Ubiquitin Pathway Proteins Influence the Mechanism of Action of the Novel Immunosuppressive Drug FTY720 in Saccharomyces cerevisiae

Abstract

FTY720 is an immunosuppressive drug in clinical development for transplant graft protection in humans.This agent is of particular interest because, unlike currently available regimes, it acts to sequester lymphocytes without causing cytotoxicity or blocking differentiation and growth potential. In an effort to elucidate the mechanism of action of FTY720, and identify its downstream effectors, we have screened genomic libraries and spontaneous mutants of the model system Saccharomyces cerevisiae for resistance to FTY720. We identified several proteins and pathways as being involved in the mechanism of action of FTY720. We show specifically that the two amino acid transporters TAT1 and TAT2, the two ubiquitin proteases UBP5 and UBP11, and the heat shock protein CAJ1 confer growth resistance to FTY720 when overexpressed. Another amino acid transporter, GNP1, and the ubiquitin structural gene UBI4 as well as the ubiquitin ligase RSP5, and its binding protein BUL1 confer growth resistance in a mutated form. Supporting the importance of amino acid transport in the growth resistance phenotype of S. cerevisiae to the immunosuppressive agent FTY720, a prototrophic strain was more resistant to FTY720 than the isogenic auxotroph. To further explore these results, the effects on amino acid uptake and protein degradation were measured in the presence of FTY720. Due to the high conservation of these proteins and pathways between yeast and humans, these results may provide valuable insights into the mechanism of action of FTY720 in lymphocyte sequestration in humans.

Genetic, Biochemical, and Transcriptional Responses of Saccharomyces cerevisiae to the Novel Immunomodulator FTY720 Largely Mimic Those of the Natural Sphingolipid Phytosphingosine

“…The growth inhibitory concentration of FTY720 had to be readjusted from 30 to 15 M when we changed the batch of YPD medium (Difco). The concentration of 15 M corresponds, as in our previous work (15), to the concentration at which the growth of JK9-3d parent strain is inhibited and the bul1⌬ mutant is growth-resistant.…”

“…All plasmids are as described previously (15). Strains were maintained according to standard laboratory procedure (16).…”

“…Transformed cells were grown to an early logarithmic phase and treated with 25 M PHS for 1 h. Total proteins were extracted and quantified using a protein microassay (Pierce). 15 g of total protein was loaded per well, and immunoblotting was performed as described previously (15). The protein loading was checked by Coomassie Blue staining followed by quantitation.…”

“…Proteins-In our previous work (15) we showed that S. cerevisiae cells transfected to overexpress the ubiquitin-specific proteases UBP5 and UBP11 (mutants lacking the RSP5 ligand BULl, which is purported to be involved in ubiquitination target specificity, or lacking the ubiquitin structural gene UBI4) are resistant to FTY720. Here we show that each of these overexpressing strains and mutants are also resistant to a 25 M concentration of PHS ( Figs.…”

“…strains expressing multiple copies of tryptophan transporter genes such as TAT1 and TAT2 and the genes UBP5 and UBP11 (which specify proteases in the protein ubiquitination cascade) as well as the heat shock protein homolog gene CAJ1 are resistant to yeast growth inhibition mediated by FTY720 (15). In addition, loss-of-function mutations in the ubiquitin structural protein UBI4, a reduction of ubiquitin ligase protein RSP5, the deletion of RSP5-binding protein BUL1, and a mutation of the glutamine transporter gene GNP1 also confer resistance to FTY720-mediated growth inhibition.…”