Archives of Biochemistry and Biophysics 451 (2006) 128–140

www.elsevier.com/locate/yabbi

Molecular organization of peroxisomal enzymes: Protein–protein interactions in the membrane and in the matrix

Randhir S. Makkar 1, Miguel A. Contreras 1, Ajaib S. Paintlia, Brian T. Smith, Ehtishamul Haq, Inderjit Singh ¤

The Charles Darby Children’s Research Institute, Department of Pediatrics, Medical University of South Carolina, Charleston, SC 29425, USA

Received 23 February 2006, and in revised form 3 May 2006Available online 24 May 2006

Abstract

The �-oxidation of fatty acids in peroxisomes produces hydrogen peroxide (H2O2), a toxic metabolite, as a bi-product. Fatty acids�-oxidation activity is deWcient in X-linked adrenoleukodystrophy (X-ALD) because of mutation in ALD-gene resulting in loss of verylong chain acyl-CoA synthetase (VLCS) activity. It is also aVected in disease with catalase negative peroxisomes as a result of inactivationby H2O2. Therefore, the following studies were undertaken to delineate the molecular interactions between both the ALD-gene product(adrenoleukodystrophy protein, ALDP) and VLCS as well as H2O2 degrading enzyme catalase and proteins of peroxisomal �-oxidation.Studies using a yeast two hybrid system and surface plasmon resonance techniques indicate that ALDP, a peroxisomal membrane pro-tein, physically interacts with VLCS. Loss of these interactions in X-ALD cells may result in a deWciency in VLCS activity. The yeast two-hybrid system studies also indicated that catalase physically interacts with L-bifunctional enzyme (L-BFE). Interactions between catalaseand L-BFE were further supported by aYnity puriWcation, using a catalase-linked resin. The aYnity bound 74-kDa protein, was identiWedas L-BFE by Western blot with speciWc antibodies and by proteomic analysis. Additional support for their interaction comes from immu-noprecipitation of L-BFE with antibodies against catalase as a catalase- L-BFE complex. siRNA for L-BFE decreased the speciWc activityand protein levels of catalase without changing its subcellular distribution. These observations indicate that L-BFE might help in oligo-merization and possibly in the localization of catalase at the site of H2O2 production in the peroxisomal �-oxidation pathway.© 2006 Published by Elsevier Inc.

Keywords: Catalase; L-bifunctional enzyme; Peroxisomes; �-Oxidation; X-linked adrenoleukodystrophy; Very long chain acyl-CoA synthetase

Peroxisomes, ubiquitous subcellular organelles areknown to contain more than 60 proteins that participate ina variety of metabolic pathways [1,2]. Peroxisomes alsocontain oxidases, which produce hydrogen peroxide(H2O2),2 and catalase, which detoxiWes H2O2 to water andmolecular oxygen [3,4]. Abnormalities in these metabolicpathways either as a result of deWciency in biogenesis of

* Corresponding author. Fax: +1 843 792 7130.E-mail address: singhi@musc.edu (I. Singh).

1 These authors contributed equally to this work.2 Abbreviations used: H2O2, hydrogen peroxide; X-ALD, X-linked

adrenoleukodystrophy; VLCS, very long chain acyl-CoA synthetase;L-BFE, L-bifunctional enzyme; PTSs, peroxisomal targeting signals;ABSF, 2-aminobenzenesulfonyl Xuoride.

0003-9861/$ - see front matter © 2006 Published by Elsevier Inc.doi:10.1016/j.abb.2006.05.003

peroxisomes (known as peroxisomal biogenesis disorders;PBDs) or from a single enzyme deWciency lead to severeneurological dysfunctions [5,6].

