REVIEWThe Proteasome: From Basic Mechanisms to Emerging Roles
Keiji Tanaka

Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan(Received for publication on March 29, 2012)(Revised for publication on April 6, 2012)(Accepted for publication on May 18, 2012)

The proteasome is a sophisticated, 2.5-MDa, multisubunit complex that contains a catalytic core particle (CP) and two terminal regulatory particles (RPs); the RPs associate with the termini of the central CP at opposite orientations. The CP consists of four axially stacked heptameric rings (two outer
α-rings and two inner β-rings), which are made up of seven structurally related, but not identical,
α and β subunits. The CP contains catalytic threonine residues (in β1, β2, and β5 with caspase-like,trypsin-like, and chymotrypsin-like activities, respectively) on the surface of the chamber formed by
two abutting β-rings. The RP recognizes polyubiquitylated substrate proteins and unfolds and translocates these proteins to the interior of the CP for degradation. The RP comprises 19 different subunits,
which are thought to form two subcomplexes called the lid and the base. One longstanding questionis how the complex structure of the proteasome is organized with high fidelity. Recently, we proposeda novel assembly mechanism that is assisted by multiple proteasome-dedicated chaperones. In addition, we discovered two immuno-type proteasomes, the immunoproteasome and the thymoproteasome,whose catalytic subunits are replaced by homologous counterparts. These two isoforms perform specialized functions that help discriminate self from non-self in cell-mediated immunity (i.e., they function as enzymes that process intracellular antigens for cytotoxic T lymphocyte responses and thymic
positive selection). Moreover, emerging evidence suggests that the proteasome is crucially involvedin the pathophysiology of various intractable diseases that are increasing in today’s aging society.
(doi: 10.2302/kjm.2012-0006-RE; Keio J Med 62 (1) : 1–12, March 2013)
Keywords: assembly, diversity, immunity, proteasome, ubiquitin

Introduction

which ATP hydrolysis (i.e., the consumption of energy)
greatly stimulated protein breakdown in vitro. This finding helped open a new area of research in extra-lysosomal proteolysis pathways in eukaryotes. Remarkably, thefollowing year, Hershko’s group identified a small heatstable protein named ATP-dependent proteolysis factor(AFP-1), which was subsequently found to be identicalto ubiquitin.3 By the early 1990s, the delineated ubiquitin hypothesis, which proposed that ubiquitin functions
as a degradation signal by covalent attachment to a target protein through an energy-dependent mechanism,
had developed significantly (reviewed by Hershko andCiechanover4,5). Studies on the ubiquitin system havesince clarified the molecular mechanistic actions6,7 andIn accordance with the second law of thermodynamics,
in living organisms, energy produced by ATP hydrolysis
was believed to be exclusively required for biosynthetic
reactions, but not for degradative reactions. However, in
1953, Simpson showed that metabolic energy is requiredfor proteolysis in living cells by measuring the releaseof amino acids from radio-labeled proteins in rat liverslices.1 This surprising result then remained largely unexplored because of its counterintuitive nature. However, in 1977, Goldberg’s laboratory focused attention on
Simpson’s hypothesis by identifying an ATP-dependent
proteolytic pathway in rabbit reticulocyte extracts,2 in

Dr. Tanaka is the recipient of the 2011 Keio Medical Science Prize.
Reprint requests to: Keiji Tanaka, PhD, Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Kamikitazawa
2-1-6, Setagaya, Tokyo 156-8506, Japan, E-mail: tanaka-kj@igakuken.or.jp
Copyright © 2013 by The Keio Journal of Medicine

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Tanaka K: The Proteasome

Fig. 1 Three-dimensional structure of the 26S proteasome.
Red, 20S proteasome (CP); blue, ATPase ring of the 19S RP; brown, lid and base (all except the ATPase ring) of 19S RP (courtesy of
Dr. Wolfgang Baumeister, Max-Planck-Institute of Biochemistry, Germany). See text for details.

addressed the importance of the pathophysiological rolesof this system.8,9 The importance of this discovery was
highlighted when the 2004 Nobel Prize in Chemistry wasawarded jointly to Aaron Ciechanover, Avram Hershko,
and Irwin Rose for the discovery of ubiquitin-mediatedprotein degradation.
I joined Goldberg’s laboratory in 1981, and during my2 years in Boston, I was able to demonstrate a new energy-dependent mechanism in proteolysis, in which ATP
serves two distinct roles in protein degradation in reticulocytes: one which requires ubiquitin and one that is independent of ubiquitin.10 A second, ubiquitin-independent
energy-requiring step was further clarified by the discovery of a eukaryotic ATP-dependent protease, called theproteasome, that selectively degrades ubiquitin-tagged
proteins (reviewed by Finley, 7 Coux et al., 11 and Baumeister et al.12). The proteasome is a large and complex
proteolytic machine that regulates a variety of physiologically and pathologically important cellular processes byselectively breaking down unnecessary proteins in eukaryotes13 (Fig. 1). We recently reviewed the pathophysiology of the proteasome in vivo, with particular emphasison mammals.14 In addition, many excellent comprehensive review articles have been recently published on the
dynamic aspects and mechanistic actions of the ubiquitin−proteasome system (for details, see Dohmen et al.15)
Basic Mechanisms
Core particle
The catalytic core particle (CP, also known as the 20S
proteasome) is a well-organized multisubunit complex

