The results of this study show that the amino acid substitution in PNPLA3 that confers susceptibility to nonalcoholic fatty liver disease (I148M) inhibits catalytic activity of the enzyme. Structural modeling indicated that the substitution does not perturb the position or orientation of the catalytic dyad residues (Ser-47 and Asp-166). Rather, the side chain of the methionine at residue 148 extends into the catalytic site, shielding the serine side chain from access to substrate. In vitro assays using recombinant PNPLA3 were consistent with the predictions of the structural model; whereas the wild type enzyme hydrolyzed emulsified triglycerides, the mutant enzyme was inactive against this substrate. Studies in mice indicated that the accumulation of triglyceride associated with the Met148 isoform is due to expression of the mutant protein, rather than loss of the wild type enzyme activity. Overexpression of wild type human PNPLA3 did not affect liver triglyceride content in mice, suggesting that PNPLA3 may not be a major triglyceride hydrolase in this organ. In contrast, expression of the I148M isoform in the liver of mice increased liver triglyceride content. These findings were recapitulated in cultured hepatoma cells (HuH-7), even when de novo triglyceride formation from acyl-CoAs was inhibited by triacsin C. These data are consistent with the notion that expression of the mutant protein promotes triglyceride accumulation by inhibiting triglyceride hydrolysis in the cell.
The predicted structure of the patain domain of PNPLA3 is highly congruent with the crystal structure of heartleaf horsenettle patatin (18
) (). Patatins differ from the classical lipases in two key respects. First, patatins use a catalytic dyad (Ser-Asp), rather than the catalytic triad (Ser-His-Asp) usually present in lipases. The predicted catalytic serine of PNPLA3 (Ser47
) lies in a highly conserved hydrolase motif (Gly-X
-Gly) at a hairpin turn between a β-strand and an α-helix. The patatin fold brings this serine into close apposition to Asp166
at the edge of a putative substrate-binding groove formed by the side chains of several hydrophobic residues, including Ile148
. In the wild type protein, the side chains of Ser47
project into the groove. When methionine is substituted for isoleucine at residue 148, the longer side chain of methionine is predicted to occlude access of substrates to the catalytic dyad (). These structural predictions are consistent with the observation that the I148M substitution abolishes cleavage of emulsified triglycerides ().
Second, the patatin structure does not contain a lid domain, which mediates interfacial activation in classical lipases. Therefore, it has been proposed that patatins act on solubilized lipids rather than on micelles like the lid domain-containing lipases (18
). In contrast to plant patatins, which are soluble proteins (9
), PNPLA3 is tightly associated with membranes and with lipid droplets. Constructs comprising the patatin domain alone were also firmly anchored to the membrane (). Whereas these experiments do not preclude additional sites of interaction between PNPLA3 and membranes, they indicate that the patatin domain of PNPLA3 interacts with the membrane. Previous studies proposed that PNPLA3 contains membrane spanning domains on the basis of its predicted secondary structure (8
). However, our model of the tertiary structure of the enzyme indicates that all of the helices form part of the globular structure of the protein and do not span the membrane. The active site of PNPLA3 is surrounded by hydrophobic and lysine residues that are exposed on the protein surface and probably mediate interaction with the membrane. The close proximity of the active site to the membrane suggests that PNPLA3 is active on lipids in membranes and/or lipid droplets rather than solubilized lipids. Experiments are in progress to define the site of attachment of PNPLA3 to membranes.
The association of PNPLA3 with lipid droplets, together with the finding that the I148M substitution inactivates triglyceride hydrolysis by PNPLA3, suggests that the association between the I148M allele and hepatic triglyceride content in humans (26
) may be explained by a simple model in which PNPLA3 normally serves to hydrolyze hepatic triglycerides. In this model, the loss of enzymatic activity associated with the I148M substitution leads to triglyceride accumulation in this organ. However unlike PNPLA2 and hormone-sensitive lipase, which reduced liver triglycerides when overexpressed in this organ (31
), overexpression of wild type PNPLA3 in mouse liver failed to lower triglyceride content, whereas overexpression of the mutant PNPLA3 actually increased hepatic triglyceride levels. These data suggest that PNPLA3 is not usually rate-limiting for triglyceride hydrolysis in the liver and that the increased liver triglyceride content associated with the I148M allele is due to the presence of the mutant protein rather than the absence of the wild type enzyme activity.
The lack of reduction in hepatic triglyceride content in the livers of mice expressing high levels of the wild type human PNPLA3 may reflect interspecies differences in the role of PNPLA3. This explanation seems unlikely because high level expression of PNPLA3 did not alter triglyceride levels in cultured human hepatocytes. An alternative explanation is that PNPLA3 stimulates fatty acid re-esterification, either directly, as proposed by Jenkins et al.
), or indirectly by activating a signaling molecule that promotes triglyceride accumulation. If PNPLA3 promotes both hydrolysis and transacylation of triglycerides, then inactivation of the catalytic site may disrupt hydrolytic activity but spare the transacylase function of the enzyme, promoting triglyceride formation. In preliminary studies using recombinant PNPLA3, we failed to show appreciable transesterification of mono- and diglycerides by either the wild type or the mutant enzyme (data not shown). Additional studies will be required to clarify the role of PNPLA3 in hepatic triglyceride metabolism relative to the other lipases.
It remains possible that the inactive PNPLA3 isoforms sequester a cofactor required for hydrolysis or restrict access of the active allele (or another lipase) to the substrate, thus causing triglyceride accumulation. Many triglyceride hydrolases, including PNPLA2, the major triglyceride hydrolase in adipose tissue, require protein co-factors for activity (32
), but a similar co-factor for PNPLA3 has not been identified. The mutant enzyme did not interfere with the wild type protein in our in vitro
), but access to the substrate or to essential co-factors may be limited in vivo
The localization of PNPLA3 to both the membrane and lipid droplet fractions in cultured hepatocytes mirrors the distribution reported previously for PNPLA2, the major triglyceride hydrolase of adipose tissue (27
), and may represent partitioning of the protein between a membrane reservoir and an active pool on the surfaces of lipid droplets. Substitution of isoleucine 148 with methionine did not affect the partitioning of the protein between the membrane and cytosol or its localization into lipid droplets (E
). To exclude the possibility that PNPLA3 associates nonspecifically with lipid droplets during the isolation procedure, a recognized artifact of lipid droplet isolation (30
), we confirmed the localization of both the wild type and mutant proteins using immunofluorescence microscopy. Thus, it is unlikely that the mutation results in mistargeting the protein away from its normal sites of action.
Despite the apparent absence of a membrane-spanning domain, PNPLA3 is tightly associated with membranes; harsh treatment with high salt, high pH, or Triton X-100 failed to elute the protein from the membrane fraction. PNPLA3 may be targeted specifically to regions of the ER membrane that are destined to become nascent lipid droplets. Alternatively, PNPLA3 may be trafficked from the ER membrane to preexisting lipid droplets by components of the ER-Golgi transport machinery, as has recently been described for PNPLA2 (27
). It is also possible that PNPLA3 performs distinct functions in membranes and lipid droplets. Elucidating the physiological substrate(s) of the enzyme will be essential to unraveling its role in lipid metabolism and in the pathogenesis of fatty liver disease.