Molecular and functional characterization of the ceramide synthase from Trypanosoma cruzi.
In this study, we characterized ceramide synthase (CerS) of the protozoan parasite Trypanosoma cruzi at the molecular and functional levels. TcCerS activity was detected initially in a cell-free system using the microsomal fraction of epimastigote forms of T. cruzi, [(3)H]dihydrosphingosine or [(3)H]sphingosine, and fatty acids or acyl-CoA derivatives as acceptor or donor substrates, respectively. TcCerS utilizes both sphingoid long-chain bases, and its activity is exclusively dependent on acyl-CoAs, with palmitoyl-CoA being preferred. In addition, Fumonisin B(1), a broad and well-known acyl-CoA-dependent CerS inhibitor, blocked the parasite's CerS activity. However, unlike observations in fungi, the CerS inhibitors Australifungin and Fumonisin B(1) did not affect the proliferation of epimastigotes in culture, even after exposure to high concentrations or after extended periods of treatment. A search of the parasite genome with the conserved Lag1 motif from Lag1p, the yeast acyl-CoA-dependent CerS, identified a T. cruzi candidate gene (TcCERS1) that putatively encodes the parasite's CerS activity. The TcCERS1 gene was able to functionally complement the lethality of a lag1Δ lac1Δ double deletion yeast mutant in which the acyl-CoA-dependent CerS is not detectable. The complemented strain was capable of synthesizing normal inositol-containing sphingolipids and is 10 times more sensitive to Fumonisin B(1) than the parental strain.
Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Centro de Ciências da Saúde Bloco G-019, Av. Carlos Chagas Filho 373, Cidade Universitária, Ilha do Fundão, Rio de Janeiro, RJ 21941-902, Brazil.
Juliana M
JM
Figueiredo
Deivid C
DC
Rodrigues
Rafael C M C
RC
Silva
Carolina M
CM
Koeller
James C
JC
Jiang
S Michal
SM
Jazwinski
José O
JO
Previato
Lucia
L
Mendonça-Previato
Turán P
TP
Urményi
Norton
N
Heise
D000214:Acyl Coenzyme A / Q000378:metabolism / Q000494:pharmacology; D003001:Cloning, Molecular; D003455:Cryptococcus neoformans / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D003470:Culture Media; D004789:Enzyme Activation; D057075:Enzyme Assays; D037341:Fumonisins / Q000494:pharmacology; D017125:Genes, Protozoan; D005816:Genetic Complementation Test; D018503:Genome, Protozoan; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010767:Phosphorylcholine / Q000031:analogs & derivatives / Q000494:pharmacology; D010802:Phylogeny; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D017384:Sequence Deletion; D013110:Sphingosine / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D013764:Tetrahydronaphthalenes / Q000494:pharmacology; D013997:Time Factors; D014349:Trypanosoma cruzi / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics
D000214:Acyl Coenzyme A; D003470:Culture Media; D037341:Fumonisins; D015800:Protozoan Proteins; D013764:Tetrahydronaphthalenes; C094344:australifungin; D010767:Phosphorylcholine; C056933:fumonisin B1; C039128:miltefosine; D010088:Oxidoreductases; C108138:dihydroceramide desaturase; D013110:Sphingosine; C005682:safingol