Limits...
A parasite cysteine protease is key to host protein degradation and iron acquisition.

O'Brien TC, Mackey ZB, Fetter RD, Choe Y, O'Donoghue AJ, Zhou M, Craik CS, Caffrey CR, McKerrow JH - J. Biol. Chem. (2008)

Bottom Line: TbcatB deficiency results in a decreased growth rate and dysmorphism of the flagellar pocket and the subjacent endocytic compartment.Western blot and microscopic analysis indicate that deficiency in tbcatB results in accumulation of both host and parasite proteins, including the lysosomal marker p67.Because even a modest deficiency in tbcatB is lethal for the parasite, tbcatB is a logical target for the development of new anti-trypanosomal chemotherapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Sandler Center for Basic Research in Parasitic Diseases, University of California, San Francisco, California 94158-2550, USA.

ABSTRACT
Cysteine proteases of the Clan CA (papain) family are the predominant protease group in primitive invertebrates. Cysteine protease inhibitors arrest infection by the protozoan parasite, Trypanosoma brucei. RNA interference studies implicated a cathepsin B-like protease, tbcatB, as a key inhibitor target. Utilizing parasites in which one of the two alleles of tbcatb has been deleted, the key role of this protease in degradation of endocytosed host proteins is delineated. TbcatB deficiency results in a decreased growth rate and dysmorphism of the flagellar pocket and the subjacent endocytic compartment. Western blot and microscopic analysis indicate that deficiency in tbcatB results in accumulation of both host and parasite proteins, including the lysosomal marker p67. A critical function for parasitism is the degradation of host transferrin, which is necessary for iron acquisition. Substrate specificity analysis of recombinant tbcatB revealed the optimal peptide cleavage sequences for the enzyme and these were confirmed experimentally using FRET-based substrates. Degradation of transferrin was validated by SDS-PAGE and the specific cleavage sites identified by N-terminal sequencing. Because even a modest deficiency in tbcatB is lethal for the parasite, tbcatB is a logical target for the development of new anti-trypanosomal chemotherapy.

Show MeSH

Related in: MedlinePlus

Transmission electron microscopy of abnormal flagellar pocket and associated endocytic compartment morphology in pZJMTbCB and tbcatb+/– clones. Clones of pZJMTbCB were maintained as controls (A and B) or induced with tetracycline for 24 h (D). Swollen flagellar pockets and lysosomes were observed in tbcatb+/– clones (C) and pZJMTbCB parasites induced with tetracycline (D). Bar, 0.5 μm. Arrows indicate flagellum (A–C) and multivesicular body at the edge of the lysosome (D). FP indicates the boundaries of the flagellar pocket in uninduced parasites.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2570886&req=5

fig3: Transmission electron microscopy of abnormal flagellar pocket and associated endocytic compartment morphology in pZJMTbCB and tbcatb+/– clones. Clones of pZJMTbCB were maintained as controls (A and B) or induced with tetracycline for 24 h (D). Swollen flagellar pockets and lysosomes were observed in tbcatb+/– clones (C) and pZJMTbCB parasites induced with tetracycline (D). Bar, 0.5 μm. Arrows indicate flagellum (A–C) and multivesicular body at the edge of the lysosome (D). FP indicates the boundaries of the flagellar pocket in uninduced parasites.

Mentions: Electron microscopy was used to further delineate the phenotype of induced RNAi clones and tbcatb+/– parasites. In contrast to controls (Fig. 3, A and B), both the heterozygous knock-out (Fig. 3C) and RNAi knockdown (Fig. 3D) parasites had swollen flagellar pockets and swollen lysosomal compartments. The flagellar pockets of both tbcatB-deficient parasites were ∼5 times larger in diameter than the flagellar pocket of the controls.


A parasite cysteine protease is key to host protein degradation and iron acquisition.

O'Brien TC, Mackey ZB, Fetter RD, Choe Y, O'Donoghue AJ, Zhou M, Craik CS, Caffrey CR, McKerrow JH - J. Biol. Chem. (2008)

Transmission electron microscopy of abnormal flagellar pocket and associated endocytic compartment morphology in pZJMTbCB and tbcatb+/– clones. Clones of pZJMTbCB were maintained as controls (A and B) or induced with tetracycline for 24 h (D). Swollen flagellar pockets and lysosomes were observed in tbcatb+/– clones (C) and pZJMTbCB parasites induced with tetracycline (D). Bar, 0.5 μm. Arrows indicate flagellum (A–C) and multivesicular body at the edge of the lysosome (D). FP indicates the boundaries of the flagellar pocket in uninduced parasites.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2570886&req=5

fig3: Transmission electron microscopy of abnormal flagellar pocket and associated endocytic compartment morphology in pZJMTbCB and tbcatb+/– clones. Clones of pZJMTbCB were maintained as controls (A and B) or induced with tetracycline for 24 h (D). Swollen flagellar pockets and lysosomes were observed in tbcatb+/– clones (C) and pZJMTbCB parasites induced with tetracycline (D). Bar, 0.5 μm. Arrows indicate flagellum (A–C) and multivesicular body at the edge of the lysosome (D). FP indicates the boundaries of the flagellar pocket in uninduced parasites.
Mentions: Electron microscopy was used to further delineate the phenotype of induced RNAi clones and tbcatb+/– parasites. In contrast to controls (Fig. 3, A and B), both the heterozygous knock-out (Fig. 3C) and RNAi knockdown (Fig. 3D) parasites had swollen flagellar pockets and swollen lysosomal compartments. The flagellar pockets of both tbcatB-deficient parasites were ∼5 times larger in diameter than the flagellar pocket of the controls.

Bottom Line: TbcatB deficiency results in a decreased growth rate and dysmorphism of the flagellar pocket and the subjacent endocytic compartment.Western blot and microscopic analysis indicate that deficiency in tbcatB results in accumulation of both host and parasite proteins, including the lysosomal marker p67.Because even a modest deficiency in tbcatB is lethal for the parasite, tbcatB is a logical target for the development of new anti-trypanosomal chemotherapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Sandler Center for Basic Research in Parasitic Diseases, University of California, San Francisco, California 94158-2550, USA.

ABSTRACT
Cysteine proteases of the Clan CA (papain) family are the predominant protease group in primitive invertebrates. Cysteine protease inhibitors arrest infection by the protozoan parasite, Trypanosoma brucei. RNA interference studies implicated a cathepsin B-like protease, tbcatB, as a key inhibitor target. Utilizing parasites in which one of the two alleles of tbcatb has been deleted, the key role of this protease in degradation of endocytosed host proteins is delineated. TbcatB deficiency results in a decreased growth rate and dysmorphism of the flagellar pocket and the subjacent endocytic compartment. Western blot and microscopic analysis indicate that deficiency in tbcatB results in accumulation of both host and parasite proteins, including the lysosomal marker p67. A critical function for parasitism is the degradation of host transferrin, which is necessary for iron acquisition. Substrate specificity analysis of recombinant tbcatB revealed the optimal peptide cleavage sequences for the enzyme and these were confirmed experimentally using FRET-based substrates. Degradation of transferrin was validated by SDS-PAGE and the specific cleavage sites identified by N-terminal sequencing. Because even a modest deficiency in tbcatB is lethal for the parasite, tbcatB is a logical target for the development of new anti-trypanosomal chemotherapy.

Show MeSH
Related in: MedlinePlus