Poster abstracts
Poster number 23 submitted by Dr. Lei Pan
Characterization of TGFβ-inducible global-proteomic and phosphor-proteomic changes in activated immortalized human cardiac fibroblasts
Lei Pan (Department of Biology, University of Rochester), Debojyoti Das (Aab Cardiovascular Research Institute, University of Rochester Medical Center), Jack Lawrence Schwartz (Department of Biochemistry and Biophysics, University of Rochester Medical Center), Peng Yao (Department of Biochemistry & Biophysics and Department of Medicine, Aab Cardiovascular Research Institute, University of Rochester Medical Center)
Abstract:
Increased protein synthesis of profibrotic genes is a common feature in cardiac fibrosis and heart failure. However, protein post-translational modifications such as phosphorylation and changes in critical RNA-binding proteins (RBPs) for regulating alternative splicing and translation of profibrotic genes during cardiac fibrosis remain unclear. We found previously that TGFβ induced expression of extracellular matrix proteins and translation factors in human immortalized cardiac fibroblasts (IHCF). By normalizing phosphorylation level with global protein level, we have defined the phosphorylation status of individual proteins across the proteome in TGFβ-activated IHCF compared to untreated cells. We identified 744 genes with significantly changed phosphorylated peptides across 174 RBPs, including many key splicing factors and translation regulatory factors. Among the most significantly changed phosphorylated peptides, we observed that the phosphorylation of Ser63 residue, but not the total protein level of a canonical RBP and splicing factor, HNRNPH1, was significantly upregulated after TGFβ treatment of IHCF. We found that HNRNPH1 phosphorylation site Ser63 is conserved across evolution, from zebrafish to humans. After 24 hours of TGFβ treatment of IHCF, we validated that HNRNPH1 mRNA and protein levels remain unchanged. Using confocal immunofluorescence microscopy, we proved that HNRNPH1 was localized in the nucleus and did not shuttle into the cytoplasm of IHCF after TGFβ treatment. To investigate the function of phosphorylation of HNRNPH1 at Ser63, we attempted to generate “phosphor-dead” HNRNPH1 S63A and “phosphor-on” HNRNPH1 S63D knock-in cell lines of HEK293T via CRISPR-Cas9-mediated homology-directed repair. Our preliminary data from Sanger sequencing showed a 5-10% bulk editing efficiency. In conclusion, our results indicate that TGFβ-induced phosphorylation of a novel site of HNRNPH1, Ser63, among other RBPs, may play a critical role in cardiac fibrosis.
References:
Baum, J. and H.S. Duffy, Fibroblasts and Myofibroblasts: What Are We Talking About? Journal of Cardiovascular Pharmacology, 2011. 57(4).
Hall, C., et al., Complex Relationship Between Cardiac Fibroblasts and Cardiomyocytes in Health and Disease. Journal of the American Heart Association, 2021. 10(5): p. e019338.
Wu, J., et al., Glutamyl-Prolyl-tRNA Synthetase Regulates Proline-Rich Pro-Fibrotic Protein Synthesis During Cardiac Fibrosis. Circ Res, 2020. 127(6): p. 827-846.
Yao, P., et al., The HILDA Complex Coordinates a Conditional Switch in the 3′-Untranslated Region of the VEGFA mRNA. PLOS Biology, 2013. 11(8): p. e1001635.
Keywords: RNA-binding protein, HNRNPH1, fibrosis