The present data demonstrate that C-terminal cleavage of alpha A-crystallin not only alters its conformation and thermal stability, but also its susceptibility to degradation by the UPP. The rapid degradation of alpha A(1-162) by the UPP may prevent its accumulation in the lens.
Assuntos
Complexo de Endopeptidases do Proteassoma/fisiologia , Complexos Ubiquitina-Proteína Ligase/fisiologia , Ubiquitina/fisiologia , Cadeia A de alfa-Cristalina/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Dicroísmo Circular , Inibidores de Cisteína Proteinase/farmacologia , Expressão Gênica , Cristalino/metabolismo , Conformação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes , Cadeia A de alfa-Cristalina/químicaRESUMO
PURPOSE: To identify new mouse models for studying roles of alphaAlpha-crystallin in vivo and to investigate why and how different mutations of the alphaAlpha-crystallin gene lead to dominant or recessive cataracts. METHODS: Using mouse genetic approaches and slit lamp screening, we identified two mouse cataractous mutant lines. Causative genes were mapped by a genome-wide linkage analysis. DNA sequencing verified missense mutations of alphaA-crystallin gene in both mutant lines. Histology, imaging of green fluorescent protein (GFP)-positive lenses, and protein 2-DE gel were used to determine the morphologic and biochemical properties of mutant lenses. RESULTS: Two new alphaA-crystallin gene mutations were identified, alphaA-R54C (alphaA-Cys) and alphaA-Y118D, which cause recessive whole cataracts and dominant nuclear cataracts, respectively. In homozygous alphaA-Cys mutant mice, lens epithelial and fiber cells lost their characteristic cellular features and developed disrupted subcellular structures, such as actin filaments and mitochondria. The nuclear cataract caused by alphaA-Y118D mutation was associated with increased water-insoluble crystallins (alpha, beta, and gamma classes). These results suggest that the Arg54 residue in the N-terminal region is crucial for alphaA-crystallin to perform its roles in lens epithelial and fiber cells during development, whereas the Y118D mutation in the central alpha-crystallin domain impairs alphaA-crystallin's ability to maintain the solubility of crystallin proteins in the lens. CONCLUSIONS: This work demonstrates that different regions of alphaA-crystallin mediate distinct functions in vivo. These two mutant mouse lines provide useful animal models for further investigating the multiple roles of alphaA-crystallin in the lens.
Assuntos
Arginina/fisiologia , Catarata/genética , Cristalino/fisiologia , Mutação Puntual , Tirosina/fisiologia , Cadeia A de alfa-Cristalina/genética , Actinas/metabolismo , Animais , Catarata/patologia , Mapeamento Cromossômico , Modelos Animais de Doenças , Eletroforese em Gel Bidimensional , Células Epiteliais/fisiologia , Feminino , Genes Dominantes , Genes Recessivos , Ligação Genética , Proteínas de Fluorescência Verde , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Mitocôndrias/patologia , FenótipoRESUMO
alpha-Crystallin is the principal lens protein which, in addition to its structural role, also acts as a molecular chaperone, to prevent aggregation and precipitation of other lens proteins. One of its two subunits, alphaB-crystallin, is also expressed in many nonlenticular tissues, and a natural missense mutation, R120G, has been associated with cataract and desmin-related myopathy, a disorder of skeletal muscles [Vicart P, Caron A, Guicheney P, Li Z, Prevost MC, Faure A, Chateau D, Chapon F, Tome F, Dupret JM, Paulin D & Fardeau M (1998) Nat Genet20, 92-95]. In the present study, real-time 1H-NMR spectroscopy showed that the ability of R120G alphaB-crystallin to stabilize the partially folded, molten globule state of alpha-lactalbumin was significantly reduced in comparison with wild-type alphaB-crystallin. The mutant showed enhanced interaction with, and promoted unfolding of, reduced alpha-lactalbumin, but showed limited chaperone activity for other target proteins. Using NMR spectroscopy, gel electrophoresis, and MS, we observed that, unlike the wild-type protein, R120G alphaB-crystallin is intrinsically unstable in solution, with unfolding of the protein over time leading to aggregation and progressive truncation from the C-terminus. Light scattering, MS, and size-exclusion chromatography data indicated that R120G alphaB-crystallin exists as a larger oligomer than wild-type alphaB-crystallin, and its size increases with time. It is likely that removal of the positive charge from R120 of alphaB-crystallin causes partial unfolding, increased exposure of hydrophobic regions, and enhances its susceptibility to proteolysis, thus reducing its solubility and promoting its aggregation and complexation with other proteins. These characteristics may explain the involvement of R120G alphaB-crystallin with human disease states.
Assuntos
Cristalinas/fisiologia , Lactalbumina/metabolismo , Animais , Bovinos , Cromatografia em Gel , Lactalbumina/química , Luz , Espectrometria de Massas , Ressonância Magnética Nuclear Biomolecular , Desnaturação Proteica , Espalhamento de RadiaçãoRESUMO
PURPOSE: The vertebrate small heat shock proteins alphaA- and alphaB-crystallin contribute to the transparency and refractive power of the lens and may also prevent the aggregation of non-native proteins that would otherwise lead to cataracts. We previously showed that zebrafish (Danio rerio) and human alphaB-crystallin have diverged far more in primary structure and expression pattern than the orthologous alphaA-crystallins. In this current study we further compare the structure and function of zebrafish and mammalian alpha-crystallins. METHODS: Near UV CD spectroscopy was used to analyze the tertiary structure and thermal stability of recombinant zebrafish alpha-crystallins. The chaperone-like activities of zebrafish and human alpha-crystallins were compared by assaying their ability to prevent the chemically induced aggregation of several target proteins at temperatures between 25 degrees C and 40 degrees C. RESULTS: Zebrafish and human alphaA-crystallin showed very similar tertiary structures, while the alphaB-crystallin orthologs showed differences related to the presence of additional aromatic amino acids in the zebrafish protein. The denaturation temperatures of zebrafish crystallins were lower than those of mammals. The chaperone-like activities of the two zebrafish alpha-crystallins were highly divergent, with alphaA-crystallin showing much greater activity than alphaB-crystallin. CONCLUSIONS: alphaA-crystallin serves a similar physiological function in both zebrafish and mammals as a lens specific chaperone-like molecule. The reduced chaperone-like function of zebrafish alphaB-crystallin and its lack of extralenticular expression indicates that it plays a different physiological role from its mammalian ortholog. Future comparative studies of alpha-crystallin from closely related vertebrate species can help identify specific structural changes that lead to alterations in chaperone-like activity.
Assuntos
Chaperonas Moleculares/metabolismo , Cadeia A de alfa-Cristalina/química , Cadeia B de alfa-Cristalina/química , Animais , Western Blotting , Bovinos , Dicroísmo Circular , Eletroforese em Gel de Poliacrilamida , Humanos , Desnaturação Proteica , Estrutura Terciária de Proteína , Coelhos , Espectrofotometria Ultravioleta , Relação Estrutura-Atividade , Peixe-Zebra , Cadeia A de alfa-Cristalina/metabolismo , Cadeia B de alfa-Cristalina/metabolismoRESUMO
PURPOSE: To identify lens epithelial genes with altered expression levels in age-related human cataract. METHODS: Epithelia from age-related cataracts and from normal lenses were microdissected and RNA was extracted. RNAs were compared for gene expression differences by RT-PCR differential display. Transcripts exhibiting altered levels of gene expression were cloned and identified by sequencing. The expression levels of identified clones were confirmed by semiquantitative RT-PCR with three separately isolated RNA preparations. Specific primers were designed and used to examine the mRNA levels of other genes important in protein synthesis. RESULTS: Numerous transcripts exhibited altered levels of gene expression. One transcript exhibiting a decreased level of expression in cataract compared with normal lenses was identified as encoding ribosomal protein L21. Three additional ribosomal proteins, L15, L13a, and L7a, also exhibited decreased expression in cataract compared with normal human lenses. By contrast, the levels of elongation factor (EF)-1alpha1 and eucaryotic initiation factor (eIF)-4E remained unchanged. CONCLUSIONS: The results provide evidence that human age-related cataract is associated with decreased expression of L21 and other ribosomal proteins. The results suggest that modulation of protein synthesis and/or other functions mediated by ribosomal proteins is associated with age-related cataract.
