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.
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.
A sequence polymorphism (rs738409, I148M) in patatin‐like phospholipid domain containing protein 3 (PNPLA3) is strongly associated with nonalcoholic fatty liver disease (NAFLD), but the mechanistic ...basis for this association remains enigmatic. Neither ablation nor overexpression of wild‐type PNPLA3 affects liver fat content in mice, whereas hepatic overexpression of the human 148M transgene causes steatosis. To determine whether the 148M allele causes fat accumulation in the liver when expressed at physiological levels, we introduced a methionine codon at position 148 of the mouse Pnpla3 gene. Knockin mice had normal levels of hepatic fat on a chow diet, but when challenged with a high‐sucrose diet their liver fat levels increased 2 to 3‐fold compared to wild‐type littermates without any associated changes in glucose homeostasis. The increased liver fat in the knockin mice was accompanied by a 40‐fold increase in PNPLA3 on hepatic lipid droplets, with no increase in hepatic PNPLA3 messenger RNA (mRNA). Similar results were obtained when the catalytic dyad of PNPLA3 was inactivated by substituting the catalytic serine with alanine (S47A). Conclusion: These data provide the first direct evidence that physiological expression of PNPLA3 148M variant causes NAFLD, and that the accumulation of catalytically inactive PNPLA3 on the surfaces of lipid droplets is associated with the accumulation of TG in the liver. (Hepatology 2015;61:108–118)
Macroautophagy (or autophagy) is a conserved degradative pathway that breaks down sequestered cytoplasmic proteins and organelles in specialized double-membrane compartments called autophagosomes ...that fuse with lysosomes. Several proteins orchestrate this process, specifically Rab GTPases that are master regulators of molecular trafficking. RAB18 GTPase, a known mediator of stellate cell activation, is known to modulate autophagic flux in fibroblasts. However, its role in autophagy is unexplored in hepatic stellate cells.
The aim of this study was to investigate the role of RAB18 in modulating autophagy in hepatic stellate cells.
Role of RAB18 was determined by genetic depletion, pharmacologic inhibition, and overexpression studies to monitor autophagy flux and proteostasis in human LX2 stellate cell line.
RAB18 knockdown increases autophagy flux and regulates proteostasis. LX2 cells stimulated with transforming growth factor-beta robustly increases expression of profibrotic genes such as COL1A1 and ACTA2 along with RAB18 and its guanine nucleotide exchange factor, RAB3GAP1.
The study elucidates a role for RAB18 in autophagy and regulation of proteostasis in human stellate cells. Molecular insights into this process can provide therapeutic opportunities for intervention in liver fibrosis.
•RAB18 is a regulator of autophagy in hepatic stellate cells.•RAB18 knockdown increases autophagy flux, and modestly inhibits proteostasis.•Transforming growth factor-beta robustly increases gene expression of RAB3GAP1 encoding a RAB18 guanine exchange factor.•RAB18 is an attractive therapeutic target for liver fibrosis.
Overactive GTPases have often been linked to human diseases. The available inhibitors are limited and have not progressed far in clinical trials. We report here a first-in-class small molecule ...pan-GTPase inhibitor discovered from a high throughput screening campaign. The compound CID1067700 inhibits multiple GTPases in biochemical, cellular protein and protein interaction, as well as cellular functional assays. In the biochemical and protein interaction assays, representative GTPases from Rho, Ras, and Rab, the three most generic subfamilies of the GTPases, were probed, while in the functional assays, physiological processes regulated by each of the three subfamilies of the GTPases were examined. The chemical functionalities essential for the activity of the compound were identified through structural derivatization. The compound is validated as a useful molecular probe upon which GTPase-targeting inhibitors with drug potentials might be developed.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Missense mutants in the late endosomal Rab7 GTPase cause the autosomal dominant peripheral neuropathy Charcot-Marie-Tooth disease type 2B (CMT2B). As yet, the pathological mechanisms connecting ...mutant Rab7 protein expression to altered neuronal function are undefined. Here, we analyze the effects Rab7 CMT2B mutants on nerve growth factor (NGF) dependent intracellular signaling in PC12 cells. The nerve growth factor receptor TrkA interacted similarly with Rab7 wild-type and CMT2B mutant proteins, but the mutant proteins significantly enhanced TrkA phosphorylation in response to brief NGF stimulation. Two downstream signaling pathways (Erk1/2 and Akt) that are directly activated in response to phospho-TrkA were differentially affected. Akt signaling, arising in response to activated TrkA at the plasma membrane was unaffected. However Erk1/2 phosphorylation, triggered on signaling endosomes, was increased. Cytoplasmic phospho-Erk1/2 persisted at elevated levels relative to control samples for up to 24 h following NGF stimulation. Nuclear shuttling of phospho Erk1/2, which is required to induce MAPK phosphatase expression and down regulate signaling, was greatly reduced by the Rab7 CMT2B mutants and explains the previously reported inhibition in PC12 neurite outgrowth. In conclusion, the data demonstrate a mechanistic link between Rab7 CMT2B mutants and altered TrkA and Erk1/2 signaling from endosomes.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
A sequence variation (I148M) in patatin‐like phospholipase domain‐containing protein 3 (PNPLA3) is strongly associated with fatty liver disease, but the underlying mechanism remains obscure. In this ...study, we used knock‐in (KI) mice (Pnpla3148M/M) to examine the mechanism responsible for accumulation of triglyceride (TG) and PNPLA3 in hepatic lipid droplets (LDs). No differences were found between Pnpla3148M/M and Pnpla3+/+ mice in hepatic TG synthesis, utilization, or secretion. These results are consistent with TG accumulation in the Pnpla3148M/M mice being caused by impaired TG mobilization from LDs. Sucrose feeding, which is required to elicit fatty liver in KI mice, led to a much larger and more persistent increase in PNPLA3 protein in the KI mice than in wild‐type (WT) mice. Inhibition of the proteasome (bortezomib), but not macroautophagy (3‐methyladenine), markedly increased PNPLA3 levels in WT mice, coincident with the appearance of ubiquitylated forms of the protein. Bortezomib did not increase PNPLA3 levels in Pnpla3148M/M mice, and only trace amounts of ubiquitylated PNPLA3 were seen in these animals. Conclusion: These results are consistent with the notion that the 148M variant disrupts ubiquitylation and proteasomal degradation of PNPLA3, resulting in accumulation of PNPLA3‐148M and impaired mobilization of TG from LDs. (Hepatology 2017;66:1111‐1124).
A missense mutation (E167K) in TM6SF2 (transmembrane 6 superfamily member 2), a polytopic protein of unknown function, is associated with the full spectrum of fatty liver disease. To investigate the ...role of TM6SF2 in hepatic triglyceride (TG) metabolism, we inactivated the gene in mice. Chronic inactivation of Tm6sf2 in mice is associated with hepatic steatosis, hypocholesterolemia, and transaminitis, thus recapitulating the phenotype observed in humans. No dietary challenge was required to elicit the phenotype. Immunocytochemical and cell fractionation studies revealed that TM6SF2 was present in the endoplasmic reticulum and Golgi complex, whereas the excess neutral lipids in the Tm6sf2−/− mice were located in lipid droplets. Plasma VLDL-TG levels were reduced in the KO animals due to a 3-fold decrease in VLDL-TG secretion rate without any associated reduction in hepatic apoB secretion. Both VLDL particle size and plasma cholesterol levels were significantly reduced in KO mice. Despite levels of TM6SF2 protein being 10-fold higher in the small intestine than in the liver, dietary lipid absorption was only modestly reduced in the KO mice. Our data, taken together, reveal that TM6SF2 is required to mobilize neutral lipids for VLDL assembly but is not required for secretion of apoB-containing lipoproteins. Despite TM6SF2 being located in the endoplasmic reticulum and Golgi complex, the lipids that accumulate in its absence reside in lipid droplets.
Fatty liver disease (FLD) affects more than one-third of the population in the western world and an increasing number of children in the United States. It is a leading cause of obesity and liver ...transplantation. Mechanistic insights into the causes of FLD are urgently needed since no therapeutic intervention has proven to be effective. A sequence variation in patatin like phospholipase domain-containing protein 3 (PNPLA3), rs 738409, is strongly associated with the progression of fatty liver disease. The resulting mutant causes a substitution of isoleucine to methionine at position 148. The underlying mechanism of this disease remains unsolved although several studies have illuminated key insights into its pathogenesis. This review highlights the progress in our understanding of PNPLA3 function in lipid droplet dynamics and explores possible therapeutic interventions to ameliorate this human health hazard.
PDF
