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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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Biochemical characterization and kinetic parameters of recombinant tbcatB. A, recombinant tbcatB was expressed in P. pastoris. Crude protein was desalted, concentrated, and purified by anion exchange chromatography. Crude protein (lane 1), column flow-through (lane 2), and fractions (lanes 3–5) were resolved by SDS-PAGE and visualized by silver stain. Note both the zymogen at 42 kDa and more abundant mature form at 31 kDa (lane 4). B, pH-dependent activity of recombinant tbcatB. Protease was preincubated in 50 mm sodium citrate buffers (pH 3–8) containing 4 mm DTT. Then the same volume of respective buffer containing Z-FR-AMC was added. Activity was determined by hydrolysis of the substrate, measured by the fluorescence reporter AMC. y axis represents the rate of AMC production expressed as relative fluorescent units (RFU) per second. x axis represents the pH of the assay buffer.
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fig5: Biochemical characterization and kinetic parameters of recombinant tbcatB. A, recombinant tbcatB was expressed in P. pastoris. Crude protein was desalted, concentrated, and purified by anion exchange chromatography. Crude protein (lane 1), column flow-through (lane 2), and fractions (lanes 3–5) were resolved by SDS-PAGE and visualized by silver stain. Note both the zymogen at 42 kDa and more abundant mature form at 31 kDa (lane 4). B, pH-dependent activity of recombinant tbcatB. Protease was preincubated in 50 mm sodium citrate buffers (pH 3–8) containing 4 mm DTT. Then the same volume of respective buffer containing Z-FR-AMC was added. Activity was determined by hydrolysis of the substrate, measured by the fluorescence reporter AMC. y axis represents the rate of AMC production expressed as relative fluorescent units (RFU) per second. x axis represents the pH of the assay buffer.

Mentions: Expression, Purification, and Biochemical Properties of Recombinant TbcatB from P. pastoris—Expression of the tbcatB zymogen lead to secretion of both zymogen and mature forms of tbcatB (42 and 31 kDa, respectively) from P. pastoris (Fig. 5A). The mature form was labeled by the cathepsin B-specific active site label 125I-MB074 (11), indicating that the recombinant protease is indeed a cathepsin B-like enzyme (data not shown). We also observed 125I-MB074 labeling with the zymogen because the label is small enough to access the active site even with the pro-domain intact (11). Purification of active tbcatB was carried out by lyophilization, desalting, and Mono Q anion exchange chromatography. Protease activity was monitored using the fluorogenic peptide substrate Z-FR-AMC. When the purified enzyme was subjected to deglycosylation with peptide:N-glycosidase F, a decrease in the molecular masses of both the zymogen and mature forms of the enzyme was observed: the zymogen and mature enzyme had increased mobility of ∼8 and ∼4 kDa, respectively, consistent with two glycosylation sites in the pro region (at amino acids Asn58-Ile-Thr and Asn76-Ala-Ser) and one in the mature region of tbcatB (Asn216-Tyr-Thr). The optimal pH range for recombinant tbcatB with Z-FR-AMC was measured between pH 5.0 and 5.5 (Fig. 5B), consistent with the localization of the enzyme in an acidic organelle. TbcatB activity was inhibited by the Clan CA cysteine protease inhibitors E-64 and K11777, but not by the serine protease inhibitor phenylmethylsulfonyl fluoride. The cathepsin B cysteine protease inhibitor, CA074, inhibited tbcatB better or as well as the generic clan CA cysteine protease inhibitors (E64 and K11777), confirming the cathepsin B-like activity of the recombinant enzyme.


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)

Biochemical characterization and kinetic parameters of recombinant tbcatB. A, recombinant tbcatB was expressed in P. pastoris. Crude protein was desalted, concentrated, and purified by anion exchange chromatography. Crude protein (lane 1), column flow-through (lane 2), and fractions (lanes 3–5) were resolved by SDS-PAGE and visualized by silver stain. Note both the zymogen at 42 kDa and more abundant mature form at 31 kDa (lane 4). B, pH-dependent activity of recombinant tbcatB. Protease was preincubated in 50 mm sodium citrate buffers (pH 3–8) containing 4 mm DTT. Then the same volume of respective buffer containing Z-FR-AMC was added. Activity was determined by hydrolysis of the substrate, measured by the fluorescence reporter AMC. y axis represents the rate of AMC production expressed as relative fluorescent units (RFU) per second. x axis represents the pH of the assay buffer.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig5: Biochemical characterization and kinetic parameters of recombinant tbcatB. A, recombinant tbcatB was expressed in P. pastoris. Crude protein was desalted, concentrated, and purified by anion exchange chromatography. Crude protein (lane 1), column flow-through (lane 2), and fractions (lanes 3–5) were resolved by SDS-PAGE and visualized by silver stain. Note both the zymogen at 42 kDa and more abundant mature form at 31 kDa (lane 4). B, pH-dependent activity of recombinant tbcatB. Protease was preincubated in 50 mm sodium citrate buffers (pH 3–8) containing 4 mm DTT. Then the same volume of respective buffer containing Z-FR-AMC was added. Activity was determined by hydrolysis of the substrate, measured by the fluorescence reporter AMC. y axis represents the rate of AMC production expressed as relative fluorescent units (RFU) per second. x axis represents the pH of the assay buffer.
Mentions: Expression, Purification, and Biochemical Properties of Recombinant TbcatB from P. pastoris—Expression of the tbcatB zymogen lead to secretion of both zymogen and mature forms of tbcatB (42 and 31 kDa, respectively) from P. pastoris (Fig. 5A). The mature form was labeled by the cathepsin B-specific active site label 125I-MB074 (11), indicating that the recombinant protease is indeed a cathepsin B-like enzyme (data not shown). We also observed 125I-MB074 labeling with the zymogen because the label is small enough to access the active site even with the pro-domain intact (11). Purification of active tbcatB was carried out by lyophilization, desalting, and Mono Q anion exchange chromatography. Protease activity was monitored using the fluorogenic peptide substrate Z-FR-AMC. When the purified enzyme was subjected to deglycosylation with peptide:N-glycosidase F, a decrease in the molecular masses of both the zymogen and mature forms of the enzyme was observed: the zymogen and mature enzyme had increased mobility of ∼8 and ∼4 kDa, respectively, consistent with two glycosylation sites in the pro region (at amino acids Asn58-Ile-Thr and Asn76-Ala-Ser) and one in the mature region of tbcatB (Asn216-Tyr-Thr). The optimal pH range for recombinant tbcatB with Z-FR-AMC was measured between pH 5.0 and 5.5 (Fig. 5B), consistent with the localization of the enzyme in an acidic organelle. TbcatB activity was inhibited by the Clan CA cysteine protease inhibitors E-64 and K11777, but not by the serine protease inhibitor phenylmethylsulfonyl fluoride. The cathepsin B cysteine protease inhibitor, CA074, inhibited tbcatB better or as well as the generic clan CA cysteine protease inhibitors (E64 and K11777), confirming the cathepsin B-like activity of the recombinant enzyme.

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