The most common peroxisomal disorder caused by anindividual protein deWciency is X-ALD, a childhood disor-der characterized by abnormality in very long chain (VLC)fatty acid catabolism. X-ALD results in loss of myelin andoligodendrocytes and, Wnally loss of life [5,7]. X-ALD iscaused by mutations/deletions in the ALD gene thatencodes a 74-kDa peroxisomal membrane protein, ALDP[8]. ALDP has signiWcant homology to the members of theABC family of transporters, but its precise function in thefatty acid �-oxidation pathway is not understood at pres-ent. However, its absence leads to a reduced activity of thevery long chain acyl-CoA synthetase (VLCS) [9], an enzyme

R.S. Makkar et al. / Archives of Biochemistry and Biophysics 451 (2006) 128–140 129

that activates VLC fatty acids to CoA derivatives [10,11].This results in pathognomonic accumulation of the VLCfatty acids in X-ALD patients [7]. Results of a yeast two-hybrid system and surface plasmon resonance studiesdescribed in this manuscript document that VLCS localizedon the luminal face of peroxisomal membranes interactswith ALDP, and that loss of these interactions may causedysfunction of VLCS in X-ALD.

In the peroxisomal �-oxidation system, acyl-CoA deriv-atives are converted to enoyl-CoA plus H2O2 by acyl-CoAoxidase, and catalase degrades the H2O2 produced by thevarious types of H2O2-generating oxidases in the peroxi-somal matrix. The physiological importance of the catalaseactivity is highlighted by the existence of a genetic disease(Zellweger-like syndrome) characterized by the mislocaliza-tion of catalase to cytoplasm that impairs the peroxisomalfunction [12,13]. Peroxisomal functions were restored inWbroblasts of Zellweger-like syndrome patients treated witha natural antioxidant (Vitamin E), thus underscoring theimportance of peroxisomal redox homeostasis in a neuro-logical disorder [14].

Peroxisomal proteins are targeted to peroxisomes via vir-tue of peroxisomal targeting signals (PTSs). Two types ofthe PTSs have been identiWed: PTS-1 is a C-terminal tripep-tide (-SKL) or a conservative variant thereof [15]. PTS-2 is a9-amino acid presequence located at the N-terminus [16].The high majority of matrix proteins are equipped with aPTS1, and a few are targeted via the PTS2. Both types ofperoxisomal targeting signals have cytosolic receptors calledPex5 and Pex7, respectively, which mediate their associationwith the peroxisomal membrane import system [17].

Catalase, a tetrameric protein, largely localized in per-oxisomes is devoid of a conserved -SKL sequence; insteadcontains the variant sequence -KANL [18]. Reportsindicate that catalase folds and oligomerize in cytoplasmand then transported to peroxisomes in yeast and humans[19–21]. Our laboratory has reported that mistargeting ofcatalase to peroxisomes causes severe metabolic dysfunc-tion in human skin Wbroblasts cell lines from patients inwhich the import of other PTS1 and PTS2 signal contain-ing proteins was normal [13]. Since targeting of catalase toperoxisomes and thus alteration of peroxisomal functionsin these mutant cell lines could not be restored by transfec-tion of a vector that express normal catalase (catalase-KANL) but could be restored by chimeric catalase contain-ing PTS1or PTS2, we hypothesize that alterations in otherprotein(s) might interfere with catalase transport in thesepatients’ Wbroblasts cell lines. Therefore, to search forpotential macromolecules that might facilitate catalaseassembly/transport, we investigated for proteins that mightinteract with catalase. In this study, we describe that cata-lase interacts with L-BFE both in cytoplasm as well as inthe matrix of peroxisomes. These observations indicate thatthe described interactions between catalase and L-BFE mayfunction in assembly/oligomerization of catalase, and ulti-mately in the eYcient detoxiWcation of H2O2 produced byoxidases in peroxisomes.

Material and methods

Chemicals

Dulbecco’s modiWed Eagle’s minimum essential medium(DMEM) was from Cellgro (Mediatech, Herndon, VA,USA); fetal calf serum and antibiotic–antimycotic mix werefrom Atlanta Biologicals (Norcross, GA); Trypsin, andHank’s balanced solution (HBSS) were from Gibco (GrandIsland, NY, USA); polyclonal antibodies against catalaseand anti-rabbit IgG were from Research Diagnostics, Inc.(Fitzgerald Industries Intl., Concord, MA, USA); Triton X-100, digitonin, Tween 20, aprotinin, leupeptin, pepstatin A,hexamethylphosphoric triamide, �-mercaptoethanol, ATP,n-heptyl-D-thioglucoside, polyethylene glycol 6000, 2-amin-obenzenesulfonyl Xuoride (ABSF), and antipain were fromSigma (St. Louis, MO, USA). PuriWed beef liver catalasewas from Roche Applied Science (Indianapolis, IN, USA).All chemicals and reagents used were of analytical grade orof the highest purity commercially available.

