The Metabolomic Response of Crucian Carp (Carassius carassius) to Anoxia and Reoxygenation Differs between Tissues and Hints at Uncharacterized Survival Strategies.

The anoxia-tolerant crucian carp (Carassius carassius) has been studied in detail for numerous years, with particular focus on unravelling the underlying physiological mechanisms of anoxia tolerance. However, relatively little work has been focused on what occurs beyond anoxia, and often the focus is a single organ or tissue type. In this study, we quantified more than 100 metabolites by capillary electrophoresis-mass spectrometry (CE-MS) in brain, heart, liver, and blood plasma from four experimental groups, being normoxic (control) fish, anoxia-exposed fish, and two groups that had been exposed to anoxia followed by reoxygenation for either 3 h or 24 h. The heart, which maintains cardiac output during anoxia, unexpectedly, was slower to recover compared to the brain and liver, mainly due to a slower return to control concentrations of the energy-carrying compounds ATP, GTP, and phosphocreatine. Crucian carp accumulated amino acids in most tissues, and also surprisingly high levels of succinate in all tissues investigated during anoxia. Purine catabolism was enhanced, leading to accumulation of uric acid during anoxia and increasing urea formation that continued into 24 h of reoxygenation. These tissue-specific differences in accumulation and distribution of the metabolites may indicate an intricate system of transport between tissues, opening for new avenues of investigation of possible mechanisms aimed at reducing the generation of reactive oxygen species (ROS) and resultant tissue damage during reoxygenation.

The METTL20 Homologue from Agrobacterium tumefaciens Is a Dual Specificity Protein-lysine Methyltransferase That Targets Ribosomal Protein L7/L12 and the ß Subunit of Electron Transfer Flavoprotein (ETFß).

Human METTL20 is a mitochondrial, lysine-specific methyltransferase that methylates the ß-subunit of electron transfer flavoprotein (ETFß). Interestingly, putative METTL20 orthologues are found in a subset of α-proteobacteria, including Agrobacterium tumefaciens Using an activity-based approach, we identified in bacterial extracts two substrates of recombinant METTL20 from A. tumefaciens (AtMETTL20), namely ETFß and the ribosomal protein RpL7/L12. We show that AtMETTL20, analogous to the human enzyme, methylates ETFß on Lys-193 and Lys-196 both in vitro and in vivo ETF plays a key role in mediating electron transfer from various dehydrogenases, and we found that its electron transferring ability was diminished by AtMETTL20-mediated methylation of ETFß. Somewhat surprisingly, AtMETTL20 also catalyzed monomethylation of RpL7/L12 on Lys-86, a common modification also found in many bacteria that lack METTL20. Thus, we here identify AtMETTL20 as the first enzyme catalyzing RpL7/L12 methylation. In summary, here we have identified and characterized a novel bacterial lysine-specific methyltransferase with unprecedented dual substrate specificity within the seven ß-strand class of lysine-specific methyltransferases, as it targets two apparently unrelated substrates, ETFß and RpL7/L12. Moreover, the present work establishes METTL20-mediated methylation of ETFß as the first lysine methylation event occurring in both bacteria and humans.

Human METTL20 is a mitochondrial lysine methyltransferase that targets the ß subunit of electron transfer flavoprotein (ETFß) and modulates its activity.

Proteins are frequently modified by post-translational methylation of lysine residues, catalyzed by S-adenosylmethionine-dependent lysine methyltransferases (KMTs). Lysine methylation of histone proteins has been extensively studied, but it has recently become evident that methylation of non-histone proteins is also abundant and important. The human methyltransferase METTL20 belongs to a group of 10 established and putative human KMTs. We here found METTL20 to be associated with mitochondria and determined that recombinant METTL20 methylated a single protein in extracts from human cells. Using an methyltransferase activity-based purification scheme, we identified the ß-subunit of the mitochondrially localized electron transfer flavoprotein (ETFß) as the substrate of METTL20. Furthermore, METTL20 was found to specifically methylate two adjacent lysine residues, Lys(200) and Lys(203), in ETFß both in vitro and in cells. Interestingly, the residues methylated by METTL20 partially overlap with the so-called "recognition loop" in ETFß, which has been shown to mediate its interaction with various dehydrogenases. Accordingly, we found that METTL20-mediated methylation of ETFß in vitro reduced its ability to receive electrons from the medium chain acyl-CoA dehydrogenase and the glutaryl-CoA dehydrogenase. In conclusion, the present study establishes METTL20 as the first human KMT localized to mitochondria and suggests that it may regulate cellular metabolism through modulating the interaction between its substrate ETFß and dehydrogenases. Based on the previous naming of similar enzymes, we suggest the renaming of human METTL20 to ETFß-KMT.