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A lipid-droplet-targeted O-GlcNAcase isoform is a key regulator of the proteasome
Chithra N. Keembiyehetty, Anna Krzeslak, Dona C. Love, John A. Hanover


Protein-O-linked N-Acetyl-β-D-glucosaminidase (O-GlcNAcase, OGA; also known as hexosaminidase C) participates in a nutrient-sensing, hexosamine signaling pathway by removing O-linked N-acetylglucosamine (O-GlcNAc) from key target proteins. Perturbations in O-GlcNAc signaling have been linked to Alzheimer's disease, diabetes and cancer. Mammalian O-GlcNAcase exists as two major spliced isoforms differing only by the presence (OGA-L) or absence (OGA-S) of a histone-acetyltransferase domain. Here we demonstrate that OGA-S accumulates on the surface of nascent lipid droplets with perilipin-2; both of these proteins are stabilized by proteasome inhibition. We show that selective downregulation of OGA-S results in global proteasome inhibition and the striking accumulation of ubiquitinylated proteins. OGA-S knockdown increased levels of perilipin-2 and perilipin-3 suggesting that O-GlcNAc-dependent regulation of proteasomes might occur on the surface of lipid droplets. By locally activating proteasomes during maturation of the nascent lipid droplet, OGA-S could participate in an O-GlcNAc-dependent feedback loop regulating lipid droplet surface remodeling. Our findings therefore suggest a mechanistic link between hexosamine signaling and lipid droplet assembly and mobilization.


The post-translational modification of cellular proteins by adding a single β-N-acetylglucosamine (GlcNAc) moiety is a highly evolutionarily conserved process. The turnover rate of this modification is much faster than for the protein itself, affecting many downstream cellular activities, and thus serving as a previously unidentified signaling mechanism. Two enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) regulate the O-GlcNAc modification, by adding and removing O-GlcNAc, respectively. UDP-GlcNAc, the substrate of OGT, is derived from the hexosamine biosynthetic pathway, a pathway modulated by cellular nutrient levels. Although the target sequence of this modification does not conform to a clear consensus, O-GlcNAcylation occurs at serine or threonine residues and on many phosphoproteins (Wang et al., 2008; Love and Hanover, 2005; Slawson and Hart, 2003). More than 600 proteins have been identified as O-GlcNAc-modifiable targets. The functional roles of this modification are not yet fully understood, but growing evidence indicates that O-GlcNAc impacts multiple cellular activities. O-GlcNAcylated proteins might modulate a variety of downstream signaling pathways by influencing gene transcription, protein stability, transport and proteasome activity (Love et al., 2010; Marshall, 2006; Zachara and Hart, 2004). The O-GlcNAc modification is known to be responsive to metabolic status, and perturbations in O-GlcNAc cycling have been linked to major chronic metabolic diseases such as Alzheimer's, diabetes and cancer (Love et al., 2010; Lazarus et al., 2006; Love and Hanover, 2005). Additionally, a growing body of evidence suggests that elevated levels of O-GlcNAc, associated with cellular injury, are protective in several models of stress (Zachara et al., 2010).

The present study focuses on splice variants of OGA, which removes O-GlcNAc on target proteins. OGA was first identified as meningioma-expressed antigen 5 (MGEA5) and is found in all tissues examined, with the highest expression in brain, placenta and pancreas (Heckel et al., 1998; Comtesse et al., 2001). Human MGEA5 is present on chromosome 10 at cytological position 10q24.1, which has been identified as a diabetes susceptibility locus in the Mexican-American population (Duggirala et al., 1999; Lehman et al., 2005). The product of this gene, OGA (E.C., protein O-Glc NAcase), has been referred to as hexosaminidase C, because it is distinct from the lysosomal β hexosaminidases A and B. Compared with the acidic hexosaminidases, OGA has a neutral pH optimum and shows selectivity for the removal of GlcNAc rather than GalNAc (Gao et al., 2001). Two splice variants derived from the MGEA5 gene were originally identified in human tissues (Comtesse et al., 2001). The full-length OGA long form (OGA-L) has 916 amino acids transcribed from 16 exons (NM_012215) and a short isoform (OGA-S) contains 677 amino acids transcribed from first 10 exons (AF307332). Both isoforms are identical at the N-terminal hyaluronidase domain, but differ in that OGA-L has a histone acetyl transferase (HAT)-like domain at the C-terminus (Schultz and Pils, 2002; Toleman et al., 2004; Whisenhunt et al., 2006). The OGA-S isoform continues transcription through exon 10, skipping the splice junction and terminating with a unique 14 amino acid tail. The crystal structure of the human O-GlcNAcase has not been resolved, but two groups have reported the three-dimensional structure of two bacterial O-GlcNAcase homologs and have elucidated their catalytic mechanism (Dennis et al., 2006; Rao et al., 2006).

The functional activities of the hyaluronidase and HAT domains in OGA-L were described by Wells et al., Toleman et al. and Butkinaree et al. (Wells et al., 2002; Toleman et al., 2004; Butkinaree et al., 2008). The OGA-L isoform has been shown to be a part a gene co-repressor complex and is subject to O-GlcNAcylation by OGT (Lazarus et al., 2006; Whisenhunt et al., 2006). Until recently, the OGA-S isoform was not extensively studied. Previous reports using subcellular fractionation and antigenicity described OGA-L as a cytoplasmic–nuclear protein and OGA-S as a nuclear variant (Comtesse et al., 2001). The O-GlcNAcase activity of OGA-S was demonstrated in our laboratory using a highly sensitive and specific fluorogenic substrate (Kim et al., 2006) as was the selective inhibition of the enzyme by α-GlcNAc thiosulfonate (Kim et al., 2007). The O-GlcNAcase activity of the short isoform was confirmed independently (Macauley and Vocadlo, 2009) but its functional significance and its cellular localization remain to be established.

The present study explores the localization and functional activities of the two major OGA isoforms, OGA-L and OGA-S. Our results indicate that they both play key roles in modulating proteasome activity, although the isoforms have distinct intracellular localizations. We also demonstrate that OGA-S is targeted to the surface of nascent lipid droplets, thereby revealing a possible link between hexosamine signaling and the proteasome-dependent remodeling of the surface of lipid droplets.


