Identification of p115 as a novel ACSL4 interacting protein and its role in regulating ACSL4 degradation.

Long-chain acyl-CoA synthetase 4 (ACSL4) is an ACSL family member that exhibits unique substrate preference for arachidonic acid. ACSL4 has a functional role in hepatic lipid metabolism, and is dysregulated in non-alcoholic fatty liver disease. Our previous studies demonstrated AA-induced ACSL4 degradation via the ubiquitin-proteasomal pathway (UPP). To characterize this unique mechanism, we applied proteomic approaches coupled with LC-MS/MS and identified the intracellular general vesicular trafficking protein p115 as the prominent ACSL4 interacting protein in HepG2 cells. Importantly, we found that AA greatly enhanced p115-ACSL4 association. Combined AA treatment with p115 knockdown suggested an additive role for p115 in AA-driven ACSL4 degradation. Furthermore, in vivo studies revealed a significant upregulation of p115 protein in the liver of mice fed a high fat diet that has been previously reported to induce downregulation of ACSL4 protein expression. This new finding has revealed a novel inverse correlation between ACSL4 and p115 proteins in the liver. p115 is crucial for ER-Golgi trafficking and Golgi biogenesis. Thus far, p115 has not been reported to interact with UPP proteins nor with FA metabolism enzymes. Overall, our current study provides a novel insight into the connection between ER-Golgi trafficking and UPP machinery with p115 as a critical mediator. SIGNIFICANCE: ACSL4 is uniquely regulated by its own substrate AA, and in this study, we have found that AA leads to an enhanced interaction of ACSL4 with a novel interacting partner, the intracellular vesicle trafficking protein p115. The latter is crucial for Golgi biogenesis and ER-Golgi transport and is not known to be associated with the ubiquitin-proteasome machinery or protein stability regulation until now. This study is the first report of a possible coordination of the protein secretion pathway and the UPP in regulating a key metabolic enzyme. Our study lays the foundation to this unique crosstalk between the two major cellular pathways- secretion and protein degradation and opens up a new avenue to explore this partnership in controlling hepatic lipid metabolism. Overall, the complete elucidation of the AA-mediated ACSL4 regulation will help identify key targets in participating pathways that can be further studied for the development of therapeutics against diseases such as NAFLD, NASH and hepatocarcinoma, which are associated with dysregulated ACSL4 function.

The Cellular Distribution of RanGAP1 Is Regulated by CRM1-Mediated Nuclear Export in Mammalian Cells.

The Ran GTPase activating protein RanGAP1 plays an essential role in nuclear transport by stimulating RanGTP hydrolysis in the cytoplasmic compartment. In mammalian cells, unmodified RanGAP1 is predominantly cytoplasmic, whereas modification by small ubiquitin-related modifier protein (SUMO) targets RanGAP1 to the cytoplasmic filaments of nuclear pore complex (NPC). Although RanGAP1 contains nine putative nuclear export signals and a nuclear localization signal, little is known if RanGAP1 shuttles between the nuclear and cytoplasmic compartments and how its primary localization in the cytoplasm and at the NPC is regulated. Here we show that inhibition of CRM1-mediated nuclear export using RNAi-knockdown of CRM1 and inactivation of CRM1 by leptomycin B (LMB) results in nuclear accumulation of RanGAP1. LMB treatment induced a more robust redistribution of RanGAP1 from the cytoplasm to the nucleoplasm compared to CRM1 RNAi and also uniquely triggered a decrease or loss of RanGAP1 localization at the NPC, suggesting that LMB treatment is more effective in inhibiting CRM1-mediated nuclear export of RanGAP1. Our time-course analysis of LMB treatment reveals that the NPC-associated RanGAP1 is much more slowly redistributed to the nucleoplasm than the cytoplasmic RanGAP1. Furthermore, LMB-induced nuclear accumulation of RanGAP1 is positively correlated with an increase in levels of SUMO-modified RanGAP1, suggesting that SUMOylation of RanGAP1 may mainly take place in the nucleoplasm. Lastly, we demonstrate that the nuclear localization signal at the C-terminus of RanGAP1 is required for its nuclear accumulation in cells treated with LMB. Taken together, our results elucidate that RanGAP1 is actively transported between the nuclear and cytoplasmic compartments, and that the cytoplasmic and NPC localization of RanGAP1 is dependent on CRM1-mediated nuclear export.