Richard J. Pease

Mobilisation of triacylglycerol stores.

Triacylglycerol (TAG) is an energy dense substance which is stored by several body tissues, principally adipose tissue and the liver. Utilisation of stored TAG as an energy source requires its mobilisation from these depots and transfer into the blood plasma. The means by which TAG is mobilised differs in adipose tissue and liver although the regulation of lipid metabolism in each of these organs is interdependent and synchronised in an integrated manner. This review deals principally with the mechanism of hepatic TAG mobilisation since this is a rapidly expanding area of research and may have important implications for the regulation of plasma very-low-density lipoprotein metabolism. TAG mobilisation plays an important role in fuel selection in non-hepatic tissues such as cardiac muscle and pancreatic islets and these aspects are also reviewed briefly. Finally, studies of certain rare inherited disorders of neutral lipid storage and mobilisation may provide useful information about the normal enzymology of TAG mobilisation in healthy tissues.

The human hydroxyacylglutathione hydrolase (HAGH) gene encodes both cytosolic and mitochondrial forms of glyoxalase II

In yeast and higher plants, separate genes encode the cytosolic and mitochondrial forms of glyoxalase II. In contrast, although glyoxalase II activity has been detected both in the cytosol and mitochondria of mammals, only a single gene encoding glyoxalase II has been identified. Previously it was thought that this gene (the hydroxyacylglutathione hydrolase gene), comprised 8 exons that are transcribed into mRNA and that the resulting mRNA species encoded a single cytosolic form of glyoxalase II. Here we show that this gene gives rise to two distinct mRNA species transcribed from 9 and 10 exons, respectively. The 9-exon-derived transcript encodes two protein species: mitochondrially targeted glyoxylase II, which is initiated from an AUG codon in a previously uncharacterized part of the mRNA sequence, and cytosolic glyoxalase II, which is initiated by internal ribosome entry at a downstream AUG codon. The transcript deriving from 10 exons has an in-frame termination codon between the two initiating AUG codons and hence only encodes the cytosolic form of the protein. Confocal fluorescence microscopy indicates that the mitochondrially targeted form of glyoxalase II is directed to the mitochondrial matrix. Analysis of glyoxalase II mRNA sequences from a number of species indicates that dual initiation from alternative AUG codons is conserved throughout vertebrates.

Complement C3 is a novel plasma clot component with anti-fibrinolytic properties

Background and method: Increased plasma clot density and prolonged lysis times are associated with cardiovascular disease. In this study, we employed a functional proteomics approach to identify novel clot components which may influence clot phenotypes. Results: Analysis of perfused, solubilised plasma clots identified inflammatory proteins, including complement C3, as novel clot components. Analysis of paired plasma and serum samples confirmed concentration-dependent incorporation of C3 into clots. Surface plasmon resonance indicated high-affinity binding interactions between C3 and fibrinogen and fibrin. Turbidimetric clotting and lysis assays indicated C3 impaired fibrinolysis in a concentration-dependent manner, both in vitro and ex vivo. Conclusion: These data indicate functional interactions between complement C3 and fibrin leading to prolonged fibrinolysis. These interactions are physiologically relevant in the context of protection following injury and suggest a mechanistic link between increased plasma C3 concentration and acute cardiovascular thrombotic events.

Association of coagulation factor XIII-A with Golgi proteins within monocyte-macrophages: implications for subcellular trafficking and secretion.

Factor XIII-A (FXIII-A) is present in the cytosol of platelets, megakaryocytes, monocytes, osteoblasts, and macrophages and may be released from cells by a nonclassical pathway. We observed that plasma FXIII-A levels were unchanged in thrombocytopenic mice (Bcl-x(Plt20/Plt20) and Mpl(-/-)), which implicates nonclassical secretion from nucleated cells as the source of plasma FXIII-A. We, therefore, examined the intracellular targeting of FXIII-A in the THP-1 (monocyte/macrophage) cell line and in human monocyte-derived macrophages. Metabolic labeling of THP-1 cells did not show release of (35)S-FXIII-A either under basal conditions or when interleukin 1-beta was released in response to cell stress. However, immunofluorescence of THP-1 cells and primary macrophages showed that FXIII-A associated with podosomes and other structures adjacent to the plasma membrane, which also contain trans-Golgi network protein-46 and Golgi matrix protein-130 (GM130) but not the endoplasmic reticulum luminal protein, protein disulphide isomerase. Further, FXIII-A was present in GM130-positive intracellular vesicles that could mediate its transport, and in other contexts GM130 and its binding partner GRASP have been implicated in the delivery of nonclassically secreted proteins to the plasma membrane. Hence, this mechanism may precede FXIII-A release into the extracellular matrix from macrophages and its release into plasma from the cell type of origin.

