ABHD5 stimulates PNPLA1-mediated ω-O-acylceramide biosynthesis essential for a functional skin permeability barrier
2018; Elsevier BV; Volume: 59; Issue: 12 Linguagem: Inglês
10.1194/jlr.m089771
ISSN1539-7262
AutoresBenedikt Kien, Susanne Grond, Guenter Haemmerle, Achim Lass, Thomas O. Eichmann, Franz P.W. Radner,
Tópico(s)Plant Reproductive Biology
ResumoMutations in the genes coding for patatin-like phospholipase domain-containing 1 (PNPLA1) and α/β-hydrolase domain-containing 5 (ABHD5), also known as comparative gene identification 58, are causative for ichthyosis, a severe skin barrier disorder. Individuals with mutations in either of these genes show a defect in epidermal ω-O-acylceramide (AcylCer) biosynthesis, suggesting that PNPLA1 and ABHD5 act in the same metabolic pathway. In this report, we identified ABHD5 as a coactivator of PNPLA1 that stimulates the esterification of ω-hydroxy ceramides with linoleic acid for AcylCer biosynthesis. ABHD5 interacts with PNPLA1 and recruits the enzyme to its putative triacylglycerol substrate onto cytosolic lipid droplets. Conversely, alleles of ABHD5 carrying point mutations associated with ichthyosis in humans failed to accelerate PNPLA1-mediated AcylCer biosynthesis. Our findings establish an important biochemical function of ABHD5 in interacting with PNPLA1 to synthesize crucial epidermal lipids, emphasizing the significance of these proteins in the formation of a functional skin permeability barrier. Mutations in the genes coding for patatin-like phospholipase domain-containing 1 (PNPLA1) and α/β-hydrolase domain-containing 5 (ABHD5), also known as comparative gene identification 58, are causative for ichthyosis, a severe skin barrier disorder. Individuals with mutations in either of these genes show a defect in epidermal ω-O-acylceramide (AcylCer) biosynthesis, suggesting that PNPLA1 and ABHD5 act in the same metabolic pathway. In this report, we identified ABHD5 as a coactivator of PNPLA1 that stimulates the esterification of ω-hydroxy ceramides with linoleic acid for AcylCer biosynthesis. ABHD5 interacts with PNPLA1 and recruits the enzyme to its putative triacylglycerol substrate onto cytosolic lipid droplets. Conversely, alleles of ABHD5 carrying point mutations associated with ichthyosis in humans failed to accelerate PNPLA1-mediated AcylCer biosynthesis. Our findings establish an important biochemical function of ABHD5 in interacting with PNPLA1 to synthesize crucial epidermal lipids, emphasizing the significance of these proteins in the formation of a functional skin permeability barrier. The skin forms a permeability barrier between the external environment and the individual's internal milieu, thereby protecting the individual from invasion of pathogens and harmful substances such as chemicals and allergens as well as from the loss of water and electrolytes (1.Madison K.C. Barrier function of the skin: "la raison d'etre" of the epidermis.J. Invest. Dermatol. 2003; 121: 231-241Abstract Full Text Full Text PDF PubMed Scopus (812) Google Scholar, 2.Proksch E. Brandner J.M. Jensen J. The skin: an indispensable barrier.Exp. Dermatol. 2008; 17: 1063-1072Crossref PubMed Scopus (1172) Google Scholar). This barrier resides in the stratum corneum (SC), the outermost layer of the epidermis, and consists of terminally differentiated keratinocytes (corneocytes) that are embedded in a lipid-enriched matrix consisting of ceramides, cholesterol, and nonesterified FAs (3.Feingold K.R. Thematic review series: skin lipids. The role of epidermal lipids in cutaneous permeability barrier homeostasis.J. Lipid Res. 2007; 48: 2531-2546Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, 4.Feingold K.R. Elias P.M. Role of lipids in the formation and maintenance