Proteasome-mediated protein degradation is enhanced by fusion ubiquitin with unstructured degron.

Methods to induce proteasomal degradation of unwanted proteins are valuable in biomedical studies and thus receive increasing attention. For efficient degradation, the proteasome requires both a ubiquitin tag, which delivers substrates to the proteasome, and an unstructured region, where the proteasome engages the substrate for unfolding and degradation. We fused two degron components into a single molecule to create a fusion protein comprising ubiquitin and Rpn4-derived unstructured region. We demonstrated that the fusion protein retained its function to polyubiquitinate target proteins, thereby inducing more efficient proteasomal target degradation than wild-type ubiquitin in vitro and in cells. These results provide novel strategies for robust degradation enhancement of polyubiquitinated proteins.

Conserved Sequence Preferences Contribute to Substrate Recognition by the Proteasome.

The proteasome has pronounced preferences for the amino acid sequence of its substrates at the site where it initiates degradation. Here, we report that modulating these sequences can tune the steady-state abundance of proteins over 2 orders of magnitude in cells. This is the same dynamic range as seen for inducing ubiquitination through a classic N-end rule degron. The stability and abundance of His3 constructs dictated by the initiation site affect survival of yeast cells and show that variation in proteasomal initiation can affect fitness. The proteasome's sequence preferences are linked directly to the affinity of the initiation sites to their receptor on the proteasome and are conserved between Saccharomyces cerevisiae, Schizosaccharomyces pombe, and human cells. These findings establish that the sequence composition of unstructured initiation sites influences protein abundance in vivo in an evolutionarily conserved manner and can affect phenotype and fitness.

Proteasomal degradation of damaged polyubiquitin.

Ubiquitination is one of the most important post-translational modifications of proteins and is involved in various cellular activities, such as proteasomal protein degradation. Ubiquitination is performed via sequential reactions of three enzymes producing polyubiquitin chains, while deubiquitination enzymes can reverse this process, making it possible to recycle ubiquitin molecules. However, such repeated use may seriously damage ubiquitin molecules and result in cell toxicity. Here we show efficient, selective proteasomal degradation of damaged polyubiquitin chains both in vitro and in vivo. However, the degradation efficiency of the damaged polyubiquitin strongly depends on the extent and location of damage to polyubiquitin. Moderate damage at the C-terminal ubiquitin moiety accelerates the degradation of polyubiquitin chains, whereas other damaged ubiquitin escapes from proteasomal degradation. We suggest that the cell can cope with damaged ubiquitin by the cooperative actions of the proteasome and autophagy.

Inhibition of the 26S proteasome by peptide mimics of the coiled-coil region of its ATPase subunits.

Regulation of proteasomal degradation is an indispensable tool for biomedical studies. Thus, there is demand for novel proteasome inhibitors. Proteasomal degradation requires formation of coiled-coil structure by the N-terminal region of ATPase subunits of the proteasome cap. Here we show that peptides that mimic the N-terminal coiled-coil region of ATPase subunits interfere with proteasome function. These results suggest that coiled-coil peptides represent promising new proteasome inhibitors and that N-terminal coiled-coil regions of ATPase subunits are targets for proteasome inhibition.

Artificial regulation of p53 function by modulating its assembly.

The tumor suppressor p53, a 393-amino acid transcription factor with four domains, induces cell cycle arrest, senescence, and apoptosis in response to diverse stress. Tetramer formation is critical for the function of p53. The tetramerization domain permits the tetramerization of p53, where bundled four DNA-binding domains recognize the specific target DNA sequences and activate hundreds of genes, which lead to the various cell fates. Here we show that tumor suppressive functions of p53 can be regulated by manipulating tetramer formation of an engineered p53, in which tetramerization domain of p53 is replaced with an inducible tetramer forming protein. This result suggests that artificial regulation of p53 activity by the engineered p53 is a useful tool to investigate the tumor suppression mechanism of p53 and to combat cancer.

Defining the geometry of the two-component proteasome degron.

