cDNA cloning, expression, and functional characterization of PI31, a proline-rich inhibitor of the proteasome

Sandra L. McCutchen-Maloney, Koichi Matsuda, Naoki Shimbara, Derk D. Binns, Keijii Tanaka, Clive A. Slaughter, George N. DeMartino

Research output: Contribution to journalArticle

88 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)18557-18565
Number of pages9
JournalJournal of Biological Chemistry
Volume275
Issue number24
DOIs
StatePublished - Jun 16 2000
Externally publishedYes

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Proteasome Inhibitors
Cloning
Proteasome Endopeptidase Complex
Proline
Organism Cloning
Complementary DNA
Proteins
Ubiquitin
Electrophoresis
Escherichia coli
Proteolysis
Polyacrylamide Gel Electrophoresis
Hydrolysis
Blood
Erythrocytes
Molecular Weight
Chemical activation
Molecular weight
Cells
Databases

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

McCutchen-Maloney, S. L., Matsuda, K., Shimbara, N., Binns, D. D., Tanaka, K., Slaughter, C. A., & DeMartino, G. N. (2000). cDNA cloning, expression, and functional characterization of PI31, a proline-rich inhibitor of the proteasome. Journal of Biological Chemistry, 275(24), 18557-18565. https://doi.org/10.1074/jbc.M001697200

cDNA cloning, expression, and functional characterization of PI31, a proline-rich inhibitor of the proteasome. / McCutchen-Maloney, Sandra L.; Matsuda, Koichi; Shimbara, Naoki; Binns, Derk D.; Tanaka, Keijii; Slaughter, Clive A.; DeMartino, George N.

In: Journal of Biological Chemistry, Vol. 275, No. 24, 16.06.2000, p. 18557-18565.

Research output: Contribution to journalArticle

McCutchen-Maloney, SL, Matsuda, K, Shimbara, N, Binns, DD, Tanaka, K, Slaughter, CA & DeMartino, GN 2000, 'cDNA cloning, expression, and functional characterization of PI31, a proline-rich inhibitor of the proteasome', Journal of Biological Chemistry, vol. 275, no. 24, pp. 18557-18565. https://doi.org/10.1074/jbc.M001697200
McCutchen-Maloney, Sandra L. ; Matsuda, Koichi ; Shimbara, Naoki ; Binns, Derk D. ; Tanaka, Keijii ; Slaughter, Clive A. ; DeMartino, George N. / cDNA cloning, expression, and functional characterization of PI31, a proline-rich inhibitor of the proteasome. In: Journal of Biological Chemistry. 2000 ; Vol. 275, No. 24. pp. 18557-18565.
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