Effect of donor HSD17B13 genotype on patient survival after liver transplant: a retrospective cohort study.

Background: Several genetic variants are associated with chronic liver disease. The role of these variants in outcomes after liver transplantation (LT) is uncertain. The aim of this study was to determine if donor genotype at risk-associated variants in PNPLA3 (rs738409 C>G, p.I148M) and HSD17B13 (rs72613567 T>TA; rs80182459, p.A192Lfs∗8) influences post-LT survival. Methods: In this retrospective cohort study, data on 2346 adults who underwent first-time LT between January 1, 1999 and June 30, 2020 and who had donor DNA samples available at five large Transplant Immunology Laboratories in Texas, USA, were obtained from the United Network for Organ Sharing (UNOS). Duplicates, patients with insufficient donor DNA for genotyping, those who were <18 years of age at the time of transplant, had had a previous transplant or had missing genotype data were excluded. The primary outcomes were patient and graft survival after LT. The association between donor genotype and post-LT survival was examined using Kaplan-Meier method and multivariable-adjusted Cox proportional hazards models. Findings: Median age of LT recipients was 57 [interquartile range (IQR), 50-62] years; 837 (35.7%) were women; 1362 (58.1%) White, 713 (30.4%) Hispanic, 182 (7.8%) Black/African-American. Median follow-up time was 3.95 years. Post-LT survival was not affected by donor PNPLA3 genotype but was significantly reduced among recipients of livers with two HSD17B13 loss-of-function (LoF) variants compared to those receiving livers with no HSD17B13 LoF alleles (unadjusted one-year survival: 82.6% vs 93.9%, P < 0.0001; five-year survival: 73.1% vs 82.9%, P = 0.0017; adjusted hazard ratio [HR], 2.25; 95% CI, 1.61-3.15 after adjustment for recipient age, sex, and self-reported ethnicity). Excess mortality was restricted to those receiving steroid induction immunosuppression (crude 90-day post-LT mortality, 9.3% [95% CI, 1.9%-16.1%] vs 1.9% [95% CI, 0.9%-2.9%] in recipients of livers with two vs no HSD17B13 LoF alleles, P = 0.0012; age, sex, and ethnicity-adjusted HR, 2.85; 95% CI, 1.72-4.71, P < 0.0001). No reduction was seen among patients who did not receive steroid induction (90-day mortality 3.1% [95% CI, 0%-7.3%] vs 2% [95% CI, 0.9%-3.1%], P = 0.65; adjusted HR, 1.17; 95% CI, 0.66-2.08, P = 0.60). Interpretation: Donor HSD17B13 genotype adversely affects post-LT survival in patients receiving steroid induction. Additional studies are required to confirm this association. Funding: The National Institutes of Health and American Society of Transplant Surgeons Collaborative Scientist Grant.

The ubiquitin E3 ligase BFAR promotes degradation of PNPLA3.

A missense variant in patatin-like phospholipase domain-containing protein 3 [PNPLA3(I148M)] is the most impactful genetic risk factor for fatty liver disease (FLD). We previously showed that PNPLA3 is ubiquitylated and subsequently degraded by proteasomes and autophagosomes and that the PNPLA3(148M) variant interferes with this process. To define the machinery responsible for PNPLA3 turnover, we used small interfering (si)RNAs to inactivate components of the ubiquitin proteasome system. Inactivation of bifunctional apoptosis regulator (BFAR), a membrane-bound E3 ubiquitin ligase, reproducibly increased PNPLA3 levels in two lines of cultured hepatocytes. Conversely, overexpression of BFAR decreased levels of endogenous PNPLA3 in HuH7 cells. BFAR and PNPLA3 co-immunoprecipitated when co-expressed in cells. BFAR promoted ubiquitylation of PNPLA3 in vitro in a reconstitution assay using purified, epitope-tagged recombinant proteins. To confirm that BFAR targets PNPLA3, we inactivated Bfar in mice. Levels of PNPLA3 protein were increased twofold in hepatic lipid droplets of Bfar-/- mice with no associated increase in PNPLA3 mRNA levels. Taken together these data are consistent with a model in which BFAR plays a role in the post-translational degradation of PNPLA3. The identification of BFAR provides a potential target to enhance PNPLA3 turnover and prevent FLD.

Ligand-specific changes in conformational flexibility mediate long-range allostery in the lac repressor.

Biological regulation ubiquitously depends on protein allostery, but the regulatory mechanisms are incompletely understood, especially in proteins that undergo ligand-induced allostery with few structural changes. Here we used hydrogen-deuterium exchange with mass spectrometry (HDX/MS) to map allosteric effects in a paradigm ligand-responsive transcription factor, the lac repressor (LacI), in different functional states (apo, or bound to inducer, anti-inducer, and/or DNA). Although X-ray crystal structures of the LacI core domain in these states are nearly indistinguishable, HDX/MS experiments reveal widespread differences in flexibility. We integrate these results with modeling of protein-ligand-solvent interactions to propose a revised model for allostery in LacI, where ligand binding allosterically shifts the conformational ensemble as a result of distinct changes in the rigidity of secondary structures in the different states. Our model provides a mechanistic basis for the altered function of distal mutations. More generally, our approach provides a platform for characterizing and engineering protein allostery.

Saturation mutagenesis of a predicted ancestral Syk-family kinase.

Many tyrosine kinases cannot be expressed readily in Escherichia coli, limiting facile production of these proteins for biochemical experiments. We used ancestral sequence reconstruction to generate a spleen tyrosine kinase (Syk) variant that can be expressed in bacteria and purified in soluble form, unlike the human members of this family (Syk and zeta-chain-associated protein kinase of 70 kDa [ZAP-70]). The catalytic activity, substrate specificity, and regulation by phosphorylation of this Syk variant are similar to the corresponding properties of human Syk and ZAP-70. Taking advantage of the ability to express this novel Syk-family kinase in bacteria, we developed a two-hybrid assay that couples the growth of E. coli in the presence of an antibiotic to successful phosphorylation of a bait peptide by the kinase. Using this assay, we screened a site-saturation mutagenesis library of the kinase domain of this reconstructed Syk-family kinase. Sites of loss-of-function mutations identified in the screen correlate well with residues established previously as critical to function and/or structure in protein kinases. We also identified activating mutations in the regulatory hydrophobic spine and activation loop, which are within key motifs involved in kinase regulation. Strikingly, one mutation in an ancestral Syk-family variant increases the soluble expression of the protein by 75-fold. Thus, through ancestral sequence reconstruction followed by deep mutational scanning, we have generated Syk-family kinase variants that can be expressed in bacteria with very high yield.

Contribution of a genetic risk score to ethnic differences in fatty liver disease.

BACKGROUND AND AIMS: Susceptibility to fatty liver disease (FLD) varies among individuals and between racial/ethnic groups. Several genetic variants influence FLD risk, but whether these variants explain racial/ethnic differences in FLD prevalence is unclear. We examined the contribution of genetic risk factors to racial/ethnic-specific differences in FLD. METHODS: A case-control study comparing FLD patients (n = 1194) and population-based controls (n = 3120) was performed. Patient characteristics, FLD risk variants (PNPLA3-rs738409 + rs6006460, TM6SF2-rs58542926, HSD17B13-rs80182459 + rs72613567, MBOAT7/TMC4-rs641738, and GCKR-rs1260326) and a multi-locus genetic risk score (GRS) were examined. The odds of FLD for individuals with different risk factor burdens were determined. RESULTS: Hispanics and Whites were over-represented (56% vs. 38% and 36% vs. 29% respectively) and Blacks under-represented (5% vs. 23%) among FLD patients, compared to the population from which controls were selected (p < .001). Among cases and controls, Blacks had a lower and Hispanics a greater, net number of risk alleles than Whites (p < .001). GRS was associated with increased odds of FLD (ORQ5vsQ1  = 8.72 [95% CI = 5.97-13.0], p = 9.8 × 10-28 ), with the association being stronger in Hispanics (ORQ5vsQ1  = 14.8 [8.3-27.1]) than Blacks (ORQ5vsQ1  = 3.7 [1.5-11.5], P-interaction = 0.002). After accounting for GRS, the odds of FLD between Hispanics and Whites did not differ significantly (OR = 1.06 [0.87-1.28], p = .58), whereas Blacks retained much lower odds of FLD (OR = 0.21, [0.15-0.30], p < .001). CONCLUSIONS: Blacks had a lower and Hispanics a greater FLD risk allele burden than Whites. These differences contributed to, but did not fully explain, racial/ethnic differences in FLD prevalence. Identification of additional factors protecting Blacks from FLD may provide new targets for prevention and treatment of FLD.

