Biochemical analysis of the 20 S proteasome of Trypanosoma brucei.
We describe here biochemical characterization of the 20 S proteasome from the parasitic protozoan Trypanosoma brucei. Similar to the mammalian proteasome, the T. brucei proteasome is made up of seven alpha- and seven beta-subunits. Of the seven beta-type subunits, five contain pro-sequences that are proteolytically removed during assembly, and three of them are predicted to be catalytic based on primary sequence. Affinity labeling studies revealed that, unlike the mammalian proteasome where three beta-subunits were labeled by the affinity reagents, only two beta-subunits of the T. brucei proteasome were labeled in the complex. These two subunits corresponded to beta2 and beta5 subunits responsible for the trypsin-like and chymotrypsin-like proteolytic activities, respectively. Screening of a library of 137,180 tetrapeptide fluorogenic substrates against the T. brucei 20 S proteasome confirmed the nominal beta1-subunit (caspase-like or PGPH) activity and identified an overall substrate preference for hydrophobic residues at the P1 to P4 positions in a substrate. This overall stringency is relaxed in the 11 S regulator (PA26)-20 S proteasome complex, which shows both appreciable activities for cleavage after acidic amino acids and a broadened activity for cleavage after basic amino acids. The 20 S proteasome from T. brucei also shows appreciable activity for cleavage after P1-Gln that is minimally observed in the human counterpart. These results demonstrate the importance of substrate sequence specificity of the T. brucei proteasome and highlight its biochemical divergence from the human enzyme.
Wang
Ching C
CC
Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94143-0446, USA. ccwang@ugl.ucsf.edu
Bozdech
Zbynek
Z
Liu
Chao-lin
CL
Shipway
Aaron
A
Backes
Bradley J
BJ
Harris
Jennifer L
JL
Bogyo
Matthew
M
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D020134:Catalytic Domain; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D021122:Protein Subunits; D015800:Protozoan Proteins / Q000737:chemistry*; D013379:Substrate Specificity; D014346:Trypanosoma brucei brucei / Q000201:enzymology*
D009097:Multienzyme Complexes; D021122:Protein Subunits; D015800:Protozoan Proteins; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex