Characterization of the human patatin-like phospholipase family
2006; Elsevier BV; Volume: 47; Issue: 9 Linguagem: Inglês
10.1194/jlr.m600185-jlr200
ISSN1539-7262
AutoresPaul A. Wilson, Scott D. Gardner, Natalie M. Lambie, Stéphane Commans, Daniel Crowther,
Tópico(s)Ubiquitin and proteasome pathways
ResumoSeveral publications have described biological roles for human patatin-like phospholipases (PNPLAs) in the regulation of adipocyte differentiation. Here, we report on the characterization and expression profiling of 10 human PNPLAs. A variety of bioinformatics approaches were used to identify and characterize all PNPLAs encoded by the human genome. The genes described represent a divergent family, most with a highly conserved ortholog in several mammalian species. In silico characterization predicts that two of the genes function as integral membrane proteins and are regulated by cAMP/cGMP. A structurally guided protein alignment of the patatin-like domain identifies a number of conserved residues in all family members. Quantitative PCR was used to determine the expression profile of each family member. Affymetrix-based profiling of a human preadipocyte cell line identified several members that are differentially regulated during cell differentiation. Cumulative data suggest that patatin-like genes normally expressed at very low levels are induced in response to environmental signals. Given the observed conservation of the patatin fold and lipase motif in all human PNPLAs, a single nomenclature to describe the PNPLA family is proposed. Several publications have described biological roles for human patatin-like phospholipases (PNPLAs) in the regulation of adipocyte differentiation. Here, we report on the characterization and expression profiling of 10 human PNPLAs. A variety of bioinformatics approaches were used to identify and characterize all PNPLAs encoded by the human genome. The genes described represent a divergent family, most with a highly conserved ortholog in several mammalian species. In silico characterization predicts that two of the genes function as integral membrane proteins and are regulated by cAMP/cGMP. A structurally guided protein alignment of the patatin-like domain identifies a number of conserved residues in all family members. Quantitative PCR was used to determine the expression profile of each family member. Affymetrix-based profiling of a human preadipocyte cell line identified several members that are differentially regulated during cell differentiation. Cumulative data suggest that patatin-like genes normally expressed at very low levels are induced in response to environmental signals. Given the observed conservation of the patatin fold and lipase motif in all human PNPLAs, a single nomenclature to describe the PNPLA family is proposed. The patatin glycoprotein is a nonspecific lipid acyl hydrolase that is found in high concentrations in mature potato tubers (1Rydel T.J. Williams J.M. Krieger E. Moshiri F. Stallings W.C. Brown S.M. Pershing J.C. Purcell J.P. Alibhai M.F. The crystal structure, mutagenesis, and activity studies reveal that patatin is a lipid acyl hydrolase with a Ser-Asp catalytic dyad..Biochemistry. 2003; 42: 6696-6708Crossref PubMed Scopus (222) Google Scholar). Patatin is reported to play a role in plant signaling (2Holk A. Rietz S. Zahn M. Quader H. Scherer G.F.E. Molecular identification of cytosolic, patatin-related phospholipases A from Arabidopsis with potential functions in plant signal transduction..Plant Physiol. 2002; 130: 90-101Crossref PubMed Scopus (123) Google Scholar), to cleave fatty acids from membrane lipids (2Holk A. Rietz S. Zahn M. Quader H. Scherer G.F.E. Molecular identification of cytosolic, patatin-related phospholipases A from Arabidopsis with potential functions in plant signal transduction..Plant Physiol. 2002; 130: 90-101Crossref PubMed Scopus (123) Google Scholar), and to act as defense against plant parasites (3Strickland J.A. Orr G.L. Walsh T.A. Inhibition of Diabrotica larval growth by patatin, the lipid acyl hydrolase from potato tubers..Plant Physiol. 