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

N-Terminal Coiled-Coil Structure of ATPase Subunits of 26S Proteasome Is Crucial for Proteasome Function.

The proteasome is an essential proteolytic machine in eukaryotic cells, where it removes damaged proteins and regulates many cellular activities by degrading ubiquitinated proteins. Its heterohexameric AAA+ ATPase Rpt subunits play a central role in proteasome activity by the engagement of substrate unfolding and translocation for degradation; however, its detailed mechanism remains poorly understood. In contrast to AAA+ ATPase domains, their N-terminal regions of Rpt subunits substantially differ from each other. Here, to investigate the requirements and roles of the N-terminal regions of six Rpt subunits derived from Saccharomyces cerevisiae, we performed systematic mutational analysis using conditional knockdown yeast strains for each Rpt subunit and bacterial heterologous expression system of the base subcomplex. We showed that the formation of the coiled-coil structure was the most important for the N-terminal region of Rpt subunits. The primary role of coiled-coil structure would be the maintenance of the ring structure with the defined order. However, the coiled-coil region would be also be involved in substrate recognition and an interaction between lid and base subcomplexes

Expression of mouse and yeast Rpt subunits rescues the growth of yeast with conditional suppression of the wild-type yeast Rpt subunit.

<p>(A) Rpt tet-off strains were transformed with the pAUR123 yeast expression vector encoding yeast (sc) and mouse (m) Rpt subunits, grown to early log phase and individually spotted in duplicate as ten-fold serial dilutions on plates either without (right panel) or with (left panel) doxycycline. Plates were incubated at 30°C for 2 days and then photographed. (B) Multiple sequence alignment of the N-terminal regions of Rpt subunits derived from yeast, worm, fly, frog, and mouse. The coiled-coil regions of Rpt subunits predicted by PairCoil2 are indicated above the sequences (ref. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134056#pone.0134056.s006" target="_blank">S6 Fig</a>). Sequence conservation is indicated beneath the alignment, and conserved residues are marked and color-coded according to the default ClustalX settings. Multiple sequence alignments of full-length Rpt subunits are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134056#pone.0134056.s003" target="_blank">S3 Fig</a>.</p

Structure of yeast Rpt subunits and construction of Rpt tet-off strains.

<p>(A) A schematic representation of structural domains of the Rpt subunits. Multiple sequence alignment of yeast Rpt subunits are indicated beneath the domain structure. Rpt1–6 were derived from <i>Saccharomyces cerevisiae</i>. Sequence conservation is indicated beneath the alignment, and conserved residues are marked and color-coded according to the default ClustalX settings. Enlarged sequence alignment is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134056#pone.0134056.s001" target="_blank">S1 Fig</a>. (B) Structure of 26S proteasome. Molecular surface of the 19S activator particle bound to the 20S core particle (CP; PDB ID 4B4T) (left). The 19S regulatory particle, which contains Rpt AAA+ ATPase subunits (green) and non-ATPase subunits (yellow), caps either end of the 20S CP (gray). Enlarged view of the Rpt AAA+ ATPase subunits are shown as a ribbon (right). N-terminal coiled coils formed by Rpt1–Rpt2 (light red), Rpt4–Rpt5 (red), and Rpt3–Rpt6 (dark red) are colored. Structures are produced by PyMOL. (C) Strain construction by one-step homologous replacement of native promoters with a TetO₇-containing cassette. (D) Culture of Rpt tet-off strains was grown to early log phase (OD₆₀₀ of approximately 0.6–0.8). Ten-fold serial dilutions of these cultures were spotted on YPDA medium agar plates or YPDA medium agar plates containing 10 μg/ml doxycycline. Plates were incubated at 30°C for 3 days and then photographed.</p

Coiled-coil mutations reveal that destabilization of Rpt subunits hampers yeast growth.

<p>(A) Coiled-coil probability for wild-type (black lines) and CC− mutants (blue lines) of Rpt subunits calculated by PairCoil2. The thickness of the red lines represents the confidence of the prediction (p-scores) by Paircoil2. (B) Rpt tet-off strains were transformed with the pAUR123 yeast expression vector encoding wild-type and CC− mutants of Rpt subunits, grown to early log phase, and individually spotted in duplicate as a ten-fold serial dilution on plates either without (right panel) or with (left panel) doxycycline. Plates were incubated at 30°C for 2 days and then photographed. (C) Disruption of the coiled-coil conformation of Rpt subunit induces the accumulation of polyubiquitinated proteins. The accumulation of polyubiquitinated proteins in the Rpt tet-off yeast cells expressing wild-type and CC− mutants (Rpt1CC−, Rpt2CC−, Rpt3CC−, Rpt4CC−, Rpt5CC−, and Rpt6CC−) were analyzed using western blot with an anti-polyubiquitin antibody. After yeast cells at early log phase were treated with 20 μg/mL Dox for 3 h, cells were harvested and lysed with glass beads in the presence of 10% TCA to preserve ubiquitination patterns. PGK1 was used as a loading control.</p

Base subcomplex formation by mutant Rpt subunits.

