Cloning, heterologous expression, and substrate specificities of protein farnesyltransferases from Trypanosoma cruzi and Leishmania major.
Chagas disease and leishmaniasis are tropical diseases caused by the protozoan parasites, Trypanosoma cruzi and Leishmania species, respectively. Protein farnesyltransferase (PFT) is being investigated as a target for anti-trypanosomatid agents because inhibitors of this enzyme are highly toxic to these parasites compared to mammalian cells. Here, we report the cloning of the alpha- and beta-subunit genes of PFT from T. cruzi and Leishmania major. The proteins encoded by these genes are considerably larger than those of mammalian PFTs due to the presence of a number of inserts of >25 amino acids that map to junctions between helical structural elements. These inserts are not part of the active site or the interface between the two subunits. Northern blots demonstrate expression of messenger RNA for the PFT subunits in both mammalian and insect life-cycle stages of these parasites. The T. cruzi, Trypanosoma brucei, and L. major PFTs were overexpressed in the Sf9 cell/baculovirus system as active enzyme forms. Kinetic studies with a panel of CALX-containing peptides with all 20 amino acids in the X-position show that trypanosomatid PFTs have similar substrate specificities and these are different from the mammalian PFT substrate specificity patterns.
Buckner
Frederick S
FS
Department of Medicine, University of Washington, Seattle 98195, USA. fbukner@u.washington.edu
Eastman
Richard T
RT
Nepomuceno-Silva
José L
JL
Speelmon
Emily C
EC
Myler
Peter J
PJ
Van Voorhis
Wesley C
WC
Yokoyama
Kohei
K
D019883:Alkyl and Aryl Transferases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000981:Antiprotozoal Agents / Q000494:pharmacology; D016367:Baculoviridae / Q000235:genetics; D003001:Cloning, Molecular; D016054:DNA, Protozoan / Q000032:analysis; D015195:Drug Design; D018320:Leishmania major / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D018411:Spodoptera; D013379:Substrate Specificity; D014349:Trypanosoma cruzi / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics
D000981:Antiprotozoal Agents; D016054:DNA, Protozoan; D011994:Recombinant Proteins; D019883:Alkyl and Aryl Transferases; C064911:p21(ras) farnesyl-protein transferase