Oligomeric Hsp33 with enhanced chaperone activity

Hsp33, an Escherichia coli cytosolic chaperone, is inactive under normal conditions but becomes active upon oxidative stress. It was previously shown to dimerize upon activation in a concentration- and temperature-dependent manner. This dimer was thought to bind to aggregation-prone target proteins, preventing their aggregation. In the present study, we report small angle x-ray scattering (SAXS), steady state and time-resolved fluorescence, gel filtration, and glutaraldehyde cross-linking analysis of full-length Hsp33. Our circular dichroism and fluorescence results show that there are significant structural changes in oxidized Hsp33 at different temperatures. SAXS, gel filtration, and glutaraldehyde cross-linking results indicate, in addition to the dimers, the presence of oligomeric species. Oxidation in the presence of physiological salt concentration leads to significant increases in the oligomer population. Our results further show that under conditions that mimic the crowded milieu of the cytosol, oxidized Hsp33 exists predominantly as an oligomeric species. Interestingly, chaperone activity studies show that the oligomeric species is much more efficient compared with the dimers in preventing aggregation of target proteins. Taken together, these results indicate that in the cell, Hsp33 undergoes conformational and quaternary structural changes leading to the formation of oligomeric species in response to oxidative stress. Oligomeric Hsp33 thus might be physiologically relevant under oxidative stress

When does the switch from hydrotropy to micellar behavior occur?

Lower alkanoates, alkyl sulfates, and alkylbenzenesulfonates behave as hydrotropes, while those with longer alkyl chains are micellar. We address the question of how long should the alkyl chain be in order for alkylbenzenesulfonates to switch from hydrotropy to micellar behavior. While surface tension and solubilization properties show a transition into micellar-type behavior beyond pentylbenzenesulfonate, microenvironmental features do not show any clear break between hydrotropes and micelles. The answer to the question thus seems to depend on the method used to monitor the properties

Q2N and S65D substitutions of ubiquitin unravel functional significance of the invariant residues Gln2 and Ser65

Ubiquitin is a small, globular protein, structure of which has been perfected and conserved through evolution to manage diverse functions in the macromolecular metabolism of eukaryotic cells. Several non-homologous proteins interact with ubiquitin through entirely different motifs. Though the roles of lysines in the multifaceted functions of ubiquitin are well documented, very little is known about the contribution of other residues. In the present study, the importance of two invariant residues, Gln2 and Ser65, have been examined by substituting them with Asn and Asp, respectively, generating single residue variants of ubiquitin UbQ2N and UbS65D. Gln2 and Ser65 form part of parallel G1 β-bulge adjacent to Lys63, a residue involved in DNA repair, cell-cycle regulated protein synthesis and imparting resistance to protein synthesis inhibitors. The secondary structure of variants is similar to that of UbF45W, a structural homologue of wild-type ubiquitin (UbWt). However, there are certain functional differences observed in terms of resistance to cycloheximide, while there are no major differences pertaining to growth under normal conditions, adherence to N-end rule and survival under heat stress. Further, expression of UbQ2N impedes protein degradation by ubiquitin fusion degradation (UFD) pathway. Such differential responses with respect to functions of ubiquitin produced by mutations may be due to interference in the interactions of ubiquitin with selected partner proteins, hint at biomedical implications

Glutamate64 to glycine substitution in G1 β-bulge of ubiquitin impairs function and stabilizes structure of the protein

Ubiquitin is a globular protein with a highly conserved sequence. Sequence conservation and compact structure make it an ideal protein for structure-function studies. One of the atypical secondary structural features found in ubiquitin is a parallel G1 β-bulge. Glutamate at 64 is the first residue of this β-bulge and the third residue in a type II turn. However, glycine is seen in these positions in several proteins. To understand the effects of substitution of glutamate64 by glycine on the structure, stability and function of ubiquitin, mutant UbE64G has been constructed and characterized in Saccharomyces cerevisiae. The secondary and tertiary structures of UbE64G mutant protein are only marginally different from wild-type protein (UbWt) and fluorescent form of ubiquitin (UbF45W). The earlier studies have shown that the structure and stability of UbWt and UbF45W were similar. However, UbE64G has less surface hydrophobicity than UbWt. UbE64G is found to be more stable compared with UbF45W towards guanidinium chloride induced denaturation. In vivo, complementation shows substrate proteins with Pro as the N-terminal residue, which undergo ubiquitination, have extended half-lives with UbE64G. This altered preference for Pro as opposed to Met might be related to natural preference of glutamate at 64th position in ubiquitin

