Search

Modification of proteins with small ubiquitin-related modifier (SUMO; SUMOylation) is involved in the regulation of various biological processes. Recent studies have demonstrated that noncovalent associations between SUMOylated proteins and co-operative proteins containing SUMO-interacting motifs (SIMs) are important for the spatiotemporal organization of many protein complexes. In this study, we demonstrate that interactions between lamin A, a major component of the nuclear lamina, and SUMO isoforms are dependent on one of the four SIMs (SIM3) resided in lamin A polypeptide in vitro. Live cell imaging and immunofluorescence staining showed that SIM3 is required for accumulation of lamin A on the chromosomes during telophase, and subsequent evaluation of a panel of deletion mutants determined that a 156-amino acid region spanning the carboxyl-terminal Ig-fold domain of lamin A is sufficient for this accumulation. Notably, mutation of SIM3 abrogated the dephosphorylation of mitosis-specific phosphorylation at Ser-22 of lamin A, which normally occurs during telophase, and the subsequent nuclear lamina reorganization. Furthermore, expression of a conjugation-defective SUMO2 mutant, which was previously shown to inhibit endogenous SUMOylation in a dominant-negative manner, also impaired the accumulation of wild type lamin A on telophase chromosomes. These findings suggest that interactions between SIM3 of lamin A and a putative SUMO2-modified protein plays an important role in the reorganization of the nuclear lamina at the end of mitosis.

The microenvironment of cells, which includes basement proteins, shear stress, and extracellular stimuli, should be taken into consideration when examining physiological cell behavior. Although microfluidic devices allow cellular responses to be analyzed with ease at the single-cell level, few have been designed to recover cells. We herein demonstrated that a newly developed microfluidic device helped to improve culture conditions and establish a clonality-validated human pluripotent stem cell line after tracing its growth at the single-cell level. The device will be a helpful tool for capturing various cell types in the human body that have not yet been established in vitro.

Lipid droplet (LD) is a ubiquitous organelle that stores triacylglycerol and other neutral lipids. Perilipin 5 (Plin5), a member of the perilipin protein family that is abundantly expressed in the heart, is essential to protect LDs from attack by lipases, including adipose triglyceride lipase. Plin5 controls heart metabolism and performance by maintaining LDs under physiological conditions. Aberrant lipid accumulation in the heart leads to organ malfunction, or cardiomyopathy. To elucidate the role of Plin5 in a metabolically disordered state and the mechanism of lipid-induced cardiomyopathy, we studied the effects of streptozotocin-induced type 1 diabetes in Plin5-knockout (KO) mice. In contrast to diabetic wild-type mice, diabetic Plin5-KO mice lacked detectable LDs in the heart and did not exhibit aberrant lipid accumulation, excessive reactive oxygen species (ROS) generation, or heart malfunction. Moreover, diabetic Plin5-KO mice exhibited lower heart levels of lipotoxic molecules, such as diacylglycerol and ceramide, than wild-type mice. Membrane translocation of protein kinase C and the assembly of NADPH oxidase 2 complex on the membrane were also suppressed. The results suggest that diabetic Plin5-KO mice are resistant to type 1 diabetes-induced heart malfunction due to the suppression of the diacylglycerol/ceramide-protein kinase C pathway and of excessive ROS generation by NADPH oxidase.

Widely tunable terahertz (THz) waves were successfully generated from 0.5 to 10 THz via difference frequency generation (DFG) in a configurationally locked polyene 2-[3-(4-hydroxystyryl)-5,5-dimethylcyclohex-2-enylidene] malononitrile (OH1) crystal. Potassium titanium oxide phosphate optical parametric oscillator pumped by nanosecond Q-switched Nd:YAG laser was used to generate two waves, which were then used to irradiate OH1 crystal. The maximum energy of the generated THz wave was about 461 pJ/pulse. We investigated the dependency of generated THz energy to the excitation pump power density and OH1 crystal thickness. In addition, we compared the THz energy generated by OH1 crystal to 4-N,N-dimethylamino-4′-N′-methyl-4-stilbazolium tosylate (DAST) crystal using DFG, and we achieved 560 times higher energy using OH1 crystal than DAST crystal at around 1.1 THz.

Summary The regulation of lipolysis in adipocytes involves coordinated actions of many lipid droplet (LD)-associated proteins such as perilipin, hormone sensitive lipase (HSL), adipose triglyceride lipase (ATGL), and its activator protein, CGI-58. Here, we describe the cellular origin and physiological significance of micro LDs (mLDs) that emerge in the cytoplasm during active lipolysis, as well as the roles of key lipolytic proteins on mLDs in differentiated 3T3-L1 adipocytes. Multiplex coherent anti-Stokes Raman scattering (CARS) microscopy demonstrated that mLDs receive the fatty acid (FA) moiety of triglyceride from pre-existing LDs during lipolysis. However, when FA re-esterification was blocked, mLDs did not emerge. Time-lapse imaging of GFP-tagged LD-associated proteins and immunocytochemical analyses showed that particulate structures carrying LD-associated proteins emerged throughout the cells upon lipolytic stimulation, but not when FA re-esterification was blocked. Overall lipolysis, as estimated by glycerol release, was significantly lowered by blocking re-esterification, whereas release of free FAs was enhanced. ATGL was co-immunoprecipitated with CGI-58 from the homogenates of lipolytically stimulated cells. Following CGI-58 knockdown or ATGL inhibition with bromoenol lactone, release of both glycerol and FA was significantly lowered. AICAR, an activator of AMP-activated protein kinase, significantly increased FA release, in accordance with increased expression of ATGL, even in the absence of CGI-58. These results suggest that, besides on the surface of pre-existing central LDs, LD-associated proteins are actively involved in lipolysis on mLDs that are formed by FA re-esterification. Regulation of mLDs and LD-associated proteins may be an attractive therapeutic target against lipid-associated metabolic diseases.

We fabricated an integrated blood cell counting system composed of planar blood preparation and blood cell counting devices. The planar blood preparation and counting devices were made of injection-molded polystyrene. 2 μL and 0.125 μL of whole blood were separated by hydrophobic passive valves and mixed with 50 μL of diluting fluid in the preparation device to accurately dilute the blood 25-fold and 400-fold, as primary and secondary dilution. We found that the hydrophobic fluoric resin surface treatments for the passive valves and the wall of the channels were effective for diluting whole blood. After the diluted blood was transferred into the blood counting devices, the number of blood cells was counted based on the change in electrode conductance at flow rate about 1 μL/s (50–500 cells/s). The coefficient of variation for the reproducibility of the blood cell counting was

Lipid droplets (LDs) are ubiquitous organelles storing neutral lipids, including triacylglycerol (TAG) and cholesterol ester. The properties of LDs vary greatly among tissues, and LD-binding proteins, the perilipin family in particular, play critical roles in determining such diversity. Overaccumulation of TAG in LDs of non-adipose tissues may cause lipotoxicity, leading to diseases such as diabetes and cardiomyopathy. However, the physiological significance of non-adipose LDs in a normal state is poorly understood. To address this issue, we generated and characterized mice deficient in perilipin 5 (Plin5), a member of the perilipin family particularly abundant in the heart. The mutant mice lacked detectable LDs, containing significantly less TAG in the heart. Particulate structures containing another LD-binding protein, Plin2, but negative for lipid staining, remained in mutant mice hearts. LDs were recovered by perfusing the heart with an inhibitor of lipase. Cultured cardiomyocytes from Plin5-null mice more actively oxidized fatty acid than those of wild-type mice. Production of reactive oxygen species was increased in the mutant mice hearts, leading to a greater decline in heart function with age. This was, however, reduced by the administration of N-acetylcysteine, a precursor of an antioxidant, glutathione. Thus, we conclude that Plin5 is essential for maintaining LDs at detectable sizes in the heart, by antagonizing lipase(s). LDs in turn prevent excess reactive oxygen species production by sequestering fatty acid from oxidation and hence suppress oxidative burden to the heart. Background: Perilipin family proteins are important in determining the properties of lipid droplets (LDs). Results: Perilipin 5-deficient mice lack detectable LDs, exhibit enhanced fatty acid oxidation, and suffer increased ROS production in the heart. Conclusion: Perilipin 5 protects the heart from oxidative burden by sequestering fatty acid from excessive oxidation. Significance: These findings may help to increase understanding of the functions of non-adipose LDs.

Chanarin–Dorfman syndrome (CDS) is a rare autosomal recessive disease of lipid metabolism; it is associated with congenital ichthyosis typed as non-bullous congenital ichthyosiform erythroderma (NCIE). CDS is characterized by the presence of an abnormally large number of cytosolic lipid droplets containing triacylglycerol (TG) in various tissues such as the skin, liver, and leukocytes. Mutations in the CGI-58 (also called ABHD5) gene encoding a 39-kDa protein of the α/β hydrolase domain subfamily have been shown to be responsible for this disorder. In adipocytes, CGI-58 is involved in TG degradation on lipid droplets; in doing so, it coordinates with several lipolytic factors including perilipin, a member of the PAT protein family, and ATGL, a putative rate-limiting lipase in adipocytes. In quiescent adipocytes, CGI-58 interacts with perilipin on the surfaces of lipid droplets. Upon hormonal stimulation, CGI-58 facilitates massive lipolysis by activating ATGL. Some CGI-58 mutations found in CDS patients cancel the ability to interact with perilipin or activate ATGL, indicating that the loss of these interactions is physiologically important. However, based on the tissue distributions of these lipolytic factors, there are likely multiple molecular targets of CGI-58 actions. This in turn gives rise to the multiple phenotypes of CDS, such as ichthyosis, liver steatosis, or neurosensory diseases.

Heme acts as a ligand for transcription factors and regulates the expression of several genes. The nuclear receptor retinoid X receptor alpha (RXRalpha) plays important roles in various nuclear receptor-dependent signaling pathways. We here show that heme binds to RXRalpha and impairs its DNA-binding activity. Deletion and mutation studies of RXRalpha revealed that the binding region of hemin corresponded to the ligand binding domain of mouse RXRalpha and cysteine 374 was involved in the binding. The DNA-binding activity using the DR-1 consensus sequence of RXRalpha in electrophoretic mobility shift assays was inhibited by heme. The reporter assay also showed a decrease of RXRalpha-dependent transcriptional activity. It was reported that hemin enhanced the adipocyte differentiation of mouse 3T3-L1 cells, where the functions of several nuclear receptors including RXRalpha and peroxisome proliferator-activated receptor-gamma (PPAR-gamma) are activated. However, the inductions of adipogenic factor mRNAs including PPAR-gamma, fatty acid binding protein-4 and glucose transporter-4 were markedly repressed by heme during adipocyte differentiation. These results suggest that heme causes the impairment of RXRalpha-dependent signal pathways and inhibits the adipocyte differentiation of 3T3-L1 cells.

A lipid droplet (LD)-associated protein, perilipin, is a critical regulator of lipolysis in adipocytes. We previously showed that Comparative Gene Identification-58 (CGI-58), a product of the causal gene of Chanarin-Dorfman syndrome, interacts with perilipin on LDs. In this study, we investigated the function of CGI-58 using RNA interference. Notably, CGI-58 knockdown caused an abnormal accumulation of LDs in both 3T3-L1 preadipocytes and Hepa1 hepatoma cells. CGI-58 knockdown did not influence the differentiation of 3T3-L1 adipocytes but reduced the activity of both basal and cAMP-dependent protein kinase-stimulated lipolysis. In vitro studies showed that CGI-58 itself does not have lipase/esterase activity, but it enhanced the activity of adipose triglyceride lipase. Upon lipolytic stimulation, endogenous CGI-58 was rapidly dispersed from LDs into the cytosol along with small particulate structures. This shift in localization depends on the phosphorylation of perilipin, because phosphorylated perilipin lost the ability to bind CGI-58. During lipolytic activation, LDs in adipocytes vesiculate into micro-LDs. Using coherent anti-Stokes Raman scattering microscopy, we pursued the formation of micro-LDs in single cells, which seemed to occur in cytoplasmic regions distant from the large central LDs. CGI-58 is not required for this process. Thus, CGI-58 facilitates lipolysis in cooperation with perilipin and other factors, including lipases.