1995; 109: 667-674Crossref PubMed Scopus (82) Google Scholar). Proteins encoding a patatin-like domain are ubiquitously distributed across all life forms, including eukaryotes and prokaryotes [see the species distribution tree of the PFAM (protein family) database (4Bateman A. Coin L. Durbin R. Finn R.D. Hollich V. Griffiths-Jones S. Khanna A. Marshall M. Moxon S. Sonnhammer E.L.L. et al.The PFAM protein families database..Nucleic Acids Res. 2004; 32: D138-D141Crossref PubMed Google Scholar), accession number PF01734 (http://www.sanger.ac.uk//cgi-bin/Pfam/getacc?PF01734), for further details], and are observed to participate in a miscellany of biological roles, including sepsis induction (5Allewelt M. Coleman F.T. Grout M. Priebe G.P. Pier G.B. Acquisition of expression of the Pseudomonas aeruginosa ExoU cytotoxin leads to increased bacterial virulence in a murine model of acute pneumonia and systemic spread..Infect. Immun. 2000; 68: 3998-4004Crossref PubMed Scopus (154) Google Scholar), host colonization (6La Camera S. Geoffroy P. Samaha H. Ndiaye A. Rahim G. Legrand M. Heitz T. A pathogen-inducible patatin-like lipid acyl hydrolase facilitates fungal and bacterial host colonization in Arabidopsis..Plant J. 2005; 44: 810-825Crossref PubMed Scopus (122) Google Scholar), triglyceride metabolism (7Kurat C.F. Natter K. Petschnigg J. Wolinski H. Scheuringer K. Scholz H. Zimmermann R. Leber R. Zechner R. Kohlwein S.D. Obese yeast: triglyceride lipolysis is functionally conserved from mammals to yeast..J. Biol. Chem. 2006; 281: 491-500Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar), and membrane trafficking (8Shohdy N. Efe J.A. Emr S.D. Shuman H.A. Pathogen effector protein screening in yeast identifies Legionella factors that interfere with membrane trafficking..Proc. Natl. Acad. Sci. USA. 2005; 102: 4866-4871Crossref PubMed Scopus (179) Google Scholar). Of interest is the observation that prokaryotic patatin-like proteins appear more similar to eukaryotic paralogues than any other bacterial lipases (9Banerji S. Flieger A. Patatin-like proteins: a new family of lipolytic enzymes present in bacteria?.Microbiology. 2004; 150: 522-525Crossref PubMed Scopus (96) Google Scholar). This suggests that distant homologs may have arisen from a common ancestor. Together, these observations indicate that the patatin-like domain represents a unique superfamily quite distinct from all other lipolytic families. Recent studies investigating the role of the hormone-sensitive lipase in mammalian adipose tissue identified a novel patatin-like lipase (TTS-2.2) that was significantly upregulated in differentiating murine adipocytes and appeared to act coordinately with hormone-sensitive lipase in the catabolism of triglycerides (10Zimmermann R. Strauss J.G. Haemmerle G. Schoiswohl G. Birner-Gruenberger R. Riederer M. Lass A. Neuberger G. Eisenhaber F. Hermetter A. et al.Fat mobilization in adipose tissue is promoted by adipose triglyceride lipase..Science. 2004; 306: 1383-1386Crossref PubMed Scopus (1533) Google Scholar). The rat ortholog of this gene was also found to be upregulated in differentiating adipocytes and transiently induced during fasting (11Villena J.A. Roy S. Sarkadi-Nagy E. Kim K.H. Sul H.S. Desnutrin, an adipocyte gene encoding a novel patatin domain-containing protein, is induced by fasting and glucocorticoids: ectopic expression of desnutrin increases triglyceride hydrolysis..J. Biol. Chem. 2004; 279: 47066-47075Abstract Full Text Full Text PDF PubMed Scopus (510) Google Scholar). The yeast protein Tgl4 has since been reported to be a functional ortholog of the mammalian TTS-2.2 gene (7Kurat C.F. Natter K. Petschnigg J. Wolinski H. Scheuringer K. Scholz H. Zimmermann R. Leber R. Zechner R. Kohlwein S.D. Obese yeast: triglyceride lipolysis is functionally conserved from mammals to yeast..J. Biol. Chem. 2006; 281: 491-500Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). Jenkins et al. (12Jenkins C.M. Mancuso D.J. Yan W. Sims H.F. Gibson B. Gross R.W. Identification, cloning, expression, and purification of three novel human calcium-independent phospholipase A2 family members possessing triacylglycerol lipase and acylglycerol transacylase activities..J. Biol. Chem. 2004; 279: 48968-48975Abstract Full Text Full Text PDF PubMed Scopus (685) Google Scholar) reported details of three murine patatin-like proteins [adiponutrin (ADPN), TTS-2.2, and GS2] with abundant triacylglyerol (catabolic) and