A Parasite Cysteine Protease Is Key to Host Protein Degradation and Iron Acquisition
2008; Elsevier BV; Volume: 283; Issue: 43 Linguagem: Inglês
10.1074/jbc.m805824200
ISSN1083-351X
AutoresTheresa C. O’Brien, Zachary B. Mackey, Richard D. Fetter, Youngchool Choe, Anthony J. O’Donoghue, Min Zhou, Charles S. Craik, Conor R. Caffrey, James H. McKerrow,
Tópico(s)Research on Leishmaniasis Studies
ResumoCysteine 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. 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. Proteases are ubiquitous enzymes that function in virtually all biological phenomena. Two of the major groups of proteases, Clan CA (papain-like) cysteine proteases and Clan SA (trypsin-like) serine proteases, underwent an evolutionary inversion whereby the more abundant cysteine proteases of primitive eukaryotes gave way to serine proteases with the evolution of arthropods (1Rawlings N.D. Tolle D.P. Barrett A.J. Nucleic Acids Res. 2004; 32: D160-D164Crossref PubMed Google Scholar) (merops.sanger.ac.uk/). Therefore, an analysis of the role of cysteine proteases in protozoa can provide insights into differential use and molecular evolution of this protease class. Trypanosoma brucei is a protozoan parasite and the causative agent of human African trypanosomiasis, a fatal disease that is transmitted by the bite of the tsetse fly. Only four drugs are available to treat human African trypanosomiasis: two for the first stage of the disease when parasites proliferate in the blood (pentamidine and suramin) and two for the second stage when parasites have established infection in the cerebrospinal fluid (melarsoprol and eflornithine). These drugs cause serious side effects and are expensive to manufacture and administer (2Luscher A. de Koning H.P. Maser P. Curr. Pharm. Des. 2007; 13: 555-567Crossref PubMed Scopus (69) Google Scholar). There is an obvious and urgent need to develop new chemotherapies to treat human African trypanosomiasis. Two Clan CA cysteine proteases have been identified in T. brucei: rhodesain, which is cathepsin L-like and most abundant (3Caffrey C.R. Hansell E. Lucas K.D. Brinen L.S. Alvarez Hernandez A. Cheng J. Gwaltney S.L. 2nd, Roush W.R. Stierhof Y.D. Bogyo M. Steverding D. McKerrow J.H. Mol. Biochem. Parasitol. 2001; 118: 61-73Crossref PubMed Scopus (154) Google Scholar), and tbcatB, a cathepsin B-like enzyme (4Mackey Z.B. O'Brien T.C. Greenbaum D.C. Blank R.B. McKerrow J.H. J. Biol. Chem. 2004; 279: 48426-48433Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Treatment of parasites in culture with the nonspecific cysteine protease inhibitor, benzyloxycarbonyl-phenylalanyl-alanyl diazomethane (Z-Phe-Ala-CHN2) 2The abbreviations used are: Z-Phe-Ala-CHN2, benzyloxycarbonylphenylalanyl-alanyl diazomethane; RNAi, RNA interference; FR, flanking region; D-PBS, Dulbecco's phosphate-buffered saline; Z-FR-AMC, benzyloxycarbonyl-phenylalanyl-arginyl-7-amido-4-methyl coumarin; DTT, dithiothreitol; PS-SCL, positional scanning combinatorial synthetic combinatorial library; ACC, 7-amino-4-carbamoylmethylcoumarin; Tf-R, transferrin receptor; BSA, bovine serum albumin; MES, 4-morpholineethanesulfonic acid; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol. is lethal to cultured parasites at 10 μm (5Scory S. Caffrey C.R. Stierhof Y.D. Ruppel A. Steverding D. Exp. Parasitol. 1999; 91: 327-333Crossref PubMed Scopus (83) Google Scholar). Parasites treated with this inhibitor exhibit altered cell morphology, are unable to undergo cytokinesis, and are defective in host protein degradation (5Scory S. Caffrey C.R. Stierhof Y.D. Ruppel A. Steverding D. Exp. Parasitol. 1999; 91: 327-333Crossref PubMed Scopus (83) Google Scholar, 6Scory S. Stierhof Y.D. Caffrey C.R. Steverding D. Kinetoplastid. Biol. Dis. 2007; 6: 1-6Crossref PubMed Scopus (25) Google Scholar). Knockdown of tbcatB expression by RNA interference (RNAi) is also lethal in T. brucei, causing phenotypic defects similar to those seen with the inhibitor (4Mackey Z.B. O'Brien T.C. Greenbaum D.C. Blank R.B. McKerrow J.H. J. Biol. Chem. 2004; 279: 48426-48433Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). In contrast, knockdown of rhodesain expression produced no abnormal phenotype in cultured parasites (4Mackey Z.B. O'Brien T.C. Greenbaum D.C. Blank R.B. McKerrow J.H. J. Biol. Chem. 2004; 279: 48426-48433Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). These results led to the hypothesis that whereas tbcatB is less abundant, it is, nonetheless, essential to T. brucei survival in culture and is a key target of the inhibitor. Furthermore, when RNAi targeting tbcatB is induced in a mouse model of T. brucei infection, mice are cured of their infection. 3Abdulla, M. (2008) PLoS Negl. Trop. Dis., in press. One clue to the function of tbcatB comes from the observation that a host iron-transporting protein, transferrin, accumulates in Z-Phe-Ala-CHN2-treated and tbcatB RNAi knockdown parasites (4Mackey Z.B. O'Brien T.C. Greenbaum D.C. Blank R.B. McKerrow J.H. J. Biol. Chem. 2004; 279: 48426-48433Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 5Scory S. Caffrey C.R. Stierhof Y.D. Ruppel A. Steverding D. Exp. Parasitol. 1999; 91: 327-333Crossref PubMed Scopus (83) Google Scholar). Transferrin serves as the sole source of iron for T. brucei and is rapidly degraded in an endosomal or lysosomal compartment in the parasite (7Steverding D. Parasitol. Int. 2000; 48: 191-198Crossref PubMed Scopus (66) Google Scholar). Thus, accumulation of transferrin implicates tbcatB in the process of iron acquisition and suggests that transferrin may be a natural substrate of the protease. The RNAi studies showed only modest knockdown of tbcatB mRNA and protein, yet the phenotype was dramatic (4Mackey Z.B. O'Brien T.C. Greenbaum D.C. Blank R.B. McKerrow J.H. J. Biol. Chem. 2004; 279: 48426-48433Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Therefore, to validate the previous RNAi data and further our understanding of the functional role of tbcatB, we generated a single allele deletion strain of T. brucei and, together with the inducible-RNAi strain, analyzed the effects of tbcatB deficiency on cell morphology, protease localization, and the iron acquisition pathway. We also expressed recombinant tbcatB and analyzed its substrate specificity profile both for clues as to the identity of natural substrates and as a means to determine subsite characterization that will aid future inhibitor design. Culturing of T. brucei—All bloodstream form strains of T. brucei were incubated in 5% carbon dioxide at 37 °C in HMI-9 medium containing 10% heat-inactivated fetal bovine serum (Omega Scientific), 10% Serum Plus (JRH Biosciences), 1× penicillin/streptomycin. The pZJMTbCB clones were cultured in media containing, 5.0 μg/ml hygromycin B and 2.5 μg/ml G418, as well as 2.5 μg/ml phleomycin as previously described (4Mackey Z.B. O'Brien T.C. Greenbaum D.C. Blank R.B. McKerrow J.H. J. Biol. Chem. 2004; 279: 48426-48433Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Induction of RNAi was carried out by adding tetracycline to a final concentration of 100 ng/ml. Generation of tbcatb Single-allele Knock-out T. brucei Clones (tbcatb+/–)—To generate the targeting vector, a cassette containing the phleomycin resistance marker flanked by the 5′- and 3′-flanking regions (FR) of the tbcatb gene was constructed. The following primers were used: 5′-FR forward primer, 5′-gcggccgccagaagctccactgcctcgcattg-3′; 5′-FR reverse primer, 5′-gatatccatgtgtcaccggatttggggtctgca-3′; 3′-FR forward primer, 5′-tctagataggttgcacatcgttaaacctagag-3′; 3′-FR reverse primer, 5′-gggcccacatccttatcccttccccgagggcg-3′. The cassette was cloned into the pCR2.1 vector (Invitrogen) at NotI and ApaI restriction endonuclease