Park Yong-Sung, Kim Tae-Hyoung, Yun Cheol-Won
School of Life Sciences and Biotechnology, Korea University, Anam-dong, Sungbuk-gu, Seoul, Republic of Korea.
Department of Biochemistry, Chosun University School of Medicine, Dong-Gu, Gwang-Ju, Republic of Korea.
Biochem J. 2017 Jul 3;474(14):2365-2378. doi: 10.1042/BCJ20170191.
Although copper functions as a cofactor in many physiological processes, copper overload leads to harmful effects in living cells. Thus, copper homeostasis is tightly regulated. However, detailed copper metabolic pathways have not yet been identified in filamentous fungi. In this report, we investigated the copper transcription factor AfMac1 ( homolog) and identified its regulatory mechanism in AfMac1 has domains homologous to the DNA-binding and copper-binding domains of Mac1 from , and AfMac1 efficiently complemented Mac1 in Expression of resulted in up-regulation, and mutation of the DNA-binding domain of failed to activate expression in The deletion strain of failed to grow in copper-limited media, and its growth was restored by introducing We found that AfMac1 specifically bound to the promoter region of based on EMSA. The AfMac1-binding motif 5'-TGTGCTCA-3' was identified from the promoter region of , and the addition of mutant lacking the AfMac1-binding motif failed to up-regulate in Furthermore, deletion of significantly reduced strain virulence and activated conidial killing activity by neutrophils and macrophages. Taken together, these results suggest that AfMac1 is a copper transcription factor that regulates cellular copper homeostasis in .
尽管铜在许多生理过程中作为辅因子发挥作用，但铜过载会对活细胞产生有害影响。因此，铜稳态受到严格调控。然而，丝状真菌中详细的铜代谢途径尚未确定。在本报告中，我们研究了铜转录因子AfMac1（同源物），并确定了其在 中的调控机制。AfMac1具有与来自 的Mac1的DNA结合和铜结合结构域同源的结构域，并且AfMac1在 中有效地补充了Mac1。 的表达导致 上调，并且 的DNA结合结构域的突变未能在 中激活 表达。 的 缺失菌株在铜限制培养基中无法生长，通过引入 恢复了其生长。基于电泳迁移率变动分析（EMSA），我们发现AfMac1特异性结合到 的启动子区域。从 的启动子区域鉴定出AfMac1结合基序5'-TGTGCTCA-3'，并且添加缺乏AfMac1结合基序的突变体未能在 中上调 。此外， 的缺失显著降低了菌株毒力，并激活了中性粒细胞和巨噬细胞的分生孢子杀伤活性。综上所述，这些结果表明AfMac1是一种调节 中细胞铜稳态的铜转录因子。
