Structural and functional characterizations of the proteasome-activating protein PA26 from Trypanosoma brucei.
The activated 20 S proteasome, which has been found only in mammalian cells, is composed of two heptamer rings of an activator protein on each end of the 20 S proteasome and is inducible by interferon-gamma. A 20 S proteasome has been recently identified in a protozoan pathogen Trypanosoma brucei, but there has been no experimental evidence yet for the presence of a 26 S proteasome. Instead, an activated form of 20 S proteasome was isolated from this organism, which has significantly enhanced peptidase activities. It consists of an additional activator protein with an estimated molecular mass of 26 kDa (PA26) (To, W. Y., and Wang, C. C. (1997) FEBS Lett. 404, 253-262). The profile and sequences of tryptic peptides from PA26 were determined by mass spectrometry; no matches were found in the data base. The peptide sequences were used in reverse transcriptase-polymerase chain reaction to isolate a full-length cDNA clone encoding PA26. The protein sequence thus derived from it indicates little sequence identity with those of mammalian activator proteins PA28 alpha, beta, or gamma. There is only a single copy of PA26 gene in T. brucei. Purified recombinant PA26 polymerizes spontaneously to form heptamer ring with an outer diameter of 8.5 nm. The ring binds and activates 20 S proteasomes from T. brucei as well as rat, whereas human PA28alpha can neither bind nor activate T. brucei 20 S proteasome. The former is thus apparently more ubiquitous than PA28 in its capability of binding to and activating 20 S proteasomes. Its presence in T. brucei may also suggest a more ancient origin of proteasome activator proteins and a much wider involvement in protein degradation among other eukaryotic organisms than was originally envisaged.
Yao
Y
Y
Department of Pharmaceutical Chemistry, Howard Hughes Medical Institute, University of California, San Francisco, California 94143, USA.
Huang
L
L
Krutchinsky
A
A
Wong
M L
ML
Standing
K G
KG
Burlingame
A L
AL
Wang
C C
CC
D000595:Amino Acid Sequence; D000818:Animals; D015139:Blotting, Southern; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D016054:DNA, Protozoan / Q000032:analysis / Q000235:genetics; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D018628:Gene Dosage; D006639:Histidine / Q000235:genetics; D015151:Immunoblotting; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D011487:Protein Conformation; D020033:Protein Isoforms / Q000235:genetics; D015800:Protozoan Proteins; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D014346:Trypanosoma brucei brucei / Q000737:chemistry / Q000201:enzymology / Q000235:genetics*
D018076:DNA, Complementary; D016054:DNA, Protozoan; D019476:Insect Proteins; D009097:Multienzyme Complexes; C402204:PA26 protein, Trypanosoma brucei; D020033:Protein Isoforms; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D006639:Histidine; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex