P. P. Van Veldhoven

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.

The deficient degradation of synthetic 2- and 3-methyl-branched fatty acids in fibroblasts from patients with peroxisomal disorders

SummaryThe oxidation of pristanic and phytanic acids by human skin fibroblasts was compared to that of their synthetic analogues, 2-methylpalmitic and 3-methylmargaric acids. The synthetic compounds and natural substrates were degraded at comparable rates in control and X-linked adrenoleukodystrophy fibroblasts. The α-decarboxylation of 3-methylmargaric acid, similarly to that of phytanic acid, was affected in Refsum disease and Zellweger syndrome, but not in X-linked adrenoleukodystrophy. The β-oxidation of 2-methylpalmitic acid, similarly to that of pristanic acid, was deficient in fibroblasts derived from patients suffering from Zellweger syndrome, confirming the importance of peroxisomes in the breakdown of 2-methyl-branched fatty acids. No deficiency was observed in fibroblasts from X-linked adrenoleukodystrophy patients. The 1-14C-labelled 2- and 3-methyl-branched fatty acids, which are easier to synthesize that the natural analogues, are therefore valuable tools for the diagnosis of human peroxisomal disorders.

The role of 2-hydroxyacyl-CoA lyase, a thiamin pyrophosphate-dependent enzyme, in the peroxisomal metabolism of 3-methyl- branched fatty acids and 2-hydroxy straight-chain fatty acids

2-Hydroxyphytanoyl-CoA lyase (abbreviated as 2-HPCL), renamed to 2-hydroxyacyl-CoA lyase (abbreviated as HACL1), is the first peroxisomal enzyme in mammals that has been found to be dependent on TPP (thiamin pyrophosphate). It was discovered in 1999, when studying alpha-oxidation of phytanic acid. HACL1 has an important role in at least two pathways: (i) the degradation of 3-methyl-branched fatty acids like phytanic acid and (ii) the shortening of 2-hydroxy long-chain fatty acids. In both cases, HACL1 catalyses the cleavage step, which involves the splitting of a carbon-carbon bond between the first and second carbon atom in a 2-hydroxyacyl-CoA intermediate leading to the production of an (n-1) aldehyde and formyl-CoA. The latter is rapidly converted into formate and subsequently to CO(2). HACL1 is a homotetramer and has a PTS (peroxisomal targeting signal) at the C-terminal side (PTS1). No deficiency of HACL1 has been described yet in human, but thiamin deficiency might affect its activity.

Fibroblast studies documenting a case of peroxisomal 2-methylacyl-CoA racemase deficiency: possible link between racemase deficiency and malabsorption and vitamin K deficiency

Background 2‐Methylacyl‐CoA racemase interconverts the 2‐methyl group of pristanoyl‐CoA or the 25‐methyl group of hydroxylated cholestanoyl‐CoAs, allowing further peroxisomal desaturation of these compounds in man by the branched chain acyl‐CoA oxidase, which recognise only the S‐isomers. Hence, oxidation studies in fibroblasts, currently based on the use of racemic substrates such as [1–14C] pristanic acid, do not allow us to distinguish between a deficient racemase or an impaired oxidase.

Farnesylation of Pex19p is not essential for peroxisome biogenesis in yeast and mammalian cells

Abstract.Pex19p exhibits a broad binding specificity for peroxisomal membrane proteins (PMPs), and is essential for the formation of functional peroxisomal membranes. Pex19p orthologues contain a C-terminal CAAX motif common to prenylated proteins. In addition, Saccharomyces cerevisiae and Chinese hamster Pex19p are at least partially farnesylated in vivo. Whether farnesylation of Pex19p plays an essential or merely ancillary role in peroxisome biogenesis is currently not clear. Here, we show that (i) nonfarnesylated and farnesylated human Pex19p display a similar affinity towards a select set of PMPs, (ii) a variant of Pex19p lacking a functional farnesylation motif is able to restore peroxisome biogenesis in Pex19p-deficient cells, and (iii) peroxisome protein import is not affected in yeast and mammalian cells defective in one of the enzymes involved in the farnesylation pathway. Summarized, these observations indicate that the CAAX box-mediated processing steps of Pex19p are dispensable for peroxisome biogenesis in yeast and mammalian cells.

Peroxisome targeting of porcine 17beta-hydroxysteroid dehydrogenase type IV/D-specific multifunctional protein 2 is mediated by its C-terminal tripeptide AKI.

