Biochimica et Biophysica Acta (BBA) - General Subjects ( IF 3 ) Pub Date : 2020-11-07 , DOI: 10.1016/j.bbagen.2020.129781 Aslihan Ekim Kocabey , Gerhard Rödel , Uta Gey
Background
Besides their role in copper metabolism, Sco proteins from different organisms have been shown to play a defensive role against oxidative stress. In the present study, we set out to identify crucial amino acid residues for the antioxidant activity.
Methods
Native and mutated Sco proteins from human, Arabidopsis thaliana and the yeast Kluyveromyces lactis were expressed in the model organism Saccharomyces cerevisiae. The oxidative stress resistance of the respective transformants was determined by growth and lipid peroxidation assays.
Results
A functionally important site, located 15 amino acids downstream of the well-conserved copper binding CxxxC motif, was identified. Mutational analysis revealed that a positive charge at this position has a detrimental effect on the antioxidant capacity. Bioinformatic analysis predicts that this site is surface-exposed, and according to Co-IP data it is required for binding of proteins that are connected to known antioxidant pathways.
Conclusion
This study shows that the antioxidant capacity of eukaryotic Sco proteins is conserved and depends on the presence of functional site(s) rather than the extent of overall sequence homology.
General significance
These findings provide an insight into the conserved functional sites of eukaryotic Sco proteins that are crucial for combating oxidative stress. This capacity is probably not due to an enzymatic activity but rather is indirectly mediated by interaction with other proteins.
中文翻译:
Sco蛋白的抗氧化功能取决于关键的表面残留物
背景
除了它们在铜代谢中的作用外,来自不同生物体的Sco蛋白还显示出对氧化应激的防御作用。在本研究中,我们着手确定抗氧化活性的关键氨基酸残基。
方法
来自人,拟南芥和酵母克鲁维酵母的天然和突变的Sco蛋白在模型生物酿酒酵母中表达。通过生长和脂质过氧化测定来确定各个转化体的抗氧化应激性。
结果
确定了一个功能重要的位点,位于保守的铜结合CxxxC基序下游15个氨基酸。突变分析表明,该位置的正电荷对抗氧化能力有不利影响。生物信息学分析预测该位点是表面暴露的,根据Co-IP数据,结合与已知抗氧化剂途径相关的蛋白质是必需的。
结论
这项研究表明,真核Sco蛋白的抗氧化能力是保守的,取决于功能位点的存在而不是整个序列同源性的程度。
一般意义
这些发现为真核Sco蛋白质的保守功能位点提供了见解,而这些功能位点对抵抗氧化应激至关重要。该能力可能不是由于酶促活性,而是通过与其他蛋白质的相互作用间接介导的。