of about 750 kDa. Based on electron microscopy (EM)and quaternary structure analyses by X-ray crystallography, the overall configuration of the CP is conserved invarious eukaryotes from yeast to mammals.16,17 The CP
is barrel shaped as a result of the axial stacking of twoouter α-rings and two inner β-rings. These rings respectively comprise seven structurally similar α and β subunits, resulting in an α1 − 7β1 − 7β1 − 7α1 − 7 structure (Fig.
2, left panel). Of the seven β-type subunits that make up
the inner β-rings, three types (β1, β2, and β5) containcatalytically active threonine residues at their N terminiand show N-terminal nucleophile hydrolase activity thatcleaves peptide bonds. The β1, β2, and β5 subunits are associated with caspase-like, trypsin-like, and chymotrypsin-like activities, respectively, that cleave peptide bondsat post-acidic, -basic, and -hydrophobic amino-acid residues, respectively. Two pairs of these three active sites
reside in the chamber formed by the two abutting β-rings,
facing the interior of the cavity. Its multifaceted featuresand proteolytic specificities indicate that the proteasome
can rapidly hydrolyze substrate proteins through an exhaustive or limited digestion mechanism (see below).
The crystal structure of the CP reveals that the center of the α-ring is almost completely closed, preventing
substrate proteins from penetrating into the inner cavityof the β-ring where the proteolytically active sites arelocated. Indeed, the N-termini of the α subunits form aphysical barrier that prevents substrate proteins from accessing the active sites in the β-ring, indicating that the
CP is latent in cells. Thus, a substrate is able to access theactive sites only after passing through a narrow opening
in the closed gate at the center of the α-rings.

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Fig. 2 Structural features of the CP complex.
Left panel, the crystal structure of the 20S proteasome from a bovine liver. Upper left panel, top view; lower left panel, side view. The
active threonine residues in β1, β2, and β5 appear in blue, purple, and red, respectively. For details, see Unno et al.17 Middle panel,model of immuno-type proteasome isoforms. The standard proteasome (top), immunoproteasome (middle), and thymoproteasome(bottom) are depicted. Right panel, diagrams of the electrostatic surface potential of the S1 pockets for the chymotryptic active sites ofthe standard proteasome (top), immunoproteasome (middle), and thymoproteasome (bottom). The surface is colored according to theelectrostatic potential (blue, positive; red, negative). The catalytic threonine residue is surrounded by a dotted circle (top panel). The
models of the immunoproteasome and thymoproteasome were predicted from standard structures.

Regulatory particles
The regulatory particle (RP) was initially found biochemically to serve as a proteasome activator and to havea molecular mass of approximately 700,000 (it is often
called PA700). Two symmetrically disposed RPs (whose
exact sizes are 900 kDa each) are attached to the endsof the central CP to form the so-called 26S proteasome,although its correct sedimentation coefficient determinedby a physical technique is approximately 30S; the 26S
proteasome is likely the functional unit that selectively digests a wide array of polyubiquitylated substrate proteinsin eukaryotic cells.14 The RP recognizes polyubiquitylated proteins, deubiquitylates these proteins to recycle theubiquitin moieties, and then unfolds and translocates theproteins into the interior of the CP (Fig. 3). Moreover, theRP is also involved in opening the α-ring that forms theclosed gate of the CP. Thus, the RP essentially contributes to the above-mentioned four steps that occur prior to
the destruction of target proteins by the CP.

The RP consists of a set of distinct integral subunits
that can be subclassified into two groups: a regulatory
particle of six triple-ATPase (Rpt) subunits and a regulatory particle of thirteen non-ATPase (Rpn) subunits. The
RP can be separated into two subcomplexes: the lid andthe base. The base includes six different AAA+ ATPasesubunits (Rpt1−Rpt6) and three non-ATPase subunits(Rpn1, Rpn2, and Rpn13), while the lid consists of nine
non-ATPase subunits (Rpn3, 5−9, 11, 12 and 15).13 The
concept of the lid and base was founded on biochemical studies in which these subcomplexes could be easily
and consistently isolated from extracts of yeast that genetically lacked the Rpn10 subunit.18 Accordingly, it has
been long assumed that the Rpn10 subunit connects thebase to the lid and stabilizes this complex. Intriguingly,
a recent single-particle analysis using cryo-EM revealed
that Rpn10 and Rpn13 are located at a distant site on theapical part of the RP complex near the periphery19 (seeFig. 3). This orientation is rational because both subunits
serve as ubiquitin receptors; Rpn10 and Rpn13 function

