Nobuyuki Tanahashi

Primary structures of two homologous subunits of PA28, a γ-interferon-inducible protein activator of the 20S proteasome

The primary structures of two proteins that comprise PA28, an activator of the 20S proteasome, have been determined by cDNA cloning and sequencing. These protein subunits, termed PA28α and PA28β, are about 50% identical to one another and are highly conserved between rat and human. PA28α and PA28β are homologous to a previously described protein, Ki antigen, whose function is unknown. PA28α, but neither PA28β nor Ki antigen, contains a ‘KEKE motif’, which has been postulated to promote the binding of proteins having this structural feature. PA28α and PA28β were coordinately regulated by γ‐interferon, which greatly induced mRNA levels of both proteins in cultured cells. The mRNA level of the Ki antigen also increased in response to γ‐interferon treatment, but the magnitude of the increase was less than that for the PA28s, and the effect was transient. These results demonstrate the existence of a new protein family, at least two of whose members are involved in proteasome activation. They also provide the basis for future structure/function studies of PA28 subunits and the determination of their relative physiological roles in the regulation of proteasome activity.

Immunoproteasome assembly and antigen presentation in mice lacking both PA28α and PA28β

Two members of the proteasome activator, PA28α and PA28β, form a heteropolymer that binds to both ends of the 20S proteasome. Evidence in vitro indicates that this interferon‐γ (IFN‐γ)‐inducible heteropolymer is involved in the processing of intracellular antigens, but its functions in vivo remain elusive. To investigate the role of PA28α/β in vivo, we generated mice deficient in both PA28α and PA28β genes. The ATP‐dependent proteolytic activities were decreased in PA28α−/−/β−/− cells, suggesting that ‘hybrid proteasomes’ are involved in protein degradation. Treatment of PA28α−/−/β−/− cells with IFN‐γ resulted in sufficient induction of the ‘immunoproteasome’. Moreover, splenocytes from PA28α−/−/β−/− mice displayed no apparent defects in processing of ovalbumin. These results are in marked contrast to the previous finding that immunoproteasome assembly and immune responses were impaired in PA28β−/− mice. PA28α−/−/β−/− mice also showed apparently normal immune responses against infection with influenza A virus. However, they almost completely lost the ability to process a melanoma antigen TRP2‐derived peptide. Hence, PA28α/β is not a prerequisite for antigen presentation in general, but plays an essential role for the processing of certain antigens.

Molecular properties of the proteasome activator PA28 family proteins and γ-interferon regulation

Background: Recent cDNA cloning of two homologous proteasome activators, PA28α and PA28β, indicated the presence of a structurally related third protein, Ki antigen, but a functional relationship between Ki antigen and the two PA28 proteins is unknown. Accumulating evidence has implicated an important role for PA28 in the major histocompatibility complex (MHC) class I‐restricted antigen processing pathway. Recently, an immunomodulatory cytokine γ‐interferon (γ‐IFN) was found to increase greatly the messages for PA28α and PA28β, but not Ki antigen, in human cells.

Proteasomes and Antigen Processing

Publisher Summary This chapter discusses the recent findings related to the roles of proteasomes in the major histocompatibility complex (MHC) class I-restricted antigen-processing pathway. One of the most important responses in this antigen-specific immune system is to distinguish correctly non-self-antigens from self-antigens for selective elimination because deviation from this recognition would lead to a variety of opportunistic infections or autoimmune diseases. The chapter focuses on the roles of proteasomes and their regulators in antigen processing with special reference to the influence of γ-interferon (γ-IFN) on both the structure and the functions of the proteasome. Proteasomes play a central role in various biological processes, one of which is the generation of the peptides presented by MHC class I molecules to the circulating T lymphocytes. Proteasomes have been implicated as the processing enzyme for the generation of the ligand peptides for MHC class I receptors. Future studies should address how proteasome genes have evolved. Acquisition of the γ-IFN-responsive proteasomal and PA28 family genes may be related to that of multiple MHC and TAP genes during evolution. Studies on molecular evolution may provide new insight into the alternative roles of proteasome genes in immunity.

Nin1p, a regulatory subunit of the 26S proteasome, is necessary for activation of Cdc28p kinase of Saccharomyces cerevisiae

The nin1‐1 mutant of Saccharomyces cerevisiae cannot perform the G1/S and G2/M transitions at restrictive temperatures. At such temperatures, nin1‐1 strains fail to activate histone H1 kinase after release from alpha factor‐imposed G1 block and after release from hydroxyurea‐imposed S block. The nin1‐1 mutation shows synthetic lethality with certain cdc28 mutant alleles such as cdc28‐IN. Two lines of evidence indicate that Nin1p is a component of the 26S proteasome complex: (i) Nin1p, as well as the known component of the 26S proteasome, shifted to the 26S proteasome peak in the glycerol density gradient after preincubation of crude extract with ATP‐Mg2+, and (ii) nin1‐1 cells accumulated polyubiquitinated proteins under restrictive conditions. These results suggest that activation of Cdc28p kinase requires proteolysis. We have cloned a human cDNA encoding a regulatory subunit of the 26S proteasome, p31, which was found to be a homolog of Nin1p.