sites. For electroporation, 108 strain trypanosomes were pelleted by centrifugation, washed twice with 10 ml of cytomix (8van den Hoff M.J. Christoffels V.M. Labruyere W.T. Moorman A.F. Lamers W.H. Methods Mol. Biol. 1995; 48: 185-197PubMed Google Scholar), and finally resuspended in 0.5 ml of cytomix. One hundred micrograms of the targeting vector was linearized with NotI restriction endonuclease, precipitated with ethanol, and resuspended in 100 μl of cytomix. The parasites and DNA suspensions were mixed in a 4-mm electroporator cuvette and pulsed with 1.7 kV and 25 microfarads. After pulsing, the parasites were transferred to 24 ml of complete medium and incubated overnight at 37 °C with 5% carbon dioxide. Phleomycin was added to the medium to select for clones having the targeting vector integrated into the genome. Proper integration into the tbcatb locus was verified by PCR. T. brucei Growth Assay—Trypanosomes were cultured at a density of 1 × 104 cells/ml and counted using a hemocytometer after 24, 48, and 72 h. Immunofluorescence Microscopy—T. brucei were harvested by centrifugation at 4 °C, washed in cold Dulbecco's phosphate-buffered saline (D-PBS), and fixed in 4% paraformaldehyde/D-PBS for 1 h at 4 °C. All subsequent washes were carried out with excess D-PBS. Fixed cells were washed and applied to 25-mm round coverslips that had been coated with polylysine (0.1% w/v in water, Sigma) and allowed to settle for 20 min at room temperature. The cells were permeabilized in D-PBS containing 0.1% Triton X-100 (Sigma) for 10 min, washed, and blocked for 1 h with 1% bovine serum albumin (BSA) prepared in D-PBS. After blocking, cells were incubated in rabbit anti-p67 antiserum (a gift from J. D. Bangs) (9Kelley R.J. Alexander D.L. Cowan C. Balber A.E. Bangs J.D. Mol. Biochem. Parasitol. 1999; 98: 17-28Crossref PubMed Scopus (59) Google Scholar) (diluted 1:400 in 1% BSA/D-PBS) for 1 h, washed, incubated in Texas Red goat anti-rabbit IgG (Molecular Probes) (diluted 1:400 in 1% BSA/D-PBS) for 1 h, washed, and mounted on slides with Prolong Gold Antifade Reagent with 4,6-diamidino-2-phenylindole (Invitrogen). The cells were visualized on an Axio-Imager M1 microscope (Zeiss), equipped with an X-Cite 120 fluorescence illumination system (EXFP Life Sciences). Transmission Electron Microscopy—Approximately 40 million T. brucei were harvested by centrifugation at 4 °C. Pelleted parasites were resuspended in 1 ml of media for high pressure freezing using either a Bal-Tec HPM 010 or Wohlwend HPF Compact 01 high-pressure freezer (University of California, Berkeley Electron Microscopy Laboratory). The parasites were processed for conventional EM by freeze substitution in 1% OsO4, 0.1% uranyl acetate in acetone using a Leica AFS2 and embedded in Eponate 12 resin. Sections were cut with a Leica Ultracut E ultramicrotome using a diamond knife and picked up on Pioloform films on slot grids. Sections were stained with uranyl acetate and Sato's lead, and photographed using a Gatan 4k × 4k camera on an FEI Spirit transmission electron microscope operated at 120 kV. Immunoelectron Microscopy—T. brucei samples for immuno-EM were prepared by high pressure freezing as indicated above, except freeze substitution was conducted in 0.2% glutaraldehyde, 0.1% uranyl acetate in acetone. Following substitution, the samples were infiltrated in LR White resin at 4 °C, and UV-polymerized at –20 °C using benzoin methyl ether as the UV catalyst in the LR White. Sections were picked up on carbon-coated Pioloform films on nickel grids. Sections were incubated with primary antibodies (rabbit anti-tbcatB (4Mackey Z.B. O'Brien T.C. Greenbaum D.C. Blank R.B. McKerrow J.H. J. Biol. Chem. 2004; 279: 48426-48433Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar) or rabbit anti-transferrin receptor (a gift from P. Borst) (10Mussmann R. Janssen H. Calafat J. Engstler M. Ansorge I. Clayton C. Borst P. Mol. Microbiol. 