Dihydrotanshinone I preconditions myocardium against ischemic injury via PKM2 glutathionylation sensitive to ROS

Ischemic preconditioning(IPC)is a potential intervention known to protect the heart against ischemia/reperfusion injury,but its role in the no-reflow phenomenon that follows reperfusion is unclear.Dihydrotanshinone I(DT)is a natural compound and this study illustrates its role in cardiac ischemic injury from the aspect of IPC.Pretreatment with DT induced modest ROS production and protected cardiomyocytes against oxygen and glucose deprivation(OGD),but the protection was prevented by a ROS scavenger.In addition,DT administration protected the heart against isoprenaline challenge.Mechanistically,PKM2 reacted to transient ROS via oxidization at Cys423/Cys424,leading to glutathionylation and nuclear translocation in dimer form.In the nucleus,PKM2 served as a co-factor to promote HIF-1a-dependent gene induction,contributing to adaptive responses.In mice subjected to permanent coronary ligation,cardiac-specific knockdown of Pkm2 blocked DT-mediated preconditioning protection,which was rescued by overexpression of wild-type Pkm2,rather than Cys423/424-mutated Pkm2.In conclusion,PKM2 is sensitive to oxidation,and subsequent glutathionylation promotes its nuclear translocation.Although IPC has been viewed as a protective means against reperfusion injury,our study reveals its potential role in protection of the heart from no-reflow ischemia.

Methods for Analysis of Protein Glutathionylation and their Application to Photosynthetic Organisms

Protein S-glutathionylation, the reversible formation of a mixed-disulfide between glutathione and protein thiols, is involved in protection of protein cysteines from irreversible oxidation, but also in protein redox regulation. Recent studies have implicated S-glutathionylation as a cellular response to oxidative/nitrosative stress, likely playing an important role in signaling. Considering the potential importance of glutathionylation, a number of methods have been developed for identifying proteins undergoing glutathionylation. These methods, ranging from analysis of purified proteins in vitro to large-scale proteomic analyses in vivo, allowed identification of nearly 200 targets in mammals. By contrast, the number of known glutathionylated proteins is more limited in photosynthetic organisms, although they are severely exposed to oxidative stress. The aim of this review is to detail the methods available for identification and analysis of glutathionylated proteins in vivo and in vitro. The advantages and drawbacks of each technique will be discussed as well as their application to photosynthetic organisms. Furthermore, an overview of known glutathionylated proteins in photosynthetic organisms is provided and the physiological importance of this post-translational modification is discussed.

Redox-dependent regulation of end-binding protein 1 activity by glutathionylation

Cytoskeletal proteins are susceptible to glutathionylation under oxidizing conditions,and oxidative damage has been implicated in several neurodegenerative diseases.End-binding protein 1(EB1)is a master regulator of microtubule plus-end tracking proteins(+TIPs)and is critically involved in the control of microtubule dynamics and cellular processes.However,the impact of glutathionylation on EB1 functions remains unknown.Here we reveal that glutathionylation is important for controlling EB1 activity and protecting EB1 from irreversible oxidation.In vitro biochemical and cellular assays reveal that EB1 is glutathionylated.Diamide,a mild oxidizing reagent,reduces EB1 comet number and length in cells,indicating the impairment of microtubule dynamics.Three cysteine residues of EB1 are glutathionylated,with mutations of these three cysteines to serines attenuating microtubule dynamics but buffering diamide-induced decrease in microtubule dynamics.In addition,glutaredoxin 1(Grx1)deglutathionylates EB1,and Grx1 depletion suppresses microtubule dynamics and leads to defects in cell division orientation and cell migration,suggesting a critical role of Grx1-mediated deglutathionylation in maintaining EB1 activity.Collectively,these data reveal that EB1 glutathionylation is an important protective mechanism for the regulation of microtubule dynamics and microtubule-based cellular activities.

The role of glutathione-mediated triacylglycerol synthesis in the response to ultra-high cadmium stress in Auxenochlorella protothecoides

Under ultra-high cadmium(Cd)stress,large amounts of glutathione are produced in Auxenochlorella protothecoides UTEX 2341,and the lipid content increases significantly.Glutathione is the best reductant that can effectively remove Cd,but the relationship between lipid accumulation and the cellular response to Cd stress has not been ascertained.Integrating analyses of the transcriptomes and lipidomes,the mechanism of lipid accumulation to Cd tolerance were studied from the perspectives of metabolism,transcriptional regulation and protein glutathionylation.Under Cd stress,basic metabolic pathways,such as purine metabolism,translation and pre-m RNA splicing process,were inhibited,while the lipid accumulation pathway was significantly activated.Further analysis revealed that the transcription factors(TFs)and genes related to lipid accumulation were also activated.Analysis of the TF interaction sites showed that ABI5,MYBrel and NF-YB could further regulate the expression of diacylglycerol acyltransferase through glutathionylation/deglutathionylation,which led to increase of the triacylglycerol(TAG)content.Lipidomes analysis showed that TAG could help maintain lipid homeostasis by adjusting its saturation/unsaturation levels.This study for the first time indicated that glutathione could activate TAG synthesis in microalga A.protothecoides,leading to TAG accumulation and glutathione accumulation under Cd stress.Therefore,the accumulation of TAG and glutathione can confer resistance to high Cd stress.This study provided insights into a new operation mode of TAG accumulation under heavy metal stress.