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Tanaka K: The Proteasome

Fig. 3 Schematic presentation of the RP complex.
The Pru and UIM motifs interact with ubiquitin (Ub) chains. For details, see text.19

as integral ubiquitin receptors that efficiently trap polyubiquitylated substrates, which is required for the initialstage of degradation. Indeed, Rpn10 achieves this function via a C-terminal ubiquitin-interacting motif (UIM).
In contrast, the N-terminal domain of Rpn13 shows nohomology to known ubiquitin-binding motifs, but instead contains a novel pleckstrin-like receptor for ubiquitin (Pru) domain.20,21 The Pru domain in human Rpn13
shows high affinity for diubiquitin.22
To date, in contrast to yeast and mammalian 20S CPs,
all efforts to define the atomic structure of the 26S holoproteasome have failed, presumably because of the fragile nature and dynamics of the RP complex. Nevertheless,very recently, two groups independently delineated thecomplete subunit architecture of the RP using cryo-EM,
X-ray crystallography of the individual subunits, residuespecific chemical cross-linking, and a new heterologous
expression system for the lid, and provided insight into
the sequence of events prior to the degradation of polyubiquitylated substrates23,24 (Fig. 3). One function of thelid is to deubiquitylate captured substrates, a process inwhich the metalloisopeptidase Rpn11 helps recycle theubiquitin.25 Importantly, Rpn11 deubiquitylating enzyme(DUB) cleaves the polyubiquitin chain at a site proximal to the substrate, coupled with substrate degradation. Rpn11 was found to be located near the substrateentry position in the ATPase ring, elucidating how thepolyubiquitin chain is cleaved just before the substrate

is incorporated into the Rpt complex channel. In addition, in mammalian cells, two DUBs [Usp14 and Uch37(also known as UCHL5)] that are physically associatedwith the base complex sequentially cleave the ubiquitin
moiety at a distal site to the substrate and regulate protein degradation.26,27 Usp14 (equivalent to yeast Ubp6)
is directly associated with Rpn1, and Uch37 binds to thebase Rpn2 via Rpn13.13 Interestingly, the C-terminal domain of Rpn13 stimulates the deubiquitylating activityof Uch37 that is found only in higher eukaryotes, which
seems to collaborate with the N-terminal ubiquitin receptor site to facilitate proteolysis. In addition, it was recently found that the subunit Rpn6 with as-yet-unknown
function plays a pivotal role in stabilizing the otherwise
weak interaction between the CP and the RP.28 Considering these follow-up studies, it is noteworthy that the
aforementioned RP architecture is not compatible with
the notion of base and lid subcomplexes of the RP, andthus these concepts should be reconsidered.
The base complex is composed of six homologous
AAA-ATPase subunits (Rpt1−Rpt6) forming a crownlike shape, and three non-ATPase subunits (Rpn1, Rpn2,
and Rpn13). The ATPase ring serves the following threefunctions: (1) it promotes substrate unfolding after the
Rpt ring pulls substrates into the central pore, (2) it opens
the channel in the closed α-ring, and (3) it translocatesthe substrates into the CP, thus enhancing their destruction.29–31 Among the six Rpt subunits, only Rpt2, Rpt3,

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and Rpt5 contain a C-terminal hydrophobic-tyrosine-X(HbYX) motif, and the Rpt2 and Rpt5 subunits specifically facilitate the opening of the CP gate.14 However, it
is noteworthy that the energy generated by the Rpt ringis sufficient to promote unfolding of substrate structuredomains, but not for any other processes mediated by theRP, i.e., ATP is utilized only to unfold the target proteins.
Further studies are required to determine the crystal
structures of the RP and/or the 26S proteasome to understand at the atomic resolution how the proteasome mechanistically catalyzes the destruction of target proteins.
Assembly
A growing body of evidence has revealed the molecularmechanisms underlying proteasome assembly (reviewed
by Murata et al.,32 Tomko and Hochstrasser,33 and Saeki
and Tanaka34). One important goal is to discover a restricted set of proteasome-dedicated assembling chaperones that assist in CP formation. First, we identified
four novel chaperones, named proteasome assembly
chaperone (PAC) 1−4 in mammals, that assist in α-ringformation; all four were subsequently identified in yeast.
These chaperones form a pair of functional heterodimers,
PAC1–PAC2 and PAC3−PAC4.32,33 Although the PAC1−
PAC2 and PAC3−PAC4 complexes perform distinct rolesin the assembly pathway, they cooperate in the efficient
assembly of α-rings. Intriguingly, knocking down PAC1
and/or PAC2 expression in mammalian cells decreases
the number of α-rings and results in the accumulation ofabnormal products that cause unusual α-ring dimers, implying that the PAC1−PAC2 complex prevents accidental
formation of non-productive intermediates (i.e., off-pathway assembly). In contrast, the PAC3−PAC4 complex
was found to specifically bind the α5 subunit. Additional
co-crystal structure and mutational analyses revealed
that this complex helps to successively bind the neighboring subunits, contributing to the ordered assembly of
α-ring subunits.
After completion of α-ring assembly, the β subunits areincorporated onto the α-ring at the correct positions in adefined order, forming the 20S proteasome (reviewed by
Murata et al.32). Indeed, we clarified the mechanism bywhich β-rings form on α-rings by characterizing assembly intermediates that accumulated in mammalian cells
in which each β subunit was successively knocked down.
Starting from β2, incorporation of the other β subunits
occurs in an orderly manner that depends on the propeptides of β2 and β5 and the β2 C-terminus, which function
as so-called intramolecular chaperones. Unexpectedly,
Ump1 (also called POMP, proteasome maturation protein, or proteassemblin), which is a chaperone that functions at the final assembly step in yeast, is incorporated
as early as β2 and is required for the structural integrityof early assembly intermediates in mammals. Based on

