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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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Tetrapeptide substrate specificity profiling of tbcatB determined using complete diverse PS-SCL and comparison with related enzymes. A, the library contained P1, P2, P3, and P4 libraries in which the P position is fixed with one of 20 amino acids (norleucine is used in place of isoleucine) and the other three positions are occupied by the 20 amino acids in an equimolar mixture. All of the substrates contained an ACC fluorogenic leaving group. Protease specificity was determined by hydrolysis of substrates, as measured by ACC fluorescence intensity. Assays were performed in triplicate and error bars denote the mean ± S.D. y axis represents the rate of ACC production expressed as a percentage of the maximum rate observed in each experiment. x axis indicates the amino acid held constant at position, designated by the one-letter code (with n representing norleucine), and amino acids are grouped along the axis to reflect the chemical properties of their side chains (acidic, basic, polar, aromatic, and aliphatic amino acids). B, a comparison of preferred substrate motifs for recombinant tbcatB, papain (14), human cathepsin B (14), human cathepsin L (14), rhodesain (14), and cruzain (14) determined using the complete diverse library.
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fig6: Tetrapeptide substrate specificity profiling of tbcatB determined using complete diverse PS-SCL and comparison with related enzymes. A, the library contained P1, P2, P3, and P4 libraries in which the P position is fixed with one of 20 amino acids (norleucine is used in place of isoleucine) and the other three positions are occupied by the 20 amino acids in an equimolar mixture. All of the substrates contained an ACC fluorogenic leaving group. Protease specificity was determined by hydrolysis of substrates, as measured by ACC fluorescence intensity. Assays were performed in triplicate and error bars denote the mean ± S.D. y axis represents the rate of ACC production expressed as a percentage of the maximum rate observed in each experiment. x axis indicates the amino acid held constant at position, designated by the one-letter code (with n representing norleucine), and amino acids are grouped along the axis to reflect the chemical properties of their side chains (acidic, basic, polar, aromatic, and aliphatic amino acids). B, a comparison of preferred substrate motifs for recombinant tbcatB, papain (14), human cathepsin B (14), human cathepsin L (14), rhodesain (14), and cruzain (14) determined using the complete diverse library.

Mentions: Substrate Specificity Profiling of Recombinant TbcatB Identifies an Optimal Substrate Motif Preference—Substrate specificity profiling, using PS-SCLs, has been used to determine the P1–P4 preference of a protease for peptide substrates (20). Substrate specificity data obtained from screening a PS-SCL has facilitated natural substrate identification for Schistosoma mansoni legumain (21) and granzyme B (20, 22). Here, a complete diverse PS-SCL was used, containing 160,000 tetrapeptide substrates. This library completely randomizes each of the P1, P2, P3, and P4 positions with 20 amino acids (14). The PS-SCL demonstrated that tbcatB has a strong preference for arginine or lysine residues at P1 and for amino acids with large hydrophobic side chains at P2 (Fig. 6A). TbcatB was also more selective at P3 and P4 than its orthologs (14) (Fig. 6B) and P3 specificity differs significantly from that of human cathepsin B (Fig. 6B). The optimal substrate motif for tbcatB, as predicted by substrate specificity profiling, was: P4, Arg/Lys; P3, Arg/Lys; P2, X; P1, Arg/Lys.


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)

Tetrapeptide substrate specificity profiling of tbcatB determined using complete diverse PS-SCL and comparison with related enzymes. A, the library contained P1, P2, P3, and P4 libraries in which the P position is fixed with one of 20 amino acids (norleucine is used in place of isoleucine) and the other three positions are occupied by the 20 amino acids in an equimolar mixture. All of the substrates contained an ACC fluorogenic leaving group. Protease specificity was determined by hydrolysis of substrates, as measured by ACC fluorescence intensity. Assays were performed in triplicate and error bars denote the mean ± S.D. y axis represents the rate of ACC production expressed as a percentage of the maximum rate observed in each experiment. x axis indicates the amino acid held constant at position, designated by the one-letter code (with n representing norleucine), and amino acids are grouped along the axis to reflect the chemical properties of their side chains (acidic, basic, polar, aromatic, and aliphatic amino acids). B, a comparison of preferred substrate motifs for recombinant tbcatB, papain (14), human cathepsin B (14), human cathepsin L (14), rhodesain (14), and cruzain (14) determined using the complete diverse library.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig6: Tetrapeptide substrate specificity profiling of tbcatB determined using complete diverse PS-SCL and comparison with related enzymes. A, the library contained P1, P2, P3, and P4 libraries in which the P position is fixed with one of 20 amino acids (norleucine is used in place of isoleucine) and the other three positions are occupied by the 20 amino acids in an equimolar mixture. All of the substrates contained an ACC fluorogenic leaving group. Protease specificity was determined by hydrolysis of substrates, as measured by ACC fluorescence intensity. Assays were performed in triplicate and error bars denote the mean ± S.D. y axis represents the rate of ACC production expressed as a percentage of the maximum rate observed in each experiment. x axis indicates the amino acid held constant at position, designated by the one-letter code (with n representing norleucine), and amino acids are grouped along the axis to reflect the chemical properties of their side chains (acidic, basic, polar, aromatic, and aliphatic amino acids). B, a comparison of preferred substrate motifs for recombinant tbcatB, papain (14), human cathepsin B (14), human cathepsin L (14), rhodesain (14), and cruzain (14) determined using the complete diverse library.
Mentions: Substrate Specificity Profiling of Recombinant TbcatB Identifies an Optimal Substrate Motif Preference—Substrate specificity profiling, using PS-SCLs, has been used to determine the P1–P4 preference of a protease for peptide substrates (20). Substrate specificity data obtained from screening a PS-SCL has facilitated natural substrate identification for Schistosoma mansoni legumain (21) and granzyme B (20, 22). Here, a complete diverse PS-SCL was used, containing 160,000 tetrapeptide substrates. This library completely randomizes each of the P1, P2, P3, and P4 positions with 20 amino acids (14). The PS-SCL demonstrated that tbcatB has a strong preference for arginine or lysine residues at P1 and for amino acids with large hydrophobic side chains at P2 (Fig. 6A). TbcatB was also more selective at P3 and P4 than its orthologs (14) (Fig. 6B) and P3 specificity differs significantly from that of human cathepsin B (Fig. 6B). The optimal substrate motif for tbcatB, as predicted by substrate specificity profiling, was: P4, Arg/Lys; P3, Arg/Lys; P2, X; P1, Arg/Lys.

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