ABSTRACT
The Trypanosoma brucei cysteine protease cathepsin B (TbCatB), which is involved in host protein degradation, is a promising target to develop new treatments against sleeping sickness, a fatal disease caused by this protozoan parasite. The structure of the mature, active form of TbCatB has so far not provided sufficient information for the design of a safe and specific drug against T. brucei. By combining two recent innovations, in vivo crystallization and serial femtosecond crystallography, we obtained the room-temperature 2.1 angstrom resolution structure of the fully glycosylated precursor complex of TbCatB. The structure reveals the mechanism of native TbCatB inhibition and demonstrates that new biomolecular information can be obtained by the "diffraction-before-destruction" approach of x-ray free-electron lasers from hundreds of thousands of individual microcrystals.
Subject(s)
Cathepsin B/chemistry , Protozoan Proteins/chemistry , Trypanosoma brucei brucei/enzymology , Amino Acid Sequence , Animals , Catalytic Domain , Cathepsin B/antagonists & inhibitors , Crystallization , Crystallography, X-Ray , Enzyme Precursors/chemistry , Glycosylation , Models, Molecular , Molecular Sequence Data , Protein Conformation , Protozoan Proteins/antagonists & inhibitors , Sf9 Cells , Spodoptera , X-RaysABSTRACT
Phylogenetic analyses based on defined proteins or different RNA species have revealed that the order kinetoplastida belongs to the early-branching eukaryotes and may thus contain organisms in which complex cellular events are easier to analyze. This view was further supported by results from a bioinformatic survey that suggested that nearly half of the autophagy-related proteins existent in yeast are missing in trypanosomatids. On the other hand, these organisms have evolved a highly sophisticated machinery to escape from the different host immune-response strategies and have learned to cope with extremely variable environmental conditions by morphological and functional reorganization of the cell. For both the stress response and the differentiation processes, autophagy seems to be an indispensable prerequisite. So far autophagy has not been systematically investigated in trypanosomatids. Here we present technical information on how to handle the different parasites belonging to this order and give an overview of the current status of autophagy research in these organisms.