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LPS Reduces SCF-Fbxo9 to Elevate PRMT4 Protein Levels in Lymphocytes in an Acute Lung Injury Model
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A2248 - LPS Reduces SCF-Fbxo9 to Elevate PRMT4 Protein Levels in Lymphocytes in an Acute Lung Injury Model
Author Block: C. Zou1, Y. Lai1, X. Li1, J. Li2; 1Medicine, University of Pittsburgh, Pittsburgh, PA, United States, 2University of Pittsburgh, Pittsburgh, PA, United States.
RATIONAL: Protein Arginine N-methyltransferase 4/Coactivator-Associated Arginine Methyltransferase 4 (PRMT4/CARM1), a type I methyltransferase enzyme of the 11 members in protein arginine methyltransferase family, regulates crucial life processes including gene transcription, proliferation, DNA splicing, and development. Dysregulation of PRMT4 has been reported to be involved in the pathogenesis of many human diseases including lung cancers, antigen-induced pulmonary inflammation and other respiratory diseases. However, the mechanism (s) of how PRMT4 is regulated and the role of PRMT4 in acute lung injury is yet illusive. METHODES: Molecular and cellular biological and biochemical techniques such as molecular cloning, PCR, site-directed mutagenesis, co-IP, cell culture, and Immunoblotting, etc, were applied in this study. RESULTS: By using molecular and biochemical approaches, we demonstrated that gram-negative bacteria derived endotoxin lipopolysaccharide (LPS) elevates PRMT4 protein levels in a range of cell types including lung epithelial cell, monocytes, Jurkat T-lymphocytes, and SKW6.4 B-lymphocytes. PRMT4 protein is unstable and is degraded via ubiquitin proteasomal machinery. By screening an E3 ubiquitin ligase library, we identified that SCF-Fbxo9 E3 ubiquitin ligase specifically catalyzes PRMT4 polyubiquitination at K228. Fbxo9 docks to a phosphodegron 132-TXXXS within PRMT4. The phosphorylation/dephosphrylation status of the T/S residues in the degron is critical for Fbo9 binding. For an optimal Fbxo9 binding, T132 must be dephosphorylated and S136 must be phosphorylated. Interestingly, LPS down-regulates Fbxo9 to accumulate PRMT4 in a range of cells described above. Exploration of the pathophysiological significance of the elevated PRMT4 in lymphocytes in an acute lung injury model is undergoing.
CONCLUSIONS: This study dissected the molecular mechanism that LPS down-regulates an SCF-Fbxo9 E3 ubiquitin ligase to elevate PRMT4 protein level in a range of cell types. This study adds a paradigm for how LPS determines a protein cellular abundance by directly manipulation of the availability of an E3 ubiquitin ligase.
Author Block: C. Zou1, Y. Lai1, X. Li1, J. Li2; 1Medicine, University of Pittsburgh, Pittsburgh, PA, United States, 2University of Pittsburgh, Pittsburgh, PA, United States.
RATIONAL: Protein Arginine N-methyltransferase 4/Coactivator-Associated Arginine Methyltransferase 4 (PRMT4/CARM1), a type I methyltransferase enzyme of the 11 members in protein arginine methyltransferase family, regulates crucial life processes including gene transcription, proliferation, DNA splicing, and development. Dysregulation of PRMT4 has been reported to be involved in the pathogenesis of many human diseases including lung cancers, antigen-induced pulmonary inflammation and other respiratory diseases. However, the mechanism (s) of how PRMT4 is regulated and the role of PRMT4 in acute lung injury is yet illusive. METHODES: Molecular and cellular biological and biochemical techniques such as molecular cloning, PCR, site-directed mutagenesis, co-IP, cell culture, and Immunoblotting, etc, were applied in this study. RESULTS: By using molecular and biochemical approaches, we demonstrated that gram-negative bacteria derived endotoxin lipopolysaccharide (LPS) elevates PRMT4 protein levels in a range of cell types including lung epithelial cell, monocytes, Jurkat T-lymphocytes, and SKW6.4 B-lymphocytes. PRMT4 protein is unstable and is degraded via ubiquitin proteasomal machinery. By screening an E3 ubiquitin ligase library, we identified that SCF-Fbxo9 E3 ubiquitin ligase specifically catalyzes PRMT4 polyubiquitination at K228. Fbxo9 docks to a phosphodegron 132-TXXXS within PRMT4. The phosphorylation/dephosphrylation status of the T/S residues in the degron is critical for Fbo9 binding. For an optimal Fbxo9 binding, T132 must be dephosphorylated and S136 must be phosphorylated. Interestingly, LPS down-regulates Fbxo9 to accumulate PRMT4 in a range of cells described above. Exploration of the pathophysiological significance of the elevated PRMT4 in lymphocytes in an acute lung injury model is undergoing.
CONCLUSIONS: This study dissected the molecular mechanism that LPS down-regulates an SCF-Fbxo9 E3 ubiquitin ligase to elevate PRMT4 protein level in a range of cell types. This study adds a paradigm for how LPS determines a protein cellular abundance by directly manipulation of the availability of an E3 ubiquitin ligase.
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