Structural perturbation and enhancement of the chaperone-like activity of α-crystallin by arginine hydrochloride

Structural perturbation of α-crystallin is shown to enhance its molecular chaperone-like activity in preventing aggregation of target proteins. We demonstrate that arginine, a biologically compatible molecule that is known to bind to the peptide backbone and negatively charged side-chains, increases the chaperone-like activity of calf eye lens α-crystallin as well as recombinant human αA- and αB-crystallins. Arginine-induced increase in the chaperone activity is more pronounced for αB-crystallin than for αA-crystallin. Other guanidinium compounds such as aminoguanidine hydrochloride and guanidine hydrochloride also show a similar effect, but to different extents. A point mutation, R120G, in αB-crystallin that is associated with desmin-related myopathy, results in a significant loss of chaperone-like activity. Arginine restores the activity of mutant protein to a considerable extent. We have investigated the effect of arginine on the structural changes of α-crystallin by circular dichroism, fluorescence, and glycerol gradient sedimentation. Far-UV CD spectra show no significant changes in secondary structure, whereas near-UV CD spectra show subtle changes in the presence of arginine. Glycerol gradient sedimentation shows a significant decrease in the size of α-crystallin oligomer in the presence of arginine. Increased exposure of hydrophobic surfaces of α-crystallin, as monitored by pyrene-solubilization and ANS-fluorescence, is observed in the presence of arginine. These results show that arginine brings about subtle changes in the tertiary structure and significant changes in the quaternary structure of α-crystallin and enhances its chaperone-like activity significantly. This study should prove useful in designing strategies to improve chaperone function for therapeutic applications

Inhibition of Cu<SUP>2+</SUP>- mediated generation of reactive oxygen species by the small heat shock protein &#945;&#946;-crystallin: The relative contributions of the N- and C-terminal domains

Oxidative stress, Cu2+ homeostasis, and small heat shock proteins (sHsp's) have important implications in several neurodegenerative diseases. The ubiquitous sHsp aB-crystallin is an oligomeric protein that binds Cu2+. We have investigated the relative contributions of the N- and C-terminal (C-TD&#945;&#946;-crystallin) domains of &#945;&#946;-crystallin to its Cu2+-binding and redox-attenuation properties and mapped the Cu2+-binding regions. C-TD&#945; &#946;-crystallin binds Cu2+ with slightly less affinity and inhibits Cu2+-catalyzed, ascorbate-mediated generation of ROS to a lesser extent than &#945;&#946; -crystallin. [Cu2+]/[subunit] stoichiometries for redox attenuation by &#945;&#946;-crystallin and C-TD&#945;&#946;-crystallin are 5 and 2, respectively. Both &#945;&#946;-crystallin and C-TD&#945;&#946;-crystallin also inhibit the Fenton reaction of hydroxyl radical formation. Trypsinization of &#945;&#946; -crystallin bound to a Cu2+–NTA column and MALDI-TOF analysis of column-bound peptides yielded three peptides located in the N-terminal domain, and in-solution trypsinization of aB-crystallin followed by Cu2+–NTA column chromatography identified four additional Cu2+-binding peptides located in the C-terminal domain. Thus, Cu2+-binding regions are distributed in the N- and C-terminal domains. Small-angle X-ray scattering and sedimentation-velocity measurements indicate quaternary structural changes in &#945;&#946;-crystallin upon Cu2+ binding. Our study indicates that an oligomer of &#945;&#946;-crystallin can sequester a large number (&#8764; 150) of Cu2+ ions. It acts like a “Cu2+ sponge,” exhibits redox attenuation of Cu2+, and has potential roles in Cu2+ homeostasis and in preventing oxidative stress

Thermal Stress Induced Aggregation of Aquaporin 0 (AQP0) and Protection by α-Crystallin <i>via</i> Its Chaperone Function

<div><p>Aquaporin 0 (AQP0) formerly known as membrane intrinsic protein (MIP), is expressed exclusively in the lens during terminal differentiation of fiber cells. AQP0 plays an important role not only in the regulation of water content but also in cell-to-cell adhesion of the lens fiber cells. We have investigated the thermal stress-induced structural alterations of detergent (octyl glucoside)-solubilized calf lens AQP0. The results show an increase in the amount of AQP0 that aggregated as the temperature increased from 40°C to 65°C. α-Crystallin, molecular chaperone abundantly present in the eye lens, completely prevented the AQP0 aggregation at a 1∶1 (weight/weight) ratio. Since α-crystallin consists of two gene products namely αA- and αB-crystallins, we have tested the recombinant proteins on their ability to prevent thermal-stress induced AQP0 aggregation. In contrast to the general observation made with other target proteins, αA-crystallin exhibited better chaperone-like activity towards AQP0 compared to αB-crystallin. Neither post-translational modifications (glycation) nor C-terminus truncation of AQP0 have any appreciable effect on its thermal aggregation properties. α-Crystallin offers similar protection against thermal aggregation as in the case of the unmodified AQP0, suggesting that αcrystallin may bind to either intracellular loops or other residues of AQP0 that become exposed during thermal stress. Far-UV circular dichroism studies indicated a loss of αhelical structures when AQP0 was subjected to temperatures above 45°C, and the presence of α-crystallin stabilized these secondary structures. We report here, for the first time, that α-crystallin protects AQP0 from thermal aggregation. Since stress-induced structural perturbations of AQP0 may affect the integrity of the lens, presence of the molecular chaperone, α-crystallin (particularly αA-crystallin) in close proximity to the lens membrane is physiologically relevant.</p></div

Chaperone-like activity of human α-crystallin gene products against target proteins.

<p><b>A</b>: (•) Recombinant human αA-crystallin (0.1 mg/ml) (homooligomer) and (▴) recombinant human αB-crystallin (0.1 mg/ml) (homooligomer), against (▪) thermal aggregation of ξ-crystallin (0.1 mg/ml) at 43°C. <b>B</b>: (•) Recombinant human αA-crystallin (0.2 mg/ml) (homooligomer) and (▴) recombinant human αB-crystallin (0.2 mg/ml) (homooligomer), against (▪) DTT-induced aggregation of insulin (0.2 mg/ml) at 43°C.</p

Copper alters aggregation behavior of prion protein and induces novel interactions between its N-and C-terminal regions

Copper is reported to promote and prevent aggregation of prion protein. Conformational and functional consequences of Cu(2+)-binding to prion protein (PrP) are not well understood largely because most of the Cu(2+)-binding studies have been performed on fragments and truncated variants of prion protein. In this context, we set out to investigate the conformational consequences of Cu(2+)-binding to full-length prion protein (PrP) by ITC, NMR and small angle X-Ray scattering (SAXS). In the present study, we report altered aggregation behavior of full-length PrP upon binding to Cu(2+). At physiological temperature, Cu(2+) did not promote aggregation suggesting that Cu(2+) may not play a role in the aggregation of PrP at physiological temperature (37°C). However, Cu(2+)-bound PrP aggregated at lower temperatures. This temperature-dependent process is reversible. Our results show two novel intra-protein interactions upon Cu(2+)-binding. The N-terminal region (residues 90-120 which contains the site H96/H111) becomes proximal to helix-1 (residues 144-147) and its nearby loop region (residues 139-143), which may be important in preventing amyloid fibril formation in the presence of Cu(2+). In addition, we observed another novel interaction between the N-terminal region comprising the octapeptide repeats (residues 60-91) and helix-2 (residues 174-185) of PrP. Small Angle X-Ray Scattering studies of full length PrP show significant compactness upon Cu(2+)-binding. Our results demonstrate novel long-range inter-domain interactions of the N- and C-terminal regions of PrP upon Cu(2+)-binding which might have physiological significance