BBA - Molecular and Cell Biology of Lipids (v.1831, #9)

Porcine pancreatic lipase related protein 2 has high triglyceride lipase activity in the absence of colipase by Xunjun Xiao; Leah E. Ross; Wednesday A. Sevilla; Yan Wang; Mark E. Lowe (1435-1441).
Efficient dietary fat digestion is essential for newborns who consume more dietary fat per body weight than at any other time of life. In many mammalian newborns, pancreatic lipase related protein 2 (PLRP2) is the predominant duodenal lipase. Pigs may be an exception since PLRP2 expression has been documented in the intestine but not in the pancreas. Because of the differences in tissue-specific expression, we hypothesized that the kinetic properties of porcine PLRP2 would differ from those of other mammals. To characterize its properties, recombinant porcine PLRP2 was expressed in HEK293T cells and purified to homogeneity. Porcine PLRP2 had activity against tributyrin, trioctanoin and triolein. The activity was not inhibited by bile salts and colipase, which is required for the activity of pancreatic triglyceride lipase (PTL), minimally stimulated PLRP2 activity. Similar to PLRP2 from other species, PLRP2 from pigs had activity against galactolipids and phospholipids. Importantly, porcine PLRP2 hydrolyzed a variety of dietary substrates including pasteurized human mother's milk and infant formula and its activity was comparable to that of PTL. In conclusion, porcine PLRP2 has broad substrate specificity and has high triglyceride lipase activity even in the absence of colipase. The data suggest that porcine PLRP2 would be a suitable lipase for inclusion in recombinant preparations for pancreatic enzyme replacement therapy.
Keywords: Galactolipid; Colipase; Lipase; Phospholipid; Triglyceride;

Aging affects high-density lipoprotein composition and function by Michael Holzer; Markus Trieb; Viktoria Konya; Christian Wadsack; Akos Heinemann; Gunther Marsche (1442-1448).
Most coronary deaths occur in patients older than 65 years. Age associated alterations in the composition and function of high-density lipoproteins (HDL) may contribute to cardiovascular mortality. The effect of advanced age on the composition and function of HDL is not well understood.HDL was isolated from healthy young and elderly subjects. HDL composition, cellular cholesterol efflux/uptake, anti-oxidant properties and paraoxonase activity were assessed. We observed a 3-fold increase of the acute phase protein serum amyloid A, an increased content of complement C3 and proteins involved in endopeptidase/protease inhibition in HDL of elderly subjects, whereas levels of apolipoprotein E were significantly decreased. HDL from elderly subjects contained less cholesterol but increased sphingomyelin. Most importantly, HDL from elderly subjects showed defective antioxidant properties, lower paraoxonase 1 activity and was more rapidly taken up by macrophages, whereas cholesterol efflux capability was not altered.These findings suggest that aging alters HDL composition, resulting in functional impairment that may contribute to the onset/progression of cardiovascular disease.Display Omitted
Keywords: Proteome; Cardiovascular disease; Paraoxonase; Anti-oxidative activity;

A structural model of PpoA derived from SAXS-analysis—Implications for substrate conversion by Christian Koch; Giancarlo Tria; Alistair J. Fielding; Florian Brodhun; Oliver Valerius; Kirstin Feussner; Gerhard H. Braus; Dmitri I. Svergun; Marina Bennati; Ivo Feussner (1449-1457).
In plants and mammals, oxylipins may be synthesized via multi step processes that consist of dioxygenation and isomerization of the intermediately formed hydroperoxy fatty acid. These processes are typically catalyzed by two distinct enzyme classes: dioxygenases and cytochrome P450 enzymes. In ascomycetes biosynthesis of oxylipins may proceed by a similar two-step pathway. An important difference, however, is that both enzymatic activities may be combined in a single bifunctional enzyme. These types of enzymes are named Psi-factor producing oxygenases (Ppo). Here, the spatial organization of the two domains of PpoA from Aspergillus nidulans was analyzed by small-angle X-ray scattering and the obtained data show that the enzyme exhibits a relatively flat trimeric shape. Atomic structures of the single domains were obtained by template-based structure prediction and docked into the enzyme envelope of the low resolution structure obtained by SAXS. EPR-based distance measurements between the tyrosyl radicals formed in the activated dioxygenase domain of the enzyme supported the trimeric structure obtained from SAXS and the previous assignment of Tyr374 as radical-site in PpoA. Furthermore, two phenylalanine residues in the cytochrome P450 domain were shown to modulate the specificity of hydroperoxy fatty acid rearrangement.
Keywords: Cytochrome P450; Dioxygenase; Double electron–electron resonance; Oxylipin; Psi-factor producing oxygenase; Small-angle X-ray scattering;

Biochemical changes accompanying apoptotic cell death in retinoblastoma cancer cells treated with lipogenic enzyme inhibitors by Suryanarayanan Vandhana; Karunakaran Coral; Udayakumar Jayanthi; Perinkulam Ravi Deepa; Subramanian Krishnakumar (1458-1466).
Retinoblastoma (RB) is a malignant intra-ocular neoplasm that affects children (usually below the age of 5 years). In addition to conventional chemotherapy, novel therapeutic strategies that target metabolic pathways such as glycolysis and lipid metabolism are emerging. Fatty acid synthase (FASN), a lipogenic multi-enzyme complex, is over-expressed in retinoblastoma cancer. The present study evaluated the biochemical basis of FASN inhibition induced apoptosis in cultured Y79 RB cells. FASN inhibitors (cerulenin, triclosan and orlistat) significantly inhibited FASN enzyme activity (P < 0.05) in Y79 RB cells. This was accompanied by a decrease in palmitate synthesis (end-product depletion), and increased malonyl CoA levels (substrate accumulation). Differential lipid profile was biochemically estimated in neoplastic (Y79 RB) and non-neoplastic (3T3) cells subjected to FASN inhibition. The relative proportion of phosphatidyl choline to neutral lipids (triglyceride + total cholesterol) in Y79 RB cancer cells was found to be higher than the non-neoplastic cells, indicative of altered lipid distribution and utilization in tumor cells. FASN inhibitor treated Y79 RB and fibroblast cells showed decrease in the cellular lipids (triglyceride, cholesterol and phosphatidyl choline) levels. Apoptotic DNA damage induced by FASN inhibitors was accompanied by enhanced lipid peroxidation.
Keywords: Fatty acid synthase; Retinoblastoma; Malonyl CoA; Apoptosis; Phospholipid; Oxidative stress;

Peroxisomes contribute to the acylcarnitine production when the carnitine shuttle is deficient by Sara Violante; Lodewijk IJlst; Heleen te Brinke; Janet Koster; Isabel Tavares de Almeida; Ronald J.A. Wanders; Fátima V. Ventura; Sander M. Houten (1467-1474).
Fatty acid β-oxidation may occur in both mitochondria and peroxisomes. While peroxisomes oxidize specific carboxylic acids such as very long-chain fatty acids, branched-chain fatty acids, bile acids, and fatty dicarboxylic acids, mitochondria oxidize long-, medium-, and short-chain fatty acids. Oxidation of long-chain substrates requires the carnitine shuttle for mitochondrial access but medium-chain fatty acid oxidation is generally considered carnitine-independent. Using control and carnitine palmitoyltransferase 2 (CPT2)- and carnitine/acylcarnitine translocase (CACT)-deficient human fibroblasts, we investigated the oxidation of lauric acid (C12:0). Measurement of the acylcarnitine profile in the extracellular medium revealed significantly elevated levels of extracellular C10- and C12-carnitine in CPT2- and CACT-deficient fibroblasts. The accumulation of C12-carnitine indicates that lauric acid also uses the carnitine shuttle to access mitochondria. Moreover, the accumulation of extracellular C10-carnitine in CPT2- and CACT-deficient cells suggests an extramitochondrial pathway for the oxidation of lauric acid. Indeed, in the absence of peroxisomes C10-carnitine is not produced, proving that this intermediate is a product of peroxisomal β-oxidation. In conclusion, when the carnitine shuttle is impaired lauric acid is partly oxidized in peroxisomes. This peroxisomal oxidation could be a compensatory mechanism to metabolize straight medium- and long-chain fatty acids, especially in cases of mitochondrial fatty acid β-oxidation deficiency or overload.
Keywords: Fatty acid β-oxidation; Mitochondria; Medium-chain fatty acids; Carnitine palmitoyltransferase 1; Carnitine palmitoyltransferase 2; Carnitine/acylcarnitine translocase;

