title: A parasite cysteine protease is key to host protein degradation and iron acquisition.
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.
authors.affiliation: Department of Pathology and Sandler Center for Basic Research in Parasitic Diseases, University of California, San Francisco, California 94158-2550, USA.
authors.forename: Theresa C
authors.initials: TC
authors.lastname: O'Brien
authors.forename: Zachary B
authors.initials: ZB
authors.lastname: Mackey
authors.forename: Richard D
authors.initials: RD
authors.lastname: Fetter
authors.forename: Youngchool
authors.initials: Y
authors.lastname: Choe
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: O'Donoghue
authors.forename: Min
authors.initials: M
authors.lastname: Zhou
authors.forename: Charles S
authors.initials: CS
authors.lastname: Craik
authors.forename: Conor R
authors.initials: CR
authors.lastname: Caffrey
authors.forename: James H
authors.initials: JH
authors.lastname: McKerrow
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D020134:Catalytic Domain; D003545:Cysteine / Q000737:chemistry*; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D004705:Endocytosis; D031541:Fluorescence Resonance Energy Transfer; D007501:Iron / Q000737:chemistry / Q000378:metabolism*; D008247:Lysosomes / Q000378:metabolism; D046529:Microscopy, Electron, Transmission; D008954:Models, Biological; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D014168:Transferrin / Q000737:chemistry; D014346:Trypanosoma brucei brucei / Q000378:metabolism
chemical_list: D014168:Transferrin; D007501:Iron; D003546:Cysteine Endopeptidases; C533458:TbcatB protein, Trypanosoma brucei; D003545:Cysteine