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Sunitinib is the most commonly used first-line therapy for the treatment of advanced renal cell carcinoma (RCC), but intrinsic and extrinsic resistance to targeted therapies dramatically compromise the benefit of clinical outcome. Dissecting the underlying mechanisms and discovering reliable predictive biomarkers are urgently needed in clinic. Here, we discovered miR-885-5p was notably decreased after sunitinib treatment and associated with poor disease progression in clear cell renal cell carcinoma (ccRCC). In vitro and in vivo studies identified miR-885-5p inhibition contributed to sunitinib resistance. Mechanistically, sunitinib treatment reduced GATA1 expression, which in turn reduced its binding to MIR885 promoter and resulted in miR-885-5p downregulation in transcriptional level. In addition, PLIN3 was confirmed to be directly targeted by miR-885-5p and its upregulation significantly increased lipid droplets formation to decrease sunitinib sensitivity. Therefore, GATA1/miR-885-5p/ PLIN3 pathway may serve as a potential therapeutic strategy and a biomarker for sunitinib treatment in ccRCC.

Intramuscular triglycerides (IMTG) are a key substrate during prolonged exercise, but little is known about the rate of IMTG resynthesis in the postexercise period. We investigated the hypothesis that the distribution of the lipid droplet (LD)-associated perilipin (PLIN) proteins is linked to IMTG storage following exercise. Fourteen elite male triathletes (27 ± 1 yr, 66.5 ± 1.3 mL·kg−1·min−1) completed 4 h of moderate-intensity cycling. During the first 4 h of recovery, subjects received either carbohydrate or H2O, after which both groups received carbohydrate. Muscle biopsies collected pre- and postexercise and 4 and 24 h postexercise were analyzed using confocal immunofluorescence microscopy for fiber type-specific IMTG content and PLIN distribution with LDs. Exercise reduced IMTG content in type I fibers (−53%, P = 0.002), with no change in type IIa fibers. During the first 4 h of recovery, IMTG content increased in type I fibers ( P = 0.014), but was not increased more after 24 h, where it was similar to baseline levels in both conditions. During recovery the number of LDs labeled with PLIN2 (70%), PLIN3 (63%), and PLIN5 (62%; all P < 0.05) all increased in type I fibers. Importantly, the increase in LDs labeled with PLIN proteins only occurred at 24 h postexercise. In conclusion, IMTG resynthesis occurs rapidly in type I fibers following prolonged exercise in highly trained individuals. Furthermore, increases in IMTG content following exercise preceded an increase in the number of LDs labeled with PLIN proteins. These data, therefore, suggest that the PLIN proteins do not play a key role in postexercise IMTG resynthesis.

Cytoplasmic lipid droplets are highly dynamic storage organelles that are critical for cellular lipid homeostasis. While the molecular details of lipid droplet dynamics are a very active area of investigation, this work has been primarily performed in cultured cells. Taking advantage of the powerful transgenic and in vivo imaging opportunities available in zebrafish, we built a suite of tools to study lipid droplets in real time from the subcellular to the whole organism level. Fluorescently tagging the lipid droplet-associated proteins, perilipin 2 and perilipin 3, in the endogenous loci permits visualization of lipid droplets in the intestine, liver, and adipose tissue. Using these tools, we found that perilipin 3 is rapidly loaded on intestinal lipid droplets following a high-fat meal and later replaced by perilipin 2. These powerful new tools will facilitate studies on the role of lipid droplets in different tissues, under different genetic and physiological manipulations, and in a variety of human disease models.

Objective To explore the effect of cordycepin on reducing lipid droplets in adipocytes. Methods Rats were fed a 60% high-fat diet to construct a hyperlipidaemia animal model and then treated with cordycepin at different concentrations for 8 weeks. Adipocytes were extracted, and BODIPY staining was used to detect the size of the lipid droplets. The adipocyte membrane proteins ASC-1, PAT2 and P2RX5 were assessed to determine the transformation of white adipocytes to beige and brown adipocytes. In an in vitro study, 3T3-L1 cells were cultured, and Western blotting was used to determine the expression of the lipid droplet-related genes Fsp27, perilipin 3, perilipin 2, PPAR-γ, Rab5, Rab7, Rab11, perilipin 1, ATGL and CGI-58. Results We found that cordycepin could promote the transformation of white adipocytes into beige and brown adipocytes. Cordycepin also downregulated the lipid droplet-associated genes Fsp27, perilipin 3, perilipin 2, Rab5, Rab11 and perilipin 1. Moreover, cordycepin reduced the expression of protein CGI-58, which inhibits lipid droplet degradation. In addition, cordycepin significantly increased the expression of ATGL, suggesting that cordycepin might stimulate lipolysis by upregulating the expression of ATGL instead of CGI-58 and by downregulating the expression of perilipin 1. Conclusions Cordycepin could blockade lipid droplet formation and promote lipid droplet degradation.

Lipids are secreted into milk as bilayer-coated structures: milk fat globules (MFGs). Adipophilin (ADRP) and perilipin 3 (TIP47) are associated with MFGs in human breast milk; however, the role of these proteins in milk lipid secretion is not fully understood. The study aimed to investigate levels of ADRP, TIP47 and total lipid content in human breast milk, their mutual correlations, and dynamics during lactation. Milk samples from 22 healthy lactating women (Caucasian, Central European) were collected at five time points during lactation (1–3, 12–14, 29–30, 88–90 and 178–180 days postpartum). Mass spectrometry-based method was used for quantification of ADRP and TIP47 in the samples. The gravimetric method was used to determine milk total lipid content. We observed distinctive trends in ADRP, TIP47 levels and lipid content in human breast milk during the first six months of lactation. We also found a significant association between lipid content and ADRP, lipid content and TIP47, and ADRP and TIP47 concentrations in breast milk at all sampling points. A mass spectrometry-based method was developed for quantifying ADRP and TIP47 in human breast milk. Strong mutual correlations were found between ADRP, TIP47 and total lipid content in human breast milk.

Hepatitis C Virus (HCV) promotes lipid droplet (LD) formation and perturbs the expression of the LD associated PAT proteins ADRP and TIP47, to promote its own lifecycle. HCV enhances TIP47 and suppresses ADRP by displacing it from LD surface in infected cell models. We have previously shown that suppression of TIP47 by miR-148a and miR-30a decreased intracellular LDs and HCV RNA. Thus, this study aimed at examining whether this microRNA-mediated suppression of HCV would limit HCV-dependent displacement of ADRP from LDs. ADRP expression was examined in 21 HCV-infected liver biopsies and 9 healthy donor liver tissues as well as in HCV-infected Huh7 cells using qRT-PCR. miR-148a and miR-30a expression was manipulated using specific oligos in JFH-1 infected, oleic acid treated cells, to study their impact on ADRP expression using qRT-PCR, and immunofluorescence microscopy. Intracellular HCV RNA was assessed using qRT-PCR. ADRP is down regulated in patients as well as HCVcc-JFH-I infected cell models. Forcing the expression of both miRNAs induced ADRP on the mRNA and protein levels. This study shows that HCV suppresses hepatic ADRP expression in infected patients and cell lines. Forcing the expression of miR-148a and miR-30a limits the suppressive effect of HCV on ADRP. J. Med. Virol. 89:653-659, 2017. © 2016 Wiley Periodicals, Inc.