Well-Defined Regions of Apolipoprotein B-100 Undergo Conformational Change During Its Intravascular Metabolism

Apolipoprotein B (apoB)-100-containing lipoproteins are secreted from the liver as large triglyceride-rich very low density lipoproteins (VLDLs) into the circulation, where they are transformed, through the action of lipases and plasma lipid transfer proteins, into smaller, less buoyant, cholesteryl ester-rich low density lipoproteins (LDLs). As a consequence of this intravascular metabolism, apoB-containing lipoproteins are heterogeneous in size, in hydrated density, in surface charge, and in lipid and apolipoprotein composition. To identify specific regions of apoB that may undergo conformational changes during the intravascular transformation of VLDLs into LDLs, we have used a panel of 29 well-characterized anti-apoB monoclonal antibodies to determine whether individual apoB epitopes are differentially expressed in VLDL, intermediate density lipoprotein (IDL), and LDL subfractions isolated from 6 normolipidemic subjects. When analyzed in a solid-phase radioimmunoassay, the expression of most epitopes was remarkably similar in VLDLs, IDLs, and LDLs. Two epitopes that are close to the apoB LDL receptor-binding site show an increased expression in large (1.019 to 1.028 g/mL), medium (1.028 to 1.041 g/mL), and small (1.041 to 1.063 g/mL) LDLs compared with VLDLs and IDLs, and 2 epitopes situated between apoB residues 4342 and 4536 are significantly more immunoreactive in small and medium-sized LDLs compared with VLDLs, IDLs, and large LDLs. Therefore, as VLDL is converted to LDL, conformational changes identified by monoclonal antibodies occur at precise points in the metabolic cascade and are limited to well-defined regions of apoB structure. These conformational changes may correspond to alterations in apoB functional activities.

Metabolic characteristics of a human hepatoma cell line stably transfected with hormone-sensitive lipase.

Clones of HepG2 cells were selected that stably express the cDNA for hormone-sensitive lipase (HSL). When cells were cultured in the presence of labelled extracellular oleate, accumulation of labelled fatty acid as cellular triacylglycerol (TAG) was significantly lower in the transfectants compared with the wild-type cells. There was no change in the net rate of phospholipid (PL) synthesis. Culture of cells containing isotopically prelabelled TAG resulted in a greater net loss of TAG from the transfected cells than from the wild-type cells. The excess loss of labelled TAG was primarily due to an increased TAG fatty acid oxidation. Free fatty acid release into the medium was not increased in the transfectants, nor was the very low rate of lipoprotein lipid secretion. Also, there was no increased net trafficking of fatty acids from TAG into PLs. Changes in the 3H:14C ratio of TAG prelabelled with [3H]glycerol and [14C]oleate suggested that none of excess TAG fatty acid released in the transfected cells underwent intracellular re-esterification to TAG prior to oxidation. The results suggest that fatty acids mobilized by HSL are directed immediately into the oxidative pathway and are not available for biosynthetic processes. It appears likely, therefore, that intracellular TAG-derived fatty acids which enter the oxidative pathway exist in a different compartment from those that are directed towards synthesis.

The activation peptide cleft exposed by thrombin cleavage of FXIII-A 2 contains a recognition site for the fibrinogen α chain

Formation of a stable fibrin clot is dependent on interactions between factor XIII and fibrin. We have previously identified a key residue on the αC of fibrin(ogen) (Glu396) involved in binding activated factor XIII-A(2) (FXIII-A(2)*); however, the functional role of this interaction and binding site(s) on FXIII-A(2)* remains unknown. Here we (1) characterized the functional implications of this interaction; (2) identified by liquid-chromatography-tandem mass spectrometry the interacting residues on FXIII-A(2)* following chemical cross-linking of fibrin(ogen) αC389-402 peptides to FXIII-A(2)*; and (3) carried out molecular modeling of the FXIII-A(2)*/peptide complex to identify contact site(s) involved. Results demonstrated that inhibition of the FXIII-A(2)*/αC interaction using αC389-402 peptide (Pep1) significantly decreased incorporation of biotinamido-pentylamine and α2-antiplasmin to fibrin, and fibrin cross-linking, in contrast to Pep1-E396A and scrambled peptide controls. Pep1 did not inhibit transglutaminase-2 activity, and incorporation of biotinyl-TVQQEL to fibrin was only weakly inhibited. Molecular modeling predicted that Pep1 binds the activation peptide cleft (AP-cleft) within the β-sandwich domain of FXIII-A(2)* localizing αC cross-linking Q366 to the FXIII-A(2)* active site. Our findings demonstrate that binding of fibrin αC389-402 to the AP-cleft is fundamental to clot stabilization and presents this region of FXIII-A(2)* as a potential site involved in glutamine-donor substrate recognition.

