L-丝氨酸和硫化物引发IscS中醌类累积变红色机理及对铁硫簇合成的调控机制

Human IscS, a cysteine desulfurase, and its active auxiliary protein ISD11 are located in mitochondrion, and defects of them can lead to severe mitochondrion-related rare diseases. The L-cysteine can be catalyzed by cysteine desulfurase to generate sulfide, which is considered to be the only source of sulfide for the iron-sulfur cluster assembly. The function of cysteine desulfurase is highly conserved from prokaryotes to eukaryotes and even humans. However, the regulatory mechanism of cysteine desulfurase is still not fully elaborated. We recently demonstrated the color of E. coli IscS could turn red from yellow if expressed and purified from iscA/sufA double mutant, which was published on the JOURNAL OF BIOLOGICAL CHEMISTRY. We further found that this red IscS protein can also be produced with L-serine and sulfide in vivo and in vitro, and the red IscS was less active. Also, a stable red quinonoid intermediate can also be generateed in serine-glyoxylate aminotransferase, a PLP-binding protein, which similar to IscS. It is speculated that the red substances in IscS may be the similar quinonoid intermediates and their accumulation can regulate IscS enzyme activities with L-serine and sulfide, which also regulate the biosynthesis of iron-sulfur clusters. To test this hypothesis, we aim to: 1) systematically characterize the function of L-serine and sulfides in production of IscS red; 2) try to isolate and identify the component of red IscS, explore the binding sites and binding ways in IscS protein; 3) analyze the effect of the accumulation of red IscS on the enzyme activity; 4) research on the generation of red IscS how to affect iron-sulfur cluster biogenesis. The synthesis mechanism of red quinones in IscS and the regulation mechanism of iron-sulfur cluster assembly are expected. If the project can be successfully carried out, it will lay a theoretical foundation for the research on the mechanism and prevention of rare diseases caused by defect of synthesis and regulation of iron-sulfur clusters.

人IscS或其辅助蛋白缺陷均导致严重的线粒体罕见病，IscS为半胱氨酸脱硫酶，具有为铁硫簇合成提供硫的保守功能，而其活性调节机制不完全清楚。课题组前期阐述了IscS在iscA/sufA双敲除菌中表达由黄色变为红色的现象和可能的机制（发表于JBC）。后续体内、体外研究发现L-丝氨酸和硫化物导致IscS变红且活性降低，综合类似酶能生成稳定红色醌类物质等现有研究推测IscS中的红色物质可能为醌类且可以在L-丝氨酸和硫化物的作用下累积并调节自身酶活进而调控铁硫簇的生物合成。为此将①系统分析L-丝氨酸和硫化物对IscS红色生成的作用②尝试分离、分析红色物质，探究与蛋白结合位点和方式③分析红色的累积对酶活的影响④分析红色的生成对铁硫簇组装的调节。预期阐明IscS中红色醌类物质的合成机理和对铁硫簇组装调控机制。本项目的顺利展开将为铁硫簇生物合成调控、合成障碍引起罕见病的致病机理和防治等研究奠定理论基础。

人IscS或其辅助蛋白缺陷均导致严重的线粒体罕见病，IscS为半胱氨酸脱硫酶，具有为铁硫簇合成提供硫的保守功能，而其活性调节机制不完全清楚。课题组前期阐述了IscS在iscA/sufA双敲除菌中表达由黄色变为红色的现象和可能的机制。后续体内、体外研究发现L-丝氨酸和硫化物导致IscS变红且活性降低，综合类似酶能生成稳定红色醌类物质等现有研究推测IscS中的红色物质可能为醌类且可以在L-丝氨酸和硫化物的作用下累积并调节自身酶活进而调控铁硫簇的生物合成。为此将①系统分析L-丝氨酸和硫化物对IscS红色生成的作用②尝试分离、分析红色物质，探究与蛋白结合位点和方式③分析红色的累积对酶活的影响④分析红色的生成对铁硫簇组装的调节。本项目执行期间取得明显成果，在中科院二区Top期刊发表相关论文2篇，申请国家专利2个（其中1项已授权,目前状态待公告），培养了1名博士研究生，并资助其前往美国路易斯安那州立大学 Huangen Ding 教授课题组学术进修。该课题的顺利进展为铁硫簇生物合成调控、合成障碍引起罕见病的致病机理和防治等研究奠定了理论基础。