The SARS-CoV-2 spike reversibly samples an open-trimer conformation exposing novel epitopes.

Current COVID-19 vaccines and many clinical diagnostics are based on the structure and function of the SARS-CoV-2 spike ectodomain. Using hydrogen-deuterium exchange monitored by mass spectrometry, we have uncovered that, in addition to the prefusion structure determined by cryo-electron microscopy, this protein adopts an alternative conformation that interconverts slowly with the canonical prefusion structure. This new conformation-an open trimer-contains easily accessible receptor-binding domains. It exposes the conserved trimer interface buried in the prefusion conformation, thus exposing potential epitopes for pan-coronavirus antibody and ligand recognition. The population of this state and kinetics of interconversion are modulated by temperature, receptor binding, antibody binding, and sequence variants observed in the natural population. Knowledge of the structure and populations of this conformation will help improve existing diagnostics, therapeutics, and vaccines.

Hepatic TM6SF2 Is Required for Lipidation of VLDL in a Pre-Golgi Compartment in Mice and Rats.

BACKGROUND & AIMS: Substitution of lysine for glutamic acid at residu 167 in Transmembrane 6 superfamily member 2 (TM6SF2) is associated with fatty liver disease and reduced plasma lipid levels. Tm6sf2-/- mice replicate the human phenotype but were not suitable for detailed mechanistic studies. As an alternative model, we generated Tm6sf2-/- rats to determine the subcellular location and function of TM6SF2. METHODS: Two lines of Tm6sf2-/- rats were established using gene editing. Lipids from tissues and from newly secreted very low density lipoproteins (VLDLs) were quantified using enzymatic assays and mass spectrometry. Neutral lipids were visualized in tissue sections using Oil Red O staining. The rate of dietary triglyceride (TG) absorption and hepatic VLDL-TG secretion were compared in Tm6sf2-/- mice and in their wild-type littermates. The intracellular location of TM6SF2 was determined by cell fractionation. Finally, TM6SF2 was immunoprecipitated from liver and enterocytes to identify interacting proteins. RESULTS: Tm6sf2-/- rats had a 6-fold higher mean hepatic TG content (56.1 ± 28.9 9 vs 9.8 ± 3.9 mg/g; P < .0001) and lower plasma cholesterol levels (99.0 ± 10.5 vs 110.6 ± 14.0 mg/dL; P = .0294) than their wild-type littermates. Rates of appearance of dietary and hepatic TG into blood were reduced significantly in Tm6sf2-/- rats (P < .001 and P < .01, respectively). Lipid content of newly secreted VLDLs isolated from perfused livers was reduced by 53% (TG) and 62% (cholesterol) (P = .005 and P = .01, respectively) in Tm6sf2-/- mice. TM6SF2 was present predominantly in the smooth endoplasmic reticulum and endoplasmic reticulum-Golgi intermediate compartments, but not in Golgi. Both apolipoprotein B-48 and acyl-CoA synthetase long chain family member 5 physically interacted with TM6SF2. CONCLUSIONS: TM6SF2 acts in the smooth endoplasmic reticulum to promote bulk lipidation of apolipoprotein B-containing lipoproteins, thus preventing fatty liver disease.

Differences in the dynamics of the tandem-SH2 modules of the Syk and ZAP-70 tyrosine kinases.

The catalytic activity of Syk-family tyrosine kinases is regulated by a tandem Src homology 2 module (tSH2 module). In the autoinhibited state, this module adopts a conformation that stabilizes an inactive conformation of the kinase domain. The binding of the tSH2 module to phosphorylated immunoreceptor tyrosine-based activation motifs necessitates a conformational change, thereby relieving kinase inhibition and promoting activation. We determined the crystal structure of the isolated tSH2 module of Syk and find, in contrast to ZAP-70, that its conformation more closely resembles that of the peptide-bound state, rather than the autoinhibited state. Hydrogen-deuterium exchange by mass spectrometry, as well as molecular dynamics simulations, reveal that the dynamics of the tSH2 modules of Syk and ZAP-70 differ, with most of these differences occurring in the C-terminal SH2 domain. Our data suggest that the conformational landscapes of the tSH2 modules in Syk and ZAP-70 have been tuned differently, such that the autoinhibited conformation of the Syk tSH2 module is less stable. This feature of Syk likely contributes to its ability to more readily escape autoinhibition when compared to ZAP-70, consistent with tighter control of downstream signaling pathways in T cells.

