Immaculeta Osuji

methylation reactions using recombinant mammalian PRMT7 and PRMT1 at 37, 30, 21, 18, and 4 °C. Various fragments of PGC-1α corresponding to the C-terminus were used as substrates, and the methylation reactions were analyzed by fluorography and mass spectrometry to determine the extent of methylation throughout the substrates, the location of the methylated PGC-1α arginine residues, and finally, whether temperature affects the deposition of methyl groups. We also employed two prediction programs, PRmePRed and MePred-RF, to search for putative methyltransferase sites. Methylation reactions show that arginine residues R548 and R753 in PGC-1α are methylated at or below 30 °C by PRMT7, while methylation by PRMT1 was detected at these same residues at 30 °C. Computational approaches yielded additional putative methylarginine sites, indicating that since PGC-1α is an intrinsically disordered protein, additional methylated arginine residues have yet to be experimentally verified. We conclude that temperature affects the extent of arginine methylation, with more methylation by PRMT7 occurring below physiological temperature, uncovering an additional control point for PGC-1α.

Background Cardiac complications in patients with hypereosinophilia cause significant morbidity and mortality. However, mechanisms of how eosinophilic inflammation causes heart damage are poorly understood. Methods We developed a model of hypereosinophilia-associated heart disease by challenging hypereosinophilic mice with a peptide from the cardiac myosin heavy chain. Disease outcomes were measured by histology, immunohistochemistry, flow cytometry, and measurement of cells and biomarkers in peripheral blood. Eosinophil dependence was determined by using eosinophil-deficient mice (ΔdblGATA). Single cells from the heart were subjected to single-cell RNA sequencing to assess cell composition, activation states, and expression profiles. In vitro studies used bone marrow-derived eosinophils (BMDeos) and stimulated them with cytokines and pathogen-associated molecular patterns, followed by assessment of activation markers by flow cytometry. Results Mice challenged with the myocarditic and control peptide had peripheral blood leukocytosis, but only those challenged with the myocarditic peptide had heart inflammation. Heart tissue was infiltrated by eosinophil-rich inflammatory infiltrates associated with cardiomyocyte damage. Disease penetrance and severity were decreased in eosinophil-deficient mice. Single-cell RNA sequencing showed the enrichment of myeloid cells, T cells, and granulocytes (neutrophils and eosinophils) in myocarditic mice. Focusing on eosinophils, there was increased expression of genes associated with type 1 cell activation (such as CD274/PDL1), complement activation, and pathogen-associated molecular pattern recognition. To verify findings generated by single-cell RNA sequencing on a protein level, we performed flow cytometry analysis and assessed the level of type 1 and type 2 biomarkers CD274 and CD101, respectively. The proportion of cells expressing surface CD274 increased on both neutrophils and eosinophils, particularly in mice that showed inflammation by histology. There was no significant increase in expression of CD101. Finally, we assessed whether activation markers can be induced on eosinophils in vitro . Interferon γ (IFNγ) markedly increased expression of CD274, consistent with type 1 polarization. Furthermore, BMDeos stimulated with LPS showed a concentration-dependent increase in the level of CD274 expression. Conclusion Eosinophils are required for heart damage in hypereosinophilia-associated heart disease. Heart-infiltrating eosinophils in an inflammatory condition show type 1 activation, which can be recapitulated in vitro .

Peroxisome proliferator‐activated receptor γ coactivator 1α (PGC‐1α) participates in the regulation of both carbohydrate and lipid metabolism and is activated by insulin. For these reasons, it has been implicated in obesity and diabetes. PGC‐1α is subjected to many post‐translational modifications including phosphorylation of Ser570 by protein kinase B (Akt/PKB), which stops gluconeogenesis and fatty acid oxidation. In addition, PGC‐1α is methylated by protein arginine methyltransferase 1 (PRMT1) at several arginine residues, including Arg665, Arg667 and Arg669 which enhance its regulation. Evidence for additional arginine methylated sites have been found, but the exact sites remain unknown. We set out to identify methylated arginine residues on PGC‐1α. PRMT1 and truncated constructs corresponding to PGC‐1α were expressed and purified. Next, in vitro methylation reactions were carried out, followed by detection of methylated arginine residues by various methods including immunoblot and autoradiography. Identification of the methylated arginine residue(s), will enhance our understanding of PGC‐1α with the broader goal of determining its regulation in the context of obesity and diabetes. Support or Funding Information Minority Biomedical Research Support‐Research Initiative for Scientific Enhancement (MBRS‐RISE) Fellowship. Grant: R25 GM061331