Ostrich crystallins. Structural characterization of delta-crystallin with enzymic activity.
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ABSTRACT:
SUBMITTER: Chiou SH
PROVIDER: S-EPMC1149845 | biostudies-other | 1991 Jan
REPOSITORIES: biostudies-other
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Project description:In addition to contributing to lens optical properties, the α-crystallins are small heat shock proteins that possess chaperone activity and are predicted to bind and sequester destabilized proteins to delay cataract formation. The current model of α-crystallin chaperone mechanism envisions a transition from the native oligomer to an activated form that has higher affinity to non-native states of the substrate. Previous studies have suggested that this oligomeric plasticity is encoded in the primary sequence and controls access to high affinity binding sites within the N-terminal domain. Here, we further examined the role of sequence variation in the context of species-specific α-crystallins from rat and zebrafish. Alternative splicing of the αA gene in rodents produces αA(ins), which is distinguished by a longer N-terminal domain. The zebrafish genome includes duplicate αB-crystallin genes, αBa and αBb, which display divergent primary sequence and tissue expression patterns. Equilibrium binding experiments were employed to quantitatively define chaperone interactions with a destabilized model substrate, T4 lysozyme. In combination with multiangle light scattering, we show that rat αA(ins) and zebrafish α-crystallins display distinct global structural properties and chaperone activities. Notably, we find that αA(ins) and αBa demonstrate substantially enhanced chaperone function relative to other α-crystallins, binding the same substrate more than 2 orders of magnitude higher affinity and mimicking the activity of fully activated mammalian small heat shock proteins. These results emphasize the role of sequence divergence as an evolutionary strategy to tune chaperone function to the requirements of the tissues and organisms in which they are expressed.
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Project description:Delta-Crystallin, the most abundant crystallin in the avian species, was isolated and purified from duck lenses. It was shown to possess endogenous argininosuccinate lyase activity catalysing the reversible cleavage of argininosuccinate to give fumarate and arginine with an equilibrium constant of 1.8 +/- 0.23 mM. In contrast, chicken lens delta-crystallin showed only 0.4-0.8% of the enzyme activity of duck delta-crystallin under identical assay conditions. Biochemical comparison of delta-crystallins from these two species revealed distinct differences in their structural and kinetic properties. An activity-staining method was developed for the easy detection of endogenous enzyme activity of delta-crystallin from crude lens extracts of different avian species. Two-dimensional gel electrophoresis of lens homogenates indicated that, in the chicken lens, delta-crystallin is composed mainly of a subunit with a pI of 5.9 and a subunit mass of 50 kDa, whereas that of duck lens possesses various 50 kDa subunits in a pI range of 5.9-6.8. Activity staining corroborated the fact that all charge isoenzymes of duck delta-crystallin possess enzyme activity whereas that of chicken delta-crystallin is devoid of activity. For duck delta-crystallin, variation of the enzyme activity with argininosuccinate concentration in the forward reaction followed saturation kinetics with an apparent Michaelis constant for the substrate of 17 +/- 5 microM. In the reverse reaction, initial-velocity studies showed intersecting patterns. Inhibitions of the forward reaction by products (fumarate and arginine) were both non-competitive with respect to argininosuccinate. Citrulline, an analogue of arginine, inhibited the enzyme activity in both directions and was competitive with respect to arginine but non-competitive with respect to fumarate or argininosuccinate. Succinate, which inhibited the bovine argininosuccinate lyase, did not affect the delta-crystallin enzyme activity in a concentration range between 1 and 300 mM. These results suggest a random Uni Bi kinetic mechanism for the argininosuccinate lyase activity of duck delta-crystallin with the formation of various abortive delta-crystallin-argininosuccinate-arginine, delta-crystallin-argininosuccinate-fumarate and delta-crystallin-argininosuccinate-citrulline ternary complexes.
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Project description:Interaction among crystallins is required for the maintenance of lens transparency. Deamidation is one of the most common post-translational modifications in crystallins, which results in incorrect interaction and leads to aggregate formation. Various studies have established interaction among the ?- and ?-crystallins. Here, we investigated the effects of the deamidation of ?A- and ?B-crystallins on their interaction with ?A3-crystallin using surface plasmon resonance (SPR) and fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer (FLIM-FRET) methods. SPR analysis confirmed adherence of WT ?A- and WT ?B-crystallins and their deamidated mutants with ?A3-crystallin. The deamidated mutants of ?A-crystallin (?A N101D and ?A N123D) displayed lower adherence propensity for ?A3-crystallin relative to the binding affinity shown by WT ?A-crystallin. Among ?B-crystallin mutants, ?B N78D displayed higher adherence propensity whereas ?B N146D mutant showed slightly lower binding affinity for ?A3-crystallin relative to that shown by WT ?B-crystallin. Under the in vivo condition (FLIM-FRET), both ?A-deamidated mutants (?A N101D and ?A N123D) exhibited strong interaction with ?A3-crystallin (32±4% and 36±4% FRET efficiencies, respectively) compared to WT ?A-crystallin (18±4%). Similarly, the ?B N78D and ?B N146D mutants showed strong interaction (36±4% and 22±4% FRET efficiencies, respectively) with ?A3-crystallin compared to 18±4% FRET efficiency of WT ?B-crystallin. Further, FLIM-FRET analysis of the C-terminal domain (CTE), N-terminal domain (NTD), and core domain (CD) of ?A- and ?B-crystallins with ?A3-crystallin suggested that interaction sites most likely reside in the ?A CTE and ?B NTD regions, respectively, as these domains showed the highest FRET efficiencies. Overall, results suggest that similar to WT ?A- and WT?B-crystallins, the deamidated mutants showed strong interactionfor ?A3-crystallin. Variable in vitro and in vivo interactions are most likely due to the mutant's large size oligomers, reduced hydrophobicity, and altered structures. Together, the results suggest that deamidation of ?-crystallin may facilitate greater interaction and the formation of large oligomers with other crystallins, and this may contribute to the cataractogenic mechanism.
