Rob Ofman

Proteomics characterization of mouse kidney peroxisomes by tandem mass spectrometry and protein correlation profiling.

The peroxisome represents a ubiquitous single membrane-bound key organelle that executes various metabolic pathways such as fatty acid degradation by α- and β-oxidation, ether-phospholipid biosynthesis, metabolism of reactive oxygen species, and detoxification of glyoxylate in mammals. To fulfil this vast array of metabolic functions, peroxisomes accommodate ∼50 different enzymes at least as identified until now. Interest in peroxisomes has been fueled by the discovery of a group of genetic diseases in humans, which are caused by either a defect in peroxisome biogenesis or the deficient activity of a distinct peroxisomal enzyme or transporter. Although this research has greatly improved our understanding of peroxisomes and their role in mammalian metabolism, deeper insight into biochemistry and functions of peroxisomes is required to expand our knowledge of this low abundance but vital organelle. In this work, we used classical subcellular fractionation in combination with MS-based proteomics methodologies to characterize the proteome of mouse kidney peroxisomes. We could identify virtually all known components involved in peroxisomal metabolism and biogenesis. Moreover through protein localization studies by using a quantitative MS screen combined with statistical analyses, we identified 15 new peroxisomal candidates. Of these, we further investigated five candidates by immunocytochemistry, which confirmed their localization in peroxisomes. As a result of this joint effort, we believe to have compiled the so far most comprehensive protein catalogue of mammalian peroxisomes.

The role of ELOVL1 in very long-chain fatty acid homeostasis and X-linked adrenoleukodystrophy.

X‐linked adrenoleukodystrophy (X‐ALD) is caused by mutations in the ABCD1 gene encoding the peroxisomal ABC transporter adrenoleukodystrophy protein (ALDP). X‐ALD is characterized by the accumulation of very long‐chain fatty acids (VLCFA; ≥C24) in plasma and tissues. In this manuscript we provide insight into the pathway underlying the elevated levels of C26:0 in X‐ALD. ALDP transports VLCFacyl‐CoA across the peroxisomal membrane. A deficiency in ALDP impairs peroxisomal β‐oxidation of VLCFA but also raises cytosolic levels of VLCFacyl‐CoA which are substrate for further elongation. We identify ELOVL1 (elongation of very‐long‐chain‐fatty acids) as the single elongase catalysing the synthesis of both saturated VLCFA (C26:0) and mono‐unsaturated VLCFA (C26:1). ELOVL1 expression is not increased in X‐ALD fibroblasts suggesting that increased levels of C26:0 result from increased substrate availability due to the primary deficiency in ALDP. Importantly, ELOVL1 knockdown reduces elongation of C22:0 to C26:0 and lowers C26:0 levels in X‐ALD fibroblasts. Given the likely pathogenic effects of high C26:0 levels, our findings highlight the potential of modulating ELOVL1 activity in the treatment of X‐ALD.

Proteomic analysis of mouse kidney peroxisomes : identification of RP2p as a peroxisomal nudix hydrolase with acyl-CoA diphosphatase activity

Proteomic analysis of mouse kidney peroxisomes resulted in the identification of a novel nudix hydrolase designated RP2p, which is encoded by the D7RP2e gene. RP2p consists of 357 amino acids and contains two conserved domains: a nudix hydrolase domain and a CoA-binding domain. In addition, a PTS (peroxisomal targeting signal) type 1 (Ala-His-Leu) was found at the C-terminus. Analysis of the enzyme characteristics revealed that RP2p is a CoA diphosphatase with activity towards CoA, oxidized CoA and a wide range of CoA esters, including choloyl-CoA and branched-chain fatty-acyl-CoA esters. The enzymatic properties of RP2p indicate that at low substrate concentrations medium and long-chain fatty-acyl-CoA esters are the primary substrates. Enzyme activity was optimal at pH 9 or above, and required the presence of Mg2+ or Mn2+ ions. Subcellular fractionation studies revealed that all CoA diphosphatase activity in mouse kidney is restricted to peroxisomes.

