The Role of Lipoxidation in Regulating Phosphatase and Tensin Homolog (PTEN) and its Downstream Signalling

Abstract

Lipoxidation of proteins by lipid peroxidation products (LPPs) has emerged as an important mechanism regulating protein function, stability, and subcellular localization. Some LPPs can inhibit the crucial redox-sensitive tumour-suppressor Phosphatase and Tensin Homolog (PTEN). PTEN regulates metabolism, cell survival and proliferation, mainly by inhibition of the PI3K/Akt signalling pathway, and through non-canonical functions, depending on its subcellular localization. Nonetheless, the effects of only a few LPPs, including acrolein, 4-hydoxynonenal (HNE), and oxidized prostaglandins on PTEN and its downstream signalling have been reported and potential effects on its subcellular localization remain unknown. The project therefore aimed to determine the effects of the LPPs 4-hydroxyhexenal (HHE) and 1-palmitoyl-2-(9-oxo-nonanoyl)-sn-glycero-3-phosphocholine (PONPC), a reactive truncated phospholipid, on PTEN function, localization and its downstream signalling. In this work, the in vitro assessment of human recombinant PTEN activity using 3-O-methylfluorescein phosphate (OMFP) showed a time- and concentration-dependent decrease in PTEN phosphatase activity, which correlated with PTEN aggregation. Although sites of adduction of PONPC could not be determined, tandem mass spectrometry (LC-MS/MS) analysis of HHE-treated human recombinant PTEN identified sixteen sites of adduction on PTEN cysteine, lysine, and histidine residues, suggesting that inhibition of PTEN activity could be the consequence of its lipoxidation. Immunofluorescence of PTEN in MCF-7 cells suggested that HHE and PONPC could lead to accumulation of PTEN in the nucleus and at the membrane, respectively. While preliminary data suggested that PTEN expression levels remained unchanged and that Akt expression levels were unchanged with HHE but decreased with PONPC, levels of phospho-Akt (Ser473 and Thr308) were generally elevated in a time- and concentration-dependent manner with HHE and PONPC treatments in HCT116 and MCF-7 cells, with a few exceptions. This suggested that HHE and PONPC could regulate cell signalling in an Akt-dependent manner and have important functional and biological effects, possibly via lipoxidation of PTEN.