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


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.

Download full-text


Available from: Chao-Lin Liu, Feb 10, 2014
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Trypanosoma brucei develops chronic infection in mammalian hosts due to a sophisticated strategy of antigenic variation of variant surface glycoprotein (VSG) coat to escape antibody-mediated lysis. MicroRNAs are a class of non-coding RNAs with presumed post-transcriptional regulatory activity. Homology based informatic approach is used to identify the microRNA (miRNA) genes of T. brucei and their target mRNAs. Our observation reveals a set of microRNAs targeting mRNAs corresponding to VSGs. Further, a number of miRNA hairpins have been found in clusters of multiple identical copies. The target proteins, 20S proteosome, GM6 and GRESAG 4.2 corresponding to these clustered miRNAs play essential role in trypanosomiasis. These snippets can act as genetic switches modulating host-parasite interaction and provide useful clue toward treatment of trypanosomiasis.
    Biochemical and Biophysical Research Communications 09/2008; 372(3):459-63. DOI:10.1016/j.bbrc.2008.05.084 · 2.30 Impact Factor
  • Source
    • "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 . "
    [Show abstract] [Hide abstract]
    ABSTRACT: Proteases are specific modulators of signaling molecules and their underlying pathways in addition to their degradative roles. However, proteases do not act alone, but form cascades, circuits and networks that all dynamically interconnect to form the protease web, which defines the proteolytic potential of a cell or tissue in a defined condition. To describe the protease web and its net activity several novel high-throughput proteomic techniques, in the field termed degradomics, have been developed. Emerging systems biology methods to evaluate the expression, activity and substrate discovery of proteases are presented. Understanding the protease web and its perturbations in pathology will help to develop new therapeutics for the treatment of diseases, such as cancer, arthritis and chronic obstructive pulmonary diseases.
    Biological Chemistry 12/2007; 388(11):1159-62. DOI:10.1515/BC.2007.146 · 3.27 Impact Factor
  • Source
    • "High-resolution crystal structures have demonstrated that the PA28 homolog, PA26, induces a conformational change in the N-termini of 20S ␣-subunits that opens the entrance of the catalytic chamber (Whitby et al., 2000; Forster et al., 2005). PA28 (PA26) also alters the proteolytic properties of 20S and/or 19S-20S proteasomes by modifying the pattern, but not the overall size of cleaved products (Harris et al., 2001; Li et al., 2001; Cascio et al., 2002; Wang et al., 2003). PA28␣␤ subunits are particularly abundant in immune tissues and are coordinately regulated by IFN␥ together with ␤1i, ␤2i, and ␤5i subunits of the immunoproteasome , ER peptide transporters (TAP1, TAP2), and MHC class I molecules (Frü h and Yang, 1999; Rechsteiner et al., 2000; Kloetzel and Ossendorp, 2004). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Proteolytic activity of the 20S proteasome is regulated by activators that govern substrate movement into and out of the catalytic chamber. However, the physiological relationship between activators, and hence the relative role of different proteasome species, remains poorly understood. To address this problem, we characterized the total pool of cytosolic proteasomes in intact and functional form using a single-step method that bypasses the need for antibodies, proteasome modification, or column purification. Two-dimensional Blue Native(BN)/SDS-PAGE and tandem mass spectrometry simultaneously identified six native proteasome populations in untreated cytosol: 20S, singly and doubly PA28-capped, singly 19S-capped, hybrid, and doubly 19S-capped proteasomes. All proteasome species were highly dynamic as evidenced by recruitment and exchange of regulatory caps. In particular, proteasome inhibition with MG132 markedly stimulated PA28 binding to exposed 20S alpha-subunits and generated doubly PA28-capped and hybrid proteasomes. PA28 recruitment virtually eliminated free 20S particles and was blocked by ATP depletion. Moreover, inhibited proteasomes remained stably associated with distinct cohorts of partially degraded fragments derived from cytosolic and ER substrates. These data establish a versatile platform for analyzing substrate-specific proteasome function and indicate that PA28 and 19S activators cooperatively regulate global protein turnover while functioning at different stages of the degradation cycle.
    Molecular Biology of the Cell 01/2007; 17(12):4962-71. DOI:10.1091/mbc.E06-04-0311 · 4.47 Impact Factor
Show more