Molecular and biochemical characterization of rat gamma-trimethylaminobutyraldehyde dehydrogenase and evidence for the involvement of human aldehyde dehydrogenase 9 in carnitine biosynthesis.

The penultimate step in carnitine biosynthesis is mediated by γ-trimethylaminobutyraldehyde dehydrogenase (EC1.2.1.47), a cytosolic NAD+-dependent aldehyde dehydrogenase that converts γ-trimethylaminobutyraldehyde into γ-butyrobetaine. This enzyme was purified from rat liver, and two internal peptide fragments were sequenced by Edman degradation. The peptide sequences were used to search the Expressed Sequence Tag data base, which led to the identification of a rat cDNA containing an open reading frame of 1485 base pairs encoding a polypeptide of 494 amino acids with a calculated molecular mass of 55 kDa. Expression of the coding sequence in Escherichia coli confirmed that the cDNA encodes γ-trimethylaminobutyraldehyde dehydrogenase. The previously identified human aldehyde dehydrogenase 9 (EC 1.2.1.19) has 92% identity with rat trimethylaminobutyraldehyde dehydrogenase and has been reported to convert substrates that resemble γ-trimethylaminobutyraldehyde. When aldehyde dehydrogenase 9 was expressed in E. coli, it exhibited high trimethylaminobutyraldehyde dehydrogenase activity. Furthermore, comparison of the enzymatic characteristics of the heterologously expressed human and rat dehydrogenases with those of purified rat liver trimethylaminobutyraldehyde dehydrogenase revealed that the three enzymes have highly similar substrate specificities. In addition, the highest V max/K m values were obtained with γ-trimethylaminobutyraldehyde as substrate. This indicates that human aldehyde dehydrogenase 9 is the γ-trimethylaminobutyraldehyde dehydrogenase, which functions in carnitine biosynthesis.

Mitochondrial protein acetylation is driven by acetyl-CoA from fatty acid oxidation

Mitochondria integrate metabolic networks for maintaining bioenergetic requirements. Deregulation of mitochondrial metabolic networks can lead to mitochondrial dysfunction, which is a common hallmark of many diseases. Reversible post-translational protein acetylation modifications are emerging as critical regulators of mitochondrial function and form a direct link between metabolism and protein function, via the metabolic intermediate acetyl-CoA. Sirtuins catalyze protein deacetylation, but how mitochondrial acetylation is determined is unclear. We report here a mechanism that explains mitochondrial protein acetylation dynamics in vivo. Food withdrawal in mice induces a rapid increase in hepatic protein acetylation. Furthermore, using a novel LC-MS/MS method, we were able to quantify protein acetylation in human fibroblasts. We demonstrate that inducing fatty acid oxidation in fibroblasts increases protein acetylation. Furthermore, we show by using radioactively labeled palmitate that fatty acids are a direct source for mitochondrial protein acetylation. Intriguingly, in a mouse model that resembles human very-long chain acyl-CoA dehydrogenase (VLCAD) deficiency, we demonstrate that upon food-withdrawal, hepatic protein hyperacetylation is absent. This indicates that functional fatty acid oxidation is necessary for protein acetylation to occur in the liver upon food withdrawal. Furthermore, we now demonstrate that protein acetylation is abundant in human liver peroxisomes, an organelle where acetyl-CoA is solely generated by fatty acid oxidation. Our findings provide a mechanism for metabolic control of protein acetylation, which provides insight into the pathophysiogical role of protein acetylation dynamics in fatty acid oxidation disorders and other metabolic diseases associated with mitochondrial dysfunction.

Rhizomelic chondrodysplasia punctata. Deficiency of 3-oxoacyl-coenzyme A thiolase in peroxisomes and impaired processing of the enzyme.