PuriWcation of rat liver peroxisomes and cytosolic fraction

All the experiments involving the use of animals wereperformed according to a protocol approved by the MedicalUniversity of South Carolina Animal Care and Use Com-mittee. Animals were provided with food and waterad libitum. Liver peroxisomes were prepared from Sprague–Dawley rats fed with a food chow supplemented with0.025% (w/w) ciproWbrate for 2 weeks [22]. BrieXy, liverswere homogenized using a TeXon-glass homogenizer in 10volumes of homogenization buVer (3 mM imidazole, pH 7.4,containing 0.25 M sucrose, 1 mM EDTA, 1 mg/ml antipain,2 mg/ml aprotinin, 2 mg/ml leupeptin, 0.7 mg/ml pepstatinA, and 0.2 mM ABSF) at 4 °C. The homogenates were frac-tionated by diVerential centrifugation to prepare thelambda fraction (light mitochondrial fraction enriched withperoxisomes and lysosomes) and the supernatant was usedto obtain the soluble (cytosolic) fractions [23]. The lambdafraction was further subjected to isopycnic equilibrium cen-trifugation in a continuous 0–50% (w/vol) Nycodenz gradi-ent overlying a cushion of 55% (w/vol) of Nycodenz asdescribed previously [23]. After centrifugation, the fractionswere collected and analyzed for speciWc marker enzymeactivities. Fractions enriched in catalase (peroxisomal frac-tions) were pooled and used for further studies.

Assay for subcellular marker enzymes

The location of the subcellular organelles in the gradientwas determined by the assay of speciWc marker enzymes ineach fraction: catalase for peroxisomes, cytochrome c oxi-dase for mitochondria, and NADPH cytochrome c reduc-tase for endoplasmic reticulum (ER, microsomes), asdescribed elsewhere [23]. The protein concentrations weredetermined by the procedure of Bradford using �-globulinas the standard protein.

130 R.S. Makkar et al. / Archives of Biochemistry and Biophysics 451 (2006) 128–140

PuriWcation of very long chain acyl-CoA synthetase (VLCS)

Extraction and puriWcation of VLCS was performedaccording to the method described by Uchida et al. [24].BrieXy, VLCS was extracted from pooled peroxisomal frac-tion and was puriWed using a series of chromatographic col-umns: Blue Dextran-Sepharose 4B and Ultra gel AcA 34(Sigma), calcium phosphate gel/cellulose (Bio-Rad), BlueDextran-Sepharose 4B and DEAE-Toyopearl (Tosoh Bio-science, Tokyo, Japan). The puriWcation of the enzyme wasmonitored by the determination of VLCS enzyme activity[9]. The puriWed VLCS peak was pooled and dialyzedagainst 10 mM Hepes, 150 mM NaCl, pH 7.4, and was usedfor the study of molecular interactions (Biacore experi-ments).

PCR recombinant techniques

Human catalase gene DNA (Accession No.NM_001752) was ampliWed from human liver cDNAlibrary (Clontech, Palo Alto, CA, USA) by using PCR(Bio-Rad) followed by its subcloning into the NcoI andSalI restriction sites of the expression vector pGBKT7(Clontech). The cloning plasmid, pGBKT7 had the codingsequence for c-Myc. Integrity of catalase gene in CAT-pGBKT7 was conWrmed by DNA sequencing. Likewise,human ALDP (Accession No. BC015541) gene was ampli-Wed by using PCR from human liver cDNA library. Thefragments of ALDP encoding gene such as membranespanning domain, N�-terminal domain (ALDn: 1–74amino acids sequence), membrane spanning domain(ALDm: 75–290 amino acids sequence), and C�-terminaldomain (ALDc: 291–745 amino acids sequence) were gen-erated for interaction studies (Fig. 1A). Cloning site forXhoI and EcoRI were introduced in the ampliWed prod-ucts of ALDP full-length or fragments for cloning inpGBKT7 plasmids at XhoI and EcoRI site. Cloned ALDPfragments were conWrmed for gene integrity by DNAsequencing. Human VLCS gene (Accession No. D88308)was PCR ampliWed from human liver cDNA library andcloned at BamHI and NcoI site in pGADT7 (Fig. 1B) anddetermined its integrity. Transformation of these recom-binant plasmids was carried out in Escherichia coli DH-5�cells and then puriWed by using a plasmid puriWcation kit(Qiagen, Valencia, CA, USA).

