19S Regulator Research Articles

Proteasome activator (PA28) subunits, α, β and γ (Ki antigen) in NT2 neuronal precursor cells and HeLa S3 cells

The catalytic activity of the 20S proteasome can be modulated by endogenous proteins. A proteasome activator protein termed PA28 or 11S regulator, composed of two homologous subunits (alpha and beta) and a separate but related protein termed Ki antigen or PA28gamma have been characterized. To explore the functional relationship of these proteins, NT2 clone D1 human neuronal precursor cells, as well as HeLa S3 cells were labeled by immunofluorescence and immunoelectron microscopy with three different antisera directed against peptides derived from their sequences. It was found that both PA28alpha and PA28beta antisera label the cytoplasm and the nucleoli. In contrast, the PA28gamma antiserum labels the nucleus but not the nucleoli while in the cytoplasm it labels two different classes of structures identified as microtubular-like extensions and inclusion bodies that are most likely autophagosomes. The latter do not contain proteasome delta subunit antigen. The microtubular-like structures colocalize with beta-tubulin, are dispersed by nocodazole and are not affected by brefeldin A treatment. PA28alpha and PA28beta are co-localized in the cell whereas PA28gamma has a different distribution. PA28gamma complexed with the proteasome may serve a function other than or in addition to activation and may also have a proteasome-independent function.

Structure of the proteasome activator REGalpha (PA28alpha).

The specificity of the 20S proteasome, which degrades many intracellular proteins, is regulated by protein complexes that bind to one or both ends of the cylindrical proteasome structure. One of these regulatory complexes, the 11S regulator (known as REG or PA28), stimulates proteasome peptidase activity and enhances the production of antigenic peptides for presentation by class I molecules of the major histocompatibility complex (MHC). The three REG subunits that have been identified, REGalpha, REGbeta and REGgamma (also known as the Ki antigen), share extensive sequence similarity, apart from a highly variable internal segment of 17-34 residues which may confer subunit-specific properties. REGalpha and REGbeta preferentially form a heteromeric complex, although purified REGalpha forms a heptamer in solution and has biochemical properties similar to the heteromeric REGalpha/REGbeta complex. We have now determined the crystal structure of human recombinant REGalpha at 2.8 A resolution. The heptameric barrel-shaped assembly contains a central channel that has an opening of 20 A diameter at one end and another of 30 A diameter at the presumed proteasome-binding surface. The binding of REG probably causes conformational changes that open a pore in the proteasome alpha-subunits through which substrates and products can pass.

The proteasome 11S regulator subunit REG alpha (PA28 alpha) is a heptamer.

Activity of the 20S proteasome, which performs much of the cytosolic and nuclear proteolysis in eukaryotic cells, is controlled by regulatory complexes that bind to one or both ends of the cylindrical proteasome. One of these complexes, the 11S regulator (REG), is a complex of 28 kDa subunits that is thought to activate proteasomes toward the production of antigenic peptides. REG, purified from red blood cells, is a complex of REG alpha and REG beta subunits. We have crystallized recombinant REG alpha (rREG alpha) and collected diffraction data to 3.0 A resolution. The self-rotation function indicates that rREG alpha forms a heptameric ring in the crystal. Equilibrium sedimentation demonstrates that rREG alpha is a heptamer in solution also.

Identification and characterization of an activated 20S proteasome in Trypanosoma brucei

Recently, we have reported the isolation and purification of 20S proteasomes from both the procyclic and bloodstream forms of Trypanosoma brucei, but no 26S proteasome was identified under those experimental conditions (Hua et al., Mol. Biochem. Parasitol. (1996) 78, 33–46). Subsequent attempts to identify a 26S proteasome in T. brucei led to the discovery of another form of the 20S proteasome designated the activated 20S proteasome because it exhibited much higher peptidase activities than the original 20S proteasome on all the fluorogenic peptides tested, and it crossreacted with the rabbit antisera against the 20S proteasomes purified from T. brucei. The activated 20S proteasome can be isolated from both procyclic and bloodstream forms of T. brucei and has a slightly higher molecular weight than the 20S proteasome. It is stable in the absence of ATP but susceptible to elution through a DE52 column. Analysis of the activated 20S proteasome in SDS-PAGE showed the presence of all the subunit proteins from the 20S proteasome plus an extra protein with an estimated molecular mass of 26 kDa. This protein, designated PA26, is not a degradation product of the 20S proteasomal subunit proteins. It could be a homolog of the bovine PA28 and human 11S regulator protein which form complexes with the 20S proteasomes resulting in activation of their peptidase activities. This likelihood was confirmed in a reconstitution experiment in which PA26 separated from the proteasome by a DE52 column chromatography was re-introduced into the purified 20S proteasome, and resulted in the emergence of a new protein band with the same mobility and peptidase activities as the activated 20S proteasome in native polyacrylamide gel electrophoresis. The presence of an activated 20S proteasome rather than a homolog of the 26S proteasome in T. brucei suggests that PA26 may play an important role in regulating the proteasome-mediated protein degradations in trypanosomes. © 1997 Federation of European Biochemical Societies.

The 26S proteasome: a dynamic structure.

The proteasomal system consists of a proteolytic core, the 20S proteasome, which associates in ATP-dependent and independent reactions with endogenous regulators providing specific substrate binding sites, chaperone function and regulation of activity to the protease. The best known regulators of the 20S proteasome are the 11S and the 19S complexes. Three subunits of the 20S proteasome and the two subunits of the 11S regulator are induced by gamma-Interferon. However, there are no indications for an influence of gamma-interferon on the subunit composition of the 19S regulator and only a few data exist about the dynamics of this complex. The analysis of 19S regulator subunits from yeast mutants reveals that the ATPases appear to be stringently organized in the 26S complex, while peripheral non-ATPases, such as S5a, might serve as subunits which shuttle substrates to the enzyme. A novel non-ATPase has been cloned, sequenced and identified in a complex besides the 19S regulator, the function of which is presently unknown. The dynamic structure of the 26S proteasome is also characterized by transient associations with components such as the modulator and isopeptidases. Certain viral proteins can also be associated with components of the proteasomal system and alter enzymatic activities.

Structural and functional properties of proteasome activator PA28.

The proteasome activator PA28 or 11S regulator is a protein complex composed of two different but homologous polypeptides, termed PA28alpha and PA28beta. The purified activator protein (approximately 200 kDa) is a ring-shaped heteromultimer containing the two polypeptides, possibly with an (alpha3beta3 stoichiometry. The activator, which by itself shows no hydrolytic activity elicits activation of the proteasome's multiple peptidase activities by binding to the terminal rings of the proteinase. In vitro, active PA28 can be reconstituted from isolated alpha and beta subunits, yielding two different oligomers: with the single alpha subunit, PA28alpha homomultimers with moderate stimulatory activity toward 20S proteasomes are obtained whereas isolated beta-subunits are unable to form oligomers and are devoid of stimulatory activity. However, in the presence of both subunits, alphabeta heteromultimers form, concomitant with restoration of full stimulatory activity. The recent finding that PA28 modulates the proteasome-catalyzed production of antigenic peptides presented to the immune system on MHC class I molecules indicates a cellular function of the activator in antigen processing.

Phosphorylation of the Proteasome Activator PA28 Is Required for Proteasome Activation

PA28, also referred to as 11S regulator, is a potent activator of the peptidase activities of the proteasome (multicatalytic proteinase complex). Although the role(s) of PA28-20S proteasome complexes in cellular proteolytic processes remain to be defined, these particles have been implicated in antigen processing of major histocompatibility complex (MHC) class I molecules. Our results demonstrate that PA28 is phosphorylated as evidenced by32P incorporation into a single PA28 species in rabbit reticulocytes. In reticulocytes as well as human erythrocytes, PA28 is normally found in a phosphorylated state as detected by phosphoserine antibody. In human erythrocytes, this antibody recognizes three polypeptides which are also detected by antibody to PA28 on Western blot analysis. Dephosphorylation with alkaline phosphatase treatment completely abolishes the ability of PA28 to activate hydrolysis of Suc-Leu-Leu-Val-Tyr by proteasomes. After exposure to phosphatase, the three polypeptides are no longer recognized by phosphoserine antibody, although binding to PA28 antibody is unaffected. These results suggest that phosphorylation may function in transduction of cytokine and growth factor signals that, in turn, modulate antigen presentation and other processes which involve PA28-20S proteasome complexes.

The proteasome subunit, C2, contains an important site for binding of the PA28 (11S) activator.

The PA28, or 11S regulatory complex, stimulates the peptidase activities of the 20S proteasome. Monoclonal antibodies were screened for their ability to inhibit the activation by PA28 of proteasomes from rabbit reticulocytes. We identified one antibody that inhibited proteasome activation by PA28 and dissociated formed proteasome-PA28 complexes. A fourfold molar excess of antibody to proteasome markedly reduced the PA28 activation of three peptidase activities. Examination of proteasome-antibody mixtures by electron microscopy revealed that the antibody formed chains of proteasomes, and digital image analysis of individual proteasomes demonstrated that the antibody binds to the outer alpha rings. This antibody recognizes proteasome subunit C2, which we conclude contains an important contact site for the PA28 activator. However, the antibody did not block proteasome activation by PA700, or 19S regulator, which also associates with the alpha rings. Thus, these two regulators appear to bind to the proteasome at different sites.

Human lymphoblast and erythrocyte multicatalytic proteases: differential peptidase activities and responses to the 11S regulator

The multicatalytic protease (MCP) or 20S proteasome was purified from human red blood cells and two lymphoblastoid cell lines, 721.45 which constitutively expresses protease subunits LMP2 and LMP7, and 721.174 in which genes for these subunits are deleted. Each MCP was assayed using a series of fluorogenic peptides. The hydrophobic peptides gGGF-MCA, sRPFHLLVY-MCA and sLY-MCA were particularly good substrates for 721.45 MCP as compared to the enzyme from 721.174 and red blood cells. In addition, hydrolysis of gGGF-MCA and sLY-MCA was activated by human red blood cell and recombinant regulators to a greater extent using MCP from 721.45 lymphoblasts. Thus, LMP2/LMP7 and regulator appear to act synergistically in the enhanced degradation of gGGF-MCA and sLY-MCA by the multicatalytic protease.