RESUMEN
The fish Astyanax mexicanus presents, within the same species, populations of river-dwelling surface fish (SF) and blind cave-living fish. In cavefish (CF), the eyes develop almost normally during embryogenesis. But 40 h after fertilization, the lens enters apoptosis, triggering the progressive degeneration of the entire eye. Before apoptosis, the CF lens expresses early differentiation factors correctly. Here, we searched for possible late differentiation defects that would be causal in CF lens degeneration. We reasoned that crystallins, the major lens structural proteins, could be defective or misregulated. We surveyed the CF and SF transcriptomes and uncovered 14 Astyanax crystallins from the beta, gamma, lambda, mu, and zeta families. These proteins are less polymorphic and accumulate more fixed mutations, some at highly conserved positions, in CF than in SF, suggesting relaxed selection at these loci in CF. In situ hybridizations and qPCR show that crybb1c, crybgx, crygm5 are expressed at much lower levels or are not expressed in the CF lens. For the best crystallin candidates, we tested a potential causal role in CF lens apoptosis. Crybgx, crybb1c (not expressed in CF from very early on), and cryaa (previously shown to be faintly expressed in CF) failed to induce any defect when knocked-down in zebrafish embryos. However, the anti-apoptotic cryaa protected lens cells from apoptosis when reexpressed by transgenesis in CF, suggesting a cell-autonomous effect of cryaa on lens cell survival. Altogether, these data suggest that crystallin sequence evolution and expression defects may contribute to the loss of eyes in CF.
Asunto(s)
Evolución Molecular , Regulación del Desarrollo de la Expresión Génica/fisiología , Cristalino/metabolismo , Pez Cebra/metabolismo , alfa-Cristalinas/metabolismo , Animales , Apoptosis/fisiología , Diferenciación Celular/fisiología , Supervivencia Celular/fisiología , Cristalino/anomalías , Transcriptoma/fisiología , Pez Cebra/embriología , Pez Cebra/genéticaRESUMEN
This report describes the characterization of a member of the alpha-crystallin small heat shock protein family in a trypanosomatid, which was isolated from the human pathogen Trypanosoma cruzi. One alpha-crystallin small heat shock protein gene was identified in a database search. The coding region is located in an open reading frame of 429bp encoding a protein of 142 amino acids. The amino acid sequence was deduced from the isolated gene. The protein has an alpha-crystallin domain characteristic of the alpha-crystallin small heat shock proteins and a molecular weight of 15.9kDa, so the protein was designated SHSP16. Analysis of the nucleotide sequences of four different T. cruzi strains showed two different sequences, which correspond to the two main T. cruzi genetic groups. Gene expression analysis by RT-PCR showed increased transcription of the gene after the parasite was exposed to heat stress. Recombinant SHSP16 showed molecular chaperone activity in vitro, because it inhibited the thermal aggregation of the mitochondrial malate dehydrogenase enzyme.
Asunto(s)
Proteínas de Choque Térmico Pequeñas/química , Proteínas Protozoarias/química , Trypanosoma cruzi/química , alfa-Cristalinas/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Secuencia Conservada , ADN Protozoario/química , Electroforesis en Gel de Poliacrilamida , Expresión Génica , Proteínas de Choque Térmico Pequeñas/genética , Proteínas de Choque Térmico Pequeñas/metabolismo , Calor , Humanos , Datos de Secuencia Molecular , Polimorfismo Genético , Estructura Secundaria de Proteína , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , ARN Mensajero/metabolismo , ARN Protozoario/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Trypanosoma cruzi/genética , alfa-Cristalinas/genética , alfa-Cristalinas/metabolismoRESUMEN
Giardia lamblia is a medically important protozoan parasite with a basal position in the eukaryotic lineage and is an interesting model to explain the evolution of biochemical events in eukaryotic cells. G. lamblia trophozoites undergo significant changes in order to survive outside the intestine of their host by differentiating into infective cysts. In the present study, we characterize the previously identified Orf-C4 (G. lamblia open reading frame C4) gene, which is considered to be specific to G. lamblia. It encodes a 22 kDa protein that assembles into high-molecular-mass complexes during the entire life cycle of the parasite. ORF-C4 localizes to the cytoplasm of trophozoites and cysts, and forms large spherical aggregates when overexpressed. ORF-C4 overexpression and down-regulation do not affect trophozoite viability; however, differentiation into cysts is slightly delayed when the expression of ORF-C4 is down-regulated. In addition, ORF-C4 protein expression is modified under specific stress-inducing conditions. Neither orthologous proteins nor conserved domains are found in databases by conventional sequence analysis of the predicted protein. However, ORF-C4 contains a region which is similar structurally to the alpha-crystallin domain of sHsps (small heat-shock proteins). In the present study, we show the potential role of ORF-C4 as a small chaperone which is involved in the response to stress (including encystation) in G. lamblia.
Asunto(s)
Giardia lamblia/fisiología , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Animales , Regulación de la Expresión Génica , Giardia lamblia/genética , Proteínas de Choque Térmico Pequeñas/genética , Proteínas de Choque Térmico Pequeñas/metabolismo , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Estrés Fisiológico , alfa-Cristalinas/genética , alfa-Cristalinas/metabolismoRESUMEN
The results of this study describe the identification and characterization of the Toxoplasma gondii alpha-crystallin/small heat shock protein (sHsp) family. By database (www.toxodb.org) search, five parasite sHsps (Hsp20, Hsp21, Hsp28, Hsp29, and the previously characterized Hsp30/Bag1) were identified. As expected, they share the homologous alpha-crystallin domain, which is the key characteristic of sHsps. However, the N-terminal segment of each protein contains unique characteristics in size and sequence. Most T. gondii sHsps are constitutively expressed in tachyzoites and fully differentiated bradyzoites, with the exception of Hsp30/Bag1. Interestingly, by subcellular localization we observed that T. gondii sHsps are located in different compartments. Hsp20 is located at the apical end of the cell, Hsp28 is located inside the mitochondrion, Hsp29 showed a membrane-associated labeling, and Hsp21 appeared throughout the cytosol of the parasites. These particular differences in the immunostaining patterns suggest that their targets and functions might be different.