Guy P. Mannaerts

Inorganic and organic phosphate measurements in the nanomolar range

A procedure, based on the complex formation of malachite green with phosphomolybdate under acidic conditions, to measure inorganic orthophosphate in the nanomolar range is described. The addition of polyvinyl alcohol is required to stabilize the dye-phosphomolybdate complex. The advantages of the assay are simplicity, stability of the reagents, and high sensitivity. Due to the high permissible acidity in the assay (0.9 N H2SO4), the method can be adapted easily to measure nanomolar amounts of phosphate, liberated from organic compounds like phosphoproteins and phospholipids after wet digestion.

A mouse model for Zellweger syndrome

The cerebro-hepato-renal syndrome of Zellweger is a fatal inherited disease caused by deficient import of peroxisomal matrix proteins. The pathogenic mechanisms leading to extreme hypotonia, severe mental retardation and early death are unknown. We generated a Zellweger animal model through inactivation of the murine Pxr1 gene (formally known as Pex5) that encodes the import receptor for most peroxisomal matrix proteins. Pxr1−/− mice lacked morphologically identifiable peroxisomes and exhibited the typical biochemical abnormalities of Zellweger patients. They displayed intrauterine growth retardation, were severely hypotonic at birth and died within 72 hours. Analysis of the neocortex revealed impaired neuronal migration and maturation and extensive apoptotic death of neurons.

Inactivation of the Peroxisomal Multifunctional Protein-2 in Mice Impedes the Degradation of Not Only 2-Methyl-branched Fatty Acids and Bile Acid Intermediates but Also of Very Long Chain Fatty Acids

According to current views, peroxisomal β-oxidation is organized as two parallel pathways: the classical pathway that is responsible for the degradation of straight chain fatty acids and a more recently identified pathway that degrades branched chain fatty acids and bile acid intermediates. Multifunctional protein-2 (MFP-2), also called d-bifunctional protein, catalyzes the second (hydration) and third (dehydrogenation) reactions of the latter pathway. In order to further clarify the physiological role of this enzyme in the degradation of fatty carboxylates, MFP-2 knockout mice were generated. MFP-2 deficiency caused a severe growth retardation during the first weeks of life, resulting in the premature death of one-third of the MFP-2−/− mice. Furthermore, MFP-2-deficient mice accumulated VLCFA in brain and liver phospholipids, immature C27 bile acids in bile, and, after supplementation with phytol, pristanic and phytanic acid in liver triacylglycerols. These changes correlated with a severe impairment of peroxisomal β-oxidation of very long straight chain fatty acids (C24), 2-methyl-branched chain fatty acids, and the bile acid intermediate trihydroxycoprostanic acid in fibroblast cultures or liver homogenates derived from the MFP-2 knockout mice. In contrast, peroxisomal β-oxidation of long straight chain fatty acids (C16) was enhanced in liver tissue from MFP-2−/− mice, due to the up-regulation of the enzymes of the classical peroxisomal β-oxidation pathway. The present data indicate that MFP-2 is not only essential for the degradation of 2-methyl-branched fatty acids and the bile acid intermediates di- and trihydroxycoprostanic acid but also for the breakdown of very long chain fatty acids.

Human Pex19p Binds Peroxisomal Integral Membrane Proteins at Regions Distinct from Their Sorting Sequences

ABSTRACT The molecular machinery underlying peroxisomal membrane biogenesis is not well understood. The observation that cells deficient in the peroxins Pex3p, Pex16p, and Pex19p lack peroxisomal membrane structures suggests that these molecules are involved in the initial stages of peroxisomal membrane formation. Pex19p, a predominantly cytosolic protein that can be farnesylated, binds multiple peroxisomal integral membrane proteins, and it has been suggested that it functions as a soluble receptor for the targeting of peroxisomal membrane proteins (PMPs) to the peroxisome. An alternative view proposes that Pex19p functions as a chaperone at the peroxisomal membrane. Here, we show that the peroxisomal sorting determinants and the Pex19p-binding domains of a number of PMPs are distinct entities. In addition, we extend the list of peroxins with which human Pex19p interacts to include the PMP Pex16p and show that Pex19ps CaaX prenylation motif is an important determinant in the affinity of Pex19p for Pex10p, Pex11pβ, Pex12p, and Pex13p.

