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J. Biol. Chem., Vol. 279, Issue 46, 48426-48433, November 12, 2004

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A Cathepsin B-like Protease Is Required for Host Protein Degradation in Trypanosoma brucei^*

Zachary B. Mackey, Theresa C. O'Brien, Doron C. Greenbaum, Rebecca B. Blank, and James H. McKerrow

From the Department of Pathology Tropical Disease Research Unit, University of California, San Francisco, California 94143

Identification and analysis of Clan CA (papain) cysteine proteases in primitive protozoa and metazoa have suggested that this enzyme family is more diverse and biologically important than originally thought. The protozoan parasite Trypanosoma brucei is the etiological agent of African sleeping sickness. The cysteine protease activity of this organism is a validated drug target as first recognized by the killing of the parasite with the diazomethane inhibitor Z-Phe-Ala-CHN₂ (where Z is benzyloxycarbonyl). Whereas the presumed target of this inhibitor was rhodesain (also brucipain, trypanopain), the major cathepsin L-like cysteine protease of T. brucei, genomic analysis has now identified tbcatB, a cathepsin B-like cysteine protease as a possible inhibitor target. The mRNA of tbcatB is more abundantly expressed in the bloodstream versus the procyclic form of the parasite. Induction of RNA interference against rhodesain did not result in an abnormal phenotype in cultured T. brucei. However, induction of RNA interference against tbcatB led to enlargement of the endosome, accumulation of fluorescein isothiocyanate-transferrin, defective cytokinesis after completion of mitosis, and ultimately the death of cultured parasites. Therefore, tbcatB, but not rhodesain, is essential for T. brucei survival in culture and is the most likely target of the diazomethane protease inhibitor Z-Phe-Ala-CHN₂ in T. brucei.

Received for publication, March 4, 2004 , and in revised form, August 13, 2004.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY508515.

^* This work was supported by National Institute of Allergy and Infectious Diseases Tropical Disease Research Unit Grant AI35707, the University of California-San Francisco Liver Center Microscopy and Imaging Core, a University of California-San Francisco/Merck scientific initiative fellowship (to Z. B. M.), and the Sandler Family Supporting Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Pathology TDRU, University of California, 513 Parnassus HSW 501, San Francisco, CA 94143. Tel.: 415-514-3052; Fax: 415-514-3165; E-mail: zmackey{at}itsa.ucsf.edu.

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