Analysis of Drosophila 26 S proteasome using RNA interference

被引:88
作者
Wójcik, C [1 ]
DeMartino, GN [1 ]
机构
[1] Univ Texas, SW Med Ctr, Dept Physiol, Dallas, TX 75235 USA
关键词
D O I
10.1074/jbc.M109996200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
We have utilized double-stranded RNA interference (RNAi) to examine the effects of reduced expression of individual subunits of the 26 S proteasome in Drosophila S2 cells. RNAi significantly decreased mRNA and protein levels of targeted subunits of both the core 20 S proteasome and the PA700 regulatory complex. Cells deficient in any of several 26 S proteasome subunits (e.g. dbeta5, dRpt1, dRpt2, dRpt5, dRpn2, and dRpn12) displayed decreased proteasome activity (as judged by hydrolysis of succinyl-Leu-Leu-Val-Tyr-aminomethyl-coumarin), increased apoptosis, decreased cell proliferation without a specific block of the cell cycle, and accumulation of ubiquitinated cellular proteins. RNAi of many individual 26 S proteasome subunits promoted increased expression of many non-targeted subunits. This effect was not mimicked by chemical proteasome inhibitors such as lactacystin. Reduced expression of most targeted subunits disrupted the assembly of the 26 S proteasome. RNAi of six of eight targeted PA700 subunits disrupted that structure and caused accumulation of increased levels of uncapped 20 S proteasome. Notable exceptions included RNAi of dRpn10, a polyubiquitin binding subunit, and dUCH37, a ubiquitin isopeptidase. dRpn10-deficient cells showed a significant increase in suceinyl-Leu-Leu-Val-Tyr-aminomethyl-coumarin hydrolyzing activity of the 26 S proteasomes but accumulated polyubiquitinated proteins. dbeta5-Deficient cells had a phenotype similar to that of most PA700-deficient cells but also acclumulated low molecular mass complexes containing subunits of the 20 S proteasome, probably representing unassembled precursors of the 20 S proteasomes. Cells deficient in several of the 26 S proteasome subunits were more resistant to otherwise toxic concentrations of various proteasome inhibitors. Our data suggest that those cells adapted to grow in conditions of impaired ubiquitin and proteasome-dependent protein degradation.
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收藏
页码:6188 / 6197
页数:10
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共 68 条
[1]   The genome sequence of Drosophila melanogaster [J].
Adams, MD ;
Celniker, SE ;
Holt, RA ;
Evans, CA ;
Gocayne, JD ;
Amanatides, PG ;
Scherer, SE ;
Li, PW ;
Hoskins, RA ;
Galle, RF ;
George, RA ;
Lewis, SE ;
Richards, S ;
Ashburner, M ;
Henderson, SN ;
Sutton, GG ;
Wortman, JR ;
Yandell, MD ;
Zhang, Q ;
Chen, LX ;
Brandon, RC ;
Rogers, YHC ;
Blazej, RG ;
Champe, M ;
Pfeiffer, BD ;
Wan, KH ;
Doyle, C ;
Baxter, EG ;
Helt, G ;
Nelson, CR ;
Miklos, GLG ;
Abril, JF ;
Agbayani, A ;
An, HJ ;
Andrews-Pfannkoch, C ;
Baldwin, D ;
Ballew, RM ;
Basu, A ;
Baxendale, J ;
Bayraktaroglu, L ;
Beasley, EM ;
Beeson, KY ;
Benos, PV ;
Berman, BP ;
Bhandari, D ;
Bolshakov, S ;
Borkova, D ;
Botchan, MR ;
Bouck, J ;
Brokstein, P .
SCIENCE, 2000, 287 (5461) :2185-2195
[2]   Gapped BLAST and PSI-BLAST: a new generation of protein database search programs [J].
Altschul, SF ;
Madden, TL ;
Schaffer, AA ;
Zhang, JH ;
Zhang, Z ;
Miller, W ;
Lipman, DJ .
NUCLEIC ACIDS RESEARCH, 1997, 25 (17) :3389-3402
[3]   Identification of the yeast 20S proteasome catalytic centers and subunit interactions required for active-site formation [J].
Arendt, CS ;
Hochstrasser, M .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1997, 94 (14) :7156-7161
[4]   The proteasome:: Paradigm of a self-compartmentalizing protease [J].
Baumeister, W ;
Walz, J ;
Zühl, F ;
Seemuller, E .
CELL, 1998, 92 (03) :367-380
[5]   The proteasome [J].
Bochtler, M ;
Ditzel, L ;
Groll, M ;
Hartmann, C ;
Huber, R .
ANNUAL REVIEW OF BIOPHYSICS AND BIOMOLECULAR STRUCTURE, 1999, 28 :295-+
[6]  
BRADFORD MM, 1976, ANAL BIOCHEM, V72, P248, DOI 10.1016/0003-2697(76)90527-3
[7]   The base of the proteasome regulatory particle exhibits chaperone-like activity [J].
Braun, BC ;
Glickman, M ;
Kraft, R ;
Dahlmann, B ;
Kloetzel, PM ;
Finley, D ;
Schmidt, M .
NATURE CELL BIOLOGY, 1999, 1 (04) :221-226
[8]   dsRNA-mediated gene silencing in cultured Drosophila cells:: a tissue culture model for the analysis of RNA interference [J].
Caplen, NJ ;
Fleenor, J ;
Fire, A ;
Morgan, RA .
GENE, 2000, 252 (1-2) :95-105
[9]   SINGLE-STEP METHOD OF RNA ISOLATION BY ACID GUANIDINIUM THIOCYANATE PHENOL CHLOROFORM EXTRACTION [J].
CHOMCZYNSKI, P ;
SACCHI, N .
ANALYTICAL BIOCHEMISTRY, 1987, 162 (01) :156-159
[10]  
Ciechanover A, 2000, BIOESSAYS, V22, P442, DOI 10.1002/(SICI)1521-1878(200005)22:5<442::AID-BIES6>3.0.CO