The rhizomelic form of chondrodysplasia punctata (RCDP) is a peroxisomal disorder characterized biochemically by an impairment of plasmalogen biosynthesis and phytanate catabolism. We have now found that the maturation of peroxisomal 3-oxoacyl-CoA thiolase is impaired in fibroblasts from RCDP patients. To establish the subcellular localization of the 3-oxoacyl-CoA thiolase precursor protein, cultured skin fibroblasts were fractionated on a continuous Nycodenz gradient. Only a small amount of 3-oxoacyl-CoA thiolase activity was present in the catalase-containing (peroxisomal) fractions of RCDP fibroblasts in comparison with control fibroblasts. Moreover, the amount of thiolase protein in immunoblots of the catalase-containing fractions was below the limit of detection. Finally, the beta-oxidation of [14C]palmitoyl-CoA was found to be reduced in these fractions. We conclude that the mutation in RCDP leads to a partial deficiency of 3-oxoacyl-CoA thiolase activity in the peroxisomes and, concomitantly, an impairment in the ability to convert the precursor of this protein to the mature form. The reduction of 3-oxoacyl-CoA thiolase activity results in a decrease in the rate of peroxisomal beta-oxidation of palmitoyl-CoA. However, the capacity of the peroxisomes to oxidize very-long-chain fatty acids must be sufficient to prevent excessive accumulation of these compounds in vivo.

Characterization of the human omega-oxidation pathway for omega-hydroxy-very-long-chain fatty acids.

Very‐long‐chain fatty acids (VLCFAs) have long been known to be degraded exclusively in peroxisomes via β‐oxidation. A defect in peroxisomal β‐oxidation results in elevated levels of VLCFAs and is associated with the most frequent inherited disorder of the central nervous system white matter, X‐linked adre‐ noleukodystrophy. Recently, we demonstrated that VL CFAs can also undergo ω‐oxidation, which may provide an alternative route for the breakdown of VLCFAs. The ω‐oxidation of VLCFA is initiated by CYP4F2 and CYP4F3B, which produce ω‐hydroxy‐VLCFAs. In this article, we characterized the enzymes involved in the formation of very‐long‐chain dicarboxylic acids from ω‐hydroxy‐VLCFAs. We demonstrate that very‐long‐ chain dicarboxylic acids are produced via two indepen dent pathways. The first is mediated by an as yet unidentified, microsomal NAD+‐dependent alcohol de hydrogenase and fatty aldehyde dehydrogenase, which is encoded by the ALDH3A2 gene and is deficient in patients with Sjögren‐Larsson syndrome. The second pathway involves the NADPH‐dependent hydroxylation of ω‐hydroxy‐VLCFAs by CYP4F2, CYP4F3B, or CYP4F3A. Enzyme kinetic studies show that oxidation of ω‐hydroxy‐VLCFAs occurs predominantly via the NAD+‐dependent route. Overall, our data demonstrate that in humans all enzymes are present for the com plete conversion of VLCFAs to their corresponding very‐long‐chain dicarboxylic acids.— Sanders, R.‐J., Ofman, R., Dacremont, G., Wanders, R. J. A., Kemp, S. Characterization of the human ω‐oxidation pathway for ω‐hydroxy‐very‐long‐chain fatty acids. FASEB J. 22, 2064–2071 (2008)

Purification of peroxisomal acyl-CoA: dihydroxyacetonephosphate acyltransferase from human placenta