Belonging to the PLIN family, PLIN2 associates with lipid storage droplets (LSDs), but other functions of PLIN2 remain unclear. Here, we suggest that PLIN2 mediates Wnt signaling because PLIN2 small interfering RNA (siRNA) suppresses activation of Wnt/coreceptor pathways. The mediation in the Wnt/Frizzled pathway seems to occur from Dishevelleds to axin/glycogen synthase kinase 3(GSK3)/β-catenin complexes (AGβC) as Wnt decreases Dishevelled/PLIN2 but increases AGβC/PLIN2 associations. Augmenting cellular LSDs that affect PLIN2 associations with these proteins, oleic acid (OA) treatment inhibits Wnt-increased AGβC/PLIN2 associations and β-catenin T-cell factor signaling (β-CTS). Revealing that PLIN2 is a GSK3-associated protein, the study explored PLIN2-mediated effects on GSK3/GSK3 substrates. PLIN2 siRNA reduces inhibitory GSK3 levels and lithium chloride (LiCl)-upregulated β-catenin or CCAAT/enhancer binding protein α (c/EBPα) expression. OA treatment decreases LiCl-increased c/EBPα via PLIN2-c/EBPα dissociation. In addition to PLIN2 overexpression increasing β-CTS, PLIN2 depletion or overexpression drops or adds expression of GSK3 substrates, such as β-catenin, c/EBPα,c-Myc, cyclin D1, and insulin receptor substrate 1, and cell growth/survival. PLIN2 N or C terminus overexpression that is associated with higher levels of the substrates suggests that those substrates bind to specific regions of PLIN2. Mimicking the possible high lipid concentrations in cells in the human body under conditions of hyperlipidemia/obesity, OA-treated cells gain or reduce GSK3 substrate expression in parallel with a decrease (a Wnt-like effect) or increase in GSK3 activity, likely regulated by GSK3/PLIN2/GSK3 substrate associations.

The origin and physiological significance of lipid droplets (LDs) in the nucleus is not clear. Here we show that nuclear LDs in hepatocytes are derived from apolipoprotein B (ApoB)-free lumenal LDs, a precursor to very low-density lipoproprotein (VLDL) generated in the ER lumen by microsomal triglyceride transfer protein. ApoB-free lumenal LDs accumulate under ER stress, grow within the lumen of the type I nucleoplasmic reticulum, and turn into nucleoplasmic LDs by disintegration of the surrounding inner nuclear membrane. Oleic acid with or without tunicamycin significantly increases the formation of nucleoplasmic LDs, to which CTP:phosphocholine cytidylyltransferase α (CCTα) is recruited, resulting in activation of phosphatidylcholine (PC) synthesis. Perilipin-3 competes with CCTα in binding to nucleoplasmic LDs, and thus, knockdown and overexpression of perilipin-3 increases and decreases PC synthesis, respectively. The results indicate that nucleoplasmic LDs in hepatocytes constitute a feedback mechanism to regulate PC synthesis in accordance with ER stress.

Although autophagy is classically viewed as a non-selective degradation system, recent studies have revealed that various forms of selective autophagy also play crucial physiological roles. However, the induction of selective autophagy is not well understood. In this study, we established a forced selective autophagy system using a fusion of an autophagy adaptor and a substrate-binding protein. In both mammalian cells and fertilized mouse embryos, efficient forced lipophagy was induced by expression of a fusion of p62 (Sqstm1) and a lipid droplet (LD)-binding domain. In mouse embryos, induction of forced lipophagy caused a reduction in LD size and number, and decreased the triglyceride level throughout embryonic development, resulting in developmental retardation. Furthermore, lipophagy-induced embryos could eliminate excess LDs and were tolerant of lipotoxicity. Thus, by inducing forced lipophagy, expression of the p62 fusion protein generated LD-depleted cells, revealing an unexpected role of LD during preimplantation development.

Mediators such as cytokines, eicosanoids, nitric oxide and growth factors may regulate adipogenesis as well as inflammation. It is well documented that production of some form of eicosanoids activates lipid synthesis during adipogenesis but also contributes to the formation of factors maintaining low-level systemic inflammation. Developing nutrients for reduction of adipogenesis and inflammation can enhance preventive efficacy of daily diet. This study examined the effects of free fatty acid influence on changes in lipid biosynthesis and corresponding gene expression during differentiation of human subcutaneous adipose tissue stromal vascular fraction (SVF) cells. Proadipogenic conditions promoted SVF cell differentiation and lipid droplet (LD) formation up to 15 days. This correlated with gene expression of adipocyte differentiation markers as well as inflammatory cytokines and their receptors. Addition of free fatty acids to differentiation medium increased their incorporation during the first period of differentiation (48 h). Presence of eicosanoid acid (EPA) during the initial period of differentiation by elevation of Perilipin 3 protein (TIP47), may be responsible for smaller LD formation. Presence of arachidonic acid (AA) tends to deposit lipids in large form of LDs. Prolongation of differentiation up to 15 days decreased AA or EPA in cellular lipids. PUFA through up-regulation of both phospholipase 2 and enzymes related to eicosanoid production influenced type and quantity of eicosanoids which regulated the extent of SVF cell differentiation. Formation of small LDs and reduction of pro-inflammatory mediators in adipose tissue are the consequence of eicosanoid production with anti-inflammatory potential from EPA.

Chaperone-mediated autophagy (CMA) selectively degrades a subset of cytosolic proteins in lysosomes. A potent physiological activator of CMA is nutrient deprivation, a condition in which intracellular triglyceride stores or lipid droplets (LDs) also undergo hydrolysis (lipolysis) to generate free fatty acids for energetic purposes. Here we report that the LD-associated proteins perilipin 2 (PLIN2) and perilipin 3 (PLIN3) are CMA substrates and their degradation through CMA precedes lipolysis. In vivo studies revealed that CMA degradation of PLIN2 and PLIN3 was enhanced during starvation, concurrent with elevated levels of cytosolic adipose triglyceride lipase (ATGL) and macroautophagy proteins on LDs. CMA blockage both in cultured cells and mouse liver or expression of CMA-resistant PLINs leads to reduced association of ATGL and macrolipophagy-related proteins with LDs and the subsequent decrease in lipid oxidation and accumulation of LDs. We propose a role for CMA in LD biology and in the maintenance of lipid homeostasis.

ObjectivePolycystic ovary syndrome (PCOS) is associated with reduced adipose tissue lipolysis that can be rescued by aerobic exercise. We aimed to identify differences in the gene expression of perilipins and associated targets in adipose tissue in women with PCOS before and after exercise.Design and methodsWe conducted a cross-sectional study in eight women with PCOS and eight women matched for BMI and age with normal cycles. Women with PCOS also completed a 16-week prospective aerobic exercise-training study. Abdominal subcutaneous adipose tissue biopsies were collected, and primary adipose-derived stromal/stem cell cultures were established from women with PCOS before 16 weeks of aerobic exercise training (n=5) and controls (n=5). Gene expression was measured using real-time PCR, in vitro lipolysis was measured using radiolabeled oleate, and perilipin 3 (PLIN3) protein content was measured by western blotting analysis.ResultsThe expression of PLIN1, PLIN3, and PLIN5, along with coatomers ARF1, ARFRP1, and βCOP was ∼80% lower in women with PCOS (all PPPin vitro adipose oleate oxidation, glycerol secretion, and PLIN3 protein expression were increased, along with reductions in triglyceride content and absence of large lipid droplet morphology.ConclusionsThese findings suggest that PLIN3 and coatomer GTPases are important regulators of lipolysis and triglyceride storage in the adipose tissue of women with PCOS.

Background A fusion gene is a hybrid gene consisting of parts from two previously independent genes. Chromosomal rearrangements leading to gene breakage are frequent in high-grade serous ovarian carcinomas and have been reported as a common mechanism for inactivating tumor suppressor genes. However, no fusion genes have been repeatedly reported to be recurrent driver events in ovarian carcinogenesis. We combined genomic and transcriptomic information to identify novel fusion gene candidates and aberrantly expressed genes in ovarian carcinomas. Methods Examined were 19 previously karyotyped ovarian carcinomas (18 of the serous histotype and one undifferentiated). First, karyotypic aberrations were compared to fusion gene candidates identified by RNA sequencing (RNA-seq). In addition, we used exon-level gene expression microarrays as a screening tool to identify aberrantly expressed genes possibly involved in gene fusion events, and compared the findings to the RNA-seq data. Results We found a DPP9-PPP6R3 fusion transcript in one tumor showing a matching genomic 11;19-translocation. Another tumor had a rearrangement of DPP9 with PLIN3. Both rearrangements were associated with diminished expression of the 3′ end of DPP9 corresponding to the breakpoints identified by RNA-seq. For the exon-level expression analysis, candidate fusion partner genes were ranked according to deviating expression compared to the median of the sample set. The results were collated with data obtained from the RNA-seq analysis. Several fusion candidates were identified, among them TMEM123-MMP27, ZBTB46-WFDC13, and PLXNB1-PRKAR2A, all of which led to stronger expression of the 3′ genes. In view of our previous findings of nonrandom rearrangements of chromosome 19 in this cancer type, particular emphasis was given to changes of this chromosome and a DDA1-FAM129C fusion event was identified. Conclusions We have identified novel fusion gene candidates in high-grade serous ovarian carcinoma. DPP9 was involved in two different fusion transcripts that both resulted in deregulated expression of the 3′ end of the transcript and thus possible loss of the active domains in the DPP9 protein. The identified rearrangements might play a role in tumorigenesis or tumor progression.

Background & Aims Hepatocellular steatosis is the most frequent liver disease in the western world and may develop further to steatohepatitis, liver cirrhosis and hepatocellular carcinoma. We have previously shown that lipid droplet (LD)-associated proteins of the perilipin/PAT-family are differentially expressed in hepatocyte steatosis and that perilipin is expressed de novo . The aim of this study was to determine the conditions for the temporal regulation of de novo synthesis of perilipin in vitro and in vivo . Methods Immunohistochemical PAT-analysis was performed with over 120 liver biopsies of different etiology and duration of steatosis. Steatosis was induced in cultured hepatocytic cells with combinations of lipids, steatogenic substances and DMSO for up to 40days under conditions of stable down-regulation of adipophilin and/or TIP47. Results Whereas perilipin and adipophilin were expressed in human chronic liver disease irrespective of the underlying etiology, in acute/microvesicular steatosis TIP47, and MLDP were recruited from the cytoplasm to LDs, adipophilin was strongly increased, but perilipin was virtually absent. In long-term steatosis models in vitro , TIP47, MLDP, adipophilin, and finally perilipin were gradually induced. Perilipin and associated formation of LDs were intricately regulated on the transcriptional (PPARs, C/EBPs, SREBP), post-transcriptional, and post-translational level (TAG-amount, LD-fusion, phosphorylation-dependent lipolysis). In long-term steatosis models under stable down-regulation of adipophilin and/or TIP47, MLDP substituted for TIP47, and perilipin for adipophilin. Conclusions LD-maturation in hepatocytes in vivo and in vitro involves sequential expression of TIP47, MLDP, adipophilin and finally perilipin. Thus, perilipin might be used for the differential diagnosis of chronic vs. acute steatosis.

Cytosolic lipid storage droplets are primary functional organelles that regulate cellular lipid metabolism and homeostasis. Paradoxically, excess lipid stores are linked to both adaptive (fasting and chronic exercise) and mal-adaptive (obesity and related health complications) conditions. Thus, collective metabolic and physiological processes must balance lipid storage and utilization with prevention of lipocytotoxicity and compounding tissue dysfunctions, urging the need to further define the connection of mammalian lipid droplet function and lipid homeostasis. The perilipins are a multi-protein family that targets lipid droplet surfaces and regulates lipid storage and hydrolysis. Study of perilipin functions has provided insight into the physiological roles of cytosolic lipid droplets and their relationship with obesity-related pathologies. Here, we review the current knowledge of the multiple perilipin proteins in regulating tissue-specific lipid droplets and associations with tissue and systemic energetics.

Hepatitis C virus (HCV) morphogenesis and release are closely linked to lipid metabolism. It has been described recently by our group that TIP47 plays an essential role for the targeting of the NS5A-complexed RNA genome from the replicon complex to the lipid droplet. Moreover, apolipoprotein (apo) E was found to be associated with the viral particle. In light of the fact, that TIP47 harbors an apoE like domain and has a high affinity to lipoproteins, the interaction of TIP47 with the viral particle and the potential relevance for the release of the viral particle were investigated. Coimmunoprecipitations and electron microscopy analysis using immunogold labeling revealed that TIP47 binds to the viral particle and stays associated with the released HCV particle. Silencing of the TIP47 binding partner Rab9 by lentiviral transduction abolishes the viral replication. However, destruction of TIP47-Rab9 interactions by deletion/mutation of the Rab9 binding does not abolish the genome replication domain but prevents the release of HCV particles. The binding of these TIP47 mutants to the viral particle is not affected by destruction of the Rab9 binding domain. Moreover, we found that these TIP47 mutants lacking the binding site for Rab9 misdirect the de novo synthesized viral particles to the autophagosomal/lysosomal compartment where the particles are degraded. From this we conclude that the Rab9-complexed TIP47 plays an essential role for the proper release of hepatitis C viral particles.

Objective Perilipin (PLIN) 3, an intracellular lipid droplet (LD)-associated protein, is implicated in foam cell formation. Since metabolic derangements found in metabolic syndrome, such as high serum levels of glucose, insulin and free fatty acids (FFAs), are major risk factors promoting atherosclerosis, we investigated whether PLIN3 expression is affected by glucose, insulin and oleic acid (OA) using RAW264.7 cells. Methods Real-time PCR and Western blotting were performed to detect PLIN3 or PLIN2 expression. Oil-red O staining and Lipid Analysis were employed to measure cellular content of triacylglycerides (TAG) and cholesterol. Results PLIN3 mRNA was stimulated by high glucose or insulin concentrations individually, but not by OA. A combination of any two factors did not enhance PLIN3 expression any more than that evoked by glucose alone at 24 h. Interestingly, however, simultaneous addition of all three factors synergistically enhanced the PLIN3 expression. This synergistic effect was not apparent for PLIN2 mRNA expression. Inhibitors of Src family tyrosine kinase and/or phosphatidylinositol 3-kinase, both of which are activated by insulin and FFA signaling, partially suppressed PLIN3 expression induced by the combination of the three factors. While simultaneous addition of glucose, insulin and OA remarkably increased the cellular content of TAG and cholesterol, knocking-down of PLIN3 predominantly reduced TAG content. Conclusions These results indicate that PLIN3 expression is synergistically stimulated by high glucose, insulin and FFA concentrations, in parallel with TAG accumulation in macrophages. This finding raises new evidence of PLIN3 involvement in conversion of macrophages into foam cells

Background & Aims Hepatitis C virus (HCV) replication/morphogenesis takes place at the membranous web. Viral genome replication occurs in replicon complexes on the cytoplasmic face of the ER whereas HCV assembly is located on the surface of lipid droplets (LDs). This raises the question about targeting of de novo synthesized viral genomes from the replicon complex to LDs and cellular proteins involved in this process such as the LD-associated protein TIP47, also known as cytoplasmic sorting factor. Methods Viral replication was studied in HuH7.5 cells using the infectious HCV JHF1 culture system. Proteome analysis was performed by 2D gel electrophoresis and mass spectrometry. Expression of target genes was modulated by siRNA or lentiviral transduction. Confocal microscopy was performed for analysis of subcellular compartments. Protein/protein interactions were studied by co-immunoprecipitations, affinity chromatography, and yeast two-hybrid screens. Results Proteome based analysis revealed that HCV replicating cells contain less TIP47 compared to control cells. However, expression analyses demonstrated an increased TIP47 expression in HCV replicating cells. TIP47 binds to RNA-loaded NS5A. Mapping of the binding domain revealed that NS5A binds to the N-terminal PAT domain of TIP47. Overexpression of TIP47 increases the amount of released viruses, while silencing of TIP47 decreases the amount of released infectious particles. Complete knockdown of TIP47 expression abolishes virus replication. Conclusions TIP47 plays an essential role in the HCV life cycle.

Proteins that coat the lipid droplets (also known as PAT proteins or perilipin (PLIN) family proteins) have diverse functions that are not well elucidated in many tissues. In skeletal muscle, there is even less known about the functions or characteristics of these proteins or how they might change in response to perturbations that alter both intramyocellular lipid (IMCL) content and fat utilization and oxidation. Therefore, the purpose of this study was to examine the human muscle content and gene expression of the four skeletal muscle PLIN proteins in both lean and obese men and women and how this was changed following a 12-week endurance training protocol. PLIN2–PLIN5 proteins were all more abundant in women than in men (p = 0.037 and p < 0.0001, respectively), consistent with higher IMCL content observed in female skeletal muscle. PLIN5 (previously known as OXPAT) is of particular interest because it has previously been associated primarily with oxidative tissues that rely heavily on fat oxidation for energy production. Although PLIN5 was not different between lean and obese subjects, it was the only PLIN protein to increase in response to endurance training in both sexes. PLIN5 correlated with IMCL volume (p < 0.0001), but in general, the other PLIN proteins did not correlate well with IMCL volume, suggesting that the relationship between lipid accumulation and PLIN family protein content is not a simple one. Although more work is necessary, it is clear that PLIN5 likely plays an important role in IMCL accumulation and oxidation, both of which increase with endurance training in human skeletal muscle.

Lipid droplets in the liver are coated with the perilipin family of proteins, notably adipocyte differentiation-related protein (ADRP) and tail-interacting protein of 47 kDa (TIP47). ADRP is increased in hepatic steatosis and is associated with hyperlipidemia, insulin resistance, and glucose intolerance. We have shown that reducing ADRP in the liver via antisense oligonucleotide (ASO) treatment attenuates steatosis and improves insulin sensitivity and glucose tolerance. We hypothesized that TIP47 has similar effects on hepatic lipid and glucose metabolism. We found that TIP47 mRNA and protein levels were increased in response to a high-fat diet (HFD) in C57BL/6J mice. TIP47 ASO treatment decreased liver TIP47 mRNA and protein levels without altering ADRP levels. Low-dose TIP47 ASO (15 mg/kg) and high-dose TIP47 ASO (50 mg/kg) decreased triglyceride content in the liver by 35% and 52%, respectively. Liver histology showed a drastic reduction in hepatic steatosis following TIP47 ASO treatment. The high dose of TIP47 ASO significantly blunted hepatic triglyceride secretion, improved glucose tolerance, and increased insulin sensitivity in liver, adipose tissue, and muscle. These findings show that TIP47 affects hepatic lipid and glucose metabolism and may be a target for the treatment of nonalcoholic fatty liver and related metabolic disorders.

The PAT family of lipid storage droplet proteins comprised five members, each of which has become an established regulator of cellular neutral lipid metabolism. Perilipin 5 (also known as lsdp-5, MLDP, PAT-1, and OXPAT), the most recently discovered member of the family, has been shown to localize to two distinct intracellular pools: the lipid storage droplet (LD), and a poorly characterized cytosolic fraction. We have characterized the denser of these intracellular pools and find that a population of perilipin 5 not associated with large LDs resides in complexes with a discrete density (~ 1.15 g/ml) and size (~ 575 kDa). Using immunofluorescence, western blotting of isolated sucrose density fractions, native gradient gel electrophoresis, and co-immunoprecipitation, we have shown that these small (~ 15 nm), perilipin 5-encoated structures do not contain the PAT protein perilipin 2 (ADRP), but do contain perilipin 3 and several other as of yet uncharacterized proteins. The size and density of these particles as well as their susceptibility to degradation by lipases suggest that like larger LDs, they have a neutral lipid rich core. When treated with oleic acid to promote neutral lipid deposition, cells ectopically expressing perilipin 5 experienced a reorganization of LDs in the cell, resulting in fewer, larger droplets at the expense of smaller ones. Collectively, these data demonstrate that a portion of cytosolic perilipin 5 resides in high density lipid droplet complexes that participate in cellular neutral lipid accumulation.

Author, Five proteins of the perilipin (Plin) family such as Plin1 (perilipin) Plin2 (adipose differentiation-related protein), Plin3 (tail-interacting protein of 47kDa), Plin4 (S3-12), and Plin5 (myocardial lipid droplet protein) are characterized as lipid droplet (LD) proteins in adipocytes. Recent reports have demonstrated that fat-specific protein 27 (FSP27) and hypoxia-inducible protein 2 (HIG2) are also thought to be novel LD proteins in addition to proteins of the Plin family. Growing evidence have shown that LD proteins play a role in the pathophysiology in the fatty liver disease which is characterized by hepatocytes containing LD with excessive neutral lipid. Studies showed LD proteins such as Plin1, Plin2, Plin3, Plin5, FSP27, and HIG2 are expressed in the liver steatosis. Among them, a high fat diet increases expression of Plin2 and/or FSP27 through activation of peroxisome proliferator-activated receptor γ to develop fatty liver. In this article, recent advances on the role of LD proteins in pathophysiology of fatty liver diseases are summarized.

Lipid droplets, also called lipid bodies (LB) in inflammatory cells, are important cytoplasmic organelles. However, little is known about the molecular characteristics and functions of LBs in human mast cells (MC). Here, we have analyzed the genesis and components of LBs during differentiation of human peripheral blood-derived CD34(+) progenitors into connective tissue-type MCs. In our serum-free culture system, the maturing MCs, derived from 18 different donors, invariably developed triacylglycerol (TG)-rich LBs. Not known heretofore, the MCs transcribe the genes for perilipins (PLIN)1-4, but not PLIN5, and PLIN2 and PLIN3 display different degrees of LB association. Upon MC activation and ensuing degranulation, the LBs were not cosecreted with the cytoplasmic secretory granules. Exogenous arachidonic acid (AA) enhanced LB genesis in Triacsin C-sensitive fashion, and it was found to be preferentially incorporated into the TGs of LBs. The large TG-associated pool of AA in LBs likely is a major precursor for eicosanoid production by MCs. In summary, we demonstrate that cultured human MCs derived from CD34(+) progenitors in peripheral blood provide a new tool to study regulatory mechanisms involving LB functions, with particular emphasis on AA metabolism, eicosanoid biosynthesis, and subsequent release of proinflammatory lipid mediators from these cells.

