Onnebo Sara Maria Nancy, Saiardi Adolfo
Department of Cell and Developmental Biology, University College London, UK.
Biochem J. 2009 Sep 14;423(1):109-18. doi: 10.1042/BJ20090241.
Inositol pyrophosphates are involved in a variety of cellular functions, but the specific pathways and/or downstream targets remain poorly characterized. In the present study we use Saccharomyces cerevisiae mutants to examine the potential roles of inositol pyrophosphates in responding to cell damage caused by ROS (reactive oxygen species). Yeast lacking kcs1 [the S. cerevisiae IP6K (inositol hexakisphosphate kinase)] have greatly reduced IP7 (diphosphoinositol pentakisphosphate) and IP8 (bisdiphosphoinositol tetrakisphosphate) levels, and display increased resistance to cell death caused by H2O2, consistent with a sustained activation of DNA repair mechanisms controlled by the Rad53 pathway. Other Rad53-controlled functions, such as actin polymerization, appear unaffected by inositol pyrophosphates. Yeast lacking vip1 [the S. cerevisiae PP-IP5K (also known as IP7K, IP7 kinase)] accumulate large amounts of the inositol pyrophosphate IP7, but have no detectable IP8, indicating that this enzyme represents the physiological IP7 kinase. Similar to kcs1Delta yeast, vip1Delta cells showed an increased resistance to cell death caused by H2O2, indicating that it is probably the double-pyrophosphorylated form of IP8 [(PP)2-IP4] which mediates the H2O2 response. However, these inositol pyrophosphates are not involved in directly sensing DNA damage, as kcs1Delta cells are more responsive to DNA damage caused by phleomycin. We observe in vivo a rapid decrease in cellular inositol pyrophosphate levels following exposure to H2O2, and an inhibitory effect of H2O2 on the enzymatic activity of Kcs1 in vitro. Furthermore, parallel cysteine mutagenesis studies performed on mammalian IP6K1 are suggestive that the ROS signal might be transduced by the direct modification of this evolutionarily conserved class of enzymes.
肌醇焦磷酸参与多种细胞功能，但具体途径和/或下游靶点仍未得到充分表征。在本研究中，我们使用酿酒酵母突变体来研究肌醇焦磷酸在应对由活性氧（ROS）引起的细胞损伤中的潜在作用。缺乏kcs1[酿酒酵母IP6K（肌醇六磷酸激酶）]的酵母，其IP7（二磷酸肌醇五磷酸）和IP8（双二磷酸肌醇四磷酸）水平大幅降低，并表现出对H2O2引起的细胞死亡的抗性增加，这与由Rad53途径控制的DNA修复机制的持续激活一致。其他由Rad53控制的功能，如肌动蛋白聚合，似乎不受肌醇焦磷酸的影响。缺乏vip1[酿酒酵母PP-IP5K（也称为IP7K，IP7激酶）]的酵母积累大量的肌醇焦磷酸IP7，但没有可检测到的IP8，表明该酶代表生理性IP7激酶。与kcs1Δ酵母类似，vip1Δ细胞对H2O2引起的细胞死亡表现出抗性增加，表明可能是IP8的双焦磷酸化形式[(PP)2-IP4]介导了H2O2反应。然而，这些肌醇焦磷酸并不直接参与感知DNA损伤，因为kcs1Δ细胞对博来霉素引起的DNA损伤更敏感。我们在体内观察到暴露于H2O2后细胞肌醇焦磷酸水平迅速下降，并且H2O2在体外对Kcs1的酶活性有抑制作用。此外，对哺乳动物IP6K1进行的平行半胱氨酸诱变研究表明，ROS信号可能通过对这一进化保守的酶类的直接修饰来转导。
