Regulation of Myelin Basic Protein (Arginine) Methyltransferase by Thyroid Hormone in Myelinogenic Cultures of Cells Dissociated from Embryonic Mouse Brain

Abstract: The ontogenetic expression of myelin basic protein (arginine) methyltransferase in myelinogenic cultures of cells dissociated from embryonic mouse brain is highly dependent on the presence of thyroid hormone. Restoration of myelin basic protein methyltransferase to normal activities occurred 16 h after the addition of 100 n M l -3,5,3'-triiodothyronine to hypothyroid medium. These data demonstrate that thyroid hormone can regulate a posttranslational event. On the other hand, histone (arginine) methyltransferase has a different temporal activity pattern, which is not coordinated with myelination, and is not influenced by the lack of thyroid hormone. These data, which suggest the existence of two methyltransferases, were substantiated by demonstrating that the total amount of methylation of added myelin basic protein and histone is the same whether they are incubated together or separately. The requirement of thyroid hormone for the expression of the myelin basic protein methyltransferase and not for histone methyltransferase suggests that thyroid hormone preferentially regulates myelin-associated events in these cultures.     

Theoretical Insights into Catalytic Mechanism of Protein Arginine Methyltransferase 1

Protein arginine methyltransferase 1 (PRMT1), the major arginine asymmetric dimethylation enzyme in mammals, is emerging as a potential drug target for cancer and cardiovascular disease. Understanding the catalytic mechanism of PRMT1 will facilitate inhibitor design. However, detailed mechanisms of the methyl transfer process and substrate deprotonation of PRMT1 remain unclear. In this study, we present a theoretical study on PRMT1 catalyzed arginine dimethylation by employing molecular dynamics (MD) simulation and quantum mechanics/molecular mechanics (QM/MM) calculation. Ternary complex models, composed of PRMT1, peptide substrate, and S-adenosyl-methionine (AdoMet) as cofactor, were constructed and verified by 30-ns MD simulation. The snapshots selected from the MD trajectory were applied for the QM/MM calculation. The typical S_N2-favored transition states of the first and second methyl transfers were identified from the potential energy profile. Deprotonation of substrate arginine occurs immediately after methyl transfer, and the carboxylate group of E144 acts as proton acceptor. Furthermore, natural bond orbital analysis and electrostatic potential calculation showed that E144 facilitates the charge redistribution during the reaction and reduces the energy barrier. In this study, we propose the detailed mechanism of PRMT1-catalyzed asymmetric dimethylation, which increases insight on the small-molecule effectors design, and enables further investigations into the physiological function of this family.     

Redox Control of Protein Arginine Methyltransferase 1 (PRMT1) Activity

Elevated levels of asymmetric dimethylarginine (ADMA) correlate with risk factors for cardiovascular disease. ADMA is generated by the catabolism of proteins methylated on arginine residues by protein arginine methyltransferases (PRMTs) and is degraded by dimethylarginine dimethylaminohydrolase. Reports have shown that dimethylarginine dimethylaminohydrolase activity is down-regulated and PRMT1 protein expression is up-regulated under oxidative stress conditions, leading many to conclude that ADMA accumulation occurs via increased synthesis by PRMTs and decreased degradation. However, we now report that the methyltransferase activity of PRMT1, the major PRMT isoform in humans, is impaired under oxidative conditions. Oxidized PRMT1 displays decreased activity, which can be rescued by reduction. This oxidation event involves one or more cysteine residues that become oxidized to sulfenic acid (-SOH). We demonstrate a hydrogen peroxide concentration-dependent inhibition of PRMT1 activity that is readily reversed under physiological H₂O₂ concentrations. Our results challenge the unilateral view that increased PRMT1 expression necessarily results in increased ADMA synthesis and demonstrate that enzymatic activity can be regulated in a redox-sensitive manner.     

Metabolic Regulation of CaMKII Protein and Caspases in Xenopus laevis Egg Extracts

The metabolism of the Xenopus laevis egg provides a cell survival signal. We found previously that increased carbon flux from glucose-6-phosphate (G6P) through the pentose phosphate pathway in egg extracts maintains NADPH levels and calcium/calmodulin regulated protein kinase II (CaMKII) activity to phosphorylate caspase 2 and suppress cell death pathways. Here we show that the addition of G6P to oocyte extracts inhibits the dephosphorylation/inactivation of CaMKII bound to caspase 2 by protein phosphatase 1. Thus, G6P sustains the phosphorylation of caspase 2 by CaMKII at Ser-135, preventing the induction of caspase 2-mediated apoptotic pathways. These findings expand our understanding of oocyte biology and clarify mechanisms underlying the metabolic regulation of CaMKII and apoptosis. Furthermore, these findings suggest novel approaches to disrupt the suppressive effects of the abnormal metabolism on cell death pathways.     

