RESUMEN
Amelotin is an intrinsically disordered protein (IDP) rich in Pro residues and is involved in hydroxyapatite mineralization. It rapidly oligomerizes under physiological conditions of pH and pressure but reverts to its monomeric IDP state at elevated pressure. We identified a 105-residue segment of the protein that becomes ordered upon oligomerization, and we used pressure-jump NMR spectroscopy to measure long-range NOE contacts that exist exclusively in the oligomeric NMR-invisible state. The kinetics of oligomerization and dissociation were probed at the residue-specific level, revealing that the oligomerization process is initiated in the C-terminal half of the segment. Using pressure-jump NMR, the degree of order in the oligomer at the sites of Pro residues was probed by monitoring changes in cis/trans equilibria relative to the IDP state after long-term equilibration under oligomerizing conditions. Whereas most Pro residues revert to trans in the oligomeric state, Pro-49 favors a cis configuration and three Pro residues retain an unchanged cis fraction, pointing to their local lack of order in the oligomeric state. NOE contacts and secondary 13C chemical shifts in the oligomeric state indicate the presence of an 11-residue α-helix, preceded by a small intramolecular antiparallel ß-sheet, with slower formation of long-range intermolecular interactions to N-terminal residues. Although none of the models generated by AlphaFold2 for the amelotin monomer was consistent with experimental data, subunits of a hexamer generated by AlphaFold-Multimer satisfied intramolecular NOE and chemical shift data and may provide a starting point for developing atomic models for the oligomeric state.
Asunto(s)
Prolina , Proteínas , Conformación Proteica , Isomerismo , Prolina/química , Espectroscopía de Resonancia Magnética/métodosRESUMEN
Vision requires the transport and recycling of the pigment 11-cis retinaldehyde (retinal) between the retinal pigment epithelium (RPE) and photoreceptors. 11-cis retinal is also required for light-mediated photoreceptor death in dark-adapted mouse eye, probably through overstimulation of rod cells adapted for low light. Retbindin is a photoreceptor-specific protein, of unclear function, that is localized between the RPE and the tips of the photoreceptors. Unexpectedly, young Rtbdn-KO mice, with targeted deletion (KO) of retbindin, showed delayed regeneration of retinal function after bleaching and were strongly resistant to light-induced photoreceptor death. Furthermore, bio-layer interferometry binding studies showed recombinant retbindin had significant affinity for retinoids, most notably 11-cis retinal. This suggests that retbindin mediates light damage, probably through a role in transport of 11-cis retinal. In Rtbdn-KO mice, retinal development was normal, as were amplitudes of rod and cone electroretinograms (ERG) up to 4 months, although implicit times and c-waves were affected. However, with aging, both light- and dark-adapted ERG amplitudes declined significantly and photoreceptor outer segments became disordered, However, in contrast to other reports, there was little retinal degeneration or drop in flavin levels. The RPE developed vacuoles and lipid, protein and calcium deposits reminiscent of age-related macular degeneration. Other signs of premature aging included loss of OPN4+ retinal ganglion cells and activation of microglia. Thus, retbindin plays an unexpected role in the mammalian visual cycle, probably as an adaptation for vision in dim light. It mediates light damage in the dark-adapted eye, but also plays a role in light-adapted responses and in long term retinal homeostasis.
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Envejecimiento Prematuro/genética , Proteínas del Ojo/genética , Regulación de la Expresión Génica , ARN/genética , Células Fotorreceptoras Retinianas Conos/metabolismo , Enfermedades de la Retina/genética , Epitelio Pigmentado de la Retina/metabolismo , Envejecimiento Prematuro/metabolismo , Animales , Adaptación a la Oscuridad/fisiología , Modelos Animales de Enfermedad , Electrorretinografía , Proteínas del Ojo/biosíntesis , Ratones , Microscopía Electrónica de Transmisión , Células Fotorreceptoras Retinianas Conos/ultraestructura , Enfermedades de la Retina/metabolismo , Enfermedades de la Retina/patología , Epitelio Pigmentado de la Retina/ultraestructuraRESUMEN
The polycistronic miR-183/96/182 cluster is preferentially and abundantly expressed in terminally differentiating sensory epithelia. To clarify its roles in the terminal differentiation of sensory receptors in vivo, we deleted the entire gene cluster in mouse germline through homologous recombination. The miR-183/96/182 null mice display impairment of the visual, auditory, vestibular, and olfactory systems, attributable to profound defects in sensory receptor terminal differentiation. Maturation of sensory receptor precursors is delayed, and they never attain a fully differentiated state. In the retina, delay in up-regulation of key photoreceptor genes underlies delayed outer segment elongation and possibly mispositioning of cone nuclei in the retina. Incomplete maturation of photoreceptors is followed shortly afterward by early-onset degeneration. Cell biologic and transcriptome analyses implicate dysregulation of ciliogenesis, nuclear translocation, and an epigenetic mechanism that may control timing of terminal differentiation in developing photoreceptors. In both the organ of Corti and the vestibular organ, impaired terminal differentiation manifests as immature stereocilia and kinocilia on the apical surface of hair cells. Our study thus establishes a dedicated role of the miR-183/96/182 cluster in driving the terminal differentiation of multiple sensory receptor cells.
