Peroxisomal Metabolite and Cofactor Transport in Humans

Abstract

Peroxisomes are membrane-bound organelles involved in many metabolic pathways and essential for human health. They harbor a large number of enzymes involved in the different pathways, thus requiring transport of substrates, products and cofactors involved across the peroxisomal membrane. Although much progress has been made in understanding the permeability properties of peroxisomes, there are still important gaps in our knowledge about the peroxisomal transport of metabolites and cofactors. In this review, we discuss the different modes of transport of metabolites and essential cofactors, including CoA, NAD⁺, NADP⁺, FAD, FMN, ATP, heme, pyridoxal phosphate, and thiamine pyrophosphate across the peroxisomal membrane. This transport can be mediated by non-selective pore-forming proteins, selective transport proteins, membrane contact sites between organelles, and co-import of cofactors with proteins. We also discuss modes of transport mediated by shuttle systems described for NAD⁺/NADH and NADP⁺/NADPH. We mainly focus on current knowledge on human peroxisomal metabolite and cofactor transport, but also include knowledge from studies in plants, yeast, fruit fly, zebrafish, and mice, which has been exemplary in understanding peroxisomal transport mechanisms in general.

Keywords: carrier; cofactor; exchanger; membrane contact sites; metabolism; peroxisomes; transporter.

FIGURE 1

Currently known CoA-dependent enzymatic reactions and transporters in human peroxisomes. (A) Fatty acids undergo beta-oxidation after import as acyl-CoA esters into peroxisomes. During beta-oxidation, acyl-CoAs are shortened to acyl(n-2)-CoA and acetyl-CoA molecules are produced. Peroxisomal beta-oxidation is mediated by the enzymes acyl-CoA oxidase 1, 2, and 3 (ACOX1, ACOX2, and ACOX3), L- and D-bifunctional protein (LBP and DBP), acetyl-CoA acyltransferase 1 (ACAA1), and sterol carrier protein x-related thiolase (SCPx). One CoA molecule is required for each circle of beta-oxidation. Acyl-CoA molecules produced during beta-oxidation may be hydrolyzed by thioesterases into fatty acids or acetate or converted to carnitine esters by CRAT and CROT, thereby producing free CoA. Free fatty acids and acetate can probably exit peroxisome directly after which ACSL, located on the cytosolic side of peroxisomal membrane, may be involved in the reactivation of the fatty acid and acetate. It is unclear which transporter is responsible for the export of acyl-carnitines. The peroxisomal enzyme GNPAT uses acyl-CoA esters for the acylation of DHAP during ether-linked lipid biosynthesis. During this reaction free CoA is released. Phytanoyl-CoA undergoes alpha-oxidation inside peroxisomes, resulting in the formation of formyl-CoA, which spontaneously splits into formic acid and CoA. Another product of alpha-oxidation – pristanic acid - is reactivated into pristanoyl-CoA by peroxisomal ACSVL. The bile acids THCA-CoA and DHCA-CoA are subjected to one cycle of beta-oxidation in peroxisomes during which choloyl-CoA and deoxycholoyl-CoA are produced and subsequently converted to glycine or taurine conjugates by BAAT. It is unknown how the conjugates are exported from the peroxisomes. It has been suggested that PMP34 imports free CoA into peroxisomes. The CoA diphosphohydrolases NUDT19 and NUDT7 degrade CoA into 3′,5′-ADP, and 4′-phosphopantetheine, which are subsequently exported from peroxisomes, possibly by PMP34. Proteins of the ABCD subfamily import acyl-CoA molecules into peroxisomes. During the import, the ester bond of acyl-CoA is hydrolyzed, and fatty acids subsequently undergo re-esterification by the intraperoxisomal ACSVL proteins. It is unknown whether the CoA molecule from acyl-CoA is also imported into the peroxisomal matrix after hydrolysis. (B) ABCD1, ABCD2, and ABCD3 transporters have different substrate affinities, as shown on the right. After the import of fatty acids, they are esterified by the ACSVL proteins in a CoA- and ATP-dependent reaction. ABCD proteins were shown to hydrolyze the ester bond in the CoA esters during import, although it has also been suggested that ABCD transporters import acyl-CoA molecules without hydrolysis of the ester bond (see text for more information). Enzymatic reactions or molecules belonging to the same metabolic pathway are marked with background color and listed at the bottom.

FIGURE 2

Currently known NAD⁺/NADH- and NADP⁺/NADPH-dependent peroxisomal enzymatic reactions, transport of NAD⁺/NADH, NADP⁺/NADPH, NMNH⁺, AMP, and 2′,5′-ADP, and role of shuttle systems. NAD⁺/NADH and NADP⁺/NADPH may be imported into peroxisomes by specific transporters or co-imported with fully folded NAD(H)/NADP(H)-dependent enzymes. During peroxisomal beta-oxidation, D- and L-bifunctional proteins (DBP and LBP) catalyze the second (hydration) and third (dehydrogenation) step of beta oxidation. During the dehydrogenation, NAD⁺ is reduced to NADH. Subsequent reoxidation of NADH may be mediated by the three different dehydrogenases located inside peroxisomes: LDHB, which converts pyruvate into lactate; MDH1, which converts oxaloacetate into malate; GPD1, which converts DHAP into G3P. During beta-oxidation, the double bound(s) of mono-/polyunsaturated fatty acids are reduced by the peroxisomal enzyme 2,4- dienoyl-CoA reductase (DECR2). During the reduction, NADPH is oxidized to NADP⁺. NADPH is also oxidized by TER during convertion of phytenoyl-CoA into phytanoyl-CoA. NADP⁺ is reduced back to NADPH by IDH1, which converts isocitrate into 2-OG. The import and export of small metabolites [pyruvate, lactate, oxaloacetate, malate, DHAP, G3P, isocitrate, and 2-oxoglutarate (2-OG)] is probably mediated by the peroxisomal pore-forming proteins (PXMP2, Pex11α, Pex11β, and Pex11γ) that allow passage of molecules with a size below 600 Da. The role of another pore-forming protein BAK in the permeability of the peroxisomal membrane remains unclear. Monocarboxylate transporters MCT1 and MCT2 may be involved in the transport of lactate and pyruvate. The pyrophosphatase NUDT12 mediates NADH degradation (to NMNH and AMP) and NADPH degradation (to NMNH and 2’,5′-ADP). The products of NADH/NADPH degradation are subsequently exported from peroxisomes, probably by PMP34. Enzymatic reactions or molecules belonging to the same metabolic pathway are marked with background color and listed at the bottom.

FIGURE 3

Currently known FAD, PLP, ThPP, FMN, and heme-dependent enzymatic reactions in the peroxisomes and transport of FAD, PLP, ThPP, heme, FMN and AMP. Most of the peroxisomal matrix proteins are imported into peroxisomes via a PTS1-specific pore. Proteins may be imported as monomers or in a fully folded form complexed with their cofactor. Cofactors that may be co-imported with proteins are FAD (with ACOX and ADHAPS proteins), FMN (HAO1, HAO2, HAO3), ThPP (with HACL1), PLP (with AGT), and heme (with catalase). It has also been suggested that the PMP34 transporter can import free FAD into peroxisomes. In the peroxisome, FAD forms an active enzyme with apoenzyme ACOX. The pyrophosphatase NUDT12 may mediate the degradation of FAD to FMN and AMP. FMN and AMP are subsequently exported from peroxisomes, probably by PMP34. Acyl-CoA oxidases (ACOX) mediate the first (dehydrogenation) reaction of the beta-oxidation. During dehydrogenation, FAD is reduced to FADH₂. FADH₂ is re-oxidized by direct transfer of electrons to O₂, resulting in the production of H₂O₂. H₂O₂ is degraded by the heme-dependent enzyme catalase to H₂O and O₂. It is unclear whether free ThPP or PLP are imported into peroxisomes. Alkyl-dihydroxyacetonephosphate synthase (ADHAPS) catalyzes the exchange in acyl-dihydroxyacetonephosphate (acyl-DHAP) of the acyl chain with long-chain alcohol through a non-redox mechanism. The ThPP-dependent enzyme 2-hydroxyacyl-CoA lyase (HACL1) catalyzes the cleavage of 2-hydroxyphytanoyl-CoA into pristanal and formyl-CoA during peroxisomal alpha-oxidation of phytanoyl-CoA. The PLP-dependent enzyme alanine-glyoxylate aminotransferase (AGT) catalyzes the transamination of glyoxylate to glycine during glyoxylate detoxification. The FMN-dependent enzymes 2-hydroxyacid oxidases (HAO1, HAO2, HAO3) catalyze oxidation of glycolate to glyoxylate. Enzymatic reactions or molecules belonging to the same metabolic pathway are marked with background color and listed at the bottom.

FIGURE 4

Currently known ATP-dependent enzymatic reactions in the peroxisomes and transport of ATP, AMP and PP. Intraperoxisomal ATP is required for the ACSVL-mediated activation of fatty acids after their import by the ABCD proteins. Pristanic acid formed during alpha-oxidation is activated inside peroxisomes to the corresponding CoA ester in an ATP-dependent reaction. Finally, the peroxisomal protease LonP2 hydrolyzes ATP. It is unknown which transporter mediates the import of ATP into peroxisomes. The products of ATP hydrolysis – AMP and PP are exported from peroxisomes most probably by PMP34 and PXMP2, respectively.