transacylation (anabolic) activities. All three were observed to associate with the cell membrane. The ADPN gene transcript was found to be virtually absent in a fasted state and dramatically upregulated in response to feeding. A more recent report (13Lake A.C. Sun Y. Li J. Kim J. Johnson J.W. Li D. Revett T. Shih H.H. Liu W. Paulsen J.E. et al.Expression, regulation, and triglyceride hydrolase activity of adiponutrin family members..J. Lipid Res. 2005; 46: 2477-2487Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar) characterized four human ADPN-like lipases (ADPN, TTS-2.2, GS2, and GS2-like) and detailed changes in gene expression during murine adipocyte differentiation. Both TTS-2.2 and GS2 were highly expressed in metabolically active tissues. It was also reported that, except for ADPN, overexpression of the ADPN-like proteins resulted in decreased intracellular triglyceride levels (13Lake A.C. Sun Y. Li J. Kim J. Johnson J.W. Li D. Revett T. Shih H.H. Liu W. Paulsen J.E. et al.Expression, regulation, and triglyceride hydrolase activity of adiponutrin family members..J. Lipid Res. 2005; 46: 2477-2487Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). Given the significant association of the patatin-like proteins in adipocyte differentiation and their response to metabolic stimuli, the purpose of this investigation was to use a variety of bioinformatics approaches to identify and characterize all human proteins encoding a patatin-like domain. Combined with gene expression data, the collected observations will be used to further assess and characterize the role of these phospholipases in mammalian lipid metabolism. A redundant human sequence set was identified from the PFAM (4Bateman A. Coin L. Durbin R. Finn R.D. Hollich V. Griffiths-Jones S. Khanna A. Marshall M. Moxon S. Sonnhammer E.L.L. et al.The PFAM protein families database..Nucleic Acids Res. 2004; 32: D138-D141Crossref PubMed Google Scholar) patatin family alignment (accession number PF01734). Each of these sequences was used as input to a Position-Specific Iterated Basic Local Alignment Search Tool (PSI-BLAST) (14Koretke K.K. Russell R.B. Lupas A.N. Folds without a fold..Protein Sci. 2002; 11: 1575-1579Crossref PubMed Scopus (21) Google Scholar) search to query a nonredundant (>94% identity between sequences) database composed of all available human gene and gene prediction sequences (included were the TREMBL, Swiss-Protein, GenPept, ENSEMBL, GeneSeqP, PIR, and PDB databases). Equivalent full-length transcript sequences of each patatin-like gene were used to query a redundant human expressed sequence tag (EST) database (GenBank release 151, December 2005) in an effort to verify the expression of the transcripts. Gene expression was further verified using the GeneLogic Genesis Enterprise database (http://www.genelogic.com/genomics/genesis.cfm). The patatin domain encoded in each protein was delineated using the SMART database (15Schultz J. Milpetz F. Bork P. Ponting C.P. SMART, a simple modular architecture research tool: identification of signalling domains..Proc. Natl. Acad. Sci. USA. 1998; 95: 5857-5864Crossref PubMed Scopus (3029) Google Scholar). Secondary structure was predicted using JNET (16Cuff J.A. Barton G.J. Application of enhanced multiple sequence alignment profiles to improve protein secondary structure prediction..Proteins. 1999; 40: 502-511Crossref Scopus (663) Google Scholar), and fold prediction was completed using the FUGUE application (17Shi J. Blundell T.L. Mizuguchi K. FUGUE: sequence-structure homology recognition using environment-specific substitution tables and structure-dependent gap penalties..J. Mol. Biol. 2001; 310: 243-257Crossref PubMed Scopus (1085) Google Scholar). Low-resolution homology models were generated using the Swiss-PDB Viewer application (18Guex N. Peitsch M.C. SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling..Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9641) Google Scholar). Sequence alignments were completed using ClustalW (19Thompson J.D. Higgins D.G. Gibson T.J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice..Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (56002) Google Scholar). The resulting alignment was used as input for PhyloWin (20Galtier N. Gouy M. Gautier C. SeaView and Phylo_win, two graphic tools for sequence alignment and molecular phylogeny..Comput. Appl. Biosci. 