Structural basis for the disaggregase activity and regulation of Hsp104.

The Hsp104 disaggregase is a two-ring ATPase machine that rescues various forms of non-native proteins including the highly resistant amyloid fibers. The structural-mechanistic underpinnings of how the recovery of toxic protein aggregates is promoted and how this potent unfolding activity is prevented from doing collateral damage to cellular proteins are not well understood. Here, we present structural and biochemical data revealing the organization of Hsp104 from Chaetomium thermophilum at 3.7 Å resolution. We show that the coiled-coil domains encircling the disaggregase constitute a 'restraint mask' that sterically controls the mobility and thus the unfolding activity of the ATPase modules. In addition, we identify a mechanical linkage that coordinates the activity of the two ATPase rings and accounts for the high unfolding potential of Hsp104. Based on these findings, we propose a general model for how Hsp104 and related chaperones operate and are kept under control until recruited to appropriate substrates.

The redox-sensing quinone reductase Lot6p acts as an inducer of yeast apoptosis.

Mammalian NAD(P)H:quinone oxidoreductases such as human NQO1 act as inducers of apoptosis. Quinone reductases generated interest over the last decade due to their proposed function in the oxidative stress response. Furthermore, human NQO1 was reported to regulate p53 stability and p53-dependent apoptosis through regulation of cellular oxidation-reduction events. In this study, we have used low concentrations of hydrogen peroxide (0.4 and 0.6 mM) to induce apoptosis-like cell death in wild type, an LOT6 overexpressing and a Deltalot6 yeast strain to monitor cell survival. Using this approach, we demonstrate that yeast quinone reductase Lot6p, an orthologue of mammalian quinone reductases, plays a pivotal role in apoptosis-like cell death in Saccharomyces cerevisiae. Overexpression of LOT6 results in enhanced cell death, as shown by an investigation of the morphological hallmarks of apoptosis-like fragmentation of DNA and externalization of phosphatidylserine, whereas the deletion strain displays a deficiency in apoptosis-like cell death as compared with the wild type. Thus, we propose that Lot6p is directly involved in the control of the apoptosis-like cell death in yeast.

New roles of flavoproteins in molecular cell biology: an unexpected role for quinone reductases as regulators of proteasomal degradation.

Quinone reductases are ubiquitous soluble enzymes found in bacteria, fungi, plants and animals. These enzymes utilize a reduced nicotinamide such as NADH or NADPH to reduce the flavin cofactor (either FMN or FAD), which then affords two-electron reduction of cellular quinones. Although the chemical nature of the quinone substrate is still a matter of debate, the reaction appears to play a pivotal role in quinone detoxification by preventing the generation of potentially harmful semiquinones. In recent years, an additional role of quinone reductases as regulators of proteasomal degradation of transcription factors and possibly intrinsically unstructured protein has emerged. To fulfil this role, quinone reductase binds to the core particle of the proteasome and recruits certain transcription factors such as p53 and p73alpha to the complex. The latter process appears to be governed by the redox state of the flavin cofactor of the quinone reductase, thus linking the stability of transcription factors to cellular events such as oxidative stress. Here, we review the current evidence for protein complex formation between quinone reductase and the 20S proteasome in eukaryotic cells and describe the regulatory role of this complex in stabilizing transcription factors by acting as inhibitors of their proteasomal degradation.

Mechanism of flavin reduction and oxidation in the redox-sensing quinone reductase Lot6p from Saccharomyces cerevisiae.

Quinone reductases are flavin-containing enzymes that have been implicated in protecting organisms from redox stress and, more recently, as redox switches controlling the action of the proteasome. The reactions of the catalytic cycle of the dimeric quinone reductase Lot6p from Saccharomyces cerevisiae were studied in anaerobic stopped-flow experiments at 4 degrees C. Both NADH and NADPH reacted similarly, reducing the FMN prosthetic group rapidly at saturation but binding with very low affinity. The enzyme stereospecifically transferred the proS-hydride of NADPH with an isotope effect of 3.6, indicating that hydride transfer, and not an enzyme conformational change, is rate-determining in the reductive half-reaction. No intermediates such as charge-transfer complexes were detected. In the oxidative half-reaction, reduced enzyme reacted in a single phase with the six quinone substrates tested. The observed rate constants increased linearly with quinone concentration up to the limits allowed by solubility, indicating either a bimolecular reaction or very weak binding. The logarithm of the bimolecular rate constant increases linearly with the reduction potential of the quinone, consistent with the notion that quinone reductases strongly disfavor radical intermediates. Interestingly, both half-reactions of the catalytic cycle strongly resemble bioorganic model reactions; the reduction of Lot6p by NAD(P)H is moderately faster than nonenzymatic models, while the oxidation of Lot6p by quinones is actually slower than nonenzymatic reactions. This curious situation is consistent with the structure of Lot6p, which has a crease we propose to be the binding site for pyridine nucleotides and a space, but no obvious catalytic residues, near the flavin allowing the quinone to react. The decidedly suboptimized catalytic cycle suggests that selective pressures other than maximizing quinone consumption shaped the evolution of Lot6p. This may reflect the importance of suppressing other potentially deleterious side reactions, such as oxygen reduction, or it may indicate that the role Lot6p plays as a redox sensor in controlling the proteasome is more important than its role as a detoxifying enzyme.

Quinone reductase acts as a redox switch of the 20S yeast proteasome.

The proteasome has an essential function in the intracellular degradation of protein in eukaryotic cells. We found that the dimeric quinone reductase Lot6 uses the flavin mononucleotide (FMN)-binding site to bind to the 20S proteasome with a 1:2 stoichiometry-that is, one 20S proteasome molecule can associate with two quinone reductases. Furthermore, reduction of the FMN cofactor by either NADH or light irradiation results in the binding of the b-Zip transcription factor Yap4 to the Lot6-proteasome complex, indicating that recruitment of the transcription factor depends on the redox state of the quinone reductase. Here, we show that binding of Yap4 to the complex not only protects it from ubiquitin-independent proteasomal degradation, but also regulates its cellular localization. In non-stressed wild-type cells, we did not detect any Yap4 in the nucleus, whereas Yap4 was present in the nuclei from quinone-stressed yeast cultures. Thus, the Lot6-proteasome complex can be regarded as a redox switch in which the quinone reductase acts as a sensor for oxidative stress.

Lot6p from Saccharomyces cerevisiae is a FMN-dependent reductase with a potential role in quinone detoxification.

NAD(P)H:quinone acceptor oxidoreductases are flavoenzymes expressed in the cytoplasm of many tissues and afford protection against the cytotoxic effects of electrophilic quinones by catalyzing a strict two-electron reduction. Such enzymes have been reported from several mammalian sources, e.g. human, mouse and rat, and from plant species. Here, we report identification of Lot6p (YLR011wp), the first soluble quinone reductase from the unicellular model organism Saccharomyces cerevisiae. Localization studies using an antibody raised against Lot6p as well as microscopic inspection of Lot6p-GFP demonstrated accumulation of the enzyme in the cytosol of yeast cells. Despite sharing only 23% similarity to type 1 human quinone reductase, Lot6p possesses biochemical properties that are similar to its human counterpart. The enzyme catalyzes a two-electron reduction of a series of natural and artificial quinone substrates at the expense of either NADH or NADPH. The kinetic mechanism follows a ping-pong bi-bi reaction scheme, with K(M) values of 1.6-11 microm for various quinones. Dicoumarol and Cibacron Marine, two well-known inhibitors of the quinone reductase family, bind to Lot6p and inhibit its activity. In vivo experiments demonstrate that the enzymatic activity of Lot6p is consistent with the phenotype of both Deltalot6 and Lot6p overexpressing strains, suggesting that Lot6p may play a role in managing oxidative stress in yeast.

Characterization of a thermostable NADPH:FMN oxidoreductase from the mesophilic bacterium Bacillus subtilis.

The gene yhdA from Bacillus subtilis encoding a putative flavin mononucleotide (FMN)-dependent oxidoreductase was cloned and heterologously expressed in Escherichia coli. The purified enzyme has a noncovalently bound FMN cofactor, which is preferentially reduced by NADPH, indicating that YhdA is a NADPH:FMN oxidoreductase. The rate of NADPH oxidation is enhanced by the addition of external FMN, and analysis of initial rate measurements reveals the occurrence of a ternary complex in a bi-bi reaction mechanism. YhdA has also been shown to reductively cleave the -N=N- bond in azo dyes at the expense of NADPH, and hence, it possesses azoreductase activity, however, at a rate 100 times slower than that found for FMN. Using Cibacron Marine as a model compound, we could demonstrate that the dye is a competitive inhibitor of NADPH and FMN. The utilization of NADPH and the absence of a flavin semiquinone radical distinguish YhdA from flavodoxins, which adopt the same structural fold, i.e., a five-stranded beta sheet sandwiched by five alpha helices. The native molecular-mass of YhdA was determined to be 76 kDa, suggesting that the protein occurs as a tetramer, whereas the YhdA homologue in Saccharomyces cerevisiae (YLR011wp) forms a dimer in solution. Interestingly, the different oligomerization of these homologous proteins correlates to their thermostability, with YhdA exhibiting a melting point of 86.5 degrees C, which is 26.3 degrees C higher than that for the yeast protein. This unusually high melting point is proposed to be the result of increased hydrophobic packing between dimers and the additional presence of four salt bridges stabilizing the dimer-dimer interface.

Structure and function of YcnD from Bacillus subtilis, a flavin-containing oxidoreductase.

YcnD from the gram-positive bacterium Bacillus subtilis is a member of a family of bacterial proteins that act as NADH- and/or NADPH-dependent oxidoreductases. Here, we report for the first time on the biochemical characterization of the purified protein, demonstrating that YcnD is an FMN-containing enzyme that can be reduced by NADH or NADPH (Km = 6.4 and 4.4 microM, respectively). In the presence of free FMN as the electron-accepting substrate, the latter reductant showed a ping-pong Bi-Bi reaction mechanism, whereas utilization of NADH is competitively inhibited by this substrate. This finding suggests that NADPH is the physiological reductant of the enzyme. We also show that YcnD reduces nitro-organic compounds, chromate, and a series of azo dyes. The reduction of azo dyes appears to be mediated by free reduced FMN because the reaction is considerably slower in its absence. Structure determination by X-ray crystallography revealed that YcnD folds into a three layer alpha-beta-alpha sandwich strongly resembling the topology of the NADH oxidase superfamily. Similar to homologous bacterial oxidoreductase, YcnD forms homodimers with an extended dimer interface. The biochemical data and the structure are discussed in light of the putative physiological function of YcnD as an oxidoreductase delivering reduced FMN to enzymes that require the reduced cofactor for activity.