Molecular basis of cholesterol efflux via ABCG subfamily transporters.

The ABCG1 homodimer (G1) and ABCG5-ABCG8 heterodimer (G5G8), two members of the adenosine triphosphate (ATP)-binding cassette (ABC) transporter G family, are required for maintenance of cellular cholesterol levels. G5G8 mediates secretion of neutral sterols into bile and the gut lumen, whereas G1 transports cholesterol from macrophages to high-density lipoproteins (HDLs). The mechanisms used by G5G8 and G1 to recognize and export sterols remain unclear. Here, we report cryoelectron microscopy (cryo-EM) structures of human G5G8 in sterol-bound and human G1 in cholesterol- and ATP-bound states. Both transporters have a sterol-binding site that is accessible from the cytosolic leaflet. A second site is present midway through the transmembrane domains of G5G8. The Walker A motif of G8 adopts a unique conformation that accounts for the marked asymmetry in ATPase activities between the two nucleotide-binding sites of G5G8. These structures, along with functional validation studies, provide a mechanistic framework for understanding cholesterol efflux via ABC transporters.

Missense variant in insulin receptor (Y1355H) segregates in family with fatty liver disease.

A missense variant in the cytoplasmic domain of the insulin receptor (INSR) was identified by exome sequencing in affected members of a four-generation family with fatty liver disease (FLD). The variant (rs766457461, c.4063T>C, p.Y1355H) results in the substitution of histidine for a tyrosine that undergoes autophosphorylation in response to insulin stimulation in vitro. Because insulin promotes lipogenesis in hepatocytes, we hypothesized that the variant was causally linked to FLD in the family. To test this hypothesis, we used CRISPR/Cas9 technology to replace the corresponding tyrosine in mouse INSR with histidine (Y1345H). No significant differences were found in hepatic insulin signaling, as assessed by phosphorylation of INSR or AKT levels or in activation of the insulin-responsive transcription factor SREBP-1c. Glucose tolerance and hepatic triglyceride (TG) content in Insr1345H/H mice fed a chow diet or diets rich in fat, sucrose or fructose did not differ significantly from WT littermates. Thus, our studies in mice failed to support the notion that INSR (Y1355H) is causally related to FLD in the family or that phosphorylation of this residue alters hepatic TG metabolism.

Genetic and Metabolic Determinants of Plasma Levels of ANGPTL8.

CONTEXT: ANGPTL8 (A8) plays a key role in determining the tissue fate of circulating triglycerides (TGs). Plasma A8 levels are associated with several parameters of glucose and TG metabolism, but the causality of these relationships and the contribution of genetic variants to differences in A8 levels have not been explored. OBJECTIVE: To characterize the frequency distribution of plasma A8 levels in a diverse population using a newly-developed enzyme-linked immunosorbent assay (ELISA) and to identify genetic factors contributing to differences in plasma A8 levels. METHODS: We studied a population-based sample of Dallas County, comprising individuals in the Dallas Heart Study (DHS-1, n = 3538; DHS-2, n = 3283), including 2131 individuals with repeated measurements 7 to 9 years apart (age 18-85 years; >55% female; 52% Black; 29% White; 17% Hispanic; and 2% other). The main outcome measures were associations of A8 levels with body mass index (BMI), plasma levels of glucose, insulin, lipids, and hepatic TGs, as well as DNA variants identified by exome-wide sequencing. RESULTS: A8 levels varied over a 150-fold range (2.1-318 ng/mL; median, 13.3 ng/mL) and differed between racial/ethnic groups (Blacks > Hispanics > Whites). A8 levels correlated with BMI, fasting glucose, insulin, and TG levels. A variant in A8, R59W, accounted for 17% of the interindividual variation in A8 levels but was not associated with the metabolic parameters correlated with plasma A8 concentrations. CONCLUSIONS: A8 levels were strongly associated with indices of glucose and TG metabolism, but the lack of association of genetic variants at the A8 locus that impact A8 levels with these parameters indicates that differences in A8 levels are not causally related to the associated metabolic phenotypes.

Effects of robotic-assisted gait training on the central vascular health of individuals with spinal cord injury: A pilot study.