Substrate Specificities of 3-Oxoacyl-CoA Thiolase A and Sterol Carrier Protein 2/3-Oxoacyl-CoA Thiolase Purified from Normal Rat Liver Peroxisomes STEROL CARRIER PROTEIN 2/3-OXOACYL-CoA THIOLASE IS INVOLVED IN THE METABOLISM OF 2-METHYL-BRANCHED FATTY ACIDS AND BILE ACID INTERMEDIATES

The two main thiolase activities present in isolated peroxisomes from normal rat liver were purified to near homogeneity. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the first enzyme preparation displayed a single band of 41 kDa that was identified as 3-oxoacyl-CoA thiolase A (thiolase A) by N-terminal amino acid sequencing. The second enzyme preparation consisted of a 58- and a 46-kDa band. The 58-kDa polypeptide reacted with antibodies raised against either sterol carrier protein 2 or the thiolase domain of sterol carrier protein 2/3-oxoacyl-CoA thiolase (SCP-2/thiolase), formerly also called sterol carrier protein X, whereas the 46-kDa polypeptide reacted only with the antibodies raised against the thiolase domain. Internal peptide sequencing confirmed that the 58-kDa polypeptide is SCP-2/thiolase and that the 46-kDa polypeptide is the thiolase domain of SCP-2/thiolase. Thiolase A catalyzed the cleavage of short, medium, and long straight chain 3-oxoacyl-CoAs, medium chain 3-oxoacyl-CoAs being the best substrates. The enzyme was inactive with the 2-methyl-branched 3-oxo-2-methylpalmitoyl-CoA and with the bile acid intermediate 24-oxo-trihydroxycoprostanoyl-CoA. SCP-2/thiolase was active with medium and long straight chain 3-oxoacyl-CoAs but also with the 2-methyl-branched 3-oxoacyl-CoA and the bile acid intermediate. In peroxisomal extracts, more than 90% of the thiolase activity toward straight chain 3-oxoacyl-CoAs was associated with thiolase A. Kinetic parameters (K m and V max) were determined for each enzyme with the different substrates. Our results indicate the following: 1) the two (main) thiolases present in peroxisomes from normal rat liver are thiolase A and SCP-2/thiolase; 2) thiolase A is responsible for the thiolytic cleavage of straight chain 3-oxoacyl-CoAs; and 3) SCP-2/thiolase is responsible for the thiolytic cleavage of the 3-oxoacyl-CoA derivatives of 2-methyl-branched fatty acids and the side chain of cholesterol.

Human sphingosine-1-phosphate lyase: cDNA cloning, functional expression studies and mapping to chromosome 10q22(1).

Sphingosine-1-phosphate lyase catalyzes the last step in sphingolipid breakdown, the cleavage of phosphorylated sphingoid bases such as sphingenine-1-phosphate. The latter lipid is not only a catabolite, but can influence as an inter- and/or intracellular second messenger many cellular processes. To allow for the diagnosis of human disorders that might be linked to a deficient lyase, the human sphingosine-1-phosphate lyase cDNA was cloned. The obtained cDNA encoded a protein of 568 amino acids with a calculated molecular mass of 63492 Da. Hydropathy plots revealed the presence of one membrane span near the amino-terminal which is however not required for enzyme activity since recombinant poly-His-tagged lyase, lacking this membrane span, was functionally active. Site-directed mutagenesis disclosed the importance of the cysteine residues 218 and 317 for the cleavage reaction. Northern analysis showed the presence of rare large-sized mRNAs of 6.7, 5.8 and 4 kb and the highest expression in liver. By fluorescent in situ hybridization, the gene was mapped to chromosome 10q22.

Peroxisome dynamics in cultured mammalian cells.

Despite the identification and characterization of various proteins that are essential for peroxisome biogenesis, the origin and the turnover of peroxisomes are still unresolved critical issues. In this study, we used the HaloTag technology as a new approach to examine peroxisome dynamics in cultured mammalian cells. This technology is based on the formation of a covalent bond between the HaloTag protein–a mutated bacterial dehalogenase which is fused to the protein of interest–and a synthetic haloalkane ligand that contains a fluorophore or affinity tag. By using cell‐permeable ligands of distinct fluorescence, it is possible to image distinct pools of newly synthesized proteins, generated from a single genetic HaloTag‐containing construct, at different wavelengths. Here, we show that peroxisomes display an age‐related heterogeneity with respect to their capacity to incorporate newly synthesized proteins. We also demonstrate that these organelles do not exchange their protein content. In addition, we present evidence that the matrix protein content of pre‐existing peroxisomes is not evenly distributed over new organelles. Finally, we show that peroxisomes in cultured mammalian cells, under basal growth conditions, have a half‐life of approximately 2 days and are mainly degraded by an autophagy‐related mechanism. The implications of these findings are discussed.