5these findings, we proposed a model in which β-ring formation is assisted by both intramolecular and extrinsic
chaperones.32
Knocking down PAC1 does not cause complete loss of
the CP in mammalian cells, suggesting the presence ofboth chaperone-dependent and -independent assembly
pathways, although the contribution of the chaperonedependent pathway remains unclear. To elucidate thephysiological significance of PAC1-mediated proteasome
assembly in vivo, we generated PAC1-conditional knockout (KO) mice in which this chaperone is systemicallydeleted.35 In-depth studies revealed that the absence of
PAC1 was embryonically lethal and PAC1-null mice diedat approximately E6.0. The half-life of PAC1 is approximately 30−40 min, and the PAC1 protein was almost
completely lost within a few hours in PAC1-KO cells,
and thus PAC1 was also soon lost in the experimental setting in vivo. However, this phenotype is still milder thanthat of mice deficient in the proteasome base ATPases
Rpt3 and Rpt5; such mice died before they developed
into blastocysts (~E3.5). Accordingly, proteasome biogenesis is not entirely dependent on the PAC1 assembly
chaperone, and a PAC1-independent assembly pathway
also operates during embryogenesis. Nonetheless, efficient PAC1-based proteasome biogenesis is required fordevelopment beyond the blastocyst stage and thus is essential for late embryonic development in mice, which
require a large number of proteasomes. Brain-specific
PAC1-deficient mice exhibited a large decrease in the levels of both 20S and 26S proteasomes after birth, which
resulted in severely impaired development of the central
nervous system, particularly in the cerebellum, which develops significantly after birth. Taken together, the study
clearly demonstrated that PAC1 is essential for mammalian development, especially for explosive cell proliferation. In contrast, PAC1 is also responsible for producing
the majority of CPs in the liver, where hepatocytes arenormally quiescent.35 Free latent CPs almost completely
disappeared several weeks after PAC1 ablation, but, surprisingly, the levels of the 26S proteasome remained unchanged for at least 6−12 months under PAC1-deficientconditions, indicating that normal 26S proteasome levels
were potentially maintained by efficient production ina PAC1-independent assembly pathway. However, micelacking PAC1 in the liver exhibited premature senescence, suggesting that chaperone-assisted proteasomebiogenesis is essential to maintain homeostasis in the liver. These findings indicate the physiologically importantroles of the PAC1-dependent assembly pathway in rapidly
proliferative cells and of PAC1, and thereby the CP itself,
in the maintenance of cell integrity in quiescent cells.
Base assembly is also a highly organized process and is
assisted by four base-dedicated chaperone proteins: Nas2/
p27, Nas6/gankyrin/p28, Rpn14/PAAF1, and Hsm3/S5b(yeast/human: miscellaneous nomenclature).33–35 Recent
studies have proposed renaming Rpn14, Nas6, Nas2, and6

