Michael A. Billett

The assembly of triacylglycerol-rich lipoproteins: an essential role for the microsomal triacylglycerol transfer protein

Raised plasma triacylglycerol is an independent risk factor for cardiovascular disease, and an understanding of factors which regulate the synthesis and degradation of lipoproteins which carry triacylglycerol in the blood may lead to novel approaches to the treatment of hypertriacylglycerolaemia. An active microsomal triacylglycerol transfer protein (MTP) is essential for the assembly of particles which transport triacylglycerol through the circulation. After absorption in the intestine, dietary fat and fat-soluble vitamins are incorporated into chylomicrons in the intestinal epithelial cells, and these lipoproteins reach the bloodstream via the lymphatic system. Patients with the rare genetic disorder, abetalipoproteinaemia, in which MTP activity is absent, present clinically with fat-soluble vitamin and essential fatty acid deficiency, indicating a key role for MTP in the movement of fat into the body. The triacylglycerol-rich lipoprotein found in fasting blood, VLDL, is assembled in the liver by an MTP-dependent process similar to chylomicron assembly, and transports triacylglycerol to extra-hepatic tissues such as adipose tissue and heart. In the absence of MTP activity, VLDL are not synthesized and only extremely low levels of triacylglycerol are present in the blood. Dietary components, including fat, cholesterol and ethanol, can modify the expression of the MTP gene and, hence, MTP activity. The present review summarizes current knowledge of the role of MTP in the assembly and secretion of triacylglycerol-rich lipoproteins, and the regulation of its activity in both animal and cell systems.

The effect of different dietary fatty acids on lipoprotein metabolism: concentration-dependent effects of diets enriched in oleic, myristic, palmitic and stearic acids

While it is well established that the fatty acid composition of dietary fat is important in determining plasma lipoprotein cholesterol concentrations, the effects of changing the absolute quantities of the individual fatty acids are less clear. In the present study Golden Syrian hamsters were fed on isoenergetic, low cholesterol (0.05 g/kg) diets containing 100, 150 or 200 g added fat/kg. This consisted of triolein (TO) alone, or equal proportions of TO and either trimyristin (TM), tripalmitin (TP) or tristearin (TS). Each trial also included a control group fed on a diet containing 50 g TO/kg. As the mass of TO in the diet increased, plasma VLDL-cholesterol concentrations rose. The TM-rich diets produced a concentration-dependent increase in total plasma cholesterol which was a result of significant increases in both VLDL and HDL levels. The TP-rich diets increased plasma LDL- and HDL-cholesterol levels in a concentration-dependent manner. TS-containing diets did not increase the cholesterol content of any of the major lipoprotein fractions. Hepatic LDL-receptor mRNA concentrations were significantly decreased in animals fed on TP, while apolipoprotein B mRNA concentrations were significantly increased. Thus, on a low-cholesterol diet, increasing the absolute amount of dietary palmitic acid increases LDL-cholesterol more than either myristic or stearic acid. These effects on lipoprotein metabolism may be exerted through specific modulation of the expression of the LDL receptor and apolipoprotein B genes.

Stimulation of maize invertase activity following infection by Ustilago maydis

Abstract The effect of U. maydis infection on the invertase activity of maize leaves has been studied. Infection causes a specific stimulation of an acid invertase in soluble and pellet fractions of homogenized tissue. Invertase activity is stimulated within one day after infection, and is maintained at a high level until 10 days after infection, in contrast to the progressive decline in activity with age, in healthy leaves. Analyses of soluble extracts by gel electrophoresis suggest that the invertase showing this increase is derived from the host and not the pathogen.

Hepatic microsomal triglyceride transfer protein messenger RNA concentrations are increased by dietary cholesterol in hamsters

In hamsters fed high fat diets enriched in trimyristin, tripalmitin or tristearin, increased dietary cholesterol content was associated with increased plasma concentrations of very low density lipoprotein (VLDL) cholesterol and triacylglycerol (p < 0.0001 and p = 0.0017, respectively). Hepatic microsomal triglyceride transfer protein (MTP) mRNA concentration also increased (p < 0.0001), independent of the nature of dietary fat, and was significantly correlated with the plasma VLDL lipid concentrations (p = 0.0002 and p = 0.0106 for cholesterol and triacylglycerol, respectively) and hepatic cholesterol concentrations. Increased expression of the MTP gene may be part of a coordinated response to hepatic cholesterol accumulation leading to increased VLDL lipid secretion.

DNA repair : Kinetics and thresholds

DNA damage is a critical factor in the initiation of chemically induced toxicities (including cancer), and the repair of this damage represents the cells first line of defense against the deleterious effects of these agents. The various mechanisms of DNA repair are reviewed briefly and the actions of the DNA repair protein O6-alkylguanine DNA alkyltransferase (ATase) are used to illustrate how DNA repair can protect cells against alkylating agent-induced toxicities, mutagenesis, clastogenesis, and carcinogenesis. The effectiveness of this repair protein can be measured based on its ability to deplete levels of its promutagenic substrate O6-methylguanine (O6-meG) in the DNA of cells. These studies reveal that the repair of O6-meG from DNA occurs heterogeneously, both intra- and intercellularly. Even in cells that repair O6-meG hyperefficiently, certain regions of chromatin DNA are repaired with difficulty, and in other regions they are not repaired at all; most likely this lack of repair is a result of the location of the lesion in the DNA sequence. When individual cells are compared within a tissue, some cells are clearly repair deficient, because the O6-meG can persist in DNA for many weeks, whereas in other cells, it is removed within a matter of hours. The role of these repair-deficient cells as targets for alkylating agent induced carcinogenesis is considered. The mechanisms of the homeostatic control of DNA repair function in mammalian cells are not yet well understood. Because there are now indications of the mechanisms by which the level of DNA damage may be sensed (and so influence the activity of the ATase repair protein), this is an important area for future study.DNA damage is a critical factor in the initiation of chemically induced toxicities (including cancer), and the repair of this damage represents the cells first line of defense against the deleterious effects of these agents. The various mechanisms of DNA repair are reviewed briefly and the actions of the DNA repair protein 06-alkylguanine-DNA alkyltransferase (ATase) are used to illustrate how DNA repair can protect cells against alkylating agent-induced toxicities, mutagenesis, clastogenesis, and carcinogenesis. The effectiveness of this repair protein can be measured based on its ability to deplete levels of its promutagenic substrate O 6-methylguanine (O 6-meG) in the DNA of cells. These studies reveal that the repair of O 6-meG from DNA occurs heterogeneously, both intra- and intercellularly. Even in cells that repair O 6-meG hyperefficiently, certain regions of chromatin DNA are repaired with difficulty, and in other regions they are not repaired at all; most likely this lack of repair is a result of the location of the lesion in the DNA sequence. When individual cells are compared within a tissue, some cells are clearly repair deficient, because the O 6-meG can persist in DNA for many weeks, whereas in other cells, it is removed within a matter of hours. The role of these repair-deficient cells as targets for alkylating agent-induced carcinogenesis is considered. The mechanisms of the homeostatic control of DNA repair function in mammalian cells are not yet well understood. Because there are now indications of the mechanisms by which the level of DNA damage may be sensed (and so influence the activity of the ATase repair protein), this is an important area for future study.

Interactive effects of dietary cholesterol and different saturated fatty acids on lipoprotein metabolism in the hamster

The present study examines the interactive effects of three fatty acids: myristic, palmitic and stearic acids, with dietary cholesterol, on lipoprotein metabolism in the hamster. Each saturated fatty acid was fed at a concentration of 100 g pure synthetic triacylglycerol/kg in the presence of 100 g triolein/kg and was fed in the presence of 0.05, 1.2 or 2.4 g dietary cholesterol/kg. Dietary cholesterol increased the concentration of cholesterol in each of the major plasma lipoprotein fractions. The largest effects on VLDL and LDL were seen in the presence of tripalmitin where the increase between the lowest and highest dietary cholesterol groups were 129% and 38% respectively. In contrast, HDL showed the greatest change in the tristearin group when the equivalent increase was 59%. No interactive effects of dietary cholesterol and fat were seen on hepatic mRNA concentrations for the LDL receptor, hydroxymethylglutaryl-CoA reductase or the microsomal triacylglycerol transfer protein. As the amount of cholesterol in the diet increased, large differences were seen in the storage of hepatic cholesterol ester. At the highest dietary cholesterol intake the amount of hepatic cholesterol ester was 1.7-fold higher in the animals fed trimyristin compared with those fed tripalmitin. These results suggest that, as the amount of cholesterol in the diet is increased, palmitic acid becomes more hypercholesterolaemic. This is associated with a reduced ability to store cholesterol ester in the liver.

Localisation of 26S proteasomes with different subunit composition in insect muscles undergoing programmed cell death.

The 26S proteasome is a large multisubunit complex involved in degrading both cytoplasmic and nuclear proteins. We have investigated the subcellular distribution of four regulatory ATPase subunits (S6 (TBP7/MS73), S6′ (TBP1), S7 (MSS1), and S10b (SUG2)) together with components of 20S proteasomes in the intersegmental muscles (ISM) of Manduca sexta during developmentally programmed cell death (PCD). Immunogold electron microscopy shows that S6 is located in the heterochromatic part of nuclei of ISM fibres. S6′ is present in degraded material only outside intact fibres. S7 can be detected in nuclei, cytoplasm and also in degraded material. S10b, on the other hand, is initially found in nuclei and subsequently in degraded cytoplasmic locations during PCD. 20S proteasomes are present in all areas where ATPase subunits are detected, consistent with the presence of intact 26S proteasomes. These results are discussed in terms of heterogeneity of 26S proteasomes, 26S proteasome disassembly and the possible role of ATPases in non-proteasome complexes in the process of PCD.

The 26S-proteasome: regulation and substrate recognition

There is extensive reprogramming of the ATPase regulators of the 26S proteasome before the programmed elimination of the abdominal intersegmental muscles (ISM) after eclosion in Manduca sexta [1]. This extensive ATPase reprogramming only occurs in ISM which are destined to die and not in flight muscle (FM). The MS73 ATPase also increases in the proleg retractor muscles which die at a developmentally different stage to ISM. The non-ATPase regulator S5a shows a similar increase to the ATPase regulators. We have cloned the Manduca SUG2 ATPase and shown that this ATPase is a component of the 26S proteasome. This ATPase shows a similar increase in concentration to the other ATPases in 26S proteasomes before muscle death. The SUG2 ATPase is also associated with other smaller complexes besides the 26S proteasome which act as activators of the 26S proteasome. Finally, in a yeast two-hybrid genetic screen we have identified a protein in human brain which interacts with the MS73 ATPase (and human S6). The interacting protein contains 6 ankyrin repeats and is co-immunoprecipitated with anti-MS73 antiserum after in vitro transcription/translation. The ankyrin repeat protein may interact with the MS73 ATPase as part of the substrate recognition process by the 26S proteasome. Many proteins degraded by the 26S proteasome contain ankyrin repeats, e.g. IkB and some cyclins: binding through ankyrin repeats to an ATPase regulator may complement protein ubiquitination and S5a binding as recognition signals by the 26S proteasome.

Increased Expression of Genes for Platelet-Derived Growth Factor in Circulating Mononuclear Cells of Hypercholesterolemic Patients

Platelet-derived growth factor (PDGF) is implicated in the accumulation of smooth muscle cells in atherosclerotic lesions following monocyte migration through the vascular endothelium. We show here a 15- to 20-fold increase in expression of PDGF-A and -B genes (as measured by a quantitative reverse transcription-polymerase chain reaction assay of mRNA concentration) in circulating monocytes of hypercholesterolemic and hyperlipidemic patients compared with normocholesterolemic individuals. Strong positive correlations between PDGF-A and -B mRNA concentrations indicate that the two genes are coordinately regulated in mononuclear cells in both normal and hypercholesterolemic individuals. PDGF gene expression in patients correlates with concentrations of plasma total cholesterol and low-density lipoprotein cholesterol, a proven risk factor for atherosclerosis. Activation of monocyte PDGF expression may be an important component of the atherosclerotic risk associated with raised cholesterol levels and may represent an essential step in the early stages of atherogenesis. However, the marked increases in PDGF mRNA levels in patients with modest hypercholesterolemia compared with normal subjects suggest that other factors are involved. The relationship of monocyte PDGF expression to other atherosclerotic risk factors and to the different stages of atherosclerosis needs to be carefully evaluated.

A 220-kDa Activator Complex of the 26 S Proteasome in Insects and Humans A ROLE IN TYPE II PROGRAMMED INSECT MUSCLE CELL DEATH AND CROSS-ACTIVATION OF PROTEASOMES FROM DIFFERENT SPECIES

The S10b (SUG2) ATPase cDNA has been cloned by reverse transcription-polymerase chain reaction/rapid amplification of cDNA ends from mRNA of intersegmental muscles of the tobacco horn moth (Manduca sexta). The S10b ATPase is a component of the 26 S proteasome, and its concentration and that of its mRNA increase dramatically during development in a manner similar to other ATPases of the 19 S regulator of the 26 S proteasome. The S10b and S6′ (TBP1) ATPases are also present in a complex of ∼220 kDa in intersegmental muscles. The 220-kDa complex markedly activates (2–10-fold) the 26 S proteasome, even when bound to anti-S10b antibodies immobilized on Sepharose, and increases in concentration ∼5-fold like the 26 S proteasome in the intersegmental muscles in preparation for the programmed death of the muscle cells. A similar activator complex is present in human brain and placenta. Free activator complexes cross-activate: the Manduca complex activates rat skeletal muscle 26 S proteasomes, and the placental complex activates Manduca 26 S proteasomes. The placental activator complex contains S10b and S6′, but not p27. This 220-kDa activator complex has been evolutionarily conserved between species from insect to man and may have a fundamental role in proteasome regulation.