Cancer Immune Therapy Edited by G. Stuhler and P. Walden ...

Although it is still not entirely clear how immuno-subunits influence the antigenprocessing
capacity of the proteasome, one emerging concept is that incorporation
of immuno-subunits may result in subtle structural changes of the whole 20S complex
and thus, in turn, influence its processing properties [28]. For example,
although the concerted presence of all three immuno-subunits was essential for the
generation of an epitope from the HBV core antigen, an inactive b5 i subunit also
supported epitope generation. This result indicates that incorporation of b5 i influences
the structural architecture of the 20S proteasomes, and consequently affects
the cleavage-site preferences of the b1 i and b2 i active sites.
3.2.2
The Role of the Proteasome Activator PA28 in Antigen Processing
3.2 Immuno-Proteasomes
Biochemical screens performed by the groups of DeMartino and Rechsteiner have
identified several additional regulatory molecules [9, 10]. The best characterized is
the proteasome activator PA28/11S regulator, a heptameric 180±200 kDa complex
composed of a and b subunits [11, 34]. PA28 attaches in an ATP-independent way to
the outer a rings of the 20S proteasome and replaces the 19S regulator on at least
one side of the 20S core, resulting in the formation of so-called hybrid proteasomes
[35]. PA28 is expressed in all cells types of higher eukaryotes, suggesting that it displays
a basal cellular function [36]. On the other hand, PA28 gene-deficient mice are
viable, which may argue in favor of a more specialized function [37]. In support, the
expression of both of its subunits is controlled by IFN-g and professional antigenpresenting
cells generally express PA28 at high levels, which is in agreement with
the described function of this complex in MHC class I antigen processing [38].
The same biochemical screens that allowed the identification of PA28 also resulted
in the characterization of a molecule that inhibits proteasome function, PI31 [39].
PI31 is a protein of approximately 31 kDa that presumably functions as homodimer.
In vitro PI31 inhibits 20S mediated cleavage of short fluorogenic substrates and of
polypeptides and competes the binding of PA28 to 20S proteasomes. This suggests
that PI31 binds the a rings of the 20S proteasome and thereby obstructs the access
to the catalytic cavity [40, 41]. Nevertheless, transfection of PI31 into intact cells does
not interfere with cell cycle progression or protein degradation, indication that the in
vivo function of PI31 may differ from the proposed function as a general inhibitor of
proteasome activity (own unpublished observation).
Cell systems that express PA28 independently of IFN-g showed that PA28 enhances
the presentation of several viral antigens without increasing overall protein turnover
or turnover of viral protein substrates [31]. Furthermore, this enhanced peptide presentation
is independent of the presence of immuno-subunits in the 20S proteasome
[30, 31], excluding the possibility that PA28 might exert its function by increasing
immuno-proteasome formation as proposed recently [37].
Similar to the immuno-proteasomes, PA28 does not affect presentation of all MHC
class I epitopes equally (Table 3.1). Kinetic studies imply that binding of PA28 to the
20S core increases substrate affinity without changing the maximal activity of the enzyme
complex and that PA28 activates the proteasome by enhancing either the up-
33