2003; 47: 23-35Crossref PubMed Scopus (34) Google Scholar), both diluted 1:400) and 10 nm gold secondary antibodies (complete antibodies absorbed to gold) (British BioCell International, distributed by Ted Pella) diluted in 1% BSA, 0.1% Tween 20 in PBS. After labeling, the sections were fixed 5 min with 1% glutaraldehyde in phosphate-buffered saline, rinsed with distilled water, and stained with uranyl acetate followed by Sato's lead as indicated above. Control sections were immunostained using this protocol but with omission of the primary antibody. Sections were photographed as indicated above. Cloning and Expression of TbcatB in Pichia pastoris—Methods for cloning and expression of tbcatB in P. pastoris have been described previously. 4Mallari, J. P., Shelat, A., Kosinski, A., Caffrey, C. R., Connelly, M., Zhu, F., McKerrow, J. H., and Guy, R. K. (2008) Bioorg. Med. Chem. Lett. 18, 2883–2885. Epub 2008 April 8 Briefly, the sequence encoding the tbcatB zymogen (pro and mature regions of the protease) was amplified from a cDNA vector that contained the entire open reading frame of tbcatB (4Mackey Z.B. O'Brien T.C. Greenbaum D.C. Blank R.B. McKerrow J.H. J. Biol. Chem. 2004; 279: 48426-48433Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). The forward and reverse primers amplified genomic regions 5′-gagtaaacgccgccctcgttgct-3′ and 5′-cgccgtgttgggtgcaagagg-3′, respectively. The amplified DNA was purified and ligated into expression vector pPICZαB (Invitrogen) and subsequent transfection and expression techniques were modified from those given by the manufacturer as previously described (3Caffrey C.R. Hansell E. Lucas K.D. Brinen L.S. Alvarez Hernandez A. Cheng J. Gwaltney S.L. 2nd, Roush W.R. Stierhof Y.D. Bogyo M. Steverding D. McKerrow J.H. Mol. Biochem. Parasitol. 2001; 118: 61-73Crossref PubMed Scopus (154) Google Scholar). Purification of Recombinant tbcatB—Following a 48-h induction, P. pastoris cultures were centrifuged at 3000 × g for 10 min and the resulting supernatant containing recombinant tbcatB was lyophilized. The crude lyophilized protein was resuspended in 10% of the original volume in 50 mm sodium citrate buffer (pH 5.5) and desalted using PD-10 columns (GE Healthcare/Amersham Biosciences) by equilibrating in the same buffer. The solution was loaded onto a Mono Q 5/50 anion exchange column using an Akta Purifier-900 chromatography system (both GE Healthcare/Amersham Biosciences). A 50 mm MES (pH 6.5) buffer was used for column equilibration, sample loading, and protein elution, with a flow rate of 1 ml/min. Protein was eluted with a linear gradient of 0 to 1 m sodium chloride concentration over 20 min. Fractions of 0.5 ml were collected and subsequently checked for purity by SDS-PAGE and activity by hydrolysis of the fluorogenic peptide substrate benzyloxycarbonyl-phenylalanyl-arginyl-7-amido-4-methyl coumarin (Z-FR-AMC) (Peptides International, Inc.) (see enzyme activity assay below). Radiolabeling of Cysteine Protease Active Sites with 125I-Labeled Inhibitors—Ten microliters of Mono Q fractions containing recombinant tbcatB were labeled with the cathepsin B-specific active site label 125I-MB-074 (11Bogyo M. Verhelst S. Bellingard-Dubouchaud V. Toba S. Greenbaum D. Chem. Biol. 2000; 7: 27-38Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar) for 1 h at room temperature in buffer containing 5 mm dithiothreitol (DTT) and subjected to SDS-PAGE. Once the gels were dried, labeling was visualized by autoradiography. Deglycosylation of Recombinant TbcatB—A New England Biolabs PNGase F kit was used for deglycosylation of Mono Q-purified recombinant tbcatB. Methods were followed as described in the instruction manual. Protease Activity Assay—Protease activity was measured using the fluorogenic peptide substrate Z-FR-AMC, which is cleaved by