Yeast two-hybrid system

The Matchmaker GAL4 Yeast Two-Hybrid System 3was used for the yeast two hybrid experiments, asinstructed by the manufacturer (Clontech). Cloned plas-mid CAT-pGBKT7 was used to transform yeast Saccha-romyces cerevisiae AH109 using standard protocolsdescribed in the product manual. Transformed yeast cellswere selected on 100-mm SD (-Trp) media plates. Humanliver cDNA library in pACT2 vector (Clontech) was WrstampliWed and then transformed into yeast cells carrying

CAT-pGBKT7 plasmids using standard protocolsdescribed in the instruction manual. Once the cDNAlibrary was transformed to the yeast cells, double trans-formants were selected on plates lacking Leu and Trp.Next, selected colonies from Leu and Trp drop out mediawere plated on high-stringency condition on SD/-Ade/-His/-Leu/-Trp/X-�-gal medium. Double transformantspresenting blue color were picked up and grown for plas-mid puriWcation for further screening.

For ALDP and VLCS interaction studies, VLCS-pGADT7 plasmids were Wrst transformed into yeast cellsAH109 followed by second transformation with diVerentconstructs described above i.e., ALDPf-pGBKT7 (full-ALDP), ALDn-pGBKT7 (N�-terminal), ALDm-pGBKT7(membrane spanning region), and ALDc-pGBKT7 (C�-ter-minal). The interactions between VLCS and ALDPfragments were conWrmed after growing the double trans-formed yeast cells at diVerent stringency conditionsbeginning with low-stringency condition (Leu and Trp drop

Fig. 1. Schematic representation of DNA cloning and construction ofrecombinant plasmids used for yeast two-hybrid system based protein–protein interaction studies. Cloning of ALD-gene fragments (A), verylong chain acyl-CoA synthetase (VLCS) gene (B) and catalase (C) genes.

R.S. Makkar et al. / Archives of Biochemistry and Biophysics 451 (2006) 128–140 131

out media) to high-stringency condition (SD/-Ade/-His/-Leu/-Trp/X-�-gal media). Double transformants presenting bluecolor were used for �- and �- galactosidase assay studies.

Quantitative �- and �-galactosidase assays

Positive transformed yeast colonies were cultured inselective culture medium, incubated overnight at 30 °C,and the liquid media assayed for �- and �-galactosidaseactivities. These activities were assayed using p-nitro-phenyl �-D-galactopyranoside (PNP-�-gal; Sigma) andO-nitrophenyl �-D-galactopyranoside (ONPG, Sigma) assubstrate, respectively, as described by the manufacturer(Clontech).

Plasmid rescue from yeast and DNA sequencing

Plasmids from positive colonies in screening of catalaseinteracting proteins by yeast two-hybrid studies were puri-Wed using a Matchmaker yeast plasmid isolation kit (Clon-tech). Bacterial competent cells, E. coli DH-5�, were usedfor transformation of puriWed plasmids from yeast coloniesand transformed E. coli colonies selected in the presence ofantibiotic were used for further puriWcation of plasmids.DNA sequencing was performed to identify the proteinsinteracting with catalase and to conWrm homology withGene Bank genes.