Metabolic pathways in mammalian peroxisomes

This article summarizes our current knowledge of the metabolic pathways present in mammalian peroxisomes. Emphasis is placed on those aspects that are not covered by other articles in this issue: peroxisomal enzyme content and topology; the peroxisomal beta-oxidation system; substrates of peroxisomal beta-oxidation such as very-long-chain fatty acids, branched fatty acids, dicarboxylic fatty acids, prostaglandins and xenobiotics; the role of peroxisomes in the metabolism of purines, polyamines, amino acids, glyoxylate and reactive oxygen products such as hydrogen peroxide, superoxide anions and epoxides.

Peroxisomal lipid degradation via beta- and alpha-oxidation in mammals.

Peroxisomal β-oxidation is involved in the degradation of long chain and very long chain fatty acyl-(coenzyme A)CoAs, long chain dicarboxylyl-CoAs, the CoA esters of eicosanoids, 2-methyl-branched fatty acyl-CoAs (e.g. pristanoyl-CoA), and the CoA esters of the bile acid intermediates di- and trihydroxycoprostanic acids (side chain of cholesterol).In the rat, straight chain acyl-CoAs (including the CoA esters of dicarboxylic fatty acids and eicosanoids) are β-oxidized via palmitoyl-CoA oxidase, multifunctional protein-1 (which displays 2-enoyl-CoA hydratase and L-3-hydroxyacyl-CoA, dehydrogenase activities) and peroxisomal thiolase. 2-Methyl-branched acyl-CoAs are degraded via pristanoyl-CoA oxidase, multifunctional protein-2 (MFP-2) (which displays 2-enoyl-CoA hydratase and D-3-hydroxyacyl-CoA dehydrogenase activities) and sterol carrier protein-X (SCPX; displaying 2-methyl-3-oxoacyl-CoA thiolase activity). The side chain of the bile acid intermediates is shortened via one cycle of β-oxidation catalyzed by trihydroxycoprostanoyl-CoA oxidase, MFP-2 and SCPX. In the human, straight chain acyl-CoAs are oxidized via palmitoyl-CoA oxidase, multifunctional protein-1, and peroxisomal thiolase, as is the case in the rat. The CoA esters of 2-methyl-branched acyl-CoAs and the bile acid intermediates, which also possess a 2-methyl substitution in their side chain, are shortened, via branched chain acyl-CoA oxidase (which is the human homolog of trihydroxycoprostanoyl-CoA oxidase), multifunctional protein-2, and SCPX. The rat and the human enzymes have been purified, cloned, and kinetically and stereochemically characterized.3-Methyl-branched fatty acids such as phytanic acid are not directly β-oxidizable because of the position of the methyl-branch. They are first shortened by one carbon atom through the a-oxidation process to a 2-methyl-branched fatty acid (pristanic acid in the case of phytanic acid), which is then degraded via peroxisomal β-oxidation. In the human and the rat, α-oxidation is catalyzed by an acyl-CoA synthetase (producing a 3-methylacyl-CoA), a 3-methylacyl-CoA 2-hydroxylase (resulting in a 2-hydroxy-3-methylacyl-CoA), and a 2-hydroxy-3-methylacyl-CoA lyase that cleaves the 2-hydroxy-3-methylacyl-CoA into a 2-methyl-branched fatty aldehyde and formyl-CoA. The fatty aldehyde is dehydrogenated by an aldehyde dehydrogenase to a 2-methyl-branched fatty acid while formyl-CoA is hydrolyzed to formate, which is then converted to CO2. The activation, hydroxylation and cleavage reactions and the hydrolysis of formyl-CoA are performed by peroxisomal enzymes; the aldehyde dehydrogenation remains to be localized whereas the conversion of formate to CO2 occurs mainly in the cytosol.

D-aspartate oxidase, a peroxisomal enzyme in liver of rat and man

By means of subcellular fractionation D-aspartate oxidase was shown to be localized in peroxisomes in rat and human liver. The oxidase from both sources was most active on D-aspartate and N-methyl-D-aspartate. In different rat tissues, the highest enzyme activity was found in kidney, followed by liver and brain. In these tissues, oxidase activities became detectable 1-4 days after birth, reaching adult values after 4 weeks. Analysis of liver samples from patients with Zellweger syndrome, a generalized peroxisomal dysfunction, demonstrated no significant deficiency of this particular oxidase.