the protease to release free 7-amino-4-methyl coumarin fluorogenic leaving group. Enzyme samples were preincubated for 10 min in sodium citrate buffer (50 mm, pH 5.5) containing 4 mm DTT, giving a total volume of 100 μl. Following the preincubation period, 100 μl of dilute substrate (20 μm, prepared in the same buffer) was added to the enzyme solution to give a final concentration of 10 μm Z-FR-AMC and a final volume of 200 μl. Hydrolysis was measured at 25 °C using an automated microtiter plate spectrofluorimeter (Molecular Devices Flex Station). Excitation and emission wavelengths were 355 and 460 nm, respectively. One unit of activity was defined as that releasing 1 μmol of AMC min–1. To determine the Michaelis constant, Km, a range of substrate concentrations from 0.14 to 300 μm was used and the value estimated using graphics software (Prism4, GraphPad). pH Activity Profiling of Recombinant TbcatB—Mono Q-purified tbcatB was assayed for protease activity with Z-FR-AMC using 50 mm sodium citrate buffers that ranged in pH from 3 to 8, with 0.5 pH unit increments. Protease activity was assayed as described above. Initial velocities were taken and assays were performed in duplicate. Protease Inhibitor Profiling—Mono Q-purified tbcatB was assayed for protease activity with Z-FR-AMC in the presence of various protease-class selective inhibitors. The inhibitors used were K11777 (N-methylpiperazine-Phe-homoPhe-vinylsulfonephenyl) (12Engel J.C. Doyle P.S. Hsieh I. McKerrow J.H. J. Exp. Med. 1998; 188: 725-734Crossref PubMed Scopus (368) Google Scholar), CA074 (N-(l-3-trans-propylcarbamoyloxirane-2carbonyl)-l-isoleucyl-l-pro line) (13Buttle D.J. Murata M. Knight C.G. Barrett A.J. Arch. Biochem. Biophys. 1992; 299: 377-380Crossref PubMed Scopus (178) Google Scholar), phenylmethylsulfonyl fluoride, and E64 (N-[N-(l-3-trans-carboxyoxirane-2-carbonyl)-l-leucyl]-agmatine). Enzyme samples (in 10 μl volume) were preincubated with inhibitor for 5 min in 90 μl of sodium citrate buffer (50 mm, pH 5.5) containing 4 mm DTT to give a total volume of 100 μl. The inhibitor concentration range used was 0.01 to 10 μm. Following incubation, the substrate was added in the same buffer to give a final concentration of 10 μm Z-FR-AMC and final volume of 200 μl. Protease activity was assayed as described above. Substrate Specificity Profiling and Substrate Screen—The P1–P4 substrate specificity profile for tbcatB was determined using a completely diverse positional scanning synthetic combinatorial library (PS-SCL) (14Choe Y. Leonetti F. Greenbaum D.C. Lecaille F. Bogyo M. Bromme D. Ellman J.A. Craik C.S. J. Biol. Chem. 2006; 281: 12824-12832Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar). The library contained substrates that are N-terminal acetylated and possessed 7-amino-4-carbamoyl-methylcoumarin (ACC) as the fluorogenic leaving group. The procedures used to screen the library were modified from those previously described (14Choe Y. Leonetti F. Greenbaum D.C. Lecaille F. Bogyo M. Bromme D. Ellman J.A. Craik C.S. J. Biol. Chem. 2006; 281: 12824-12832Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar). Crude lyophilized tbcatB was assayed at 25 °C in buffer containing 50 mm sodium citrate buffer (pH 5.5), 100 mm NaCl, 10 mm DTT, 1 mm EDTA, 0.01% Brij-35, and 1% Me2SO (from the substrates). The concentration of tbcatB in the assays was ∼10 nm. Assays were performed in triplicate. To test the predictive capability of the substrate specificity profile obtained, authentic 7-amino-4-methyl coumarin (AMC) peptide substrates that were either "good" or "poor" matches to the profile were selected for screening in the protease activity assay described above. The range of substrate concentrations used was 3.5 to 300μm. The substrates were: Z-FR-AMC (Peptides International, Inc.), Z-Arg-Arg-AMC (Z-RR-AMC) (Peptides International, Inc.), Z-Arg-Glu-Lys-Arg-AMC (Z-REKR-AMC) (Bachem), Ac-Ile-Glu-Pro-Asp-AMC (Ac-IEPD-AMC) (Bachem), Ac-Ala-Ser-Thr-Asp-AMC (Ac-ASTD-AMC) (Bachem), Z-Leu-Arg-Gly-Gly-AMC (Z-LRGG-AMC) (Bachem), H-Arg-Gln-Arg-Arg-AMC (H-RQRR-AMC) (Bachem), Ac-Lys-Gln-Lys-Leu-Arg-AMC (Ac-KQKLR-AMC) (AnaSpec, Inc.), and Z-Arg-Arg-Leu-Arg-AMC (Z-RRLR-AMC), which was custom synthesized by SynPep. Michaelis constants were estimated for each substrate as described above. Human Serum and T. brucei Protein Database Search—A database of host serum proteins was created by searching the Protein Data Bank (PDB) (15Berman H.M. Westbrook J. Feng Z. Gilliland G. Bhat T.N. Weissig H. Shindyalov I.N. Bourne P.E. Nucleic Acids Res. 2000; 28: 235-242Crossref PubMed Scopus (28686) Google Scholar), using the keyword "serum" and limiting the search to "Homo sapiens" proteins. The results of the search were downloaded in FASTA format. The database of serum proteins was then screened using the EMBOSS fuzzpro protein pattern program (16Rice P. Longden I. Bleasby A. Trends Genet. 2000; 16: 276-277Abstract Full Text Full Text PDF PubMed Scopus (6688) Google Scholar) for the amino acid motif: P4, Arg/Lys; P3, Arg/Lys; P2, X; P1, Arg/Lys. T. brucei proteins were also screened for the presence of the optimal substrate motif preference of tbcatB. The screen was carried out using the"Search Using Quick Matrix Method"Database Search option on Scansite's website (scansite.mit.edu) (17Obenauer J.C. Cantley L.C. Yaffe M.B. Nucleic Acids Res. 2003; 31: 3635-3641Crossref PubMed Scopus (1364) Google Scholar). Once the motif (P4, Arg/Lys; P3, Arg/Lys; P2, X; P1, Arg/Lys) was entered, the following parameters were selected: organism class, invertebrates; database, SWISS-PROT; and single species, brucei. No limits were set for other parameters in the program. In Vitro Digestion of Transferrin by TbcatB—Bovine transferrin was obtained from Invitrogen. 10 nm tbcatB was assayed with 200 nm transferrin in 50 mm sodium citrate buffer (pH 5.5), 100 mm NaCl, 10 mm DTT, 1 mm EDTA. After 24 and 48 h incubation at 25 °C, digestive products were separated on a 10% BisTris gel, adsorbed onto polyvinylidene difluoride and stained with PageBlue (Fermentas). Cleavage products were excised and subjected to N-terminal sequencing (Molecular Structure Facility, University of California, Davis, CA). Three internally quenched fluorescent peptides derived from transferrin, MZ1 (K(Mca)KCACSNHEK(Dnp)), MZ2 (K(Mca)EYVTALQNK(Dnp)), and MZ3 (K(Mca)SRKDKAT CK(Dnp)) were synthesized by Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry on a Symphony Quartet (Protein Technologies). Fluorescent assays were performed as outlined previously except excitation and emission wavelengths were 328 and 393 nm, respectively. Lysate Preparation for Two-dimensional PAGE—T. brucei strain 221 and tbcatb+/– were harvested by centrifugation, washed once in D-PBS, and resuspended at ∼1 × 108 cells/ml in lysis buffer (50 mm sodium acetate, pH 5.5, 1 mm EDTA, 1% Triton X-100, 5 mm DTT, and 5 μl/ml Protease Inhibitor Mixture Set II (CalBiochem, stock solution contains 20 mm 4-(2-aminoethyl)benzenesulfonyl fluoride-hydrochloride, 1.7 mm bestatin, 200 μm E-64 protease inhibitor, 85 mm EDTA-disodium, and 2 mm pepstatin)) that had been supplemented with 0.1 unit/μl of DNase (RNase-free, Roche), 45 μm RNase A (Roche), and 30 mm MgCl2. The lysates were incubated on ice for 20 min and then cleared by centrifugation at 16,000 × g for 50 min at 4 °C. The protein concentration of the cleared lysates was determined by Bradford assay (18Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (219809) Google Scholar). If not used immediately, lysates were stored at –80 °C. Equal amounts of each lysate (200 μg) were prepared for isoelectric focusing and two-dimensional gel electrophoresis using the ReadyPrep Two-dimensional Cleanup Kit (Bio-Rad). The