Surface plasmon resonance protein–peptide analysis

The interaction of puriWed VLCS with the luminal Wrstloop of ALDP on the internal face of peroxisomal mem-brane was studied using surface plasmon resonance (SPR)measurements made on a Biacore 3000 system (BiacoreInc., Piscataway, NJ, USA). Biacore sensor chips (typeCM5) were activated with a 1:1 mixture of 0.2 M N-ethyl-N-3-(3-dimethylaminopropyl) carbodiimide and 0.05 MN-hydroxysuccinimide in water. Peptides (0.1 mg/ml,10 mM sodium acetate, pH 5.5) were immobilized on aCM5 sensor chip using the amine coupling kit (Biacore)as described by the supplier. Un-reacted sites wereblocked with 1 M ethanolamine (pH 8.5). Control Xowcells were activated and blocked in the absence of peptide.Two peptides (sequences L1: 104RTFLSVYVARLDGRLARCIVRKDPRA and L1m: same sequence as L1 butcarrying R113P mutation; Arg to Pro at position 113) [25]were custom synthesized with a purity of 98% or higher bySigma-Genosys (The Woodlands, TX, USA). Each pep-tide (L1 and L1m) was immobilized to a sensor chip andthe dissociation constants (KD) of VLCS binding to theindividual peptides (L1 and L1m) were obtained from theSPR sensorgrams proWles in a given series by simulta-neous Wt to a 1:1 binding site model using BIAevaluationversion 3.1 software provided by the manufacturer. Thebinding was evaluated over a range of puriWed VLCS con-centrations (6–200 nM). Baseline was obtained by passingthe VLCS over a control Xow cell (without peptide).

IdentiWcation of catalase-binding protein

Catalase-binding proteins were analyzed by catalaseaYnity chromatography, overlay assay with nitrocellulose-immobilized catalase and catalase immunoprecipitationtechniques.

Catalase aYnity chromatography

PuriWed catalase from beef liver (Roche) was immobi-lized to amino-link resin (Pierce, Rockford, IL, USA)according to manufacturers speciWcations at a Wnal proteinconcentration of 20 mg catalase/ml of amino-link couplinggel. The gel free binding sites were blocked with quenchingbuVer (1 M Tris) in the presence of cyanoborohydride solu-tion and were washed with 1 M NaCl followed by bindingbuVer (phosphate-buVered saline, pH 7.4, PBS), and storedat 4 °C until use.

Resin-linked catalase was equilibrated in PBS and mixedwith equal volumes of PBS-dialyzed cytosol for diVerentintervals of time at 4 °C. After incubation, the resin waswashed three times with PBS, centrifuged at 12,000 rpm for10 min and the pellet was resuspended and heated for 3 minin electrophoresis sample buVer (100 mM Tris–Cl (pH 6.8),4% (w/v) SDS, 0.2% (w/v) bromophenol blue, 20% (v/v)glycerol, and 200 mM �-mercaptoethanol). The sampleswere analyzed by SDS–PAGE (4–20% Tris–HCl criteriongradient gel; Bio-Rad). A precision plus protein standard(Bio-Rad) was loaded as the protein standard for SDS–PAGE. The gels were stained with Coomassie blue solutionas described elsewhere [23], and their protein proWles wereacquired by scanning the stained gel and rendered usingAdobe Photoshop.

Protein interactions on nitrocellulose membrane

PuriWed catalase (Roche) at a concentration of 1 mg/mlwas subjected to electrophoresis under denatured condi-tions on a preparative SDS gel (4–20% Tris–HCl criteriongradient gel IPG + 1; Bio-Rad) and the proteins were trans-ferred to nitrocellulose membrane (Amersham BiosciencesCorp., Piscataway, NJ, USA). Catalase band of 60-kDa wasvisualized with Ponceu-S staining (Sigma). This band wascut into a 2 mm length by 5 mm width pieces, and stored at4 °C until use. For binding studies pieces of the catalase onnitrocellulose membrane were blocked overnight in 5% (w/v)milk powder (Bio-Rad) in TBST (Tris buVer saline–Tween20; 20 mM Tris, 500 mM NaCl, and 0.1% Tween 20 (v/v),pH 7.5). The nitrocellulose pieces were then incubated withor without rat liver cytosol (5 mg/ml) for diVerent intervalsof time. After binding, the pieces were washed 3 times withTBST, and heated for 3 min in electrophoresis samplebuVer and analyzed by SDS–PAGE (4–20% Tris–HCl crite-rion gradient gel). The protein proWle was determined bystaining the gel as indicated above. Alternatively, for immu-nologic detection, the proteins were transferred to nitrocel-lulose membrane and analyzed using speciWc polyclonal

132 R.S. Makkar et al. / Archives of Biochemistry and Biophysics 451 (2006) 128–140

antibodies against L-BFE (kind gift of Dr. TakashiHashimoto, Japan) as indicated elsewhere [26].

Co-immunoprecipitation with antibodies against catalase

Peroxisomal proteins (200 �g) were incubated at 4 °Cwith 50�g of anti-catalase antibody (RDI) in a total vol-ume of 500�l for 12 h. Following addition of protein-A/G-agarose (Santa Cruz Biotechnology, Inc., Santa Cruz, CA,USA) the solution was incubated for additional 4 h at 4 °C.Isolated immuno-agarose complexes were extensivelywashed with phosphate-buVered saline containing 0.05%(v/v) Tween 20 (PBST). The immunoprecipitated proteinswere analyzed by SDS–PAGE (4–20%, Tris–HCl criteriongradient gel, Bio-Rad) and visualized by Coomassie bluestaining.

Proteomics analysis

The protein bands of interest were excised from thestained gel after electrophoresis (gel plug) and submittedfor analysis at the Medical University of South CarolinaMass Spectrometry Research Resource Facility. Gel plugswere placed in wells of ZipPlates (Millipore, Billerica, MA,USA) washed twice with 25 mM ammonium bicarbonate/5% ACN and dehydrated with 100% ACN. Trypsin diges-tion was carried out with sequence grade modiWed trypsin(Sigma) by adding 15 �l of a 100 ng/mL solution (in 25 mMammonium bicarbonate). Tryptic peptides were extractedfrom the ZipPlate wells after washing with 5% ACN/0.2%triXuoroacetic acid (TFA) and a Wnal wash with 0.2% TFA.Peptides were eluted oV the ZipPlate with 1.5�l of 5 mg/ml�-cyano-4-hydroxy-cinnamic acid in 50% ACN/0.1% TFAdirectly onto a MALDI plate insert. Samples were analyzedusing MALDI TOF-TOF (4700 Proteomics Analyzer massspectrometer (Applied Biosystems, Foster City, CA)) andthe MASCOT search engine and database (www.matrix-science.com).

mRNA interference assay for L-bifunctional enzyme

The Silencer siRNA Transfection kit 11 (Ambion, Aus-tin, TX, USA) was used for L-BFE silencing in PC3 cells.BrieXy, PC3 cells were transfected with siRNA for L-BFEusing siPORT NeoFX transfection agent (Ambion). ThreesiRNA for the peroxisomal protein L-BFE (Ambion) wereused (siRNA 1, ID 146224; siRNA 2, ID 112833; siRNA 3,ID 146225). The siRNAs were mixed (T2: siRNA 1 and 2;T3: siRNA 1 and 3; T4: siRNA 1, 2 and 3) and diluted inOPTI-MEM1 medium to Wnal concentration of 30 nM/well.siRNA/transfection agent was dispensed into culture platesas directed by manufacturer. A positive control (TH) usingGAPDH siRNA (Ambion) and transfection agent (T1)were included. For protein analysis of the transfected cells,two wells per plate were lysed and used for protein mea-surements (Bradford Reagent; Bio-Rad) and protein levels(Western blot).

