ABSTRACT
Glycolysis is essential to Trypanosoma brucei, the causative agent of African sleeping sickness, suggesting enzymes in the pathway could be targets for drug development. Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one, EbSe) was identified in a screen as a potent inhibitor of T. brucei hexokinase 1 (TbHK1), the first enzyme in the pathway. EbSe has a history of promiscuity as an enzyme inhibitor, inactivating proteins through seleno-sulfide conjugation with Cys residues. Indeed, dilution of TbHK1 and inhibitor following incubation did not temper inhibition suggesting conjugate formation. Using mass spectrometry to analyze EbSe-based modifications revealed that two Cys residues (C327 and C369) were oxidized after treatment. Site-directed mutagenesis of C327 led to enzyme inactivation indicating that C327 was essential for catalysis. C369 was not essential, suggesting that EbSe inhibition of TbHK1 was the consequence of modification of C327 via thiol oxidation. Additionally, neither EbSe treatment nor mutation of the nine TbHK1 Cys residues appreciably altered enzyme quaternary structure.
ABSTRACT
We report the serendipitous development of the first cultured cell models of Hirano bodies. Myc-epitope-tagged forms of the 34 kDa actin bundling protein (amino acids 1-295) and the CT fragment (amino acids 124-295) of the 34 kDa protein that exhibits activated actin binding and calcium-insensitive actin filament crosslinking activity were expressed in Dictyostelium and mammalian cells to assess the behavior of these modified forms in vivo. Dictyostelium cells expressing the CT-myc fragment: (1) form ellipsoidal regions that contain ordered assemblies of F-actin, CT-myc, myosin II, cofilin and alpha-actinin; (2) grow and develop more slowly than wildtype, but produce normal morphogenetic structures; (3) perform pinocytosis and phagocytosis normally; and (4) produce a level of total actin equivalent to wildtype, but a higher level of F-actin. The paracrystalline inclusions bear a striking resemblance to Hirano bodies, which are associated with a number of pathological conditions. Furthermore, expression of the CT fragment in murine L cells results in F-actin rearrangements characterized by loss of stress fibers, accumulation of numerous punctate foci, and large perinuclear aggregates, the Hirano bodies. Thus, failure to regulate the activity and/or affinity of an actin crosslinking protein can provide a signal for formation of Hirano bodies. More generally, formation of Hirano bodies is a cellular response to or a consequence of aberrant function of the actin cytoskeleton. The results reveal that formation of Hirano bodies is not necessarily related to cell death. These cultured cell models should facilitate studies of the biochemistry, genetics and physiological effects of Hirano bodies.