1996; 12: 543-548PubMed Google Scholar) to determine putative evolutionary relationships between members of the human patatin-like gene family. Full-length protein sequences were used as input to TMHMM (21Sonnhammer E.L. von Heijne G. Krogh A. A hidden Markov model for predicting transmembrane helices in protein sequences..Proc. Int. Conf. Intell. Syst. Mol. Biol. 1998; 6: 175-182PubMed Google Scholar) and SignalP (22Bendtsen J.D. Nielsen H. von Heijne G. Brunak S. Improved prediction of signal peptides: SignalP 3.0..J. Mol. Biol. 2004; 340: 783-795Crossref PubMed Scopus (5660) Google Scholar) to predict transmembrane domains and signal peptide sequences, respectively. Putative orthologs in mouse and rat were identified by reverse BLASTing a nonredundant nucleotide database derived from the EMBL nucleotide database (23Kanz C. Aldebert P. Althorpe N. Baker W. Baldwin A. Bates K. Browne P. van den Broek A. Castro M. Cochrane G. et al.The EMBL nucleotide sequence database..Nucleic Acids Res. 2005; 33: D29-D33Crossref PubMed Scopus (221) Google Scholar). Tissue processing and sample preparation were described previously (24Sarau H.M. Ames R.S. Chambers J. Ellis C. Elshourbagy N. Foley J.J. Schmidt D.B. Muccitelli R.M. Jenkins O. Murdock P.R. et al.Identification, molecular cloning, expression, and characterization of a cysteinyl leukotriene receptor..Mol. Pharmacol. 1999; 56: 657-663Crossref PubMed Scopus (306) Google Scholar). Briefly, cDNA prepared from 0.4 ng of poly(A+) RNA extracted from each tissue sample was loaded into each well on a 384-well optical microplate. Gene-specific primers were designed using Primer Express software (Applied Biosystems) and confirmed by BLAST searches of public and propriety sequence databases. Details of each primer are as follows (gene name/forward/reverse/probe): patatin-like phospholipase 3 (PNPLA3)/ADPN/AAATGCCAGTGAGCAGCCAA/TCTCTGCTGGACAGCCCTTG/FAM-CTCCCCATGCACACCTGAGCAGGACT-TAMRA; PNPLA7/FLJ43070/GCCTCTGTACCTGCCCTGCT/CTGTATGCAGGGCTGCTGGT/FAM-CCCAGAGAACCCTAACACAGCCTGGGG-TAMRA; PNPLA6/NTE/AGCCACAGATGCCTGAGGAC/GGGCAGGTCAGTCCAGTGTG/FAM-CTCACTCCCCCTCCTGCTGCTATGCCT-TAMRA; PNPLA9/PLA2G6/ACCGCGAGGAGTTCCAGAAG/AATGGACGAGGTCAGCTGGG/FAM-TCATCCACCTGCTGCTCTCACCCTGAG-TAMRA; PNPLA8/iPLA2γ/GCTAGACCCTGTTGCCCAGA/GGTTCTTGCAGCAAAGGCAG/FAM-TTGAACCACATCTCACAGCCTCTGTGA-TAMRA; PNPLA1/GCAGTGGGAGATTGGGCTTT/TGTCCCAACCATGATCCCTG/FAM-AAAATTCCTGCTCTGCCACAGCTCCAC-TAMRA; PNPLA4/GS2/AACGTGTGGCAATTGTGGGA/CCCTAACTGCACTGGGAGCA/FAM-TTGGCTGTGTCCCCACCCAAATCTCAT-TAMRA; PNPLA5/GS2-like/GGCGGACTTGTGGTGGATG/GATGGGCCCAAGGAGCTG/FAM-AAACCTCGAGGGCCATGTTCCTCAGC-TAMRA. The concentrations of the forward and reverse primer and probe for each assay were 900, 900, and 100 nM, respectively. Quantitative PCR was carried out using the 7900HT Sequence Detector System (Applied Biosystems) in a 10 μl reaction volume. TaqMan Universal PCR Master Mix 2X (Applied Biosystems) and universal PCR conditions recommended by the vendor (Applied Biosystems) were used. Abundance values for each tissue sample were calculated using a genomic DNA standard curve to convert TaqMan fluorescent output to a known number of sequence copies. Differentiation of multipotent adipose-derived stem cells, isolated from human adipose tissue, was completed as described previously (25Rodriguez A. Elabd C. Delteil F. Astier J. Vernochet C. Saint-Marc P. Guesnet J. Guezennec A. Amri E. Dani C. et al.Adipocyte differentiation of multipotent cells established from human adipose tissue..Biochem. Biophys. Res. Commun. 2004; 315: 255-263Crossref PubMed Scopus (255) Google Scholar). Total RNA was extracted using Trizol (Invitrogen, Carlsbad, CA). RNA purity and integrity were evaluated using the Agilent Bioanalyzer and Optical Density spectrophotometer. All samples were prepared according to the Affymetrix (Palo Alto, CA) protocol (http://www.affymetrix.com/support/). Fifteen micrograms of labeled copy RNA samples was hybridized onto U133plus2 microarrays (Affymetrix) using standard Affymetrix protocols. Quality-control parameters were as follows: noise (RawQ) < 3, consistent scale factors, background signal < 50, consistent number of genes called as “present” across arrays, and