A plastidial lysophosphatidic acid acyltransferase from oilseed rape.
The biosynthesis of phosphatidic acid, a key intermediate in the biosynthesis of lipids, is controlled by lysophosphatidic acid (LPA, or 1-acyl-glycerol-3-P) acyltransferase (LPAAT, EC 220.127.116.11). We have isolated a cDNA encoding a novel LPAAT by functional complementation of the Escherichia coli mutant plsC with an immature embryo cDNA library of oilseed rape (Brassica napus). Transformation of the acyltransferase-deficient E. coli strain JC201 with the cDNA sequence BAT2 alleviated the temperature-sensitive phenotype of the plsC mutant and conferred a palmitoyl-coenzyme A-preferring acyltransferase activity to membrane fractions. The BAT2 cDNA encoded a protein of 351 amino acids with a predicted molecular mass of 38 kD and an isoelectric point of 9.7. Chloroplast-import experiments showed processing of a BAT2 precursor protein to a mature protein of approximately 32 kD, which was localized in the membrane fraction. BAT2 is encoded by a minimum of two genes that may be expressed ubiquitously. These data are consistent with the identity of BAT2 as the plastidial enzyme of the prokaryotic glycerol-3-P pathway that uses a palmitoyl-ACP to produce phosphatidic acid with a prokaryotic-type acyl composition. The homologies between the deduced protein sequence of BAT2 with prokaryotic and eukaryotic microsomal LAP acytransferases suggest that seed microsomal forms may have evolved from the plastidial enzyme. (+info)
Overexpression of 1-acyl-glycerol-3-phosphate acyltransferase-alpha enhances lipid storage in cellular models of adipose tissue and skeletal muscle.
Plasma nonesterified fatty acids (NEFA) at elevated concentrations antagonize insulin action and thus may play a critical role in the development of insulin resistance in type 2 diabetes. Plasma NEFA and glucose concentrations are regulated, in part, by their uptake into peripheral tissues. Cellular energy uptake can be increased by enhancing either energy transport or metabolism. The effects of overexpression of 1-acylglycerol-3-phosphate acyltransferase (AGAT)-alpha, which catalyzes the second step in triglyceride formation from glycerol-3-phosphate, was studied in 3T3-L1 adipocytes and C2C12 myotubes. In myotubes, overexpression of AGAT-alpha did not affect total [14C]glucose uptake in the presence or absence of insulin, whereas insulin-stimulated [14C]glucose conversion to cellular lipids increased significantly (33%, P = 0.004) with a concomitant decrease (-30%, P = 0.005) in glycogen formation. [3H]oleic acid (OA) uptake in AGAT-overexpressing myotubes increased 34% (P = 0.027) upon insulin stimulation. AGAT-alpha overexpression in adipocytes increased basal (130%, P = 0.04) and insulin-stimulated (27%, P = 0.01) [3H]OA uptake, increased insulin-stimulated glucose uptake (56%, P = 0.04) and conversion to cellular lipids (85%, P = 0.007), and suppressed basal (-44%, P = 0.01) and isoproterenol-stimulated OA release (-45%, P = 0.03) but not glycerol release. Our data indicate that an increase in metabolic flow to triglyceride synthesis can inhibit NEFA release, increase NEFA uptake, and promote insulin-mediated glucose utilization in 3T3-L1 adipocytes. In myotubes, however, AGAT-alpha overexpression does not increase basal cellular energy uptake, but can enhance NEFA uptake and divert glucose from glycogen synthesis to lipogenesis upon insulin stimulation. (+info)
Mutations in CGI-58, the gene encoding a new protein of the esterase/lipase/thioesterase subfamily, in Chanarin-Dorfman syndrome.
Chanarin-Dorfman syndrome (CDS) is a rare autosomal recessive form of nonbullous congenital ichthyosiform erythroderma (NCIE) that is characterized by the presence of intracellular lipid droplets in most tissues. We previously localized a gene for a subset of NCIE to chromosome 3 (designated "the NCIE2 locus"), in six families. Lipid droplets were found in five of these six families, suggesting a diagnosis of CDS. Four additional families selected on the basis of a confirmed diagnosis of CDS also showed linkage to the NCIE2 locus. Linkage-disequilibrium analysis of these families, all from the Mediterranean basin, allowed us to refine the NCIE2 locus to an approximately 1.3-Mb region. Candidate genes from the interval were screened, and eight distinct mutations in the recently identified CGI-58 gene were found in 13 patients from these nine families. The spectrum of gene variants included insertion, deletion, splice-site, and point mutations. The CGI-58 protein belongs to a large family of proteins characterized by an alpha/beta hydrolase fold. CGI-58 contains three sequence motifs that correspond to a catalytic triad found in the esterase/lipase/thioesterase subfamily. Interestingly, CGI-58 differs from other members of the esterase/lipase/thioesterase subfamily in that its putative catalytic triad contains an asparagine in place of the usual serine residue. (+info)
Exploration of novel motifs derived from mouse cDNA sequences.