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Células Ciliadas Auditivas/citología , Células Ciliadas Vestibulares/citología , MicroARNs/genética , Mucosa Olfatoria/citología , Células Fotorreceptoras Retinianas Conos/citología , Células Fotorreceptoras Retinianas Bastones/citología , Animales , Regulación del Desarrollo de la Expresión Génica/genética , Células Ciliadas Auditivas/metabolismo , Células Ciliadas Vestibulares/metabolismo , Trastornos de la Audición/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Familia de Multigenes , Trastornos del Olfato/genética , Mucosa Olfatoria/metabolismo , Equilibrio Postural/genética , Células Fotorreceptoras Retinianas Conos/metabolismo , Células Fotorreceptoras Retinianas Bastones/metabolismo , Trastornos de la Sensación/genética , Trastornos de la Visión/genéticaRESUMEN
Ultra-weak self-association can govern the macroscopic solution behavior of concentrated macromolecular solutions ranging from food products to pharmaceutical formulations and the cytosol. For example, it can promote dynamic assembly of multi-protein signaling complexes, lead to intracellular liquid-liquid phase transitions, and seed crystallization or pathological aggregates. Unfortunately, weak self-association is technically extremely difficult to study, as it requires very high protein concentrations where short intermolecular distances cause strongly correlated particle motion. Additionally, protein samples near their solubility limit in vitro frequently show some degree of polydispersity. Here we exploit the strong mass-dependent separation of assemblies in the centrifugal field to study ultra-weak binding, using a sedimentation velocity technique that allows us to determine particle size distributions while accounting for colloidal hydrodynamic interactions and thermodynamic non-ideality (Chaturvedi, S. K.; et al. Nat. Commun. 2018, 9, 4415; DOI: 10.1038/s41467-018-06902-x ). We show that this approach, applied to self-associating proteins, can reveal a time-average association state for rapidly reversible self-associations from which the free energy of binding can be derived. The method is label-free and allows studying mid-sized proteins at millimolar protein concentrations in a wide range of solution conditions. We examine the performance of this method with hen egg lysozyme as a model system, reproducing its well-known ionic-strength-dependent weak self-association. The application to chicken γS-crystallin reveals weak monomer-dimer self-association with KD = 24 mM, corresponding to a standard free energy change of approximately -9 kJ/mol, which is a large contribution to the delicate balance of forces ensuring eye lens transparency.
Asunto(s)
Muramidasa/química , Multimerización de Proteína , Animales , Pollos , Muramidasa/metabolismo , Ultracentrifugación , gamma-Cristalinas/química , gamma-Cristalinas/metabolismoRESUMEN
KLPH/lctl belongs to the Klotho family of proteins. Expressed sequence tag analyses unexpectedly revealed that KLPH is highly expressed in the eye lens while northern blots showed that expression is much higher in the eye than in other tissues. In situ hybridization in mouse localized mRNA to the lens, particularly in the equatorial epithelium. Immunofluorescence detected KLPH in lens epithelial cells with highest levels in the germinative/differentiation zone. The gene for KLPH in mouse was deleted by homologous recombination. Littermate knockout (KO) and wild type (WT) mice were compared in a wide panel of pathology examinations and were all grossly normal, showing no systemic effects of the deletion. However, the lens, while superficially normal at young ages, had focusing defects and exhibited age-related cortical cataract by slit lamp examination. Whole-lens imaging showed that KO mice had disorganized lens sutures, forming a loose double-y or x instead of the tight y formation of WT. RNA-seq profiles for KO and WT littermates confirmed the absence of KLPH mRNA in KO lens and also showed complete absence of transcripts for Clic5, a protein associated with cilium/basal body related auditory defects in a mouse model. Immunofluorescence of lens epithelial flat mounts showed that Clic5 localized to cilia/centrosomes. Mice mutant for Clic5 (jitterbug) also had defective sutures. These results suggest that KLPH is required for lens-specific expression of Clic5 and that Clic5 has an important role in the machinery that controls lens fiber cell extension and organization.
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Canales de Cloruro/metabolismo , Regulación de la Expresión Génica/fisiología , Glicósido Hidrolasas/genética , Cristalino/metabolismo , Proteínas de la Membrana/genética , Animales , Northern Blotting , Western Blotting , Catarata/metabolismo , Catarata/patología , Diferenciación Celular , Células Epiteliales/citología , Células Epiteliales/metabolismo , Proteínas del Ojo/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Eliminación de Gen , Inmunoquímica , Hibridación in Situ , Cristalino/citología , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Mensajero/genéticaRESUMEN
Retinal pigment epithelium (RPE) has been implicated as key source of cholesterol-rich deposits at Bruch's membrane (BrM) and in drusen in aging human eye. We have shown that serum-deprivation of confluent RPE cells is associated with upregulation of cholesterol synthesis and accumulation of unesterified cholesterol (UC). Here we investigate the cellular processes involved in this response. We compared the distribution and localization of UC and esterified cholesterol (EC); the age-related macular degeneration (AMD) associated EFEMP1/Fibulin3 (Fib3); and levels of acyl-coenzyme A (CoA): cholesterol acyltransferases (ACAT) ACAT1, ACAT2 and Apolipoprotein B (ApoB) in ARPE-19 cells cultured in serum-supplemented and serum-free media. The results were compared with distributions of these lipids and proteins in human donor eyes with AMD. Serum deprivation of ARPE-19 was associated with increased formation of FM dye-positive membrane vesicles, many of which co-labeled for UC. Additionally, UC colocalized with Fib3 in distinct granules. By day 5, serum-deprived cells grown on transwells secreted Fib3 basally into the matrix. While mRNA and protein levels of ACTA1 were constant over several days of serum-deprivation, ACAT2 levels increased significantly after serum-deprivation, suggesting increased formation of EC. The lower levels of intracellular EC observed under serum-deprivation were associated with increased formation and secretion of ApoB. The responses to serum-deprivation in RPE-derived cells: accumulation and secretion of lipids, lipoproteins, and Fib3 are very similar to patterns seen in human donor eyes with AMD and suggest that this model mimics processes relevant to disease progression.
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Colesterol/metabolismo , Medio de Cultivo Libre de Suero/farmacología , Proteínas de la Matriz Extracelular/genética , Degeneración Macular/metabolismo , Modelos Biológicos , Epitelio Pigmentado de la Retina/efectos de los fármacos , Acetil-CoA C-Acetiltransferasa/genética , Acetil-CoA C-Acetiltransferasa/metabolismo , Acilcoenzima A/metabolismo , Apolipoproteínas B/genética , Apolipoproteínas B/metabolismo , Línea Celular , Ésteres del Colesterol/metabolismo , Cámaras de Difusión de Cultivos , Proteínas de la Matriz Extracelular/metabolismo , Regulación de la Expresión Génica , Humanos , Degeneración Macular/genética , Degeneración Macular/patología , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/metabolismo , Transducción de Señal , Esterol O-Aciltransferasa/genética , Esterol O-Aciltransferasa/metabolismo , Esterol O-Aciltransferasa 2RESUMEN
PURPOSE: Having observed that confluent ARPE-19 cells (derived from human RPE) survive well in high-glucose serum-free medium (SFM) without further feeding for several days, we investigated the expression profile of RPE cells under the same conditions. METHODS: Expression profiles were examined with microarray and quantitative PCR (qPCR) analyses, followed by western blot analysis of key regulated proteins. The effects of low-density lipoprotein (LDL) and zinc supplementation were examined with qPCR. Immunofluorescence was used to localize the LDL receptor and to examine LDL uptake. Cellular cholesterol levels were measured with filipin binding. Expression patterns in primary fetal RPE cells were compared using qPCR. RESULTS: Microarray analyses of gene expression in ARPE-19, confirmed with qPCR, showed upregulation of lipid and cholesterol biosynthesis pathways in SFM. At the protein level, the cholesterol synthesis control factor SRBEF2 was activated, and other key lipid synthesis proteins increased. Supplementation of SFM with LDL reversed the upregulation of lipid and cholesterol synthesis genes, but not of cholesterol transport genes. The LDL receptor relocated to the plasma membrane, and LDL uptake was activated by day 5-7 in SFM, suggesting increased demand for cholesterol. Confluent ARPE-19 cells in SFM accumulated intracellular cholesterol, compared with cells supplemented with serum, over 7 days. Over the same time course in SFM, the expression of metallothioneins decreased while the major zinc transporter was upregulated, consistent with a parallel increase in demand for zinc. Supplementation with zinc reversed expression changes for metallothionein genes, but not for other zinc-related genes. Similar patterns of regulation were also seen in primary fetal human RPE cells in SFM. CONCLUSIONS: ARPE-19 cells respond to serum deprivation and starvation with upregulation of the lipid and cholesterol pathways, accumulation of intracellular cholesterol, and increased demand for zinc. Similar trends are seen in primary fetal RPE cells. Cholesterol accumulation basal to RPE is a prominent feature of age-related macular degeneration (AMD), while dietary zinc is protective. It is conceivable that accumulating defects in Bruch's membrane and dysfunction of the choriocapillaris could impede transport between RPE and vasculature in AMD. Thus, this pattern of response to serum deprivation in RPE-derived cells may have relevance for some aspects of the progression of AMD.