of the cutaneous permeability barrier.Biochim. Biophys. Acta. 2014; 1841: 280-294Crossref PubMed Scopus (242) Google Scholar). To provide permeability barrier function, the extracellular lipids form lamellar membranes in the SC, which requires a scaffold on the external surface of the corneocytes consisting of protein-bound ultra-long-chain (ULC) ω-hydroxy ceramides known as the corneocyte-bound lipid envelope (CLE) (5.Elias P.M. Gruber R. Crumrine D. Menon G. Williams M.L. Wakefield J.S. Holleran W.M. Uchida Y. Formation and functions of the corneocyte lipid envelope (CLE).Biochim. Biophys. Acta. 2014; 1841: 314-318Crossref PubMed Scopus (100) Google Scholar). The formation of this hydrophobic lipid monolayer largely depends on the presence of ω-O-acylceramides (AcylCers) in the SC. The biosynthesis of these hydrophobic barrier lipids occurs in the stratum granulosum by condensation of three different carbon chains, a long-chain base (mainly sphingosine), an ω-hydroxylated ULC FA (28–36 carbon atoms), and linoleic acid (Fig. 1A), and requires the action of at least five different enzymes (Fig. 1B) (6.Uchida Y. Holleran W.M. Omega-O-acylceramide, a lipid essential for mammalian survival.J. Dermatol. Sci. 2008; 51: 77-87Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 7.Akiyama M. Corneocyte lipid envelope (CLE), the key structure for skin barrier function and ichthyosis pathogenesis.J. Dermatol. Sci. 2017; 88: 3-9Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). For CLE formation, AcylCers are transferred to the external SC where the ω-esterified linoleoyl moiety is further processed, resulting in ULC ω-hydroxy ceramides covalently bound to specific amino acid residues of structural proteins of the corneocytes (8.Krieg P. Fürstenberger G. The role of lipoxygenases in epidermis.Biochim. Biophys. Acta. 2014; 1841: 390-400Crossref PubMed Scopus (83) Google Scholar, 9.Muñoz-Garcia A. Thomas C.P. Keeney D.S. Zheng Y. Brash A.R. The importance of the lipoxygenase-hepoxilin pathway in the mammalian epidermal barrier.Biochim. Biophys. Acta. 2014; 1841: 401-408Crossref PubMed Scopus (82) Google Scholar). Mutations in genes that are involved in either the synthesis, secretion, or processing of barrier lipids lead to a defective skin permeability barrier and concomitantly to the pathophysiology of ichthyosis (10.Elias P.M. Williams M.L. Holleran W.M. Jiang Y.J. Schmuth M. Pathogenesis of permeability barrier abnormalities in the ichthyoses: inherited disorders of lipid metabolism.J. Lipid Res. 2008; 49: 697-714Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 11.Oji V. Tadini G. Akiyama M. Blanchet Bardon C. Bodemer C. Bourrat E. Coudiere P. Digiovanna J.J. Elias P. Fischer J. et al.Revised nomenclature and classification of inherited ichthyoses: Results of the First Ichthyosis Consensus Conference in Sorze 2009.J. Am. Acad. Dermatol. 2010; 63: 607-641Abstract Full Text Full Text PDF PubMed Scopus (527) Google Scholar). This rare scaling disorder is characterized by epidermal hyperplasia that results in the formation of multiple layers of corneocytes (hyperkeratosis) and abnormal desquamation leading to the detachment of large SC flakes (10.Elias P.M. Williams M.L. Holleran W.M. Jiang Y.J. Schmuth M. Pathogenesis of permeability barrier abnormalities in the ichthyoses: inherited disorders of lipid metabolism.J. Lipid Res. 2008; 49: 697-714Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 11.Oji V. Tadini G. Akiyama M. Blanchet Bardon C. Bodemer C. Bourrat E. Coudiere P. Digiovanna J.J. Elias P. Fischer J. et al.Revised nomenclature and classification of inherited ichthyoses: Results of the First Ichthyosis Consensus Conference in Sorze 2009.J. Am. Acad. Dermatol. 2010; 63: 607-641Abstract Full Text Full Text PDF PubMed Scopus (527) Google Scholar, 12.Traupe H. Fischer J. Oji V. Nonsyndromic types of ichthyoses - an update.J. Dtsch. Dermatol. Ges. 2014; 12: 109-121PubMed Google Scholar, 13.Schmuth M. Gruber R. Elias P.M. Williams M.L. Ichthyosis update: towards a function-driven model of pathogenesis of the disorders of cornification and the role of corneocyte proteins in these disorders.Adv. Dermatol. 2007; 23: 231-256Crossref PubMed Scopus (75) Google Scholar, 14.Schmuth M. Martinz V. Janecke A.R. Fauth C. Schossig A. Zschocke J. Gruber R. Inherited ichthyoses/generalized Mendelian disorders of cornification.Eur. J. Hum. Genet. 2013; 21: 123-133Crossref PubMed Scopus (61) Google Scholar). Although ichthyoses always display this visible scaling, they are phenotypically and genetically extremely heterogeneous. Since the first ichthyosis consensus conference (11.Oji V. Tadini G. Akiyama M. Blanchet Bardon C. Bodemer C. Bourrat E. Coudiere P. Digiovanna J.J. Elias P. Fischer J. et al.Revised nomenclature and classification of inherited ichthyoses: Results of the First Ichthyosis Consensus Conference in Sorze 2009.J. Am. Acad. Dermatol. 2010; 63: 607-641Abstract Full Text Full Text PDF PubMed Scopus (527) Google Scholar), the various forms of the disease are now classified into nonsyndromic ichthyoses, in which the disease is limited to the skin, and syndromic ichthyoses, where the disorder additionally affects other tissues and organs. Humans carrying mutant alleles of the lipolytic coactivator α/β-hydrolase domain-containing 5 (ABHD5), also designated as comparative gene identification 58, develop neutral lipid storage disease with ichthyosis [NLSDI; also referred to as Chanarin-Dorfman syndrome (OMIM: 275630)] (15.Lefèvre C. Jobard F. Caux F. Bouadjar B. Karaduman A. Heilig R. Lakhdar H. Wollenberg A. Verret J.L. Weissenbach J. et al.Mutations in CGI-58, the gene encoding a new protein of the esterase/lipase/thioesterase subfamily, in Chanarin-Dorfman syndrome.Am. J. Hum. Genet. 2001; 69: 1002-1012Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar, 16.Radner F.P.W. Fischer J. The important role of epidermal triacylglycerol metabolism for maintenance of the skin permeability barrier function.Biochim. Biophys. Acta. 2014; 1841: 409-415Crossref PubMed Scopus (41) Google Scholar). This rare genetic disorder belongs to the subgroup of syndromic ichthyoses that also affect lipid and energy homeostasis in the liver and brain. In mice, the phenotype of ABHD5 deficiency is even more severe, leading to premature death soon after birth due to increased transepidermal water loss and desiccation (17.Radner F.P.W. Streith I.E. Schoiswohl G. Schweiger M. Kumari M. Eichmann T.O. Rechberger G. Koefeler H.C. Eder S. Schauer S. et al.Growth retardation, impaired triacylglycerol catabolism, hepatic steatosis, and lethal skin barrier defect in mice lacking comparative gene identification-58 (CGI-58).J. Biol. Chem. 2010; 285: 7300-7311Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 18.Grond S. Radner F.P.W. Eichmann T.O. Kolb D. Grabner G.F. Wolinski H. Gruber R. Hofer P. Heier C. Schauer S. et al.Skin barrier development depends on CGI-58 protein expression during late-stage keratinocyte differentiation.J. Invest. Dermatol. 2017; 137: 403-413Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). ABHD5 is known as a coactivating protein required for the stimulation of the hydrolytic activity of adipose triglyceride lipase (ATGL; also known as patatin-like phospholipase domain-containing 2). ATGL is a key enzyme in the catabolism of intracellular, nonlysosomal triacylglycerol (TAG) depositions that are present in virtually all tissues of the body (19.Lass A. Zimmermann R. Haemmerle G. Riederer M. Schoiswohl G. Schweiger M. Kienesberger P. Strauss J.G. Gorkiewicz G. Zechner R. Adipose triglyceride lipase-mediated lipolysis of cellular fat stores is activated by CGI-58 and defective in Chanarin-Dorfman Syndrome.Cell Metab. 