β-actin and GAPDH 5′/3′ signal ratios < 3. Signal intensities were collated and analyzed using Rosetta's Resolver Biosoftware (http://www.rosettabio.com/products/resolver/default.htm). Extracting a nonredundant set of human sequences from the PFAM PNPLA alignment (accession number PF01734) generated eight unique patatin-like sequences (ADPN, TTS-2.2, Neuropathy Target Esterase (NTE), FLJ43070, IPA2γ, PNPLA1, PNPLA4, and PNPLA5). Each sequence was used to search a nonredundant human transcript database using a PSI-BLAST-based algorithm (14Koretke K.K. Russell R.B. Lupas A.N. Folds without a fold..Protein Sci. 2002; 11: 1575-1579Crossref PubMed Scopus (21) Google Scholar). This identified two additional patatin-like sequences, XM_066899 and Phospholipase A2, group VI (PLA2G6), not included in the PFAM alignment. BLAST alignments indicated that both sequences are most homologous to intracellular membrane-associated calcium-independent phospholipase A2γ (iPLA2γ) (data not shown). All 10 human sequences that encoded a significant patatin-like domain were compared using the PFAM motif search tool and the FUGUE profile search tool. Both the PFAM and FUGUE similarity scores for each of the human patatin-like domains are detailed in Table 1 . Both techniques were in broad agreement and indicated that the NTE, NTE-like, and iPLA2γ sequences had higher homology to the patatin domain than did the ADPN-like subfamily.TABLE 1Summary of predicted key characteristics of the human PNPLA familyGene NamePeptide LengthPFAM E Value (PF01734)FUGUE Similarity Score (hs1oxwa)Predicted Signal PeptidePredicted Transmembrane DomainOther Predicted MotifsPNPLA14372.7e-9 (5–81)6.91Tyrosine kinase phosphorylation site (29–36 and 169–177)PNPLA87822e-52 (445–640)19.56Tyrosine kinase phosphorylation site (217–223)PNPLA71,3174.5e-56 (928–1,094)16.8713–32Cyclic nucleotide binding sites (145–694) Glycosaminoglycan attachment site (1,027–1,030) Tyrosine kinase phosphorylation site (1,013–1,020, 1,127–1,135, and 1,181–1,188) Cell attachment sequence (658–660)PNPLA25041.8e-25 (10–179)13.367–26Prokaryotic membrane lipoprotein lipid attachment site (36–46)PNPLA61,3272.9e-63 (933–1,099)17.539–31Cyclic nucleotide binding sites (147–701) Glycosaminoglycan attachment site (735–738) Tyrosine kinase phosphorylation site (403–410) Cell attachment sequence (663–665) Uncharacterized protein family UPF0028 signature (954–986)PNPLA98064.1e-55 (481–665)13.34Ankyrin repeats (151–412)PNPLA54295.7e-13 (12–181)16.19Glycosaminoglycan site (33–36 and 353–356)PNPLA34813.7e-25 (10–179)14.90PNPLA42539.8e-41 (6–176)15.73YesTyrosine kinase phosphorylation site (246–253)PNPLA, patatin-like phospholipase. In silico characterization of the human patatin-like family of phospholipases. Summary of in silico predictions: column 2 indicates the length of the longest representative human peptide sequence in the public domain. Columns 3 and 4 list the scoring outputs for each of the predicted patatin-like domains from the PFAM and FUGUE algorithms, respectively. Columns 5, 6, and 7 summarize predicted signal peptides, transmembrane domains, and significant functional motifs as determined by the SignalP, TMHMM, and SMART applications. The relative positions of the predicted motifs shown in parentheses refer to the full-length peptide sequence used as input. Open table in a new tab PNPLA, patatin-like phospholipase. In silico characterization of the human patatin-like family of phospholipases. Summary of in silico predictions: column 2 indicates the length of the longest representative human peptide sequence in the public domain. Columns 3 and 4 list the scoring outputs for each of the predicted patatin-like domains from the PFAM and FUGUE algorithms, respectively. Columns 5, 6, and 7 summarize predicted signal peptides, transmembrane domains, and significant functional motifs as determined by the SignalP, TMHMM, and SMART applications. The relative positions of the predicted motifs shown in parentheses refer to the full-length peptide sequence used as input. Attempts to align the human protein sequences resulted in low-quality alignments as a result of minimal homology between input sequences. The residues that determined homology to the patatin domain were identified using the PFAM