Targeting and activity of OGA isoforms

The mammalian Mgea5 gene encodes two major isoforms, which we have designated OGA-S and OGA-L (Kim et al., 2006). OGA-L utilizes all 16 exons, contains a N-terminal hyaluronidase domain (Comtesse et al., 2001) and a C-terminal putative histone acetyl transferase domain (Fig. 1A). The shorter isoform comprises 10 exons with an N-terminal hyaluronidase domain and a unique 14 amino acid C-terminus. Because antibodies raised against OGA do not identify both isoforms (supplementary material Fig. S1), plasmid vectors were constructed with green fluorescent protein (GFP) fusions of both isoforms (OGA-L–GFP and OGA-S–GFP) to determine their intracellular targeting. The isoforms were expressed as full-length GFP fusions in HeLa cells (Fig. 1B, arrows), however, the number of cells expressing OGA-S–GFP was substantially lower than that expressing OGA-L–GFP, suggesting differences in RNA or protein stability between the two isoforms. Western blot analysis revealed a significant, yet differential decrease in O-GlcNAc-modified proteins upon over expression of either OGA-L–GFP or OGA-S–GFP, indicating that both enzymes are catalytically active (Fig. 1C). The levels of enzyme activity correspond to the levels of expression of both enzymes; quantitative western blot analysis demonstrated that OGA-L–GFP lowered global O-GlcNAc levels by approximately 72% and OGA-S–GFP lowed the O-GlcNAc levels by 35%. Next, an increase in OGA enzymatic activity was measured directly using a specific fluorogenic substrate (Fig. 1D). Overexpression of OGA-L–GFP increased total OGA activity by 446% compared with control, whereas OGA-S–GFP increased activity by 136%. We examined the intracellular localization of the two isoforms in HeLa cells. OGA-L–GFP localized diffusely throughout the nucleus and cytoplasm (Fig. 1E, top panel, green channel) whereas OGA-S-GFP was found associated with structures suggestive of lipid droplets (Fig. 1E, bottom panel, green channel). We monitored the in situ activities of the overexpressed enzymes by staining HeLa cells with the O-GlcNAc-specific antibody, RL2 (Fig. 1E, red channel). OGA-L was present in the nucleus and cytoplasm (Fig. 1E, top row) and efficiently depleted cytosolic O-GlcNAc. We also noted that the nuclear O-GlcNAc compartment (denoted by an asterisk) was somewhat refractory to overexpression of the enzyme. By contrast, OGA-S was associated with circular structures reminiscent of lipid droplets (Fig. 1E, bottom row). We noted that although the steady-state distribution of OGA-S was distinct from that of OGA-L, overexpression of this isoform dramatically reduced O-GlcNAc levels in the cell, including the nuclear and cytoplasmic compartments (Fig. 1E, bottom row). These data suggested that OGA-S was uniquely targeted within the cell and, like OGA-L, was catalytically active.

Fig. 1.

O-GlcNAcase is expressed as two differentially targeted and active isoforms. (A) Genomic organization and schematic exon diagram of the O-GlcNAcase isoforms. The OGA long isoform (OGA-L) is encoded by 16 exons and contains a N-terminal hyaluronidase catalytic domain and a C-terminal HAT domain. The OGA short isoform (OGA-S) is encoded by 10 exons and contains a N-terminal hyaluronidase catalytic domain. The HAT domain is replaced by a unique 14 amino acid C-terminal tail. (B) Western blot immunostained with a GFP-specific antibody showing the expression of OGA-L–GFP (~150 kDa) and OGA-S–GFP (~120 kDa). Each isoform migrated to the expected molecular mass. (C) Overexpression of OGA-L–GFP or OGA-S–GFP causes a decrease in O-GlcNAc immunoreactivity compared with untransfected cells (control). Using densitometry, a 4-fold and 1.34-fold decrease in O-GlcNAcylation was detected for OGA-L–GFP and OGA-S–GFP, respectively. (D) Total OGA enzymatic activity was measured from OGA-L–GFP and OGA-S–GFP cellular lysates using the fluorogenic substrate, FD-GlcNAc. Values are means ± standard deviation; s.d. of three separate experiments. (E) Representative confocal images of OGA-L–GFP and OGA-S–GFP overexpression in HeLa cells showing nucleocytoplasmic location of OGA-L (upper panel) and exclusive cytoplasmic staining of OGA-S isoform (lower panel). Overexpression of both isoforms reduced O-GlcNAc levels as shown by diminished O-GlcNAc antibody staining. Cells expressing the fusion proteins are outlined for comparison. Asterisks indicate the nucleus of each cell.

Fig. 2.

OGA-S accumulates at the surface of lipid droplets and its activity is elevated by increased lipid stores. (A) Transfected HeLa cells expressing either OGA-L–GFP or OGA-S–GFP were treated with 0.4 mM oleic acid to increase lipid stores. OGA-L–GFP is located diffusely in the nucleus and cytoplasm (green channel) with no specific connection with lipid droplets (red channel). By contrast, OGA-S–GFP targets to the surface of lipid droplets. Neutral lipid was stained using Nile Red (red channel). Co-staining the cells for perilipin-2 demonstrates that OGA-S–GFP colocalizes with this resident lipid droplet protein. The degree of colocalization is shown in the merged panel. A higher magnification of the area denoted by the arrows is shown in the insets. (B) Oleic acid increases the amount of OGA activity. OGA activity was measured using the fluorogenic substrate, FD-GlcNAc, in cells overexpressing OGA-L–GFP or OGA-S–GFP and treated with and without 0.4 mM oleic acid. OGA-S–GFP activity increased approximately 30% in the presence of 0.4 mM oleic acid, whereas OGA-L–GFP activity increased by 6% with oleic acid treatment. Data shown are the means of duplicate samples and are representative of two separate experiments.

To confirm the targeting of OGA-S to the lipid droplet, HeLa cells were treated with 0.4 mM oleic acid to stimulate lipid droplet formation. These cells were infected with adenoviral vectors expressing either OGA-L–GFP or OGA-S–GFP. OGA-L–GFP was found throughout the cell in both nucleus and cytoplasm, and was not enriched on neutral lipid droplets stained with Nile Red (Fig. 2A; top row; red channel). By contrast, the majority of OGA-S–GFP concentrated at the periphery of lipid droplets (Fig. 2A, middle row; green channel). OGA-S–GFP localization to lipid droplets in HeLa cells was further verified by staining of perilipin-2 (also called ADRP, ADFP and adipophilin), a lipid-droplet-associated protein. Here, OGA-S–GFP colocalized with perilipin-2 at the lipid droplet surface (Fig. 2A, bottom row; arrows and inset). To confirm that the targeting was specific and not due to the C-terminal fusion of GFP, we generated a construct fusing a hemagglutinin (HA) tag at the N-terminus of OGA-L and OGA-S (HA–OGA-S and HA–OGA-L). The localization of these HA-tagged proteins duplicated the localization of the GFP-tagged proteins, confirming that OGA-L and OGA-S are differentially targeted within the cell and that OGA-S preferentially accumulates at lipid-rich structures (supplementary material Fig. S2).

Lipid accumulation enhances OGA activity

The specific targeting and apparent stabilization of OGA-S by lipid accumulation might be expected to alter O-GlcNAcase activity. Using the highly sensitive fluorogenic O-GlcNAcase substrate, di(N-acetyl-β-D-glucosaminide; FD-GlcNAc), O-GlcNAcase activity was measured in cell lysates from HeLa cells that were overexpressing either OGA-L–GFP or OGA-S–GFP, with and without oleic acid. Total activity of OGA-L–GFP was greater than twofold higher than that of OGA-S–GFP, presumably because of markedly lower expression efficiency of OGA-S–GFP. Upon treatment of cells with oleic acid, the highest relative increase in enzyme activity was seen in the OGA-S–GFP-expressing cells (35%) compared with the OGA-L–GFP-expressing cells (6%; Fig. 2B). Thus lipid accumulation appears to enhance OGA-S–GFP activity preferentially. To test whether oleic acid supplementation also enhances endogenous OGA activity, we performed a similar experiment using untransfected cells. Cells were either serum depleted or treated with 0.4 mM oleic acid for 24 hours to modulate lipid stores (Fig. 3A). Compared with serum depletion, oleic acid supplementation increased OGA activity more than twofold (Fig. 3B). Based on the localization of OGA-S, a substantial portion of activity should be associated with the lipid droplet fraction. The lipid droplet fraction from the oleic-acid-treated cells contained greater than 50% of the total OGA activity (Fig. 3B). The modulation of OGA activity by increasing lipid droplet stores might be the result of enhanced stability of OGA-S at the lipid droplet surface, in a manner similar to that reported for perilipin-2 (Xu et al., 2005).

The unique targeting of OGA-S was tested in a physiologically relevant system using the 3T3 L1 adipocyte model. 3T3 L1 pre-adipocytes were differentiated to mature adipocytes using a standard protocol (Student et al., 1980) and whole-cell lysates from each step in differentiation were probed for changes in O-GlcNAcylation. Different subsets of proteins were detected by O-GlcNAc-specific antibodies over the course of adipogenic differentiation, indicating a variation in O-GlcNAc cycling on protein targets during this process (Fig. 4A; and see also supplementary material Fig. S3). During the differentiation process, we also detected a modulation in OGA-S targeting. The PAT proteins (perilipin, ADRP, TIP47; the last two are alternative names for other perilipins) are known as bona fide resident proteins at the lipid droplet surface and are essential for maintaining lipid stores (Londos et al., 1999; Brasaemle, 2007). Pre-adipocytes contain small lipid droplets that are enriched with perilipin-2. HA-OGA-S colocalized with the perilipin-2 in these nascent lipid droplets (Fig. 4B, merged panel). As the adipocytes differentiate, the lipid droplets increase in size and perilipin-2 is replaced with perilipin-1 (Bickel et al., 2009). Localization of OGA-S–GFP was examined in mature adipocytes. In differentiated 3T3 L1 adipocytes, the lipid droplets became markedly larger and completely coated with perilipin-1 (Fig. 4C, red channel). OGA-S–GFP localization, however, remained restricted to the smaller diameter lipid droplets (Fig. 4C, green channel; arrows) and was markedly reduced at the larger droplets containing increased amounts of perilipin-1 (Fig. 4C, red channel). There was very little colocalization between OGA-S–GFP and perilipin-1 (Fig. 4C, merged panel). These data suggested that OGA-S associates with nascent, but not mature, perilipin-1-coated lipid droplets indicating that it might play a role in the earlier stages of the adipocyte differentiation.

Fig. 3.

Lipid accumulation enhances endogenous OGA activity. (A) Schematic diagram of lipid augmentation. HeLa cells, either serum starved for 24 hours or treated with 0.4 mM oleic acid, were assayed using FD-GlcNAc to measure the OGA activity in the total lysate from each condition shown plus the lipid fraction from the oleic-acid-supplemented cells. (B) Bar graph representing the measured OGA activity from each sample in arbitrary fluorescence units. Data shown are the means of duplicate samples and are representative of three separate experiments.

Isoform-specific knockdown of OGA by siRNA

Given the unique targeting characteristics of the OGA isoforms and the potential interaction with lipid droplets, we examined the functional specificity of the endogenous OGA isoforms. Unique siRNAs were designed to selectively downregulate each of the isoforms in HeLa and HEK-293 cells (see supplementary material Table S1 for oligonucleotide sequences; oligos). The selectivity of the isoform-specific siRNAs was confirmed by several techniques. First, cells transfected with either OGA-L–GFP or OGA-S–GFP were treated with the isoform-specific siRNA oligos, and controls were treated with scrambled siRNA. Incubating transfected cells with oligos specific for either OGA-L–GFP or OGA-S–GFP resulted in dramatic reduction in GFP fluorescence for each expression vector when compared with scrambled siRNA treatments (Fig. 5A, compare right and left panels). Second, untransfected HeLa cells were treated for 48 hours with oligos selective for each OGA isoform. Semi-quantitative PCR was used to confirm mRNA knockdown of endogenous transcripts in (Fig. 5B). Transcripts for OGA-L were diminished by 50% and for OGA-S by >60%. In addition, the fluorogenic substrate FD-GlcNAc was used to measure total O-GlcNAcase activity in lysates from cells treated with the specific oligos. Downregulation of OGA-L resulted in a lowering of overall OGA activity to 33% of control cells, whereas 77% of total activity remained after downregulation of OGA-S (Fig. 5C). Modulation of O-GlcNAcase activity by downregulation of each isoform is reflected also in modified total O-GlcNAc protein levels. RNAi of OGA-L resulted in a 3-fold increase in total protein O-GlcNAcylation, whereas RNAi of OGA-S only modestly altered O-GlcNAc levels (1.4-fold; Fig. 5D, graph). Treatment with both siRNAs raised O-GlcNAc levels in an additive manner compared with that of the siRNA specific for a single isoform alone. Taken together, these data suggest that OGA-L is responsible for roughly two-thirds of the cellular O-GlcNAcase activity with OGA-S contributing approximately one-third.

O-GlcNAcase activity regulates proteasome function

Although OGA-S appears to contribute modestly to global O-GlcNAc cycling, we sought to determine if OGA-S might have specific targets. The proteasome presents an ideal target for several reasons: the activity of the 26S subunit of the proteasome is inhibited by O-GlcNAc modification (Zhang et al., 2007) and OGA-S is targeted specifically to the lipid droplet surface where proteasomal activity is required for lipid droplet remodeling (Ohsaki et al., 2006).

To identify the relative contribution of the OGA isoforms in modulating proteasomal functions, we analyzed both live cells and cell lysates for proteasome activity. HEK-293 ZsGreen Proteasome Sensor cells were used as our live cell model. These cells stably express a GFP fusion protein that is targeted for 26S proteasomal degradation allowing quantitative assay of proteasomal perturbation by following the accumulation of the GFP fusion protein. Under control conditions little fluorescence was detected because the GFP fusion protein was rapidly and continuously degraded (Fig. 6A). However, targeting either the long form or the short form of OGA for downregulation by RNAi resulted in a marked increase in steady-state GFP fluorescence, indicating that the proteasomal activity was compromised upon downregulation of OGA. As a positive control, cells were treated with the proteasome inhibitor ALLN. The number of cells with GFP accumulation was directly quantified by flow cytometry (Fig. 6B). The cells exhibited a shift of the curve to the right, indicating a tenfold increase in GFP-positive cells when treated with OGA-S (red line) or OGA-L (blue line) siRNA compared with control siRNA (green line). We noted that downregulation of OGA-S was equally effective at perturbing proteasomal activity, as was OGA-L. Maximum proteasome inhibition by ALLN further right-shifted the curve.

Fig. 4.

3T3 L1 adipocyte differentiation alters O-GlcNAcylated protein profiles and OGA-S localization. (A) O-GlcNAcylated protein profile in 3T3 L1 adipocytes changed during differentiation. Total cell lysates were collected from 3T3 L1 cells at the confluence stage (D2) and at 2-day intervals during the differentiation process. Equal amounts of protein (30 μ g/lane) from each sample were resolved using a 4–12% Bis-Tris gel and immunoblotted with an O-GlcNAc specific antibody (CTD110.6). To test the specificity of the O-GlcNAc antibody, immunoreactivity was competed with excess N-acetylglucosamine (supplementary material Fig. S1). Lysates were probed for perilipin-2 as a positive control for differentiation. (B) In 3T3 L1 pre-adipocytes, HA–OGA-S (green channel) colocalized with nascent, perilipin-2-positive lipid droplets. (C) In differentiated adipocytes, OGA-S–GFP (green) associated with the smaller lipid droplets and did not colocalize with the larger lipid droplets completely coated with perilipin-1 (red). Insets show a higher magnification of the area denoted by the arrows.

Previous reports suggested that the O-GlcNAc modification has a greater influence on the chymotrypsin-like activity of the proteasome (Zhang et al., 2003; Zhang et al., 2007). Both chymotrypsin and trypsin-like activity of the proteasome were measured in HeLa and HEK-293 cell lysates. Isoform-specific knockdown of OGA was performed before testing for proteasome activity in the lysates. Although chymotrypsin-like activity of the proteasome, as measured by the degradation of the synthetic fluorogenic peptide (Suc–LLVY–AMC), was reduced by depleting either OGA isoform, statistical significance was reached only with depletion of OGA-S (Fig. 6C, upper panel). Trypsin-like activity was not significantly altered by downregulation of either isoform (Fig. 6C, lower panel). Interestingly, the knockdown of OGA-S was equally effective (75% of control vs 83% of control) as OGA-L at perturbing proteasomal activity, which is in agreement with results from proteasome sensor cells described above. Similar results were obtained using HeLa cells (data not shown). Proteasome functional assays in two different cell models suggest that both isoforms target proteasome activity.

Fig. 5.

Selective downregulation of the O-GlcNAcase isoforms using siRNAs. (A) RNAi inhibition of OGA-L–GFP and OGA-S–GFP expression. OGA-L–GFP or OGA-S–GFP was expressed in HeLa cells and subsequently co-transfected with scrambled dsRNA (control) or isoform-specific siRNAs. Cells were fixed and analyzed by fluorescence microscopy. siRNA treatment reduced the expression of each isoform as shown by a reduction in GFP expression. GFP expression is shown in green and DAPI-stained nuclei are shown in blue. (B) RNAi reduced mRNA transcription. Semi-quantitative RT-PCR for endogenous OGA-L and OGA-S was performed using total RNA isolated from HeLa cells treated for 48 hours with scrambled dsRNA (con) and siRNAs specific for long (sirL), short (sirS) or both (sirS/L) O-GlcNAcase isoforms. GAPDH transcript levels are shown as a control. (C) OGA enzymatic activity is reduced with siRNA treatment (con, scrambled siRNA; sirL, siRNA for OGA-L; sirS, siRNA for OGA-S). OGA activity was assayed using the fluorogenic substrate (values are the percentage activity normalized to control ± s.e.m.). (D) Decreasing OGA levels by siRNA treatment increased the total amount of O-GlcNAc-modification in the HeLa cell lysate as shown by western blot analysis using an O-GlcNAc-specific antibody. Below is a bar graph showing the cumulative band intensity (percentage of the control ± s.e.m.) from two different experiments.

OGA downregulation results in increased polyubiquitinylation

Proteasomal inhibition is normally associated with accumulation of polyubiquitinylation on proteins. As a further confirmation of proteasomal inhibition by OGA, siRNA-treated cell lysates were immunoblotted with anti-ubiquitin to quantify global polyubiquitin levels. HeLa cells treated with control siRNA showed little accumulation of polyubiquitinylated proteins (Fig. 7A, con). However, by selectively lowering OGA-L with siRNA treatment, a threefold increase in cumulative levels of polyubiquitinylation was observed (Fig. 7B, sirL). Ubiquitinylated protein accumulation was fivefold higher with OGA-S (Fig. 7B, sirS). The characteristic immunoreactive ubiquitin smear is shown with a longer exposure (Fig. 7A). Downregulation of either OGA isoform inhibits proteasomal activity as measured by a synthetic sensor (Fig. 6A, ZsProSonsor1) or accumulation of polyubiquitin (Fig. 7). These data suggest that the lipid-droplet-targeted OGA-S could influence proteasome-mediated protein remodeling of the lipid droplet surface (see below).

Fig. 6.

Knockdown of the O-GlcNAcase isoforms decreases proteasome activity in cells and cell lysates. (A) ZsGreen Proteasome Sensor cells were treated with scrambled dsRNA (Con), or dsRNAs specific for OGA-L (sirL) or OGA-S (sirS) for 48 hours. ZsGreen Proteasome Sensor cells express a GFP reporter protein (ZsProSensor-1) targeted for degradation; accumulation of GFP fluorescence indicates impaired proteasome activity. Downregulation of either OGA isoform increased the number of GFP-positive cells (top row, green channel), indicating reduced proteasome activity. DAPI-stained nuclei are shown (bottom row, blue). As a positive control, the proteasome inhibitor ALLN was used at 5 μg/ml for 12 hours. (B) The accumulation of ZsProSensor-1 protein was analyzed quantitatively using FACS analysis. Histogram plots show differences in fluorescence intensity between cells treated with scrambled dsRNA (green line), ALLN (black line), siRNA for long (blue line) and short (red line) isoforms. (C) OGA downregulation modulates proteasomal activities. The proteasome peptidase activities in HEK-293 cell lysates were measured by fluorescence from the cleavage of Suc–LLVY–AMC (chymotrypsin-like activity) and Boc–LSTR–AMC (trypsin-like activity). The proteasome activity was measured 48 hours after transfection with the indicated siRNAs. Treatment with ALLN (5 μg/ml) for 12 hours is shown for comparison. Values are the mean of triplicate samples (± s.e.m.). *Statistical difference compared with control or sirL (P<0.05, paired Student's t-test).

Complex interplay of the proteasome and OGA on lipid-droplet-associated proteins

Expression of OGA-S fusion proteins was always markedly lower than that of OGA-L in our experiments, regardless of the tag used (HA or GFP) or the location of the tag (i.e. N- or C-terminal end of the enzyme). Typically, only 20–30% of the cells showed OGA-S expression, whereas tagged versions of OGA-L were robustly expressed within the monolayer. This difference in expression levels was confirmed by western blot analysis (Fig. 1B) and measured total O-GlcNAcase activity (Fig. 1D). Because rapid degradation of OGA-S could cause the low expression level, we examined how proteasome activity could alter the stability of the OGA isoforms. Proteasome inhibition by ALLN resulted in a dose-dependent accumulation of both isoforms, but had the largest impact on OGA-S accumulation (Fig. 8A). Without ALLN, OGA-S–GFP was nearly undetectable, compared with OGA-L–GFP (Fig. 8A and Fig. 1B). Measurement of the integrated intensity of the western blots showed that the expression of OGA-S–GFP increased approximately 10- and 14-fold with 25 and 50 μM ALLN treatment, respectively, compared with 3- and 5-fold for OGA-L–GFP, suggesting that OGA-S levels are tightly regulated by the proteasome. Similar results were obtained with the HA-tagged enzymes (data not shown). As a control for ALLN action, the stabilization of perilipin-2 is shown (Fig. 8A, bottom row).

Fig. 7.

OGA downregulation results in an accumulation of polyubiquitinyled proteins. (A) Western blot analysis was performed using anti-ubiquitin antibodies as described in the Materials and Methods. Total cellular lysates from cells treated with scrambled siRNA (lanes 1and 2, con), siRNA for OGA-L (lanes 3 and 4, sirL) and siRNA for OGA-S (lanes 5 and 6, sirS) are shown. A longer exposure of lanes 5 and 6 is shown on the right to demonstrate the characteristic ubiquitin immunoreactive smear. The amount of actin is shown as a loading control. (B) Bar graph of total band intensity relative to actin. Values are mean percentages ± s.e.m.

If OGA-S is selectively acting on the proteasome at lipid droplets, then downregulation of OGA-S, but not OGA-L should perturb proteasome function at the lipid droplet surface and result in an increase in lipid-droplet-associated proteins. To test this hypothesis, we selectively downregulated each isoform and measured the accumulation of perilipin-2 and perilipin-3 (also known as TIP47; a related lipid-droplet-associated protein). OGA-S-specific RNAi treatment stabilized perilipin-2 levels (Fig. 8B, upper panels). Such stabilization was not observed when OGA-L was downregulated. Quantitatively similar results were obtained with perilipin-3 (Fig. 8B, lower panels). Our results suggest that a highly regulated feedback control of OGA-S might modulate lipid-droplet protein stability (Fig. 8C). In this model, proteasome activity tightly regulates the levels of OGA-S and its associated O-GlcNAcase activity, present at the surface of the lipid droplet. O-GlcNAc cycling is also known to regulate proteasome activity by inhibiting the Rtp2 domain of the 26S proteasome (Zhang et al., 2007). Therefore, OGA-S activity, acting through proteasome activation at the surface of the lipid droplet, is ideally positioned to modulate the stability of other lipid-droplet-associated proteins. This regulation might serve to control the size and surface composition of the maturing lipid droplet.

Fig. 8.

OGA-S–GFP expression is stabilized by proteasome inactivation and modulates Perilipin-2 and perilipin-3 levels in HeLa cells. (A) Western blot analysis showing the stabilization of OGA-S–GFP, OGA-L–GFP and perilipin-2 with proteasome inhibition. Whole cell lysates from ALLN-treated (25 and 50 μg/ml) cells expressing either OGA-S–GFP or OGA-L–GFP were probed with an anti-GFP to identify the fusion protein. The amount of OGA-S–GFP increased by 10- (25 μg/ml ALLN) and 14-fold (25 μg/ml ALLN) compared with the control, whereas OGA-L–GFP increased by 3- (25 μg/ml ALLN) and 5-fold (25 μg/ml ALLN) over the control. An increase in perilipin-2 is shown as a positive control for proteasome inhibition. The amount of actin is shown as a loading control. (B) Depletion of OGA-S, but not OGA-L stabilizes perilipin-2 and -3. Western blot analysis showing siRNA-mediated OGA-S downregulation (sir-S) selectively stabilized perilipin-2 and perilipin-3 expression in HeLa cells but OGA-L siRNA (sir-L) had no effect. Treatment with scrambled siRNA (con) is shown as a negative control. (C) Model showing the possible regulatory role of OGA-S on lipid droplet surface PAT proteins. As depicted, OGT inhibits the proteasome by O-GlcNAc modification of the Rpt2 domain whereas OGA-S activates the proteasome by removing the modification. Activated proteasomes in turn facilitate the degradation of lipid droplet surface proteins (PAT) and also create a negative feedback loop by degrading OGA-S to limit its availability in a manner dependent upon lipid stores.


O-GlcNAc cycling on a multitude of diverse substrates is carried out by a single set of opposing enzymes (OGT and OGA). A key question concerning O-GlcNAc cycling is how a single set of enzymes can regulate a multitude of substrates in a coordinated manner. We speculate that isoform specificity or enzyme compartmentalization could play a role in regulating multiple substrates independently. For OGT, differentially spliced isoforms were described and shown to localize at different cellular compartments (Love et al., 2003). For the O-GlcNAcase two major isoforms have been reported, but the detailed properties of the shorter isoform were not studied (Comtesse et al., 2001). Knowledge of isoform-specific effects of OGA are important in developing inhibitors as potential drug targets in metabolic diseases. Therefore, we have examined the localization and distinct activities of the O-GlcNAcase isoforms.

Results demonstrate that both isoforms are active and are transcribed in numerous cell types, although the protein stability might affect the relative abundance of each isoform. We further demonstrate that the isoforms are differentially localized. The longer isoform resides in the nucleus and cytoplasm, whereas the shorter isoform is found concentrated at the surface of lipid droplets. The increase of lipid stores correlates positively with OGA activity, and isolated lipid droplets contain a major fraction of total OGA activity. Finally, we show that the two isoforms differentially modulate proteasome activity at the lipid droplet surface. Because proteasome activity is essential for the remodeling and assembly of lipid droplets, we propose a role for the shorter OGA isoform in regulating lipid droplet assembly and the mobilization of neutral lipids.

OGA isoforms exhibit unique catalytic properties

The relative catalytic efficiency of the two major O-GlcNAcase isoforms has recently come under scrutiny (Macauley and Vocadlo, 2009; Kim et al., 2007). Upon overexpression, OGA-S and OGA-L are each capable of dramatically lowering the amounts of modified O-GlcNAc proteins at the single cell level, as shown by reduced staining with anti O-GlcNAc antibody (Fig. 1B). The number of cells with detectable OGA-S protein, however, was greatly reduced compared with cells transfected with OGA-L. Consistent with this result, OGA activity in siRNA-treated total cell lysates indicated that OGA-S makes only a small contribution to the total pool of O-GlcNAcase activity in HeLa cells. In addition to lower expression levels, the Km for OGA-S is approximately 20-fold lower than that for OGA-L (Kim et al., 2006). The data reported in this manuscript are in agreement with the several-fold lower activity of OGA-S compared with OGA-L. Macauley and Vocadlo also reported lower efficiency of O-GlcNAcase activity for OGA-S (OGA-NV) compared with OGA-L (Macauley and Vocadlo, 2009). The known difference of the C-terminal tail of the two isoforms (long isoform having a ‘HAT-like’ domain and the short form a unique 14 amino acid tail) might offer a basis for the difference in catalytic behavior at the N-terminus of the protein.

OGA isoforms might have unique intracellular targets

The distinctive intracellular localization of the OGA isoforms suggests that they have specific functional targets. OGA-L–GFP expression is, in both nucleus and cytoplasm, consistent with its activity in many nuclear and cytoplasmic proteins. OGA-S–GFP, however, preferentially accumulates at the surface of lipid droplets. Because OGA-S is colocalized with perilipin-2 in HeLa cells and 3T3 L1 adipocytes, the short isoform of O-GlcNAcase could play a distinct role in modulating the function of lipid-droplet-associated proteins. Perilipin-2 is one of the PAT proteins on the lipid droplet surface actively involved in lipid droplet formation (Bickel et al., 2009; Brasaemle, 2007; Londos et al., 1999). Oleic acid is known to induce synthesis of perilipin-2, initiating nascent lipid droplets, whereas it is gradually replaced by perilipin-1 during lipid droplet maturation in adipocytes (Bickel et al., 2009). This transition from perilipin-2 to perilipin-1 is regulated by a proteasome-sensitive mechanism (Xu et al., 2005; Xu et al., 2006; Masuda et al., 2006). Interestingly, OGA-S demonstrates a robust effect on proteasomal activity and we have pursued the hypothesis that OGA-S could regulate perilipin-2 stability by activating proteasomes located at the lipid droplet surface.

OGA isoforms differentially regulate the proteasome

The 26S proteasome is a well-established target of O-GlcNAc cycling, as documented in several studies. O-GlcNAc modification of the proteasome in neuronal cells has been reported to inhibit proteasomal activity (Zhang et al., 2007; Zhang et al., 2003; Liu et al., 2004). O-GlcNAc modification of the Rpt2 ATPase located at the 19S proteasome regulatory cap inhibited the access of ubiquitinylated proteins to the proteasomal core for degradation (Zhang et al., 2007). Here, we provide evidence that O-GlcNAcase modulates proteasome activity in living cells, using isoform-specific siRNA treatment. Although OGA-S appears to be a minor contributor to total O-GlcNAcase activity, downregulation of this isoform had a similar impact on proteasome activity as did downregulation of OGA-L. Additionally, increased accumulation of ubiquitinylated proteins upon OGA-S siRNA treatment suggests that the proteasome is a preferred OGA-S target.

The chymotrypsin-like, trypsin-like and caspase-like activities are among the several different catalytic functions of proteasomes. Our in vitro assays demonstrated that OGA isoform downregulation has a more dramatic effect on the chymotrypsin-like activity than trypsin-like activity. This corroborates the observations of Zhang et al. showing that cleavage of the LLVY (chymotrypsin-like activity) peptide was altered by OGT, whereas there was no effect observed on a tryptic peptide LSTR (Zhang et al., 2003). The present findings suggest that O-GlcNAcylation might not inhibit global proteasome function, but could alter activity towards certain specific targets. Similarly, specific inhibition of chymotrypsin-like activity of the proteasome has been reported with long chain fatty acids (Hamel, 2009).

OGA-S functions at the surface of maturing lipid droplets

Given the localization of OGA-S on the surface of lipid droplets and its dramatic effect on proteasome function, OGA-S appears to play a key role in lipid droplet metabolism. The proteasome is known to play a key role in the remodeling of the lipid droplet surface. Perilipin-2 and perilipin-1 are ultimately degraded by the ubiquitin–proteasome pathway (Xu et al., 2005; Xu et al., 2006). Ubiquitin ligases, necessary for proteasomal function, are targeted to the lipid droplet surface and influence lipid droplet size and number (Eastman et al., 2009). Additionally, protein degradation by the proteasome-ubiquitin pathway and autophagy converge at the lipid droplet surface to regulate lipid metabolism (Ohsaki et al., 2006; Singh et al., 2009). Because these two catabolic pathways are interdependent and act at the surface of the lipid droplet, OGA-S activity and O-GlcNAc cycling probably influences both pathways to regulate lipid homeostasis.

Our data suggest a feedback mechanism between the proteasomal degradation pathway and O-GlcNAcase activity at the surface of the lipid droplet. Proteasome activity is modulated by the action of OGA-S, whereas relative abundance of the O-GlcNAc isoforms is maintained through proteasomal degradation. Thus OGA-S and the proteasome regulate each other, suggesting a feedback mechanism often observed with nutritionally responsive, metabolic regulation. The localization of OGA-S to nascent lipid droplets co-occupied by perilipin-2 and the ability of OGA-S to regulate proteasome activity suggests a complex interplay between the presence of lipid stores and the relative abundance of the OGA-S isoform. As such, OGA-S, perilipin-2, oleic acid and the proteasome might all participate in the intricate mechanism required to maintain cellular lipid stores. This notion is consistent with previous findings on lipid metabolism. C. elegans mutants with either ogt-1 or oga-1 null alleles were shown to have decreased lipid stores and increased dauer formation associated with defective O-GlcNAc cycling (Hanover et al., 2005; Forsythe et al., 2006). Luo et al. have suggested that increased O-GlcNAc flux activates AMPK and stimulates fatty acid oxidation (Luo et al., 2007).

Summary and conclusion

The differently targeted isoforms of OGA analyzed in this study are likely to perform distinct intracellular functions. The nuclear and cytoplasmic OGA-L contains a HAT domain, suggesting it participates in chromatin-related regulation of gene expression. By contrast, the lipid-droplet-associated OGA-S is likely to play a role in regulating the process of lipid storage and mobilization. By modulating the local activity of proteasomes associated with lipid droplets, OGA-S might actively participate in the perilipin-2 regulation and remodeling of the protein on the lipid droplet surface (Fig. 8C). The results of this study strongly suggest that OGA-S might be a key enzyme in maintaining the delicate balance between carbohydrate and lipid metabolism.

Materials and Methods

Chemicals and antibodies

The proteasome inhibitor Ac-leucine-leucine-norleucinal (ALLN; calpain inhibitor I) and proteasome substrates Suc–LLVY–AMC and Boc–LSTR–AMC were purchased from Sigma-Aldrich. Primary antibodies RL2 (cat no. MAI-072) and perilipin A (perilipin-1; cat no. PAI-1051) were from Affinity Bioreagents; CTD110.6 from Covance (cat no. MMS-248R); and ubiquitin from EBiosciences. Antibodies to mouse perilipin-2, perilipin-1 and perilipin-3 were gifts from the late Dr Constantine Londos (NIDDK, NIH). Pan actin, GFP and HA antibodies were from Cell Signaling Technology. Mouse and rabbit IgG and IgM secondary antibodies conjugated with IRDye 800, or IRDye700 were from Rockland Immunochemicals (Rockland, PA).

Plasmid and siRNA

A cassette containing either human OGA long (NM_012215) or short isoform (AF307332) was incorporated into the pEGFP-N1 vector (Clontech) between the KpnI and PinAI sites. OGA-L–GFP and OGA-S–GFP clones have GFP fused at the C-terminus. An adenovirus vector was constructed using the same plasmid construct (adenoviral-type 5 (dE1/E3, Vector Biolabs). Two other OGA constructs were made with HA-tag fusions using the pCMV-HA vector (Clontech). HA-OGA-L and HA-OGA-S were generated with the HA tag at the N-terminus of the OGA isoforms by inserting the OGA sequence between the BglII and KpnI sites, in frame with the HA tag.

Specific siRNA was designed, using web-based software, to silence OGA-L and OGA-S isoforms. Selected 20-mers, were purchased from Qiagen as annealed double-strand DNA. Initial experiments were conducted to select siRNA that efficiently targeted OGA isoforms, and to determine the duration of the treatments. Selected sequences of siRNA specific for human OGA-L and OGA-S are shown in supplementary material Table S1.

Cell culture

HeLa cells (ATCC) and HEK-293 ZsGreen Proteasome Sensor cells (BD Bioscience, cat. no 631535) were grown in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum and 1% of streptavidin and penicillin. 3T3 L1 pre-adipocytes were obtained from ATCC and differentiated into adipocytes according to a standard protocol (Student et al., 1980) Briefly, confluent cells were treated with 3-isobutyl-1-methylxanthine (IBMX), dexamethasone and insulin combined medium for 2 days. Insulin was added to the medium for an additional 2 days, and then the medium was changed to regular growth medium. Mature adipocytes were used for transfections. Lipid droplets were isolated as described previously (Brasaemle and Wolins, 2006).

Plasmid and siRNA treatments

HeLa and HEK-293 cells were seeded in six-well plates or in two-well chamber slides for 24 hours before transfection. Cells were transfected with 1–2 μg of plasmid DNA using Lipofectamine™ 2000 (Invitrogen) according to the manufacturer's instructions.

A mixture of equal molar amounts of siRNA was used for each siRNA target (OGA-L or OGA-S) at a final concentration of 2 μg/ml with HeLa cells and 1 μg/ml with HEK-293 cells. Cells were transfected with siRNAs using RNAiFect reagent (Qiagen) according to the manufacturer's instructions. Medium was changed 24 hours after transfection and experiments were initiated at 48 hours. These time frames and DNA concentrations were selected from preliminary experiments.

To observe the siRNA-mediated downregulation, HeLa cells were grown on glass coverslips and first transfected with OGA-L–GFP or OGA-S–GFP, followed by the respective siRNA transfection for 48 hours. Cells were fixed with 4% formaldehyde in BSA and examined by fluorescence microscopy to detect the downregulation of GFP-fused protein expression.

Cellular localization of OGA isoforms by confocal microscopy

Differentiated 3T3 L1 mouse adipocytes were cultured in two-well chamber slides or on glass coverslips and infected with adenoviral vectors encoding OGA-L–GFP or OGA-S–GFP at an MOI of 50. HeLa cell culture medium was augmented with 0.4 mM oleic acid (Sigma) to form the lipid droplets. The cells were fixed and stained with perilipin-2, perilipin-1 or LipidTOX (a neutral lipid-specific dye conjugated with Alexa Fluor 568; Invitrogen) and DAPI for nuclear staining. Cells were imaged using a Zeiss Axiovert 200M confocal microscope and processed with Volocity software (Perkin Elmer).

