Prolyl hydroxylation regulates protein degradation, synthesis, and splicing in human induced pluripotent stem cell-derived cardiomyocytes.
Protein hydroxylases are oxygen- and α-ketoglutarate-dependent enzymes that catalyse hydroxylation of amino acids such as proline, thus linking oxygen and metabolism to enzymatic activity. Prolyl hydroxylation is a dynamic post-translational modification that regulates protein stability and protein-protein interactions; however, the extent of this modification is largely uncharacterized. The goals of this study are to investigate the biological consequences of prolyl hydroxylation and to identify new targets that undergo prolyl hydroxylation in human cardiomyocytes.



We used human induced pluripotent stem cell-derived cardiomyocytes in combination with pulse-chase amino acid labelling and proteomics to analyse the effects of prolyl hydroxylation on protein degradation and synthesis. We identified 167 proteins that exhibit differences in degradation with inhibition of prolyl hydroxylation by dimethyloxalylglycine (DMOG); 164 were stabilized. Proteins involved in RNA splicing such as serine/arginine-rich splicing factor 2 (SRSF2) and splicing factor and proline- and glutamine-rich (SFPQ) were stabilized with DMOG. DMOG also decreased protein translation of cytoskeletal and sarcomeric proteins such as α-cardiac actin. We searched the mass spectrometry data for proline hydroxylation and identified 134 high confidence peptides mapping to 78 unique proteins. We identified SRSF2, SFPQ, α-cardiac actin, and cardiac titin as prolyl hydroxylated. We identified 29 prolyl hydroxylated proteins that showed a significant difference in either protein degradation or synthesis. Additionally, we performed next-generation RNA sequencing and showed that the observed decrease in protein synthesis was not due to changes in mRNA levels. Because RNA splicing factors were prolyl hydroxylated, we investigated splicing ± inhibition of prolyl hydroxylation and detected 369 alternative splicing events, with a preponderance of exon skipping.



This study provides the first extensive characterization of the cardiac prolyl hydroxylome and demonstrates that inhibition of α-ketoglutarate hydroxylases alters protein stability, translation, and splicing.
Systems Biology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA.
Andrea
A
Stoehr
DNA Sequencing and Genomics Core, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA.
Yanqin
Y
Yang
Proteomics Core Facility, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA.
Sajni
S
Patel
Systems Biology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA.
Alicia M
AM
Evangelista
Proteomics Core Facility, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA.
Angel
A
Aponte
Proteomics Core Facility, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA.
Guanghui
G
Wang
DNA Sequencing and Genomics Core, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA.
Poching
P
Liu
Systems Biology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA.
Jennifer
J
Boylston
Systems Biology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA.
Philip H
PH
Kloner
iPS Cell Core Facility, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA.
Yongshun
Y
Lin
Proteomics Core Facility, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA.
Marjan
M
Gucek
DNA Sequencing and Genomics Core, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA.
Jun
J
Zhu
Systems Biology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA murphy1@nhlbi.nih.gov.
Elizabeth
E
Murphy
D017398:Alternative Splicing; D000600:Amino Acids, Dicarboxylic / Q000494:pharmacology; D002454:Cell Differentiation; D002460:Cell Line; D064211:Connectin / Q000378:metabolism; D006801:Humans; D006900:Hydroxylation; D057026:Induced Pluripotent Stem Cells / Q000187:drug effects / Q000201:enzymology*; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000201:enzymology*; D000072670:PTB-Associated Splicing Factor / Q000378:metabolism; D011392:Proline / Q000737:chemistry*; D064828:Prolyl Hydroxylases / Q000378:metabolism*; D064800:Prolyl-Hydroxylase Inhibitors / Q000494:pharmacology; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D059748:Proteolysis; D040901:Proteomics / Q000379:methods; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D000068103:Serine-Arginine Splicing Factors / Q000378:metabolism
D000600:Amino Acids, Dicarboxylic; D064211:Connectin; D000072670:PTB-Associated Splicing Factor; D064800:Prolyl-Hydroxylase Inhibitors; D012333:RNA, Messenger; C577285:TTN protein, human; C073288:SRSF2 protein, human; D000068103:Serine-Arginine Splicing Factors; D011392:Proline; D064828:Prolyl Hydroxylases; C040947:oxalylglycine
Hypoxia; Prolyl hydroxylation; Protein degradation; Proteomics; Splicing