Asunto(s)
Proteínas Portadoras/metabolismo , Colesterol/metabolismo , Medio de Cultivo Libre de Suero , Epitelio Pigmentado de la Retina/metabolismo , Zinc/metabolismo , Células Cultivadas , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Immunoblotting , Lipoproteínas LDL/genética , Lipoproteínas LDL/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de LDL/genética , Receptores de LDL/metabolismoRESUMEN
The vertebrate eye lens contains high concentrations of crystallins. The dense lenses of fish are particularly abundant in a class called γM-crystallin whose members are characterized by an unusually high methionine content and partial loss of the four tryptophan residues conserved in all γ-crystallins from mammals which are proposed to contribute to protection from UV-damage. Here, we present the structure and dynamics of γM7-crystallin from zebrafish (Danio rerio). The solution structure shares the typical two-domain, four-Greek-key motif arrangement of other γ-crystallins, with the major difference noted in the final loop of the N-terminal domain, spanning residues 65-72. This is likely due to the absence of the conserved tryptophans. Many of the methionine residues are exposed on the surface but are mostly well-ordered and frequently have contacts with aromatic side chains. This may contribute to the specialized surface properties of these proteins that exist under high molecular crowding in the fish lens. NMR relaxation data show increased backbone conformational motions in the loop regions of γM7 compared to those of mouse γS-crystallin and show that fast internal motion of the interdomain linker in γ-crystallins correlates with linker length. Unfolding studies monitored by tryptophan fluorescence confirm results from mutant mouse γS-crystallin and show that unfolding of a ßγ-crystallin domain likely starts from unfolding of the variable loop containing the more fluorescently quenched tryptophan residue, resulting in a native-like unfolding intermediate.
Asunto(s)
Proteínas de Pez Cebra/química , gamma-Cristalinas/química , Secuencia de Aminoácidos , Animales , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Pliegue de Proteína , Relación Estructura-Actividad , Proteínas de Pez Cebra/metabolismo , gamma-Cristalinas/metabolismoRESUMEN
Crystallins are the abundant, long-lived proteins of the eye lens. The major human crystallins belong to two different superfamilies: the small heat-shock proteins (α-crystallins) and the ßγ-crystallins. During evolution, other proteins have sometimes been recruited as crystallins to modify the properties of the lens. In the developing human lens, the enzyme betaine-homocysteine methyltransferase serves such a role. Evolutionary modification has also resulted in loss of expression of some human crystallin genes or of specific splice forms. Crystallin organization is essential for lens transparency and mutations; even minor changes to surface residues can cause cataract and loss of vision.
Asunto(s)
Cristalinas/genética , Familia de Multigenes , Cadena B de alfa-Cristalina/genética , Betaína-Homocisteína S-Metiltransferasa/genética , Betaína-Homocisteína S-Metiltransferasa/metabolismo , Catarata/genética , Catarata/fisiopatología , Cristalinas/metabolismo , Proteínas de Choque Térmico Pequeñas/genética , Proteínas de Choque Térmico Pequeñas/metabolismo , Humanos , Cristalino/metabolismo , Mutación , Cadena B de alfa-Cristalina/metabolismoRESUMEN
Misfolding and aggregation of proteins occur in many pathological states. Because of the inherent disorder involved, these processes are difficult to study. We attempted to capture aggregation intermediates of γS-crystallin, a highly stable, internally symmetrical monomeric protein, by crystallization under mildly acidic and oxidizing conditions. Here we describe novel oligomerization through strained domain-swapping and partial intermolecular disulfide formation. This forms an octamer built from asymmetric tetramers, each of which comprises an asymmetric pair of twisted, domain-swapped dimers. Each tetramer shows patterns of acquired disorder among subunits, ranging from local loss of secondary structure to regions of intrinsic disorder. The octamer ring is tied together by partial intermolecular disulfide bonds, which may contribute to strain and disorder in the octamer. Oligomerization in this structure is self-limited by the distorted octamer ring. In a more heterogeneous environment, the disordered regions could serve as seeds for cascading interactions with other proteins. Indeed, solubilized protein from crystals retain many features observed in the crystal and are prone to further oligomerization and precipitation. This structure illustrates modes of loss of organized structure and aggregation that are relevant for cataract and for other disorders involving deposition of formerly well-folded proteins.
