Role of the ubiquitin–proteasome system in the regulation of P2Y13 receptor expression: impact on hepatic HDL uptake

Abstract

The protective effect of high density lipoproteins (HDL) against atherosclerosis is mainly attributed to their capacity to transport excess cholesterol from peripheral tissues back to the liver for further elimination into the bile, a process called reverse cholesterol transport (RCT). Recently, the importance of the P2Y13 receptor (P2Y13-R) was highlighted in HDL metabolism since HDL uptake by the liver was decreased in P2Y13-R deficient mice, which translated into impaired RCT. Here, we investigated for the first time the molecular mechanisms regulating cell surface expression of P2Y13-R. When transiently expressed, P2Y13-R was mainly detected in the endoplasmic reticulum (ER) and strongly subjected to proteasome degradation while its homologous P2Y12 receptor (P2Y12-R) was efficiently targeted to the plasma membrane. We observed an inverse correlation between cell surface expression and ubiquitination level of P2Y13-R in the ER, suggesting a close link between ubiquitination of P2Y13-R and its efficient targeting to the plasma membrane. The C-terminus tail exchange between P2Y13-R and P2Y12-R strongly restored plasma membrane expression of P2Y13-R, suggesting the involvement of the intra-cytoplasmic tail of P2Y13-R in expression defect. Accordingly, proteasomal inhibition increased plasma membrane expression of functionally active P2Y13-R in hepatocytes, and consequently stimulated P2Y13-R-mediated HDL endocytosis. Importantly, proteasomal inhibition strongly potentiated HDL hepatic uptake (>200 %) in wild-type but not in P2Y13-R-deficient mice, thus reinforcing the role of P2Y13-R expression in regulating HDL metabolism. Therefore, specific inhibition of the ubiquitin-proteasome system might be a novel powerful HDL therapy to enhance P2Y13-R expression and consequently promote the overall RCT.

The two-hit hypothesis for neuroinflammation: role of exogenous ATP in modulating inflammation in the brain

“…Ubiquitination at its C-terminal end leads to proteasomal degradation and poor surface expression (Pons et al, 2014). However its surface expression increases in response to oxidative stress and genotoxins such as cisplatin or UV irradiation (Morente et al, 2014).…”

P2Y13 receptors regulate microglial morphology, surveillance, and resting levels of interleukin 1β release

“…Haynes et al () and Stefani et al () found that, despite confirming P2Y 13 mRNA expression in microglia (Zhang et al, ), its expression could not be detected at the protein level. The inability to antibody‐label P2Y 13 may reflect a high rate of constitutive proteasomal degradation (Pons et al, ), or may alternatively suggest that P2Y 13 protein is only expressed at high levels in microglial culture (Quintas, Vale, Goncalves, & Queiroz, ) or upon microglial activation in situ (Niu et al, ).…”

“…Second, almost all of these studies relied highly upon the use of MRS 2211 to block P2Y 13 receptors, but we have now shown this is not a selective P2Y 13 antagonist. Furthermore, the failure, to date, to demonstrate microglial P2Y 13 protein expression in vivo suggests either that the commercially available P2Y 13 antibodies lack specificity or that the constitutive proteasomal degradation of the receptor shown to regulate the surface expression of the receptor in hepatocytes (Pons et al, ) also happens in microglia in vivo (which would imply that a low level of surface receptors can still have a significant effect on cell function).…”

“…The inability to antibody-label P2Y 13 may reflect a high rate of constitutive proteasomal degradation (Pons et al, 2014), or may alternatively suggest that P2Y 13 protein is only expressed at high levels in microglial culture (Quintas, Vale, Goncalves, & Queiroz, 2018) or upon microglial activation in situ .…”

“…A recent study supports these data showing that microglia in the brain, and not neurons, astrocytes, or neural progenitor cells, express P2Y 13 mRNA under basal conditions, using fluorescence in situ hybridization (Stefani et al, ). However, P2Y 13 protein expression may be low (Haynes et al, ), possibly due to constitutive ubiquitination and proteasomal degradation (Pons et al, ). P2Y 13 mRNA is upregulated in pathological conditions such as demyelination (Bedard, Tremblay, Chernomoretz, & Vallieres, ) and neuropathic pain (Kobayashi, Yamanaka, Yanamoto, Okubo, & Noguchi, ; Niu et al, ), suggesting a role of this receptor in neuroinflammation.…”