<p>(A) Schematic representation of the TAP procedure used to isolate the assembled base subcomplex from <i>E</i>. <i>coli</i> lysate. (B) Total lysates (top panels) and eluted fraction (middle and bottom panels) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). (Top and middle panels) Western blotting analysis: FLAG-tagged Rpt1 (red) and His₆-tagged Rpt3 (green) were revealed with anti-FLAG and anti-His₆ antibodies, respectively. (Bottom panels) The eluted fraction was separated by SDS-PAGE and visualized using Oriole staining. Deletion mutants (Rpt1Δ65, Rpt2Δ65, Rpt3Δ65, Rpt4Δ65, Rpt5Δ40, and Rpt6Δ50; left panels) and coiled-coil destabilizing (CC−) mutants (Rpt1CC−, Rpt2CC−, Rpt3CC−, Rpt4CC−, Rpt5CC−, and Rpt6CC−; right panels) that caused the defective growth of yeast cells were analyzed as above. In the input of (B), FLAG-Rpt1Δ65 appeared to have lower molecular weight due to its N-terminal deletion because FLAG-tag was attached to Rpt1. With the similar reason, His₆-Rpt3Δ65 shows smaller molecular weight. In contrast, His₆-Rpt3CC− appeared slightly higher in position than wild-type His₆-Rpt3 probably because CC− mutation (Pro replacement) lowered the migration of His₆-Rpt3CC−. For the same reason, HA-Rpt3CC− also appeared slightly higher in position than wild-type HA-Rpt3 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134056#pone.0134056.s008" target="_blank">S8 Fig</a>).</p

Coiled-coil mutations without destabilization of coiled-coils.

<p>(A) Rpt tet-off strains were transformed with the pAUR123 yeast expression vector encoding wild-type and CC0 and CC00 mutants of Rpt subunits, grown to early log phase, and individually spotted in duplicate as a ten-fold serial dilutions on plates either without (right panel) or with (left panel) doxycycline. Plates were incubated at 30°C for 2 days and then photographed. (B) Accumulation of polyubiquitinated proteins in the Rpt tet-off yeast cells expressing wild-type and CC0 or CC00 mutants (Rpt1CC00, Rpt2CC00, Rpt3CC00, Rpt4CC00, Rpt5CC0, and Rpt6CC0) were analyzed using western blot with an anti-polyubiquitin antibody. After the yeast cell at early log phase were treated with 20 μg/mL Dox for 3 h, cells were harvested and lysed with glass beads in the presence of 10% TCA. PGK1 was used as a loading control.</p

Φ Value Analysis of an Allosteric Transition of GroEL based on a Single-pathway Model

There are currently two contradictory models for the kinetics of the ATP-induced GroEL allosteric transition occurring around 20 μM ATP. One model, proposed by Horovitz et al. demonstrates the existence of two parallel pathways for the allosteric transition and an abrupt ATP-dependent switch from one pathway to the other. The other model, which was proposed by the present authors, shows no need to assume the parallel pathways, and a combination of the transition-state theory and the Monod–Wyman–Changeux model of allostery can explain the kinetics as well as the equilibrium of the transition. The discrepancy appears to be due to whether we regard the transition as reversible or irreversible. Thus, here we have investigated the reversibility of the allosteric transition between 0 μM and 70 μM ATP by the use of a stopped-flow double-jump technique, which has allowed us to monitor the kinetics of the reverse reaction from the relaxed state at a high ATP concentration to the tense state at a low ATP concentration. The tryptophan fluorescence of a tryptophan-inserted variant of GroEL was used to follow the kinetics. As a result, the allosteric transition was shown to be a reversible process, supporting the validity of our model. We also show that the structural environment around the ATP-binding site of GroEL in the transition state is very similar to that in the relaxed state (Φ=0.9) by using a Φ value analysis in the kinetic Monod–Wyman–Changeux model, which is analogous to the mutational Φ value analysis in protein folding

Regulation of Proteasomal Degradation by Modulating Proteasomal Initiation Regions

Methods for regulating the concentrations of specific cellular proteins are valuable tools for biomedical studies. Artificial regulation of protein degradation by the proteasome is receiving increasing attention. Efficient proteasomal protein degradation requires a degron with two components: a ubiquitin tag that is recognized by the proteasome and a disordered region at which the proteasome engages the substrate and initiates degradation. Here we show that degradation rates can be regulated by modulating the disordered initiation region by the binding of modifier molecules, <i>in vitro</i> and <i>in vivo</i>. These results suggest that artificial modulation of proteasome initiation is a versatile method for conditionally inhibiting the proteasomal degradation of specific proteins