protein query tool (accession number PF01734/patatin domain), and the alignment procedure was repeated using the restricted sequence as input. Although this approach demonstrated an improvement relative to the full-length protein alignment, much of the sequence remained poorly aligned. Information derived from secondary structure prediction (JNET) and fold prediction (FUGUE) was used to generate a manual alignment of the patatin-like sequences. The resulting alignment highlighted several regions of high conservation, including the characteristic glycine-rich region, the nucleophilic elbow, and the aspartate-glycine residues of the catalytic center. When shaded by physiochemical characteristics (Fig. 1 ), the protein alignment clearly illustrated the conservation of similar residues at several defined positions dispersed throughout the patatin-like domain. Such residues are obvious candidates for future mutagenesis studies. The alignment also highlighted blocks of physiochemical conservation across those regions predicted to fold as α-helices and β-sheets interspersed by highly divergent loop regions. The alignment also highlights the fact that the PNPLA1 sequence does not include the N-terminal region of the patatin domain. In particular, it is missing both the glycine-rich region and the G-X-S-X-G motif, which includes the nucleophilic serine, an essential component of the catalytic dyad. Without these residues, it is highly unlikely that PNPLA1 could function as a lipolytic enzyme. Extensive searches of the EMBL nucleotide sequence database (release 85, December 2005) did not identify additional human sequences that could be used to extend the N-terminal coding sequence of PNPLA1. However, predicted ortholog sequences from rat (XP_342106), mouse (XP_484618), dog (XP_538884), and chimpanzee (XP_527370) all demonstrate a highly conserved N-terminal sequence that encodes the lipase signature motifs. Use of the predicted PNPLA1 sequences to query the human genome sequence with BLASTP (data not shown) identified what appears to be an additional, upstream exon that would encode both the glycine-rich region and the G-X-S-X-G motif, immediately upstream of the human PNPLA1 locus. The predicted full-length sequence of the human PNPLA1 gene has been submitted to GenBank (accession number AM182887). PNPLA4 includes an alanine substitution for the first conserved glycine in the G-X-G-X-X-G motif. The alanine substitution is seen in all available human transcripts and is also the equivalent residue in the dog, mouse, cow, and frog ortholog sequences (alignment not shown). A serine residue occurs in the equivalent position in the rat ortholog sequence. The functional implications of this substitution are currently unclear, but it does not negate lipolytic activity, as PNPLA4 has previously demonstrated both lipase (13Lake A.C. Sun Y. Li J. Kim J. Johnson J.W. Li D. Revett T. Shih H.H. Liu W. Paulsen J.E. et al.Expression, regulation, and triglyceride hydrolase activity of adiponutrin family members..J. Lipid Res. 2005; 46: 2477-2487Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar) and transacylation (12Jenkins C.M. Mancuso D.J. Yan W. Sims H.F. Gibson B. Gross R.W. Identification, cloning, expression, and purification of three novel human calcium-independent phospholipase A2 family members possessing triacylglycerol lipase and acylglycerol transacylase activities..J. Biol. Chem. 2004; 279: 48968-48975Abstract Full Text Full Text PDF PubMed Scopus (685) Google Scholar) activities. Sequence comparisons indicated that the putative human patatin-like family could be divided into four subfamilies: an ADPN-like family, as proposed by Lake et al. (13Lake A.C. Sun Y. Li J. Kim J. Johnson J.W. Li D. Revett T. Shih H.H. Liu W. Paulsen J.E. et al.Expression, regulation, and triglyceride hydrolase activity of adiponutrin family members..J. Lipid Res. 2005; 46: 2477-2487Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar), an NTE-like subfamily, and two unique members represented by PNPLA9 (PLA2G6) and PNPLA8 (iPLA2γ). This observation was reinforced by a phylogenetic comparison of the patatin-like domain sequences (Fig. 2 ). PNPLA1 was found to cluster with the ADPN-like subfamily (data not shown). Because the method incorporated a