Patatin-domain lipid-droplet protein of the PNPLA family, most closely related to PNPLA2/ATGL, expressed mainly in liver and adipose tissue and strongly induced by feeding, insulin and carbohydrate. It is a single-pass type II membrane protein that partitions between the endoplasmic reticulum and the surface of lipid droplets, and it carries a Ser47/Asp166 catalytic dyad. Purified PNPLA3 hydrolyses triacylglycerol, diacylglycerol and monoacylglycerol with a preference for oleate and for polyunsaturated acyl chains, and also performs CoA-independent transacylation between acylglycerols; in hepatocytes it remodels the fatty-acid composition of the triacylglycerol pool rather than driving net lipolysis. Its triglyceride hydrolase activity is stimulated by ABHD5/CGI-58, the same co-activator that ATGL requires, and PNPLA3 binds ABHD5 more avidly than ATGL does and competes with ATGL for it, so PNPLA3 on the droplet surface restrains ATGL-mediated lipolysis. What the protein's dominant physiological reaction is remains unsettled: one group reports a CoA-dependent lysophosphatidic acid acyltransferase activity that another group, assaying the same reaction with a positive control, could not detect, and mice lacking PNPLA3 have no hepatic phenotype. The common I148M substitution is the strongest common genetic risk factor for steatotic liver disease. It reduces triglyceride hydrolase activity, but it also escapes ubiquitin-mediated degradation and accumulates on hepatic lipid droplets, and the three current mechanistic accounts of it differ fundamentally - loss of a polyunsaturated-triglyceride lipase that supplies VLDL secretion, gain of function through sequestration of ABHD5 away from ATGL, or a neomorphic activity that obstructs both ABHD5-dependent lipolysis and apolipoprotein B secretion.
Definition: Binding to a protein that activates a lipase, preventing that activator from interacting with the lipase and thereby reducing lipase activity.
Justification: PNPLA3 reduces ATGL/PNPLA2-mediated triglyceride hydrolysis without binding ATGL, by out-competing it for the co-activator ABHD5/CGI-58 on the lipid droplet surface. Neither existing term is a good fit on its own - GO:0055102 lipase inhibitor activity implies binding the lipase itself, while GO:0140311 protein sequestering activity says nothing about what the sequestration achieves. A term with both as parents would let this widely replicated mechanism be annotated as a molecular function instead of being invisible in GOA, and would also serve other droplet proteins reported to titrate ABHD5, such as the perilipins.
Parent term: protein sequestering activity
Supporting Evidence:
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005811 lipid droplet | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic assertion that PNPLA3 acts at the lipid droplet. Reason: Core location. Direct localisation in several systems, and LD targeting is mechanistically load-bearing - both Wang et al. and Teskey et al. show that PNPLA3 must be on the droplet for the ABHD5 interaction and the steatotic effect. Supporting Evidence: PMID:39814233 Here, we demonstrate that LD targeting of both ABHD5 and PNPLA3 I148M is required for the interaction. |
| GO:0006654 phosphatidic acid biosynthetic process | IBA GO_REF:0000033 | MARK AS OVER ANNOTATED | Summary: Phosphatidic acid synthesis inherited from the PNPLA family node, resting on the disputed acyltransferase activity. Reason: This process annotation is downstream of the disputed lysophosphatidic acid acyltransferase activity (see the GO:0003841 entries). Huang et al. found no LPAAT, GPAT or DGAT activity in purified PNPLA3 with CGI-58 as a positive control, and concluded that the enzyme does not promote de novo TAG synthesis. Flagged as over-annotated rather than removed, since the underlying experimental report stands unretracted. Propagation Review Root cause: SOURCE WEAK OR INFERRED Failure modes: SOURCE EVIDENCE WEAK Sources checked: MGI:MGI:2151796 · Pnpla3 (mouse) SOURCE WEAK OR INFERRED The mouse annotation's only experimental grounding for this term is IDA from PMID:22560221, the same contested LPAAT study; the rest of its rows for the term are ISO from human PNPLA3 and ARBA IEA. PANTHER:PTN000274462 · PTN000274462 SOURCE WEAK OR INFERRED Node is seeded only by the mouse and human PNPLA3 annotations, both tracing to PMID:22560221. UniProtKB:Q9NST1 · PNPLA3 (human, the target) SOURCE WEAK OR INFERRED The target's own IDA for this term also comes from PMID:22560221. Its presence in WITH/FROM is the normal marker that the node has experimental grounding on this gene; the problem is the strength of that grounding, not circularity. Supporting Evidence: PMID:21878620 Purified PNPLA3 has no detectable GPAT, LPAAT, or DGAT activity. |
| GO:0051265 diolein transacylation activity | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic assertion of CoA-independent transacylase activity. Reason: Directly measured for purified PNPLA3 by Jenkins et al. and consistent with the cellular acyl-chain remodelling phenotype reported by Ruhanen et al. This is a CoA-independent acyl transfer between acylglycerols, and is therefore not in conflict with the hydrolase activity the way the acyl-CoA-dependent LPAAT claim is; both can be run by the same Ser47 nucleophile with water or an acylglycerol hydroxyl as the acceptor. Secondary to TAG hydrolysis but not an over-annotation. The node is seeded by PNPLA3's own experimental annotation, which is the expected marker of experimental grounding rather than circularity. Supporting Evidence: PMID:15364929 iPLA2epsilon, iPLA2zeta, and iPLA2eta also possess acylglycerol transacylase activity utilizing mono-olein as an acyl donor which, in the presence of mono-olein or diolein acceptors, results in the synthesis of diolein and triolein, respectively. |
| GO:0019433 triglyceride catabolic process | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic assertion of a role in triglyceride breakdown. Reason: Core biological process, and the direct consequence of the triacylglycerol lipase activity. Note that the net cellular effect of PNPLA3 is bidirectional - it hydrolyses and re-esterifies acyl chains - so this term should be read together with the acyl-chain remodelling terms rather than as net lipolysis. |
| GO:0055088 lipid homeostasis | IBA GO_REF:0000033 | ACCEPT | Summary: General phylogenetic assertion that PNPLA family members maintain lipid balance. Reason: Correct but very general. It is the one term in GOA that survives all three current models of what PNPLA3 does, so it is retained; the specific terms below carry the information. |
| GO:0004806 triacylglycerol lipase activity | IBA GO_REF:0000033 | ACCEPT | Summary: Triacylglycerol lipase activity, the activity PNPLA3 shares with its closest paralogue PNPLA2/ATGL. Reason: Core molecular function. TG hydrolysis by purified PNPLA3 has been measured independently by four groups (Jenkins 2004, He 2010, Huang 2011, Wang 2025); the I148M substitution reduces it by ~80% and the activity is stimulated by ABHD5/CGI-58 exactly as ATGL's is. Johnson et al. add a substrate preference for polyunsaturated triacylglycerols and an in vivo readout. What is contested is not whether PNPLA3 is a lipase but how much the lipase step matters - Pnpla3-null mice show no hepatic phenotype even on lipogenic diets, and Wang et al. argue that the loss of catalytic activity in 148M is not what causes steatosis. Retained as core because the activity is directly and repeatedly demonstrated. Supporting Evidence: PMID:20034933 In vitro assays using recombinant PNPLA3 partially purified from Sf9 cells confirmed that the wild type enzyme hydrolyzes emulsified triglyceride and that the I148M substitution abolishes this activity. |
| GO:0001676 long-chain fatty acid metabolic process | IEA GO_REF:0000117 | KEEP AS NON CORE | Summary: ARBA-generated general fatty acid metabolism term. Reason: Correct in kind - PNPLA3 releases and transfers long-chain acyl groups - but a very general process term that adds little beyond the triglyceride terms. Peripheral rather than core. |
| GO:0003841 1-acylglycerol-3-phosphate O-acyltransferase activity | IEA GO_REF:0000120 | MARK AS OVER ANNOTATED | Summary: 1-acylglycerol-3-phosphate O-acyltransferase (LPAAT) activity, one side of a direct experimental contradiction. Reason: PNPLA3 was reported to be a CoA-dependent lysophosphatidic acid acyltransferase (LPA + acyl-CoA -> phosphatidic acid) by Kumari et al., who also found the activity increased rather than decreased by I148M. Huang et al. assayed the same reaction with purified and with immunoisolated PNPLA3, using CGI-58 as a positive control, and detected none. A protein cannot physiologically be both the hydrolase and the synthetase of the same acyl linkage, and GOA currently asserts both. Separately, the GOA row carrying this term with evidence code EXP cites PMID:21878620 - the paper reporting the negative result - with a plain enables qualifier; that annotation looks like it should either carry NOT or cite a different reference, and it is traceable to UniProt's EC 2.3.1.51 block, which tags both PubMed:21878620 and PubMed:22560221 as ECO:0000269 evidence although only the latter is positive. Not removed, because Kumari et al. is a real experimental report that has not been retracted, but flagged as an over-annotation and raised in suggested_questions. Supporting Evidence: PMID:21878620 Neither the wild-type nor mutant enzyme catalyzed transfer of oleic acid from oleoyl-CoA to glycerophosphate, lysophosphatidic acid, or diacylglycerol, suggesting that the enzyme does not promote de novo TAG synthesis. PMID:22560221 Here we show that ADPN promotes cellular lipid synthesis by converting lysophosphatidic acid (LPA) into phosphatidic acid. The ADPN-catalyzed LPA acyltransferase (LPAAT) reaction is specific for LPA and long-chain acyl-CoAs. |
| GO:0004623 A2-type glycerophospholipase activity | IEA GO_REF:0000120 | MARK AS OVER ANNOTATED | Summary: Phospholipase A2 activity assigned from the EC 3.1.1.4 / RHEA:15801 mapping UniProt carries for PNPLA3. Reason: A2-type glycerophospholipase activity traces to the naming of PNPLA3 as 'calcium-independent phospholipase A2-epsilon' in Jenkins et al., which is a statement about sequence motifs (the dual GXGXXG nucleotide-binding and GXSXG lipase signatures) rather than a measured PLA2 rate; the activities that paper actually reports for the three proteins are TAG lipase and acylglycerol transacylase. Huang et al. later assayed phospholipids directly and found little or no hydrolysis. Kept rather than removed because the full text of Jenkins et al. is not available here and the curator may have seen a phospholipid assay, but flagged as a probable name-driven over-annotation. Supporting Evidence: PMID:21878620 The enzyme had little or no hydrolytic activity against the other lipid substrates tested, including phospholipids, cholesteryl ester, and retinyl esters. |
| GO:0004806 triacylglycerol lipase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Triacylglycerol lipase activity, the activity PNPLA3 shares with its closest paralogue PNPLA2/ATGL. Reason: Core molecular function. TG hydrolysis by purified PNPLA3 has been measured independently by four groups (Jenkins 2004, He 2010, Huang 2011, Wang 2025); the I148M substitution reduces it by ~80% and the activity is stimulated by ABHD5/CGI-58 exactly as ATGL's is. Johnson et al. add a substrate preference for polyunsaturated triacylglycerols and an in vivo readout. What is contested is not whether PNPLA3 is a lipase but how much the lipase step matters - Pnpla3-null mice show no hepatic phenotype even on lipogenic diets, and Wang et al. argue that the loss of catalytic activity in 148M is not what causes steatosis. Retained as core because the activity is directly and repeatedly demonstrated. |
| GO:0005811 lipid droplet | IEA GO_REF:0000120 | ACCEPT | Summary: PNPLA3 is a lipid droplet surface protein; lipid droplet targeting is required for its inhibitory effect on ATGL. Reason: Core location. Direct localisation in several systems, and LD targeting is mechanistically load-bearing - both Wang et al. and Teskey et al. show that PNPLA3 must be on the droplet for the ABHD5 interaction and the steatotic effect. |
| GO:0006629 lipid metabolic process | IEA GO_REF:0000002 | MODIFY | Summary: Root-level lipid metabolism term from the InterPro patatin signature. Reason: Correct but uninformative; the specific processes PNPLA3 participates in are established experimentally. Proposed replacements: triglyceride catabolic process triglyceride acyl-chain remodeling |
| GO:0006654 phosphatidic acid biosynthetic process | IEA GO_REF:0000117 | MARK AS OVER ANNOTATED | Summary: ARBA-generated restatement of the acyltransferase route to phosphatidic acid. Reason: This process annotation is downstream of the disputed lysophosphatidic acid acyltransferase activity (see the GO:0003841 entries). Huang et al. found no LPAAT, GPAT or DGAT activity in purified PNPLA3 with CGI-58 as a positive control, and concluded that the enzyme does not promote de novo TAG synthesis. Flagged as over-annotated rather than removed, since the underlying experimental report stands unretracted. |
