An HTRF based high-throughput screening for discovering chemical compounds that inhibit the interaction between Trypanosoma brucei Pex5p and Pex14p.

The glycosome, a peroxisome-related organelle, is essential for the growth and survival of trypanosomatid protozoa. In glycosome biogenesis, Pex5p recognizes newly synthesized glycosomal matrix proteins via peroxisome-targeting signal type-1 (PTS-1) and transports them into glycosomes through an interaction with Pex14p, a component of the matrix protein import machinery on the glycosomal membrane. Knockdown of the PEX5 or PEX14 with RNAi has been shown to inhibit the growth of Trypanosoma brucei. Thus, compounds that inhibit the interaction of TbPex5p-TbPex14p are expected to become lead compounds in the development of anti-trypanosomal drugs. Here, we report a homogenous time-resolved fluorescence (HTRF) assay for the screening of compounds that inhibit the TbPex5p-TbPex14p interaction. The binding of GST-TbPex14p and TbPex5p-His with or without additional compounds was evaluated by measuring the energy transfer of the HTRF pair, using a terbium-labeled anti GST antibody as the donor and an FITC-labeled anti His antibody as the acceptor. The assay was performed in a 384-well plate platform and exhibits a Z'-factor of 0.85-0.91, while the coefficiency of variation is 1.1-7.7%, suggesting it can be readily adapted to a high-throughput format for the automated screening of chemical libraries. We screened 20,800 compounds and found 11 compounds that inhibited energy transfer. Among them, in a pull-down assay one compound exhibited selective inhibition of TbPex5p-TbPex14p without any HsPex5p-HsPex14p interaction.

Role of NH2-terminal hydrophobic motif in the subcellular localization of ATP-binding cassette protein subfamily D: common features in eukaryotic organisms.

In mammals, four ATP-binding cassette (ABC) proteins belonging to subfamily D have been identified. ABCD1-3 possesses the NH2-terminal hydrophobic region and are targeted to peroxisomes, while ABCD4 lacking the region is targeted to the endoplasmic reticulum (ER). Based on hydropathy plot analysis, we found that several eukaryotes have ABCD protein homologs lacking the NH2-terminal hydrophobic segment (H0 motif). To investigate whether the role of the NH2-terminal H0 motif in subcellular localization is conserved across species, we expressed ABCD proteins from several species (metazoan, plant and fungi) in fusion with GFP in CHO cells and examined their subcellular localization. ABCD proteins possessing the NH2-terminal H0 motif were localized to peroxisomes, while ABCD proteins lacking this region lost this capacity. In addition, the deletion of the NH2-terminal H0 motif of ABCD protein resulted in their localization to the ER. These results suggest that the role of the NH2-terminal H0 motif in organelle targeting is widely conserved in living organisms.

ABC subfamily D proteins and very long chain fatty acid metabolism as novel targets in adrenoleukodystrophy.

Peroxisomes are involved in a variety of metabolic processes, including ß-oxidation of fatty acids, especially very long chain fatty acids. Three peroxisomal ABC proteins belonging to subfamily D have been identified in mammalian peroxisomes that have an important role in fatty acid metabolism. ABCD1/ALDP and ABCD2/ALDRP are suggested to be involved in the transport of very long chain acyl-CoA, and ABCD3/PMP70 is involved in the transport of long chain acyl-CoA. ABCD1 is known to be responsible for X-linked adrenoleukodystrophy (X-ALD); an inborn error of peroxisomal ß-oxidation of very long chain fatty acids. X-ALD is characterized biochemically by the accumulation of very long chain fatty acids in all tissues, including the brain white matter. Progressive demyelination of the central nervous system and adrenal dysfunction have been observed. The pharmacological up-regulation of peroxisomal ß-oxidation of very long chain fatty acids and the suppression of fatty acid elongation are important aspects of an optimal therapeutic approach. Attractive targets for the treatment of X-ALD patients include the ABCD2 as well as elongase that is involved in the elongation of very long chain fatty acids. In addition, stabilization of mutant ABCD1 that has retained some of its function might be another approach, since most of the mutant ABCD1s with a missense mutation are degraded rapidly by proteasomes before or after targeting to peroxisomes. Protection of the central nervous system against oxidative damage is also important in order to delay the progress of disease. We summarize recent pharmaceutical studies and consider the potential for future X-ALD therapies.