Recent reports demonstrated that 3,5-diiodo-l-thyronine (T2) was able to prevent lipid accumulation in the liver of rats fed a high-fat diet (HFD). In this study, we investigated how the rat liver responds to HFD and T2treatment by assessing the transcription profiles of some genes involved in the pathways of lipid metabolism: oxidation, storage and secretion. The mRNA levels of the peroxisome proliferator-activated receptors (PPARα, PPARγ and PPARδ), and of their target enzymes acyl-CoA oxidase and stearoyl-CoA desaturase were evaluated by real-time RT-PCR. Moreover, the expression of the adipose triglyceride lipase involved in lipid mobilisation, of the main PAT proteins acting in lipid droplet (LD) turnover, and of apoprotein B (apo B), the major protein component of very low-density lipoproteins (VLDLs) were analysed. Overall, our data demonstrated that T2administration to HFD rats counteracts most of the hepatic transcriptional changes that occurred in response to the excess exogenous fat. In particular, our results suggest that T2may prevent the pathways leading to lipid storage in LDs, promote the processes of lipid mobilisation from LDs and secretion as VLDL, in addition to the stimulation of pathways of lipid oxidation. In conclusion, our findings might give an insight into the mechanisms underlying the anti-steatotic ability of T2and help to define the potential therapeutic role of T2for preventing or treating liver steatosis.

We previously showed that adipose differentiation related protein (Adfp)-deficient mice display a 60% reduction in hepatic triglyceride (TG) content. In this study, we investigated the role of ADFP in lipid and glucose homeostasis in a genetic obesity model, Lep(ob/ob) mice. We bred Adfp(-/-) mice with Lep(ob/ob) mice to create Lep(ob/ob)/Adfp(-/-) and Lep(ob/ob)/Adfp(+/+) mice and analyzed the hepatic lipids, lipid droplet (LD) morphology, LD protein composition and distribution, lipogenic gene expression, and VLDL secretion, as well as insulin sensitivity of the two groups of mice. Compared with Lep(ob/ob)/Adfp(+/+) mice, Lep(ob/ob)/Adfp(-/-) mice displayed an increased VLDL secretion rate, a 25% reduction in hepatic TG associated with improvement in fatty liver grossly and microscopically with a change of the size of LDs in a proportion of the hepatocytes and a redistribution of major LD-associated proteins from the cytoplasmic compartment to the LD surface. There was no detectable change in lipogenic gene expression. Lep(ob/ob)/Adfp(-/-) mice also had improved glucose tolerance and insulin sensitivity in both liver and muscle. The alteration of LD size in the liver of Lep(ob/ob)/Adfp(-/-) mice despite the relocation of other LDPs to the LD indicates a nonredundant role for ADFP in determining the size and distribution of hepatic LDs.

Macrophages are, along with T CD4+lymphocytes, the major targets of HIV-1 infection and play a key role in viral pathogenesis as a significant reservoir. Identification of the cellular cofactors involved in the production of infectious HIV-1 from macrophages is thus crucial. Nevertheless molecular mechanisms allowing the production of infectious particles from macrophages are not entirely deciphered. In this study, we investigated the role of the cellular cofactor TIP47 in HIV-1 morphogenesis in primary macrophages. Our data show that TIP47 is essential for HIV-1 infectivity and propagation. Mutations in HIV-1 Gag or Envelope (Env) proteins, which abolish interaction with TIP47, impair HIV-1 propagation and infectivity preventing colocalization of Gag and Env, and thereby inhibiting Env incorporation into virions. Whereas disruption of Gag-TIP47 interaction increases slightly Gag particles production, impaired Env-TIP47 binding reduces Gag particles release and, strikingly, induces their retention in the viral assembly compartments. Thus, as in T lymphocytes, TIP47 is critical for the efficient release of infectious HIV-1 particles from the assembly compartment of primary macrophages, making TIP47 a new potential therapeutic target for limiting HIV-1 infectivity and spreading. This work is funded by ANRS, SIDACTION, ANR-07-JCJC-0102 programs and is part of the activities of the HIV-ACE research network (HEALTH-F3-2008-201095) supported by a grant of the European Commission, within the Priority 1 ''Health'' work programme of the 7th Framework Programme of the EU.

In many human cancers, lipogenic pathways are activated; in some tumors, such as hepatocellular carcinoma, this is reflected by the presence of visible lipid droplets. Yet, the biology of steatogenesis in malignant tumors is largely unknown. We have recently shown that lipid droplet-associated proteins of the PAT-family, named after their constituents perilipin (perilipin 1), adipophilin (perilipin 2), and TIP47 (perilipin 3) are differentially expressed in hepatic steatogenesis. We have comprehensively investigated PAT-expression in neoplastic steatogenesis as well as in respective normal tissues with immunohistology and electron microscopy as well as protein biochemical and molecular biological methods. By staining for PAT-proteins, we found lipid droplet accumulation to be a frequent phenomenon of carcinoma cells. Although adipophilin and TIP47 stained almost ubiquitously the rim of lipid droplets in various tumor types, especially those with clear cell phenotype, perilipin was restricted to lipid droplets of hepatocellular adenoma and carcinoma, sebaceous adenoma and carcinoma, and lipomatous tumors. In hepatocellular carcinoma, perilipin, adipophilin, and TIP47 were coexpressed, and showed regional heterogeneity with a predominantly mutually exclusive localization pattern. In step-wise carcinogenesis, adipophilin expression correlated with the proliferation rate and was upregulated during early tumorigenesis, whereas perilipin was often lost during hepatocarcinogenesis. In conclusion, expression analysis of PAT-proteins showed that by far more carcinomas contain (PAT-positive) lipid droplets than expected by conventional light microscopy. PAT-proteins, such as perilipin, are differentially expressed in different tumor types and thus may support diagnostic considerations. Because inhibition of lipogenesis has been shown to exert antineoplastic effects, PAT-proteins may represent targets for interventive strategies.

Recently, we found that enterocytes dynamically store triglycerides (TGs) in cytoplasmic lipid droplets (CLDs) during dietary fat absorption. A dynamic pool of TG in the form of CLDs which expands and depletes relative to time post dietary fat challenge is present in the absorptive cells of the small intestine, enterocytes. To identify cellular factors which may play a role in the regulation of this dynamic process we investigated the expression and localization of a lipid droplet associated protein family, PAT proteins, in enterocytes of mice chronically and acutely challenged by dietary fat. We found that adipophilin and Tip47 are the only PAT genes present in mouse intestinal mucosa and both genes are present at higher levels after high-fat challenges. We found TIP47 protein present in the intestine from chow and high-fat challenged mice; however, adipophilin protein was only present after high-fat challenges. In addition, TIP47 protein level was higher after an acute than a chronic high-fat challenge whereas adipophilin protein level was higher after a chronic than an acute high-fat challenge. We co-imaged TG in CLDs using CARS microscopy and TIP47 or adipophilin using immunocytochemistry in isolated enterocytes from mice challenged chronically and acutely by high levels of dietary fat. TIP47, but not adipophilin, coats CLDs in enterocytes after an acute high-fat challenge suggesting that TIP47 plays a role in the synthesis of CLDs from newly synthesized TG at the beginning of the process of dietary fat absorption in enterocytes. Adipophilin, on the other hand, coats CLDs only in enterocytes of chronic high-fat fed mice suggesting that adipophilin may play a role in the stabilization of TG stored in CLDs in longer term. These results suggest distinct roles for TIP47 and adipophilin in dietary fat absorption.

Lipid droplets are intracellular organelles that play central roles in lipid metabolism. In many cells, lipid droplets undergo active motion, typically along microtubules. This motion has been proposed to aid growth and breakdown of droplets, to allow net transfer of nutrients from sites of synthesis to sites of need and to deliver proteins and lipophilic signals. This review summarizes the current understanding of where, why and how lipid droplets move.

The PAT family of lipid droplet proteins includes 5 members in mammals: perilipin, adipose differentiation-related protein (ADRP), tail-interacting protein of 47 kiloDaltons (TIP47), S3-12, and OXPAT. Members of this family are also present in evolutionarily distant organisms, including insects, slime molds and fungi. All PAT proteins share sequence similarity and the ability to bind intracellular lipid droplets, either constitutively or in response to metabolic stimuli, such as increased lipid flux into or out of lipid droplets. Positioned at the lipid droplet surface, PAT proteins manage access of other proteins (lipases) to the lipid esters within the lipid droplet core and can interact with cellular machinery important for lipid droplet biogenesis. Genetic variations in the gene for the best characterized of the mammalian PAT proteins, perilipin, have been associated with metabolic phenotypes, including type 2 diabetes mellitus and obesity. In this review, we discuss how the PAT proteins regulate cellular lipid metabolism both in mammals and in model organisms.

Objective— Uptake of lipids by macrophages (MΦ) leads to lipid droplet accumulation and foam cell formation. The PAT family proteins are implicated in lipid droplet formation, but the precise function of the 47-kDa tail interacting protein (TIP47), a member of this family, is poorly defined. The present study was performed to determine the function of TIP47 in MΦ lipid metabolism. Methods and Results— Freeze-fracture cytochemistry demonstrates that TIP47 is present in the plasma membrane of MΦ and is aggregated into clusters when the cells are incubated with oleate. Suppression of adipophilin levels using siRNA knockdown leads to migration of TIP47 from a cytoplasmic pool to the lipid droplet. Further, reduction of TIP47 decreases triglyceride levels, whereas raising TIP47 levels by expression of EGFP-TIP47 shows the opposite effect. Conclusion— Our results show that the TIP47 protein levels directly correlate with triglyceride levels. We propose that TIP47 may act as a carrier protein for free fatty acids and in this way participates in conversion of MΦ into foam cells.

TIP47 (tail-interacting protein of 47 kD) was characterized as a cargo selection device for mannose 6-phosphate receptors (MPRs), directing their transport from endosomes to the trans-Golgi network. In contrast, our current analysis shows that cytosolic TIP47 is not recruited to organelles of the biosynthetic and endocytic pathways. Knockdown of TIP47 expression had no effect on MPR distribution or trafficking and did not affect lysosomal enzyme sorting. Therefore, our data argue against a function of TIP47 as a sorting device. Instead, TIP47 is recruited to lipid droplets (LDs) by an amino-terminal sequence comprising 11-mer repeats. We show that TIP47 has apolipoprotein-like properties and reorganizes liposomes into small lipid discs. Suppression of TIP47 blocked LD maturation and decreased the incorporation of triacylglycerol into LDs. We conclude that TIP47 functions in the biogenesis of LDs.

OBJECTIVE—Accumulation of intracellular lipid droplets (LDs) in non-adipose tissues is recognized as a strong prognostic factor for the development of insulin resistance in obesity. LDs are coated with perilipin, adipose differentiation–related protein, tail interacting protein of 47 kd (PAT) proteins that are thought to regulate LD turnover by modulating lipolysis. Our hypothesis is that PAT proteins modulate LD metabolism and therefore insulin resistance. RESEARCH DESIGN AND METHODS—We used a cell culture model (murine AML12 loaded with oleic acid) and small interfering RNA to directly assess the impact of PAT proteins on LD accumulation, lipid metabolism, and insulin action. PAT proteins associated with excess fat deposited in livers of diet-induced obese (DIO) mice were also measured. RESULTS—Cells lacking PAT proteins exhibited a dramatic increase in LD size and a decrease in LD number. Further, the lipolytic rate increased by ∼2- to 2.5-fold in association with increased adipose triglyceride lipase (ATGL) at the LD surface. Downregulation of PAT proteins also produced insulin resistance, as indicated by decreased insulin stimulation of Akt phosphorylation (P < 0.001). Phosphoinositide-dependent kinase-1 and phosphoinositide 3-kinase decreased, and insulin receptor substrate-1 307 phosphorylation increased. Increased lipids in DIO mice livers were accompanied by changes in PAT composition but also increased ATGL, suggesting a relative PAT deficiency. CONCLUSIONS—These data establish an important role for PAT proteins as surfactant at the LD surface, packaging lipids in smaller units and restricting access of lipases and thus preventing insulin resistance. We suggest that a deficiency of PAT proteins relative to the quantity of ectopic fat could contribute to cellular dysfunction in obesity and type 2 diabetes.

The protein-tyrosine phosphatase PTPMEG2 is located on the cytoplasmic face of the enclosing membrane of secretory vesicles, where it regulates vesicle size by promoting homotypic vesicle fusion by dephosphorylating N-ethylmaleimide-sensitive factor, a key regulator of vesicle fusion. Here we address the question of how PTPMEG2 is targeted to this subcellular location. Using a series of deletion mutants, we pinpointed the N-terminal Sec14p homology (SEC14) domain of PTPMEG2, residues 1-261, as the region containing the secretory vesicle targeting signal. This domain, alone or appended to a heterologous protein, was localized to intracellular vesicle membranes. Yeast two-hybrid screening identified a number of secretory vesicle proteins that interacted directly with the SEC14 domain of PTPMEG2, providing a mechanism for PTPMEG2 targeting to secretory vesicles. Two such proteins, mannose 6-phosphate receptor-interacting protein TIP47 and Arfaptin2, were found to alter PTPMEG2 localization when overexpressed, and elimination of TIP47 resulted in loss of PTPMEG2 function. We conclude that the N terminus of PTPMEG2 is necessary for the targeting of this phosphatase to the secretory vesicle compartment by association with other proteins involved in intracellular transport.

This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/, Supplementary information accompanies this paper at http://www.nature.com/srep, Dengue virus (DENV) causes over 500,000 hospitalizations and 20,000 deaths worldwide every year. Dengue epidemics now reach temperate regions due to globalization of trade and travel and climate changes. Currently, there are no successful therapeutic or preventive approaches. We previously developed a peptide drug lead, pep14-23, that inhibits the biologically relevant interaction of DENV capsid (C) protein with lipid droplets (LDs). Surprisingly, pep14-23 also inhibits DENV C interaction with very low-density lipoproteins (VLDL). We thus investigated the similarity between the proposed DENV C molecular targets in LDs and VLDL, respectively, the proteins perilipin 3 (PLIN3) and apolipoprotein E (APOE). APOE N-terminal and PLIN3 C-terminal regions are remarkably similar, namely APOE α -helix 4 (APOEα 4) and PLIN3 α -helix 5 (PLIN3α 5) sequences, which are also highly superimposable structurally. Interestingly, APOE α -helical N-terminal sequence and structure superimposes with DENV C α -helices α 1 and α 2. Moreover, the DENV C hydrophobic cleft can accommodate the structurally analogous APOEα 4 and PLIN3α 5 helical regions. Mirroring DENV C-LDs interaction (previously shown experimentally to require PLIN3), we experimentally demonstrated that DENV C-VLDL interaction requires APOE. Thus, the results fit well with previous data and suggest future drug development strategies targeting the above mentioned α –helical structures., We acknowledge the support of Fundação para a Ciência e Tecnologia – Ministério da Educação e Ciência (FCT-MEC, Portugal) project PTDC/SAU-ENB/117013/2010, and Calouste Gulbenkian Foundation (Portugal). AFF and ICM also acknowledge FCT-MEC fellowship SFRH/BD/77609/2011 and Investigador FCT Program research contract IF/00772/2013, respectively.

This study aimed at identifying potential microRNAs that modulate hepatic lipid droplets (LD) through targeting the Tail interacting protein of 47kDa (TIP47) in HCV infection.Bioinformatics analysis revealed that miR-148a and miR-30a potentially target TIP47. Expression profiling showed that both microRNAs were downregulated, while TIP47 was upregulated in liver biopsies of HCV-infected patients. Forcing the expression of both microRNAs in JFH-I infected, oleic acid-treated Huh7 cells, significantly suppressed TIP47 expression and reduced cellular LDs with marked decrease in viral RNA. This study shows that miR-148a and miR-30a, regulate TIP47 expression and LDs in HCV infected cells.

About 80% of patients with type 2 diabetes are classified as overweight. However, only about 1/3 of severely obese subjects have type 2 diabetes. This indicates that several se- verely obese individuals may possess certain characteristics that protect them against type 2 diabetes. We therefore hypothesized that this apparent paradox could be related to funda- mental differences in skeletal muscle lipid handling. Energy metabolism and metabolic flexi- bility were examined in human myotubes derived from severely obese subjects without (BMI 44 ± 7 kg/m 2 ) and with type 2 diabetes (BMI 43 ± 6 kg/m 2 ). Lower insulin sensitivity was observed in myotubes from severely obese subjects with type 2 diabetes. Lipolysis rate was higher, and oleic acid accumulation, triacylglycerol content, and fatty acid adaptability were lower in myotubes from severely obese subjects with type 2 diabetes compared to se- verely obese non-diabetic subjects. There were no differences in lipid distribution and mRNA and protein expression of the lipases HSL and ATGL, the lipase cofactor CGI-58, or the lipid droplet proteins PLIN2 and PLIN3. Glucose and oleic acid oxidation were also simi- lar in cells from the two groups. In conclusion, myotubes established from severely obese donors with established type 2 diabetes had lower ability for lipid accumulation and higher li- polysis rate than myotubes from severely obese donors without diabetes. This indicates that a difference in intramyocellular lipid turnover might be fundamental in evolving type 2 diabetes.

Retinoic acid at nanomolar concentrations modulates epidermal functions by serving as a transcription factor ligand. Under conditions of retinol sufficiency, it is imperative to limit retinoic acid biosynthesis from serum-derived retinol. In the epidermis, this is accomplished by esterifying retinol with long-chain fatty acids. Retinylester (RE) pools serve as a source of retinol for retinoic acid production under retinol deficiency and when required for proper differentiation. We have recently reported that GS2 lipase is expressed in keratinocytes and has the enzymatic properties of keratinocyte RE hydrolase. As GS2 lipase has a robust activity that can affect the intracellular retinol levels, we postulated that its activity must be regulated. Therefore, we screened keratinocyte cDNA expression libraries for the putative inhibitor. Herein, we report the identity of an inhibitor, TIP47, which prevents RE hydrolysis catalyzed by GS2 lipase and hormone-sensitive lipase. This protein was known to transport mannose-6-phosphate receptors from endosome to trans-Golgi and to be distributed between the cytoplasm and lipid droplets. Using a series of deletion mutants, we found two regions involved in the inhibitory activity. Residues within the carboxyl alpha3-alpha4 helices are essential in the context of the full-length protein. Residues within the amino-terminal also contribute depending on the context.

Adipose differentiation-related protein (ADRP) and TIP47 show sequence similarity, particularly in their N-terminal PAT-1 domain. Under standard culture conditions, ADRP existed in most lipid droplets (LDs), whereas TIP47 was observed only in some LDs and recruited to LDs on treatment with fatty acids. By analyzing deletion mutants, we found that the C-terminal half of TIP47, or more specifically the putative hydrophobic cleft [S.J. Hickenbottom, A.R. Kimmel, C. Londos, J.H. Hurley, Structure of a lipid droplet protein; the PAT family member TIP47, Structure (Camb) 12 (2004) 1199-1207.], was involved in LD targeting and responsiveness to fatty acids. The result contrasted with that observed for ADRP and implied a distinct LD-targeting mechanism for TIP47. Consistent with this, overexpression of Rab18 decreased ADRP, but not TIP47, from LDs, and TIP47 did not displace pre-existing ADRP from LDs. But ADRP may be a factor to control the TIP47 behavior, because TIP47 in LDs increased upon down-regulation of ADRP. The results suggested that the putative hydrophobic cleft is critical for the unique characteristics of TIP47.

The human genome encodes ∼70 Rab GTPases that localize to the surfaces of distinct membrane compartments. To investigate the mechanism of Rab localization, chimeras containing heterologous Rab hypervariable domains were generated, and their ability to bind seven Rab effectors was quantified. Two chimeras could bind effectors for two distinctly localized Rabs; a Rab5/9 hybrid bound both Rab5 and Rab9 effectors, and a Rab1/9 hybrid bound to certain Rab1 and Rab9 effectors. These unusual chimeras permitted a test of the importance of effector binding for Rab localization. In both cases, changing the cellular concentration of a key Rab9 effector, which is called tail-interacting protein of 47 kD, moved a fraction of the proteins from their parental Rab localization to that of Rab9. Thus, relative concentrations of certain competing effectors could determine a chimera's localization. These data confirm the importance of effector interactions for Rab9 localization, and support a model in which effector proteins rely on Rabs as much as Rabs rely on effectors to achieve their correct steady state localizations.

The presence of lipid in the cell cytoplasm is useful for supporting the diagnosis of sebaceous gland carcinoma (SGC). Currently this requires histochemical stains that are carried out on frozen sections of unprocessed tissue. Recently, several anti-adipocytic antibodies that recognise proteins associated with lipid vesicles have been described. These antibodies can be applied to paraffin-wax sections.To assess the ability of anti-adipocytic antibodies to identify intracytoplasmic lipid in SGC.Immunohistochemistry with a monoclonal antibody to adipophilin and polyclonal antibodies to perilipin and TIP47/PP17 was carried out on archival, formalin-fixed, paraffin-wax-embedded sections of 26 samples of SGC. The immunostaining was compared with 22 other eyelid tumours (11 basal cell carcinomas (BCC), 10 squamous cell carcinomas (SCC) and 1 Merkel cell tumour).Immunohistochemical staining was positive in 23, 10 and 2 cases of 26 SGC with adipophilin, perilipin and TIP47, respectively. The positive staining identified cytoplasmic lipid vesicles. Anti-adipophilin was positive in five other eyelid tumours (4 BCC and 1 SCC) staining small cytoplasmic granules that can be easily distinguished from the staining in SGC.Immunohistochemical staining for adipophilin and perilipin is a useful ancillary technique for the demonstration of lipid in SGC that may be applied to paraffin-wax sections.

The most common human brain tumours - gliomas - have poor prognosis with and without treatment. The current therapy conditions act sub-lethally and cannot effectively suppress the proliferation of glioma cells. Here we show differential protein expression patterns in surviving human malignant U87-MG glioma cells under clinically relevant chemo/radiotherapy. In parallel experiments, the cells underwent either irradiation (2 Gy, 200 KV X-ray) or chemotreatment with 30 microg/mL of temozolomide in the cultivation medium or combined chemo/radiation treatment. The cell cultures were treated during 5 days from day 4 until day 9 of growth. Modulated expression patterns of vimentin and RhoA GTPase indicate a potentially increasing grade of malignancy in treated cell fractions correlating well with extremely aggressive tumour phenotypes observed clinically at recidivation of treated malignant gliomas.

Lipid droplets (LDs), also called adiposomes, are found in many eukaryotic cells, and are highly upregulated in lipid-storage cells, such as adipocytes. The mechanism by which adiposomes and their component neutral lipids are degraded is an important health issue with the rapidly spreading epidemic of obesity. Recently, a novel triglyceride lipase (adipose triglyceride lipase (ATGL)) that catalyses the initial step in triglyceride hydrolysis in adipocyte LDs was identified. Here, we show that ATGL also functions in non-adipocyte cells, and has an important role in LD degradation in these cells. Overexpression of wild-type ATGL causes a marked decrease in LD size, whereas a catalytically inactive mutant retains the ability to localize to LDs, but is unable to decrease their size. Depletion of ATGL by RNA interference leads to a significant increase in the size of LDs. These results show that ATGL has an important role in LD/adiposome turnover in mammalian cells.

The number of proteins known to be associated with lipid droplets (LDs) is increasing. However, the reported distribution of a given protein in the LDs was, in some cases, found not reproduced by other groups. We report here that the choice of the fixation and permeabilization method is important in order to observe LD proteins using immunofluorescence microscopy. Formaldehyde fixation followed by treatment with Triton X-100, one of the most frequently used protocols for the immunolabeling of cultured cells, was not appropriate to label adipocyte differentiation-related protein (ADRP), TIP47, or Rab18 in LDs. Formaldehyde fixation followed by treatment with digitonin or saponin, allowed the visualization of all these proteins in LDs. When cells were fixed with glutaraldehyde, permeabilization by Triton X-100 could also be used for ADRP. These observations suggest that LD proteins are likely to be solubilized by some detergents, and strong cross-linkage to the surrounding protein matrix or mild permeabilization is necessary for their retention on the LD surface.

One of the central reactions in bodily energy metabolism is lipolysis in adipocytes, the reaction that governs the release of stored fatty acids from the adipocyte triacylglycerol pool, which constitutes the major energy reserve in animals. These fatty acids are then transported by serum albumin to various tissues to supply their energy requirements. This reaction was previously thought to result from phosphorylation and activation of hormone-sensitive lipase by protein kinase A (PKA) but is now known to be governed by a translocation of the lipase from the cytosol to the surface of the intracellular lipid droplet that houses the reservoir of TAG. This droplet is coated with perilipin A, which is also phosphorylated by PKA in response to lipolytic stimuli, and phosphorylation of perilipin A is essential for HSL translocation and stimulated lipolysis.

We present a new method for the rapid identification of amino acid residues that contribute to protein-protein interfaces. Tail-interacting protein of 47 kDa (TIP47) binds Rab9 GTPase and the cytoplasmic domains of mannose 6-phosphate receptors and is required for their transport from endosomes to the Golgi apparatus. Cysteine mutations were incorporated randomly into TIP47 by expression in Escherichia coli cells harboring specific misincorporator tRNAs. We made use of the ability of the native TIP47 protein to protect 48 cysteine probes from chemical modification by iodoacetamide as a means to obtain a surface map of TIP47, revealing the identity of surface-localized, hydrophobic residues that are likely to participate in protein-protein interactions. Direct mutation of predicted interface residues confirmed that the protein had altered binding affinity for the mannose 6-phosphate receptor. TIP47 mutants with enhanced or diminished affinities were also selected by affinity chromatography. These methods were validated in comparison with the protein's crystal structure, and provide a powerful means to predict protein-protein interaction interfaces.

Identification of antibody targets associated with malignant disease is indispensable to developing passive and active antibody-based therapeutics or diagnostic agents. We have developed a novel technique combining Western blotting, genetic profiling, and mass spectroscopy that allows for the rapid and unambiguous identification of such antigens in a high-throughput manner. Herein, we demonstrate this technique, designated Ab SCAN, by deducing the known target of a monoclonal antibody and by identifying a new antigen that was observed to be the frequent target of humoral immune responses in prostate cancer patients. In both instances, a specific antigen emerged as the sole protein candidate. The newly identified antigen, mannose-6-phosphate/IGF II receptor, may be an important naturally immunogenic antigen involved in prostate cancer. The Ab SCAN technique is uniquely suited to the analysis of longitudinal serum samples from clinical studies and could be a powerful tool to correlate humoral immune responses directed against discreet antigens with clinical events.

Intracellular cycling of the cation-dependent mannose 6-phosphate receptor (CD-MPR) between different compartments is directed by signals localized in its cytoplasmic tail. A di-aromatic motif (Phe18-Trp19 with Trp19 as the key residue) in its cytoplasmic tail is required for the sorting of the receptor from late endosomes back to the Golgi apparatus. However, the cation-independent mannose 6-phosphate receptor (CI-MPR) lacks such a di-aromatic motif. Therefore the ability of amino acids other than aromatic residues to replace Trp19 in the CD-MPR cytoplasmic tail was tested. Mutant constructs with bulky hydrophobic residues (valine, isoleucine, or leucine) instead of Trp19 exhibited 30-60% decreases in binding to the tail interacting protein of 47 kDa (Tip47), a protein mediating this transport step, and partially prevented receptor delivery to lysosomes. Decreasing hydrophobicity of residues at position 19 resulted in further impairment of Tip47 binding and an increase of receptor accumulation in lysosomes. Intriguingly, mutants mislocalized to lysosomes did not completely co-localize with a lysosomal membrane protein, which might suggest the presence of subdomains within lysosomes. These data indicate that sorting of the CD-MPR in late endosomes requires a distinct di-aromatic motif with only limited possibilities for variations, in contrast to the CI-MPR, which seems to require a putative loop (Pro49-Pro-Ala-Pro-Arg-Pro-Gly55) along with additional hydrophobic residues in the cytoplasmic tail. This raises the possibility of two separate binding sites on Tip47 because both receptors require binding to Tip47 for endosomal sorting.

Here, we report that human immunodeficiency virus type 1 (HIV-1) Env glycoprotein is located mainly in the trans- Golgi network (TGN) due to determinants present in the cytoplasmic domain of the transmembrane gp41 glycoprotein (TMgp41). Internalization assays demonstrated that Env present at the cell surface returns to the TGN. We found that the cytoplasmic domain of TMgp41 binds to TIP47, a protein required for the transport of mannose-6-phosphate receptors from endosomes to the TGN. Overexpression of a mutant of TIP47 affected the transport of Env from endosomes to the TGN. Retrograde transport of Env to the TGN requires a Y 802 W 803 diaromatic motif present in the TMgp41 cytoplasmic domain. Mutation of this motif abolished both targeting to the TGN as well as interaction with TIP47. These data support the view that binding of TIP47 to HIV-1 Env facilitates its delivery to the TGN. Lastly, we show that virus mutated in the Y 802 W 803 motif is poorly infectious and presents a defect in Env incorporation, supporting a model in which retrograde transport of Env is implicated in the optimization of fully infectious HIV-1 production.

Intracellular neutral lipid storage droplets are essential organelles of eukaryotic cells, yet little is known about the proteins at their surfaces or about the amino acid sequences that target proteins to these storage droplets. The mammalian proteins Perilipin, ADRP, and TIP47 share extensive amino acid sequence similarity, suggesting a common function. However, while Perilipin and ADRP localize exclusively to neutral lipid storage droplets, an association of TIP47 with intracellular lipid droplets has been controversial. We now show that GFP-tagged TIP47 co-localizes with isolated intracellular lipid droplets. We have also detected a close juxtaposition of TIP47 with the surfaces of lipid storage droplets using antibodies that specifically recognize TIP47, further indicating that TIP47 associates with intracellular lipid storage droplets. Finally, we show that related proteins from species as diverse as Drosophila and Dictyostelium can also target mammalian or Drosophila lipid droplet surfaces in vivo. Thus, sequence and/or structural elements within this evolutionarily ancient protein family are necessary and sufficient to direct association to heterologous intracellular lipid droplet surfaces, strongly indicating that they have a common function for lipid deposition and/or mobilization.