| GO:0016020 membrane | IEA GO_REF:0000120 | KEEP AS NON CORE | Summary: Generic membrane location from the UniProt subcellular location vocabulary. Reason: Correct - UniProt describes PNPLA3 as a single-pass type II membrane protein, and it partitions between membranes and lipid droplets - but bare 'membrane' carries almost no information. The informative locations, lipid droplet and endoplasmic reticulum membrane, are annotated separately. |
| GO:0016042 lipid catabolic process | IEA GO_REF:0000002 | MODIFY | Summary: General lipid catabolism from the InterPro signature. Reason: Correct but too general; the specific catabolic process is triglyceride hydrolysis. Proposed replacements: triglyceride catabolic process |
| GO:0016787 hydrolase activity | IEA GO_REF:0000002 | MODIFY | Summary: Root-level hydrolase activity from the InterPro patatin signature. Reason: Correct but carries no information; the specific hydrolytic reaction is well established. Proposed replacements: triacylglycerol lipase activity |
| GO:0019432 triglyceride biosynthetic process | IEA GO_REF:0000117 | KEEP AS NON CORE | Summary: Triglyceride synthesis, correct only in the transacylation sense. Reason: PNPLA3 does make triacylglycerol, but by CoA-independent transacylation of acylglycerols (Jenkins et al.), not by de novo acylation - Huang et al. explicitly excluded GPAT, LPAAT and DGAT activity and concluded the enzyme does not promote de novo TAG synthesis. Retained as a genuine but secondary capability, and deliberately not treated as core, since the term invites the de novo reading that the data exclude. |
| GO:0019433 triglyceride catabolic process | IEA GO_REF:0000120 | ACCEPT | Summary: Triglyceride breakdown from the EC 3.1.1.3 mapping. Reason: Core biological process, and the direct consequence of the triacylglycerol lipase activity. Note that the net cellular effect of PNPLA3 is bidirectional - it hydrolyses and re-esterifies acyl chains - so this term should be read together with the acyl-chain remodelling terms rather than as net lipolysis. |
| GO:0035727 lysophosphatidic acid binding | IEA GO_REF:0000117 | MARK AS OVER ANNOTATED | Summary: Substrate binding inferred from the disputed acyltransferase activity. Reason: Lysophosphatidic acid binding was inferred from the LPAAT assays of Kumari et al. and stands or falls with that activity, which Huang et al. could not reproduce. Flagged with the same status as the acyltransferase terms rather than given independent weight. |
| GO:0036042 long-chain fatty acyl-CoA binding | IEA GO_REF:0000117 | KEEP AS NON CORE | Summary: Long-chain acyl-CoA binding. Reason: Unlike the LPA binding annotation, this one has independent support - Huang et al. showed purified PNPLA3 hydrolyses oleoyl-CoA, albeit with a Vmax 100-fold below that for triolein, which requires the enzyme to bind long-chain acyl-CoA. Real, but a minor activity rather than a core function. Supporting Evidence: PMID:21878620 Purified PNPLA3 also catalyzed the hydrolysis of oleoyl-CoA, but the V(max) was 100-fold lower for oleoyl-CoA than for triolein. |
| GO:0009744 response to sucrose | IEA GO_REF:0000107 | KEEP AS NON CORE | Summary: Nutritional induction of PNPLA3 transferred from rat. Reason: Transferred from rat by Ensembl Compara. These terms describe the transcriptional regulation of PNPLA3 by nutritional and hormonal state - UniProt records that PNPLA3 mRNA is down-regulated by a very low-calorie diet and elevated by refeeding - rather than a process the protein carries out. Real but peripheral. |
| GO:0032869 cellular response to insulin stimulus | IEA GO_REF:0000107 | KEEP AS NON CORE | Summary: Insulin-driven induction of PNPLA3 transferred from rat. Reason: Transferred from rat by Ensembl Compara. These terms describe the transcriptional regulation of PNPLA3 by nutritional and hormonal state - UniProt records that PNPLA3 mRNA is down-regulated by a very low-calorie diet and elevated by refeeding - rather than a process the protein carries out. Real but peripheral. Insulin induction is mechanistically important for the variant phenotype - 148M knock-in mice only become steatotic on diets that raise insulin and hence PNPLA3 expression - but it is an input to the gene, not an activity of the protein. |
| GO:0050872 white fat cell differentiation | IEA GO_REF:0000107 | MARK AS OVER ANNOTATED | Summary: Adipogenesis term transferred from rat. Reason: PNPLA3/adiponutrin transcript rises steeply during hormone-induced differentiation of 3T3-L1 preadipocytes, which is what this term records. Being induced during a developmental programme is not the same as executing it, and there is no evidence that PNPLA3 drives adipocyte differentiation. |
| GO:1905243 cellular response to 3,3',5-triiodo-L-thyronine | IEA GO_REF:0000107 | KEEP AS NON CORE | Summary: Thyroid hormone responsiveness of PNPLA3 transferred from rat. Reason: Transferred from rat by Ensembl Compara. These terms describe the transcriptional regulation of PNPLA3 by nutritional and hormonal state - UniProt records that PNPLA3 mRNA is down-regulated by a very low-calorie diet and elevated by refeeding - rather than a process the protein carries out. Real but peripheral. |
| GO:0036155 acylglycerol acyl-chain remodeling | TAS Reactome:R-HSA-1482883 | ACCEPT | Summary: Reactome pathway assignment for acyl-chain remodelling of DAG and TAG. Reason: Core biological process and the best single description of what PNPLA3 does to hepatocyte lipid. Ruhanen et al. showed directly that wild-type PNPLA3 accelerates turnover of labelled oleate in TAG and shifts the TAG pool towards saturated and monounsaturated species with reciprocal enrichment of polyunsaturated phosphatidylcholine, an effect lost with I148M. Remodelling by sequential deacylation and re-acylation is also what reconciles the hydrolase and transacylase activities into one job. |
| GO:0004465 lipoprotein lipase activity | TAS Reactome:R-HSA-1482811 | MODIFY | Summary: Lipoprotein lipase activity asserted from the Reactome reaction 'DAG is hydrolyzed to 2-MAG by PNPLA2/3'. Reason: The GO term requires that the triacylglycerol being hydrolysed is part of a lipoprotein. PNPLA3 is an intracellular single-pass membrane and lipid droplet protein and has never been shown to act on circulating lipoproteins; the Reactome reaction it comes from is an intracellular DAG hydrolysis step, not lipoprotein lipolysis. This is a wrong-term mapping rather than a wrong biological claim, and the intended activity is already annotated. Proposed replacements: triacylglycerol lipase activity |
| GO:0004806 triacylglycerol lipase activity | TAS Reactome:R-HSA-1482777 | ACCEPT | Summary: Triacylglycerol lipase activity, the activity PNPLA3 shares with its closest paralogue PNPLA2/ATGL. Reason: Core molecular function. TG hydrolysis by purified PNPLA3 has been measured independently by four groups (Jenkins 2004, He 2010, Huang 2011, Wang 2025); the I148M substitution reduces it by ~80% and the activity is stimulated by ABHD5/CGI-58 exactly as ATGL's is. Johnson et al. add a substrate preference for polyunsaturated triacylglycerols and an in vivo readout. What is contested is not whether PNPLA3 is a lipase but how much the lipase step matters - Pnpla3-null mice show no hepatic phenotype even on lipogenic diets, and Wang et al. argue that the loss of catalytic activity in 148M is not what causes steatosis. Retained as core because the activity is directly and repeatedly demonstrated. |
| GO:0003841 1-acylglycerol-3-phosphate O-acyltransferase activity | EXP PMID:21878620 Expression and characterization of a PNPLA3 protein isoform ... | MARK AS OVER ANNOTATED | Summary: LPAAT activity annotated with evidence code EXP to the very paper that reports the negative result for this reaction. Reason: PNPLA3 was reported to be a CoA-dependent lysophosphatidic acid acyltransferase (LPA + acyl-CoA -> phosphatidic acid) by Kumari et al., who also found the activity increased rather than decreased by I148M. Huang et al. assayed the same reaction with purified and with immunoisolated PNPLA3, using CGI-58 as a positive control, and detected none. A protein cannot physiologically be both the hydrolase and the synthetase of the same acyl linkage, and GOA currently asserts both. Separately, the GOA row carrying this term with evidence code EXP cites PMID:21878620 - the paper reporting the negative result - with a plain enables qualifier; that annotation looks like it should either carry NOT or cite a different reference, and it is traceable to UniProt's EC 2.3.1.51 block, which tags both PubMed:21878620 and PubMed:22560221 as ECO:0000269 evidence although only the latter is positive. Not removed, because Kumari et al. is a real experimental report that has not been retracted, but flagged as an over-annotation and raised in suggested_questions. Supporting Evidence: PMID:21878620 Purified PNPLA3 has no detectable GPAT, LPAAT, or DGAT activity. |
| GO:0004806 triacylglycerol lipase activity | EXP PMID:21878620 Expression and characterization of a PNPLA3 protein isoform ... | ACCEPT | Summary: Substrate profiling of purified recombinant PNPLA3 and PNPLA3-I148M. Reason: Core molecular function. TG hydrolysis by purified PNPLA3 has been measured independently by four groups (Jenkins 2004, He 2010, Huang 2011, Wang 2025); the I148M substitution reduces it by ~80% and the activity is stimulated by ABHD5/CGI-58 exactly as ATGL's is. Johnson et al. add a substrate preference for polyunsaturated triacylglycerols and an in vivo readout. What is contested is not whether PNPLA3 is a lipase but how much the lipase step matters - Pnpla3-null mice show no hepatic phenotype even on lipogenic diets, and Wang et al. argue that the loss of catalytic activity in 148M is not what causes steatosis. Retained as core because the activity is directly and repeatedly demonstrated. Supporting Evidence: PMID:21878620 Maximal hydrolytic activity of PNPLA3 was observed against the three major glycerolipids, TAG, diacylglycerol, and monoacylglycerol, with a strong preference for oleic acid as the acyl moiety. |
| GO:0016020 membrane | EXP PMID:15364929 Identification, cloning, expression, and purification of thr... | KEEP AS NON CORE | Summary: Membrane association of the purified/expressed protein. Reason: Correct - UniProt describes PNPLA3 as a single-pass type II membrane protein, and it partitions between membranes and lipid droplets - but bare 'membrane' carries almost no information. The informative locations, lipid droplet and endoplasmic reticulum membrane, are annotated separately. |
| GO:0016411 acylglycerol O-acyltransferase activity | TAS Reactome:R-HSA-1482647 | MODIFY | Summary: Acyl transfer onto an acylglycerol, from the Reactome transacylation reactions. Reason: The Reactome reactions behind these rows are CoA-independent transacylations of monoacylglycerol and diacylglycerol, i.e. the reaction Jenkins et al. measured. The GO term is therefore right in kind but general, and general enough to be read as an acyl-CoA-dependent acyltransferase, which is precisely the activity Huang et al. excluded. Replacing it with the two specific transacylation terms removes that ambiguity. Proposed replacements: mono-olein transacylation activity diolein transacylation activity |
| GO:0016411 acylglycerol O-acyltransferase activity | TAS Reactome:R-HSA-1482654 | MODIFY | Summary: Acyl transfer onto an acylglycerol, from the Reactome transacylation reactions. Reason: The Reactome reactions behind these rows are CoA-independent transacylations of monoacylglycerol and diacylglycerol, i.e. the reaction Jenkins et al. measured. The GO term is therefore right in kind but general, and general enough to be read as an acyl-CoA-dependent acyltransferase, which is precisely the activity Huang et al. excluded. Replacing it with the two specific transacylation terms removes that ambiguity. Proposed replacements: mono-olein transacylation activity diolein transacylation activity |
| GO:0004806 triacylglycerol lipase activity | IDA PMID:20034933 A sequence variation (I148M) in PNPLA3 associated with nonal... | ACCEPT | Summary: Hydrolysis of emulsified triglyceride by recombinant PNPLA3, abolished by I148M. Reason: Core molecular function. TG hydrolysis by purified PNPLA3 has been measured independently by four groups (Jenkins 2004, He 2010, Huang 2011, Wang 2025); the I148M substitution reduces it by ~80% and the activity is stimulated by ABHD5/CGI-58 exactly as ATGL's is. Johnson et al. add a substrate preference for polyunsaturated triacylglycerols and an in vivo readout. What is contested is not whether PNPLA3 is a lipase but how much the lipase step matters - Pnpla3-null mice show no hepatic phenotype even on lipogenic diets, and Wang et al. argue that the loss of catalytic activity in 148M is not what causes steatosis. Retained as core because the activity is directly and repeatedly demonstrated. Supporting Evidence: PMID:20034933 In vitro assays using recombinant PNPLA3 partially purified from Sf9 cells confirmed that the wild type enzyme hydrolyzes emulsified triglyceride and that the I148M substitution abolishes this activity. |
| GO:0005811 lipid droplet | IDA PMID:20034933 A sequence variation (I148M) in PNPLA3 associated with nonal... | ACCEPT | Summary: PNPLA3 is a lipid droplet surface protein; lipid droplet targeting is required for its inhibitory effect on ATGL. Reason: Core location. Direct localisation in several systems, and LD targeting is mechanistically load-bearing - both Wang et al. and Teskey et al. show that PNPLA3 must be on the droplet for the ABHD5 interaction and the steatotic effect. Supporting Evidence: PMID:20034933 Cell fractionation studies revealed that approximately 90% of wild type PNPLA3 partitioned between membranes and lipid droplets |
| GO:0003841 1-acylglycerol-3-phosphate O-acyltransferase activity | IDA PMID:22560221 Adiponutrin functions as a nutritionally regulated lysophosp... | MARK AS OVER ANNOTATED | Summary: Direct report that adiponutrin converts lysophosphatidic acid to phosphatidic acid. Reason: PNPLA3 was reported to be a CoA-dependent lysophosphatidic acid acyltransferase (LPA + acyl-CoA -> phosphatidic acid) by Kumari et al., who also found the activity increased rather than decreased by I148M. Huang et al. assayed the same reaction with purified and with immunoisolated PNPLA3, using CGI-58 as a positive control, and detected none. A protein cannot physiologically be both the hydrolase and the synthetase of the same acyl linkage, and GOA currently asserts both. Separately, the GOA row carrying this term with evidence code EXP cites PMID:21878620 - the paper reporting the negative result - with a plain enables qualifier; that annotation looks like it should either carry NOT or cite a different reference, and it is traceable to UniProt's EC 2.3.1.51 block, which tags both PubMed:21878620 and PubMed:22560221 as ECO:0000269 evidence although only the latter is positive. Not removed, because Kumari et al. is a real experimental report that has not been retracted, but flagged as an over-annotation and raised in suggested_questions. Supporting Evidence: PMID:22560221 Notably, the I148M variant of human ADPN exhibits increased LPAAT activity leading to increased cellular lipid accumulation. |
| GO:0005811 lipid droplet | IDA PMID:23398201 PNPLA3/adiponutrin functions in lipid droplet formation. | ACCEPT | Summary: PNPLA3 is a lipid droplet surface protein; lipid droplet targeting is required for its inhibitory effect on ATGL. Reason: Core location. Direct localisation in several systems, and LD targeting is mechanistically load-bearing - both Wang et al. and Teskey et al. show that PNPLA3 must be on the droplet for the ABHD5 interaction and the steatotic effect. Supporting Evidence: PMID:23398201 We demonstrate that PNPLA3 is targetted to LDs in a process that requires an intact Brummer box domain |
| GO:0034389 lipid droplet organization | IMP PMID:23398201 PNPLA3/adiponutrin functions in lipid droplet formation. | ACCEPT | Summary: Changing PNPLA3 levels changes lipid droplet size in both directions. Reason: Well supported and mechanistically central - the disease-relevant behaviour of PNPLA3 is expressed through the droplet surface. Chamoun et al. also saw that co-expressed ABHD5/CGI-58 reversed the droplet enlargement caused by PNPLA3, an early observation of the ABHD5 relationship that later work made central. Supporting Evidence: PMID:23398201 We show that increased levels of the NAFLD-linked PNPLA3 isoform leads to larger LDs, whereas decreased levels of PNPLA3 had the opposite effect. |
| GO:0001676 long-chain fatty acid metabolic process | IDA PMID:22560221 Adiponutrin functions as a nutritionally regulated lysophosp... | KEEP AS NON CORE | Summary: Long-chain acyl handling observed in the adiponutrin acyltransferase study. Reason: Correct in kind - PNPLA3 releases and transfers long-chain acyl groups - but a very general process term that adds little beyond the triglyceride terms. Peripheral rather than core. |
| GO:0004806 triacylglycerol lipase activity | IDA PMID:22560221 Adiponutrin functions as a nutritionally regulated lysophosp... | ACCEPT | Summary: Triacylglycerol lipase activity, the activity PNPLA3 shares with its closest paralogue PNPLA2/ATGL. Reason: Core molecular function. TG hydrolysis by purified PNPLA3 has been measured independently by four groups (Jenkins 2004, He 2010, Huang 2011, Wang 2025); the I148M substitution reduces it by ~80% and the activity is stimulated by ABHD5/CGI-58 exactly as ATGL's is. Johnson et al. add a substrate preference for polyunsaturated triacylglycerols and an in vivo readout. What is contested is not whether PNPLA3 is a lipase but how much the lipase step matters - Pnpla3-null mice show no hepatic phenotype even on lipogenic diets, and Wang et al. argue that the loss of catalytic activity in 148M is not what causes steatosis. Retained as core because the activity is directly and repeatedly demonstrated. Kumari et al. themselves detected only very low TG hydrolase activity, which UniProt records as making the hydrolase assignment controversial; the weight for this term comes from the other four studies. |
| GO:0005811 lipid droplet | IDA PMID:22560221 Adiponutrin functions as a nutritionally regulated lysophosp... | ACCEPT | Summary: PNPLA3 is a lipid droplet surface protein; lipid droplet targeting is required for its inhibitory effect on ATGL. Reason: Core location. Direct localisation in several systems, and LD targeting is mechanistically load-bearing - both Wang et al. and Teskey et al. show that PNPLA3 must be on the droplet for the ABHD5 interaction and the steatotic effect. |
| GO:0006650 glycerophospholipid metabolic process | IDA PMID:22560221 Adiponutrin functions as a nutritionally regulated lysophosp... | MARK AS OVER ANNOTATED | Summary: Glycerophospholipid metabolism via the disputed LPA-to-PA step. Reason: This process annotation is downstream of the disputed lysophosphatidic acid acyltransferase activity (see the GO:0003841 entries). Huang et al. found no LPAAT, GPAT or DGAT activity in purified PNPLA3 with CGI-58 as a positive control, and concluded that the enzyme does not promote de novo TAG synthesis. Flagged as over-annotated rather than removed, since the underlying experimental report stands unretracted. |
| GO:0006654 phosphatidic acid biosynthetic process | IDA PMID:22560221 Adiponutrin functions as a nutritionally regulated lysophosp... | MARK AS OVER ANNOTATED | Summary: Phosphatidic acid synthesis via the disputed LPAAT activity. Reason: This process annotation is downstream of the disputed lysophosphatidic acid acyltransferase activity (see the GO:0003841 entries). Huang et al. found no LPAAT, GPAT or DGAT activity in purified PNPLA3 with CGI-58 as a positive control, and concluded that the enzyme does not promote de novo TAG synthesis. Flagged as over-annotated rather than removed, since the underlying experimental report stands unretracted. |
