S-Nitrosylation-mediated coupling of DJ-1 with PTEN induces PI3K/AKT/mTOR pathway-dependent keloid formation

Background:Keloids are aberrant dermal wound healing characterized by invasive growth,extracellular matrix deposition,cytokine overexpression and easy recurrence.Many factors have been implicated as pathological causes of keloids,particularly hyperactive inflammation,tension alignment and genetic predisposition.S-Nitrosylation(SNO),a unique form of protein modification,is associated with the local inflammatory response but its function in excessive fibrosis and keloid formation remains unknown.We aimed to discover the association between protein SNO and keloid formation.Methods:Normal and keloid fibroblasts were isolated from collected normal skin and keloid tissues.The obtained fibroblasts were cultured in DMEM supplemented with 10%fetal bovine serum and 1%penicillin/streptomycin.The effects of DJ-1 on cell proliferation,apoptosis,migration and invasion,and on the expression of proteins were assayed.TurboID-based proximity labelling and liquid chromatography-mass spectrometry were conducted to explore the potential targets of DJ-1.Biotin-switch assays and transnitrosylation reactions were used to detect protein SNO.Quantitative data were compared by two-tailed Student’s t test.Results:We found that DJ-1 served as an essential positive modulator to facilitate keloid cell proliferation,migration and invasion.A higher S-nitrosylated DJ-1(SNO-DJ-1)level was observed in keloids,and the effect of DJ-1 on keloids was dependent on SNO of the Cys106 residue of the DJ-1 protein.SNO-DJ-1 was found to increase the level of phosphatase and tensin homolog(PTEN)S-nitrosylated at its Cys136 residue via transnitrosylation in keloids,thus diminishing the phosphatase activity of PTEN and activating the PI3K/AKT/mTOR pathway.Furthermore,Cys106-mutant DJ-1 is refractory to SNO and abrogates DJ-1-PTEN coupling and the SNO of the PTEN protein,thus repressing the PI3K/AKT/mTOR pathway and alleviating keloid formation.Importantly,the biological effect of DJ-1 in keloids is dependent on the SNO-DJ-1/SNO-PTEN/PI3K/AKT/mTOR axis.Conclusions:For the first time,this study demonstrated the effect of transnitrosylation from DJ-1 to PTEN on promoting keloid formation via the PI3K/AKT/mTOR signaling pathway,suggesting that SNO of DJ-1 may be a novel therapeutic target for keloid treatment.

High-Resolution Crystal Structure and Redox Properties of Chloroplastic Triosephosphate Isomerase from Chlamydomonas reinhardtii

Triosephosphate isomerase （TPI） catalyzes the interconversion of glyceraldehyde-3-phosphate to dihydroxyacetone phosphate. Photosynthetic organisms generally contain two isoforms of TPI located in both cytoplasm and chloroplasts. While the cytoplasmic TPI is involved in the glycolysis, the chloroplastic isoform participates in the Calvin-Benson cycle, a key photosynthetic process responsible for carbon fixation. Compared with its cytoplasmic counterpart, the functional features of chloroplastic TPI have been poorly investigated and its three-dimensional structure has not been solved. Recently, several studies proposed TPI as a potential target of different redox modifications including dithiol/disulfide interchanges, glutathionylation, and nitrosylation. However, neither the effects on protein activity nor the molecular mechanisms underlying these redox modifications have been investigated. Here, we have produced recombinantly and purified TPI from the unicellular green alga Chlamydomonas reinhardtii （Cr）. The biochemical properties of the enzyme were delineated and its crystallographic structure was determined at a resolution of 1.1 A. CrTPI is a homodimer with subunits containing the typical （β/α）8-barrel fold. Although no evidence for TRX regulation was obtained, CrTPI was found to undergo glutathionylation by oxidized glutathione and trans-nitrosylation by nitrosoglutathione, confirming its sensitivity to multiple redox modifications.