Assuntos
Envelhecimento/metabolismo , Catarata/metabolismo , Cristalino/metabolismo , Proteínas Ribossômicas/metabolismo , Idoso , Sequência de Bases , Células Epiteliais/metabolismo , Feminino , Amplificação de Genes , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , RNA/isolamento & purificação , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Ribossômicas/genéticaRESUMO
PURPOSE: To determine the nucleotide sequence, amino acid sequence and tissue specificity of zebrafish alphaA-crystallin. METHODS: RACE, both 3' and 5', was used to clone the zebrafish alphaA-crystallin gene. The peptide sequence of the encoded protein was deduced and compared to cavefish, shark, amphibian, bird and human orthologues using the CLUSTAL W algorithm. alphaA-crystallin transcript was evaluated in brain, heart, lens, liver, skeletal muscle/skin, and spleen by semi-quantitative RT-PCR. RESULTS: The 173 amino acid sequence of zebrafish alphaA-crystallin was determined to be 73% and 86% similar to its human and cavefish orthologues, respectively. We detected high expression of zebrafish alphaA-crystallin in the lens and very low expression in liver and spleen. CONCLUSIONS: Few amino acids identified as being functionally important to chaperone function differ between zebrafish and mammalian alphaA-crystallin. The expression of alphaA-crystallin is mainly confined to the lens in both taxa. These data suggest that zebrafish alphaA-crystallin plays a physiologically limited role outside of the zebrafish lens, similar to its mammalian orthologues.
Assuntos
Cristalinas/genética , Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Cristalinas/isolamento & purificação , Cristalinas/metabolismo , Regulação da Expressão Gênica , Humanos , Cristalino/química , Cristalino/metabolismo , Dados de Sequência Molecular , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de AminoácidosRESUMO
PURPOSE: Age-related cataract is a multi-factorial disease with a poorly understood etiology. Numerous studies provide evidence that the human eye lens has evolved specific regulatory and protective systems to ameliorate lens damage associated with cataract. Other studies suggest that the presence of cataract is associated with the altered expression of specific genes including metallothionein IIa, osteonectin, transglutaminase 2, betaig-h3, multiple ribosomal proteins, ADAM9, and protein phosphatase 2A. Here, we sought to identify further gene expression changes that are associated with cataract and to cluster the identified genes into specific biological pathways. METHODS: Oligonucleotide microarray hybridization was used to analyze the full complement of gene expression differences between lens epithelia isolated from human age-related cataract relative to clear lenses. The expression levels of a subset of the identified genes were further evaluated by semi-quantitative RT-PCR. The identified genes were functionally clustered into specific categories and the probability of over-representation of each category was determined using the computer program EASE. RESULTS: 412 transcripts were observed to be increased and 919 transcripts were observed to be decreased by 2 fold or more in lens epithelia isolated from age-related cataract relative to clear lenses. Of these, 74 were increased and 241 were decreased at the 5 fold level or greater. Seventeen genes selected for further confirmation exhibited similar trends in expression when examined by RT-PCR using both the original and separately prepared clear and cataract RNA populations. Functional clustering of the identified genes using the EASE bioinformatics software package revealed that, among others, transcripts increased in cataract are associated with transcriptional control, chromosomal organization, ionic and cytoplasmic transport, and extracellular matrix components while transcripts decreased in cataract are associated with protein synthesis, defense against oxidative stress, heat-shock/chaperone activity, structural components of the lens, and cell cycle control. CONCLUSIONS: These data suggest that cataract is associated with multiple previously identified and novel changes in lens epithelial gene expression and they point to numerous pathways likely to play important roles in lens protection, maintenance, and age-related cataract.