| GO:0016020 membrane | IDA PMID:22560221 Adiponutrin functions as a nutritionally regulated lysophosp... | KEEP AS NON CORE | Summary: Membrane fraction localisation. Reason: Correct - UniProt describes PNPLA3 as a single-pass type II membrane protein, and it partitions between membranes and lipid droplets - but bare 'membrane' carries almost no information. The informative locations, lipid droplet and endoplasmic reticulum membrane, are annotated separately. |
| GO:0019432 triglyceride biosynthetic process | IDA PMID:22560221 Adiponutrin functions as a nutritionally regulated lysophosp... | KEEP AS NON CORE | Summary: Increased cellular lipid accumulation attributed to acyltransferase activity. Reason: PNPLA3 does make triacylglycerol, but by CoA-independent transacylation of acylglycerols (Jenkins et al.), not by de novo acylation - Huang et al. explicitly excluded GPAT, LPAAT and DGAT activity and concluded the enzyme does not promote de novo TAG synthesis. Retained as a genuine but secondary capability, and deliberately not treated as core, since the term invites the de novo reading that the data exclude. |
| GO:0019433 triglyceride catabolic process | IDA PMID:22560221 Adiponutrin functions as a nutritionally regulated lysophosp... | ACCEPT | Summary: Triglyceride hydrolysis measured alongside the acyltransferase work. Reason: Core biological process, and the direct consequence of the triacylglycerol lipase activity. Note that the net cellular effect of PNPLA3 is bidirectional - it hydrolyses and re-esterifies acyl chains - so this term should be read together with the acyl-chain remodelling terms rather than as net lipolysis. |
| GO:0035727 lysophosphatidic acid binding | IDA PMID:22560221 Adiponutrin functions as a nutritionally regulated lysophosp... | MARK AS OVER ANNOTATED | Summary: Substrate binding inferred from the LPAAT assays. Reason: Lysophosphatidic acid binding was inferred from the LPAAT assays of Kumari et al. and stands or falls with that activity, which Huang et al. could not reproduce. Flagged with the same status as the acyltransferase terms rather than given independent weight. |
| GO:0036042 long-chain fatty acyl-CoA binding | IDA PMID:22560221 Adiponutrin functions as a nutritionally regulated lysophosp... | KEEP AS NON CORE | Summary: Acyl donor specificity for long-chain acyl-CoAs. Reason: Unlike the LPA binding annotation, this one has independent support - Huang et al. showed purified PNPLA3 hydrolyses oleoyl-CoA, albeit with a Vmax 100-fold below that for triolein, which requires the enzyme to bind long-chain acyl-CoA. Real, but a minor activity rather than a core function. |
| GO:0042171 lysophosphatidic acid acyltransferase activity | IDA PMID:22560221 Adiponutrin functions as a nutritionally regulated lysophosp... | MARK AS OVER ANNOTATED | Summary: The same disputed acyltransferase reaction under its substrate-specific term. Reason: PNPLA3 was reported to be a CoA-dependent lysophosphatidic acid acyltransferase (LPA + acyl-CoA -> phosphatidic acid) by Kumari et al., who also found the activity increased rather than decreased by I148M. Huang et al. assayed the same reaction with purified and with immunoisolated PNPLA3, using CGI-58 as a positive control, and detected none. A protein cannot physiologically be both the hydrolase and the synthetase of the same acyl linkage, and GOA currently asserts both. Separately, the GOA row carrying this term with evidence code EXP cites PMID:21878620 - the paper reporting the negative result - with a plain enables qualifier; that annotation looks like it should either carry NOT or cite a different reference, and it is traceable to UniProt's EC 2.3.1.51 block, which tags both PubMed:21878620 and PubMed:22560221 as ECO:0000269 evidence although only the latter is positive. Not removed, because Kumari et al. is a real experimental report that has not been retracted, but flagged as an over-annotation and raised in suggested_questions. |
| GO:0036153 triglyceride acyl-chain remodeling | IDA PMID:24511104 PNPLA3 mediates hepatocyte triacylglycerol remodeling. | ACCEPT | Summary: Stable-isotope tracing in hepatocytes showing fatty-acid-selective TAG remodelling by PNPLA3. Reason: Core biological process and the best single description of what PNPLA3 does to hepatocyte lipid. Ruhanen et al. showed directly that wild-type PNPLA3 accelerates turnover of labelled oleate in TAG and shifts the TAG pool towards saturated and monounsaturated species with reciprocal enrichment of polyunsaturated phosphatidylcholine, an effect lost with I148M. Remodelling by sequential deacylation and re-acylation is also what reconciles the hydrolase and transacylase activities into one job. Supporting Evidence: PMID:24511104 This study reveals a function of PNPLA3 in FA-selective TAG remodeling, resulting in increased TAG saturation. |
| GO:0005789 endoplasmic reticulum membrane | TAS Reactome:R-HSA-1482647 | ACCEPT | Summary: Endoplasmic reticulum membrane location from Reactome. Reason: Supported beyond Reactome - Ruhanen et al. found that PNPLA3 distributes between lipid droplets and the endoplasmic reticulum/cytosol and that I148M shifts it towards droplets, and UniProt classifies PNPLA3 as a single-pass type II membrane protein. A real secondary location. |
| GO:0005789 endoplasmic reticulum membrane | TAS Reactome:R-HSA-1482654 | ACCEPT | Summary: Endoplasmic reticulum membrane location from Reactome. Reason: Supported beyond Reactome - Ruhanen et al. found that PNPLA3 distributes between lipid droplets and the endoplasmic reticulum/cytosol and that I148M shifts it towards droplets, and UniProt classifies PNPLA3 as a single-pass type II membrane protein. A real secondary location. |
| GO:0005789 endoplasmic reticulum membrane | TAS Reactome:R-HSA-1482777 | ACCEPT | Summary: Endoplasmic reticulum membrane location from Reactome. Reason: Supported beyond Reactome - Ruhanen et al. found that PNPLA3 distributes between lipid droplets and the endoplasmic reticulum/cytosol and that I148M shifts it towards droplets, and UniProt classifies PNPLA3 as a single-pass type II membrane protein. A real secondary location. |
| GO:0005789 endoplasmic reticulum membrane | TAS Reactome:R-HSA-1482811 | ACCEPT | Summary: Endoplasmic reticulum membrane location from Reactome. Reason: Supported beyond Reactome - Ruhanen et al. found that PNPLA3 distributes between lipid droplets and the endoplasmic reticulum/cytosol and that I148M shifts it towards droplets, and UniProt classifies PNPLA3 as a single-pass type II membrane protein. A real secondary location. |
| GO:0051264 mono-olein transacylation activity | IDA PMID:15364929 Identification, cloning, expression, and purification of thr... | ACCEPT | Summary: CoA-independent transacylation measured with purified protein. Reason: Directly measured with affinity-purified protein, using mono-olein as acyl donor to build diolein and triolein. CoA-independent transacylation is chemically compatible with the same Ser47 nucleophile that performs hydrolysis (the acyl-enzyme intermediate is resolved by an acylglycerol hydroxyl instead of water), so unlike the acyl-CoA-dependent LPAAT claim it does not contradict the lipase activity. Kept, and read together with the acyl-chain remodelling terms. Supporting Evidence: PMID:15364929 iPLA2epsilon, iPLA2zeta, and iPLA2eta also possess acylglycerol transacylase activity utilizing mono-olein as an acyl donor which, in the presence of mono-olein or diolein acceptors, results in the synthesis of diolein and triolein, respectively. |
| GO:0051265 diolein transacylation activity | IDA PMID:15364929 Identification, cloning, expression, and purification of thr... | ACCEPT | Summary: CoA-independent transacylation measured with purified protein. Reason: Directly measured with affinity-purified protein, using mono-olein as acyl donor to build diolein and triolein. CoA-independent transacylation is chemically compatible with the same Ser47 nucleophile that performs hydrolysis (the acyl-enzyme intermediate is resolved by an acylglycerol hydroxyl instead of water), so unlike the acyl-CoA-dependent LPAAT claim it does not contradict the lipase activity. Kept, and read together with the acyl-chain remodelling terms. |
| GO:0016020 membrane | ISS GO_REF:0000024 | KEEP AS NON CORE | Summary: Membrane location projected from the mouse orthologue. Reason: Correct - UniProt describes PNPLA3 as a single-pass type II membrane protein, and it partitions between membranes and lipid droplets - but bare 'membrane' carries almost no information. The informative locations, lipid droplet and endoplasmic reticulum membrane, are annotated separately. |
| GO:0004806 triacylglycerol lipase activity | IDA PMID:15364929 Identification, cloning, expression, and purification of thr... | ACCEPT | Summary: The founding biochemical demonstration of TAG lipase activity for adiponutrin/PNPLA3. Reason: Core molecular function. TG hydrolysis by purified PNPLA3 has been measured independently by four groups (Jenkins 2004, He 2010, Huang 2011, Wang 2025); the I148M substitution reduces it by ~80% and the activity is stimulated by ABHD5/CGI-58 exactly as ATGL's is. Johnson et al. add a substrate preference for polyunsaturated triacylglycerols and an in vivo readout. What is contested is not whether PNPLA3 is a lipase but how much the lipase step matters - Pnpla3-null mice show no hepatic phenotype even on lipogenic diets, and Wang et al. argue that the loss of catalytic activity in 148M is not what causes steatosis. Retained as core because the activity is directly and repeatedly demonstrated. Supporting Evidence: PMID:15364929 we demonstrate that each possesses abundant TAG lipase activity |
| GO:0019432 triglyceride biosynthetic process | IDA PMID:15364929 Identification, cloning, expression, and purification of thr... | KEEP AS NON CORE | Summary: Triolein synthesis by transacylation from mono-olein and diolein. Reason: PNPLA3 does make triacylglycerol, but by CoA-independent transacylation of acylglycerols (Jenkins et al.), not by de novo acylation - Huang et al. explicitly excluded GPAT, LPAAT and DGAT activity and concluded the enzyme does not promote de novo TAG synthesis. Retained as a genuine but secondary capability, and deliberately not treated as core, since the term invites the de novo reading that the data exclude. |
| GO:0019433 triglyceride catabolic process | IDA PMID:15364929 Identification, cloning, expression, and purification of thr... | ACCEPT | Summary: Triglyceride hydrolysis by purified PNPLA3. Reason: Core biological process, and the direct consequence of the triacylglycerol lipase activity. Note that the net cellular effect of PNPLA3 is bidirectional - it hydrolyses and re-esterifies acyl chains - so this term should be read together with the acyl-chain remodelling terms rather than as net lipolysis. |
| GO:0004623 A2-type glycerophospholipase activity | IDA PMID:15364929 Identification, cloning, expression, and purification of thr... | MARK AS OVER ANNOTATED | Summary: Phospholipase A2 activity attributed to PNPLA3 in the paper that named it iPLA2-epsilon. Reason: A2-type glycerophospholipase activity traces to the naming of PNPLA3 as 'calcium-independent phospholipase A2-epsilon' in Jenkins et al., which is a statement about sequence motifs (the dual GXGXXG nucleotide-binding and GXSXG lipase signatures) rather than a measured PLA2 rate; the activities that paper actually reports for the three proteins are TAG lipase and acylglycerol transacylase. Huang et al. later assayed phospholipids directly and found little or no hydrolysis. Kept rather than removed because the full text of Jenkins et al. is not available here and the curator may have seen a phospholipid assay, but flagged as a probable name-driven over-annotation. |
| GO:0140311 protein sequestering activity | IDA PMID:39550037 PNPLA3(148M) is a gain-of-function mutation that promotes he... | NEW | Summary: Proposed new annotation. PNPLA3 binds ABHD5/CGI-58 in preference to ATGL and competes with ATGL for it, retaining the co-activator on the lipid droplet surface and so limiting ATGL-mediated triglyceride hydrolysis. Reason: This activity is absent from GOA entirely, yet it is the molecular event on which all three current models of the I148M variant converge, and it has been demonstrated independently by two laboratories using luciferase complementation, co-immunoprecipitation, imaging and fluorescence cross-correlation spectroscopy. Wild-type and I148M protein bind ABHD5 with similar characteristics, so this is a property of the gene product rather than only of the variant; what the variant adds is roughly fortyfold greater abundance on hepatic droplets. GO:0140311 is the closest existing term - it describes binding a protein to prevent it interacting with other partners - but it does not record that the sequestered protein is a lipase activator, so a more specific child is proposed in proposed_new_terms. An enzyme-regulator term of this kind, rather than a hydrolase term, may be the correct representation of PNPLA3's physiologically decisive molecular function. Supporting Evidence: PMID:39550037 ABHD5 interacted preferentially with PNPLA3 relative to ATGL in cultured hepatocytes. PMID:39550037 luciferase reconstitution assays were used in cultured hepatocytes to show that ABHD5 binds preferentially to PNPLA3 over ATGL PMID:39814233 Here, we demonstrate that LD targeting of both ABHD5 and PNPLA3 I148M is required for the interaction. |