2006; 3: 309-319Abstract Full Text Full Text PDF PubMed Scopus (676) Google Scholar, 20.Lass A. Zimmermann R. Oberer M. Zechner R. Lipolysis - a highly regulated multi-enzyme complex mediates the catabolism of cellular fat stores.Prog. Lipid Res. 2011; 50: 14-27Crossref PubMed Scopus (451) Google Scholar). Remarkably, humans and mice with mutant ATGL alleles also suffer from neutral lipid storage disease (OMIM: 610717) but show normal skin development and function, which indicates that ABHD5 exerts an ATGL-independent role in skin physiology (17.Radner F.P.W. Streith I.E. Schoiswohl G. Schweiger M. Kumari M. Eichmann T.O. Rechberger G. Koefeler H.C. Eder S. Schauer S. et al.Growth retardation, impaired triacylglycerol catabolism, hepatic steatosis, and lethal skin barrier defect in mice lacking comparative gene identification-58 (CGI-58).J. Biol. Chem. 2010; 285: 7300-7311Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 21.Fischer J. Lefèvre C. Morava E. Mussini J-M. Laforêt P. Negre-Salvayre A. Lathrop M. Salvayre R. The gene encoding adipose triglyceride lipase (PNPLA2) is mutated in neutral lipid storage disease with myopathy.Nat. Genet. 2007; 39: 28-30Crossref PubMed Scopus (368) Google Scholar). Functional analyses of ABHD5-deficient skin from mice and humans revealed a lack of AcylCer synthesis, resulting in defective CLE formation (17.Radner F.P.W. Streith I.E. Schoiswohl G. Schweiger M. Kumari M. Eichmann T.O. Rechberger G. Koefeler H.C. Eder S. Schauer S. et al.Growth retardation, impaired triacylglycerol catabolism, hepatic steatosis, and lethal skin barrier defect in mice lacking comparative gene identification-58 (CGI-58).J. Biol. Chem. 2010; 285: 7300-7311Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 18.Grond S. Radner F.P.W. Eichmann T.O. Kolb D. Grabner G.F. Wolinski H. Gruber R. Hofer P. Heier C. Schauer S. et al.Skin barrier development depends on CGI-58 protein expression during late-stage keratinocyte differentiation.J. Invest. Dermatol. 2017; 137: 403-413Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 22.Uchida Y. Cho Y. Moradian S. Kim J. Nakajima K. Crumrine D. Park K. Ujihara M. Akiyama M. Shimizu H. et al.Neutral lipid storage leads to acylceramide deficiency, likely contributing to the pathogenesis of Dorfman-Chanarin syndrome.J. Invest. Dermatol. 2010; 130: 2497-2499Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). In parallel, mutant skin accumulated free extractable ULC ω-hydroxy ceramides, the precursor lipids for AcylCer formation, which indicates a role of ABHD5 in the final step of AcylCer biosynthesis (Fig. 1B) (18.Grond S. Radner F.P.W. Eichmann T.O. Kolb D. Grabner G.F. Wolinski H. Gruber R. Hofer P. Heier C. Schauer S. et al.Skin barrier development depends on CGI-58 protein expression during late-stage keratinocyte differentiation.J. Invest. Dermatol. 2017; 137: 403-413Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Important progress on elucidating the ATGL-independent function of ABHD5 in epidermal lipid metabolism and skin barrier formation was made when we identified mutations in the ATGL-homologous gene patatin-like phospholipase domain-containing 1 (PNPLA1) that were linked to ichthyosis development in humans and golden retriever dogs (23.Grall A. Guaguère E. Planchais S. Grond S. Bourrat E. Hausser I. Hitte C. Le Gallo M. Derbois C. Kim G.J. et al.PNPLA1 mutations cause autosomal recessive congenital ichthyosis in golden retriever dogs and humans.Nat. Genet. 