An Internal Standard-Assisted Synthesis and Degradation Proteomic Approach Reveals the Potential Linkage between VPS4B Depletion and Activation of Fatty Acid β-Oxidation in Breast Cancer Cells
The endosomal/lysosomal system, in particular the endosomal sorting complexes required for transport (ESCRTs), plays an essential role in regulating the trafficking and destination of endocytosed receptors and their associated signaling molecules. Recently, we have shown that dysfunction and down-regulation of vacuolar protein sorting 4B (VPS4B), an ESCRT-III associated protein, under hypoxic conditions can lead to the abnormal accumulation of epidermal growth factor receptor (EGFR) and aberrant EGFR signaling in breast cancer. However, the pathophysiological consequences of VPS4B dysfunction remain largely elusive. In this study, we used an internal standard-assisted synthesis and degradation mass spectrometry (iSDMS) method, which permits the direct measurement of protein synthesis, degradation and protein dynamic expression, to address the effects of VPS4B dysfunction in altering EGF-mediated protein expression. Our initial results indicate that VPS4B down-regulation decreases the expression of many proteins involved in glycolytic pathways, while increased the expression of proteins with roles in mitochondrial fatty acid β-oxidation were up-regulated in VPS4B-depleted cells. This observation is also consistent with our previous finding that hypoxia can induce VPS4B down-regulated, suggesting that the adoption of fatty acid β-oxidation could potentially serve as an alternative energy source and survival mechanism for breast cancer cells in response to hypoxia-mediated VPS4B dysfunction. 1. Introduction VPS4B, a member of the AAA (ATPases associated with diverse cellular activities) protein family, plays an important role in the lysosomal degradation pathway, which functions in ligand-induced membrane receptor downregulation. In lysosomal degradation, endocytosed receptors are sorted into multivesicular bodies (MVBs), which requires the sequential assembly of endosomal sorting complex required for transport I, II, and III (ESCRT-I, -II, and -III) on the endosomal membrane [1]. VPS4B functions to dissociate ESCRT-III from endosomes for further rounds of endosomal sorting [2]. Expression of functionally inactive VPS4B results in the accumulation of receptors on abnormally enlarged endosomes (class E compartments) and the abolishment of MVB biogenesis [3–6]. As an essential ESCRT-III interacting protein, VPS4B is also involved in protein trafficking among membrane compartments and in signal transduction [4, 5, 7, 8]. Epidermal growth factor- (EGF-) mediated signaling is one of the most important signaling pathways for cell growth, proliferation, invasion,
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