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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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Fluorescence localization of the lysosomal membrane glycoprotein p67 in pZJMTbCB and tbcatb+/–clones. A, B, E, and F, clones of pZJMTbCB were maintained as controls (A and B) or induced with tetracycline for 24 h (E and F). Cells were stained with rabbit anti-p67 antiserum and Texas Red goat anti-rabbit secondary antibody (red channel). 4,6-Diamidino-2-phenylindole was used to visualize DNA in the nucleus and kinetoplast by fluorescence microscopy (blue channel). B and C, tbcatb+/– clones (B and C) were prepared in an identical manner. Increased p67 labeling was observed in large, distorted organelles in tbcatb+/– clones (D) and parasites subjected to RNAi against tbcatB mRNA (F).
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fig2: Fluorescence localization of the lysosomal membrane glycoprotein p67 in pZJMTbCB and tbcatb+/–clones. A, B, E, and F, clones of pZJMTbCB were maintained as controls (A and B) or induced with tetracycline for 24 h (E and F). Cells were stained with rabbit anti-p67 antiserum and Texas Red goat anti-rabbit secondary antibody (red channel). 4,6-Diamidino-2-phenylindole was used to visualize DNA in the nucleus and kinetoplast by fluorescence microscopy (blue channel). B and C, tbcatb+/– clones (B and C) were prepared in an identical manner. Increased p67 labeling was observed in large, distorted organelles in tbcatb+/– clones (D) and parasites subjected to RNAi against tbcatB mRNA (F).

Mentions: Abnormal Lysosomal and Flagellar Pocket Morphology in TbcatB-deficient Parasites—Treatment of wild-type T. brucei with the diazomethyl ketone cysteine protease inhibitor Z-Phe-Ala-CHN2 lead to enlargement of the lysosome and an increase in total protein content, presumably as a result of decreased protein degradation (6). RNAi knockdown of tbcatB lead to similar lysosomal swelling (4). To compare the tbcatB-deficient phenotype with the phenotype of inhibitor-treated parasites, we first examined the lysosomal compartment by the localization of p67, a lysosomal type I membrane glycoprotein (19). In control parsasites, p67 staining was limited to a discrete organelle focus between the kinetoplast and nucleus (Fig. 2, A and B). In contrast, both induced RNAi clones (Fig. 2, E and F) and tbcatb+/– clones (Fig. 2, C and D) showed enhanced p67 labeling in large distorted organelles.


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)

Fluorescence localization of the lysosomal membrane glycoprotein p67 in pZJMTbCB and tbcatb+/–clones. A, B, E, and F, clones of pZJMTbCB were maintained as controls (A and B) or induced with tetracycline for 24 h (E and F). Cells were stained with rabbit anti-p67 antiserum and Texas Red goat anti-rabbit secondary antibody (red channel). 4,6-Diamidino-2-phenylindole was used to visualize DNA in the nucleus and kinetoplast by fluorescence microscopy (blue channel). B and C, tbcatb+/– clones (B and C) were prepared in an identical manner. Increased p67 labeling was observed in large, distorted organelles in tbcatb+/– clones (D) and parasites subjected to RNAi against tbcatB mRNA (F).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: Fluorescence localization of the lysosomal membrane glycoprotein p67 in pZJMTbCB and tbcatb+/–clones. A, B, E, and F, clones of pZJMTbCB were maintained as controls (A and B) or induced with tetracycline for 24 h (E and F). Cells were stained with rabbit anti-p67 antiserum and Texas Red goat anti-rabbit secondary antibody (red channel). 4,6-Diamidino-2-phenylindole was used to visualize DNA in the nucleus and kinetoplast by fluorescence microscopy (blue channel). B and C, tbcatb+/– clones (B and C) were prepared in an identical manner. Increased p67 labeling was observed in large, distorted organelles in tbcatb+/– clones (D) and parasites subjected to RNAi against tbcatB mRNA (F).
Mentions: Abnormal Lysosomal and Flagellar Pocket Morphology in TbcatB-deficient Parasites—Treatment of wild-type T. brucei with the diazomethyl ketone cysteine protease inhibitor Z-Phe-Ala-CHN2 lead to enlargement of the lysosome and an increase in total protein content, presumably as a result of decreased protein degradation (6). RNAi knockdown of tbcatB lead to similar lysosomal swelling (4). To compare the tbcatB-deficient phenotype with the phenotype of inhibitor-treated parasites, we first examined the lysosomal compartment by the localization of p67, a lysosomal type I membrane glycoprotein (19). In control parsasites, p67 staining was limited to a discrete organelle focus between the kinetoplast and nucleus (Fig. 2, A and B). In contrast, both induced RNAi clones (Fig. 2, E and F) and tbcatb+/– clones (Fig. 2, C and D) showed enhanced p67 labeling in large distorted organelles.

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