Objective: To investigate the effect of a short-term, robotic-assisted (exoskeleton) gait training (RGT) program on central and peripheral hemodynamic measures in patients with spinal cord injury (SCI).Design: Parallel group, non-randomized trial with before (baseline) and after (follow-up) assessments.Setting: Single-center, community-based neuro-physiotherapy practice.Participants: Twelve individuals with SCI (ASI A to C).Interventions: Participants completed either a 5-day RGT program plus physiotherapy (n = 6), or a usual care physiotherapy only program (control group; n = 6). The RGT program consisted of daily 60-min physiotherapy and 90-min of RGT. Outcome measures were measured before and after the rehabilitation program.Main outcome measure(s): The primary outcome measure was arterial wave reflection (Augmentation index [AIx]), with central and peripheral blood pressures also reported. Data are presented as mean (SD) and effect sizes (partial eta squared; η2p).Results: There was a significant reduction in AIx (30 ± 18-21 ± 15%; η2p=0.75) and mean arterial pressure (89 ± 11-82 ± 10 mmHg; η2p=0.47) following completion of the RGT program (both P < 0.05). There were no changes in these measures for the control group. Although not significantly different, medium to large effects were observed in favor of RGT for all other central and peripheral measures (η2p=0.06-0.21), except for heart rate and pulse pressure (η2p<0.04).Conclusions: RGT using an exoskeleton is a promising therapy for improving cardiovascular health in patients with SCI. Specifically, this study indicates decreased arterial wave reflection and supports the need for larger randomized controlled trials.Trial Registration: Clinical trials Registry (https://clinicaltrials.gov/; NCT03611803).

Rehabilitation Before and After Autologous Haematopoietic Stem Cell Transplantation (AHSCT) for Patients With Multiple Sclerosis (MS): Consensus Guidelines and Recommendations for Best Clinical Practice on Behalf of the Autoimmune Diseases Working Party, Nurses Group, and Patient Advocacy Committee of the European Society for Blood and Marrow Transplantation (EBMT).

Autologous haematopoietic stem cell transplantation (AHSCT) is increasingly used to treat people with multiple sclerosis (MS). Supported by an evolving evidence base, AHSCT can suppress active inflammation in the central nervous system and induce long-term changes in immune cell populations, thereby stabilizing, and, in some cases, reversing disability in carefully selected MS patients. However, AHSCT is an intensive chemotherapy-based procedure associated with intrinsic risks, including profound cytopenia, infection, and organ toxicity, accompanied by an on-going degree of immuno-compromise and general deconditioning, which can be associated with a transient increase in functional impairment in the early stages after transplantation. Although international guidelines and recommendations have been published for clinical and technical aspects of AHSCT in MS, there has been no detailed appraisal of the rehabilitation needed following treatment nor any specific guidelines as to how this is best delivered by hospital and community-based therapists and wider multidisciplinary teams in order to maximize functional recovery and quality of life. These expert consensus guidelines aim to address this unmet need by summarizing the evidence-base for AHSCT in MS and providing recommendations for current rehabilitation practice along with identifying areas for future research and development.

Angiopoietin-like protein 3 governs LDL-cholesterol levels through endothelial lipase-dependent VLDL clearance.

Angiopoietin-like protein (ANGPTL)3 regulates plasma lipids by inhibiting LPL and endothelial lipase (EL). ANGPTL3 inactivation lowers LDL-C independently of the classical LDLR-mediated pathway and represents a promising therapeutic approach for individuals with homozygous familial hypercholesterolemia due to LDLR mutations. Yet, how ANGPTL3 regulates LDL-C levels is unknown. Here, we demonstrate in hyperlipidemic humans and mice that ANGPTL3 controls VLDL catabolism upstream of LDL. Using kinetic, lipidomic, and biophysical studies, we show that ANGPTL3 inhibition reduces VLDL-lipid content and size, generating remnant particles that are efficiently removed from the circulation. This suggests that ANGPTL3 inhibition lowers LDL-C by limiting LDL particle production. Mechanistically, we discovered that EL is a key mediator of ANGPTL3's novel pathway. Our experiments revealed that, although dispensable in the presence of LDLR, EL-mediated processing of VLDL becomes critical for LDLR-independent particle clearance. In the absence of EL and LDLR, ANGPTL3 inhibition perturbed VLDL catabolism, promoted accumulation of atypical remnants, and failed to reduce LDL-C. Taken together, we uncover ANGPTL3 at the helm of a novel EL-dependent pathway that lowers LDL-C in the absence of LDLR.

ANGPTL8 has both endocrine and autocrine effects on substrate utilization.

The angiopoietin-like protein ANGPTL8 (A8) is one of 3 ANGPTLs (A8, A3, A4) that coordinate changes in triglyceride (TG) delivery to tissues by inhibiting lipoprotein lipase (LPL), an enzyme that hydrolyzes TG. Previously we showed that A8, which is expressed in liver and adipose tissue, is required to redirect dietary TG from oxidative to storage tissues following food intake. Here we show that A8 from liver and adipose tissue have different roles in this process. Mice lacking hepatic A8 have no circulating A8, high intravascular LPL activity, low plasma TG levels, and evidence of decreased delivery of dietary lipids to adipose tissue. In contrast, mice lacking A8 in adipose tissue have higher postprandial TG levels and similar intravascular LPL activity and plasma A8 levels and higher levels of plasma TG. Expression of A8, together with A4, in cultured cells reduced A4 secretion and A4-mediated LPL inhibition. Thus, hepatic A8 (with A3) acts in an endocrine fashion to inhibit intravascular LPL in oxidative tissues, whereas A8 in adipose tissue enhances LPL activity by autocrine/paracrine inhibition of A4. These combined actions of A8 ensure that TG stores are rapidly replenished and sufficient energy is available until the next meal.

Accumulation of PNPLA3 on lipid droplets is the basis of associated hepatic steatosis.

Fatty liver disease (FLD) is a disorder in which accumulation of triglycerides (TGs) in the liver can lead to inflammation, fibrosis, and cirrhosis. Previously, we identified a variant (I148M) in patatin-like phospholipase domain-containing protein 3 (PNPLA3) that is strongly associated with FLD, but the mechanistic basis for the association remains elusive. Although PNPLA3 has TG hydrolase activity in vitro, inactivation or overexpression of the WT protein in mice does not cause steatosis. In contrast, expression of two catalytically defective forms of PNPLA3 (I148M or S47A) in sucrose-fed mice causes accumulation of both PNPLA3 and TGs on hepatic lipid droplets (LDs). To determine if amassing PNPLA3 on LDs is a cause or consequence of steatosis, we engineered a synthetic isoform of PNPLA3 that uncouples protein accumulation from loss of enzymatic activity. Expression of a ubiquitylation-resistant form of PNPLA3 in mice caused accumulation of PNPLA3 on hepatic LDs and development of FLD. Lowering PNPLA3 levels by either shRNA knockdown or proteolysis-targeting chimera (PROTAC)-mediated degradation reduced liver TG content in mice overexpressing PNPLA3(148M). Taken together, our results show that the steatosis associated with PNPLA3(148M) is caused by accumulation of PNPLA3 on LDs.

PNPLA3, CGI-58, and Inhibition of Hepatic Triglyceride Hydrolysis in Mice.

A variant (148M) in patatin-like phospholipase domain-containing protein 3 (PNPLA3) is a major risk factor for fatty liver disease. Despite its clinical importance, the pathogenic mechanism linking the variant to liver disease remains poorly defined. Previously, we showed that PNPLA3(148M) accumulates to high levels on hepatic lipid droplets (LDs). Here we examined the effect of that accumulation on triglyceride (TG) hydrolysis by adipose triglyceride lipase (ATGL), the major lipase in the liver. As expected, overexpression of ATGL in cultured hepatoma (HuH-7) cells depleted the cells of LDs, but unexpectedly, co-expression of PNPLA3(wild type [WT] or 148M) with ATGL inhibited that depletion. The inhibitory effect of PNPLA3 was not caused by the displacement of ATGL from LDs. We tested the hypothesis that PNPLA3 interferes with ATGL activity by interacting with its cofactor, comparative gene identification-58 (CGI-58). Evidence supporting such an interaction came from two findings. First, co-expression of PNPLA3 and CGI-58 resulted in LD depletion in cultured cells, but expression of PNPLA3 alone did not. Second, PNPLA3 failed to localize to hepatic LDs in liver-specific Cgi-58 knockout (KO) mice. Moreover, overexpression of PNPLA3(148M) increased hepatic TG levels in WT, but not in Cgi-58 KO mice. Thus, the pro-steatotic effects of PNPLA3 required the presence of CGI-58. Co-immunoprecipitation and pulldown experiments in livers of mice and in vitro using purified proteins provided evidence that PNPLA3 and CGI-58 can interact directly. Conclusion: Taken together, these findings are consistent with a model in which PNPLA3(148M) promotes steatosis by CGI-58-dependent inhibition of ATGL on LDs.