Sciadonic acid (20:3 Delta-5,11,14) and juniperonic acid (20:4 Delta-5,11,14,17) are polyunsaturated fatty acids (PUFAs) that lack the Delta-8 double bond of arachidonic acid (20:4 Delta-5,8,11,14) and eicosapentaenoic acid (20:5 Delta-5,8,11,14,17), respectively. Here, we demonstrate that these conifer oil-derived PUFAs are metabolized to essential fatty acids in animal cells. When Swiss 3T3 cells were cultured with sciadonic acid, linoleic acid (18:2 Delta-9,12) accumulated in the cells to an extent dependent on the concentration of sciadonic acid. At the same time, a small amount of 16:2 Delta-7,10 appeared in the cellular lipids. Both 16:2 Delta-7,10 and linoleic acid accumulated in sciadonic acid-supplemented CHO cells, but not in peroxisome-deficient CHO cells. We confirmed that 16:2 Delta-7,10 was effectively elongated to linoleic acid in rat liver microsomes. These results indicate that sciadonic acid was partially degraded to 16:2 Delta-7,10 by two cycles of beta-oxidation in peroxisomes, then elongated to linoleic acid in microsomes. Supplementation of Swiss 3T3 cells with juniperonic acid, an n-3 analogue of sciadonic acid, induced accumulation of alpha-linolenic acid (18:3 Delta-9,12,15) in cellular lipids, suggesting that juniperonic acid was metabolized in a similar manner to sciadonic acid. This PUFA remodeling is thought to be a process that converts unsuitable fatty acids into essential fatty acids required by animals.

Promoters of Drosophila melanogaster genes related to DNA replication, such as those for the 180-kDa catalytic subunit of DNA polymerase α and proliferating cell nuclear antigen (PCNA), contain a common 8-bp palindromic sequence (5′-TATCGATA-3′), named the DNA replication-related element (DRE) (12). These DREs are required for promoter activities both in cultured cells and in flies in vivo (41). We have purified the DRE-binding factor (DREF) from cultured Drosophila cells, consisting of an 86-kDa polypeptide homodimer specifically binding to DRE, and isolated a cDNA (12, 13). The importance of Drosophila DREF in development has been demonstrated from studies using transgenic flies (11, 14, 44). For example, ectopic expression of Drosophila DREF in eye imaginal disc cells behind the morphogenetic furrow, which are normally postmitotic, induced ectopic DNA synthesis and apoptosis and abolished photoreceptor specifications (11). More recently, we and Hyun et al. have succeeded in knocking down Drosophila DREF expression in various tissues (16, 45). Decreased levels of DREF in developing wing and eye imaginal discs were associated with reduction in wing size with smaller cells and drastically aberrant small and rough eyes, respectively. These lines of evidence indicate that the Drosophila DRE/DREF system performs important roles in regulation of cell growth as well as cell proliferation during development. How many and what kind of genes other than those described above are under control of the Drosophila DRE/DREF system? Immunostaining of polytene chromosomes of salivary glands revealed that Drosophila DREF binds to hundreds of loci (8, 10), and recent computational analysis of core promoters in the Drosophila genome showed DRE to be the second most frequent motif apparent in core promoter sequences from −60 to +40, with a frequency higher than those for the TATA box and initiator sequences (4, 24). Already, we and others have demonstrated that the Drosophila DRE/DREF system regulates a number of Drosophila genes involved in DNA replication as well as those involved in cell cycle progression through S (dE2F1) (33) and G2/M (D-raf, D-myb, and cyclin A) phases (25, 29, 34). Despite much progress in understanding the Drosophila DRE/DREF system, little is known about the corresponding mammalian DRE/DREF system. We have identified a human homologue of DREF (hDREF) and a binding consensus sequence [hDRE; 5′-TGTCG(C/T)GA(C/T)A-3′] (26). Our previous study showed that the expression level of hDREF is elevated during the G1-to-S transition and reaches a maximum at S phase in normal human fibroblasts. Moreover, RNA interference experiments targeting hDREF pointed to an important role in cell cycle progression. We also demonstrated that the histone H1 gene carrying an hDRE is regulated by hDREF (26). However, other functions and target genes of hDREF remain to be clarified. The ribosome is a vital organelle which is responsible for protein synthesis in all living organisms. Production of mature ribosomes consisting of rRNAs and ribosomal proteins (RPs) requires a highly coordinated multistep process, and many reports have shown that the initiation of rRNA transcription is tightly linked to cell cycle progression, with synthesis of rRNA increasing during G1 phase, becoming maximal in S and G2 phases, and being repressed during mitosis (7, 20, 22). Similarly, coordinated synthesis of all RP genes during the cell cycle, leading to the precise and equimolar production of the 79 RPs necessary for ribosome biogenesis and translation control, is required to support adequate protein synthesis (37). Despite the obvious importance of RP gene expression for cell proliferation, only a limited number of experimental studies of mammalian RP gene promoter structures and transcriptional regulation have been performed so far. Recent determination of a complete map and nucleotide sequences now allows searches for regulatory elements common to a number of human RP genes, and Perry has identified “box A” [5′-TCTCGCGA(G/T)-3′] as one of the most conserved sequences in RP gene promoters (28). In this report, we document evidence that hDREF is a transcription factor essential for cell proliferation which binds to box A and positively regulates a set of human RP genes. We also show that mammalian RP gene expression is induced during late G1 and S phases and that fluctuation of hDREF expression during the cell cycle is in line with cell cycle-dependent expression of RP genes.

The human transcription factor DNA replication-related element-binding factor (hDREF) is essential for the transcription of a number of housekeeping genes. The mechanisms underlying constitutively active transcription by hDREF were unclear. Here, we provide evidence that hDREF possesses small ubiquitin-like modifier (SUMO) ligase activity and can specifically SUMOylate Mi2α, an ATP-dependent DNA helicase in the nucleosome remodeling and deacetylation complex. Moreover, immunofluorescent staining and biochemical analyses showed that coexpression of hDREF and SUMO-1 resulted in dissociation of Mi2α from chromatin, whereas a SUMOylation-defective Mi2α mutant remained tightly bound to chromatin. Chromatin immunoprecipitation and quantitative RT-PCR analysis demonstrated that Mi2α expression diminished transcription of the ribosomal protein genes, which are positively regulated by hDREF. In contrast, coexpression of hDREF and SUMO-1 suppressed the transcriptional repression by Mi2α. These data indicate that hDREF might incite transcriptional activation by SUMOylating Mi2α, resulting in the dissociation of Mi2α from the gene loci. We propose a novel mechanism for maintaining constitutively active states of a number of hDREF target genes through SUMOylation.

Peroxisome proliferator-activated receptors (PPARs) constitute a subfamily of nuclear receptor superfamily. A wide variety of compounds including hypolipidemic agents, antidiabetic drugs, and long-chain fatty acids are the potential ligands of PPARs. To approach the regulatory mechanisms of PPARs, we studied on two subjects in this work. First, we identified a functional PPAR-binding site in the spacer region between the PEX11alpha and perilipin genes, which are arranged in tandem on the mouse genome. By gene reporter assays and in vivo as well as in vitro binding assays, we show that these genes are regulated tissue-selectively through this common binding site: The PEX11alpha gene is activated by PPARalpha in the liver, whereas the perilipin gene by PPARgamma in the adipose tissue. As the second subject, we found that PPARgamma2 is conjugated with small ubiquitin-related modifier (SUMO) at a specific lysine residue in the amino-terminal region. By site-directed mutagenesis combined with gene reporter assays and sumoylation analyses, we show that sumoylation represses the ligand-independent transactivating function carried by this region, and hence negatively regulates the whole transactivating competence of PPARgamma2. In addition, phosphorylation at a specific site in the amino-terminal region represses the transactivation by PPARgamma2 possibly through enhancing sumoylation.

Human mesenchymal stem cells (hMSCs) are capable of differentiating into several cell types including adipocytes, osteoblasts, and chondrocytes, under appropriate culture conditions. We found that SP600125, an inhibitor of Jun N-terminal kinase (JNK), promoted adipogenesis whereas it repressed osteogenesis from hMSCs. SP600125 increased the expression of adipogenic transcription factors, CCAAT/enhancer-binding proteins alpha and beta as well as peroxisome proliferator-activated receptor gamma2, which suggested that the chemical acted on the early steps of transcriptional regulatory cascade in adipogenesis. A gene reporter assay showed that SP600125 and a dominant negative JNK promoted a transcriptional activity dependent on the cAMP-response element (CRE). Thus, JNK represses adipogenesis from hMSCs probably by, at least in part, inhibiting the transactivating function of CRE-binding protein. Another action of JNK, phosphorylation at Ser(307) of insulin receptor substrate-1, was also predicted to contribute to the repression of adipogenesis.

Lipid droplets (LDs) are a class of ubiquitous cellular organelles that are involved in lipid storage and metabolism. Although the mechanisms of the biogenesis of LDs are still unclear, a set of proteins called the PAT domain family have been characterized as factors associating with LDs. Perilipin, a member of this family, is expressed exclusively in the adipose tissue and regulates the breakdown of triacylglycerol in LDs via its phosphorylation. In this study, we used a yeast two-hybrid system to examine the potential function of perilipin. We found direct interaction between perilipin and CGI-58, a deficiency of which correlated with the pathogenesis of Chanarin-Dorfman syndrome (CDS). Endogenous CGI-58 was distributed predominantly on the surface of LDs in differentiated 3T3-L1 cells, and its expression increased during adipocyte differentiation. Overexpressed CGI-58 tagged with GFP gathered at the surface of LDs and colocalized with perilipin. This interaction seems physiologically important because CGI-58 mutants carrying an amino acid substitution identical to that found in CDS lost the ability to be recruited to LDs. These mutations significantly weakened the binding of CGI-58 with perilipin, indicating that the loss of this interaction is involved in the etiology of CDS. Furthermore, we identified CGI-58 as a binding partner of ADRP, another PAT domain protein expressed ubiquitously, by yeast two-hybrid assay. GFP-CGI-58 expressed in non-differentiated 3T3-L1 or CHO-K1 cells was colocalized with ADRP, and the CGI-58 mutants were not recruited to LDs carrying ADRP, indicating that CGI-58 may also cooperate with ADRP.

SKIP has been described as a transcriptional coregulator as well as a spliceosome component, but the relationship between these functions is not clear. We found that SKIP activated reporter gene expression from the basal promoters of viral origin. SKIP exhibited more prominent effect on the promoters with stronger activities, in an experiment employing a series of reporter constructs carrying different numbers of GC boxes. We also found that SKIP suppressed aberrant splicing at a cryptic splice donor site in the luciferase reporter gene. In addition, SKIP suppressed splicing of an extra intron created by a beta-thalassemia mutation in the human beta-globin gene. In the transfection experiment, an intronless reporter exhibited a higher level of expression, but was less significantly activated by SKIP, than the intron-containing reporter. These results indicate that SKIP affects gene expression by both transcriptional activation and regulation of pre-mRNA splicing.

Most cis-acting regulatory elements have generally been assumed to activate a single nearby gene. However, many genes are clustered together, raising the possibility that they are regulated through a common element. We show here that a single peroxisome proliferator response element (PPRE), located between the mouse PEX11 alpha and perilipin genes, confers on both genes activation by peroxisome proliferator-activated receptor alpha (PPAR alpha) and PPAR gamma. A functional PPRE 8.4 kb downstream of the promoter of PEX11 alpha, a PPAR alpha target gene, was identified by a gene transfection study. This PPRE was positioned 1.9 kb upstream of the perilipin gene and also functioned with the perilipin promoter. In addition, this PPRE, when combined with the natural promoters of the PEX11 alpha and perilipin genes, conferred subtype-selective activation by PPAR alpha and PPAR gamma 2. The PPRE sequence specifically bound to the heterodimer of RXR alpha and PPAR alpha or PPAR gamma 2, as assessed by electrophoretic gel mobility shift assays. Furthermore, tissue-selective binding of PPAR alpha and PPAR gamma to the PPRE was demonstrated in hepatocytes and adipocytes, respectively, by chromatin immunoprecipitation assay. Hence, the expression of these genes is induced through the same PPRE in the liver and adipose tissue, where the two PPAR subtypes are specifically expressed.

Background: Although phospholipase C (PLC)δ1 containing a functional nuclear export signal (NES) is normally localized at the plasma membrane and in the cytoplasm, it shuttles between the nucleus and the cytoplasm. Since nucleocytoplasmic shuttling of a molecule is generally regulated by a balance between its NES and the nuclear localization signal (NLS), we examined whether PLCδ1 contains an NLS sequence. Results: A region corresponding to the C terminus of the X domain and the XY-linker, which contains clusters of basic amino acid residues, was essential for the nuclear import of PLCδ1 in Madin–Darby canine kidney cells. A series of point mutations on lysine residues in this region revealed that K432 and K434 in combination were important for the nuclear import. A short synthetic peptide corresponding to residues 429–442, however, was not able to function as an NLS sequence when they were injected into the cytoplasm in a carrier-conjugated form. Neither a longer peptide equivalent to PLCδ1 412–498 fused to a protein tag consisting of glutathione S-transferase and green fluorescent protein was imported to the nucleus after microinjection into the cytoplasm. Conclusion: The nuclear import of PLCδ1 requires the C-terminus of the X domain, particularly the amino acid residues K432 and K434, and the XY-linker. The region alone, however, cannot serve as a functional NLS. The machinery for nuclear transport may require additional structural component(s) of the enzyme.