Hsm3 as RP assembling chaperones (RACs), or RAC1,
2, 3, and 4, respectively.33 Intriguingly, precursor intermediates consisting of two pairs of Rpt subunits with appropriate specific chaperone(s) (notably the RAC3−Rpt5−
Rpt4, RAC2−Rpt3−Rpt6−RAC1, and RAC4−Rpt1−Rpt2complexes) are formed prior to ATPase ring assembly.
Among these, RAC1 transiently binds to the C-terminal
domain of the Rpt6 subunit (Rpt6-C) during maturation of the ATPase ring. The crystal structure of yeast
RAC1 revealed that the top face of RAC1 has a highly
acidic surface, whereas the putative interacting surfaceof Rpt6-C is basic, suggesting that there are complementary charge interactions between these two proteins,36 asobserved in the interaction between RAC2 and Rpt3-C.37
On the other hand, RAC4 transiently binds to the C-terminal domain of the Rpt1 subunit and forms a tetramericcomplex, RAC4−Rpt1−Rpt2−Rpn1, during maturation of
the ATPase ring of the RP. Moreover, the crystal structure of RAC4 revealed that it has a C-shaped structure
consisting of 11 HEAT repeats.38 The structure of the
RAC4−Rpt1−C complex revealed that the interacting
surface between RAC4 and Rpt1 is a hydrophobic coreand complementary charged surface. Mutations in the
RAC4−Rpt1 surface resulted in an assembly defect in the
26S proteasome. Furthermore, a structural model of the
RAC4−Rpt ring complex and an in vitro binding assay
suggest that RAC4 can bind to Rpt2 in addition to Rpt1.
Moreover, we recently used a quantitative proteomics approach to determine the near stoichiometric binding of
Ubp6 to the base precursor(s) of the RP as well as to themature 26S proteasome and determined that Ubp6 facilitates proteasomal assembly by clearing polyubiquitylated
substrates from assembly precursors through its deubiquitylating activity.39
Regulation
Currently, it is thought that genes encoding proteasome
components, including many proteasome-interacting proteins (PIPs), comprise approximately 0.5% of all genes in
the human genome, indicating that these proteins havetremendous biological importance. Despite their potential importance, the mechanisms controlling proteasome
regulation were not fully addressed until recently. One
longstanding question was how proteasome expression is
maintained or regulated based on changes in the intracellular and/or environmental conditions. In yeast, it was established that the transcription factor Rpn4 is a transcriptional activator that binds to the promoters of proteasome
genes and is required for proteasome gene expression.40,41
Interestingly, Rpn4 is extremely short-lived, and it is rapidly degraded by the 26S proteasome with a half-life (t1/2)
of approximately 2 min, indicating that there is a feedback circuit that homeostatically controls the proteasome.
A similar transcriptional feedback loop for proteasome

Tanaka K: The Proteasome

regulation is also conserved in mammals. Surprisingly,
erythroid-derived 2-related factor 1 (Nrf1; also named
TCF11), which is a basic leucine zipper transcription factor that mediates cellular responses to many xenobiotic
and pro-oxidant stresses, was shown to be necessary to
induce proteasome gene transcription.42,43 Thus, Nrf1
is assumed to be a functional yeast Rpn4 counterpart inmammalian cells, although there are no obvious structural similarities. Under physiological conditions, Nrf1
is targeted to the endoplasmic reticulum (ER), implyingthat it is translocated into the nucleus in response to an
activating signal. Nrf1 is targeted for ER-associated protein degradation (ERAD) through a process that requires
the E3 ubiquitin ligase HRD1. Proteasome inhibitors trigger the accumulation of oxidant-damaged proteins andpromote the nuclear translocation of Nrf1 from the ER,
permitting activation of proteasome gene expression bybinding to antioxidant response elements in their promoter regions, indicating that a transcriptional control loop
regulates human proteasome-dependent protein degradation to counteract proteotoxic stress caused by proteasome inhibition.
In addition, Nrf1 is currently thought to be controlled
at multiple post-translational steps, including ubiquitylation, proteolytic processing, and proteasome-mediateddegradation, as well as by phosphorylation. Intriguingly, β-TrCP, an adaptor for the Skp1-Cul1-F-box protein(SCF) ubiquitin ligase, promotes Nrf1 degradation bycatalyzing its polyubiquitylation in the nucleus.44 On theother hand, it has also been reported that Nrf1 is a shortlived protein that is targeted by the F-box protein Fbw7,
which is another substrate-specifying component of SCFtype ubiquitin ligase that mediates degradation via theubiquitin−proteasome pathway.45 Together, these findings indicate that Nrf1 plays a central role in upregulating the proteasome system through an ERAD-dependent
feedback loop. Currently, however, the mechanism that
regulates Nrf1 expression in vivo is poorly understood.
To determine the function of Nrf1 in the brain, mice witha late-stage Nrf1 deletion in neuronal cells were generated. Loss of Nrf1 leads to impaired proteasome function
and neurodegeneration. These findings reveal that Nrf1 isa key transcriptional regulator required for the expressionof proteasome genes in neurons and suggest that perturbations in Nrf1 function may contribute to the pathogenesis of neurodegenerative diseases.46 In addition, anotherNrf1-related transcription factor, Nrf2, which is responsible for the basal and inducible expression of numerous
detoxifying and antioxidant genes, is also reported to partially control the upregulation of proteasome genes,47,48
but the details are not well understood.

