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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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Ultrathin cryosection immunogold labeling of tbcatB in strain 221 and pZJMTbCB parasites after RNA induction. A and B, cross-sections were labeled with anti-tbcatB antiserum and 10-nm gold antibodies. Immunolabeling of tbcatB is observed in lysosomes in 221 parasites (A, short arrows) and outside, but proximal to, lysosomes in parasites subjected to RNAi against tbcatB mRNA (B, long arrows). Bar, 200 nm.
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fig4: Ultrathin cryosection immunogold labeling of tbcatB in strain 221 and pZJMTbCB parasites after RNA induction. A and B, cross-sections were labeled with anti-tbcatB antiserum and 10-nm gold antibodies. Immunolabeling of tbcatB is observed in lysosomes in 221 parasites (A, short arrows) and outside, but proximal to, lysosomes in parasites subjected to RNAi against tbcatB mRNA (B, long arrows). Bar, 200 nm.

Mentions: TbcatB Localizes to the Lysosome—Rhodesain, the most abundant cysteine protease activity in T. brucei, is found in the lysosome (3). Immunoelectron microscopy revealed that tbcatB, although less abundant, also localizes to lysosomes (Fig. 4A). When tbcatB expression is disrupted by RNAi, tbcatB protein fails to enter lysosomes but instead accumulates in adjacent tubular/vesicular structures (Fig. 4B), suggestive of an arrest in a post-Golgi vesicle.


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)

Ultrathin cryosection immunogold labeling of tbcatB in strain 221 and pZJMTbCB parasites after RNA induction. A and B, cross-sections were labeled with anti-tbcatB antiserum and 10-nm gold antibodies. Immunolabeling of tbcatB is observed in lysosomes in 221 parasites (A, short arrows) and outside, but proximal to, lysosomes in parasites subjected to RNAi against tbcatB mRNA (B, long arrows). Bar, 200 nm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig4: Ultrathin cryosection immunogold labeling of tbcatB in strain 221 and pZJMTbCB parasites after RNA induction. A and B, cross-sections were labeled with anti-tbcatB antiserum and 10-nm gold antibodies. Immunolabeling of tbcatB is observed in lysosomes in 221 parasites (A, short arrows) and outside, but proximal to, lysosomes in parasites subjected to RNAi against tbcatB mRNA (B, long arrows). Bar, 200 nm.
Mentions: TbcatB Localizes to the Lysosome—Rhodesain, the most abundant cysteine protease activity in T. brucei, is found in the lysosome (3). Immunoelectron microscopy revealed that tbcatB, although less abundant, also localizes to lysosomes (Fig. 4A). When tbcatB expression is disrupted by RNAi, tbcatB protein fails to enter lysosomes but instead accumulates in adjacent tubular/vesicular structures (Fig. 4B), suggestive of an arrest in a post-Golgi vesicle.

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