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Agregación Patológica de Proteínas , gamma-Cristalinas , Catarata/metabolismo , Disulfuros/química , Humanos , Estructura Secundaria de Proteína , gamma-Cristalinas/químicaRESUMEN
The molecular mechanisms underlying morphological diversity in retinal cell types are poorly understood. We have previously reported that several members of the Copine family of Ca-dependent membrane adaptors are expressed in Retinal Ganglion Cells and transcriptionally regulated by Brn3 transcription factors. Several Copines are enriched in the retina and their over-expression leads to morphological changes -formation of elongated processes-, reminiscent of neurites, in HEK293 cells. However, the role of Copines in the retina is largely unknown. We now investigate Cpne4, a Copine whose expression is restricted to Retinal Ganglion Cells. Over-expression of Cpne4 in RGCs in vivo led to formation of large varicosities on the dendrites but did not otherwise visibly affect dendrite or axon formation. Protein interactions studies using yeast two hybrid analysis from whole retina cDNA revealed two Cpne4 interacting proteins-Host Cell Factor 1 and Morn2. Mass Spectrometry analysis of retina lysate pulled down using Cpne4 or its vonWillebrand A domain showed 207 interacting proteins. A Gene Ontology analysis of the discovered proteins suggests that Cpne4 is involved in several metabolic and signaling pathways in the retina.
Asunto(s)
Axones/metabolismo , Proteínas de Unión al Calcio/metabolismo , Retina/metabolismo , Células Ganglionares de la Retina/metabolismo , Animales , Proteínas de Unión al Calcio/genética , Células HEK293 , Humanos , Ratones , Ratones Noqueados , Neuritas/metabolismo , TransfecciónRESUMEN
Deposition of hydroxyapatite (HAP) basal to the retinal pigment epithelium (RPE) is linked to the progression of age-related macular degeneration (AMD). Serum-deprivation of RPE cells in culture mimics some features of AMD. We now show that serum-deprivation also leads to the induction of amelotin (AMTN), a protein involved in hydroxyapatite mineralization in enamel. HAP is formed in our culture model and is blocked by siRNA inhibition of AMTN expression. In situ hybridization and immunofluorescence imaging of human eye tissue show that AMTN is expressed in RPE of donor eyes with geographic atrophy ("dry" AMD) in regions with soft drusen containing HAP spherules or nodules. AMTN is not found in hard drusen, normal RPE, or donor eyes diagnosed with wet AMD. These findings suggest that AMTN is involved in formation of HAP spherules or nodules in AMD, and as such provides a new therapeutic target for slowing disease progression.
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Proteínas del Esmalte Dental/metabolismo , Durapatita/metabolismo , Atrofia Geográfica/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Anciano , Medio de Cultivo Libre de Suero , HumanosRESUMEN
BACKGROUND: The optic nerve is a pure white matter central nervous system (CNS) tract with an isolated blood supply, and is widely used in physiological studies of white matter response to various insults. We examined the gene expression profile of human optic nerve (ON) and, through the NEIBANK online resource, to provide a resource of sequenced verified cDNA clones. An un-normalized cDNA library was constructed from pooled human ON tissues and was used in expressed sequence tag (EST) analysis. Location of an abundant oligodendrocyte marker was examined by immunofluorescence. Quantitative real time polymerase chain reaction (qRT-PCR) and Western analysis were used to compare levels of expression for key calcium channel protein genes and protein product in primate and rodent ON. RESULTS: Our analyses revealed a profile similar in many respects to other white matter related tissues, but significantly different from previously available ON cDNA libraries. The previous libraries were found to include specific markers for other eye tissues, suggesting contamination. Immune/inflammatory markers were abundant in the new ON library. The oligodendrocyte marker QKI was abundant at the EST level. Immunofluorescence revealed that this protein is a useful oligodendrocyte cell-type marker in rodent and primate ONs. L-type calcium channel EST abundance was found to be particularly low. A qRT-PCR-based comparative mammalian species analysis reveals that L-type calcium channel expression levels are significantly lower in primate than in rodent ON, which may help account for the class-specific difference in responsiveness to calcium channel blocking agents. Several known eye disease genes are abundantly expressed in ON. Many genes associated with normal axonal function, mRNAs associated with axonal transport, inflammation and neuroprotection are observed. CONCLUSION: We conclude that the new cDNA library is a faithful representation of human ON and EST data provide an initial overview of gene expression patterns in this tissue. The data provide clues for tissue-specific and species-specific properties of human ON that will help in design of therapeutic models.
