Bozaquel-Morais Bruno L, Madeira Juliana B, Venâncio Thiago M, Pacheco-Rosa Thiago, Masuda Claudio A, Montero-Lomeli Monica
Instituto de Bioquímica Médica Leopoldo de Meis, Programa de Biologia Molecular e Biotecnologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
Universidade Estadual do Norte Fluminense Darcy Ribeiro, Campos dos Goytacazes, Rio de Janeiro, Brazil.
PLoS One. 2017 Jan 11;12(1):e0169682. doi: 10.1371/journal.pone.0169682. eCollection 2017.
Acetyl-CoA carboxylase (Acc1p) is a key enzyme in fatty acid biosynthesis and is essential for cell viability. To discover new regulators of its activity, we screened a Saccharomyces cerevisiae deletion library for increased sensitivity to soraphen A, a potent Acc1p inhibitor. The hits identified in the screen (118 hits) were filtered using a chemical-phenotype map to exclude those associated with pleiotropic drug resistance. This enabled the identification of 82 ORFs that are genetic interactors of Acc1p. The main functional clusters represented by these hits were "transcriptional regulation", "protein post-translational modifications" and "lipid metabolism". Further investigation of the "transcriptional regulation" cluster revealed that soraphen A sensitivity is poorly correlated with ACC1 transcript levels. We also studied the three top unknown ORFs that affected soraphen A sensitivity: SOR1 (YDL129W), SOR2 (YIL092W) and SOR3 (YJR039W). Since the C18/C16 ratio of lipid acyl lengths reflects Acc1p activity levels, we evaluated this ratio in the three mutants. Deletion of SOR2 and SOR3 led to reduced acyl lengths, suggesting that Acc1p is indeed down-regulated in these strains. Also, these mutants showed no differences in Snf1p/AMPK activation status and deletion of SNF1 in these backgrounds did not revert soraphen A sensitivity completely. Furthermore, plasmid maintenance was reduced in sor2Δ strain and this trait was shared with 18 other soraphen A sensitive hits. In summary, our screen uncovered novel Acc1p Snf1p/AMPK-independent regulators.
乙酰辅酶A羧化酶（Acc1p）是脂肪酸生物合成中的关键酶，对细胞活力至关重要。为了发现其活性的新调节因子，我们在酿酒酵母缺失文库中筛选了对索拉非尼A（一种有效的Acc1p抑制剂）敏感性增加的菌株。利用化学表型图谱对筛选出的命中基因（118个命中基因）进行过滤，以排除与多药耐药相关的基因。这使得我们能够鉴定出82个作为Acc1p遗传相互作用因子的开放阅读框（ORF）。这些命中基因所代表的主要功能簇为“转录调控”、“蛋白质翻译后修饰”和“脂质代谢”。对“转录调控”簇的进一步研究表明，索拉非尼A敏感性与ACC1转录水平的相关性较差。我们还研究了影响索拉非尼A敏感性的三个最主要的未知ORF：SOR1（YDL129W）、SOR2（YIL092W）和SOR3（YJR039W）。由于脂质酰基长度的C18/C16比值反映了Acc1p的活性水平，我们在这三个突变体中评估了该比值。SOR2和SOR3的缺失导致酰基长度缩短，这表明在这些菌株中Acc1p确实被下调。此外，这些突变体在Snf1p/AMPK激活状态上没有差异，并且在这些背景下删除SNF1并没有完全恢复索拉非尼A敏感性。此外，sor2Δ菌株中的质粒维持能力降低，这一特征也与其他18个对索拉非尼A敏感的命中基因相同。总之，我们的筛选发现了新的不依赖于Acc1p Snf1p/AMPK的调节因子。
