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![Triton X-114 extraction of geranylgeranylated Rab-3. A, lysis buffer containing unprocessed Rab-3 was extracted with Triton X-114 as described under " Experimental Procedures. " Rab-3 was immunoprecipitated from aqueous (a) (lane 1) and detergent (d) (lane 2) phases and determined by Western blotting. B and D, cells were incubated in lovastatin (2 g/ml) and [ 3 H]mevalonolactone as described under " Experimental Procedures. " After 1 h of incubation with insulin (100 nM) lysates were normalized for protein and extracted with Triton X-114. Aqueous (a) (lane 3) and detergent (d) (lane 4) phases were immunoprecipitated with anti-Rab-3 antibodies, analyzed by SDS-PAGE, and determined by either Western blotting (B) for protein or autoradiography (D) for labeled product. C and E, lysates of the insulin-treated cells were used as a source of GGTase II to geranylgeranylated Rab-3 in vitro as described under " Experimental Procedures. " The reaction mixture was then incubated with Triton X-114. Protein and labeled product were determined by Western blotting (C) and autoradiography (D), respectively, in aqueous (a) (lane 5) and detergent (d) (lane 6) phases.](profile/Marc-Goalstone/publication/13372966/figure/fig2/AS:349472073764868@1460332043346/Triton-X-114-extraction-of-geranylgeranylated-Rab-3-A-lysis-buffer-containing.png)
Triton X-114 extraction of geranylgeranylated Rab-3. A, lysis buffer containing unprocessed Rab-3 was extracted with Triton X-114 as described under " Experimental Procedures. " Rab-3 was immunoprecipitated from aqueous (a) (lane 1) and detergent (d) (lane 2) phases and determined by Western blotting. B and D, cells were incubated in lovastatin (2 g/ml) and [ 3 H]mevalonolactone as described under " Experimental Procedures. " After 1 h of incubation with insulin (100 nM) lysates were normalized for protein and extracted with Triton X-114. Aqueous (a) (lane 3) and detergent (d) (lane 4) phases were immunoprecipitated with anti-Rab-3 antibodies, analyzed by SDS-PAGE, and determined by either Western blotting (B) for protein or autoradiography (D) for labeled product. C and E, lysates of the insulin-treated cells were used as a source of GGTase II to geranylgeranylated Rab-3 in vitro as described under " Experimental Procedures. " The reaction mixture was then incubated with Triton X-114. Protein and labeled product were determined by Western blotting (C) and autoradiography (D), respectively, in aqueous (a) (lane 5) and detergent (d) (lane 6) phases.
Source publication
Rab proteins play a crucial role in the trafficking of intracellular vesicles. Rab proteins are GTPases that cycle between an inactive GDP-bound form and an active GTP-bound conformation. A prerequisite to Rab activation by GTP loading is its post-translational modification by the addition of geranylgeranyl moieties to highly conserved C-terminal c...
Citations
... The levels of GGPP and FPP were examined according to previously reported protocol by HPLC-MS/MS in AML-12 cell [34]. To evaluate RhoA prenylation, hepatocyte subcellular fractionation was collected following a Triton X-114 partition method [35]. Briefly, the cells were directly lysed in 2% Triton X-114 on ice for 30 min. ...
Background
Non-alcoholic fatty liver disease (NAFLD) has been well defined as a common chronic liver metabolism disorder. Statins as a first-line therapeutic treatment had some side effects. Here, we found that Fumigaclavine C (FC) was collected from endophytic Aspergillus terreus via the root of Rhizophora stylosa (Rhizophoraceae), had potential anti-adipogenic and hepatoprotective effects both in vitro and in vivo without obvious adverse side effects. However, the mechanisms of the prevention and management of FC for hepatic steatosis are incompletely delineated.
Methods
The pharmacodynamic effects of FC were measured in high-fat diet (HFD)-induced obese mice. Liver index and blood biochemical were examined. Histopathological examination in the liver was performed by hematoxylin & eosin or oil red O. The levels of serum TG, TC, LDL-c, HDL-c, FFA, T-bili, ALT, AST, creatinine, and creatine kinase were estimated via diagnostic assay kits. The levels of hepatic lipid metabolism-related genes were detected via qRT-PCR. The expression levels of hepatic de novo lipogenesis were quantitated with Western blot analysis.
Results
FC-treatment markedly reduced hepatic lipid accumulation in HFD-induced obese mice. FC significantly attenuated the hepatic lipid metabolism and ameliorated liver injury without obvious adverse side effects. Moreover, FC also could dose-dependently modulate the expressions of lipid metabolism-related transcription genes. Mechanically, FC notably suppressed sterol response element binding protein-1c mediated de novo lipogenesis via interfering with the RhoA/ROCK signaling pathway by decreasing the levels of geranylgeranyl diphosphate and farnesyl diphosphate.
Conclusions
These findings suggested that FC could improve hepatic steatosis through inhibiting de novo lipogenesis via modulating the RhoA/ROCK signaling pathway.
... However, this approach might also be limited by the size, conformation and composition of prenylated proteins. Besides, phase partitioning using triton X-114 is quite well documented for the purification of acylated and prenylated proteins (Goalstone et al., 1999;Mohamed et al., 2012;Yang et al., 2016;Akula et al., 2019). But, this approach does not fit with the prospect of putative protein interacting with prenylated proteins as a variability in interactant proteins polarity would induce differential separation. ...
... Cependant, cette approche pourrait également être limitée par la taille, la conformation et la composition des protéines prénylées. En outre, le partage de phase utilisant le triton X-114 est assez bien documenté pour la purification des protéines acylées et prénylées (Goalstone et al., 1999 ;Mohamed et al., 2012 ;Yang et al., 2016 ;Akula et al., 2019). Mais, cette approche ne correspond pas à la perspective d'une protéine putative interagissant avec des protéines prénylées car une variabilité de la polarité des protéines interactantes induirait une séparation l'inhibition de la prénylation des protéines influence la biosynthèse de métabolites spécialisés. ...
The protein prenylation is a key post-translational modification for the development, signaling and environmental adaptation of eukaryotic cells. Although many active and specific inhibitors of protein prenyltransferases have been isolated, the lack of knowledge on the biological mechanisms controlling protein modification by isoprenic groups makes their use inefficient in the treatment of cancer or malaria. To characterize the modification of prenylated proteins, we used a unique cell model to visualize these changes in vivo. In addition, techniques to capture the modified proteins were developed to analyze the mechanisms at the molecular level by using proteomics and metabolomics approaches. In a screening of potential inhibitors, an effective drug in vivo was identified. Moreover, the screening allowed to observe in living cells a fluorescence of vismiones (prenylated anthranoids) extracted from plants and described for their antimalarial properties. An ingenious method combining spectral imaging microscopy and metabolomic analysis was developed to track vismiones in vivo and to characterize the related metabolism in plant cells.
... However, we found that total amount of some classical Rab proteins, including Rab1, Rab4, Rab5, Rab7, Rab11, Rab27, were not influenced by CHML knockdown (Supplementary Fig. 5a). Since geranylgeranylation is required for membrane targeting of Rabs, we performed Triton X-114 partition assay, which could distinguish unprocessed Rabs in aqueous phase from geranylgeranylated Rabs in Triton X-114 phase 17 . However, CHML knockdown did not influence the distribution of Rabs in Triton X-114 phase and aqueous phase, indicating that downregulation of CHML may not affect geranylgeranylation of Rab proteins ( Supplementary Fig. 5b). ...
Metastasis-associated recurrence is the major cause of poor prognosis in hepatocellular carcinoma (HCC), however, the underlying mechanisms remain largely elusive. In this study, we report that expression of choroideremia-like (CHML) is increased in HCC, associated with poor survival, early recurrence and more satellite nodules in HCC patients. CHML promotes migration, invasion and metastasis of HCC cells, in a Rab14-dependent manner. Mechanism study reveals that CHML facilitates constant recycling of Rab14 by escorting Rab14 to the membrane. Furthermore, we identify several metastasis regulators as cargoes carried by Rab14-positive vesicles, including Mucin13 and CD44, which may contribute to metastasis-promoting effects of CHML. Altogether, our data establish CHML as a potential promoter of HCC metastasis, and the CHML-Rab14 axis may be a promising therapeutic target for HCC. Metastasis-associated recurrence is a major cause of poor prognosis in hepatocellular carcinoma. Here, the authors show that expression of choroideremia-like (CHML) is elevated and associates with poor prognosis in hepatocellular carcinoma, and mechanistically CHML promotes metastasis in a Rab14-dependent manner.
... The FPP and GGPP levels were determined in the AML-12 cell line as previously described using the HPLC-MS/MS (Wang et al., 2013). To assess RhoA prenylation, subcellular fractionation of the hepatocytes was performed using the Triton X-114 partition method (Goalstone et al., 1999). Briefly, adherent hepatocytes were lysed with 2% Triton X-114 for 30 min on ice. ...
Currently, there is no standard therapy for non-alcoholic fatty liver disease (NAFLD), and statins have been developed as a first-line pharmaceutical therapeutic option for NAFLD-associated dyslipidemia. However, prolonged statins therapy has side effects, as statins inhibit HMG-CoA reductase, an enzyme at the very beginning of the mevalonate pathway. Here, we found that zoledronic acid (ZA), an inhibitor of farnesyl diphosphate synthase in the downstream mevalonate pathway, could attenuate hepatic lipid accumulation and improve liver injury in both high-fat diet-induced C57BL/6J mice and ob/ob mice. Moreover, the hepatic lipid metabolism was largely inhibited after ZA administration in high-fat diet-induced obese mice. Mechanically, ZA inhibited SREBP-1c-mediated de novo lipogenesis through suppressing RhoA activation via decreasing farnesyl diphosphate and geranylgeranyl diphosphate levels. In conclusion, our data provide a novel application of ZA in improving hepatic steatosis.
... This led to an effort to functionally inhibit FTase and GGTase toward potential therapeutic treatments [31; 32]. Insulin has also been observed to activate GGTase-II and increase geranylgeranylated Rab-4 in a MEK/ERK dependent manner, but the physiological significance of this is still unclear [33]. Therefore, we have focused on the CAAX-type prenyl transferases as possible mediators of insulin regulated gene expression. ...
Increased activity of prenyl transferases is observed in pathological states of insulin resistance, diabetes, and obesity. Thus, functional inhibitors of farnesyl transferase (FTase) and geranylgeranyl transferase (GGTase) may be promising therapeutic treatments. We previously identified insulin responsive genes from a rat H4IIE hepatoma cell cDNA library, including β-actin, EGR1, Pip92, c-fos, and Hsp60. In the present study, we investigated whether acute treatment with FTase and GGTase inhibitors would alter insulin responsive gene initiation and/or elongation rates. We observed differential regulation of insulin responsive gene expression, suggesting a differential sensitivity of these genes to one or both of the specific protein prenylation inhibitors.
... Ras geranylgeranylation was measured as previously described. 32 Briefly, an equal volume of 4% TritonX-114 was added to the supernatant. TritonX-114 is lipid soluble and can dissolve the cell membrane more easily than the cell cytoplasm because the cell membrane is also lipid soluble. ...
Cigarette smoke activates the extracellular signal-regulated kinase (ERK) 1/2 mitogen activated-protein kinase pathway, which, in turn, is responsible for early growth response gene-1 (EGR-1) activation. Here we provide evidence that EGR-1 activation can also reactivate ERK 1/2 mitogen activated-protein kinase through a positive feedback loop through its target gene (geranylgeranyl diphosphate synthase) GGPPS. For the first time, the GGPPS gene is identified as a target of EGR-1, as EGR-1 can directly bind to the predicted consensus-binding site in the GGPPS promoter and regulate its transcription. Long-term observations show that there are two ERK 1/2 phosphorylation peaks after cigarette smoke extract stimulation in human lung epithelial Beas-2B cells. The first peak (at 10 minutes) is responsible for EGR-1 accumulation, and the second (at 4 hours) is diminished after the disruption of EGR-1 transcriptional activity. EGR-1 overexpression enhances Ras prenylation and membrane association in a GGPPS-dependent manner, and it augments ERK 1/2 activation. Likewise, a great reduction of the second peak of ERK 1/2 phosphorylation is observed during long-term cigarette smoke extract stimulation in cells where GGPPS is disrupted. Thus, we have uncovered an intricate positive feedback loop in which ERK 1/2-activated EGR-1 promotes ERK 1/2 reactivation through promoting GGPPS transcription, which might affect cigarette smoke-related lung pathological processes.
... The total protein concentration was diluted to 1 mg/ml. Ras geranylgeranylation was measured as described previously (28). Briefly, an equal volume of 4% Triton X-114 was added to the supernatant to solubilize and fractionate the lipid-rich cell membrane. ...
One of the most important characteristics of type 2 diabetes is insulin resistance, during which the patients normally experienced hyperinsulinism stress that would alter insulin signal transduction in insulin target tissues. We have previously found that early growth responsive gene-1 (Egr-1), a zinc finger transcription factor, is highly expressed in db/db mice and in the fat tissue of individuals with type 2 diabetes. In this report, we found that chronic exposure to hyperinsulinism caused persistent Erk/MAPK activity in adipocytes and enhanced insulin resistance in an Egr-1-dependent manner. An elevation in Egr-1 augmented Erk1/2 activation via geranylgeranyl diphosphate synthase (GGPPS). Egr-1-promoted GGPPS transcription increased Ras prenylation and caused Erk1/2 activation. The sustained activation of Erk1/2 resulted in the phosphorylation of insulin receptor substrate-1 at Serine 612. Phosphorylation at this site impaired insulin signaling in adipocytes and reduced glucose uptake. The loss of Egr-1 function, knockdown of GGPPS, or inhibition of Erk1/2 activity in insulin-resistant adipocytes restored insulin receptor substrate-1 tyrosine phosphorylation and increased insulin sensitivity. Our results suggest a new mechanism by which the Egr-1/GGPPS/Erk1/2 pathway is responsible for insulin resistance during hyperinsulinism. This pathway provides a new therapeutic target for increasing insulin sensitivity: inhibiting the function of Egr-1.
... Perillyl alcohol (POH) is an inhibitor of both Rab GG transferase and GG transferase-I, but it is twice as potent in inhibiting Rab GG transferase as GG transferase-I (7). GGTI-298 is a specific inhibitor of GG transferase-I (8). ...
An adverse effect of statins, cholesterol-lowering drugs, is contractile dysfunction of skeletal muscles. We investigated the mechanism underlying this effect in cultured myofibers isolated from rats. Fluvastatin (Flv) for 72 h decreased caffeine- and ionomycin-induced contraction of myofibers and Ca(2+) release from sarcoplasmic reticulum (SR). Ca(2+)-shortening curves measured in skinned myofibers indicated that myofibrillar Ca(2+) sensitivity was unaffected by Flv. A luciferin-luciferase assay revealed less ATP contents in Flv-treated myofibers. Among mevalonate metabolites, including geranylgeranylpyrophosphate (GGPP), farnesylpyrophosphate (FPP), coenzyme Q9, and coenzyme Q10, only GGPP prevented Flv-induced ATP reduction. A selective Rab geranylgeranyltransferase (GG transferase) inhibitor, perillyl alcohol (POH), and a specific GG transferase-I inhibitor, GGTI-298, both mimicked Flv in decreasing ATP and contraction. Mitochondrial membrane potential was decreased by Flv, and this effect was rescued by GGPP and mimicked by POH and GGTI-298. An endoplasmic reticulum (ER)-to-Golgi traffic inhibitor, brefeldin A, and a Rho inhibitor, membrane permeable exoenzyme C3 transferase, both decreased ATP. We conclude that statin-induced contractile dysfunction is due to reduced Ca(2+) release from SR and reduced ATP levels in myofibers with damaged mitochondria. GGPP depletion and subsequent inactivation of Rab1, possibly along with Rho, may underlie the mitochondrial damage by Flv.
... Rab is primarily geranylgeranylated by a transferase called Rab geranylgeranyl transferase (Rab GGTase; ref. 32), which could be inhibited by perillyl alcohol (33). If Rab is involved, then the effect of statins should be mimicked by perillyl alcohol but not by specific inhibitors of geranylgeranyl transferase-I (GGTase-I) or farnesyl transferase (FTase) (34), which prenylates various small GTPases other than Rab (35). ...
Three-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase inhibitors, known as statins, induce skeletal muscle injury including myalgia, myositis, and rhabdomyolysis. The mechanism of this myotoxicity remains unknown. This study examined the effect of statins on single skeletal myofibers enzymatically isolated from the rat flexor digitorum brevis muscles. Fluvastatin and pravastatin induced the formation of numerous vacuoles in the myofibers after 72 h of treatment. This effect progressed in a time- and concentration-dependent manner and, consequently, cell death occurred after 120 h. Electron micrographs revealed craters along the sarcolemma and swelling of the sarcoplasmic reticula and mitochondria, in addition to intracellular vacuoles. When caffeine was added after 72 h of fluvastatin treatment, contractile shortening of statin-treated myofibers was significantly attenuated and blebs formed on the surface of the myofibers. The coapplication of geranylgeranylpyrophosphate (GGPP) with fluvastatin prevented the morphological changes, while that of farnesylpyrophosphate (FPP) was ineffective. Furthermore, perillyl alcohol, an inhibitor of Rab geranylgeranyl transferase and geranylgeranyl transferase-I (GGTase-I), mimicked the effect of statins, while a specific GGTase-I inhibitor (GGTI-298) or a farnesyl transferase inhibitor (FTI-277) failed to do so. These results suggest that the inactivation of Rab GTPase, which involved in intracellular membrane transport, is a crucial factor in statin-induced-morphological abnormality in skeletal muscle fibers.
... Recent evidence has shown that changes in GGTase II activity can alter the levels of some geranylgeranylated Rab proteins (Goalstone et al., 1999). To study the effects of changes in the amounts of membrane-bound Ypt proteins in yeast cells without causing a full depletion of these proteins from intracellular membranes, we created thermosensitive (ts) mrs6 alleles that accumulate unprenylated Ypt proteins at 258C as well as at 378C. ...
The Rab/Ypt small G proteins are essential for intracellular vesicle trafficking in mammals and yeast. The vesicle-docking process requires that Ypt proteins are located in the vesicle membrane. C-terminal geranylgeranyl anchors mediate the membrane attachment of these proteins. The Rab escort protein (REP) is essential for the recognition of Rab/Ypt small G proteins by geranylgeranyltransferase II (GGTase II) and for their delivery to acceptor membranes. What effect an alteration in the levels of prenylated Rab/Ypt proteins has on vesicle transport or other cellular processes is so far unknown. Here, we report the characterization of a yeast REP mutant, mrs6-2, in which reduced prenylation of Ypt proteins occurs even at the permissive temperature. A shift to the restrictive temperature does not alter exponential growth during the first 3 h. The amount of Sec4p, but not Ypt1p, bound to vesicle membranes is reduced 2.5 h after the shift compared with wild-type or mrs6-2 cells incubated at 25°C. In addition, vesicles fail to be polarized towards the bud and small budded binucleate cells accumulate at this time point. Growth in 1 M sorbitol or overexpression of MLC1, encoding a myosin light chain able to bind the unconventional type V myosin Myo2, or of genes involved in cell wall maintenance, such as SLG1, GFA1 and LRE1, suppresses mrs6-2 thermosensitivity. Our data suggest that, at least at high temperature, a critical minimal level of Ypt protein prenylation is required for maintaining vesicle polarization.