subsequent procedures used for IEF and two-dimensional gel electrophoresis were carried out as described in the ReadyPrep Two-dimensional Starter Kit (Bio-Rad) instruction manual. Bio-Rad ReadyStrip IPG strips, pH 4–7, were used for IEF and 8–16% Criterion Ready Gels (Bio-Rad) were used for SDS-PAGE. Following SDS-PAGE, the gels were immediately transferred to polyvinylidene difluoride membrane (Millipore), using procedures described below. Western Blots of Two-dimensional PAGE Gels—Two-dimensional gels that had been transferred to polyvinylidene difluoride membranes were blocked for 1 h (10 mm Tris, pH 7.4, 5% dry milk, and 0.1% BSA, w/v). After blocking, membranes were incubated with rabbit anti-transferrin antiserum (1:2500 dilution) (described above) for 1 h and washed 3 times for 10 min with TBST (10 mm Tris, pH 7.4, 150 mm NaCl, 0.4% Tween 20). After washing, membranes were incubated with horseradish peroxidase-conjugated donkey anti-rabbit IgG (1:5000 dilution) (GE Healthcare/Amersham Biosciences) for 1 h. The blots were washed three times for 10 min with TBST and twice for 10 min with TBS (TBST without 0.4% Tween 20). Finally, the blots were visualized by ECL (GE Healthcare/Amersham Biosciences). Transferrin Starvation—Strain 221 and tbcatb+/– parasites were incubated at a density of 5 × 104 cells/ml for 36 h in appropriate media (see methods for culturing T. brucei above), containing 10% fetal bovine serum or, instead, dog serum (Innovative Research). After the incubation period, equal numbers of parasites (1 × 107) were resuspended in lysis buffer (50 mm sodium acetate, 1 mm EDTA, 1% Triton X-100, pH 5.5, 5 mm DTT). The lysates were incubated on ice for 20 min and cleared by centrifugation at 16,000 × g for 15 min at 4 °C. The extracts were resolved by SDS-PAGE and subjected to Western blot analysis, using procedures described above and rabbit anti-transferrin receptor antibody (a gift of P. Borst) (10Mussmann R. Janssen H. Calafat J. Engstler M. Ansorge I. Clayton C. Borst P. Mol. Microbiol. 2003; 47: 23-35Crossref PubMed Scopus (34) Google Scholar) (diluted 1:250) as primary antibody. Labeling was quantified by densitometry using Image J software (National Institute of Health). Single-allele Deletion of T. brucei Cathepsin B Gene Leads to Decreased Parasite Replication—Silencing of tbcatB by RNAi produced a marked phenotype (4Mackey Z.B. O'Brien T.C. Greenbaum D.C. Blank R.B. McKerrow J.H. J. Biol. Chem. 2004; 279: 48426-48433Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Parasites exhibited endosomal or lysosomal swelling, decreased growth rate, arrest in cytokinesis, and eventual death. However, this dramatic phenotype was associated with only a modest decrease in tbcatB mRNA, protein, and protease activity (4Mackey Z.B. O'Brien T.C. Greenbaum D.C. Blank R.B. McKerrow J.H. J. Biol. Chem. 2004; 279: 48426-48433Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Therefore, to validate and extend the RNAi results, a tbcatB heterozygous knock-out clone (tbcatb+/–) was produced by homologous recombination using a tbcatb targeting vector. The rate of replication of the tbcatb+/– clones was ∼40% less than the control 90-13 strain after 72 h (Fig. 1). tbcatb–/– clones could not be isolated due to presumed lethality. 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 (6Scory S. Stierhof Y.D. Caffrey C.R. Steverding D. Kinetoplastid. Biol. Dis. 2007; 6: 1-6Crossref PubMed Scopus (25) Google Scholar). RNAi knockdown of tbcatB lead to similar lysosomal swelling (4Mackey Z.B. O'Brien T.C. Greenbaum D.C. Blank R.B. McKerrow J.H. J. Biol. Chem. 2004; 279: 48426-48433Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). 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 (19Alexander D.L. Schwartz K.J. Balber A.E. Bangs J.D. J. Cell Sci. 2002; 115: 3253-
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