title: cDNA cloning, expression, and functional characterization of PI31, a proline-rich inhibitor of the proteasome.
abstract: The primary structure of PI31, a protein inhibitor of the 20 S proteasome, was deduced by cDNA cloning and sequencing. The human protein has a calculated molecular weight of 29,792, a value in excellent accord with 31,000, as estimated by SDS-polyacrylamide gel electrophoresis for purified bovine PI31, and is not similar to any other protein in current data bases. PI31 is a proline-rich protein, particularly within its carboxyl-terminal half where 26% of the amino acids are proline. Wild-type PI31 and various truncation mutants were expressed in Escherichia coli and purified to homogeneity. Recombinant wild-type PI31 displayed structural and functional properties similar to those of PI31 purified from bovine red blood cells and inhibited the hydrolysis of protein and peptide substrates by the 20 S proteasome. Analysis of truncation mutants demonstrated that proteasome inhibition was conferred by the carboxyl-terminal proline-rich domain of PI31, which appears to have an extended secondary structure. Inhibition of the 20 S proteasome by PI31 involved formation a proteasome-PI31 complex. In addition to its direct inhibition of the 20 S proteasome, PI31 inhibited the activation of the proteasome by each of two proteasome regulatory proteins, PA700 and PA28. These results suggest that PI31 plays an important role in control of proteasome function, including that in ubiquitin-dependent pathways of protein degradation.
authors.affiliation: Department of Physiology, The Howard Hughes Medical Institute, The University of Texas Southwestern Medical Center, Dallas, Texas 75235, USA.
authors.forename: S L
authors.initials: SL
authors.lastname: McCutchen-Maloney
authors.forename: K
authors.initials: K
authors.lastname: Matsuda
authors.forename: N
authors.initials: N
authors.lastname: Shimbara
authors.forename: D D
authors.initials: DD
authors.lastname: Binns
authors.forename: K
authors.initials: K
authors.lastname: Tanaka
authors.forename: C A
authors.initials: CA
authors.lastname: Slaughter
authors.forename: G N
authors.initials: GN
authors.lastname: DeMartino
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000235:genetics*; D018076:DNA, Complementary / Q000737:chemistry; D004912:Erythrocytes / Q000201:enzymology; D006801:Humans; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism; D009124:Muscle Proteins; D046988:Proteasome Endopeptidase Complex; D011487:Protein Conformation; D011506:Proteins / Q000378:metabolism
chemical_list: D015853:Cysteine Proteinase Inhibitors; D018076:DNA, Complementary; D009097:Multienzyme Complexes; D009124:Muscle Proteins; C085834:PA700 proteasome activator; C085047:PSME1 protein, human; C121623:PSMF1 protein, human; D011506:Proteins; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex