The Proteasome Subunit, C2, Contains an Important Site for Binding of the PA28 (11S) Activator

Abstract

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 a 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 a rings. Thus, these two regulators appear to bind to the proteasome at different sites.

Mapping and Structural Dissection of Human 20 S Proteasome Using Proteomic Approaches

“…The active proteolytic sites are located in the ␤ subunits that constitute the two inner rings of the particle (5). The two outer ␣ subunit rings participate in proteasome assembly (6), control substrate entry (7), and associate with regulatory particles such as PA28 and PA700 (8,9). Upon induction by interferon-␥, the catalytic subunits ␤1, ␤2, and ␤5 can be replaced by three other subunits ␤1i, ␤2i, and ␤5i, respectively, to form the "immunoproteasome" that exhibits a modified activity profile as compared with that of the constitutively expressed standard proteasome (3,10,11).…”

HIV-1 Tat Inhibits the 20 S Proteasome and Its 11 S Regulator-mediated Activation

“…However, because the 20 S proteasome subunit C2 is involved in 11 S regulator binding (24), it could also participate in the interaction with Tat.…”

Normal human dermal fibroblasts: Proteomic analysis of cell layer and culture medium

“…As far as the proteasome, it is a cylinder-shaped protease arranged as four axially stacked heptameric rings comprised of several a-and b-subunits [42]. The complex biological activity of this structure is dynamically controlled by regulatory complexes that bind to specific subunits of the proteasome, as in the case of the PA28 activator that was shown to bind to the C2 proteasome subunits [43]. It could be speculated that binding of regulatory elements to the a1-subunits may cause a reduced availability of the complex in the medium.…”