Short OGA is targeted to the mitochondria and regulates mitochondrial reactive oxygen species level

O-GlcNAcylation is a reversible post-translational modification involved the regulation of cytosolic, nuclear and mitochondrial proteins. Only two enzymes, OGT and OGA, control attachment and removal of O-GlcNAc on proteins, respectively. Whereas a variant OGT (mOGT) has been proposed as the main isoform that O-GlcNAcylates proteins in mitochondria, identification of a mitochondrial OGA has not been performed yet. Two splice variants of OGA (short and long isoforms) have been described previously. In this work, using cell fractionation experiments, we show that short-OGA is preferentially recovered in mitochondria-enriched fractions from HEK-293T cells as well as mouse embryonic fibroblasts. Moreover, fluorescent microscopy imaging confirmed that GFP-tagged short-OGA is addressed to mitochondria. In addition, using a BRET-based mitochondrial O-GlcNAcylation biosensor, we show that co-transfection of short-OGA markedly reduced O-GlcNAcylation of the biosensor, whereas long-OGA had no significant effect. Finally, using genetically encoded or chemical fluorescent mitochondrial probes, we showed that short-OGA overexpression increases mitochondrial ROS levels, whereas long-OGA had no significant effect. Together, our work reveals that the short-OGA isoform is targeted to the mitochondria where it regulates ROS homoeostasis.

Attenuating Effect of Chlorella Extract on NLRP3 Inflammasome Activation by Mitochondrial Reactive Oxygen Species

The NOD-like receptor family, pyrin domain-containing protein 3 (NLRP3) inflammasome has been linked to the pathogenesis of a wide variety of human diseases. Although many drugs and inhibitors have been developed to treat NLRP3-associated diseases, only limited clinical data support their efficacy and safety. Chlorella, a unicellular green alga that is widely and safely used as a food supplement, contains various antioxidants. In this study, we obtained a fat-soluble extract from Chlorella (CE) and demonstrated that it reduced NLRP3 inflammasome activation by inhibiting mitochondrial reactive oxygen species and caspase-1 activation. In addition, CE supplementation attenuated lipopolysaccharide-induced interleukin 1β transcription through activation of hypoxia-inducible factor 1α in vitro and in vivo. As Chlorella is a safe and useful food supplement, it may be a practical pharmacological approach for treating NLRP3-driven diseases.

Lysophosphatidylcholine acyltransferase 1 controls the mitochondrial reactive oxygen species generation and survival of the retinal photoreceptor cells

ABSTRACTDue to the high energy demands and characteristic morphology, retinal photoreceptor cells require a specialized lipid metabolism for survival and function. Accordingly, dysregulation of lipid metabolism leads to the photoreceptor cell death and retinal degeneration. Mice with a frameshift mutation of lysophosphatidylcholine acyltransferase 1 (Lpcat1), which produces saturated phosphatidylcholine (PC) composed of two saturated fatty acids, has been reported to cause spontaneous retinal degeneration (rd11 mice). In this study, we performed a detailed characterization of LPCAT1 in the retina and found that genetic deletion of Lpcat1 induces light-independent and photoreceptor-specific apoptosis in mice. Lipidomic analyses of the retina and isolated photoreceptor outer segment (OS) suggested that loss of Lpcat1 decreases saturated PC production and affects the proper cellular fatty acid flux, presumably by altering saturated fatty acyl-CoA availabilities. Furthermore, we demonstrated that Lpcat1 deletion increased mitochondrial reactive oxygen species (ROS) levels in photoreceptor cells, but not in other retinal cells without affecting the OS structure and trafficking of OS-localized proteins. These results suggest that the LPCAT1-dependent production of saturated PC is critical for metabolic adaptation during photoreceptor maturation. Our findings highlight the therapeutic potential of saturated fatty acid metabolism in photoreceptor cell degeneration-related retinal diseases.

Mitochondrial Permeability Transition Causes Mitochondrial Reactive Oxygen Species- and Caspase 3-Dependent Atrophy of Single Adult Mouse Skeletal Muscle Fibers

Elevated mitochondrial reactive oxygen species (mROS) and an increase in caspase-3 activity are established mechanisms that lead to skeletal muscle atrophy via the upregulation of protein degradation pathways. However, the mechanisms upstream of an increase in mROS and caspase-3 activity in conditions of muscle atrophy have not been identified. Based upon knowledge that an event known as mitochondrial permeability transition (MPT) causes an increase in mROS emission and the activation of caspase-3 via mitochondrial release of cytochrome c, as well as the circumstantial evidence for MPT in some muscle atrophy conditions, we tested MPT as a mechanism of atrophy. Briefly, treating cultured single mouse flexor digitorum brevis (FDB) fibers from adult mice with a chemical inducer of MPT (Bz423) for 24 h caused an increase in mROS and caspase-3 activity that was accompanied by a reduction in muscle fiber diameter that was able to be prevented by inhibitors of MPT, mROS, or caspase-3 (p < 0.05). Similarly, a four-day single fiber culture as a model of disuse caused atrophy that could be prevented by inhibitors of MPT, mROS, or activated caspase-3. As such, our results identify MPT as a novel mechanism of skeletal muscle atrophy that operates through mROS emission and caspase-3 activation.