Article

Biochemical Analysis of the 20 S Proteasome of Trypanosoma brucei

Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94143-0446, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 06/2003; 278(18):15800-8. DOI: 10.1074/jbc.M300195200
Source: PubMed

ABSTRACT

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.

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Available from: Chao-Lin Liu, Feb 10, 2014
    • "Alternatively, and less well studied, is the ubiquitination of obsolete matrix proteins, followed by their release from the organelle and targeting to proteasomes for degradation (reviewed in [78]). Trypanosomatids possess both proteasomes [79] and the machinery for all steps of the autophagy pathways (reviewed by [80,81] ). Homologues of genes coding for nearly half of the approximately 30 known yeast autophagy (Atg)-related proteins have been identified in the genome databases of each of the trypanosomatid parasites. "
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    • "Proteosome mediated proteolysis is essential for the transformation and proliferation of trypanosomes by evading the host immune system [31]. The biochemical differences of the T. brucei and human 20S proteosome are proposed to aid in designing specific inhibitors of this essential protease complex in trypanosome [32]. GM6, one of the targets for the second cluster are nonvariant antigens found in African trypanosomes. "
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    • "ABPs were demonstrated to react only with the active protease and not with its zymogen (Williams et al., 1989; Kidd et al., 2001) or protease complexed to an endogenous inhibitor (Liu et al., 1999). ABPs have been designed to target different protease classes, such as serine proteases (Williams et al., 1989), cysteine proteases (Thornberry et al., 1994; Greenbaum et al., 2000, 2002a; Grabarek and Darzynkiewicz, 2002), threonine proteases (Kessler et al., 2001; Wang et al., 2003) and metalloproteinases (Chan et al., 2004; Saghatelian et al., 2004). ABPs presenting an affinity tag, such as biotin or streptavidin, are used to isolate active proteases from complex mixture and allow protease identification and quantification when coupled to mass spectrometry techniques . "
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