The eukaryotic 26S proteasome controls cellular processes by degrading specific regulatory proteins. Most proteins are targeted for degradation by a signal or degron that consists of two parts: a proteasome-binding tag, typically covalently attached polyubiquitin chains, and an unstructured region that serves as the initiation region for proteasomal proteolysis. Here we have characterized how the arrangement of the two degron parts in a protein affects degradation. We found that a substrate is degraded efficiently only when its initiation region is of a certain minimal length and is appropriately separated in space from the proteasome-binding tag. Regions that are located too close or too far from the proteasome-binding tag cannot access the proteasome and induce degradation. These spacing requirements are different for a polyubiquitin chain and a ubiquitin-like domain. Thus, the arrangement and location of the proteasome initiation region affect a protein's fate and are important in selecting proteins for proteasome-mediated degradation.

Substrate selection by the proteasome during degradation of protein complexes.

The proteasome controls the turnover of many cellular proteins. Two structural features are typically required for proteins to be degraded: covalently attached ubiquitin polypeptides that allow binding to the proteasome and an unstructured region in the targeted protein that initiates proteolysis. Here, we have tested the degradation of model proteins to further explore how the proteasome selects its substrates. Using purified yeast proteasome and mammalian proteasome in cell lysate, we have demonstrated that the two structural features can act in trans when separated onto different proteins in a multisubunit complex. In such complexes, the location of the unstructured initiation site and its chemical properties determine which subunit is degraded. Thus, our findings reveal the molecular basis of subunit specificity in the degradation of protein complexes. In addition, our data provide a plausible explanation for how adaptor proteins can bind to otherwise stable proteins and target them for degradation.

Protein targeting to ATP-dependent proteases.

ATP-dependent proteases control diverse cellular processes by degrading specific regulatory proteins. Recent work has shown that protein substrates are specifically transferred to ATP-dependent proteases through different routes. These routes can function in parallel or independently. In all of these targeting mechanisms, it can be useful to separate two steps: substrate binding to the protease and initiation of degradation.

Asymmetry of the GroEL-GroES complex under physiological conditions as revealed by small-angle x-ray scattering.

Despite the well-known functional importance of GroEL-GroES complex formation during the chaperonin cycle, the stoichiometry of the complex has not been clarified. The complex can occur either as an asymmetric 1:1 GroEL-GroES complex or as a symmetric 1:2 GroEL-GroES complex, although it remains uncertain which type is predominant under physiological conditions. To resolve this question, we studied the structure of the GroEL-GroES complex under physiological conditions by small-angle x-ray scattering, which is a powerful technique to directly observe the structure of the protein complex in solution. We evaluated molecular structural parameters, the radius of gyration and the maximum dimension of the complex, from the x-ray scattering patterns under various nucleotide conditions (3 mM ADP, 3 mM ATP gamma S, and 3 mM ATP in 10 mM MgCl(2) and 100 mM KCl) at three different temperatures (10 degrees C, 25 degrees C, and 37 degrees C). We then compared the experimentally observed scattering patterns with those calculated from the known x-ray crystallographic structures of the GroEL-GroES complex. The results clearly demonstrated that the asymmetric complex must be the major species stably present in solution under physiological conditions. On the other hand, in the presence of ATP (3 mM) and beryllium fluoride (10 mM NaF and 300 microM BeCl(2)), we observed the formation of a stable symmetric complex, suggesting the existence of a transiently formed symmetric complex during the chaperonin cycle.

N-Acyldopamine induces aggresome formation without proteasome inhibition and enhances protein aggregation via p62/SQSTM1 expression.

Accumulation of ubiquitinated protein aggregates is a common pathology associated with a number of neurodegenerative diseases and selective autophagy plays a critical role in their elimination. Although aging-related decreases in protein degradation properties may enhance protein aggregation, it remains unclear whether proteasome dysfunction is indispensable for ubiquitinated-protein aggregation in neurodegenerative diseases. Here, we show that N-oleoyl-dopamine and N-arachidonyl-dopamine, which are endogenous brain substances and belong to the N-acyldopamine (AcylDA) family, generate cellular inclusions through aggresome formation without proteasome inhibition. Although AcylDA itself does not inhibit proteasome activity in vitro, it activates the rearrangement of vimentin distribution to form a vimentin cage surrounding aggresomes and sequesters ubiquitinated proteins in aggresomes. The gene transcription of p62/SQSTM1 was significantly increased by AcylDAs, whereas the transcription of other ubiquitin-dependent autophagy receptors was unaffected. Genetic depletion of p62 resulted in the loss of ubiquitinated-protein sequestration in aggresomes, indicating that p62 is a critical component of aggresomes. Furthermore, AcylDAs accelerate the aggregation of mutant huntingtin exon 1 proteins. These results suggest that aggresome formation does not require proteasome dysfunction and AcylDA-induced aggresome formation may participate in forming cytoplasmic protein inclusions.