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Physiology and Pathology inCell-mediated Immunity
To date, expanding knowledge on the ubiquitin−proteasome pathway (UPS) clearly indicates that the UPS
is involved in multiple physiologically important cellular processes, such as developmental programs and environmental responses. Consequently, it is evident that
failures in the UPS can cause pathologically intractablediseases. In addition, emerging evidence, based mainly
on murine genetic analysis of regulatory subunits in addition to catalytic subunits, points to the physiology andpathology of the proteasome in vivo (reviewed by Tanaka
et al.14). In this section, I discuss the immunological rolesof the proteasome, focusing on its diversity, but I will notinclude other pathophysiological roles that have been already comprehensively overviewed.14
Immunoproteasome
The proteasome processively degrades target proteinsto generate oligopeptides ranging in length from 3 to 15
amino acids. In vertebrates, the oligopeptides producedby the CP can be used as antigenic peptides [also often
called major histocompatibility complex (MHC) class I
ligands or cytotoxic T lymphocyte (CTL) epitopes] for
MHC class I molecules (reviewed by Tanaka and Kasahara49 and Rock et al.50). MHC class I displays intracellular/endogenous antigens to CD8+ killer cells or CTLs,
whose T-cell receptors recognize the MHC−peptide
complex with high fidelity, and subsequently activate T
cells to initiate cell-mediated immunity. In 1994, Rockand Goldberg’s group reported that proteasome inhibitors
block the generation of peptides presented on MHC class
I molecules, indicating for the first time that the proteasome is an antigen-processing enzyme in the MHC class
I pathway and solving a long-term mystery in immunology.51
Subsequently, in 1994, we found that interferon-γ(IFN-γ; also called type II IFN or immuno-cytokine) induces the expression of β1i, β2i, and β5i catalytic subunits, which are structurally related to β1, β2, and β5 ofthe standard proteasome, respectively, with more than
50% identity (Fig. 2, middle panel). The standard proteasome is constitutively expressed in all cells and tissues,
but expression decreases on administration of IFN-γ. The
expression of these three unique subunits leads to theformation of a unique CP isoform in which the catalytic
subunits β1, β2, and β5 are replaced with β1i, β2i, and β5i,
respectively, as a result of the response to IFN-γ. In 1994,
we proposed that the IFN-γ-inducible proteasome isotype
should be called the “immunoproteasome” to emphasize
its specialized roles as a dedicated antigen-processing
enzyme in cell-mediated immunity49,52,53 (Fig. 2, middle panel). The immunoproteasome can be generated by

7

preferential assembly mechanisms, but it does not replacemature proteasomes. In the preferential assembly mechanism, propeptides from immunoproteasomal subunits
contribute to their preferentially selective incorporation
into the β-ring as intramolecular chaperones.54–56 The
chymotrypsin-like activity of the proteasome is thought
to be important for the production of antigenic peptides
with high affinities for MHC class I clefts, which serveas peptide-binding pockets. Interestingly, the immunoproteasome has higher chymotrypsin-like activity than
the standard proteasome.54,57 The vertebrate proteasome
system has considerable diversity among the catalytic
subunits, which have evolved during the acquisition ofadaptive immunity.49 Mice lacking genes that encode immunoproteasome subunits (i.e., β1i, β2i, or β5i) display
defective antigen processing and consequently have compromised immune responses (review details in Sijts andKloetzel58). These initial studies emphasized the immunological importance of this immunoproteasome in mice.
Further studies using β1i, β2i, or β5i double and/or triple
KO mice more convincingly addressed the pivotal rolesof these immunosubunits in the production of MHC class
I peptides and showed that the immunoproteasome plays
a much more important role in antigen presentation than
was previously thought.58,59
A recent surprising discovery established that the immunoproteasome suppresses cell death resulting from
IFN-induced oxidative stress by blocking the accumulation of harmful protein aggregates, indicating that theimmunoproteasome has unexpected fundamental roles
other than a specific role in the production of MHC class
I ligands.60 Efficient clearance of these aggregates by the
immunoproteasome helps preserve cell viability underIFN-induced oxidative stress conditions. Other evidence
has also indicated that the immunoproteasome plays important roles beyond the antigen-processing pathway.
One landmark study determined that a specific inhibitorof β5i, namely PR-957, blocked β5i-mediated presentationof MHC class I-restricted antigens.61,62 Interestingly, thesame inhibitor resulted in profound reductions in cellular
infiltration, cytokine production, and autoantibody levels, thereby attenuating the progression of experimental
arthritis. These studies highlight a unique role for β5i incontrolling pathogenic immune responses and provide atherapeutic rationale for targeting β5i in autoimmune disorders.
More recently, several groups independently reported
that a homozygous missense mutation in the β5i gene
(PSMB8) causes an autoinflammatory disorder named
Nakajo−Nishimura syndrome (NNS) that is inherited in
an autosomal recessive fashion and is clinically characterized by autoinflammation and lipodystrophy.63,64 It is
likely that the G201V and G197V mutations in β5i are
in close proximity to the catalytic threonine residue anddisrupt the β-sheet structure. These β5i mutants are notefficiently incorporated during immunoproteasome bio-

