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.

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.

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.

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.

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.

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.

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.

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.

Sequence composition of disordered regions fine-tunes protein half-life.

The proteasome controls the concentrations of most proteins in eukaryotic cells. It recognizes its protein substrates through ubiquitin tags and initiates degradation at disordered regions within the substrate. Here we show that the proteasome has pronounced preferences for the amino acid sequence of the regions at which it initiates degradation. Specifically, proteins in which the initiation regions have biased amino acid compositions show longer half-lives in yeast than proteins with unbiased sequences in the regions. The relationship is also observed on a genomic scale in mouse cells. These preferences affect the degradation rates of proteins in vitro, can explain the unexpected stability of natural proteins in yeast and may affect the accumulation of toxic proteins in disease. We propose that the proteasome's sequence preferences provide a second component to the degradation code and may fine-tune protein half-life in cells.

Paradigms of protein degradation by the proteasome.

The proteasome is the main proteolytic machine in the cytosol and nucleus of eukaryotic cells where it degrades hundreds of regulatory proteins, removes damaged proteins, and produces peptides that are presented by MHC complexes. New structures of the proteasome particle show how its subunits are arranged and provide insights into how the proteasome is regulated. Proteins are targeted to the proteasome by tags composed of several ubiquitin moieties. The structure of the tags tunes the order in which proteins are degraded. The proteasome itself edits the ubiquitin tags and drugs that interfere in this process can enhance the clearance of toxic proteins from cells. Finally, the proteasome initiates degradation at unstructured regions within its substrates and this step contributes to substrate selection.

Sequence- and species-dependence of proteasomal processivity.

The proteasome is the degradation machine at the center of the ubiquitin-proteasome system and controls the concentrations of many proteins in eukaryotes. It is highly processive so that substrates are degraded completely into small peptides, avoiding the formation of potentially toxic fragments. Nonetheless, some proteins are incompletely degraded, indicating the existence of factors that influence proteasomal processivity. We have quantified proteasomal processivity and determined the underlying rates of substrate degradation and release. We find that processivity increases with species complexity over a 5-fold range between yeast and mammalian proteasome, and the effect is due to slower but more persistent degradation by proteasomes from more complex organisms. A sequence stretch that has been implicated in causing incomplete degradation, the glycine-rich region of the NFκB subunit p105, reduces the proteasome's ability to unfold its substrate, and polyglutamine repeats such as found in Huntington's disease reduce the processivity of the proteasome in a length-dependent manner.

Dissecting a bimolecular process of MgATP²- binding to the chaperonin GroEL.

Although allosteric transitions of GroEL by MgATP(2)(-) have been widely studied, the initial bimolecular step of MgATP(2-) binding to GroEL remains unclear. Here, we studied the equilibrium and kinetics of MgATP(2)(-) binding to a variant of GroEL, in which Tyr485 was replaced by tryptophan, via isothermal titration calorimetry (ITC) and stopped-flow fluorescence spectroscopy. In the absence of K(+) at 4-5 °C, the allosteric transitions and the subsequent ATP hydrolysis by GroEL are halted, and hence, the stopped-flow fluorescence kinetics induced by rapid mixing of MgATP(2)(-) and the GroEL variant solely reflected MgATP(2)(-) binding, which was well represented by bimolecular noncooperative binding with a binding rate constant, k(on), of 9.14×10(4) M(-1) s(-1) and a dissociation rate constant, k(off), of 14.2 s(-1), yielding a binding constant, K(b) (=k(on)/k(off)), of 6.4×10(3) M(-1). We also successfully performed ITC to measure binding isotherms of MgATP(2)(-) to GroEL and obtained a K(b) of 9.5×10(3) M(-1) and a binding stoichiometric number of 6.6. K(b) was thus in good agreement with that obtained by stopped-flow fluorescence. In the presence of 10-50 mM KCl, the fluorescence kinetics consisted of three to four phases (the first fluorescence-increasing phase, followed by one or two exponential fluorescence-decreasing phases, and the final slow fluorescence-increasing phase), and comparison of the kinetics in the absence and presence of K(+) clearly demonstrated that the first fluorescence-increasing phase corresponds to bimolecular MgATP(2)(-) binding to GroEL. The temperature dependence of the kinetics indicated that MgATP(2)(-) binding to GroEL was activation-controlled with an activation enthalpy as large as 14-16 kcal mol(-1).

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.