SUMMARY Mutations in 1-acylglycerol-3-phosphate-O-acyltransferase 2 (AGPAT2) cause congenital generalized lipodystrophy. To understand the molecular mechanisms underlying the metabolic complications associated with AGPAT2 deficiency, Agpat2 null mice were generated. Agpat2−/− mice develop severe lipodystrophy affecting both white and brown adipose tissue, severe insulin resistance, diabetes, and hepatic steatosis. The expression of lipogenic genes and rates of de novo fatty acid biosynthesis were increased ~4-fold in Agpat2−/− mouse livers. The mRNA and protein levels of monoacylglycerol acyltransferase isoform 1 were markedly increased in the livers of Agpat2−/− mice suggesting that the alternative monoacylglycerol pathway for triglyceride biosynthesis is activated in the absence of AGPAT2. Feeding a fat-free diet reduced liver triglycerides by ~50% in Agpat2−/− mice. These observations suggest that both dietary fat and hepatic triglyceride biosynthesis via a novel monoacylglycerol pathway may contribute to hepatic steatosis in Agpat2−/− mice.

Most cells synthesize their glycerophospholipids and triglycerides (TG) to maintain the cellular integrity and to provide energy for cellular functions. The phospholipids are synthesized de novo in cells through an evolutionary conserved process involving serial acylations of glycerol-3-phosphate. Several isoforms of the enzyme 1-acylglycerol-3-phosphate-O-acyltransferase (AGPAT) acylate lysophosphatidic acid at the sn-2 position to produce phosphatidic acid. We cloned a cDNA predicted to be an AGPAT isoform and designated it AGPAT9. The human AGPAT9 gene spans across 14 exons and encodes for a polypeptide of 534 amino acids. AGPAT9 is highly expressed in the lung and spleen, followed by leukocyte, omental adipose tissue, and placenta. In the Chinese Hamster Ovary (CHO), cell lysates overexpressing AGPAT9, we observed AGPAT activity but not the lysophosphatidylcholine acyltransferase activity. When AGPAT9 is coexpressed with AGPAT1 in CHO cells, both the isoforms localize to the endoplasmic reticulum (ER) and occupy the same ER domain as AGPAT1. Despite substitution of asparagine with proline in the NHX 4 D motif and arginine with cysteine in the EGTR motif, AGPAT9 retains AGPAT activity suggesting that residues asparagine and arginine in the NHX 4 D and EGTR motifs respectively are not essential for the enzymatic activity. Based on the X-ray crystallographic structure of a related acyltransferase, squash gpat, a model is proposed in which a hydrophobic pocket in AGPAT9 accommodates fatty acyl chains of both substrates in an orientation, whereas the HX 4 D motif participates in catalysis. Based on the activity and expression pattern of AGPAT9 in the lung and spleen, this novel isoform could be implicated in the biosynthesis of phospholipids and TG in these tissues.

MspA is the major porin of Mycobacterium smegmatis mediating the exchange of hydrophilic solutes across the outer membrane (OM). It is the prototype of a new family of octameric porins with a single central channel of 9.6 nm in length and consists of two hydrophobic ␤-barrels of 3.7 nm in length and a more hydrophilic, globular rim domain. The length of the hydrophobic domain of MspA does not match the thicknesses of mycobacterial OMs of 5-12 nm as derived from electron micrographs. Further, the membrane topology of MspA is unknown as it is for any other mycobacterial OM protein. We used MspA as a molecular ruler to define the boundaries of the OM of M. smegmatis by surface labeling of single cysteine mutants. Seventeen mutants covered the surface of the rim domain and were biotinylated with a membrane-impermeable reagent. The label efficiencies in vitro were remarkably similar to the predicted accessibilities of the cysteines. By contrast, six of these mutants were protected from biotinylation in M. smegmatis cells. Tryptophan 21 defines a horizontal plane that dissects the surfaceexposed versus the membrane-protected residues of MspA. The 8 phenylalanines at position 99 form a ring at the periplasmic end of the hydrophobic ␤-barrel domain. These results indicated that (i) the membrane boundaries of MspA are defined by aromatic girdles as in porins of Gram-negative bacteria and (ii) loops and a 3.4-nmlong part of the hydrophilic rim domain are embedded into the OM of M. smegmatis. This is the first report suggesting that elements other than hydrophobic ␣-helices or ␤-sheets are integrated into a lipid membrane.Mycobacteria are surrounded by an inner membrane and a giant macromolecule consisting of peptidoglycan, arabinogalactan, and very long chain fatty acids, the mycolic acids (1). Minnikin (2) originally proposed that the mycolic acids, which are covalently bound to the arabinogalactan-peptidoglycan co-polymer, form the inner leaflet of an unique outer membrane (OM).3 Experimental evidence for this model was provided by x-ray diffraction studies, which showed that the mycolic acids are oriented in parallel and perpendicular to the plane of the cell envelope (3). The presence of an organized structure of extremely low fluidity in an isolated mycobacterial cell wall was confirmed by differential scanning calorimetry (4). The vast abundance of non-covalently bound lipids in mycobacteria is assumed to constitute the outer leaflet of a supported asymmetric lipid bilayer. The analysis of spin-labeled fatty acids inserted into isolated mycobacterial cell walls by electron paramagnetic resonance supported the existence of a moderately fluid outer leaflet, whereas the inner leaflet had an extremely low fluidity (4, 5). This interpretation is consistent with observations that mutants with defects in the production of some of the major extractable lipids (glycopeptidolipids, phthiocerol dimycocerosate) showed an increased OM permeability (6, 7). Thus, the mycobacterial OM resembles a supported asymmetric lipid bilayer ...