Pex6p belongs to the AAA family of ATPases. Its CHO mutant, ZP92, lacks normal peroxisomes but contains peroxisomal membrane remnants, so called peroxisomal ghosts, which are detected with anti–70-kDa peroxisomal membrane protein (PMP70) antibody. No peroxisomal matrix proteins were detected inside the ghosts, but exogenously expressed green fluorescent protein (GFP) fused to peroxisome targeting signal-1 (PTS-1) accumulated in the areas adjacent to the ghosts. Electron microscopic examination revealed that PMP70-positive ghosts in ZP92 were complex membrane structures, rather than peroxisomes with reduced matrix protein import ability. In a typical case, a set of one central spherical body and two layers of double-membraned loops were observed, with endoplasmic reticulum present alongside the outer loop. In the early stage of complementation by PEX6 cDNA, catalase and acyl-CoA oxidase accumulated in the lumen of the double-membraned loops. Biochemical analysis revealed that almost all the peroxisomal ghosts were converted into peroxisomes upon complementation. Our results indicate that 1) Peroxisomal ghosts are complex membrane structures; and 2) The complex membrane structures become import competent and are converted into peroxisomes upon complementation with PEX6.

Mutation of the lamin A gene (LMNA) causes a diverse range of diseases referred to as laminopathies. Because most laminopathies have a dominant inheritance pattern and progress gradually, cultured cells stably expressing mutant lamin A at the same level as endogenous wild-type cells are required for chronological analysis. In this study, we showed that an expression system involving a lentiviral vector that carries the human metallothionein gene basal promoter ensures stable and basal-level expression of proteins and is thus suitable for investigating the properties of lamin A mutants. The small ubiquitin-related modifier (SUMO) modification (SUMOylation)-defective E203G mutant that is associated with familial dilated cardiomyopathy exhibited abnormal subnuclear distribution and inhibited normal localization of WT lamin A in a dominant-negative manner. Low-level and long-term expression of the E203G mutant resulted in multinucleated giant cells, aberrant lipid droplet accumulation in the cytoplasm and premature senescence. Expression of another SUMOylation-defective mutant (K201R) did not induce any phenotypes observed in cells expressing E203G. These results indicate that the E203G mutant may inhibit the normal functions of wild-type lamin A in a dominant-negative manner, but a defect in SUMOylation itself may not be involved in disease pathogenesis.

We established a Chinese hamster ovary cell line having a temperature-sensitive phenotype in peroxisome biogenesis. This mutant (65TS) was produced by transforming a PEX2-defective mutant, Z65, with a mutant PEX2 gene,PEX2 E55K, derived from a patient with infantile Refsum disease, a milder form of peroxisome biogenesis disorder. In 65TS, catalase was found in the cytosol at a nonpermissive temperature (39 °C), but upon the shift to a permissive temperature (33 °C), catalase gradually localized to the structures containing a 70-kDa peroxisomal membrane protein, PMP70. In contrast to catalase, other matrix proteins containing typical peroxisome targeting signals, acyl-CoA oxidase and peroxisomal 3-ketoacyl-CoA thiolase, were co-localized with PMP70 in most cells, even at 39 °C. We found that these structures are partially functional peroxisomes and named them “catalase-less peroxisomes.” Catalase-less peroxisomes were also observed in human fibroblasts from patients with milder forms of peroxisome biogenesis disorder, including the one from which the mutantPEX2 gene was derived. We suggest that these structures are the causes of the milder phenotypes of the patients. Temperature-dependent restoration of the peroxisomes in 65TS occurred even in the presence of cycloheximide, a protein synthesis inhibitor. Thus, we conclude that in 65TS, catalase-less peroxisomes are the direct precursors of peroxisomes.

Peroxisome biogenesis disorders (PBDs) contain various clinical phenotypes; Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD), and infantile Refsum disease (IRD), decreasing in the clinical severity in this order. We found that all IRD cell lines and some NALD lines belonging to several different complementation groups are temperature-sensitive in peroxisome assembly; that is, they lacked catalase-positive peroxisomes at 37 degrees C, but do gain the peroxisomes at 30 degrees C. We identified heterozygous mutations E55K/R119Stop in the PEX2 gene of an IRD patient of complementation group F. The E55K mutation was the direct cause of the temperature-sensitivity because similar phenotypes could be transferred to PEX2-defective CHO cells by transfecting the mutant gene. Thus, temperature-sensitive peroxisome assembly is representative of milder forms of PBDs.

We have found that peroxisome assembly is temperature-sensitive (ts) in mild forms of peroxisome biogenesis disorders (PBDs), that is all infantile Refsum disease (IRD) patients and a few neonatal adrenoleukodystrophy patients of several complementation groups. The number of peroxisomes increased daily in incubation at 30 degrees C in the ts cells. Oxidation of very long-chain fatty acids, processing of acyl-CoA oxidase and dihydroxyacetonephosphate acyltransferase activity also improved after 8 days incubation at 30 degrees C in the IRD fibroblasts. These biochemical functions of the peroxisome did not change at 30 degrees C in Zellweger fibroblasts. Number of peroxisomes gradually decreased after 4 days when the temperature shifted from 30 to 37 degrees C in the ts cells. These results indicate that the biochemical functions of peroxisome are also restored by incubation at 30 degrees C in the mild and ts phenotype of PBDs, and the results will aid to predict the severity and the prognosis of affected children.

Nuclear matrix is a complex intranuclear network supposed to be involved in the various nuclear functions. In order to identify the nuclear matrix proteins, we isolated a cDNA clone from a human placenta cDNA library. This clone was partially represented a known cDNA clone HA1237. HA1237 encoded a 631-amino-acid peptide, which we designated NXP-1. NXP-1 was related to yeast Rad21/Scc1/Mcd1, Xenopus XRAD21, and mouse PW29, and identical with HR21spA isolated from a human testis cDNA library. We developed a polyclonal antibody to the purified NXP-1 bacterially expressed as a fusion protein with GST. Western blot analysis with anti-NXP-1 polyclonal antibody showed nuclear matrix localization of NXP-1 in HeLa cells. Indirect immunofluorescence staining also showed nuclear and nuclear matrix localization of the NXP-1. Results of in vitro binding assays employing nuclear matrix preparations indicated that the N-terminal region (16–128 amino acid) of NXP-1 has an important role in nuclear matrix distribution.

The 70-kDa peroxisomal membrane protein (PMP70) is one of major components of peroxisomal membranes. In rodents, PMP70 is markedly induced by administration of hypolipidemic agents in parallel with peroxisome proliferation and the induction of peroxisomal fatty acid beta-oxidation enzymes. PMP70 is an ATP-binding cassette transporter, identified for the first time in intracellular membranes of eukaryotic cells. The authors' recent studies suggest that PMP70 is synthesized on free polysomes and posttranslationally inserted into peroxisomal membranes, and assembles as dimeric or oligomeric forms on peroxisomal membranes. PMP70 is suggested to be involved in metabolic transport of long-chain acyl-CoA across peroxisomal membranes.

The transactivating function of the A/B region of mouse peroxisome proliferator-activated receptor α (PPARα; NR1C1) was characterized. The truncated version of PPARα lacking the A/B region had 60–70% lower transactivating function than full-length PPARα in both the presence and absence of the peroxisome proliferator ciprofibrate. When tethered to the yeast Gal4 DNA-binding domain, the A/B region exhibited the significant ligand-independent transactivating function, AF-1 activity. The first 44 amino acid residues were necessary for maximal transactivation, and the minimally essential region was further delimited to amino acids 15–44. This region is highly enriched with acidic residues, but mutational analyses showed that the protein structure, rather than the negative charge itself, was important for the AF-1 activity. An α-helical configuration was predicted for this region, and a CD spectrum analysis of the synthetic peptides showed that mutant sequences with higher AF-1 activity have higher helical contents and vice versa. The most active mutant, in which Met31 was replaced with Leu, was ∼5-fold more potent than the wild-type A/B region. These findings indicate that the AF-1 region of PPARα is an acidic activation domain and that the helix-forming property is implicated in the transactivating function.

Peroxisome biogenesis disorders, including Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD) and infantile Refsum disease, are lethal hereditary diseases caused by abnormalities in peroxisomal assembly. To date, 12 genotypes have been identified. We now have evidence that the complete human cDNA encoding Pex13p, an SH3 protein of a docking factor for the peroxisome targeting signal 1 receptor (Pex5p), rescues peroxisomal matrix protein import and its assembly in fibroblasts from PBD patients of complementation group H. In addition, we detected mutations on the human PEX13 cDNA in two patients of group H. A severe phenotype of a ZS patient (H-02) was homozygous for a nonsense mutation, W234ter, which results in the loss of not only the SH3 domain but also the putative transmembrane domain of Pex13p. A more mildly affected NALD patient (H-01), whose fibroblasts showed the temperature-sensitive (TS) phenotype, was homozygous for a missense mutation in the SH3 domain of Pex13p, I326T. This mutant PEX13 cDNA expression in a PEX13-defective CHO mutant showed I326T to be a TS mutation and thus suggested that Pex13p with the I326T mutation in the SH3 domain is stable at 30 degrees C but is somewhat unstable at 37 degrees C.

The PEX6 (peroxisome assembly factor-2, PAF-2) gene encodes a member of the AAA protein (ATPases associated with diverse cellular activities) family and restores peroxisome assembly in fibroblasts from peroxisome biogenesis disorder patients belonging to complementation group C (group 4 in the United States). We have now clarified the genomic DNA structure of human PEX6 and identified mutations in patients from various ethnic groups. The human PEX6 gene consists of 17 exons and 16 introns, spanning about 14kb. The largest exon, exon 1, has at least 952 bp nucleotides. Eleven novel mutations (18 alleles) were identified by direct sequencing of the PEX6 cDNA from 10 patients. All these mutations have been confirmed in the corresponding genomic DNA. There was no common mutation, but an exon skip was identified in two unrelated Japanese patients. Most of the mutations led to premature termination or large deletions of the PEX6 protein and resulted in the most severe peroxisome biogenesis disorder phenotype of Zellweger syndrome. A patient with an atypical Zellweger syndrome had a missense mutation that was shown to disrupt the cell's ability to form peroxisomes. This mutation analysis will aid in understanding the functions of the PEX6 protein in peroxisomal biogenesis. Hum Mutat 13:487-496, 1999.

We thank T. Hashimoto and N. Usuda for anti-human and anti-rat catalase antibodies, respectively, and we thank H. W. Moser, A. B. Moser, R. J. A. Wanders, G. T. N. Besley, B. C. Paton, and A. Poulos for patients' fibroblasts. This work was supported in part by Grants-in-Aid for Scientific Research and Exploratory Research, from the Ministry of Education, Science, Sports and Culture of Japan, and by grants from the Sumitomo Foundation and Uehara Memorial Foundation.

HumanPEX1(HsPEX1) is the causative gene for peroxisome-deficiency disorders such as Zellweger syndrome of complementation group I, encoding the peroxin, Pex1p, a member of AAA family. Pex1p tagged with an epitope flag was expressed in wild-type Chinese hamster ovary (CHO) cell, CHO-K1. Pex1p was localized in the cytoplasm, as assessed by immunofluorescent microscopy. Cell-lysate ofHsPEX1-transfected CHO-K1 was incubated within vitrosynthesized35S-labelled Pex6p, an AAA family peroxin. Immunoprecipitation of Pex1p using anti-Pex1p antibody resulted in concomitant recovery of35S-Pex6p. Conversely,35S-Pex1p was obtained in immunoprecipitate from CHO-K1 expressing human Pex6p, using anti-Pex6p antibody. These results strongly suggest that Pex1p and Pex6p interact with each other.

Ten complementation groups of generalized peroxisome biogenesis disorders (PBD), (excluding rhizomelic chondrodysplasia punctata) have been identified using complementation analysis. Four of the genes involved have been identified using two different methods of (1) genetic functional complementation of peroxisome deficient CHO cell mutants and (2) homology searches for human dbEST, based on yeast genes involved in peroxisome biogenesis (PEX genes). We report here the first identification of a new complementation group which is genetically different from peroxisome deficient CHO mutants. There were no complementations by the human PEX 13 gene. The nature of the related gene is being investigated.