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Axones/metabolismo , ADN Complementario/metabolismo , Etiquetas de Secuencia Expresada/metabolismo , Oligodendroglía/metabolismo , Nervio Óptico/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Northern Blotting , Western Blotting , ADN Complementario/análisis , ADN Complementario/genética , Bases de Datos Genéticas , Femenino , Técnica del Anticuerpo Fluorescente , Perfilación de la Expresión Génica , Biblioteca de Genes , Humanos , Macaca , Masculino , Microscopía Confocal , Persona de Mediana Edad , Nervio Óptico/química , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The eye lens is packed with soluble crystallin proteins, providing a lifetime of transparency and light refraction. gamma-Crystallins are major components of the dense, high refractive index central regions of the lens and generally have high solubility, high stability and high levels of cysteine residues. Human gammaC belongs to a group of gamma-crystallins with a pair of cysteine residues at positions 78 and 79. Unlike other gamma-crystallins it has relatively low solubility, whereas mouse gammaC, which has the exposed C79 replaced with arginine, and a novel mouse splice variant, gammaCins, are both highly soluble. Furthermore, human gammaC is extremely stable, while the mouse orthologs are less stable. Evolutionary pressure may have favoured stability over solubility for human gammaC and the reverse for the orthologs in the mouse. Mutation of C79 to R79, in human gammaC, greatly increased solubility, however, neither form produced crystals. Remarkably, when the human gammaD R36S crystallization cataract mutation was mimicked in human gammaC-crystallin, the solubility of gammaC was dramatically increased, although it still did not crystallize. The highly soluble mouse gammaC-crystallin did crystallize. Its X-ray structure was solved and used in homology modelling of human gammaC, and its mutants C79R and R36S. The human gammaD R36S mutant was also modelled from human gammaD coordinates. Molecular dynamics simulation of the six molecules in the solution state showed that the human gammaCs differed from gammaDs in domain pairing, behaviour that correlates with interface sequence changes. When the fluctuations of the calculated molecular dipoles, for the six structures, over time were analysed, characteristic patterns for soluble gammaC and gammaD proteins were observed. Individual sequence changes that increase or decrease solubility correlated well with changes in the magnitude and direction of these dipoles. It is suggested that changes in surface residues have allowed adaptation for the differing needs of human and mouse lenses.
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Cristalino/química , gamma-Cristalinas/química , gamma-Cristalinas/fisiología , Secuencia de Aminoácidos , Animales , Bovinos , Perros , Cobayas , Humanos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Mutantes/química , Mutación Puntual , Estructura Terciaria de Proteína/fisiología , Homología de Secuencia de Aminoácido , Solubilidad , gamma-Cristalinas/genética , gamma-Cristalinas/aislamiento & purificaciónRESUMEN
PURPOSE: To characterize gene expression patterns in guinea pig ocular tissues and identify orthologs of human genes from NEIBank expressed sequence tags. METHODS: RNA was extracted from dissected eye tissues of 2.5-month-old guinea pigs to make three unamplified and unnormalized cDNA libraries in the pCMVSport-6 vector for the lens, retina, and eye minus lens and retina. Over 4,000 clones were sequenced from each library and were analyzed using GRIST for clustering and gene identification. Lens crystallin EST data were validated using two-dimensional electrophoresis (2-DE), matrix assisted laser desorption (MALDI), and electrospray ionization mass spectrometry (ESIMS). RESULTS: Combined data from the three libraries generated a total of 6,694 distinctive gene clusters, with each library having between 1,000 and 3,000 clusters. Approximately 60% of the total gene clusters were novel cDNA sequences and had significant homologies to other mammalian sequences in GenBank. Complete cDNA sequences were obtained for many guinea pig lens proteins, including alphaA/alphaAinsert-, gammaN-, and gammaS-crystallins, lengsin and GRIFIN. The ratio of alphaA- to alphaB-crystallin on 2-DE gels was 8: 1 in the lens nucleus and 6.5: 1 in the cortex. Analysis of ESTs, genome sequence, and proteins (by MALDI), did not reveal any evidence for the presence of gammaD-, gammaE-, and gammaF-crystallin in the guinea pig. Predicted masses of many guinea pig lens crystallins were confirmed by ESIMS analysis. For the retina, orthologs of human phototransduction genes were found, such as Rhodopsin, S-antigen (Sag, Arrestin), and Transducin. The guinea-pig ortholog of NRL, a key rod photoreceptor-specific transcription factor, was also represented in EST data. In the 'rest-of-eye' library, the most abundant transcripts included decorin and keratin 12, representative of the cornea. CONCLUSIONS: Genomic analysis of guinea pig eye tissues provides sequence-verified clones for future studies. Guinea pig orthologs of many human eye specific genes were identified. Guinea pig gene structures were similar to their human and rodent gene counterparts. Surprisingly, no orthologs of gammaD-, gammaE-, and gammaF-crystallin were found in EST, proteomic, or the current guinea pig genome data.
