ADRP/adipophilin is degraded through the proteasome-dependent pathway during regression of lipid-storing cells

Adipose differentiation-related protein (ADRP) is a major protein associated with lipid droplets in various types of cells, including macrophage-derived foam cells and liver cells. However, the role of ADRP in the processes of formation and regression of these cells is not understood. When J774 murine macrophages were incubated with either VLDL or oleic acid, their content of both ADRP and triacylglycerol (TG) increased 3- to 4-fold. Induction of ADRP during TG accumulation was also observed in oleic acid-treated HuH-7 human liver cells. Addition of triacsin C, a potent inhibitor of acyl-CoA synthase, for 6 h decreased the amount of TG in VLDL-induced foam cells and oleic acid-treated liver cells; it decreased the amount of ADRP protein in parallel, indicating the amount of ADRP reduced during regression of the lipid-storing cells. Addition of a proteasome inhibitor during triacsin C treatment abolished the ADRP decrease and accumulated polyubiquitinated ADRP. In addition, the proteasome inhibitor reversed not only the degradation of ADRP but also TG reduction by triacsin C. These results suggest that cellular amounts of ADRP and TG regulate each other and that the ubiquitin-proteasome system is involved in degradation of ADRP during regression of lipid-storing cells.     

Sterol carrier protein-2 expression modulates protein and lipid composition of lipid droplets

Despite the critical role lipid droplets play in maintaining energy reserves and lipid stores for the cell, little is known about the regulation of the lipid or protein components within the lipid droplet. Although immunofluorescence of intact cells as well as Western analysis of isolated lipid droplets revealed that sterol carrier protein-2 (SCP-2) was not associated with lipid droplets, SCP-2 expression significantly altered the structure of the lipid droplet. First, the targeting of fatty acid and cholesterol to the lipid droplets was significantly decreased. Second, the content of several proteins important for lipid droplet function was differentially increased (perilipin A), reduced severalfold (adipose differentiation-related protein (ADRP), vimentin), or almost completely eliminated (hormone-sensitive lipase and proteins >93 kDa) in the isolated lipid droplet. Third, the distribution of lipids within the lipid droplets was significantly altered. Double labeling of cells with 12-(N-methyl)-N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-octadecanoic acid (NBD-stearic acid) and antisera to ADRP showed that 70, 24, and 13% of lipid droplets contained ADRP, NBD-stearic acid, or both, respectively. SCP-2 expression decreased the level of ADRP in the lipid droplet but increased the proportion wherein ADRP and NBD-stearic acid colocalized by 3-fold. SCP-2 expression also decreased the lipid droplet fatty acid and cholesterol mass (nmol/mg protein) by 5.2- and 6.6-fold, respectively. Finally, SCP-2 expression selectively altered the pattern of esterified fatty acids in favor of polyunsaturated fatty acids within the lipid droplet. Displacement studies showed differential binding affinity of ADRP for cholesterol and fatty acids. These data suggested that SCP-2 and ADRP play a significant role in regulating fatty acid and cholesterol targeting to lipid droplets as well as in determining their lipid and protein components.     

Dynamics of lipid droplet-associated proteins during hormonally stimulated lipolysis in engineered adipocytes: stabilization and lipid droplet binding of adipocyte differentiation-related protein/adipophilin

In mature adipocytes, triglyceride is stored within lipid droplets, which are coated with the protein perilipin, which functions to regulate lipolysis by controlling lipase access to the droplet in a hormone-regulatable fashion. Adipocyte differentiation-related protein (ADRP) is a widely expressed lipid droplet binding protein that is coexpressed with perilipin in differentiating fat cells but is minimally present in fully differentiated cultured adipocytes. We find that fibroblasts ectopically expressing C/EBPalpha (NIH-C/EBPalpha cells) differentiate into mature adipocytes that simultaneously express perilipin and ADRP. In response to isoproterenol, perilipin is hyperphosphorylated, lipolysis is enhanced, and subsequently, ADRP expression increases coincident with it surrounding intracellular lipid droplets. In the absence of lipolytic stimulation, inhibition of proteasomal activity with MG-132 increased ADRP levels to those of cells treated with 10 mum isoproterenol, but ADRP does not surround the lipid droplet in the absence of lipolytic stimulation. We overexpressed a perilipin A construct in NIH-C/EBPalpha cells where the six serine residues known to be phosphorylated by protein kinase A were changed to alanine (Peri A Delta1-6). These cells show no increase in ADRP expression in response to isoproterenol. We propose that ADRP can replace perilipin on existing lipid droplets or those newly formed as a result of fatty acid reesterification, under dynamic conditions of hormonally stimulated lipolysis, thus preserving lipid droplet morphology/structure.     

Proteasome activator enhances survival of Huntington's disease neuronal model cells

In patients with Huntington's disease (HD), the proteolytic activity of the ubiquitin proteasome system (UPS) is reduced in the brain and other tissues. The pathological hallmark of HD is the intraneuronal nuclear protein aggregates of mutant huntingtin. We determined how to enhance UPS function and influence catalytic protein degradation and cell survival in HD. Proteasome activators involved in either the ubiquitinated or the non-ubiquitinated proteolysis were overexpressed in HD patients' skin fibroblasts or mutant huntingtin-expressing striatal neurons. Following compromise of the UPS, overexpression of the proteasome activator subunit PA28gamma, but not subunit S5a, recovered proteasome function in the HD cells. PA28gamma also improved cell viability in mutant huntingtin-expressing striatal neurons exposed to pathological stressors, such as the excitotoxin quinolinic acid and the reversible proteasome inhibitor MG132. These results demonstrate the specific functional enhancements of the UPS that can provide neuroprotection in HD cells.     

Degradation of survival motor neuron (SMN) protein is mediated via the ubiquitin/proteasome pathway

Homozygous deletion or mutation in the survival motor neuron (SMN)1 gene causes proximal spinal muscular atrophy (SMA), whereas SMN2 acts as a modifying gene that can influence the severity of SMA. It has been suggested that restoration of the SMN protein level in neuronal cells may prevent cell loss and may be helpful for treatment of SMA. Recent studies indicate that the ubiquitin/proteasome pathway is a major system for proteolysis of intracellular proteins. In this study, we investigate whether SMN protein is degraded via the ubiquitin/proteasome pathway. Primary fibroblasts were established from the skin biopsies of SMA patients and the effect of a proteasome inhibitor MG132 and lysosome inhibitor NH(4)Cl on SMN protein level was examined. We found that MG132, but not NH(4)Cl, significantly increased the amount and nuclear accumulation of SMN protein in SMA patient's fibroblasts. Immunoprecipitation/western blot analysis indicated that SMN protein was ubiquitinated in cells. In vitro protein ubiquitination assay also demonstrated that SMN protein could be conjugated with ubiquitin. Taken together, we have provided clear evidences that degradation of SMN protein is mediated via the ubiquitin/proteasome pathway and suggest that proteasome inhibitors may up-regulate SMN protein level and may be useful for the treatment of SMA.     

TIP47 associates with lipid droplets

Most mammalian cells package neutral lipids into droplets that are surrounded by a monolayer of phospholipids and a specific set of proteins including the adipose differentiation-related protein (ADRP; also called adipophilin), which is found in a wide array of cell types, and the perilipins, which are restricted to adipocytes and steroidogenic cells. TIP47 was initially identified in a yeast two-hybrid screen for proteins that interact with the cytoplasmic tail of the mannose 6-phosphate receptor, yet its sequence is highly similar to the lipid droplet protein, ADRP, and more distantly related to perilipins. Hence, we hypothesized that TIP47 might be associated with lipid droplets. In HeLa cells grown in standard low lipid-containing culture media, immunofluorescence microscopy revealed that the cells had few lipid droplets; however, TIP47 and ADRP were found on the surfaces of the small lipid droplets present. When the cells were grown in media supplemented with physiological levels of fatty acids, the amount of neutral lipid stored in lipid droplets increased dramatically, as did the staining of TIP47 and ADRP surrounding these droplets. TIP47 was found primarily in the cytosolic fractions of HeLa cells and murine MA10 Leydig cells grown in low lipid-containing culture medium, while ADRP was undetectable in these fractionated cell homogenates. When HeLa and MA10 Leydig cells were lipid-loaded, significant levels of ADRP were found in the floating lipid droplet fractions and TIP47 levels remained constant, but the distribution of a significant portion of TIP47 shifted from the cytosolic fractions to the lipid droplet fractions. Thus, we conclude that TIP47 associates with nascent lipid droplets and can be classified as a lipid droplet-associated protein.     

Glucagon regulates intracellular distribution of adipose differentiation-related protein during triacylglycerol accumulation in the liver

Cellular lipid droplets (LD) are organelles involved in cellular lipid metabolism. When liver cellular components were fractionated using sucrose density gradient centrifugation, adipose differentiation-related protein (ADRP) was distributed in both the top and bottom fractions, which correspond to the LD and membranous fractions, respectively, in the mouse liver under normal feeding conditions. After overnight fasting, triacylglycerol and ADRP increased nearly 2.5-fold in the mouse liver, and a portion appeared in the intermediate-density LD (iLD) fractions. ADRP in the iLD fractions was also increased in a mouse nonalcoholic steatohepatitis model induced by methione/choline-deficient diet. When HuH-7 human hepatoma cells were incubated with oleic acid for 24 h, the amount of ADRP increased, and it was distributed in both the LD and membrane fractions. However, ADRP appeared in the iLD fractions upon treatment of HuH-7 cells with glucagon. This behavior of ADRP was cAMP-dependent, as the ADRP-positive iLD fractions were induced by dibutylyl cAMP and were blocked by protein kinase A inhibitors. A portion of ADRP colocalized microscopically with calnexin, which is present in the iLD fractions, by treatment of HuH-7 cells or human primary hepatocytes with oleic acid and glucagon, but not by treatment with oleic acid alone. Glucagon has a role in the reorganization of endoplasmic reticulum membranes to generate ADRP-associated lipid-poor particles in hepatic cells, which is related to LD formation during lipid storage.     

ADRP stimulates lipid accumulation and lipid droplet formation in murine fibroblasts

Adipose differentiation-related protein (ADRP) is a lipid droplet-associated protein that is expressed early during adipose differentiation. The present study was undertaken to reveal the role of ADRP in adipose differentiation. In murine fibroblasts infected with green fluorescent protein (GFP)-ADRP fusion protein expression adenovirus vector, confocal microscopic analysis showed the number and size of lipid droplets apparently increased comparing with those of control cells. Overexpressed GFP-ADRP were mainly located at the surface of lipid droplets and appeared to be "ring-shaped." Triacylglycerol content was also significantly (P < 0.001) increased in GFP-ADRP-overexpressed cells compared with control cells. ADRP-induced lipid accumulation did not depend on adipocyte-specific gene induction, such as peroxisome proliferator-activated receptor-gamma, lipoprotein lipase, or other lipogenic genes, including acyl-CoA synthetase, fatty acid-binding protein, and fatty acid transporter. In conclusion, ADRP stimulated lipid accumulation and lipid droplet formation without induction of other adipocyte-specific genes or other lipogenic genes in murine fibroblasts. The detailed molecular mechanisms of ADRP on lipid accumulation remain to be elucidated.     

Regulation of ADRP expression by long-chain polyunsaturated fatty acids in BeWo cells, a human placental choriocarcinoma cell line

Transplacental transfer of maternal fatty acids is critical for fetal growth and development. In the placenta, a preferential uptake of fatty acids toward long-chain polyunsaturated fatty acids (LCPUFAs) has been demonstrated. Adipose differentiation-related protein (ADRP) is a lipid droplet-associated protein that has been ascribed a role in cellular fatty acid uptake and storage. However, its role in placenta is not known. We demonstrate that ADRP mRNA and protein are regulated by fatty acids in a human placental choriocarcinoma cell line (BeWo) and in primary human trophoblasts. LCPUFAs of the n-3 and n-6 series [arachidonic acid (20:4n-6), docosahexaenoic acid (22:6n-3), and eicosapentaenoic acid (20:5n-3)] were more efficient than shorter fatty acids at stimulating ADRP mRNA expression. The fatty acid-mediated increase in ADRP mRNA expression was not related to the differentiation state of the cells. Synthetic peroxisome proliferator-activated receptor and retinoic X receptor agonists increased ADRP mRNA level but had no effect on ADRP protein level in undifferentiated BeWo cells. Furthermore, we show that incubation of BeWo cells with LCPUFAs, but not synthetic agonists, increased the cellular content of radiolabeled oleic acid, coinciding with the increase in ADRP mRNA and protein level. These studies provide new information on the regulation of ADRP in placental trophoblasts and suggest that LCPUFA-dependent regulation of ADRP could be involved in the metabolism of lipids in the placenta.     

Expression of adipose differentiation-related protein (ADRP) is conjointly regulated by PU.1 and AP-1 in macrophages

ADRP is associated with intracellular lipid droplets. We demonstrate the regulatory mechanism for ADRP expression in RAW264.7 macrophages. The ADRP mRNA expression was stimulated by PMA, and synergistically enhanced in association with its protein level in the presence of lipids. A proteasome inhibitor protected the protein from degradation under the lipid-free conditions. One of the possible sites of the PMA action was proved to be an Ets/AP-1 element in the promoter, since mutations of this site reduced the PMA-induced promoter activity, and ligation of this element led to a significant increase in the PMA-responsiveness of homologous or heterologous promoters. Mutations of this site diminished the synergistic effect on the promoter activity induced by PMA and oleic acid, suggesting a possible interaction between this site and the downstream PPARdelta site. EMSA revealed that PU.1 and AP-1 conjointly bound to this site. The juxtaposition of the two sequences was requisite for full activity, since spacer sequences between them decreased the PMA-induced activity. PI3 kinase inhibitor was found to reduce the PMA-induced mRNA expression and promoter activity in parallel with PU.1/AP-1 complex formation on EMSA. From these results, we concluded that the Ets/AP-1 site is an important cis-acting element that regulates the ADRP gene expression in macrophages.     

Proteomic Analysis of MG132-Treated Germinating Pollen Reveals Expression Signatures Associated with Proteasome Inhibition

Chemical inhibition of the proteasome has been previously found to effectively impair pollen germination and tube growth in vitro. However, the mediators of these effects at the molecular level are unknown. By performing 2DE proteomic analysis, 24 differentially expressed protein spots, representing 14 unique candidate proteins, were identified in the pollen of kiwifruit (Actinidia deliciosa) germinated in the presence of the MG132 proteasome inhibitor. qPCR analysis revealed that 11 of these proteins are not up-regulated at the mRNA level, but are most likely stabilized by proteasome inhibition. These differentially expressed proteins are predicted to function in various pathways including energy and lipid metabolism, cell wall synthesis, protein synthesis/degradation and stress responses. In line with this evidence, the MG132-induced changes in the proteome were accompanied by an increase in ATP and ROS content and by an alteration in fatty acid composition.     

Specific inhibitors prevent proteolytic degradation of recombinant proteins expressed in High Five cells

The insect cell line BTI-TN-5B1-4 (High Five) is frequently used to express recombinant proteins in large amounts using the baculovirus expression system. However, extensive proteolytic degradation of recombinant proteins is often encountered. Furthermore, we have observed that recombinant proteins migrate in SDS-PAGE in agreement with poly-ubiquitinated forms of the protein, suggesting a ubiquitin/proteasome degradation pathway. Here, we describe a systematic study unraveling the effect of adding proteasome inhibitors or specific protease inhibitors to the growth medium of High Five insect cells infected with recombinant baculovirus. Furthermore, protease inhibitors were added to the lysis buffer to establish the most efficient way to inhibit proteolytic activity after lysis of baculovirus-infected cells expressing recombinant proteins. We conclude that a combination of adding protease inhibitors to the growth medium and to the lysis buffer minimizes the proteolytic activity in High Five cells. The most efficient protease inhibitors were E-64 in the growth medium together with Leupeptin in the lysis buffer at concentrations higher than with available cocktails of inhibitors. The optimal treatment of High Five cells is different from the optimal treatment of Sf9 cells. For proteins susceptible to ubiquitinylation, a treatment of insect cell cultures with the proteasome inhibitor MG132 (LLL) leads to a considerable reduction of the yield of production of recombinant protein.     

Oleic acid-induced PKC isozyme translocation in RAW 264.7 macrophages

Fatty acids are important second messengers that mediate various cellular functions, but their role in the formation of macrophage foam cells is not known. High plasma levels of oleic acid (OA) in obese patients are often associated with a high risk for atherosclerosis. In this study, we investigated the protein kinase C (PKC) isozymes involved in OA-induced lipid accumulation in RAW 264.7 macrophages. The results show that OA induces translocation of PKC alpha, beta1, and delta from the cytosol to the cell membrane 5 min after the treatment. After 16 h incubation with OA, PKC delta was found to be colocalized with adipose differentiation-related protein (ADRP) on the surface of lipid droplets, but immunoprecipitation experiments showed that PKC delta was not biochemically associated with ADRP. After 16 h incubation with OA plus phorbol 12-myristate 13-acetate (PMA), PKC delta staining on the lipid droplet surface was not seen, whereas the accumulation of lipid droplets was unaffected. Furthermore, downregulation of PKC delta was confirmed by immunoblotting. These results demonstrate possible involvement of specific PKC isozymes in the early phase of lipid accumulation, possibly during the uptake of OA.     

Quantitative analysis of global ubiquitination in HeLa cells by mass spectrometry

Ubiquitination regulates a host of cellular processes by labeling proteins for degradation, but also by functioning as a regulatory, nonproteolytic posttranslational modification. Proteome-wide strategies to monitor changes in ubiquitination profiles are important to obtain insight into the various cellular functions of ubiquitination. Here we describe generation of stable cell lines expressing a tandem hexahistidine-biotin tag (HB-tag) fused to ubiquitin for two-step purification of the ubiquitinated proteome under fully denaturing conditions. Using this approach we identified 669 ubiquitinated proteins from HeLa cells, including 44 precise ubiquitin attachment sites on substrates and all seven possible ubiquitin chain-linkage types. To probe the dynamics of ubiquitination in response to perturbation of the ubiquitin/proteasome pathway, we combined ubiquitin profiling with quantitative mass spectrometry using the stable isotope labeling with amino acids in cell culture (SILAC) strategy. We compared untreated cells and cells treated with the proteasome inhibitor MG132 to identify ubiquitinated proteins that are targeted to the proteasome for degradation. A number of proteasome substrates were identified. In addition, the quantitative approach allowed us to compare proteasome targeting by different ubiquitin chain topologies in vivo. The tools and strategies described here can be applied to detect changes in ubiquitination dynamics in response to various changes in growth conditions and cellular stress and will contribute to our understanding of the ubiquitin/proteasome system.