RNA editing in kinetoplastid mitochondria occurs by a series of enzymatic steps that is catalyzed by a macromolecular complex. Four novel proteins and their corresponding genes were identified by mass spectrometric analysis of purified editing complexes from Trypanosoma brucei. These four proteins, TbMP81, TbMP63, TbMP42, and TbMP18, contain conserved sequences to various degrees. All four proteins have sequence similarity in the C terminus; TbMP18 has considerable sequence similarity to the C-terminal region of TbMP42, and TbMP81, TbMP63, and TbMP42 contain zinc finger motif(s). Monoclonal antibodies that are specific for TbMP63 and TbMP42 immunoprecipitate in vitro RNA editing activities. The proteins are present in the immunoprecipitates and sediment at 20S along with the in vitro editing, and RNA editing ligases TbMP52 and TbMP48. Recombinant TbMP63 and TbMP52 coimmunoprecipitate. These results indicate that these four proteins are components of the RNA editing complex and that TbMP63 and TbMP52 can interact.

Trypanosoma brucei, the causative agent of African sleeping sickness, has three nearly identical genes encoding cysteine homologues of classical selenocysteine-containing glutathione peroxidases. The proteins are expressed in the mammalian and insect stages of the parasite. One of the genes, which contains a mitochondrial as well as a glycosomal targeting signal has been overexpressed. The recombinant T. brucei peroxidase has a high preference for the trypanothione/tryparedoxin couple as electron donor for the reduction of different hydroperoxides but accepts also T. brucei thioredoxin. The apparent rate constants k(2)' for the regeneration of the reduced enzyme are 2 x 10(5) m(-1) s(-1) with tryparedoxin and 5 x 10(3) m(-1) s(-1) with thioredoxin. No saturation kinetics was observed and the rate-limiting step of the overall reaction is reduction of the hydroperoxide. With glutathione, the peroxidase has marginal activity and reduction of the enzymes becomes limiting with a k(2)' value of 3 m (-1) s(-1). The T. brucei peroxidase, in contrast to the related Trypanosoma cruzi enzyme, also accepts hydrogen peroxide as substrate. The catalytic efficiency of the peroxidase studied here is comparable with that of the peroxiredoxin-like tryparedoxin peroxidases, which shows that trypanosomes possess two distinct peroxidase systems both dependent on the unique dithiol trypanothione.

We have cloned and characterized two distinct cysteine protease cDNAs from Leishmania donovani chagasi. One of the cDNAs, Ldccy2, was isolated from a cDNA library prepared from total promastigote RNA while the other cDNA, Ldccys1, was isolated from a cDNA library prepared from total amastigote RNA. Ldccys2 has an open reading frame of 471 amino acids and Ldccys1 has an open reading frame of 447 amino acids. Comparison of the predicted protein sequences of the two distinct cysteine proteases with those of cysteine proteases from Leishmania pifanoi, a member of the L. mexicana complex, showed that the cysteine proteases from the two species of Leishmania are similar in their protein sequences. Each of the two cDNAs is distinct in genomic arrangement and chromosome location. Ldccys1 belongs to a family of cysteine proteases encoded by tandemly organized genes located on chromosome 7 while Ldccys2 appears to be a single cysteine protease gene located on chromosome 10. The organization of the two families of cysteine protease genes in L. donovani donovani was also found to be similar. In this species, the Lddcys1 genes are located on chromosome 5 while the Lddcys2 gene is located on chromosome 8. The Ldccys1 genes are expressed abundantly in the amastigotes recovered from infected hamsters, but at a very low level in the promastigote stage of development. On the other hand, the Ldccys2 gene is expressed both in the promastigote and amastigote stages. We have overexpressed the two cDNAs of cysteine proteases in Leishmania cells and the over-produced cysteine proteases are biologically active and are inhibited by cysteine protease inhibitors. Furthermore, the over-produced and indigenous amastigote specific cysteine protease, Ldccys1, reacted with polyclonal antibodies raised against this protein.

Ten to twelve copies of the 83-kDa heat-shock protein gene (hsp83) from Trypanosoma brucei are arranged in a head-to-tail tandem array of 2.8-kb repeat units, which are transcribed to give 2.6-kb mature mRNAs. We have cloned and sequenced one of the repeat units. The gene encodes a putative protein of 81 kDa which is highly homologous to Hsp83 of Drosophila melanogaster (75%), Hsp90 of Saccharomyces cerevisiae (72%) and the C62.5 protein of Escherichia coli (61%). The 5' end of the mature mRNA was mapped by primer extension sequence analysis and shown to contain the spliced leader. The mapping of the 3' poly(A) addition sites by S1 analysis indicated that there is 218 nt of intergenic sequence linking the boundaries encoding the mature mRNA. Within this sequence are a number of elements conserved with the trypanosome hsp70 intergenic region, including a 14-nt sequence that also has homology to the Drosophila heat-shock consensus element.

LR: 20061115; JID: 8509412; 0 (DNA, Neoplasm); 0 (DNA, Single-Stranded); ppublish

LR: 20071114; GR: CA41025/CA/NCI NIH HHS/United States; GR: CA45583/CA/NCI NIH HHS/United States; GR: CA47512/CA/NCI NIH HHS/United States; JID: 9421547; 0 (Codon); 0 (DNA, Neoplasm); EC 3.6.5.2 (HRAS protein, human); EC 3.6.5.2 (Proto-Oncogene Proteins p21(ras)); ppublish

LR: 20061115; JID: 0374630; ppublish

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