The equilibrium unfolding intermediate observed at pH 4 and its relationship with the kinetic folding intermediates in green fluorescent protein.

Folding mechanisms of a variant of green fluorescent protein (F99S/M153T/V163A) were investigated by a wide variety of spectroscopic techniques. Equilibrium measurements on acid-induced denaturation of the protein monitored by chromophore and tryptophan fluorescence and small-angle X-ray scattering revealed that this protein accumulates at least two equilibrium intermediates, a native-like intermediate and an unfolding intermediate, the latter of which exhibits the characteristics of the molten globule state under moderately denaturing conditions at pH 4. To elucidate the role of the equilibrium unfolding intermediate in folding, a series of kinetic refolding experiments with various combinations of initial and final pH values, including pH 7.5 (the native condition), pH 4.0 (the moderately denaturing condition where the unfolding intermediate is accumulated), and pH 2.0 (the acid-denaturing condition) were carried out by monitoring chromophore and tryptophan fluorescence. Kinetic on-pathway intermediates were accumulated during the folding on the refolding reaction from pH 2.0 to 7.5. However, the signal change corresponding to the conversion from the acid-denatured to the kinetic intermediate states was significantly reduced on the refolding reaction from pH 4.0 to pH 7.5, whereas only the signal change corresponding to the above conversion was observed on the refolding reaction from pH 2.0 to pH 4.0. These results indicate that the equilibrium unfolding intermediate is composed of an ensemble of the folding intermediate species accumulated during the folding reaction, and thus support a hierarchical model of protein folding.

Rapamycin-induced oligomer formation system of FRB-FKBP fusion proteins.

Most proteins form larger protein complexes and perform multiple functions in the cell. Thus, artificial regulation of protein complex formation controls the cellular functions that involve protein complexes. Although several artificial dimerization systems have already been used for numerous applications in biomedical research, cellular protein complexes form not only simple dimers but also larger oligomers. In this study, we showed that fusion proteins comprising the induced heterodimer formation proteins FRB and FKBP formed various oligomers upon addition of rapamycin. By adjusting the configuration of fusion proteins, we succeeded in generating an inducible tetramer formation system. Proteins of interest also formed tetramers by fusing to the inducible tetramer formation system, which exhibits its utility in a broad range of biological applications.

Novel method for the high-throughput production of phosphorylation site-specific monoclonal antibodies.

Threonine phosphorylation accounts for 10% of all phosphorylation sites compared with 0.05% for tyrosine and 90% for serine. Although monoclonal antibody generation for phospho-serine and -tyrosine proteins is progressing, there has been limited success regarding the production of monoclonal antibodies against phospho-threonine proteins. We developed a novel strategy for generating phosphorylation site-specific monoclonal antibodies by cloning immunoglobulin genes from single plasma cells that were fixed, intracellularly stained with fluorescently labeled peptides and sorted without causing RNA degradation. Our high-throughput fluorescence activated cell sorting-based strategy, which targets abundant intracellular immunoglobulin as a tag for fluorescently labeled antigens, greatly increases the sensitivity and specificity of antigen-specific plasma cell isolation, enabling the high-efficiency production of monoclonal antibodies with desired antigen specificity. This approach yielded yet-undescribed guinea pig monoclonal antibodies against threonine 18-phosphorylated p53 and threonine 68-phosphorylated CHK2 with high affinity and specificity. Our method has the potential to allow the generation of monoclonal antibodies against a variety of phosphorylated proteins.

Phi 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 microM 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 microM and 70 microM 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 (Phi=0.9) by using a Phi value analysis in the kinetic Monod-Wyman-Changeux model, which is analogous to the mutational Phi value analysis in protein folding.

The allosteric transition of GroEL induced by metal fluoride-ADP complexes.