OGA mRNA expression

Total RNA was isolated from HeLa cells using a RNeasy Mini Kit (Qiagen). cDNA was synthesized from 1 μg of total RNA with Omniscript reverse transcriptase (Qiagen) in 20 μl reaction volume according to the enzyme supplier's instructions. Semi-quantitative PCR was performed in a 50 μl reaction mixture containing 5 μl cDNA, and Taq DNA polymerase (TaKaRa). The following primer sequence was used for amplifying OGA-L sense 5′-AATTGAAGAATGGCGGTCAC-3′ and antisense: 5′-CTCTAAAGGCCCAGGGTTCT-3′, and for OGA-S sense: 5′-CCCTGGAGGATTTGCAGTTA-3′ and antisense: 5′-CCTGGTGCACCTACCTAACC-3′; GAPDH served as a control. The reaction mixture was denatured at 94°C for 5 minutes and amplified by 35 cycles under the following conditions: denaturing for 30 seconds at 94°C, annealing for 30 seconds at 60°C and polymerization for 30 seconds at 72°C, followed by a single extension for 5 minutes at 72°C.

Western blot analysis

Proteins were collected using m-PER buffer (Pierce–Thermo Fisher Scientific) containing a protease inhibitor cocktail and phosphatase inhibitors (Roche Applied Sciences), according to the manufacture's protocol. Cell extracts were sonicated three times for 10 seconds each in an ice-cold water bath, centrifuged at 11,000 g for 10 minutes and supernatant was collected. Protein was quantified by the BCA (Pierce–Thermo Fisher Scientific) method. An equal amount of each protein (30–50 μg) was resolved using a 10% NUPAGE gel, and western blot analysis was performed with antibodies specific for O-GlcNAcylated protein (RL2 or CTD 110.6). To study the specificity of the antibody, half of the blot was incubated with 20 mM O-GlcNAc solution together with the CTD110.6 antibody, followed by secondary antibody. Rabbit polyclonal antibody raised against OGA (MGEA5) was obtained from ProteinTech Group (Chicago, IL). Intensity of the antibody-bound proteins was detected using IR dye secondary antibodies, and scanned with an Odyssey infrared imager (LI-COR Biosciences). The band intensity was calculated using Odyssey software and was normalized to actin expression levels.

O-GlcNAcase activity

O-GlcNAcase activity of HeLa cell lysates, transfected with different plasmids (OGA-L–GFP, OGA-S–GFP) and siRNA treatments (control, OGA-L- and OGA-S-specific siRNAs) were assayed using fluorescently labeled FD-GlcNAc substrate (1 mM) as described by Kim et al. (Kim et al., 2006). Cell lysates were prepared in 20 mM Tris-HCl at pH 7.5 with protease inhibitors. The assay was conducted in 0.5 M citrate phosphate pH 6.5 at 37°C for 30 minutes in the presences of 500 mM GalNAc to saturate the GalNAcase activity. Fluorescence was read at 435 nm excitation and 535 nm emission using a Victor2 multi-label counter (Perkin Elmer).

Live cell proteasome activity using ZsGreen Proteasome Sensor cells

To assay proteasome function in living HEK-293 cells, ZsGreen Proteasome Sensor cells (BD Biosciences) were treated with OGA-specific RNAi. The fluorescent protein Zs-ProSensor-1 is a fusion protein consisting of ZsGreen (a green fluorescent protein from Zoanthus sp., a reef coral) and amino acids 410–461 of the mouse ornithine decarboxylase (MODC). This chimeric protein is constitutively degraded by the proteasome in stably transfected HEK-293 cells and accumulates GFP under conditions that alter the activity of the proteasome. HEK-293 ZsGreen Proteasome Sensor cells were treated with siRNAs for the O-GlcNAcase isoforms, and control siRNA. ALLN is a well-characterized inhibitor of proteasome-dependent proteolysis and was used as the reference control. The green fluorescence of the chimeric protein was monitored by microscopy using an Axiovert 200M microscope; micrographs were taken with the same exposure times for all the samples, with GFP filters.

Fluorescence-activated cell sorting (FACS) analysis was performed to quantify GFP expression, which correlated with the quantitative measure of proteasome inactivation. After transfection for 48 hours with siRNAs, HEK-293 ZsGreen Proteasome Sensor cells were trypsinized and centrifuged for 5 minutes at 200 g at 4°C. The cell pellet was washed with PBS and cells were fixed with 4% formaldehyde and resuspended in PBS. Samples were analyzed with a FACS Calibur flow cytometer (Becton-Dickinson) and data were analyzed using CellQuest 3.1 software (Becton-Dickinson). For each sample, 10,000 events were collected.

Proteasome peptidase activity assay

Proteasome activity was assayed in lysates of HEK-293 and HeLa cells using peptide substrates linked to the fluorogenic reporter aminomethylcoumarin (Suc–LLVY–AMC and Boc–LSTR–AMC; Sigma). OGA siRNA-transfected cells cultured in 12-well plates were collected, washed in PBS, lysed in ice-cold buffer A (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 5 mM EDTA, 1% Triton X-100) for 30 minutes and subjected to sonication three times for 15 seconds each. Supernatant was isolated after centrifugation and the protein quantified using the BCA method before analysis of proteasome activity. Aliquots of lysates (50 μl) were suspended in 50 μl of buffer B (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 5 mM EDTA, 4 mM DTT) the reaction was started by adding 100 μl of 100 mM substrate and continued for 90 minutes at 37°C. The reaction was stopped with 800 μl of 1% SDS. Fluorescence intensity was measured (excitation 360 nm, emission 450 nm) using a F2000 fluorescence spectrophotometer (Hitachi). Enzymatic activity was normalized for protein concentration and expressed as percent activity of the lysate treated with scrambled siRNA. Each measurement was carried out using at least three independent dsRNA and plasmid transfections.

Proteasome inhibitor assay

HeLa cells were cultured in six-well plates and transfected with long and short forms of GFP-tagged or HA-tagged plasmids. After 24 hours, ALLN was added at 25 or 50 μM. This experiment was performed with and without 0.4 mM oleic acid treatment for 24 hours. Cells lysates were extracted as described above, and western immunoblot analysis performed with antibodies to GFP or HA and to perilipin-2.

Statistical analysis

Experiments were carried out in duplicate or triplicate and the data collected were normalized to the control. Paired Student's t-tests were used to test for statistical significance.


We are grateful to the Londos laboratory for providing us with antibodies to perilipins 1, 2 and 3, and assistance with the lipid droplet isolation. This research was supported by the Intramural Research Program of the National Institutes of Health, National Institute for Diabetes and Digestive and Kidney Diseases. Deposited in PMC for release after 12 months.


  • Accepted March 24, 2011.


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