Kinetic evidences for facilitation of peptide channelling by the proteasome activator PA28.
The activation kinetics of constitutive and IFNgamma-stimulated 20S proteasomes obtained with homomeric (recPA28alpha, recPA28beta) and heteromeric (recPA28alphabeta) forms of recombinant 11S regulator PA28 was analysed by means of kinetic modelling. The activation curves obtained with increasing concentrations of the individual PA28 subunits (RecP28alpha/RecP28beta/RecP28alpha + RecP28beta) exhibit biphasic characteristics which can be attributed to a low-level activation by PA28 monomers and full proteasome activation by assembled activator complexes. The dissociation constants do not reveal significant differences between the constitutive and the immunoproteasome. Intriguingly, the affinity of the proteasome towards the recPA28alphabeta complex is about two orders of magnitude higher than towards the homomeric PA28alpha and PA28beta complexes. Striking similarities can been revealed in the way how PA28 mediates the kinetics of latent proteasomes with respect to three different fluorogenic peptides probing the chymotrypsin-like, trypsin-like and peptidylglutamyl-peptide hydrolyzing like activity: (a) positive cooperativity disappears as indicated by a lack of sigmoid initial parts of the kinetic curves, (b) substrate affinity is increased, whereby (c), the maximal activity remains virtually constant. As these kinetic features are independent of the peptide substrates, we conclude that PA28 exerts its activating influence on the proteasome by enhancing the uptake (and release) of shorter peptides.
Stohwasser
R
R
Deutsches Institut für Ernährungsforschung, Abt. Präventiv-Medizinische Lebensmittelforschung, Bergholz-Rehbrücke, Bundesrepublik Deutschland. stohwass@www.dife.de
Salzmann
U
U
Giesebrecht
J
J
Kloetzel
P M
PM
Holzhütter
H G
HG
D000818:Animals; D001324:Autoantigens; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004789:Enzyme Activation; D005982:Glutathione Transferase / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D051379:Mice; D009097:Multienzyme Complexes / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D021122:Protein Subunits; D011506:Proteins / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D001324:Autoantigens; D015853:Cysteine Proteinase Inhibitors; C099558:Ki antigen; D009097:Multienzyme Complexes; D021122:Protein Subunits; D011506:Proteins; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex