ABSTRACT
'Conformational diseases' are a group of diverse disorders that have been associated with misfolding of specific proteins, leading to their aggregation in particular cell tissues. Despite their relevance, the mechanisms involved in neurodegenerative processes remains poorly understood. Mutations in Cu,Zn superoxide dismutase (SOD1) are implicated in death of motor neurons in amyotrophic lateral sclerosis. Among others, the SOD1(G93A) mutation is known to weaken the structure and this could lead to conformational variations of the protein. As an approach to understand the tissue-specific propensity of protein aggregation, we developed an experimental procedure allowing rapid extraction of variants of human SOD1 (hSOD1) produced in different tissues. Using an antibody-based affinity chromatography procedure enzymatically active hSOD was extracted, indicating preservation of its native conformation. Analysis of the eluted fractions of hSOD extracted from the brain and liver of transgenic hSOD(G93A) rats provided evidence about heterodimers rSOD-hSOD(G93A) formation in both extracts. Moreover, when characterized by 2-DE and MALDI-TOF/TOF MS, the extracted hSOD(G93A) showed a complex profile suggesting the existence of various covalent modifications of the enzyme in both tissues. Thus, this method should allow following post-translational modifications of hSOD1 produced in various tissues.
Subject(s)
Antibodies, Immobilized/metabolism , Chromatography, Affinity/methods , Superoxide Dismutase/chemistry , Animals , Blotting, Western , Brain/enzymology , Electrophoresis, Gel, Two-Dimensional , Humans , Immunosorbent Techniques , Liver/enzymology , Protein Conformation , Protein Multimerization , Protein Processing, Post-Translational , Proteomics/methods , Rats , Rats, Sprague-Dawley , Rats, Transgenic , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Silver Staining , Superoxide Dismutase/genetics , Superoxide Dismutase/isolation & purification , Superoxide Dismutase/metabolismABSTRACT
As an approach to understand how translation may affect protein folding, we analyzed structural and functional properties of the human estrogen receptor alpha synthesized by different eukaryotic translation systems. A minimum of three conformations of the receptor were detected using limited proteolysis and a sterol ligand-binding assay. The receptor in vitro translated in rabbit reticulocyte lysate was rapidly degraded by protease, produced major bands of about 34kDa and showed a high affinity for estradiol. In a wheat germ translation system, the receptor was more slowly digested. Two soluble co-existing conformations were evident by different degradation patterns and estradiol binding. Our data show that differences in the translation machinery may result in alternative conformations of the receptor with distinct sterol binding properties. These studies suggest that components of the cellular translation machinery itself might influence the protein folding pathways and the relative abundance of different receptor conformers.
Subject(s)
Estrogen Receptor alpha/chemistry , Estrogen Receptor alpha/ultrastructure , Protein Modification, Translational , Binding Sites , Protein Binding , Protein Conformation , Protein Folding , Protein Isoforms/chemistryABSTRACT
With the aim of identifying genes involved in development and parasite adaptation in cestodes, four coding sequences were isolated from the cyclophyllidean Mesocestoides corti larval stage (tetrathyridium). Genes showed significant similarity to the cysteine-rich secreted protein (CRISP) encoding genes, a large family that includes stage and tissue-specific genes from diverse organisms, many associated with crucial biological processes. The full-length McCrisp2 cDNA encodes a predicted protein of 202 residues in length, containing 10 cysteines and a putative signal peptide. The expression level of McCrisp2 was estimated by Real-time PCR, relative to GAPDH, showing an increase of 75% in segmented worms compared to tetrathyridia. By in situ hybridization, McCrisp2 expression was localized mainly at the larvae apical region of tetrathyridia and in the proglottids of segmented worms. Taken together our results suggest a possible role for M. corti CRISP proteins as ES products, potentially involved in differentiation processes as proposed for homologs in other organisms.
