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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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Accumulation of transferrin receptor in pZJMTbCB clones after RNAi induction and in tbcatb+/– parasites. A and B, strain 221 and tbcatb+/– parasites were incubated for 36 h in media containing either dog serum (Dog) or fetal calf serum (Calf) at a density of 5 × 104 cells/ml. Media for tbcatb+/– parasites was supplemented with 2.5 μg/ml phleomycin. After the incubation period, equal numbers of parasites were lysed and the extract resolved by SDS-PAGE for Western blot analysis with transferrin receptor antiserum (A). Labeling was quantified for each lane by densitometry (B). Transferrin receptor antibody labeling (70 and 52 kDa bands) is greater in dog serum samples when compared with fetal calf serum samples, and greater in tbcatb+/– extracts when compared with 221 extracts. C and D, thin sections from samples of strain 221 parasites and pZJMTbCB clones that had been induced with tetracycline for 24 h and prepared by high pressure freezing and freeze substitution. The sections were labeled with anti-transferrin receptor antiserum and 10-nm gold antibodies. Immunolabeling of transferrin receptor was observed in the swollen flagellar pockets of the RNAi-induced parasites (C) but not in controls (D). Bar, 200 nm.
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fig8: Accumulation of transferrin receptor in pZJMTbCB clones after RNAi induction and in tbcatb+/– parasites. A and B, strain 221 and tbcatb+/– parasites were incubated for 36 h in media containing either dog serum (Dog) or fetal calf serum (Calf) at a density of 5 × 104 cells/ml. Media for tbcatb+/– parasites was supplemented with 2.5 μg/ml phleomycin. After the incubation period, equal numbers of parasites were lysed and the extract resolved by SDS-PAGE for Western blot analysis with transferrin receptor antiserum (A). Labeling was quantified for each lane by densitometry (B). Transferrin receptor antibody labeling (70 and 52 kDa bands) is greater in dog serum samples when compared with fetal calf serum samples, and greater in tbcatb+/– extracts when compared with 221 extracts. C and D, thin sections from samples of strain 221 parasites and pZJMTbCB clones that had been induced with tetracycline for 24 h and prepared by high pressure freezing and freeze substitution. The sections were labeled with anti-transferrin receptor antiserum and 10-nm gold antibodies. Immunolabeling of transferrin receptor was observed in the swollen flagellar pockets of the RNAi-induced parasites (C) but not in controls (D). Bar, 200 nm.

Mentions: To explore the hypothesis that tbcatB-deficient parasites undergo iron starvation due to lack of transferrin degradation, we examined the expression and localization of the Tf-R in tbcatB heterozygous knockouts and tbcatB RNAi knockdowns, respectively. Up-regulation of Tf-R expression was first confirmed using an iron-starvation assay in which parasites were cultured in media containing 10% dog serum versus the fetal calf serum that is typically used. The T. brucei transferrin receptor expressed by strain 221 parasites has a very low affinity for dog serum transferrin (28) and the parasites respond by up-regulating Tf-R expression (26). In tbcatb+/– parasites, the Tf-R is up-regulated by more than 40% above the wild type strain, suggesting that the tbcatB-deficient parasites experience a severe starvation phenotype (Fig. 8, A and B). Tetracycline-induced pZJMTbCB parasites accumulated abundant Tf-R in swollen flagellar pockets (Fig. 8C), whereas controls did not (Fig. 8D).


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)

Accumulation of transferrin receptor in pZJMTbCB clones after RNAi induction and in tbcatb+/– parasites. A and B, strain 221 and tbcatb+/– parasites were incubated for 36 h in media containing either dog serum (Dog) or fetal calf serum (Calf) at a density of 5 × 104 cells/ml. Media for tbcatb+/– parasites was supplemented with 2.5 μg/ml phleomycin. After the incubation period, equal numbers of parasites were lysed and the extract resolved by SDS-PAGE for Western blot analysis with transferrin receptor antiserum (A). Labeling was quantified for each lane by densitometry (B). Transferrin receptor antibody labeling (70 and 52 kDa bands) is greater in dog serum samples when compared with fetal calf serum samples, and greater in tbcatb+/– extracts when compared with 221 extracts. C and D, thin sections from samples of strain 221 parasites and pZJMTbCB clones that had been induced with tetracycline for 24 h and prepared by high pressure freezing and freeze substitution. The sections were labeled with anti-transferrin receptor antiserum and 10-nm gold antibodies. Immunolabeling of transferrin receptor was observed in the swollen flagellar pockets of the RNAi-induced parasites (C) but not in controls (D). Bar, 200 nm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2570886&req=5

fig8: Accumulation of transferrin receptor in pZJMTbCB clones after RNAi induction and in tbcatb+/– parasites. A and B, strain 221 and tbcatb+/– parasites were incubated for 36 h in media containing either dog serum (Dog) or fetal calf serum (Calf) at a density of 5 × 104 cells/ml. Media for tbcatb+/– parasites was supplemented with 2.5 μg/ml phleomycin. After the incubation period, equal numbers of parasites were lysed and the extract resolved by SDS-PAGE for Western blot analysis with transferrin receptor antiserum (A). Labeling was quantified for each lane by densitometry (B). Transferrin receptor antibody labeling (70 and 52 kDa bands) is greater in dog serum samples when compared with fetal calf serum samples, and greater in tbcatb+/– extracts when compared with 221 extracts. C and D, thin sections from samples of strain 221 parasites and pZJMTbCB clones that had been induced with tetracycline for 24 h and prepared by high pressure freezing and freeze substitution. The sections were labeled with anti-transferrin receptor antiserum and 10-nm gold antibodies. Immunolabeling of transferrin receptor was observed in the swollen flagellar pockets of the RNAi-induced parasites (C) but not in controls (D). Bar, 200 nm.
Mentions: To explore the hypothesis that tbcatB-deficient parasites undergo iron starvation due to lack of transferrin degradation, we examined the expression and localization of the Tf-R in tbcatB heterozygous knockouts and tbcatB RNAi knockdowns, respectively. Up-regulation of Tf-R expression was first confirmed using an iron-starvation assay in which parasites were cultured in media containing 10% dog serum versus the fetal calf serum that is typically used. The T. brucei transferrin receptor expressed by strain 221 parasites has a very low affinity for dog serum transferrin (28) and the parasites respond by up-regulating Tf-R expression (26). In tbcatb+/– parasites, the Tf-R is up-regulated by more than 40% above the wild type strain, suggesting that the tbcatB-deficient parasites experience a severe starvation phenotype (Fig. 8, A and B). Tetracycline-induced pZJMTbCB parasites accumulated abundant Tf-R in swollen flagellar pockets (Fig. 8C), whereas controls did not (Fig. 8D).

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