global gap-removal function, the initial tree was derived from only 72 residue positions. To increase confidence in the analysis, the partial domain sequence represented by PNPLA1 was removed. The resulting phylogenetic tree was constructed from 144 residue positions and did not alter the previous relationship (i.e., when PNPLA1 was included in the analysis) of the remaining sequences. Low-resolution homology models of each of the human PNPLAs were generated using Macromolecular Structure Database (MSD) entry 1OXW (i.e., a crystal structure derived from a plant patatin isozyme) as a template. These were used to give an approximation of the three-dimensional organization of conserved residues. A representative model of the human PNPLA3 (ADPN) is shown in Fig. 3 and illustrates both the conserved α/β, patatin-like fold and the spatial orientation of the residues that compose the catalytic dyad (model coordinates are available as supplementary data). When each model was examined in conjunction with the sequence alignment, it was apparent that the catalytic residues were “framed” by several conserved hydrophobic residues (e.g., residues 8, 49, 109, 189, and 195 of the alignment in Fig. 2). When structural data were combined with the observed evolutionary relationships (Fig. 2), other positions were predicted to determine subfamily specificity (e.g., a conserved leucine residue at position 9 is specific to the ADPN subfamily, whereas an isoleucine residue at position 46 appears to be a characteristic of the NTE-like subfamily). Such observations will be the input to future mutagenesis studies that will fully characterize both enzyme function and substrate recognition. The collective specificity to the unique patatin enzyme suggested that these proteins represented a human patatin-like family. Given the numerous aliases used to describe the various family members, we derived the following consistent nomenclature: PNPLA1, PNPLA2 (TTS2.2), PNPLA3 (ADPN), PNPLA4 (GS2), PNPLA5 (GS2-like), PNPLA6 (NTE), PNPLA7 (NTE-like), PNPLA8 (iPLA2γ), PNPLA9 (PLA2G6), and PNPLA10P (pseudogene). This proposed nomenclature was submitted to the Human Genome Organization (HUGO) Gene Nomenclature Committee for approval. All protein sequences were further characterized using a variety of in silico prediction algorithms (Table 1). Briefly, PNPLA6 (NTE), PNPLA7 (NTE-like), and PNPLA2 (TTS-2.2) are all predicted to encode single transmembrane domains. PNPLA4 is predicted to be a secreted protein, which along with the PNPLA1, PNPLA8 (iPLA2γ), PNPLA6, and PNPLA7 protein sequences all encode one or more tyrosine kinase phosphorylation sites. Both the PNPLA6 and PNPLA7 sequences encode regions of cyclic nucleotide binding sites, whereas PNPLA9 is characterized by a region of ankyrin repeats. The expression profile of each gene was verified using TaqMan analysis of a representative sample set of human tissues (see supplementary data for all expression data). This indicated that PNPLA3 (ADPN) was expressed at very low levels in a number of tissues with marginally higher copy numbers detected in liver, bone, and macrophages (Fig. 4 ). Equivalent data extracted from the GeneLogic Genesis Enterprise database (probe set 233030_at) supported the observation of increased PNPLA3 expression in liver tissue (data not shown) and contradict previous descriptions of this lipase as an adipocyte-specific gene (11Villena J.A. Roy S. Sarkadi-Nagy E. Kim K.H. Sul H.S. Desnutrin, an adipocyte gene encoding a novel patatin domain-containing protein, is induced by fasting and glucocorticoids: ectopic expression of desnutrin increases triglyceride hydrolysis..J. Biol. Chem. 2004; 279: 47066-47075Abstract Full Text Full Text PDF PubMed Scopus (510) Google Scholar), although this discrepancy may reflect variation between the rat and human species.Fig. 4.Expression profiles of the human patatin-like family. Gene-specific TaqMan primers were used to determine the expression profile of each of the human PNPLAs. Equivalent RNA was extracted from the tissues of four unrelated donors as described in Materials and Methods. Abundance values for each tissue sample were calculated using a genomic DNA standard curve to convert TaqMan fluo
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