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Bases de Datos Genéticas , Etiquetas de Secuencia Expresada , Ojo/metabolismo , Cobayas/genética , Empalme Alternativo/genética , Animales , ADN Complementario/genética , Electroforesis en Gel Bidimensional , Ojo/citología , Proteínas del Ojo/química , Proteínas del Ojo/genética , Biblioteca de Genes , Genoma , Humanos , Cristalino/química , Espectrometría de Masas , Datos de Secuencia Molecular , Peso Molecular , Proteínas Nucleares/química , Proteínas Nucleares/genética , Sistemas de Lectura Abierta/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Retina/metabolismo , Análisis de Secuencia de ADN , Solubilidad , gamma-Cristalinas/química , gamma-Cristalinas/genéticaRESUMEN
NEIBank is an integrated resource for genomics and bioinformatics in vision research. It includes expressed sequence tag (EST) data and sequence-verified cDNA clones for multiple eye tissues of several species, web-based access to human eye-specific SAGE data through EyeSAGE, and comprehensive, annotated databases of known human eye disease genes and candidate disease gene loci. All expression- and disease-related data are integrated in EyeBrowse, an eye-centric genome browser. NEIBank provides a comprehensive overview of current knowledge of the transcriptional repertoires of eye tissues and their relation to pathology.
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Biología Computacional , Bases de Datos Genéticas , Genómica , Recursos en Salud , Investigación , Visión Ocular/genética , Etiquetas de Secuencia Expresada , Oftalmopatías/genética , Perfilación de la Expresión Génica , Biblioteca de Genes , Humanos , InternetRESUMEN
Lengsin is a major protein of the vertebrate eye lens. It belongs to the hitherto purely prokaryotic GS I branch of the glutamine synthetase (GS) superfamily, but has no enzyme activity. Like the taxon-specific crystallins, Lengsin is the result of the recruitment of an ancient enzyme to a noncatalytic role in the vertebrate lens. Cryo-EM and modeling studies of Lengsin show a dodecamer structure with important similarities and differences with prokaryotic GS I structures. GS homology regions of Lengsin are well conserved, but the N-terminal domain shows evidence of dynamic evolutionary changes. Compared with birds and fish, most mammals have an additional exon corresponding to part of the N-terminal domain; however, in human, this is a nonfunctional pseudoexon. Genes related to Lengsin are also present in the sea urchin, suggesting that this branch of the GS I family, supplanted by GS II enzymes in vertebrates, has an ancient role in metazoans.
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Proteínas del Ojo/fisiología , Glutamato-Amoníaco Ligasa/química , Cristalino/fisiología , Secuencia de Aminoácidos , Animales , Catálisis , Microscopía por Crioelectrón , Evolución Molecular , Proteínas del Ojo/metabolismo , Glutamato-Amoníaco Ligasa/metabolismo , Glutamato-Amoníaco Ligasa/fisiología , Humanos , Cristalino/metabolismo , Ratones , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Ratas , Erizos de Mar , Homología de Secuencia de AminoácidoRESUMEN
Variants in the skeletal muscle ryanodine receptor 1 gene (RYR1) result in a spectrum of RYR1-related disorders. Presentation during infancy is typical and ranges from delayed motor milestones and proximal muscle weakness to severe respiratory impairment and ophthalmoplegia. We aimed to elucidate correlations between genotype, protein structure and clinical phenotype in this rare disease population. Genetic and clinical data from 47 affected individuals were analyzed and variants mapped to the cryo-EM RyR1 structure. Comparisons of clinical severity, motor and respiratory function and symptomatology were made according to the mode of inheritance and affected RyR1 structural domain(s). Overall, 49 RYR1 variants were identified in 47 cases (dominant/de novo, n = 35; recessive, n = 12). Three variants were previously unreported. In recessive cases, facial weakness, neonatal hypotonia, ophthalmoplegia/paresis, ptosis, and scapular winging were more frequently observed than in dominant/de novo cases (all, p < 0.05). Both dominant/de novo and recessive cases exhibited core myopathy histopathology. Clinically severe cases were typically recessive or had variants localized to the RyR1 cytosolic shell domain. Motor deficits were most apparent in the MFM-32 standing and transfers dimension, [median (IQR) 85.4 (18.8)% of maximum score] and recessive cases exhibited significantly greater overall motor function impairment compared to dominant/de novo cases [79.7 (18.8)% vs. 87.5 (17.7)% of maximum score, p = 0.03]. Variant mapping revealed patterns of clinical severity across RyR1 domains, including a structural plane of interest within the RyR1 cytosolic shell, in which 84% of variants affected the bridging solenoid. We have corroborated genotype-phenotype correlations and identified RyR1 regions that may be especially sensitive to structural modification.
