Characterization of recombinant REGalpha, REGbeta, and REGgamma proteasome activators.
Full-length cDNAs for three human proteasome activator subunits, called REGalpha, REGbeta, and REGgamma, have been expressed in Escherichia coli, and the purified recombinant proteins have been characterized. Recombinant alpha or gamma subunits form heptameric species; recombinant beta subunits are found largely as monomers or small multimers. Each recombinant REG stimulates cleavage of fluorogenic peptides by human red cell proteasomes. The pattern of activated peptide hydrolysis is virtually identical for REGalpha and REGbeta. These two subunits, alone or in combination, stimulate cleavage after basic, acidic, and most hydrophobic residues in many peptides. Recombinant alpha and beta subunits bind each other with high affinity, and the REGalpha/beta heteromeric complex activates hydrolysis of LLVY-methylcoumaryl-7-amide (LLVY-MCA) and LLE-beta-nitroanilide (LLE-betaNA) more than REGalpha or REGbeta alone. Using filter binding and gel filtration assays, recombinant REGgamma subunits were shown to bind themselves but not alpha or beta subunits. REGgamma differs from REGalpha and REGbeta in that it markedly stimulates hydrolysis of peptides with basic residues in the P1 position but only modestly activates cleavage of LLVY-MCA or LLE-betaNA by the proteasome. REGgamma binds the proteasome with higher affinity than REGalpha or REGbeta yet with lower affinity than complexes containing both REGalpha and REGbeta. In summary, each of the three REG homologs is a proteasome activator with unique biochemical properties.
Realini
C
C
Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, Utah 84132, USA.
Jensen
C C
CC
Zhang
Z
Z
Johnston
S C
SC
Knowlton
J R
JR
Hill
C P
CP
Rechsteiner
M
M
D000595:Amino Acid Sequence; D001324:Autoantigens; D002118:Calcium / Q000378:metabolism; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004789:Enzyme Activation; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism*; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013696:Temperature
D001324:Autoantigens; C099558:Ki antigen; D009097:Multienzyme Complexes; D009687:Nuclear Proteins; D011506:Proteins; C108663:REG proteasome activator subunit beta; D011994:Recombinant Proteins; C088683:multicatalytic protease activator; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D002118:Calcium