PA28
PA28 is a ring-shaped multimeric complex (180 kDa)
that can bind to the two ends of the 20S proteasome and dramatically
stimulate its capacity to hydrolyze small peptides. In mammals, PA28
is a heteromeric complex of two homologous subunits, PA28α and PA28β,
both of which are induced by γ-interferon, which is a potent stimulator
of MHC-class I antigen presentation. Most peptides presented on surface
MHC-molecules are generated by proteasomes during the course of intracellular
protein degradation. Amongst its multiple actions, γ-interferon induces
alternative forms of the 20S proteasome (“immunoproteasomes”)
with distinct β-subunits, which alter its cleavage specificity so as
to enhance the production of antigenic peptides. PA28, therefore, is
assumed to promote antigen presentation, and transfection of PA28α alone
has been reported to enhance class I-presentation of some antigens. By
itself, PA28α in vitro forms a heptameric ring that can stimulate peptide
hydrolysis by 20S particles to the same extent as the heteromeric (3α3β)
complex. The crystal structure of PA26 (a PA28 homolog) in association
with 20S yeast particle has been solved by X-ray diffraction. In this
structure, the binding of PA26 opens the normally closed central channel
in the proteasome’s α-ring, through which substrates enter the
degradative particle and products exit. Immunoprecipitation studies indicated
that, in vivo, PA28 also exists in larger complexes that also contain
the 19S RP, which is required for ATP-ubiquitin-dependent degradation
of proteins. These complexes are however very labile and do not withstand
standard chromatographic methods or exposure to high ionic strength buffers.
In collaboration with Alfred Goldberg (Harvard Medical School), we have
avoided this problem of instability by in vitro reconstituting these
hybrid complexes from highly purified 26S proteasomes and recombinant
PA28α. By this approach, we have succeeded in defining several biochemical
and structural features of these hybrid complexes that appear likely
to be of immunological importance.
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We
used single particle electron microscopy to visualize the hybrid complex.
Electron micrographs of a negatively stained proteasome mixture revealed
the six different proteasome species: a 20S proteasome, proteasomes that
are singly-capped (19S-20S) and doubly-capped (19S-20S-19S) with 19S complex,
proteasomes that are singly-capped (PA28-20S) and doubly-capped (PA28-20S-PA28)
with PA28 rings as well as a hybrid proteasome with a 19S complex and
a PA28 ring bound to either end (19S-20S-PA28; inset). For the visualization
of the different complexes, an excess of PA28 was used due to the weak
interaction of PA28α rings with the 20S proteasome. Accordingly, the images
showed many top views of unbound PA28 rings. The hybrid complexes showed
enhanced hydrolysis of small peptides, but no significant increase in
rates of protein breakdown. Nevertheless, during breakdown of proteins,
the complex containing PA28αβ or PA28α generated a pattern of peptides
different from those generated by 26S proteasomes, without altering mean
product length. Presumably, this change in peptides produced accounts
for the capacity of PA28 to enhance antigen presentation.
 » Cascio et
al. (2002) EMBO J. 21: 2636-2645. |