Differential Ubiquitination of Profilin-1 in Hypoxia-induced Pulmonary Hypertension Pubblico
Zhao, Jingru (Spring 2018)
Abstract
Pulmonary hypertension (PH) is a chronic illness characterized by increased pulmonary arterial pressure due to constriction and thickening of the arterial walls. In part, this is due to smooth muscle cell proliferation. Protein ubiquitination is an important regulator of cellular proliferation. Ubiquitin ligases tag target proteins with ubiquitin moieties at lysine (K) residues to alter protein stability and expression. We used mass spectrometry (MS) to screen lung tissue from chronic hypoxia mice exposed to 10% O2 for 3 weeks for changes in ubiquitination. Profilin-1, a mediator of actin-polymerization, had decreased ubiquitination on K54 (fold change -1.86) and K126 (fold change -1.40). Overexpression of profilin-1 is associated with increased wall thickness in aortas of spontaneously hypertensive rats, but its role in PH is not well characterized. Profilin-1 promotes actin polymerization, a process that is known to play a key role in cellular proliferation, cell motility, and muscle contraction and is up-regulated in PH. We hypothesized that hypoxia-induced changes in ubiquitination of profilin-1 changes its regulation of actin polymerization, leading to proliferation of smooth muscle cells. In our hypoxia exposed human pulmonary artery smooth muscle cell (hPASMC) and chronic hypoxia mouse lung models, we found no difference in profilin-1 protein level when compared to normoxia controls. This suggests that ubiquitination of profilin-1 could be a novel mechanism of regulating its activity rather than stability. Knockdown of profilin-1 with siRNA or disrupting actin polymerization using 200nM latrunculin B or 0.25 µM cytochalasin D leads to significant decrease in hPASMC proliferation. Preliminary fractionation results also show an increased association of profilin-1 and G-actin in hypoxia. Together, our results indicate that reduced ubiquitination of profilin-1 may increase actin polymerization and contribute to PH pathogenesis.
Table of Contents
CHAPTER 1. Introduction 1
Pulmonary Hypertension 2
Models for Hypoxia-induced PH 10
Ubiquitin and Protein Ubiquitination 11
Profilin-1 16
Summary and Hypothesis 22
CHAPTER 2. Methods 24
CHAPTER 3. Results 34
CHAPTER 4. Discussion 51
REFERENCES 58
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