Mobilizing the proteolytic machine: cell biological roles of proteasome activators and inhibitors

Proteasomes perform the majority of proteolysis that occurs in the cytosol and nucleus of eukaryotic cells and, thereby, perform crucial roles in cellular regulation and homeostasis. Isolated proteasomes are inactive because substrates cannot access the proteolytic sites. PA28 and PA200 are activators that bind to proteasomes and stimulate the hydrolysis of peptides. Several protein inhibitors of the proteasome have also been identified, and the properties of these activators and inhibitors have been characterized biochemically. By contrast, their physiological roles – which have been reported to include production of antigenic peptides, proteasome assembly and DNA repair – are controversial. In this article, we briefly review the biochemical data and discuss the possible biological roles of PA28, PA200 and proteasome inhibitors.

Introduction

Proteasomes are large complexes that carry out crucial roles in many cellular pathways by degrading proteins in the cytosol and nucleus of eukaryotic cells to enforce quality control and to regulate many cellular processes [1]. The catalytic heart of these complexes, the 20S proteasome, has been highly conserved from yeast to humans, with simpler versions also found in some archaea and prokaryotes. The 20S proteasome is a barrel-shaped assembly of 28 protein subunits that possesses three distinct proteolytic active sites with different specificities (Figure 1). Together, the three active sites, present in the two central rings of β subunits, hydrolyze almost all peptide bonds, having trouble only with those bonds that follow glycine and proline. As revealed by structural studies performed by Huber and colleagues 2, 3, the potentially catastrophic elimination of inappropriate substrates is prevented by sequestration of active sites within the hollow structure of the 20S proteasome. Substrates access the central catalytic chamber through axial ports in the end rings of α subunits [4], although in the absence of activators these channels are closed and proteasome activity is repressed (Figure 2).

Proteasomes are activated by protein complexes that bind to the end rings of α subunits (Figure 3). The best-known activator is PA700 [proteasome activator MW 700, also known as 19S or regulatory complex (RC)], which has been highly conserved from yeast to humans and binds to the 20S proteasome to form the 26S proteasome. PA700 is the only proteasome activator that is known to stimulate degradation of protein substrates, which it generally recognizes by a polyubiquitin modification and which it processes by an ATP-dependent mechanism. Thus, PA700 is thought to mediate most of the biological effects of the proteasome by facilitating substrate degradation. This biological role is well established, and PA700 and 26S proteasomes have been reviewed extensively elsewhere [5].

In contrast to PA700, two other evolutionarily conserved protein complexes, PA28 (also known as 11S or REG) [6] and PA200 [7], that have been shown to bind specifically to and activate 20S proteasomes against model peptide substrates do not recognize ubiquitinated proteins or use ATP. PA28 family members, which are found in higher eukaryotes but are, apparently, absent from yeasts, exist as homo- or heteromeric complexes of seven ∼28-kDa subunits. PA200 is a single-chain protein of ∼200 kDa, with homologs present in yeast, worms and humans. The biological roles of PA28 and PA200 are understood less well than those of PA700, although their biochemical activities and evolutionary conservation implies that they have important roles in cellular physiology, and several important functions have been proposed. In this article, we focus on the possible biological functions of PA28 and PA200. We also discuss possible roles of protein inhibitors of the proteasome that, like PA28 and PA200, have been characterized biochemically but have controversial biological functions.