Studies on a multicatalytic, protease complex from Trypanosoma brucei brucei.
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Date
1999
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Abstract
Subcellular fractionation (together with immunocytochemical localisation studies) showed that
the parasite Trypanosoma brucei brucei possesses a multicatalytic protease complex (MCPTb).
This complex is predominantly cytosolic but some activity is also present in the nuclear
fraction. MCP-Tb was isolated from T. b. brucei and compared to the properties of other
proteasomes reported in the literature and to the 20S MCP isolated from bovine red blood cells
(MCP-rbc). The isolation procedure employed four-steps: anion exchange chromatography on
Q-Sepharose, adsorption chromatography on HA-Ultrogel, molecular exclusion
chromatography on Sephacryl S-300 and glycerol density gradient sedimentation.
The molecular mass of intact MCP-Tb was shown to be smaller than that of MCP-rbc.
Separation of the different proteasome subunits by 2D-PAGE showed that MCP-Tb has 12
different polypeptide components compared to the 28 different polypeptide components of
MCP-rbc. The N-terminal sequence of an MCP-Tb subunit showed that this subunit did not
have any obvious sequence homology with the subunits of proteasomes from other cells.
Furthermore, anti-MCP-Tb antibodies (which exhibited the in vitro inhibitory activity of
MCP-Tb) did not cross-react with MCP-rbc showing that MCP-Tb and MCP-rbc are antigenically distinct.
The basic enzymatic properties of MCP-Tb were fairly typical of other 20S proteasomes.
MCP-Tb had multiple peptidase activities (identified as chymotrypsin-like, trypsin-like and
peptidyl glutamylpeptide hydrolase activities) that are characteristic of proteasomes.
Furthermore, the characteristics of inhibition by a variety of inhibitors were similar to those of
other proteasomes, including MCP-rbc. The activities of 20S proteasomes from most cell
types are activated by endogenous high molecular mass complexes such as the bovine 19S
complex called PA700. These complexes form end-on associations with the 20S proteasome.
However, no endogenous MCP-activator was found in T. b. brucei. Nevertheless, MCP-Tb
was activated in an ATP-dependent manner by bovine PA700. Inhibition of the intrinsic
phosphatase activity of PA700 inhibited the protease enhancing effect of PA700. Electron microscopic examination of negatively stained MCP-Tb and MCP-rbc showed
particles that were morphologically indistinguishable. However, the MCP-Tb also exhibited
unique end-on associations between individual units forming long (up to 200 nm) ribbon-like
chains. Since access to the active sites of proteasomes occurs through the pores at the end of the complexes, this end-on association, when coupled to our observation of an apparent lack of an endogenous activator, suggests that T. b. brucei may have evolved an alternate mechanism
for controlling their proteasome activity.
Description
Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1999.
Keywords
Trypanosoma brucei brucei--Molecular aspects., Trypanosomiasis--Chemoprevention., Proteolytic enzymes., Theses--Biochemistry.