Structural basis for docking of peroxisomal membrane protein carrier Pex19p onto its receptor Pex3p.

Peroxisomes require peroxin (Pex) proteins for their biogenesis. The interaction between Pex3p, which resides on the peroxisomal membrane, and Pex19p, which resides in the cytosol, is crucial for peroxisome formation and the post-translational targeting of peroxisomal membrane proteins (PMPs). It is not known how Pex3p promotes the specific interaction with Pex19p for the purpose of PMP translocation. Here, we present the three-dimensional structure of the complex between a cytosolic domain of Pex3p and the binding-region peptide of Pex19p. The overall shape of Pex3p is a prolate spheroid with a novel fold, the 'twisted six-helix bundle.' The Pex19p-binding site is at an apex of the Pex3p spheroid. A 16-residue region of the Pex19p peptide forms an α-helix and makes a contact with Pex3p; this helix is disordered in the unbound state. The Pex19p peptide contains a characteristic motif, consisting of the leucine triad (Leu18, Leu21, Leu22), and Phe29, which are critical for the Pex3p binding and peroxisome biogenesis.

Identification of a substrate-binding site in a peroxisomal beta-oxidation enzyme by photoaffinity labeling with a novel palmitoyl derivative.

Peroxisomes play an essential role in a number of important metabolic pathways including beta-oxidation of fatty acids and their derivatives. Therefore, peroxisomes possess various beta-oxidation enzymes and specialized fatty acid transport systems. However, the molecular mechanisms of these proteins, especially in terms of substrate binding, are still unknown. In this study, to identify the substrate-binding sites of these proteins, we synthesized a photoreactive palmitic acid analogue bearing a diazirine moiety as a photophore, and performed photoaffinity labeling of purified rat liver peroxisomes. As a result, an 80-kDa peroxisomal protein was specifically labeled by the photoaffinity ligand, and the labeling efficiency competitively decreased in the presence of palmitoyl-CoA. Mass spectrometric analysis identified the 80-kDa protein as peroxisomal multifunctional enzyme type 2 (MFE2), one of the peroxisomal beta-oxidation enzymes. Recombinant rat MFE2 was also labeled by the photoaffinity ligand, and mass spectrometric analysis revealed that a fragment of rat MFE2 (residues Trp(249) to Arg(251)) was labeled by the ligand. MFE2 mutants bearing these residues, MFE2(W249A) and MFE2(R251A), exhibited decreased labeling efficiency. Furthermore, MFE2(W249G), which corresponds to one of the disease-causing mutations in human MFE2, also exhibited a decreased efficiency. Based on the crystal structure of rat MFE2, these residues are located on the top of a hydrophobic cavity leading to an active site of MFE2. These data suggest that MFE2 anchors its substrate around the region from Trp(249) to Arg(251) and positions the substrate along the hydrophobic cavity in the proper direction toward the catalytic center.

Multiple organelle-targeting signals in the N-terminal portion of peroxisomal membrane protein PMP70.

Most membrane proteins are recognized by a signal recognition particle and are cotranslationally targeted to the endoplasmic reticulum (ER) membrane, whereas almost all peroxisomal membrane proteins are posttranslationally targeted to the destination. Here we examined organelle-targeting properties of the N-terminal portions of the peroxisomal isoform of the ABC transporter PMP70 (ABCD3) using enhanced green fluorescent protein (EGFP) fusion. When the N-terminal 80 amino acid residue (N80)-segment preceding transmembrane segment (TM) 1 was deleted and the TM1-TM2 region was fused to EGFP, the TM1 segment induced ER-targeting and integration in COS cells. When the N80-segment was fused to EGFP, the fusion protein was targeted to the outer mitochondrial membrane. When both the N80-segment and the following TM1-TM2 region were present, the fusion located exclusively to the peroxisome. The full-length PMP70 molecule was clearly located in the ER in the absence of the N80-segment, even when multiple peroxisome-targeting signals were retained. We concluded that the TM1 segment possesses a sufficient ER-targeting function and that the N80-segment is critical for suppressing the ER-targeting function to allow the TM1-TM2 region to localize to the peroxisome. Cooperation of the organelle-targeting signals enables PMP70 to correctly target to peroxisomal membranes.

A novel immunoregulatory protein in human colostrum, syntenin-1, for promoting the development of IgA-producing cells from cord blood B cells.

Human colostrum contains many bioactive factors that must promote the development of intestinal mucosal immunity in infants. Especially, the presence of certain cytokines such as transforming growth factor (TGF)-beta or IL-10 has been of great interest for IgA production as a function of mucosal immune response. In the present study, we attempted to investigate whether unidentified factors inducing generation of IgA-producing cells from naive B cells might exist in colostrum. For this purpose, colostrum samples were directly added to a culture consisting of naive B cells and dendritic cells from cord blood and CD40 ligand-transfected L cells, comparing with recombinant IL-10 (rIL-10) and/or rTGF-beta. It was noted that most colostrum samples alone were able to induce IgA-secreting cells at higher levels than rIL-10 and/or rTGF-beta. IgA-inducing activity of colostrum was abolished by neither anti-neutralizing mAbs against IL-10 nor TGF-beta, though partially by anti-IL-6 mAb. We prepared partially purified fractions from both pooled colostrums with and without IgA-inducing activity and comparatively performed quantitative proteomic analysis by two-dimensional difference gel electrophoresis followed by liquid chromatography-mass spectrometry. As a result, syntenin-1 was identified as a candidate for IgA-inducing protein in colostrum. Western blot analysis indicated that levels of syntenin-1 in colostrum samples were correlated with their IgA-inducing activities. Moreover, we demonstrated that recombinant syntenin-1 could induce preferentially IgA production from naive B cells. These results suggest that syntenin-1 serves as one of IgA-inducing factors for B cells.

70-kDa peroxisomal membrane protein related protein (P70R/ABCD4) localizes to endoplasmic reticulum not peroxisomes, and NH2-terminal hydrophobic property determines the subcellular localization of ABC subfamily D proteins.

70-kDa peroxisomal membrane protein related protein (P70R/ABCD4) is a member of ATP-binding cassette (ABC) protein subfamily D. ABC subfamily D proteins are also known as peroxisomal ABC proteins. Therefore, P70R is thought to be a peroxisomal membrane protein. However, the subcellular localization of P70R is not extensively investigated. In this study, we transiently expressed P70R in fusion with HA (P70R-HA) in CHO cells and examined subcellular localization by immunofluorescence. Surprisingly, P70R-HA was localized to the endoplasmic reticulum (ER), not to peroxisomes. To examine the ER-targeting property of P70R, we expressed various NH(2)-terminal deletion constructs of P70R. Among the NH(2)-terminal deletion constructs, mutant proteins starting with hydrophobic transmembrane segment (TMS) were localized to ER, but the ones containing the NH(2)-terminal hydrophilic cytosolic domain were not. ABC subfamily D proteins destined for peroxisomes have NH(2)-terminal hydrophilic region adjacent to TMS1. However, only P70R lacks the region and is translated with NH(2)-terminal hydrophobic TMS1. Furthermore, attachment of the NH(2)-terminal hydrophilic domain to the NH(2)-terminus of P70R excluded P70R from the ER-targeting pathway. These data suggest that P70R resides in the ER but not the peroxisomal membranes, and the hydrophobic property of NH(2)-terminal region determines the subcellular localization of ABC subfamily D proteins.

Characterization of the interaction between recombinant human peroxin Pex3p and Pex19p: identification of TRP-104 IN Pex3p as a critical residue for the interaction.