The liver isoform of carnitine palmitoyltransferase 1 is not targeted to the endoplasmic reticulum

Liver microsomal fractions contain a malonyl-CoA-inhibitable carnitine acyltransferase (CAT) activity. It has been proposed [Fraser, Corstorphine, Price and Zammit (1999) FEBS Lett. 446, 69-74] that this microsomal CAT activity is due to the liver form of carnitine palmitoyltransferase 1 (L-CPT1) being targeted to the endoplasmic reticulum (ER) membrane as well as to mitochondria, possibly by an N-terminal signal sequence [Cohen, Guillerault, Girard and Prip-Buus (2001) J. Biol. Chem. 276, 5403-5411]. COS-1 cells were transiently transfected to express a fusion protein in which enhanced green fluorescent protein was fused to the C-terminus of L-CPT1. Confocal microscopy showed that this fusion protein was localized to mitochondria, and possibly to peroxisomes, but not to the ER. cDNAs corresponding to truncated (amino acids 1-328) or full-length L-CPT1 were transcribed and translated in the presence of canine pancreatic microsomes. However, there was no evidence of authentic insertion of CPT1 into the ER membrane. Rat liver microsomal fractions purified by sucrose-density-gradient centrifugation contained an 88 kDa protein (p88) which was recognized by an anti-L-CPT1 antibody and by 2,4-dinitrophenol-etomoxiryl-CoA, a covalent inhibitor of L-CPT1. Abundance of p88 and malonyl-CoA-inhibitable CAT activity were increased approx. 3-fold by starvation for 24 h. Deoxycholate solubilized p88 and malonyl-CoA-inhibitable CAT activity from microsomes to approximately the same extent. The microsomal fraction contained porin, which, relative to total protein, was as abundant as in crude mitochondrial outer membranes fractions. It is concluded that L-CPT1 is not targeted to the ER membrane and that malonyl-CoA CAT in microsomal fractions is L-CPT1 that is derived from mitochondria, possibly from membrane contact sites.

Effect of transglutaminase 2 (TG2) deficiency on atherosclerotic plaque stability in the apolipoprotein E deficient mouse.

BACKGROUND Transglutaminase 2 (TG2), a cross-linking enzyme that confers supra-molecular structures with extra rigidity and resistance against proteolytic degradation, is expressed in the shoulder regions of human atherosclerotic plaques. It has been proposed that TG2 prevents tearing and promotes plaque repair at these potential weak points, and also promotes ectopic calcification of arteries. TG2 is also expressed within plaques that develop within the brachiocephalic arteries of apolipoprotein E (apoE) deficient mice. OBJECTIVES To determine the role that TG2 plays in plaque development and calcification, mice were bred that were doubly deficient in apoE and TG2, and were maintained on a high-fat diet for 6 months. RESULTS Lesion size and composition were not significantly altered in the apoE/TG2 double-knockout mice, with the exception of a 9.7% decrease in the proportion of the plaque occupied by lipid (p=0.032). The frequency of buried fibrous caps within brachiocephalic plaques was significantly higher in male than in female mice, but TG2 deficiency had no effect on either gender. The extent of lesion calcification varied markedly between individual mice, but it was not decreased in the apoE/TG2 double-knockout mice. CONCLUSION These data indicate that, in the apoE knockout mouse model of atherosclerosis, TG2 does not influence plaque composition or calcification. The data further suggest that TG2 does not influence plaque stability or repair in these mice.

Normal Bone Deposition Occurs in Mice Deficient in Factor XIII-A and Transglutaminase 2

Transglutaminase activity has been widely implicated in bone deposition. A predominant role has been proposed for factor (F)XIII-A and a subsidiary role suggested for the homologous protein, transglutaminase 2. Full-length FXIII-A is an 83kDa protransglutaminase that is present both in plasma and also in haematopoietic and connective tissue lineages. Several studies have reported expression in murine cells, including osteocytes, of a 37 kDa protein that reacts with the monoclonal anti-FXIII-A antibody AC-1A1. This protein was presumed to be a catalytically active fragment of FXIII-A-83 and to play a major role in bone deposition. We detected a 37 kDa AC-1A1 reactive protein in FXIII-A mRNA negative cell lines and in tissues from FXIII-A(-/-) mice. By mass spectrometric sequencing of AC-1A1 immunoprecipitates, we identified this protein as transaldolase-1, and confirmed that recombinant transaldolase-1 is recognised by AC-1A1. We have also shown that bone deposition is normal in FXIII-A(-/-).TG2(-/-) double knockout mice, casting doubt on the role of transglutaminases in bone mineralisation. Various studies have used antibody AC-1A1 for immunohistochemistry or immunofluorescence. We observe strong FXIII-A dependent staining in paraffin embedded mouse heart sections, with relatively low background in non-expressing mouse cells. In contrast, FXIII-A independent staining predominates in cultured human cells using a standard immunofluorescence procedure. Immunofluorescence is present in membrane compartments that are expected to lack transaldolase, indicating that other off-target antigens are recognised by AC-1A1. This has significant implications for studies that have used this approach to define the subcellular trafficking of FXIII-A in osteocytes.