2012; 44: 140-147Crossref PubMed Scopus (177) Google Scholar). Functional analyses of affected skin demonstrated that humans and mice with defective PNPLA1 function develop an identical defect in AcylCer biosynthesis, as shown for ABHD5 deficiency (24.Grond S. Eichmann T.O. Dubrac S. Kolb D. Schmuth M. Fischer J. Crumrine D. Elias P.M. Haemmerle G. Zechner R. et al.PNPLA1 deficiency in mice and humans leads to a defect in the synthesis of omega-O-acylceramides.J. Invest. Dermatol. 2017; 137: 394-402Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 25.Hirabayashi T. Anjo T. Kaneko A. Senoo Y. Shibata A. Takama H. Yokoyama K. Nishito Y. Ono T. Taya C. et al.PNPLA1 has a crucial role in skin barrier function by directing acylceramide biosynthesis.Nat. Commun. 2017; 8: 14609Crossref PubMed Scopus (89) Google Scholar, 26.Pichery M. Huchenq A. Sandhoff R. Severino-Freire M. Zaafouri S. Opálka L. Levade T. Soldan V. Bertrand-Michel J. Lhuillier E. et al.PNPLA1 defects in patients with autosomal recessive congenital ichthyosis and KO mice sustain PNPLA1 irreplaceable function in epidermal omega-O-acylceramide synthesis and skin permeability barrier.Hum. Mol. Genet. 2017; 26: 1787-1800Crossref PubMed Scopus (37) Google Scholar). PNPLA1 mutant epidermis lacked AcylCer and accumulated ULC ω-hydroxy ceramides, leading to a severe dysfunction of the skin permeability barrier in affected individuals. In line with these observations, PNPLA1 has recently been suggested to act as transacylase, utilizing TAG as an acyl donor for the synthesis of AcylCer (27.Ohno Y. Kamiyama N. Nakamichi S. Kihara A. PNPLA1 is a transacylase essential for the generation of the skin barrier lipid ω-O-acylceramide.Nat. Commun. 2017; 8: 14610Crossref PubMed Scopus (78) Google Scholar). However, the measured transacylase activity of PNPLA1 was relatively low in in vitro assays, suggesting that PNPLA1 requires additional proteins to reach full enzymatic activity similar to what has been demonstrated for the homologous protein ATGL that is activated by ABHD5. Here, we show that ABHD5 interacts with PNPLA1 and stimulates AcylCer biosynthesis. Human and murine cDNAs were prepared from total RNA obtained from murine epidermis or white adipose tissue or from in vitro differentiated human primary keratinocytes as described previously (28.Radner F.P.W. Marrakchi S. Kirchmeier P. Kim G-J. Ribierre F. Kamoun B. Abid L. Leipoldt M. Turki H. Schempp W. et al.Mutations in CERS3 cause autosomal recessive congenital ichthyosis in humans.PLoS Genet. 2013; 9: e1003536Crossref PubMed Scopus (122) Google Scholar) using SuperScript Reverse Transcriptase protocol (Invitrogen Life Technologies, Carlsbad, CA). Sequences containing the complete open-reading frame of human PNPLA1, FA elongase-4 (ELOVL4), cytochrome P450 family 4 subfamily F member 22 (CYP4F22), ceramide synthase 3 (CERS3), and murine Pnpla1, Abhd5, and Atgl were amplified by PCR from respective cDNA using Phusion High-Fidelity DNA Polymerase (Finnzymes, Espoo, Finland) and the gene-specific primer pairs listed in supplemental Table S1. PCR amplification products were ligated to compatible restriction sites of the following eukaryotic expression vectors: pcDNA4/HisMax (Invitrogen Life Technologies), pFLAG-CMV-5.1 (Sigma-Aldrich, St Louis, MO), pEYFP-C1 (Takara Bio USA, Mountain View, CA), pECFP-C1 (Takara Bio USA), or pLVX-ultra-IRES-Puro (see below). Human ABHD5, murine Abhd5, and Pnpla1 were cloned into pcDNA4/HisMax vector as described previously (19.Lass A. Zimmermann R. Haemmerle G. Riederer M. Schoiswohl G. Schweiger M. Kienesberger P. Strauss J.G. Gorkiewicz G. Zechner R. Adipose triglyceride lipase-mediated lipolysis of cellular fat stores is activated by CGI-58 and defective in Chanarin-Dorfman Syndrome.Cell Metab. 2006; 3: 309-319Abstract Full Text Full Text PDF PubMed Scopus (676) Google Scholar, 23.Grall A. Guaguère E. Planchais S. Grond S. Bourrat E. Hausser I. Hitte C. Le Gallo M. Derbois C. Kim G.J. et al.PNPLA1 mutations cause autosomal recessive congenital ichthyosis in golden retriever dogs and humans.Nat. Genet. 2012; 44: 140-147Crossref PubMed Scopus (177) Google Scholar). A control pcDNA4/HisMax vector expressing β-galactosidase (β-gal) was provided by the manufacturer (Invitrogen Life Technologies). The point mutations in ABHD5, c.389A>C (p.Gln130Pro), c.778G>A (p.Glu260Lys), and c.568C>T (p.Gln190Ter), were generated as described previously (19.Lass A. Zimmermann R. Haemmerle G. Riederer M. Schoiswohl G. Schweiger M. Kienesberger P. Strauss J.G. Gorkiewicz G. Zechner R. Adipose triglyceride lipase-mediated lipolysis of cellular fat stores is activated by CGI-58 and defective in Chanarin-Dorfman Syndrome.Cell Metab. 2006; 3: 309-319Abstract Full Text Full Text PDF PubMed Scopus (676) Google Scholar). Sequence analyses were performed at Microsynth AG (Balgach, Switzerland). HEK 293T cells (ATCC CRL-3216) or COS-7 cells (ATCC CRL-1651) were cultivated in DMEM (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% FBS (Sigma-Aldrich) on cell culture dishes precoated with 0.1% gelatin (Sigma-Aldrich) in a standard humidified 7% CO2 atmosphere at 37°C. Cells were transfected with 2.5 µg plasmid DNA using Metafectene (Biontex GmbH, Munich, Germany) according to the manufacturer's instructions. HEK 293T cells were transfected with plasmids as indicated. Twenty-four hours after transfection, culture medium was changed to DMEM without FBS supplementation. After 30 min of incubation, cells were cultivated in DMEM supplemented with 20 µM nervonic acid, 10 µM linoleic acid, 2 µM sphingosine, and 10 µM [1-14C]linoleic acid (ARC 0294; American Radiolabeled Chemicals, St. Louis, MO) or 25 nM [3-3H]sphingosine (ART 0490; American Radiolabeled Chemicals) at 37°C for 6 h. FAs were complexed to essentially FA-free BSA (Sigma-Aldrich) at a molar FA-BSA ratio of 3:1. After washing cells twice with PBS, lipids were extracted three times with chloroform-methanol-glacial acetic acid (66/33/1; v/v/v). Phase separation was obtained by the addition of 1/5 vol. water and centrifugation at 1,400 g at room temperature for 5 min. The lower organic phase containing the lipids was collected and dried under a stream of nitrogen. Remaining cellular proteins were solubilized in 0.3 N NaOH and 0.1% SDS at 65°C overnight, and protein content was determined using Pierce BCA reagent (Thermo Fisher Scientific) and BSA as a standard. Lipids corresponding to equal amounts of protein were separated by normal-phase TLC as described previously (24.Grond S. Eichmann T.O. Dubrac S. Kolb D. Schmuth M. Fischer J. Crumrine D. Elias P.M. Haemmerle G. Zechner R. et al.PNPLA1 deficiency in mice and humans leads to a defect in the synthesis of omega-O-acylceramides.J. Invest. Dermatol. 2017; 137: 394-402Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Radiolabeled lipids on TLC plates were detected by spraying a fluorographic reagent containing scintillation cocktail (Rotiszint; Carl Roth GmbH, Karlsruhe, Germany), methanol, and water (4/1/1; v/v/v), followed by exposure to a light-sensitive film (Amersham Hyperfilm ECL; GE Healthcare, Chicago, IL) at −80°C for 24–48 h. For the identification of the lipid spots corresponding to AcylCer, we radiolabeled lipid extracts from wild-type and PNPLA1-deficient epidermis (the latter of which is known to lack AcylCer) with [1-14C]linoleic acid as described previously (24.Grond S. Eichmann T.O. Dubrac S. Kolb D. Schmuth M. Fischer J. Crumrine D. Elias P.M. Haemmerle G. Zechner R. et al.PNPLA1 deficiency in mice and humans leads to a defect in the synthesis of omega-O-acylceramides.J. Invest. Dermatol. 