We thank K. Hori for technical assistance, M. Ohara for helpful comments, and K. Kamijo for human PMP70 cDNA and an anti-ALDP antibody. This study was supported in part by a grant-in-aid for scientific research (08670870) from the Ministry of Education, Science, Sports, and Culture of Japan, by a research grant from the National Center of Neurology and Psychiatry of the Ministry of Health and Welfare of Japan, and by a research grant from Ono Medical Research Foundation.

Polymethylene-interrupted (PMI)-polyunsaturated fatty acids (PUFA) are fatty acids present largely in gymnosperm. Sciadonic acid (SciA, 20:3 Δ-5,11,14) and juniperonic acid (JA, 20:4 Δ-5,11,14,17) are typical C20 PMI-PUFA with an isolated double bond at Δ5. Previously, we found that SciA and JA are converted to linoleic acid (LNA) and α-linolenic acid (ΑLA), respectively. The conversion process includes chain-shortening step by peroxisomal β-oxidation for elimination a double bond at Δ5, and subsequent chain-elongation step in microsomes. In this study, we examined the substrate specificity of this metabolism in rodent and human cells. Supplementation of SciA, eicosadienoic acid (EDA, 20:2 Δ-11,14) or JA to CHO-K1 cells (wild type) induced an accumulation of LNA, LNA or ALA, respectively, in cellular lipids. These changes were not observed in the peroxisomes-deficient CHO cells, indicating involvement of peroxisomes in the metabolism. Two types of human cells (MKN74 and HepG2) also converted the C20 PMI-PUFA and EDA to the respective essential fatty acids. In contrast, no chain-shortened metabolite of pinolenic acid (18:3 Δ-5,9,12) was detected in any cell lines tested. From these results, C20 PMI-PUFA and EDA, but not C18 PMI-PUFA, are suggested as being effectively converted to essential fatty acids by the fatty acid remodeling system in rodent and human cells.

Using a probe obtained by PCR amplification, a full-length cDNA encoding squalene synthase was isolated from a mouse liver cDNA library. Its nucleotide sequence had an open reading frame for a 416 amino acid polypeptide (calculated molecular mass, 48 kDa). In vitro transcription of the cDNAfollowed by in vitro translation produced a protein of the expected size. The deduced amino acid sequence was 93%, 88% and 46% identical to those of the rat, human and budding yeast squalene synthases, respectively. Blotting analyses showed that the mRNA is 1.6 kb in size and that less than two copies of the gene are present in the mouse genome. To establish the enzyme activity, the entire coding region was subcloned into an expression plasmid so that it was in frame with the N-terminal region of β-galactosidase. Escherichia coli , which was transformed with the recombinant plasmid, expressed high activity of converting farnesyl diphosphate into squalene.

Cell-free translation products of hepatic free polysomal RNA from a clofibrate-treated rat were incubated at 26 degrees C for 0-60 min with a post-heavy mitochondrial supernatant fraction from normal rat liver. Exogenously added proteinase K-resistant precursor and mature forms of peroxisomal 3-ketoacyl-CoA thiolase were recovered in a particulate fraction and increased with time. Both forms of thiolase cosedimented with peroxisomes, when the proteinase K-treated import reaction mixture was centrifuged in a sucrose density gradient. The in vitro import and processing of thiolase precursors, types A and B, was likewise reproduced with highly purified peroxisomes. These results strongly suggest that the precursor form of 3-ketoacyl-CoA thiolase is translocated into peroxisomes, apparently without tight coupling with proteolytic processing to the mature protein.

The role of the histidine residue at position −17 of the amino-terminal signal peptide of rat peroxisomal 3-ketoacyl-CoA thiolase was studied in vivo , employing site-directed mutagenesis. Among the nine amino acids tested, only glutamine could partially substitute for the histidine. Mutants carrying basic amino acids, arginine and lysine, and hydrophobic residues, leucine and valine, in place of histidine were all translocated to mitochondria, but not to peroxisomes. These results indicate that the signal peptide of the thiolase is recognized by a mechanism totally different from that for the SKL motif, a known peroxisomal targeting signal. Relationship of the thiolase signal peptide to those of mitochondrial proteins is discussed.

We designed a polymerase chain reaction-based strategy to obtain information about the origin and distribution of a newly discovered proliferating cell nuclear antigen (PCNA) homolog. Carrot genomic segments were amplified using degenerate primers for two conserved regions of known PCNA homologs. The genes encoding the larger PCNA as well as typical PCNA contained introns. Thus, unlike processed PCNA pseudogenes in mammals, the larger homolog is not generated through reverse transcription of a typical PCNA mRNA. Moreover, introns of the larger PCNA homolog were positioned at the characteristic sites in plant PCNA genes. Attempts to amplify cDNA for an additional PCNA homolog from mammalian cells have been unsuccessful. These results suggest that the larger PCNA homolog was generated, presumably through gene duplication, after the divergence of the Planta and Animalia.

Genetic heterogeneity in peroxisome-deficient disorders, including Zellweger's cerebrohepatorenal syndrome, neonatal adrenoleukodystrophy and infantile Refsum disease, was investigated. Fibroblasts from 17 patients were fused using polyethylene glycol, cultivated on cover slips, and the formation of peroxisomes in the fused cells was visualized by immunofluorescence staining, using anti-human catalase IgG. Two distinct staining patterns were observed: (1) peroxisomes appeared in the majority of multinucleated cells, and (2) practically no peroxisomes were identified. Single step 12-(1'-pyrene) dodecanoic acid/ultraviolet (P12/UV)-selection confirmed that the former groups were resistant to this selection, most of the surviving cells contained abundant peroxisomes, and the latter cells died. In the complementary matching, [1-14C]lignoceric acid oxidation and the biosynthesis of peroxisomal proteins were also normalized. Five complementation groups were identified. Group A: Zellweger syndrome and infantile Refsum disease; Groups B, C and D: Zellweger syndrome; Group E: Zellweger syndrome, neonatal adrenoleukodystrophy and infantile Refsum disease. We compared these groupings with those of Roscher and identified eight complementation groups. There was no obvious relation between complementation groups and clinical phenotypes. These results indicate that the transport, intracellular processing and function of peroxisomal proteins were normalized in the complementary matching and that at least eight different genes are involved in the formation of normal peroxisomes and in the transport of peroxisomal enzymes.