New insights on glucosylated lipids: Metabolism and functions by Yohei Ishibashi; Ayako Kohyama-Koganeya; Yoshio Hirabayashi (1475-1485).
Ceramide, cholesterol, and phosphatidic acid are major basic structures for cell membrane lipids. These lipids are modified with glucose to generate glucosylceramide (GlcCer), cholesterylglucoside (ChlGlc), and phosphatidylglucoside (PtdGlc), respectively. Glucosylation dramatically changes the functional properties of lipids. For instance, ceramide acts as a strong tumor suppressor that causes apoptosis and cell cycle arrest, while GlcCer has an opposite effect, downregulating ceramide activities. All glucosylated lipids are enriched in lipid rafts or microdomains and play fundamental roles in a variety of cellular processes. In this review, we discuss the biological functions and metabolism of these three glucosylated lipids.
Keywords: Glycolipid; Sphingolipid; Glucosylceramide; Glucosylation; Cholesterylglucoside; Phosphatidylglucoside;

Characterization of the two intracellular lipases of Y. lipolytica encoded by TGL3 and TGL4 genes: New insights into the role of intracellular lipases and lipid body organisation by Thierry Dulermo; Brigitte Tréton; Athanasios Beopoulos; Affoué Philomène Kabran Gnankon; Ramdane Haddouche; Jean-Marc Nicaud (1486-1495).
Eukaryotes store lipids in a specialised organelle, the lipid body (LB), mainly as triglycerides (TAGs). Both the rates of synthesis and degradation contribute to the control of the accumulation of TAGs. The synthesis of TAGs in yeasts has been well documented, especially in the model yeast Saccharomyces cerevisiae and in the oleaginous yeast Yarrowia lipolytica. However, descriptions of the processes involved in TAG degradation are more scarce and mostly for S. cerevisiae. Here, we report the characterisation of two Y. lipolytica genes, YlTGL3 and YlTGL4, encoding intracellular lipases involved in TAG degradation. The two proteins are localised in lipid bodies, and YlTgl4 was mainly found at the interface between LBs. Surprisingly, the spatial organisation of YlTgl3 and YlTgl4 depends on the culture medium and on the physiological phase of the cell. Inactivation of one or both genes doubles the lipid accumulation capacity of Y. lipolytica, increasing the cell's capacity to accumulate TAGs. The amino acid sequence of YlTgl4 contains the consensus sequence motif (G/A)XSXG, typical of serine hydrolases, whereas YlTgl3 does not. Single and double mutants are unable to degrade TAGs, and higher expression of YlTgl4 correlates with TAG degradation. Therefore, we propose that YlTgl4 is the main lipase responsible for TAG degradation and that YlTgl3 may act as a positive regulator of YlTgl4 rather than a functional lipase. Thus, contrary to S. cerevisiae, Y. lipolytica possesses two intracellular lipases with distinct roles and with distinct localisations in the LB.
Keywords: Triacylglyceride; Intracellular lipase; Lipid; Yarrowia lipolytica; Lipid body; Protein localization;