Selective Proteasomal Dysfunction in the Hippocampal CA1 Region after Transient Forebrain Ischemia

Delayed neuronal death in the hippocampal CA1 region after transient forebrain ischemia may share its underlying mechanism with neurodegeneration and other modes of neuronal death. The precise mechanism, however, remains unknown. In the postischemic hippocampus, conjugated ubiquitin accumulates and free ubiquitin is depleted, suggesting impaired proteasome function. The authors measured regional proteasome activity after transient forebrain ischemia in male Mongolian gerbils. At 30 minutes after ischemia, proteasome activity was 40% of normal in the frontal cortex and hippocampus. After 2 hours of reperfusion, it had returned to normal levels in the frontal cortex, CA3 region, and dentate gyrus, but remained low for up to 48 hours in the CA1 region. Thus, the 26S proteasome was globally impaired in the forebrain during transient ischemia and failed to recover only in the CA1 region after reperfusion. The authors also measured 20S and 26S proteasome activities directly after decapitation ischemia (at 5 and 20 minutes) by fractionating the extracts with glycerol gradient centrifugation. Without adenosine triphosphate (ATP), only 20S proteasome activity was detected in extracts from both the hippocampus and frontal cortex. When the extracts were incubated with ATP in an ATP-regenerating system, 26S proteasome activity recovered almost fully in the frontal cortex but only partially in the hippocampus. Thus, after transient forebrain ischemia, ATP-dependent reassociation of the 20S catalytic and PA700 regulatory subunits to form the active 26S proteasome is severely and specifically impaired in the hippocampus. The irreversible loss of proteasome function underlies the delayed neuronal death induced by transient forebrain ischemia in the hippocampal CA1 region.

Double‐cleavage production of the CTL epitope by proteasomes and PA28: role of the flanking region

Proteasomes are known to produce major histocompatibility complex (MHC) class I ligands from endogenous antigens, and the γ‐interferon‐inducible proteasome activator PA28 has been thought to play an important role in the generation of immunodominant MHC ligands by proteasomes. Several attempts have been made to show that proteasomes have the ability to yield cytotoxic T lymphocyte (CTL) epitopes effectively from model polypeptides derived from viral and intracellular proteins in vitro, but their antigen processing mechanism is poorly understood.

ATP-Dependent Inactivation and Sequestration of Ornithine Decarboxylase by the 26S Proteasome Are Prerequisites for Degradation

ABSTRACT The 26S proteasome is a eukaryotic ATP-dependent protease, but the molecular basis of its energy requirement is largely unknown. Ornithine decarboxylase (ODC) is the only known enzyme to be degraded by the 26S proteasome without ubiquitinylation. We report here that the 26S proteasome is responsible for the irreversible inactivation coupled to sequestration of ODC, a process requiring ATP and antizyme (AZ) but not proteolytic activity. Neither the 20S proteasome (catalytic core) nor PA700 (the regulatory complex) by itself contributed to this ODC inactivation. Analysis with a C-terminal mutant ODC revealed that the 26S proteasome recognizes the C-terminal degradation signal of ODC exposed by attachment of AZ, and subsequent ATP-dependent sequestration of ODC in the 26S proteasome causes irreversible inactivation, possibly unfolding, of ODC and dissociation of AZ. These processes may be linked to the translocation of ODC into the 20S proteasomal inner cavity, centralized within the 26S proteasome, for degradation.

Nob1p, a new essential protein, associates with the 26S proteasome of growing Saccharomyces cerevisiae cells

Nob1p, which interacts with Nin1p/Rpn12, a subunit of the 19S regulatory particle (RP) of the yeast 26S proteasome, has been identified by two-hybrid screening. NOB1 was found to be an essential gene, encoding a protein of 459 amino acid residues. Nob1p was detected in growing cells but not in cells in the stationary phase. During the transition to the stationary phase, Nob1p was degraded, at least in part, by the 26S proteasome. Nob1p was found only in proteasomal fractions in a glycerol gradient centrifugation profile and immuno-coprecipitated with Rpt1, which is an ATPase component of the yeast proteasomes. These results suggest that association of Nob1p with the proteasomes is essential for the function of the proteasomes in growing cells.

The proteasome-dependent proteolytic system

The 20S proteasome is an intriguingly large complex that acts as a proteolytic catalytic machine. Accumulating evidence indicates the existence of multiple factors capable of regulating the proteasome function. They are classified into two different categories, one type of regulator is PA700 or PA28 that is reversibly associated with the 20S proteasome to form enzymatically active proteasomes and the other type including a 300-kDa modulator and PI31 indirectly influences proteasome activity perhaps by promoting or suppressing the assembly of the 20S proteasome with PA700 or PA28. Thus, there have been documented two types of proteasomes composed of a core catalytic proteasome and a pair of symmetrically disposed PA700 or PA28 regulatory particle. Moreover, the recently-identified proteasome containing both PA28 and PA700 appears to play a significant role in the ATP-dependent proteolytic pathway in cells, as can the 26S proteasome which is known as a eukaryotic ATP-dependent protease.