Missense Mutant Patatin‐Like Phospholipase Domain Containing 3 Alters Lipid Droplet Turnover in Partnership With CGI‐58
Authors: Scott McHenry, Nicholas O. Davidson
Journal: Hepatology (2019)

Supported by grants from the National Institutes of Health, Digestive Disease Research Core Center: DK‐52574 (ARAC), DK‐07130 (T32), DK‐56260, HL‐38180, and DK‐112378. Potential conflict of interest: Nothing to report. See Article on Page 2427 A common missense variant, rs738409, in patatin‐like phospholipase domain containing 3 (PNPLA3I148M) was identified more than a decade ago as a dominant genetic determinant of hepatic steatosis (HS)1 and has since been replicated in multiple studies spanning nonalcoholic fatty liver disease etiology and progression.2 But how exactly this mutant gene functions to promote HS and progressive liver injury has been the subject of intense investigation. Earlier studies demonstrated that wild‐type (WT; I148) PNPLA3 exhibited triglyceride (TG) lipase activity in vitro, whereas the mutant (I148M) variant manifested a loss of lipase activity.3 Those findings led to the suggestion that the I148M variant functioned as a loss‐of‐function allele and two corresponding predictions: first, that genetic deletion of Pnpla3 would result in HS in knockout mice and, second, that overexpression of WT PNPLA3 would decrease liver fat. However, neither of these predictions turned out to be correct. Neither germline deletion of (mouse) Pnpla34 nor forced overexpression of (human) PNPLA3WT resulted in an obvious lipid phenotype.5 On the other hand, overexpression of the mutant human PNPLA3I148M led to accumulation of lipid droplets (LDs) and an altered spectrum of TG fatty acids.5 Those findings, coupled with studies from knock‐in Pnpla3I148M mice that showed increased HS with sucrose feeding,6 suggested an alternative explanation, namely that the mutant PNPLA3 functions as a dominant‐negative allele to promote HS. More‐recent findings demonstrated that catalytically defective forms of PNPLA3 (either the I148M or S47A variant) exhibit defective ubiquitylation and, as a result, cause PNPLA3 protein accumulation on the surface of LDs.7 These key background studies raise the question of how accumulation of the mutant PNPLA3 protein on LDs exerts a dominant‐negative impact on lipase activity in hepatocytes? In the current issue of Hepatology, Wang et al.8 begin to answer this question using a combination of cell‐based and mouse studies to examine the distribution and function of adipose triglyceride lipase (ATGL), the major intracellular hepatic lipase, as well as its partner (and cofactor) protein, comparative gene identification‐58 (CGI‐58). They show that forced overexpression of ATGL in hepatoma (Huh‐7) cells depleted LDs (as expected), but then made the unexpected discovery that forced coexpression of ATGL with PNPLA3 (either WT or I148M) inhibited lipolysis. Those findings suggest that PNPLA3 overexpression (either WT or mutant) inhibits ATGL‐mediated lipolysis. An important control in those studies was to demonstrate that coexpression of ATGL with another LD protein (in this case, 17β‐hydroxysteroid dehydrogenase 13) led to an indistinguishable pattern of LD depletion as ATGL overexpression alone, suggesting that the dominant‐negative phenotype with PNPLA3 overexpression is not simply a nonspecific effect. So, how might this be mediated? In order to examine how both WT and mutant PNPLA3 inhibit ATGL, Wang et al. then asked whether overexpression of PNPLA3 modified the availability of the ATGL cofactor, CGI‐58.8 They show that overexpression of both WT PNPLA3 and CGI‐58 resulted in LD depletion (presumably as a result of activating endogenous ATGL), whereas overexpression of CGI‐58 with catalytically "dead" PNPLA3, either the PNPLA3I148M or PNPLA3S47A mutants, caused no LD depletion. They show that overexpression of PNPLA3 on LDs failed to change either the association of ATGL with CGI‐58 or the distribution of ATGL across LDs. Those findings suggest that interactions between PNPLA3 and CGI‐58 might, in turn, directly modulate ATGL function and lipase activity. To answer this question, they turned to a line of CGI‐58 liver‐specific knockout mice (CGI‐58LKO) that were shown previously to exhibit a dramatic increase in HS.9 They isolated hepatic LDs from those knockout mice and found that they contain virtually no detectable PNPLA3 protein, yet exhibit unchanged levels of ATGL, implying that CGI‐58 might be required for recruitment of PNPLA3 to LDs. To answer that question, they injected adenoviral vectors expressing either WT or mutant PNPLA3 into CGI‐58 floxed or CGI‐58LKO mice and found no localization of exogenous PNPLA3 in LDs from knockout livers. In addition, they further demonstrated that adenoviral PNPLA3I148M administration into CGI‐58LKO mice failed to increase hepatic TG content, suggesting that PNPLA3 is dependent on CGI‐58 for its recruitment and/or stabilization on the surface of hepatic LDs and also that the dominant‐negative effects of the mutant PNPLA3 require CGI‐58 expression. In a final series of experiments, the investigators also show that CGI‐58 and PNPLA3 can be coimmunoprecipitated in cell culture and also from mouse liver, strongly suggesting that these two LD proteins physically interact. Taken together, the new findings from Wang et al. represent an advance in our understanding of the mechanisms underlying the dominant‐negative function of mutant PNPLA3.8 The findings suggest a model in which the increased abundance of PNPLA3I148M results in sequestration of CGI‐58 on LDs and, as a result, limits cofactor availability for activation of ATGL. The investigators found no evidence for displacement of CGI‐58 from LDs with PNPLA3I148M overexpression, suggesting that there is a physical interaction at the LD surface that interferes with lipase activation. Those conclusions are supported by the finding that LDs from CGI‐58LKO mice contain no PNPLA3. That being said, the question remains how PNPLA3, CGI‐58, and ATGL each find their way to hepatocyte LDs in the correct stoichiometric proportions and function to regulate LD assembly and turnover under physiological conditions of fasting or nutrient excess. It is worth noting that other studies have demonstrated that another mutant PNPLA3 (E434K) tended to decrease HS in PNPLA3 I148M carriers, suggesting that yet other LD proteins may act as codominant modifiers.10 How might these findings help shape a personalized approach to therapies targeted to mitigate progression of liver disease in patients harboring the rs738409 polymorphism? One might envision antisense compounds targeting hepatic PNPLA3 as a strategy to decrease the accumulation of mutant PNPLA3I148M on LDs or alternatively develop strategies to augment CGI‐58/ATGL interactions. The current work illuminates a path from cell biological pathways and biochemical mechanisms to testable hypotheses that may benefit patients with liver disease.