Synthesis of phospholipids can occur de novo or via remodeling of the existing phospholipids. Synthesis of triglycerides, a form of energy storage in cells, is an end product of these pathways. Several 1-acylglycerol-3-phosphate-O-acyltransferases (AGPATs) acylate lysophosphatidic acid (LPA) at the sn-2 (carbon 2) position to produce phosphatidic acid (PA). These enzymes are involved in phospholipids and triglyceride synthesis through an evolutionary conserved process involving serial acylations of glycerol-3-phosphate. We cloned a cDNA predicted to be an AGPAT isoform (AGPAT10). This cDNA has been recently identified as glycerol-3-phosphate-O-acyltransferase isoform 3 (GPAT3). When this AGPAT10/GPAT3 cDNA was expressed in Chinese Hamster ovary cells, the protein product localizes to the endoplasmic reticulum. In vitro enzymatic activity using lysates of human embryonic kidney-293 cells infected with recombinant AGPAT10/GPAT3 adenovirus show that the protein has a robust AGPAT activity with an apparent Vmax of 2 nmol/min per mg protein, but lacks GPAT enzymatic activity. This AGPAT has similar substrate specificities for LPA and acyl-CoA as shown for another known isoform, AGPAT2. We further show that when overexpressed in human Huh-7 cells depleted of endogenous AGPAT activity by sh-RNA-AGPAT2-lentivirus, the protein again demonstrates AGPAT activity. These observations strongly suggest that the cDNA previously identified as GPAT3 has AGPAT activity and thus we prefer to identify this clone as AGPAT10 as well.

The folding of membrane proteins was addressed using outer membrane protein porin from the soil bacterium Paracoccus denitrificans (P. den.). IR spectroscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis were used to probe the effect of mutagenesis on the thermal stability of the protein. Secondary structure analysis by amide I ir spectroscopy showed that the wild-type protein was predominantly composed of beta-sheet, which supports the x-ray crystal structure information (A. Hirsch, J. Breed, K. Saxena, O.-M. H. Richter, B. Ludwig, K. Diederichs, and W. Welte, FEBS Letters, 1997, Vol. 404, pp. 208-210). The mutants E81Q, W74C, and E81Q/D148N were shown to have similar secondary structure composition as the wild type. Wild-type protein and the mutants in detergent micelles underwent irreversible denaturation as a result of heating. Transition temperature calculated from the amide I analysis revealed that mutant porins were slightly less stable compared to the wild type. The protein in micelles showed complete monomerization of the trimer above 85 degrees C. In native-like conditions (provided by liposomes), no change was observed in the secondary structure of the protein until 95 degrees C. This is supported by SDS-PAGE as no change in quaternary structure was observed, proving that the proteins are structurally thermostable in liposomes as compared to micelles. Our studies demonstrated that porins resistant to detergents and proteases are highly thermostable as well.

Loss-of-function mutations in 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) 2 in humans and mice result in loss of both the white and brown adipose tissues from birth. AGPAT2 generates precursors for the synthesis of glycerophospholipids and triacylglycerols. Loss of adipose tissue, or lipodystrophy, results in hyperinsulinemia, diabetes mellitus, and severe hepatic steatosis. Here, we analyzed biochemical properties of human AGPAT2 and its close homolog, AGPAT1, and we studied their role in liver by transducing their expression via recombinant adenoviruses in Agpat2 ؊/؊ mice. The in vitro substrate specificities of AGPAT1 and AGPAT2 are quite similar for lysophosphatidic acid and acyl-CoA. Protein homology modeling of both the AGPATs with glycerol-3-phosphate acyltransferase 1 (GPAT1) revealed that they have similar tertiary protein structure, which is consistent with their similar substrate specificities. When co-expressed, both isoforms co-localize to the endoplasmic reticulum. Despite such similarities, restoring AGPAT activity in liver by overexpression of either AGPAT1 or AGPAT2 in Agpat2 ؊/؊ mice failed to ameliorate the hepatic steatosis. From these studies, we suggest that the role of AGPAT1 or AGPAT2 in liver lipogenesis is minimal and that accumulation of liver fat is primarily a consequence of insulin resistance and loss of adipose tissue in Agpat2 ؊/؊ mice.

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Lipid protein interactions play a key role in the stability and function of various membrane proteins. Earlier we have reported the extreme thermal stability of porin from Paracoccus denitrificans reconstituted into liposomes. Here, we used Fourier transform infrared spectroscopy for a label free analysis of the global secondary structural changes and local changes in the tyrosine microenvironment. Our results show that a mixed lipid system (non-uniform bilayer) optimizes the thermal stability of porin as compared to the porin in pure lipids (uniform bilayer) or detergent micelles. This is in line with the fact that the bacterial outer membrane is a dynamic system made up of lipids of varying chain lengths, head groups and the barrel wall height contacting the membrane is uneven.