To understand the mechanism of a functionally important ATP-induced allosteric transition of GroEL, we have studied the effect of a series of metal fluoride-ADP complexes and vanadate-ADP on GroEL by kinetic fluorescence measurement of pyrene-labeled GroEL and by small-angle X-ray scattering measurement of wild-type GroEL. The metal fluorides and vanadate, complexed with ADP, are known to mimic the gamma-phosphate group of ATP, but they differ in geometry and size; it is expected that these compounds will be useful for investigating the strikingly high specificity of GroEL for ATP that enables the induction of the allosteric transition. The kinetic fluorescence measurement revealed that aluminium, beryllium, and gallium ions, when complexed with the fluoride ion and ADP, induced a biphasic fluorescence change of pyrenyl GroEL, while scandium and vanadate ions did not induce any kinetically observed change in fluorescence. The burst phase and the first phase of the fluorescence kinetics were reversible, while the second phase and subsequent changes were irreversible. The dependence of the burst-phase and the first-phase fluorescence changes on the ADP concentration indicated that the burst phase represents non-cooperative nucleotide binding to GroEL, and that the first phase represents the allosteric transition of GroEL. Both the amplitude and the rate constant of the first phase of the fluorescence kinetics were well understood in terms of a kinetic allosteric model, which is a combination of transition state theory and the Monod-Wyman-Changeux allosteric model. From the kinetic allosteric model analysis, the relative free energy of the transition state in the metal fluoride-ADP-induced allosteric transition of GroEL was found to be larger than the corresponding free energy of the ATP-induced allosteric transition by more than 5.5kcal/mol. However, the X-ray scattering measurements indicated that the allosteric state induced by these metal fluoride-ADP complexes is structurally equivalent to the allosteric state induced by ATP. These results suggested that both the size and coordination geometry of gamma-phosphate (and its analogs) are related to the allosteric transition of GroEL. It was therefore concluded that the tetrahedral geometry of gamma-phosphate (or its analogs) and the inter-atomic distance ( approximately 1.6A) between phosphorus (vanadium, or metal atom) and oxygen (or fluorine) are both important for inducing the allosteric transition of GroEL, leading to the high selectivity of GroEL for ATP about ligand adenine nucleotides, which function as the preferred allosteric ligand.

Equilibrium and kinetics of the allosteric transition of GroEL studied by solution X-ray scattering and fluorescence spectroscopy.

We have studied the ATP-induced allosteric structural transition of GroEL using small angle X-ray scattering and fluorescence spectroscopy in combination with a stopped-flow technique. With X-ray scattering one can clearly distinguish the three allosteric states of GroEL, and the kinetics of the transition of GroEL induced by 85 microM ATP have been observed directly by stopped-flow X-ray scattering for the first time. The rate constant has been found to be 3-5s(-1) at 5 degrees C, indicating that this process corresponds to the second phase of the ATP-induced kinetics of tryptophan-inserted GroEL measured by stopped-flow fluorescence. Based on the ATP concentration dependence of the fluorescence kinetics, we conclude that the first phase represents bimolecular non-cooperative binding of ATP to GroEL with a bimolecular rate constant of 5.8 x 10(5)M(-1)s(-1) at 25 degrees C. Considering the electrostatic repulsion between negatively charged GroEL (-18 of the net charge per monomer at pH 7.5) and ATP, the rate constant is consistent with a diffusion-controlled bimolecular process. The ATP-induced fluorescence kinetics (the first and second phases) at various ATP concentrations (< 400 microM) occur before ATP hydrolysis by GroEL takes place and are well explained by a kinetic allosteric model, which is a combination of the conventional transition state theory and the Monod-Wyman-Changeux model, and we have successfully evaluated the equilibrium and kinetic parameters of the allosteric transition, including the binding constant of ATP in the transition state of GroEL.

Fast compaction of alpha-lactalbumin during folding studied by stopped-flow X-ray scattering.

To monitor the fast compaction process during protein folding, we have used a stopped-flow small-angle X-ray scattering technique combined with a two-dimensional charge-coupled device-based X-ray detector that makes it possible to improve the signal-to-noise ratio of data dramatically, and measured the kinetic refolding reaction of alpha-lactalbumin. The results clearly show that the radius of gyration and the overall shape of the kinetic folding intermediate of alpha-lactalbumin are the same as those of the molten globule state observed at equilibrium. Thus, the identity between the kinetic folding intermediate and the equilibrium molten globule state is firmly established. The present results also suggest that the folding intermediate is more hydrated than the native state and that the hydrated water molecules are dehydrated when specific side-chain packing is formed during the change from the molten globule to the native state.

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.

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, in vitro and in vivo. These results suggest that artificial modulation of proteasome initiation is a versatile method for conditionally inhibiting the proteasomal degradation of specific proteins.