8genesis, resulting in assembly defects and reduced activities of all proteasomal peptidases within cells expressing
immunoproteasomes. The reduced proteasomal activities
cause an accumulation of ubiquitylated and oxidized proteins due to defective proteasome assembly, leading tostress-dependent activation of the p38 MAP kinase pathway. Consequently, high IL-6 expression is detected in
the skin and B cells of NNS patients. These changes may
account for the exaggerated inflammatory response andperiodic fever observed in these patients. On the other
hand, downregulation of β5i inhibited the differentiationof murine and human adipocytes in vitro. These findings
identify β5i as an essential component and regulator not
only of inflammation but also of adipocyte differentiation, indicating that immunoproteasomes have pleiotropic
functions in maintaining the homeostasis of a variety ofcell types. Until recently, NNS was thought to be a local
disease endemic to Japan, but affected patients with similar, but not identical, mutations in β5i have been foundin pedigrees from Portugal and Mexico with panniculitis-induced lipodystrophy (JMP) syndrome and chronic
atypical neutrophilic dermatosis with lipodystrophy andelevated temperature (CANDLE) syndrome,65,66 indicating that β5i mutations are a worldwide phenomenon.
Thymoproteasome
The thymus is the organ where T lymphocytes are
generated. On entering the thymus, T-lymphoid progenitor cells are translocated in the outer region of thecortex where they begin expressing CD4, CD8, and αβT-cell antigen receptor (TCR) to initiate their development into T lymphocytes; the thymocytes are referred toas CD4+CD8+ (double positive, DP) T cells. DP T cells
that interact via TCR with the peptide/MHC complex in
the cortical thymic epithelial cells (cTEC) are selected forsurvival or death depending upon the TCR-peptide/MHC
interaction. DP T cells that receive weak TCR signals are
induced to survive and continue development into mature T lymphocytes; i.e., CD4+CD8− or CD4−CD8+ single
positive (SP) T cells. These processes of TCR-mediateddevelopmental promotion of DP T cells to SP T cells arereferred to as “positive selection.” On the other hand,
DP T cells that receive strong TCR signals are destined
to die, a process termed “negative selection” (see below
for details). Whereas the molecular mechanisms underling negative selection have been studied extensively, themechanisms of positive selection remain poorly understood.
Another landmark study was the discovery of the thymoproteasome as a unique enzyme involved in the formation of the MHC class I-restricted CD8+ T-cell repertoire
during thymic positive selection. In 2007, we serendipitously identified a previously unrecognized catalytic subunit with an overall sequence that is highly similar to β5

Tanaka K: The Proteasomeand β5i, suggesting that the subunit belongs to the same
β5 family of proteins.67 Interestingly, the subunit was
expressed exclusively in the mouse thymic cortex, particularly in cTECs. Because of its unique expression, thesubunit was designated β5t. This new subunit was also
determined to have thymus-specific expression in humans.68 The molecular mechanism underlying thymusspecific expression of β5t is unknown, but it is plausiblethat the transcriptional factor Foxn1 is responsible forthis unique expression because β5t expression is impairedin the thymus of Foxn1-deficient nude mouse embryos.69
Intriguingly, β5t and the immunosubunits β1i and β2i, but
not their standard counterparts β1 and β2, are incorporated into the vertebrate-specific alternative 20S proteasome, which we named the “thymoproteasome” becauseof its thymus-specific expression (Fig. 2, middle panel).67
As mentioned above, although the chymotrypsin-like activity of proteasomes plays an essential role in MHC class
I-restricted antigen processing, the incorporation of β5t,instead of β5 or β5i, into the proteasome markedly reduces this activity, strongly indicating that the thymoproteasome may alter the quality of the peptide repertoire. A
superimposed structural model revealed that this reduced
activity is caused by changes in residues near the catalytic
site. The residues near the active sites of both β5 and β5i
are hydrophobic, which allows them to access hydrophobic peptides easily, but those of β5t are hydrophilic andantagonize the chymotryptic activity (Fig. 2, right panel).
To examine the in vivo role of β5t and the thymoproteasome, we generated β5t-KO mice, and these displayed
major defects in thymic CD8+ T-cell development. There
were no obvious abnormalities in thymic architecture,including size and histology, indicating that β5t is not essential for the differentiation and proliferation of cTECs;
specifically, there were no obvious changes in the numbers of cTECs, CD4+CD8+ (co-receptors of TCR) DP
thymocytes, CD4+ T-cell populations, or of any otherexamined cell type, including Aire-expressing medullary
thymic epithelial cells. In addition, β5t-deficient mice did
not have altered negative selection or impaired T-cell responses. In contrast, the number of immunoproteasomes
increased at the expense of thymoproteasomes (i.e., β5tchanged to β5i in the thymus cortex), which could be responsible for the production of MHC ligands that could
not fulfill positive selection. This finding is consistent
with the unchanged expression levels of MHC class I
in β5t-deficient cTECs, which indicates that most MHC
class I molecules on the cTEC surface are associated withpeptides irrespective of the β5t deficiency because empty
MHC molecules are extremely unstable and rapidly degraded via the endocytotic pathway. These results emphasize the role of β5t in the development of the MHC
class I-restricted CD8+ T-cell repertoire during thymic
selection, indicating that thymoproteasome-generated
peptides are responsible for the positive selection of developing thymocytes.67

