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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.

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Transferrin accumulation in stain 221 and tbcatb+/–T. brucei. Parasites were harvested, lysed, and protein concentration of the lysates was determined by Bradford assay. Two hundred micrograms of 221 and tbcatb+/– lysates was prepared using the ReadyPrep Two-dimensional Cleanup Kit (Bio-Rad), which removes ionic detergents, salts, nucleic acids, lipids, and other substances. Lysates were subjected to isoelectric focusing and finally resolved by SDS-PAGE. Transferrin was visualized by Western blot analysis using anti-transferrin antiserum and horseradish peroxidase-conjugated donkey anti-rabbit IgG. Antibody staining was observed only in the tbcatb+/– blot (B, arrows); no signal was observed in the 221 blot (A).
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fig7: Transferrin accumulation in stain 221 and tbcatb+/–T. brucei. Parasites were harvested, lysed, and protein concentration of the lysates was determined by Bradford assay. Two hundred micrograms of 221 and tbcatb+/– lysates was prepared using the ReadyPrep Two-dimensional Cleanup Kit (Bio-Rad), which removes ionic detergents, salts, nucleic acids, lipids, and other substances. Lysates were subjected to isoelectric focusing and finally resolved by SDS-PAGE. Transferrin was visualized by Western blot analysis using anti-transferrin antiserum and horseradish peroxidase-conjugated donkey anti-rabbit IgG. Antibody staining was observed only in the tbcatb+/– blot (B, arrows); no signal was observed in the 221 blot (A).

Mentions: Undegraded Transferrin Accumulates in tbcatb+/– Parasites—Parasites internalize transferrin via receptor-mediated endocytosis in coated vesicles (23). These vesicles form at the flagellar pocket, the site of all endocytosis and excocytosis in T. brucei, and rapidly transit the endocytic system to the lysosome (reviewed in Ref. 24). Iron release is thought to occur in acidic vesicles, where transferrin is degraded in endosomal/lysosomal compartments and the transferrin receptor (Tf-R) is recycled to the flagellar pocket (7, 25). We examined the accumulation of transferrin in tbcatb+/– and control parasites by two-dimensional PAGE and Western blot analysis. Antibody to transferrin identified two protein species of ∼64 and ∼50 kDa in lysate obtained from tbcatb+/– parasites (Fig. 7B), whereas no transferrin species were detected in lysates obtained from wild-type parasites (Fig. 7A).


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)

Transferrin accumulation in stain 221 and tbcatb+/–T. brucei. Parasites were harvested, lysed, and protein concentration of the lysates was determined by Bradford assay. Two hundred micrograms of 221 and tbcatb+/– lysates was prepared using the ReadyPrep Two-dimensional Cleanup Kit (Bio-Rad), which removes ionic detergents, salts, nucleic acids, lipids, and other substances. Lysates were subjected to isoelectric focusing and finally resolved by SDS-PAGE. Transferrin was visualized by Western blot analysis using anti-transferrin antiserum and horseradish peroxidase-conjugated donkey anti-rabbit IgG. Antibody staining was observed only in the tbcatb+/– blot (B, arrows); no signal was observed in the 221 blot (A).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig7: Transferrin accumulation in stain 221 and tbcatb+/–T. brucei. Parasites were harvested, lysed, and protein concentration of the lysates was determined by Bradford assay. Two hundred micrograms of 221 and tbcatb+/– lysates was prepared using the ReadyPrep Two-dimensional Cleanup Kit (Bio-Rad), which removes ionic detergents, salts, nucleic acids, lipids, and other substances. Lysates were subjected to isoelectric focusing and finally resolved by SDS-PAGE. Transferrin was visualized by Western blot analysis using anti-transferrin antiserum and horseradish peroxidase-conjugated donkey anti-rabbit IgG. Antibody staining was observed only in the tbcatb+/– blot (B, arrows); no signal was observed in the 221 blot (A).
Mentions: Undegraded Transferrin Accumulates in tbcatb+/– Parasites—Parasites internalize transferrin via receptor-mediated endocytosis in coated vesicles (23). These vesicles form at the flagellar pocket, the site of all endocytosis and excocytosis in T. brucei, and rapidly transit the endocytic system to the lysosome (reviewed in Ref. 24). Iron release is thought to occur in acidic vesicles, where transferrin is degraded in endosomal/lysosomal compartments and the transferrin receptor (Tf-R) is recycled to the flagellar pocket (7, 25). We examined the accumulation of transferrin in tbcatb+/– and control parasites by two-dimensional PAGE and Western blot analysis. Antibody to transferrin identified two protein species of ∼64 and ∼50 kDa in lysate obtained from tbcatb+/– parasites (Fig. 7B), whereas no transferrin species were detected in lysates obtained from wild-type parasites (Fig. 7A).

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