3-Ketothiolase deficiency (3KTD) stems from a deficiency of mitochondrial acetoacetyl-coenzyme A thiolase (T2). We analyzed the molecular basis of 3KTD in two generations of a family. A boy (patient 2, GK04), his father (patient 1, GK05), his mother, and his brother were studied; three mutant alleles of T2 gene were identified. Patient 1 is a compound heterozygote: one allele has a point mutation of G to A at position 547 on his T2 cDNA, causing Gly150 to Arg substitution of the mature T2 subunit, and the other allele has GT to TT transition at the 5' splice site of intron 8, causing exon 8's skipping of the T2 cDNA. Patient 2 is also a compound heterozygote: one allele inherited from his mother has AG to CG transition at the 3' splice site of intron 10, causing exon 11's skipping of the T2 cDNA, and the other allele derived from patient 1 has the G to A mutation (Gly to Arg). The brother of patient 2 is an obligatory carrier with the mutant allele causing the exon 8 skipping. This report seems to be the first complete molecular definition of 3KTD at the gene level.

To examine the function of the amino-terminal presequence of rat peroxisomal 3-ketoacyl-CoA thiolase precursor, fusion proteins of various amino-terminal regions of the precursor with non-peroxisomal enzymes were expressed in cultured mammalian cells. On immunofluorescence microscopy, all constructs carrying the presequence part exhibited punctate patterns of distribution, identical with that of catalase, a peroxisomal marker. Proteins lacking all or a part of the prepiece were found in the cytosol. These results indicate that the presequence of the thiolase has sufficient information for peroxisomal targeting.

To examine 3-ketothiolase deficiency at the gene level, we analyzed the structure of the human mitochondrial acetoacetyl-CoA thiolase (MAT; EC 2.3.1.9)-encoding gene ( MAT ). From the genomic library of a normal subject in λEMBL3, we isolated seven overlapping clones covering the entire length of MAT and the structural organization was determined. The gene spans approx. 27 kb and contains twelve exons interrupted by eleven introns. The 5′-flanking region of the gene lacks a conventional TATA box, but is G+C-rich and contains two CAAT boxes. Included are a putative binding site for the transcription factor, Sp1, and sequences resembling the binding sites of several other transcription factors, all features characteristics of housekeeping genes. A CAT assay revealed that a 101-bp DNA fragment immediately upstream from the cap site has promoter activity, and suggested that a DNA fragment from bp −8 to −102 probably contains a negative regulatory element(s).

The molecular basis of 3-ketothiolase deficiency (3KTD) was examined in a 3KTD family. Immunochemical analyses showed that mitochondrial acetoacetyl-CoA thiolase (T2) biosynthesized in the patient's fibroblasts (GK06) was unstable and that the parents and brother were obligatory carriers of 3KTD. When sequencing the PCR-amplified patient's T2cDNA, we noted a G to A replacement which caused 347 Ala to Thr substitution of the mature T2 subunit. Transfection analysis revealed that this substitution resulted in an instability of the T2 protein. Analyses of the T2 cDNA and gene of the family indicated that the patient was a compound heterozygote; the allele that derived from the mother had a point mutation ( 347 Ala to Thr) and the other allele from the father has a mutation which would abolish the T2 gene expression. This report is apparently the first definition of a mutant allele for 3KTD, at the gene level.

Peroxisome biogenesis disorders (PBD) comprise three phenotypes including Zellweger syndrome (ZS) (the most severe), neonatal adrenoleucodystrophy, and infantile Refsum disease (IRD) (the most mild), and can be classified into at least 12 genetic complementation groups, which are not predictive of the phenotypes. Several pathogenic genes for PBD groups have been identified, but the relationship between the defective gene products and phenotypic heterogeneity has remained unclear. We identified a mutation in the PEX2 gene in an IRD patient with compound heterozygosity for a missense mutation and the known nonsense mutation detected in ZS patients. In transfection experiments using the peroxisome deficient CHO mutant, Z65 with a nonsense mutation in the PEX2 gene, we noted the E55K mutation had mosaic activities of peroxisomal protein import machinery and residual activities of peroxisomal functions, including dihydroxyacetone phosphate acyltransferase and β oxidation of very long chain fatty acids. The nonsense mutation severely affects these peroxisomal functions as well as the protein import. These data suggest that allelic heterogeneity of the PEX gene affects the peroxisomal protein import and functions and regulates the clinical severity in PBD. Keywords: Zellweger syndrome; infantile Refsum disease; PEX gene; mosaic

White adipocytes are unique in that they contain large unilocular lipid droplets that occupy most of the cytoplasm. To identify genes involved in the maintenance of mature adipocytes, we expressed dominant-negative PPARγ in 3T3-L1 cells and performed a microarray screen. The fat-specific protein of 27 kDa (FSP27) was strongly downregulated in this context. FSP27 expression correlated with induction of differentiation in cultured preadipocytes, and the protein localized to lipid droplets in murine white adipocytes in vivo. Ablation of FSP27 in mice resulted in the formation of multilocular lipid droplets in these cells. Furthermore, FSP27-deficient mice were protected from diet-induced obesity and insulin resistance and displayed an increased metabolic rate due to increased mitochondrial biogenesis in white adipose tissue (WAT). Depletion of FSP27 by siRNA in murine cultured white adipocytes resulted in the formation of numerous small lipid droplets, increased lipolysis, and decreased triacylglycerol storage, while expression of FSP27 in COS cells promoted the formation of large lipid droplets. Our results suggest that FSP27 contributes to efficient energy storage in WAT by promoting the formation of unilocular lipid droplets, thereby restricting lipolysis. In addition, we found that the nature of lipid accumulation in WAT appears to be associated with maintenance of energy balance and insulin sensitivity.

Perilipin is a protein localized on lipid droplet surfaces in adipocytes and steroidogenic cells, playing a central role in regulated lipolysis. Expression of the perilipin gene is markedly induced during adipogenesis. We found that transcription from the perilipin gene promoter is activated by an orphan nuclear receptor, estrogen receptor-related receptor (ERR)alpha. A response element to this receptor was identified in the promoter region by a gene reporter assay, the electrophoretic-gel mobility-shift assay and the chromatin immunoprecipitation assay. Peroxisome proliferator-activated receptor gamma coactivator (PGC)-1alpha enhanced, whereas small heterodimer partner (SHP) repressed, the transactivating function of ERRalpha on the promoter. Thus, the perilipin gene expression is regulated by a transcriptional network controlling energy metabolism, substantiating the functional importance of perilipin in the maintenance of body energy balance.