The product of the porcine HSD17B4 gene is a peroxisomal 80 kDa polypeptide containing three functionally distinct domains. The N‐terminal part reveals activities of 17β‐estradiol dehydrogenase type IV and D‐specific 3‐hydroxyacyl CoA dehydrogenase, the central part shows D‐specific hydratase activity with straight and 2‐methyl‐branched 2‐enoyl‐CoAs. The C‐terminal part is similar to sterol carrier protein 2. The 80 kDa polypeptide chain ends with the tripeptide AKI, which resembles the motif SKL, the first identified peroxisome targeting signal PTS1. So far AKI, although being similar to the consensus sequence PTS1, has neither been reported to be present in mammalian peroxisomal proteins, nor has it been shown to be functional. We investigated whether the HSD17B4 gene product is targeted to peroxisomes by this C‐terminal motif. Recombinant human PTS1 binding protein Pex5p interacted with the bacterially expressed C‐terminal domain of the HSD17B4 gene product. Binding was competitively blocked by a SKL‐containing peptide. Recombinant deletion mutants of the C‐terminal domain lacking 3, 6, and 14 amino acids and presenting KDY, MIL, and IML, respectively, at their C‐termini did not interact with Pex5p. The wild‐type protein and mutants were also transiently expressed in the HEK 293 cells. Immunofluorescence analysis with polyclonal antibodies against the C‐terminal domain showed a typical punctate peroxisomal staining pattern upon wild‐type transfection, whereas all mutant proteins localized in the cytoplasm. Therefore, AKI is a functional PTS1 signal in mammals and the peroxisome targeting of the HSD17B4 gene product is mediated by Pex5p. J. Cell. Biochem. 73:70–78, 1999.

Thiamine pyrophosphate: An essential cofactor for the α - oxidation in mammals - implications for thiamine deficiencies?

Abstract.The identification of 2-hydroxyphytanoyl-CoA lyase (2-HPCL), a thiamine pyrophosphate (TPP)-dependent peroxisomal enzyme involved in the α-oxidation of phytanic acid and of 2-hydroxy straight chain fatty acids, pointed towards a role of TPP in these processes. Until then, TPP had not been implicated in mammalian peroxisomal metabolism. The effect of thiamine deficiency on 2-HPCL and α-oxidation has not been studied, nor have possible adverse effects of deficient α-oxidation been considered in the pathogenesis of diseases associated with thiamine shortage, such as thiamine-responsive megaloblastic anemia (TRMA). Experiments with cultured cells and animal models showed that α-oxidation is controlled by the thiamine status of the cell/tissue/organism, and suggested that some pathological consequences of thiamine starvation could be related to impaired α-oxidation. Whereas accumulation of phytanic acid and/or 2-hydroxyfatty acids or their α-oxidation intermediates in TRMA patients given a normal supply of thiamine is unlikely, this may not be true when malnourished.

Activation of 3-methyl-branched fatty acids in rat liver.

1. Subcellular fractionation of rat liver revealed that 3-methylmargaric acid, a monobranched phytanic acid analogue, can be activated by mitochondria, endoplasmic reticulum and peroxisomes. 2. Indirect data (effects of pyrophosphate and Triton X-100) suggested that the peroxisomal activation of 3-methylmargaric, 2-methylpalmitic and palmitic acid is catalyzed by different enzymes. 3. Despite many attempts, column chromatography of solubilized peroxisomal membrane proteins so far did not provide more conclusive data. On various matrices, lignoceroyl-CoA synthetase clearly eluted differently from the synthetases acting on 3-methylmargaric, 2-methylpalmitic and palmitic acid. The latter three however, tended to coelute together, although often not in an identical manner.

Potential limitations in the use of KillerRed for fluorescence microscopy

KillerRed, a bright red fluorescent protein, is a genetically encoded photosensitizer, which generates radicals and hydrogen peroxide upon green light illumination. The protein is a potentially powerful tool for selective light‐induced protein inactivation and cell killing, and can also be used to study downstream effects of locally increased levels of reactive oxygen species. The initial aim of this study was to investigate whether or not KillerRed‐mediated reactive oxygen species production inside peroxisomes could trigger the sequestration of these organelles into autophagosomes. Green fluorescent protein‐tagged microtubule‐associated protein 1 light chain 3 was used as autophagosome marker. We observed that KillerRed also emits weak green fluorescence upon excitation at 480 nm, and this may lead to erroneous data interpretation in conditions where green fluorophores are used. We discuss this potential pitfall of KillerRed for biological imaging and formulate recommendations to avoid misinterpretation of the data.

Peroxisomal localization of α-oxidation in human liver

It was found that α-oxidation in rat liver is a peroxisomal process, consisting of an activation, a 2-hydroxylation, and a reaction leading to the production of formate. α-Oxidation of 3-methyl-substituted fatty acids was quantified by measuring the production of formate and CO2, and the production of a 2-hydroxy-3-methylacyl-CoA-intermediate was demonstrated. We wanted to extend these findings to human liver, in view of the controversy over the subcellular localization of α-oxidation in man.In homogenates from human liver, rates of α-oxidation were highest when measured in the presence of ATP, CoA, Mg2+, 2-oxoglutarate, ascorbate and Fe2+. In subcellular fractions prepared by differential centrifugation and in fractions obtained after subfractionation of a peroxisome-enriched fraction on a Percoll gradient, production of formate and of a 2-hydroxy-3-methylacyl-CoA intermediate coincided with the peroxisomal marker catalase. In broken fractions, production of CO2 was almost negligible as compared to formate production.We conclude from our findings that in human liver, as in rat liver, α-oxidation of 3-methyl-substituted fatty acids is a peroxisomal process, consisting of an activation reaction, a 2-hydroxylation reaction and a reaction or reactions leading to the generation of formate as a primary product which is subsequently converted to CO2. Furthermore activation, 2-hydroxylation and generation of formate appear to be coupled (see Casteels et al 1996). These data demonstrate that Refsum disease should indeed be classified as a peroxisomal disease.