Proteins required for peroxisome biogenesis are termed peroxins. The peroxin Pex3p is a peroxisomal membrane protein (PMP), involved in peroxisomal membrane biogenesis. It acts as a docking receptor for another peroxin Pex19p, which is a specific carrier protein for newly synthesized PMPs. Here we have determined the physicochemical properties and binding manners of Pex3p-Pex19p interaction, in terms of the affinity, the stoichiometry, and the binding site in Pex3p. The cytosolic domain of human Pex3p was overproduced, using an Escherichia coli expression system and was highly purified by two chromatography steps. Gel filtration chromatography analyses and intrinsic tryptophan fluorescence titrations revealed that a one-to-one complex is formed between monomeric Pex3p and monomeric Pex19p. The tryptophan fluorescence spectrum of Pex3p showed a large 18-nm blue shift of the maximum emission wavelength by the binding of Pex19p. This result indicates that either one or two tryptophan residues of Pex3p (Trp-104 and Trp-224) are directly involved in binding to Pex19p. We investigated the binding activities of the wild-type and tryptophan mutants of Pex3p by pull-down assays and surface plasmon resonance analyses. As a result, the wild-type and the W104A and W104F mutants showed K(D) values of 3.4 nm, 1080 nm, and 66.2 nm, respectively. The affinity differences with mutation affected their peroxisome restoring activities in pex3 ZPG208 cells. These findings suggest that the indole ring of Trp-104 directly interacts with Pex19p to facilitate the specific peroxisomal translocation of the Pex19p-PMP complexes.

Hydrophobic regions adjacent to transmembrane domains 1 and 5 are important for the targeting of the 70-kDa peroxisomal membrane protein.

The 70-kDa peroxisomal membrane protein (PMP70) is a major component of peroxisomal membranes. Human PMP70 consists of 659 amino acid residues and has six putative transmembrane domains (TMDs). PMP70 is synthesized on cytoplasmic ribosomes and targeted posttranslationally to peroxisomes by an unidentified peroxisomal membrane protein targeting signal (mPTS). In this study, to examine the mPTS within PMP70 precisely, we expressed various COOH-terminally or NH(2)-terminally deleted constructs of PMP70 fused with green fluorescent protein (GFP) in Chinese hamster ovary cells and determined their intracellular localization by immunofluorescence. In the COOH-terminally truncated PMP70, PMP70(AA.1-144)-GFP, including TMD1 and TMD2 of PMP70, was still localized to peroxisomes. However, by further removal of TMD2, PMP70(AA.1-124)-GFP lost the targeting ability, and PMP70(TMD2)-GFP did not target to peroxisomes by itself. The substitution of TMD2 in PMP70(AA.1-144)-GFP for TMD4 or TMD6 did not affect the peroxisomal localization, suggesting that PMP70(AA.1-124) contains the mPTS and an additional TMD is required for the insertion into the peroxisomal membrane. In the NH(2)-terminal 124-amino acid region, PMP70 possesses hydrophobic segments in the region adjacent to TMD1. By the disruption of these hydrophobic motifs by the mutation of L21Q/L22Q/L23Q or I70N/L71Q, PMP70(AA.1-144)-GFP lost targeting efficiency. The NH(2)-terminally truncated PMP70, GFP-PMP70(AA.263-375), including TMD5 and TMD6, exhibited the peroxisomal localization. PMP70(AA.263-375) also possesses hydrophobic residues (Ile(307)/Leu(308)) in the region adjacent to TMD5, which were important for targeting. These results suggest that PMP70 possesses two distinct targeting signals, and hydrophobic regions adjacent to the first TMD of each region are important for targeting.

Adrenoleukodystrophy: subcellular localization and degradation of adrenoleukodystrophy protein (ALDP/ABCD1) with naturally occurring missense mutations.

Mutation in the X-chromosomal adrenoleukodystrophy gene (ALD; ABCD1) leads to X-linked adrenoleukodystrophy (X-ALD), a severe neurodegenerative disorder. The encoded adrenoleukodystrophy protein (ALDP/ABCD1) is a half-size peroxisomal ATP-binding cassette protein of 745 amino acids in humans. In this study, we chose nine arbitrary mutant human ALDP forms (R104C, G116R, Y174C, S342P, Q544R, S606P, S606L, R617H, and H667D) with naturally occurring missense mutations and examined the intracellular behavior. When expressed in X-ALD fibroblasts lacking ALDP, the expression level of mutant His-ALDPs (S606L, R617H, and H667D) was lower than that of wild type and other mutant ALDPs. Furthermore, mutant ALDP-green fluorescence proteins (S606L and H667D) stably expressed in CHO cells were not detected due to rapid degradation. Interestingly, the wild type ALDP co-expressed in these cells also disappeared. In the case of X-ALD fibroblasts from an ALD patient (R617H), the mutant ALDP was not detected in the cells, but appeared upon incubation with a proteasome inhibitor. When CHO cells expressing mutant ALDP-green fluorescence protein (H667D) were cultured in the presence of a proteasome inhibitor, both the mutant and wild type ALDP reappeared. In addition, mutant His-ALDP (Y174C), which has a mutation between transmembrane domain 2 and 3, did not exhibit peroxisomal localization by immunofluorescense study. These results suggest that mutant ALDPs, which have a mutation in the COOH-terminal half of ALDP, including S606L, R617H, and H667D, were degraded by proteasomes after dimerization. Further, the region between transmembrane domain 2 and 3 is important for the targeting of ALDP to the peroxisome.

ATP-binding and -hydrolysis activities of ALDP (ABCD1) and ALDRP (ABCD2), human peroxisomal ABC proteins, overexpressed in Sf21 cells.

The peroxisomal ATP-binding cassette (ABC) proteins, adrenoleukodystrophy protein (ALDP, ABCD1) and ALD-related protein (ALDRP, ABCD2), were expressed in Spodoptera frugiperda 21 (Sf21) insect cells using a baculovirus-mediated expression system. Immunoelectron microscopy and subcellular fractionation revealed that the overexpressed ALDP was distributed in various subcellular organelles including mitochondria, nucleus and peroxisomes. The ALDP was not extractable with Na(2)CO(3) treatment, suggesting that it integrated into membranes. ATPase activity was detected in the membrane fraction expressing ALDP. The nucleotide-binding capacities of the expressed ALDP were estimated by the binding to ATP- or ADP-agarose. ALDP exhibited an affinity to both ADP and ATP. In contrast, ALDRP exhibited an affinity to ADP but scarcely to ATP. The ALDP in the Sf21 membrane fraction was extracted with n-dodecyl-beta-maltoside and successively purified with a chelate column. The nucleotide-binding and ATPase activities of the purified ALDP were, however, not detected. It may be that certain membranous components are required for the activity. We demonstrate for the first time that the peroxisomal ABC proteins can be expressed in Sf21 membranes maintaining their nucleotide-binding abilities and ATPase activities, and the expressed proteins will be of use for further characterization.

Role of Pex19p in the targeting of PMP70 to peroxisome.

Pex19p is a protein required for the peroxisomal membrane synthesis. The 70-kDa peroxisomal membrane protein (PMP70) is synthesized on free cytosolic ribosomes and then inserted posttranslationally into peroxisomal membranes. Pex19p has been shown to play an important role in this process. Using an in vitro translation system, we investigated the role of Pex19p as a chaperone and identified the regions of PMP70 required for the interaction with Pex19p. When PMP70 was translated in the presence of purified Pex19p, a large part of PMP70 existed as soluble form and was co-immunoprecipitated with Pex19p. However, in the absence of Pex19p, PMP70 formed aggregates during translation. To identify the regions that interact with Pex19p, various truncated PMP70 were translated in the presence of Pex19p and subjected to co-immunoprecipitation. The interaction was markedly reduced by the deletion of the NH(2)-terminal 61 amino acids or the region around TMD6. Further, we expressed these deletion constructs of PMP70 in fusion with the green fluorescent protein in CHO cells. Fusion proteins lacking these Pex19p binding sites did not display any peroxisomal localization. These results suggest that Pex19p binds to PMP70 co-translationally and keeps PMP70 as a proper conformation for the localization to peroxisome.