Keio J Med 2013; 62 (1): 1–12

During positive selection, DP cells that interact with
self-peptide–MHC complexes on cTECs with sufficiently
modest avidity [i.e., affinity (MHC−TCR interaction) ×
density (surface MHC levels)] are rescued from intrathymic death and induced to differentiate into CD4+ or CD8+
SP thymocytes. In contrast, DP cells that have high affinity or avidity interactions with self-peptide–MHC complexes are eliminated through apoptosis, a process referred to as negative selection.70 In addition, thymocytesthat lack functional T-cell receptors also undergo apoptosis, a process called null selection. These selection mechanisms are commonly referred to as thymic education of
the immune system. To date, however, the mechanisms
by which cTECs provide specialized signals for positive
selection remain poorly understood. Considering that theproteasome is essential for the production of MHC class
I ligands and that β5t specifically attenuates proteasomal
chymotryptic activity without altering the caspase- andtrypsin-like activities, as mentioned above, it is possiblethat the thymoproteasome in cTECs predominantly produces MHC class I ligands with moderate avidity rather
than high-affinity ligands, which would support the positive selection hypothesis that was proposed many years
ago.70 Although the positive selection hypothesis has been
considered unreliable for more than a decade because of
the lack of solid evidence, the discovery of the thymoproteasome will enhance our understanding of positive
selection mechanisms in the thymus.
Until the discovery of the thymoproteasome, little was
known about how self-peptides displayed in the thymus
contribute to the development of immunocompetent andself-protective T cells. In contrast, the role of thymic selfpeptides in eliminating self-reactive T cells and thereby
preventing autoimmunity is well established. To date,
we know that cTECs display thymoproteasome-specificpeptide−MHC class I complexes that are essential for the
positive selection of the major and diverse repertoire of
MHC class I-restricted T cells. CD8+ T cells generated in
the absence of thymoproteasomes displayed a markedly
altered T-cell receptor repertoire that was defective inboth allogeneic and antiviral responses.71 Thus, the thymoproteasome is required to generate optimal cellularityof CD8+ T cells and plays an essential role in the development of the MHC class I-restricted CD8+ T-cell repertoire
during thymic positive selection.72
We previously proposed a chromosomal duplication
hypothesis to explain the emergence of IFN-γ-regulated
subunits. We compared the sequences of the β-type subunit genes of the standard and immunoproteasome toshow that each subunit pair is derived from a common
ancestor gene.49 The basic assumption in this model isthat all the IFN-γ-regulated immunosubunits emerged
simultaneously as a result of a duplication of the MHC
region during genome-wide duplication. As is also thecase for immunoproteasome genes, the β5t gene (officially named PSMB11) appears to result from a modification9and duplication of existing nonimmune genes, such as β5,
and this duplication may have been instrumental in theemergence of the adaptive immune system. Intriguingly,
β5t is encoded by an intronless gene, in contrast to the β5
and β5i genes, which both contain three exons. Curiously,
the β5t gene is adjacent to the PSMB5 gene encoding theβ5 subunit of the standard proteasome in mammals andlower vertebrates, such as reptiles, amphibians, and teleost fish. This fact suggests that the PSMB11 gene arosefrom a tandem duplication from the evolutionarily moreancient PSMB5 gene and then lost introns evolutionarily
by an unknown mechanism.73
OutlookIn this review, we briefly discussed the structure−function relationship of the proteasome with particular emphasis on the pathophysiological roles of the core proteasome
machinery of the CP and of the RP, the main regulatorof the CP. However, it is important to note that there areother regulators, such as PA28 and PA200, indicating that
there are additional multifaceted mechanisms that regulate the proteasome system (for details, see Tanaka et al,14
Tanaka and Kasahara,49 and Stadtmueller and Hill 74).
Over the past 25 years, the divergent functions of the proteasome have been comprehensively elucidated in the life
sciences. However, the incidences of intractable diseases
such as cancers, infectious diseases, and neurodegenerative diseases have recently been increasing, especially inthe elderly population. In this context, it is plausible thatdysfunctions in the proteasome machinery are a common
abnormality that partially contributes to these diseases.
Thus, the proteasome has been extensively studied in bothbasic and clinical fields using multipronged approaches.
Acknowledgements
This work was supported by grants from the Ministryof Education, Culture, Sports, Science and Technology(MEXT) of Japan and the Target Protein Project of MEXT.
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