| S-EPMC4691197 | biostudies-literature
Project description:In humans, ten genes encode small heat shock proteins with lens αA-crystallin and αB-crystallin representing two of the most prominent members. The canonical isoforms of αA-crystallin and αB-crystallin collaborate in the eye lens to prevent irreversible protein aggregation and preserve visual acuity. α-Crystallins form large polydisperse homo-oligomers and hetero-oligomers and as part of the proteostasis system bind substrate proteins in non-native conformations, thereby stabilizing them. Here, we analyzed a previously uncharacterized, alternative splice variant (isoform 2) of human αA-crystallin with an exchanged N-terminal sequence. This variant shows the characteristic α-crystallin secondary structure, exists on its own predominantly in a monomer-dimer equilibrium, and displays only low chaperone activity. However, the variant is able to integrate into higher order oligomers of canonical αA-crystallin and αB-crystallin as well as their hetero-oligomer. The presence of the variant leads to the formation of new types of higher order hetero-oligomers with an overall decreased number of subunits and enhanced chaperone activity. Thus, alternative mRNA splicing of human αA-crystallin leads to an additional, formerly not characterized αA-crystallin species which is able to modulate the properties of the canonical ensemble of α-crystallin oligomers.
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Project description:XT-I (xylosyltransferase I) is the initial enzyme in the post-translational biosynthesis of glycosaminoglycan chains in proteoglycans. To gain insight into the structure-function relationship of the enzyme, a soluble active form of human XT-I was expressed in High Five insect cells with an apparent molecular mass of 90 kDa. Analysis of the electrophoretic mobility of the protein under non-reducing and reducing conditions indicated that soluble XT-I does not form homodimers through disulphide bridges. In addition, the role of the cysteine residues was investigated by site-directed mutagenesis combined with chemical modifications of XT-I by N-phenylmaleimide. Replacement of Cys471 or Cys574 with alanine led to a complete loss of catalytic activity, indicating the necessity of these residues for maintaining an active conformation of soluble recombinant XT-I by forming disulphide bonds. On the other hand, N-phenylmaleimide treatment showed no effect on wild-type XT-I but strongly inactivated the cysteine mutants in a dose-dependant manner, indicating that seven intramolecular disulphide bridges are formed in wild-type XT-I. The inhibitory effect of UDP on the XT-I activity of C561A (Cys561-->Ala) mutant enzyme was significantly reduced compared with all other tested cysteine mutants. In addition, we tested for binding to UDP-agarose beads. The inactive mutants revealed no significantly different nucleotide-binding properties. Our study demonstrates that recombinant XT-I is organized as a monomer with no free thiol groups and strongly suggests that the catalytic activity does not depend on the presence of free thiol groups, furthermore, we identified five cysteine residues which are critical for enzyme activity.
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Project description:To test the hypothesis that α-crystallin chaperone activity plays a central role in maintenance of lens transparency, we investigated its interactions with γ-crystallin mutants that cause congenital cataract in mouse models. Although the two substitutions, I4F and V76D, stabilize a partially unfolded γD-crystallin intermediate, their affinities to α-crystallin are marginal even at relatively high concentrations. Detectable binding required further reduction of γD-crystallin stability which was achieved by combining the two mutations. Our results demonstrate that mutants and possibly age-damaged γ-crystallin can escape quality control by lens chaperones rationalizing the observation that they nucleate protein aggregation and lead to cataract.
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Project description:Carnivorous plants are known to secrete acid proteinases to digest prey, mainly insects, for nitrogen uptake. In the present study, we have purified, for the first time, to homogeneity two acid proteinases (nepenthesins I and II) from the pitcher fluid of Nepenthes distillatoria (a pitcher-plant known locally as badura) and investigated their enzymic and structural characteristics. Both enzymes were optimally active at pH approx. 2.6 towards acid-denatured haemoglobin; the specificity of nepenthesin I towards oxidized insulin B chain appears to be similar, but slightly wider than those of other APs (aspartic proteinases). Among the enzymic properties, however, the most notable is their unusual stability: both enzymes were remarkably stable at or below 50 degrees C, especially nepenthesin I was extremely stable over a wide range of pH from 3 to 10 for over 30 days. This suggests an evolutionary adaptation of the enzymes to their specific habitat. We have also cloned the cDNAs and deduced the complete amino acid sequences of the precursors of nepenthesins I and II (437 and 438 residues respectively) from the pitcher tissue of N. gracilis. Although the corresponding mature enzymes (each 359 residues) are homologous with ordinary pepsin-type APs, both enzymes had a high content of cysteine residues (12 residues/molecule), which are assumed to form six unique disulphide bonds as suggested by computer modelling and are supposed to contribute towards the remarkable stability of nepenthesins. Moreover, the amino acid sequence identity of nepenthesins with ordinary APs, including plant vacuolar APs, is remarkably low (approx. 20%), and phylogenetic comparison shows that nepenthesins are distantly related to them to form a novel subfamily of APs with a high content of cysteine residues and a characteristic insertion, named 'the nepenthesin-type AP-specific insertion', that includes a large number of novel, orthologous plant APs emerging in the gene/protein databases.
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Project description:The small heat shock protein αB-crystallin is an oligomeric molecular chaperone that binds aggregation-prone proteins. As a component of the proteostasis system, it is associated with cataract, neurodegenerative diseases, and myopathies. The structural determinants for the regulation of its chaperone function are still largely elusive. Combining different experimental approaches, we show that phosphorylation-induced destabilization of intersubunit interactions mediated by the N-terminal domain (NTD) results in the remodeling of the oligomer ensemble with an increase in smaller, activated species, predominantly 12-mers and 6-mers. Their 3D structures determined by cryo-electron microscopy and biochemical analyses reveal that the NTD in these species gains flexibility and solvent accessibility. These modulated properties are accompanied by an increase in chaperone activity in vivo and in vitro and a more efficient cooperation with the heat shock protein 70 system in client folding. Thus, the modulation of the structural flexibility of the NTD, as described here for phosphorylation, appears to regulate the chaperone activity of αB-crystallin rendering the NTD a conformational sensor for nonnative proteins.
| S-EPMC3791731 | biostudies-literature
Project description:Crystallins are transparent, refractive proteins that contribute to the focusing power of the vertebrate eye lens. These proteins are extremely soluble and resist aggregation for decades, even under crowded conditions. Crystallins have evolved to avoid strong interprotein interactions and have unusual hydration properties. Crystallin aggregation resulting from mutation, damage, or aging can lead to cataract, a disease state characterized by opacity of the lens.Different aggregation mechanisms can occur, following multiple pathways and leading to aggregates with varied morphologies. Studies of variant proteins found in individuals with childhood-onset cataract have provided insight into the molecular factors underlying crystallin stability and solubility. Modulation of exposed hydrophobic surface is critical, as is preventing specific intermolecular interactions that could provide nucleation sites for aggregation. Biophysical measurements and structural biology techniques are beginning to provide a detailed picture of how crystallins crowd into the lens, providing high refractivity while avoiding excessively tight binding that would lead to aggregation.Despite the central biological importance of refractivity, relatively few experimental measurements have been made for lens crystallins. Our work and that of others have shown that hydration is important to the high refractive index of crystallin proteins, as are interactions between pairs of aromatic residues and potentially other specific structural features.This Account describes our efforts to understand both the functional and disease states of vertebrate eye lens crystallins, particularly the ?-crystallins. We use a variety of biophysical techniques, notably NMR spectroscopy, to investigate crystallin stability and solubility. In the first section, we describe efforts to understand the relative stability and aggregation propensity of different ?S-crystallin variants. The second section focuses on interactions of these proteins with the holdase chaperone ?B-crystallin. The third, fourth, and fifth sections explore different modes of aggregation available to crystallin proteins, and the final section highlights the importance of refractive index and the sometimes conflicting demands of selection for refractivity and solubility.
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Project description: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 ?A-crystallin. The objective of this work was to determine the effects of wt ?A- and ?B-crystallins on the degradation of C-terminally truncated ?A-crystallin (?A(1-162)) and vice versa.Recombinant wt ?A, ?B, and ?A(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.FRET, multiangle light scattering, and coprecipitation assays showed that ?A(1-162) exchanged subunits with wt ?A- or wt ?B- crystallin to form hetero-oligomers. ?A(1-162) was more susceptible than wt ?A-crystallin to degradation by the UPP. When mixed with wt ?A-crystallin at 1:1 or 1:4 (?A(1-162) : wt) ratios to form hetero-oligomers, the degradation of ?A(1-162) was significantly decreased. Conversely, formation of hetero-oligomers with ?A(1-162) enhanced the degradation of wt ?A-crystallin. The presence of ?A(1-162), but not wt ?A-crystallin, decreased the degradation of wt ?B-crystallin.?A(1-162) forms hetero-oligomers with wt ?A- and ?B-crystallins. Oligomerization with wt ?A- or ?B-crystallins reduces the susceptibility of ?A(1-162) to degradation by the UPP. In addition, the presence of ?A(1-162) in the hetero-oligomers also affects the degradation of wt ?A- and ?B-crystallins.
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