Regulation of heart size in Xenopus laevis

The region with the potential to form the heart has traditionally been called the heart field. This region can be approximated by, but is not identical to, the expression domain of the early cardiac gene Nkx2.5. The region expressing Nkx2.5 does not change in size, although there are major shape changes and a subdivision of the region into non-myogenic and myogenic lineages. Using a variety of embryo manipulations, we have sought to determine whether cellular interactions could change the size of the initial Nkx2.5-expressing region and thus change the size of the heart. We have shown that if the heart is isolated from the dorsal half of the embryo, the volume of tissue expressing myocardial differentiation markers increases, indicating that signals restricting the size of the heart come from the dorsal side. Despite the change in myocardial volume, the non-myogenic heart lineages are still present. The ability of dorsal tissues to restrict the size of the heart is further demonstrated by fusing two Xenopus embryos shortly after gastrulation, generating twinned embryos where the heart of one embryo would develop adjacent to different tissues of the second embryo. The final size of the differentiated heart was markedly reduced if it developed in close proximity to the dorso-anterior surface of the head but not if it developed adjacent to the flank or belly. In all cases, the manipulations that restricted the size of the myocardium also restricted the expression of Nkx2.5 and GATA-4, both key regulatory genes in the cardiogenic pathway. These results provide evidence for a model in which signals from dorso-anterior tissues restrict the size of the heart after gastrulation but before neural fold closure.     

Protein Labelling Patterns in Oocytes of Xenopus laevis

Soluble and microsomal proteins synthesized at various stages of oogenesis in Xenopus laevis were compared by ³H and ¹⁴C dual-isotope labelling with subsequent mixing and analysis on sodium dodecyl sulfate (SDS)-polyacrylamide gels. The results indicated that the proteins labelled in all the stages of oogenesis studied are remarkably similar. However, the patterns in the small and medium-sized oocytes are more similar to one another than to the patterns characteristic of large oocytes. The greatest differences were found when comparing the microsomal proteins.
The labelling patterns of oocyte proteins in vitro were not significantly different from the in vivo patterns for the stages of oogenesis studied. These results indicate that (1) there is little quantitative contribution of proteins to either the soluble or microsomal fractions from extra-oocytic sources in vivo and (2) the in vitro system itself has little effect on the labelling patterns over the incubation period.     

Regulation of neurons in the suprachiasmatic nucleus of Xenopus laevis

In the amphibian Xenopus laevis, suprachiasmatic melanotrope-inhibiting neurons (SMINs) play an important role in the regulation of the background adaptation process. In this study, we investigated the innervation of the SMINs at the light- and electron- microscopical level. Immunocytochemistry in combination with confocal laser scanning microscopy revealed co-existence of neuropeptide Y (NPY) and synaptobrevin in spots in the direct vicinity of the SMINs, suggesting the existence of NPY-containing synapses on these cells. At the ultrastructural level, the SMINs showed a high degree of plasticity, containing more electron-dense vesicles and a larger extent of RER in white- than in black-adapted animals. In black-adapted animals, symmetric synapses (Gray type II) were observed on the soma of the SMINs, suggesting an inhibitory input to these cells. The synaptic profiles contained electron-lucent and electron-dense vesicles, indicating the involvement of both a classical neurotransmitter and a neuropeptide (possibly NPY) in this input. In white-adapted animals, synapses were only found at some distance from the SMIN somata. Our findings indicate a striking plasticity of the innervation of the SMINs in relation to background adaptation and support the hypothesis that the SMINs are innervated by NPY-containing interneurons that inhibit SMIN activity in black-adapted animals.     

Mapping of Post-translational Modifications of Transition Proteins,TP1 and TP2, and Identification of Protein Arginine Methyltransferase 4 and Lysine Methyltransferase 7 as Methyltransferase for TP2

In a unique global chromatin remodeling process during mammalian spermiogenesis, 90% of the nucleosomal histones are replaced by testis-specific transition proteins, TP1, TP2, and TP4. These proteins are further substituted by sperm-specific protamines, P1 and P2, to form a highly condensed sperm chromatin. In spermatozoa, a small proportion of chromatin, which ranges from 1 to 10% in mammals, retains the nucleosomal architecture and is implicated to play a role in transgenerational inheritance. However, there is still no mechanistic understanding of the interaction of chromatin machinery with histones and transition proteins, which facilitate this selective histone replacement from chromatin. Here, we report the identification of 16 and 19 novel post-translational modifications on rat endogenous transition proteins, TP1 and TP2, respectively, by mass spectrometry. By in vitro assays and mutational analysis, we demonstrate that protein arginine methyltransferase PRMT4 (CARM1) methylates TP2 at Arg⁷¹, Arg⁷⁵, and Arg⁹² residues, and lysine methyltransferase KMT7 (Set9) methylates TP2 at Lys⁸⁸ and Lys⁹¹ residues. Further studies with modification-specific antibodies that recognize TP2K88me1 and TP2R92me1 modifications showed that they appear in elongating to condensing spermatids and predominantly associated with the chromatin-bound TP2. This work establishes the repertoire of post-translational modifications that occur on TP1 and TP2, which may play a significant role in various chromatin-templated events during spermiogenesis and in the establishment of the sperm epigenome.