2017; 137: 394-402Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar) and used them as lipid standards. To obtain homogenates containing radiolabeled linoleic acid donor and ULC ω-hydroxy ceramide acceptor substrate for in vitro AcylCer synthesis assays, HEK 293T cells were transfected with a mammalian expression plasmid encoding ELOVL4, CYP4F22, and CERS3 and labeled with [1-14C]linoleic acid as described above. In parallel, cells were transfected with a PNPLA1 or ABHD5 expression plasmid but were not incubated with radiolabeled linoleic acid. After 24 h, cells were scraped off culture dishes, washed three times with PBS, and disrupted in buffer A (0.25 M sucrose, 1 mM EDTA, 1 mM dithiothreitol, 20 mg/ml leupetine, 2 mg/ml antipain, 1 mg/ml pepstatin, pH 7.0) by sonication (Virsonic 475; Virtis, Gardiner, NJ). After the removal of nuclei and unbroken cells by centrifugation at 1,000 g and 4°C for 5 min, the protein concentration of cell homogenates was determined using Bradford reagent (Bio-Rad, Hercules, CA) and BSA as a standard. To determine the in vitro AcylCer synthesis activity of PNPLA1 and ABHD5, 100 µg protein of respective cell homogenates were mixed with equal amounts of homogenates containing lipid acceptor and radiolabeled linoleic acid donor substrate in a total assay buffer volume of 240 µl (50 mM HEPES/NaOH pH 7.4, 150 mM NaCl, 10% glycerol). After incubation in a water bath at 37°C for 60 min, the reaction was stopped by adding 1.2 ml chloroform-methanol-glacial acetic acid (66/33/1; v/v/v). The organic phase containing the lipids was obtained by centrifugation at 1,400 g at room temperature for 5 min. The synthesis of radiolabeled AcylCer was analyzed by TLC as described above. HEK 293T cells were transfected with FLAG-PNPLA1 or FLAG-ATGL together with histidine (His)-ABHD5 or His-β-Gal expressing vectors using Metafectene as described above. Twenty-four hours after transfection, cells were harvested and lysed in lysis buffer (50 mM Tris/HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 20 μg/ml leupeptine, 2 μg/ml antipain, 1 μg/ml pepstatin) by incubation for 30 min at 4°C on a rotating wheel. Lysates were centrifuged at 1,000 g and 4°C for 5 min. Equal amounts of lysate proteins (1 mg) were incubated with 20 μl anti-FLAG M2-agarose beads (Sigma-Aldrich) overnight at 4°C on a rotating wheel. Thereafter, beads were extensively washed with lysis buffer and heated in 2× Laemmli buffer at 95°C for 10 min. Input and immunoprecipitation fractions were subjected to immunoblot analyses using appropriate antibodies. For localization studies, COS-7 cells were seeded in chambers mounted onto coverslips (Sarstedt, Nümbrecht, Germany) and transfected with yellow fluorescent protein (YFP)-tagged murine PNPLA1 and cyan fluorescent protein (CFP)-tagged murine ABHD5 expressing vectors using Metafectene as described above. Four hours after transfection, the culture medium was supplemented with 400 µM oleic acid complexed to essentially FA-free BSA (Sigma-Aldrich) at a molar FA-BSA ratio of 3:1 to promote lipid droplet formation. After 18 h, cells were imaged using a Leica SP5 confocal microscope equipped with a Leica HCX 63× 1.4 numerical aperture oil immersion objective. CFP and YFP fluorescence were excited at 458 and 514 nm, respectively, and emission was detected between 477 and 535 nm. Immunoblot analyses were performed according to standard protocols using anti-FLAG M2-HRP (A8592; Sigma-Aldrich) antibody or anti-His (27-4710-01; GE Healthcare), anti-Xpress (R910-25; Invitrogen) or anti-GAPDH primary antibody (2118S; Cell Signaling Technology, Danvers, MA, USA), and the appropriate HRP-conjugated secondary