We performed a systematic maximum density subgraph (MDS) detection of conserved sequence regions to discover new, biologically relevant motifs from a set of 21,050 conceptually translated mouse cDNA (FANTOM1) sequences. A total of 3202 candidate sequences, which shared similar regions over >20 amino acid residues, were screened against known conserved regions listed in Pfam, ProDom, and InterPro. The filtering procedure resulted in 139 FANTOM1 sequences belonging to 49 new motif candidates. Using annotations and multiple sequence alignment information, we removed by visual inspection 42 candidates whose members were found to be false positives because of sequence redundancy, alternative splicing, low complexity, transcribed retroviral repeat elements contained in the region of the predicted open reading frame, and reports in the literature. The remaining seven motifs have been expanded by hidden Markov model (HMM) profile searches of SWISS-PROT/TrEMBL from 28 FANTOM1 sequences to 164 members and analyzed in detail on sequence and structure level to elucidate the possible functions of motifs and members. The novel and conserved motif MDS00105 is specific for the mammalian inhibitor of growth (ING) family. Three submotifs MDS00105.1-3 are specific for ING1/ING1L, ING1-homolog, and ING3 subfamilies. The motif MDS00105 together with a PHD finger domain constitutes a module for ING proteins. Structural motif MDS00113 represents a leucine zipper-like motif. Conserved motif MDS00145 is a novel 1-acyl-SN-glycerol-3-phosphate acyltransferase (AGPAT) submotif containing a transmembrane domain that distinguishes AGPAT3 and AGPAT4 from all other acyltransferase domain-containing proteins. Functional motif MDS00148 overlaps with the kazal-type serine protease inhibitor domain but has been detected only in an extracellular loop region of solute carrier 21 (SLC21) (organic anion transporters) family members, which may regulate the specificity of anion uptake. Our motif discovery not only aided in the functional characterization of new mouse orthologs for potential drug targets but also allowed us to predict that at least 16 other new motifs are waiting to be discovered from the current SWISS-PROT/TrEMBL database. (+info)
Limnanthes douglasii lysophosphatidic acid acyltransferases: immunological quantification, acyl selectivity and functional replacement of the Escherichia coli plsC gene.
Antibodies were raised against the two membrane-bound lysophosphatidic acid acyltransferase (LPAAT) enzymes from Limnanthes douglasii (meadowfoam), LAT1 and LAT2, using the predicted soluble portion of each protein as recombinant protein antigens. The antibodies can distinguish between the two acyltransferase proteins and demonstrate that both migrate in an anomalous fashion on SDS/PAGE gels. The antibodies were used to determine that LAT1 is present in both leaf and developing seeds, whereas LAT2 is only detectable in developing seeds later than 22 daf (days after flowering). Both proteins were found exclusively in microsomal fractions and their amount was determined using the recombinant antigens as quantification standards. LAT1 is present at a level of 27 pg/microg of membrane protein in leaf tissue and +info)
Prevalence of mutations in AGPAT2 among human lipodystrophies.
Berardinelli-Seip congenital lipodystrophy (BSCL) is a heterogeneous genetic disease characterized by near absence of adipose tissue and severe insulin resistance. We have previously identified mutations in the seipin gene in a subset of our patients' cohort. Recently, disease-causing mutations in AGPAT2 have been reported in BSCL patients. In this study, we have performed mutation screening in AGPAT2 and the related AGPAT1 in patients with BSCL or other forms of lipodystrophy who have no detectable mutation in the seipin gene. We found 38 BSCL patients from 30 families with mutations in AGPAT2. Three of the known mutations were frequently found in our families. Of the eight new alterations, six are null mutations and two are missense mutations (Glu172Lys and Ala238Gly). All the patients harboring AGPAT2 mutations presented with typical features of BSCL. We did not find mutations in patients with other forms of lipodystrophies, including the syndromes of Lawrence, Dunnigan, and Barraquer-Simons, or with type A insulin resistance. In conclusion, mutations in the seipin gene and AGPAT2 are confined to the BSCL phenotype. Because we found mutations in 92 of the 94 BSCL patients studied, the seipin gene and AGPAT2 are the two major genes involved in the etiology of BSCL. (+info)
Truncation of CGI-58 protein causes malformation of lamellar granules resulting in ichthyosis in Dorfman-Chanarin syndrome.
Dorfman-Chanarin syndrome is a rare autosomal recessive inherited lipid storage disease characterized by ichthyosis, leukocyte lipid vacuoles, and involvement of several internal organs. Recently, CGI-58 mutations were identified as the cause of Dorfman-Chanarin syndrome. The physiologic roles of the CGI-58 protein and the pathomechanisms of Dorfman-Chanarin syndrome still remain to be clarified, however. The patient, a 16-y-old male, demonstrated ichthyosis, small ears, lipid vacuoles in his leukocytes, liver dysfunction, and mental retardation. Sequencing of CGI-58 revealed that the patient was homozygous for a novel nonsense mutation R184X, in exon 4. The putative truncated protein was 52.4% of the length of the normal CGI-58 polypeptide and lacked approximately 60% of the lipid binding region, 66.4% of the alpha/beta hydrolase folding segment of the polypeptide, and two of the CGI-58 catalytic triads, resulting in a significant loss of lipase/esterase/thioesterase activity. Electron microscopy revealed a large number of abnormal lamellar granules, a disturbed intercellular lamellar structure, and lipid vacuoles in the epidermis. These results suggested that CGI-58 protein is involved in the lipid metabolism of lamellar granules and that defective lipid production in lamellar granules caused by a CGI-58 protein deficiency is involved in the pathogenesis of ichthyosis in Dorfman-Chanarin syndrome. (+info)
CGI-58 interacts with perilipin and is localized to lipid droplets. Possible involvement of CGI-58 mislocalization in Chanarin-Dorfman syndrome.
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. (+info)