Assuntos
Envelhecimento/fisiologia , Catarata/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Cristalino/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Idoso , Primers do DNA/química , Células Epiteliais/metabolismo , Humanos , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para CimaRESUMO
PURPOSE: We previously demonstrated that the ubiquitin-proteasome pathway (UPP) is a general protein quality control system that selectively degrades damaged or abnormal lens proteins, including C-terminally truncated αA-crystallin. The objective of this work was to determine the effects of wt αA- and αB-crystallins on the degradation of C-terminally truncated αA-crystallin (αA(1-162)) and vice versa. METHODS: Recombinant wt αA, αB, and αA(1-162) were expressed in Escherichia coli and purified to homogeneity by chromatography. Subunit exchange and oligomerization were detected by fluorescence resonance energy transfer (FRET), multiangle-light scattering and coprecipitation assays. Protein substrates were labeled with (125)I and lens epithelial cell lysates were used as the source of the UPP for degradation assays. RESULTS: FRET, multiangle light scattering, and coprecipitation assays showed that αA(1-162) exchanged subunits with wt αA- or wt αB- crystallin to form hetero-oligomers. αA(1-162) was more susceptible than wt αA-crystallin to degradation by the UPP. When mixed with wt αA-crystallin at 1:1 or 1:4 (αA(1-162) : wt) ratios to form hetero-oligomers, the degradation of αA(1-162) was significantly decreased. Conversely, formation of hetero-oligomers with αA(1-162) enhanced the degradation of wt αA-crystallin. The presence of αA(1-162), but not wt αA-crystallin, decreased the degradation of wt αB-crystallin. CONCLUSIONS: αA(1-162) forms hetero-oligomers with wt αA- and αB-crystallins. Oligomerization with wt αA- or αB-crystallins reduces the susceptibility of αA(1-162) to degradation by the UPP. In addition, the presence of αA(1-162) in the hetero-oligomers also affects the degradation of wt αA- and αB-crystallins.
Assuntos
Complexo de Endopeptidases do Proteassoma/fisiologia , Cadeia A de alfa-Cristalina/química , Cadeia A de alfa-Cristalina/metabolismo , Cadeia B de alfa-Cristalina/química , Cadeia B de alfa-Cristalina/metabolismo , Células Cultivadas , Células Epiteliais , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes , Humanos , Cristalino , Luz , Proteólise , Espalhamento de RadiaçãoRESUMO
Small heat shock proteins alphaA and alphaB crystallin form highly polydisperse oligomers that frustrate protein aggregation, crystallization, and amyloid formation. Here, we present the crystal structures of truncated forms of bovine alphaA crystallin (AAC(59-163)) and human alphaB crystallin (ABC(68-162)), both containing the C-terminal extension that functions in chaperone action and oligomeric assembly. In both structures, the C-terminal extensions swap into neighboring molecules, creating runaway domain swaps. This interface, termed DS, enables crystallin polydispersity because the C-terminal extension is palindromic and thereby allows the formation of equivalent residue interactions in both directions. That is, we observe that the extension binds in opposite directions at the DS interfaces of AAC(59-163) and ABC(68-162). A second dimeric interface, termed AP, also enables polydispersity by forming an antiparallel beta sheet with three distinct registration shifts. These two polymorphic interfaces enforce polydispersity of alpha crystallin. This evolved polydispersity suggests molecular mechanisms for chaperone action and for prevention of crystallization, both necessary for transparency of eye lenses.