antibody. HRP-conjugated IgG was detected using Clarity Western ECL Substrate (Bio-Rad). Finally, membrane-bound proteins were stained with Coomassie blue staining solution [45% (v/v) ethanol, 10% (v/v) acetic acid, 0.25% (v/w) Coomassie Brilliant Blue R250] followed by destaining with a solution consisting of 30% (v/v) ethanol and 10% (v/v) acetic acid. To evaluate whether PNPLA1 is a potential interaction partner of ABHD5 similar to what has been previously reported for ATGL (19.Lass A. Zimmermann R. Haemmerle G. Riederer M. Schoiswohl G. Schweiger M. Kienesberger P. Strauss J.G. Gorkiewicz G. Zechner R. Adipose triglyceride lipase-mediated lipolysis of cellular fat stores is activated by CGI-58 and defective in Chanarin-Dorfman Syndrome.Cell Metab. 2006; 3: 309-319Abstract Full Text Full Text PDF PubMed Scopus (676) Google Scholar), we performed coimmunoprecipitation experiments with HEK 293T cells coexpressing murine FLAG-tagged PNPLA1 and murine His-tagged ABHD5 (Fig. 1C, D). Using this experimental setup, we observed that ABHD5 coimmunoprecipitated with FLAG-tagged PNPLA1 bound to anti-FLAG-tagged antibody-coated agarose beads. Similarly, His-tagged ABHD5 coimmunoprecipitated with FLAG-tagged ATGL, which was used as a positive control in this assay. Experimental controls confirmed a specific interaction between PNPLA1 and ABHD5. We did not observe an unspecific binding between the FLAG- and the His-tag (controls: PNPLA1-FLAG, His-β-Gal; His-ABHD5, empty FLAG vector) or binding of His-tagged ABHD5 to the anti-FLAG-tagged antibody-coupled agarose beads (control: His-ABHD5 alone). This finding demonstrates that PNPLA1 interacts with ABHD5, suggesting a direct role of ABHD5 in AcylCer biosynthesis (18.Grond S. Radner F.P.W. Eichmann T.O. Kolb D. Grabner G.F. Wolinski H. Gruber R. Hofer P. Heier C. Schauer S. et al.Skin barrier development depends on CGI-58 protein expression during late-stage keratinocyte differentiation.J. Invest. Dermatol. 2017; 137: 403-413Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). To investigate the protein interaction of PNPLA1 with ABHD5, we analyzed the intracellular localization of murine YFP-tagged PNPLA1 and CFP-tagged ABHD5 by live-cell imaging. Consistent with previous reports (29.Chang P.A. Han L.P. Sun L.X. Huang F.F. Identification mouse patatin-like phospholipase domain containing protein 1 as a skin-specific and membrane-associated protein.Gene. 2016; 591: 344-350Crossref PubMed Scopus (4) Google Scholar), fluorescent microscopy of YFP-PNPLA1-transfected COS-7 cells revealed an evenly distributed YFP signal, consistent with a cytosolic localization of the YFP-PNPLA1 fusion protein (Fig. 2A). Upon coexpression of YFP-PNPLA1 and CFP-ABHD5, we observed that both proteins colocalized at lipid droplets (Fig. 2B, merge image and inset), appearing as vesicular structures by differential interference contrast microscopy (Fig. 2B, differential interference contrast image and inset). Given that ABHD5 is a bona fide lipid droplet-associated protein (30.Subramanian V. Rothenberg A. Gomez C. Cohen A.W. Garcia A. Bhattacharyya S. Shapiro L. Dolios G. Wang R. Lisanti M.P. et al.Perilipin A mediates the reversible binding of CGI-58 to lipid droplets in 3T3–L1 adipocytes.J. Biol. Chem. 2004; 279: 42062-42071Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar, 31.Yamaguchi T. Omatsu N. Matsushita S. Osumi T. CGI-58 interacts with perilipin and is localized to lipid droplets. Possible involvement of CGI-58 mislocalization in Chanarin-Dorfman syndrome.J. Biol. Chem. 2004; 279: 30490-30497Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar) (supplemental Fig. S1), our finding demonstrates that ABHD5 also inter
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