Tokyo Metropolitan Institute of Medical Science: Laboratory of Protein Metabolism

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Protein Metabolism:
Focusing on the Ubiquitin Proteasome System

Prof. Dr. Keiji Tanaka
Chairperson of TMiMS

All proteins of the cell continually are recycled with individually distinct life-span, which is fundamentally important to keep healthy cellular activities in eukaryotes. The proteasome, in collaboration with the ubiquitin system used for choice of target proteins, selectively degrades unnecessary proteins that must be eliminated from the cells. Indeed, the ubiquitin-proteasome system (UPS) plays a pivotal role in the control of a diverse array of basic cellular functions by catalyzing a number of reactions timely, rapidly, and irreversibly. The missions of our laboratory aim to elucidate the molecular mechanisms of the UPS and to integrate it into physiology and pathology.

Research Projects

1. Structure and Assembly of Proteasomes

The 26S proteasome is a highly organized and sophisticated proteolytic nanomachine that degrades ubiquitylated proteins in an ATP-dependent fashion. The 26S proteasome consisting of a 20S core/catalytic particle (CP) and one or two 19S regulatory particles (RP). The CP (alias 20S proteasome) is composed of four heptameric rings, which are made up of seven structurally related a and b subunits, displaying an α1-7 β1-7 β1-7 α1-7 organization. The RP recognizes ubiquitylated proteins, deubiquitylates for recycling of ubiquitin, and then unfolds and translocates them into the interior of the CP for degradation (Fig. 1).

Figure 1. The atomic model of the 26S proteasome

One longstanding question is how the complex structure of the proteasome is organized with high fidelity. To surely execute the protein degradation, the 26S proteasome itself should be assembled rapidly and correctly to the sophisticated structure from more than 66 subunits. We have identified approximately 10 proteasome-dedicated assembling chaperones (i.e., CP and RP chaperones) that assist in the efficient formation of 20S and 26S proteasomes. Of them, we have determined the tertiary structures of CP and RP chaperones, such as Ump1 (natively unfolded protein), PAC3/4, Rpn14, Hsm3, Nas2, by X-ray and NMR analyses, displaying the mechanisms underlying proteasome assembly at the atomic levels. Based on these findings, we have established a model in which multiple dedicated chaperones govern proteasome assembly (Fig. 2). Recently, we unexpectedly found that the TRC proteins and Bag6, whose depletion resulted in poor CP biogenesis and the accumulation of immature CPs. This finding is an entirely novel aspect of the proteasome assembly pathway and suggests that the efficacy of proteasome assembly is linked to the cellular environment. To prove end, we further perform a genetic screening to identify new molecules/pathways related to the proteasome biogenesis.

Figure 2. The assembly pathway of the 26S proteasome

2. Roles of Specialized Proteasomes in Cell-mediated Immunity

The proteasome has acquired diversity of the catalytic β subunits, which have evolved during the acquisition of adaptive immunity. To date, we have discovered the vertebrate-specific alternative 20S CP, which we named the "immunoproteasome" and the “thymoproteasome”. The immunoproteasome has catalytic subunits β1i, β2i, and β5i replacing β1, β2, and β5 and enhances production of MHC-I ligands. The thymoproteasome contains thymus-specific subunit β5t in place of β5 or β5i and plays a pivotal role in positive selection of CD8+ T cells (Fig. 3). Whereas the immunoproteasome plays a specialized role as a professional antigen-processing enzyme in cell-mediated immunity, the thymoproteasome is involved in the development of CD8+T cells in thymus; i.e., it has a key role in the generation of MHC class I-restricted CD8+T cell repertoire during thymic selection called positive selection.

Figure 3. The immuno- and thymo-proteasomes

3. Spatio-temporal dynamics of the proteasome

Little is known about the molecular dynamics of the proteasomes in living cells. We measured the absolute concentration, dynamics, and complex formation of the proteasome by quantitative live-cell imaging and quantitative proteomics analyses. We found that the 26S proteasome is a highly mobile complex and enriches in the nucleus. The 26S proteasome appears to complete its assembly process in the cytoplasm and then translocates as a holoenzyme into the nucleus (Fig. 4). Furthermore, we also found that the proteasome dynamically changes its subcellular localization and its cofactors under various stresses. This might be a novel cellular response for adapting to stress by regulating protein homeostasis.

Figure 4. The proteasome dynamics in cells revealed by FCS

4. Developing Methods to Decipher the Ubiquitin Code

Ubiquitylation is involved in numerous important cellular processes such as proteasomal degradation, DNA repair, protein sorting, and signal transduction. The ubiquitin function is relied on eight structurally distinct ubiquitin chains of different lengths, but our knowledge of the relationship between their topology and functional outcomes is still insufficient.

To understand the ubiquitin code, it is essential to develop new methods for analyzing linkage types, chain lengths, and complexity of ubiquitylation. We have established a highly sensitive MS/MS quantification method for Ub signals, named Ub-absolute quantification/parallel reaction monitoring (Ub-AQUA/PRM) (Fig. 5).

Figure 5. Ubiquitin chain quantification by a high resolution mass spectrometry

Using Ub-AQUA/PRM, we currently perform a comprehensive analysis of major UBD proteins in yeast and human cultured cells to investigate how the ubiquitin-binding proteins recognize distinct Ub signals and sort ubiquitylated proteins to distinct cellular pathways. Recently, we have identified a major pathway targeting the K48-linked ubiquitylated substrates for proteasomal degradation in yeast (Fig. 6). We are further analyzing such decoder proteins for the proteasome in human cells.

Figure 6. The Cdc48/p97-RAD23 axis contributes K48-linkage selectivity for proteasomal degration

5. Roles of Novel Ubiquitin Code

Using quantitative mass spectrometry, we recently identified multiple chemical modifications of ubiquitin itself. Acetylation of ubiquitin inhibits the elongation of particular ubiquitin chains, whereas phosphorylation of S65 ubiquitin stimulates ubiquitin chain synthesis for mitophagy. More recently, we identified more complexed ubiquitin chains branched at K48 and K63. The K48/K63 branched chains act as a unique coding signal that specifically affects recognition by downstream reader proteins to enhance NF-κB signaling (Fig.7). These novel ubiquitin codes greatly expand ubiquitin functions. We further explore additional roles and regulatory mechanisms of these novel ubiquitin codes.

Figure 7. The K48/K63 branched ubiquitin chains regulate NF-κB signaling pathway