The peroxisomal enzyme acyl-CoA:dihydroxyacetonephosphate acyltransferase (DHAPAT) was extracted from human placental membranes using CHAPS as a detergent in the presence of 1 M KCl. Prior to assay dipalmitoylphosphatidylcholine was added to the sample as eluted from the various columns in order to stabilize the protein for subsequent enzyme activity measurements at 37 degrees C. The enzyme was purified from the placental membrane using ocytl-Sepharose CL-4B chromatography, Hydroxyapatite HTP chromatography, CM-Sepharose CL-6B, PBE 94 chromatofocusing and TSK G3000 SW size exclusion chromatography. A final purification of more than 8000-fold with respect to the placental membranes was achieved with a final yield of about 5%. Upon chromatofocusing the peak of activity eluted at a pH of 5.1-5.3 indicating a low isoelectric point. A native M(r) of 60-80 kDa was calculated from HPLC size exclusion chromatography. SDS-PAGE of the final purified fraction showed one major band with a M(r) of 65 kDa. These results suggest that DHAPAT is a monomeric protein. A polyclonal antiserum raised against the purified fraction was prepared in rabbits. Immunoprecipitation experiments showed complete precipitation of DHAPAT activity in fractions prepared from human placenta, liver and skin fibroblasts. Immunoprecipitation was also used to determine the residual amount of DHAPAT protein in liver from a patient with the Zellweger syndrome. A value of about 10% was found, which closely corresponds to the residual amount of enzyme activity.

Mutations in the Gene Encoding 3-Hydroxyisobutyryl-CoA Hydrolase Results in Progressive Infantile Neurodegeneration

Only a single patient with 3-hydroxyisobutyryl-CoA hydrolase deficiency has been described in the literature, and the molecular basis of this inborn error of valine catabolism has remained unknown until now. Here, we present a second patient with 3-hydroxyisobutyryl-CoA hydrolase deficiency, who was identified through blood spot acylcarnitine analysis showing persistently increased levels of hydroxy-C(4)-carnitine. Both patients manifested hypotonia, poor feeding, motor delay, and subsequent neurological regression in infancy. Additional features in the newly identified patient included episodes of ketoacidosis and Leigh-like changes in the basal ganglia on a magnetic resonance imaging scan. In cultured skin fibroblasts from both patients, the 3-hydroxyisobutyryl-CoA hydrolase activity was deficient, and virtually no 3-hydroxyisobutyryl-CoA hydrolase protein could be detected by western blotting. Molecular analysis in both patients uncovered mutations in the HIBCH gene, including one missense mutation in a conserved part of the protein and two mutations affecting splicing. A carefully interpreted acylcarnitine profile will allow more patients with 3-hydroxyisobutyryl-CoA hydrolase deficiency to be diagnosed.

Omega-oxidation of very long-chain fatty acids in human liver microsomes: implications for X-linked adrenoleukodystrophy?

X-linked adrenoleukodystrophy (X-ALD) is a severe neurodegenerative disorder biochemically characterized by elevated levels of very long-chain fatty acids (VLCFA). Excess levels of VLCFAs are thought to play an important role in the pathogenesis of X-ALD. Therefore, therapeutic approaches for X-ALD are focused on the reduction or normalization of VLCFAs. In this study, we investigated an alternative oxidation route for VLCFAs, namely ω-oxidation. The results described in this study show that VLCFAs are substrates for the ω-oxidation system in human liver microsomes. Moreover, VLCFAs were not only converted into ω-hydroxy fatty acids, but they were also further oxidized to dicarboxylic acids via cytochrome P450-mediated reactions. High sensitivity toward the specific P450 inhibitor 17-octadecynoic acid suggested that ω-hydroxylation of VLCFAs is catalyzed by P450 enzymes belonging to the CYP4A/F subfamilies. Studies with individually expressed human recombinant P450 enzymes revealed that two P450 enzymes, i.e. CYP4F2 and CYP4F3B, participate in the ω-hydroxylation of VLCFAs. Both enzymes belong to the cytochrome P450 4F subfamily and have a high affinity for VLCFAs. In summary, this study demonstrates that VLCFAs are substrates for the human ω-oxidation system, and for this reason, stimulation of the in vivo VLCFA ω-oxidation pathway may provide an alternative mode of treatment to reduce the levels of VLCFAs in patients with X-ALD.