| GO:0010897 negative regulation of triglyceride catabolic process | IDA PMID:39550037 PNPLA3(148M) is a gain-of-function mutation that promotes he... | NEW | Summary: Proposed new annotation. By holding ABHD5 on the droplet surface, PNPLA3 lowers ATGL-dependent triglyceride hydrolysis; overexpressing ABHD5 reverses the hepatic steatosis of Pnpla3 148M knock-in mice. Reason: The biological consequence of the sequestration activity, and the step that links the molecular event to the human phenotype. Inhibition requires ATGL to be present and PNPLA3 to be on the lipid droplet, which is what distinguishes it from a simple loss of PNPLA3's own lipase activity. Note this sits alongside, and is not a contradiction of, the GO:0019433 triglyceride catabolic process annotations - PNPLA3 hydrolyses triglyceride itself while restraining the larger ATGL-dependent flux. Supporting Evidence: PMID:39550037 These findings support the premise that PNPLA3(148M) is a gain-of-function mutation that promotes hepatic steatosis by accumulating on lipid droplets and inhibiting ATGL-mediated lipolysis in an ABHD5-dependent manner. PMID:41046517 We propose that I148M is a neomorph that exacerbates fatty liver risk by simultaneously impeding two major CGI-58-dependent pathways for liver triglyceride clearance: lipolysis and secretion. |
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Download this section (compressed HTML)Q: Is PNPLA3's physiologically relevant molecular function catalytic at all? Its triglyceride hydrolase activity is well measured in vitro but dispensable in Pnpla3-null mice, while its competition with ATGL for ABHD5/CGI-58 accounts for the human genetic signal. If the sequestration arm is the dominant one, the appropriate molecular function is an enzyme-regulator term rather than a hydrolase term.
Suggested experts: Helen H Hobbs, Jonathan C Cohen, David E Cohen, Raymond J Deshaies
Q: Is PNPLA3 an acyltransferase? Kumari et al. report a CoA-dependent lysophosphatidic acid acyltransferase activity and Huang et al. report none for the same reaction with CGI-58 as a positive control. GOA currently asserts both the hydrolysis and the synthesis of the same acyl linkage. What accounts for the discrepancy - protein preparation, detergent state, or assay configuration?
Suggested experts: Rudolf Zechner, Robert Zimmermann, Helen H Hobbs
Q: Why does GOA carry GO:0003841 1-acylglycerol-3-phosphate O-acyltransferase activity with evidence code EXP and PMID:21878620 as the reference, without a NOT qualifier, when that paper reports the reaction as undetectable? Should the annotation be negated or re-referenced, and should UniProt's EC 2.3.1.51 evidence block stop citing PubMed:21878620?
Q: Does wild-type PNPLA3, at its endogenous abundance, measurably restrain ATGL-mediated lipolysis in normal liver, or does the sequestration effect only become significant when I148M accumulates on droplets? This decides whether the ABHD5-sequestration molecular function belongs to the gene product or only to the variant.
Suggested experts: Helen H Hobbs, James G Granneman, Emilio P Mottillo
Q: How can hepatic Pnpla3 knockout aggravate steatosis and reduce VLDL-triglyceride secretion in one laboratory while Pnpla3-null mice show hepatic triglyceride comparable to wild type, even on high-carbohydrate diets, in another? Dietary composition has been suggested but not tested head to head.
Suggested experts: Jun Liu, Helen H Hobbs, Jonathan C Cohen
Q: Does PNPLA3 have measurable phospholipase A2 activity, or is the 'iPLA2-epsilon' designation purely a sequence-motif classification? Huang et al. found little or no hydrolysis of phospholipids by purified PNPLA3.
Suggested experts: Richard W Gross, Christopher M Jenkins
Experiment: Generate knock-in mice carrying a PNPLA3 allele that is catalytically dead (S47A) but retains ABHD5 binding, and a separate allele that retains full hydrolase activity but carries C-terminal substitutions that abolish lipid droplet targeting and hence the ABHD5 interaction, each on both the wild-type and the 148M background. Phenotype hepatic triglyceride, VLDL-triglyceride secretion and ATGL-dependent lipolysis on a high-sucrose diet. If steatosis tracks with ABHD5 binding and not with catalysis, the enzyme-regulator function is the physiological one.
Hypothesis: The physiologically decisive activity of PNPLA3 is sequestration of ABHD5, not its own triglyceride hydrolase activity.
Type: Separation-of-function knock-in mouse genetics
Experiment: Run a blinded head-to-head assay in which the Zechner and Hobbs laboratory preparations of recombinant PNPLA3 and PNPLA3-I148M are exchanged and each is assayed by both groups' protocols, with CGI-58 as a positive control and a catalytically dead S47A protein as a negative control, reading out phosphatidic acid formation by mass spectrometry rather than radiolabel incorporation alone. Include a parallel triglyceride hydrolase readout on the same preparations so that activity ratios rather than absolute rates are compared.
Hypothesis: The disagreement over lysophosphatidic acid acyltransferase activity is an assay artefact rather than a real difference in the protein.
Type: Inter-laboratory blinded enzymology
Experiment: In primary human hepatocytes from I148I homozygotes, titrate endogenous PNPLA3 with siRNA across a range that stays within physiological abundance, and measure ABHD5-dependent ATGL activity and triglyceride turnover with stable-isotope tracing under both fasted-like and lipogenic conditions. A dose-dependent increase in ATGL activity as wild-type PNPLA3 falls would establish the sequestration function for the wild-type protein.
Hypothesis: Wild-type PNPLA3 restrains ATGL at endogenous expression levels.
Type: Graded knockdown with stable-isotope flux measurement
Experiment: Use dual stable-isotope labelling (distinct labels on the glycerol backbone and on the acyl donor) in hepatocytes expressing wild-type PNPLA3, S47A and I148M to separate hydrolysis, re-esterification and direct acyl transfer, quantifying positional acyl-chain exchange in triacylglycerol without net change in triacylglycerol mass. This would establish whether GO:0051264 and GO:0051265 transacylation, rather than net lipolysis, describe what PNPLA3 does in cells.
Hypothesis: PNPLA3 acts as a transacylase rather than a net lipase in intact hepatocytes.
Type: Dual-label lipid flux analysis
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