Domain architecture and activity of human Pex19p, a chaperone-like protein for intracellular trafficking of peroxisomal membrane proteins.

Pex19p is a peroxin involved in peroxisomal membrane biogenesis and probably functions as a chaperone and/or soluble receptor specific for cargo peroxisomal membrane proteins (PMPs). To elucidate the functional constituents of Pex19p in terms of the protein structure, we investigated its domain architecture and binding affinity toward various PMPs and peroxins. The human Pex19p cDNA was overexpressed in Escherichia coli, and a highly purified sample of the Pex19p protein was prepared. When PMP22 was synthesized by cell-free translation in the presence of Pex19p, the PMP22 bound to Pex19p was soluble, whereas PMP22 alone was insoluble. This observation shows that Pex19p plays a role in capturing PMP and maintaining its solubility. In a similar manner, Pex19p was bound to PMP70 and Pex16p as well as the Pex3p soluble fragment. Limited proteolysis analyses revealed that Pex19p consists of the C-terminal core domain flanking the flexible N-terminal region. Separation of Pex19p into its N- and C-terminal halves abolished interactions with PMP22, PMP70, and Pex16p. In contrast, the flexible N-terminal half of Pex19p was bound to the Pex3p soluble fragment, suggesting that the binding mode of Pex3p toward Pex19p differs from that of other PMPs. This idea is supported by our detection of the Pex19p-Pex3p-PMP22 ternary complex.

Existence of catalase-less peroxisomes in Sf21 insect cells.

Catalase activity, a peroxisomal marker enzyme, was not detectable in any of the subcellular fractions of Spodoptera frugiperda (Sf) 21 insect cells, although marker enzymes in other organelles were distributed in the fractions in a manner similar to that seen in mammalian cells. When a green fluorescent protein fused with peroxisome targeting signal 1 at the C-terminal (GFP-SKL) was expressed in Sf21 cells, punctate fluorescent dots were observed in the cytoplasm. The fraction where GFP-SKL was concentrated exhibited long-chain and very-long-chain fatty acid beta-oxidation activities in the presence of KCN and the density of this fraction was slightly higher than that of mitochondria. Immunoelectron microscopy studies with anti-SKL antibody demonstrated that Sf21 cells have immunoreactive peroxisome-like organelles which are structurally distinct from mitochondria, endoplasmic reticulum, and lysosomes. In contrast to peroxisomal matrix proteins, adrenoleukodystrophy protein, a peroxisomal membrane protein, was not located to peroxisomes. This suggests that the targeting signal for PMP in insect cells is distinct from that in mammalian cells. These results demonstrate that Sf21 insect cells have unique catalase-less peroxisomes capable of beta-oxidation of fatty acids.

Nucleotide-induced conformational changes of PMP70, an ATP binding cassette transporter on rat liver peroxisomal membranes.

Nucleotide-induced conformational changes of the 70-kDa peroxisomal membrane protein (PMP70) were investigated by means of limited-trypsin digestion. Rat liver peroxisomes preincubated with various nucleotides were subsequently digested by trypsin. The digestion products were subjected to immunoblot analysis with an anti-PMP70 antibody that recognizes the carboxyl-terminal 15 amino acids of the protein. PMP70 was initially cleaved in the boundary region between the transmembrane and nucleotide-binding domains and a carboxyl-terminal 30-kDa fragment resulted. The fragment in turn was progressively digested at the helical domain between the Walker A and B motifs. The fragment, however, could be stabilized with MgATP or MgADP. In contrast to MgATP, MgATP-gammaS protected whole PMP70 as well as the fragment. The 30-kDa fragment processed by trypsin was recovered in the post-peroxisomal fraction as a complex with a molecular mass of about 60 kDa irrespective of the presence of MgATP. These results suggest that PMP70 exists as a dimer on the peroxisomal membranes and the binding and hydrolysis of ATP induce conformational changes in PMP70 close to the boundary between the transmembrane and nucleotide binding domains and the helical domain between the Walker A and B motifs.