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Enfermedades Neuromusculares/genética , Enfermedades Neuromusculares/fisiopatología , Canal Liberador de Calcio Receptor de Rianodina/genética , Acetilcisteína/uso terapéutico , Adolescente , Adulto , Estudios Transversales , Método Doble Ciego , Femenino , Estudios de Asociación Genética , Variación Genética , Humanos , Masculino , Fármacos Neuromusculares/uso terapéutico , Enfermedades Neuromusculares/tratamiento farmacológico , Enfermedades Neuromusculares/patología , Estudios Prospectivos , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Relación Estructura-Actividad , Adulto JovenRESUMEN
PURPOSE: To develop a microarray for the rabbit that can be used for ocular gene expression research. METHODS: Messenger RNA was isolated from anterior segment tissues (cornea, conjunctiva, and iris) and posterior segment tissues (lens, retina, and sclera) of rabbit eyes and used to create two independent cDNA libraries through the NEIBank project. Clones from each of these libraries were sequenced from both the 5' and 3' ends. These sequences and those from the National Center for Biotechnology Information (NCBI) taxonomy database for rabbit were combined and electronically assembled into a set of unique nonoverlapping continuous sequences (contigs). For each contig, a homology search was performed using BLASTX and BLASTN against both the NCBI NR and NT databases to provide gene annotation. Unique contigs were sent to Agilent Technologies, where 60 base oligonucleotide probes were designed and synthesized, in situ, on two different arrays in an 8 array x 1900 element format. Glaucoma filtration surgery was performed on one eye of six rabbits. After 14 days, tissue was harvested from the conjunctiva and Tenon's capsule of both the surgically treated and untreated control eyes. Total RNA from each sample was labeled with cyanine dyes and hybridized to our custom microarrays. RESULTS: Of the 3,154 total probes present on the two arrays, 2,522 had a signal value above the background. The expression of 315 genes was significantly altered by glaucoma filtration surgery. Genes whose expression was altered included proteins associated with inflammatory response, defense response, and proteins involved in synthesis of the extracellular matrix. CONCLUSIONS: The results of this rabbit microarray study are consistent with those from other wound healing studies, indicating that this array can provide valid information on broad patterns of gene expression. This is the first microarray available for rabbit studies and is a valuable tool that can be used to study molecular events in the eye.
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Investigación Biomédica/métodos , Ojo/metabolismo , Expresión Génica , Análisis de Secuencia por Matrices de Oligonucleótidos , Conejos/genética , Animales , Sistemas de Computación , Cirugía Filtrante , Biblioteca de Genes , Glaucoma/genética , Glaucoma/metabolismo , Glaucoma/cirugía , Reacción en Cadena de la PolimerasaRESUMEN
NEIBank is a project to gather and organize genomic resources for eye research. The first phase of this project covers the construction and sequence analysis of cDNA libraries from human and animal model eye tissues to develop an overview of the repertoire of genes expressed in the eye and a resource of cDNA clones for further studies. The sequence data are grouped and identified using the tools of bioinformatics and the results are displayed through a web site where they can be interrogated by keyword search, chromosome location, by Blast (sequence comparison) or by alignment on completed genomes. Many novel proteins and novel splice forms of known genes have already emerged from analysis of the accumulating data. This review provides an overview of the current state of the database for human eye tissues, with specific comparisons to some parallel data from mouse and rat, and with illustrative examples of the kinds of insights and discoveries these data can produce. One of the major themes that emerges is that at the molecular level human eye tissues have significant differences from those of rodents, encompassing species specific genes, alternative splice forms and great variation in levels of gene expression. These point to specific adaptations and mechanisms in the human eye and emphasize that care needs to be taken in the application of appropriate animal model systems.