cDNA Cloning, Expression, and Functional Characterization of PI31, a Proline-rich Inhibitor of the Proteasome

doi:10.1074/jbc.M001697200

cDNA Cloning, Expression, and Functional Characterization of PI31, a Proline-rich Inhibitor of the Proteasome*

From the ^‡Department of Physiology,^‡Department of Biochemistry,^‖Department of Pharmacology and ^§§The Howard Hughes Medical Institute, The University of Texas Southwestern Medical Center, Dallas, Texas 75235, the ^§Faculty of Nutrition, Kobe-Gakuin University, Nishi-ku, Kobe 651-21, Japan, the ^¶Department of Biomedical R & D, Sumitoma Electric Industries, 1 Tayacho, Sakae-ku, Yokohama 244, Japan, and the ^**The Tokyo Metropolitan Institute of Medical Science, and CREST, Japan Science and Technology Corporation (JST), 3-18-22 Honkomagome, Bunkyo-Ku, Tokyo 113-0021, Japan

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.

Footnotes

↵* This work was supported by grants from the Muscular Dystrophy Association (to G. N. D.), National Institutes of Health Grants DK 46181 and HL06296 (to G. N. D.) and 1 F32 GM18388-01 (to S. L. M.-M.), and by the Ministry of Education, Science and Culture of Japan (to K. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) .
↵¶¶ To whom correspondence should be addressed. Tel.: 214-648-3308; Fax: 214-648-4771; E-mail: gdemar@mednet.swmed.edu.
Published, JBC Papers in Press, April 11, 2000, DOI 10.1074/jbc.M001697200
↵2 J. Kalish and G. N. DeMartino, unpublished observations.
Abbreviations:

HPLC

high performance liquid chromatography

PCR

polymerase chain reaction

PAGE

polyacrylamide gel electrophoresis

Suc

succinyl

AMC

7-amido-methylcoumarin

IPTG

isopropyl-1-thio-β-d-galactopyranoside
- Received February 25, 2000.
- Revision received April 6, 2000.
The American Society for Biochemistry and Molecular Biology, Inc.