Search

Glyoxalases prevent the formation of advanced glycation end products by converting glycolysis-derived methylglyoxal to d-lactate with the help of glutathione. Vander Jagt and colleagues previously showed that erythrocytes release about thirty times more d-lactate after infection with the human malaria parasite Plasmodium falciparum. Functional glyoxalases in the host-parasite unit might therefore be crucial for parasite survival. Here, we determined the antimalarial and hemolytic activity of two tight-binding glyoxalase inhibitors using infected and uninfected erythrocytes. In addition, we synthesized and analyzed a set of diester derivates of both tight-binding inhibitors resulting in up to threefold lower IC50 values and an altered methemoglobin formation and hemolytic activity depending on the type of ester. Inhibitor treatments of uninfected erythrocytes revealed an extremely slow inactivation of the host cell glyoxalase, irrespective of inhibitor modifications, and a potential dispensability of the host cell enzyme for parasite survival. Our study highlights the benefits and drawbacks of different esterifications of glutathione-derived inhibitors and demonstrates the suitability of glyoxalase inhibitors as a tool for deciphering the relevance and mode of action of different glyoxalase systems in a host-parasite unit., Graphical abstract fx1

Objective Diabetic retinopathy (DR) is induced by an accumulation of reactive metabolites such as ROS, RNS, and RCS species, which were reported to modulate the activity of cation channels of the TRPC family. In this study, we use Trpc1/4/5/6−/− compound knockout mice to analyze the contribution of these TRPC proteins to diabetic retinopathy. Methods We used Nanostring- and qPCR-based analysis to determine mRNA levels of TRPC channels in control and diabetic retinae and retinal cell types. Chronic hyperglycemia was induced by Streptozotocin (STZ) treatment. To assess the development of diabetic retinopathy, vasoregression, pericyte loss, and thickness of individual retinal layers were analyzed. Plasma and cellular methylglyoxal (MG) levels, as well as Glyoxalase 1 (GLO1) enzyme activity and protein expression, were measured in WT and Trpc1/4/5/6−/− cells or tissues. MG-evoked toxicity in cells of both genotypes was compared by MTT assay. Results We find that Trpc1/4/5/6−/− mice are protected from hyperglycemia-evoked vasoregression determined by the formation of acellular capillaries and pericyte drop-out. In addition, Trpc1/4/5/6−/− mice are resistant to the STZ-induced reduction in retinal layer thickness. The RCS metabolite methylglyoxal, which represents a key mediator for the development of diabetic retinopathy, was significantly reduced in plasma and red blood cells (RBCs) of STZ-treated Trpc1/4/5/6−/− mice compared to controls. GLO1 is the major MG detoxifying enzyme, and its activity and protein expression were significantly elevated in Trpc1/4/5/6-deficient cells, which led to significantly increased resistance to MG toxicity. GLO1 activity was also increased in retinal extracts from Trpc1/4/5/6−/− mice. The TRPCs investigated here are expressed at different levels in endothelial and glial cells of the retina. Conclusion The protective phenotype in diabetic retinopathy observed in Trpc1/4/5/6−/− mice is suggestive of a predominant action of TRPCs in Müller cells and microglia because of their central position in the retention of a proper homoeostasis of the neurovascular unit., Highlights • Trpc1/4/5/6−/− mice are protected from hyperglycemia-evoked vasoregression and damage to the neurovascular unit. • The level of methylglyoxal (MG), which plays a crucial role in the development of DR is reduced in Trpc1/4/5/6−/− mice. • The protection against DR in Trpc1/4/5/6−/− mice may be mediated by an increase in GLO1 activity and protein expression.

KRIT1 (CCM1) is a disease gene responsible for Cerebral Cavernous Malformations (CCM), a major cerebrovascular disease of proven genetic origin affecting 0.3–0.5% of the population. Previously, we demonstrated that KRIT1 loss-of-function is associated with altered redox homeostasis and abnormal activation of the redox-sensitive transcription factor c-Jun, which collectively result in pro-oxidative, pro-inflammatory and pro-angiogenic effects, suggesting a novel pathogenic mechanism for CCM disease and raising the possibility that KRIT1 loss-of-function exerts pleiotropic effects on multiple redox-sensitive mechanisms. To address this possibility, we investigated major redox-sensitive pathways and enzymatic systems that play critical roles in fundamental cytoprotective mechanisms of adaptive responses to oxidative stress, including the master Nrf2 antioxidant defense pathway and its downstream target Glyoxalase 1 (Glo1), a pivotal stress-responsive defense enzyme involved in cellular protection against glycative and oxidative stress through the metabolism of methylglyoxal (MG). This is a potent post-translational protein modifier that may either contribute to increased oxidative molecular damage and cellular susceptibility to apoptosis, or enhance the activity of major apoptosis-protective proteins, including heat shock proteins (Hsps), promoting cell survival. Experimental outcomes showed that KRIT1 loss-of-function induces a redox-sensitive sustained upregulation of Nrf2 and Glo1, and a drop in intracellular levels of MG-modified Hsp70 and Hsp27 proteins, leading to a chronic adaptive redox homeostasis that counteracts intrinsic oxidative stress but increases susceptibility to oxidative DNA damage and apoptosis, sensitizing cells to further oxidative challenges. While supporting and extending the pleiotropic functions of KRIT1, these findings shed new light on the mechanistic relationship between KRIT1 loss-of-function and enhanced cell predisposition to oxidative damage, thus providing valuable new insights into CCM pathogenesis and novel options for the development of preventive and therapeutic strategies., Graphical abstract Schematic models representing adaptive redox responses associated with KRIT1 loss-of-function. KRIT1 loss-of-function causes a persistent activation of the redox-sensitive transcription factors c-Jun and Nrf2 and consequent upregulation of downstream targets, including cycloxygenase-2 (COX-2), heme oxygenase-1 (HO-1) and glyoxalase 1 (GLO1). While the c-Jun/COX-2 axis promotes pro-oxidant and pro-inflammatory effects, the Nrf2/HO-1 and Nrf2/GLO1 pathways mediate adaptive antioxidant responses that counteract these effects by limiting ROS* and MG intracellular accumulation, thus contributing to reduce a vicious cycle of oxidative stress and providing an adaptive defense for long term cell survival. However, this sustained adaptive redox homeostasis occurs at the expense of other cytoprotective mechanisms, including the MG-dependent formation of cytoprotective AP-Hsp70 and AP-Hsp27 protein adducts, leading to enhanced cell susceptibility to oxidative DNA damage and apoptosis, and sensitizing cells to additional stressful insults. Inter-individual differences in Nrf2-mediated adaptive defense mechanisms might influence susceptibility to CCM disease onset and progression. *The generic ROS term refers to O2•−and H2O2as well as to putative secondary oxidative products that might be implicated without certainty.fx1, Highlights • KRIT1 loss causes a chronic adaptive redox response based on the JNK-Nrf2-Glo1 axis. • Phospho-JNK, Nrf2 and Glo1 are upregulated in endothelial cells lining human CCMs. • Defective autophagy contributes to the sustained upregulation of the Nrf2-Glo1 axis. • Nrf2-Glo1 upregulation causes a drop of AP-modified Hsp70 and Hsp27 proteins. • Sustained Nrf2-Glo1 activation sensitizes cells to oxidative stress and apoptosis.