Novel aminoarylcysteine adducts in globin of rats dosed with naphthylamine and nitronaphthalene isomers.

Novel aminonaphthylcysteine (ANC) adducts, formed via naphthylnitrenium ions and/or their metabolic precursors in the biotransformation of naphthylamines (NA) and nitronaphthalenes (NN), were identified and quantified in globin of rats dosed intraperitoneally with 0.16 mmol/kg b.w. of 1-NA, 1-NN, 2-NA and 2-NN. Using HPLC-ESI-MS2 analysis of the globin hydrolysates, S-(1-amino-2-naphthyl)cysteine (1A2NC) together with S-(4-amino-1-naphthyl)cysteine (4A1NC) were found in rats given 1-NA or 1-NN, and S-(2-amino-1-naphthyl)cysteine (2A1NC) in those given 2-NA or 2-NN. The highest level of ANC was produced by the most mutagenic and carcinogenic isomer 2-NA (35.8 ± 5.4 nmol/g globin). The ratio of ANC adduct levels for 1-NA, 1-NN, 2-NA and 2-NN was 1:2:100:3, respectively. Notably, the ratio of 1A2NC:4A1NC in globin of rats dosed with 1-NA and 1-NN differed significantly (2:98 versus 16:84 respectively), indicating differences in mechanism of the adduct formation. Moreover, aminonaphthylmercapturic acids, formed via conjugation of naphthylnitrenium ions and/or their metabolic precursors with glutathione, were identified in the rat urine. Their amounts excreted after dosing rats with 1-NA, 1-NN, 2-NA and 2-NN were in the ratio 1:100:40:2, respectively. For all four compounds tested, haemoglobin binding index for ANC was several-fold higher than that for the sulphinamide adducts, generated via nitrosoarene metabolites. Due to involvement of electrophilic intermediates in their formation, ANC adducts in globin may become toxicologically more relevant biomarkers of cumulative exposure to carcinogenic or non-carcinogenic arylamines and nitroarenes than the currently used sulphinamide adducts.

A rapid approach for in locus overexpression of Trypanosoma brucei proteins.

Altering amounts of a protein in a cell has become a crucial tool for understanding its function. In many organisms, including the protozoan parasite Trypanosoma brucei, protein overexpression has been achieved by inserting a protein-coding sequence into an overexpression vector. Here, we have adapted the PCR only based system for tagging trypanosome proteins at their endogenous loci such that it in addition enables a tetracycline-inducible T7 RNA polymerase-mediated protein overexpression. Hence, this approach bypasses the need for molecular cloning, making it rapid and cost effective. We validated the approach for ten flagellum-associated proteins with molecular weights ranging from 40 to over 500 kDa. For a majority of the recombinant proteins a significant (3-50 fold) increase in the cellular amount was achieved upon induction of overexpression. Two of the largest proteins studied, the dynein heavy chains, were significantly overexpressed, while two were not. Our data suggest that this may reflect the extent of the T7 RNA polymerase processivity on the trypanosome genomic DNA. We further show that the overexpression is informative as to cellular functions of the studied proteins, and that these cultures can serve as an excellent source for purification of the overexpressed proteins. We believe that this rapid in locus overexpression system will become a valuable tool to interrogate cellular functions and biochemical activities of trypanosome proteins.

Inhibition of F1 -ATPase from Trypanosoma brucei by its regulatory protein inhibitor TbIF1.

Hydrolysis of ATP by the mitochondrial F-ATPase is inhibited by a protein called IF1 . In the parasitic flagellate, Trypanosoma brucei, this protein, known as TbIF1 , is expressed exclusively in the procyclic stage, where the F-ATPase is synthesizing ATP. In the bloodstream stage, where TbIF1 is absent, the F-ATPase hydrolyzes ATP made by glycolysis and compensates for the absence of a proton pumping respiratory chain by translocating protons into the intermembrane space, thereby maintaining the essential mitochondrial membrane potential. We have defined regions and amino acid residues of TbIF1 that are required for its inhibitory activity by analyzing the binding of several modified recombinant inhibitors to F1 -ATPase isolated from the procyclic stage of T. brucei. Kinetic measurements revealed that the C-terminal portion of TbIF1 facilitates homodimerization, but it is not required for the inhibitory activity, similar to the bovine and yeast orthologs. However, in contrast to bovine IF1 , the inhibitory capacity of the C-terminally truncated TbIF1 diminishes with decreasing pH, similar to full length TbIF1 . This effect does not involve the dimerization of active dimers to form inactive tetramers. Over a wide pH range, the full length and C-terminally truncated TbIF1 form dimers and monomers, respectively. TbIF1 has no effect on bovine F1 -ATPase, and this difference in the mechanism of regulation of the F-ATPase between the host and the parasite could be exploited in the design of drugs to combat human and animal African trypanosomiases.

The F1 -ATPase from Trypanosoma brucei is elaborated by three copies of an additional p18-subunit.

The F-ATPases (also called the F1 Fo -ATPases or ATP synthases) are multi-subunit membrane-bound molecular machines that produce ATP in bacteria and in eukaryotic mitochondria and chloroplasts. The structures and enzymic mechanisms of their F1 -catalytic domains are highly conserved in all species investigated hitherto. However, there is evidence that the F-ATPases from the group of protozoa known as Euglenozoa have novel features. Therefore, we have isolated pure and active F1 -ATPase from the euglenozoan parasite, Trypanosoma brucei, and characterized it. All of the usual eukaryotic subunits (α, ß, γ, δ, and ε) were present in the enzyme, and, in addition, two unique features were detected. First, each of the three α-subunits in the F1 -domain has been cleaved by proteolysis in vivo at two sites eight residues apart, producing two assembled fragments. Second, the T. brucei F1 -ATPase has an additional subunit, called p18, present in three copies per complex. Suppression of expression of p18 affected in vitro growth of both the insect and infectious mammalian forms of T. brucei. It also reduced the levels of monomeric and multimeric F-ATPase complexes and diminished the in vivo hydrolytic activity of the enzyme significantly. These observations imply that p18 plays a role in the assembly of the F1 domain. These unique features of the F1 -ATPase extend the list of special characteristics of the F-ATPase from T. brucei, and also, demonstrate that the architecture of the F1 -ATPase complex is not strictly conserved in eukaryotes.