Quantitative real-time PCR

RNA was isolated from the transfected cells using Tri-Zol reagent according to the manufacturer’s instructions(Invitrogen, Carlsbad, California, USA). cDNA was syn-thesized from RNA by using iScript cDNA synthesis kit(Bio-Rad), according to the manufacturer’s instructions.For quantitative real-time PCR, a PCR master mixture wasprepared using the SYBRGreen PCR Master Mix (AppliedBiosystems, Foster City, USA) and aliquoted together with1 �l template and 100 nM of each primer (Wnal volume of25 �l). All samples were run in triplicate. Thermal cyclingconditions were as follows: activation of iTaq DNA poly-merase at 95 °C for 10 min, followed by 40 cycles of ampliW-cation at 95 °C for 30 s and 58 °C for 1 min. The expressionof the target gene was normalized with 18S rRNA. Theprimers were designed using the Premier Biosoft Interna-tional software (Bio-Rad) and synthesized by IntegratedDNA Technologies (IDT, Coralville, IA). The sequences ofthe primers were: 18S rRNA, forward (F): 5�-CGTCTGCCCTATCAACTTTCG-3�, reverse (R): 5�-GCCTGCTGCCTTCCTTGG-3�; catalase, F: 5�-ACGTGCTGAATGAGGAACAGAGGA-3�, R: 5�-ACCTCAGTGAAGTTCTTGACCGCT-3�; L-bifunctional enzyme, F: 5�-GGCATCAAGAAGGAGGAGGAG-3� and R: 5�-CAACAACACCAACTGAGGAGA-3� and for the Reverse transcriptase-PCR ampliWcation of GAPDH the primers were F: 5�-CGGGATCGTGGAAGGGCTAATGA-3�, and R, 5�-CTTCACGAAGTTGTCATTGAGGGCA-3�.

Enzymatic assay for catalase

Catalase activity was determined in 6-well plates. BrieXy,plates were washed 3 times with serum free medium andtotal catalase activity was determined in the presence of Tri-ton X-100 detergent. Serum free medium (50�l) and 2% Tri-ton X-100 (50�l) were added to each well containingtransfected cells. After 1 min of detergent action, 1 ml ofhydrogen peroxide substrate in 0.25 M sucrose was added asdescribed elsewhere [14]. A blank well was included in eachassay. The substrate that remains after a period of incuba-tion at 4 °C was detected by formation of a colored complexby addition of 1 ml of titanium oxysulfate [14]. Sampleswere read at 405 nm (Shimadzu UV1601 spectrophotome-ter). Cytosolic catalase activity was assayed by replacingTriton X-100 with digitonin (0.2 mg/ml in 3 mM imidazole,5 mM EDTA-Na2, 0.25 M sucrose, pH 7.4). Peroxisome-associated catalase (membrane bound/or latency) is calcu-lated as the diVerence between total catalase activity and thecytosolic catalase activity, expressed as percentage [27].

Statistics

The statistical evaluation of the data was performedusing the analysis of variance (ANOVA) followed by theStudent’s t-test (InStat 3, GraphPad Software Inc., SanDiego, CA, USA). Data are presented as the

R.S. Makkar et al. / Archives of Biochemistry and Biophysics 451 (2006) 128–140 133

mean§ standard deviation (SD) for n number of experi-ments. p < 0.05 was accepted as statistically signiWcantdiVerence.

Results

Protein–protein interaction in the peroxisomes

The observed deWcient activities of VLCS and �-oxida-tion of VLC fatty acids in X-ALD [9] and these functionsnormalization following the ALDP transfection [28] indi-cate that VLCS may function by interacting with ALDP.To evaluate the possibility of physical interaction amongproteins in peroxisomes, we studied the interaction of theperoxisomal membrane protein, ALDP, and its peptides,with VLCS, by a yeast two-hybrid system and surface plas-mon resonance analysis. Additionally, the interactionbetween catalase and catalase-binding protein was studiedby the yeast two-hybrid system and catalase aYnity bindingassays.

Protein–protein interaction in the peroxisome membrane

VLCS and ALDP interactions determined by yeast two-hybrid system

VLCS and ALDP protein–protein and protein–peptideinteraction studies were conducted using the yeast two-hybrid system by using full-length VLCS and full-lengthALDP as well as ALDP peptides as described in themethod section (Fig. 1A and B). Strong interactions wereobserved between VLCS and ALDP and between VLCSand the ALDP peptide encompassing the sequence corre-sponding to the transmembrane domains of ALDP (resi-dues 75–290) (Table 1). However, such interactions werenot detected between VLCS and the peptide containing the

Table 1Molecular interaction between very long chain acyl-CoA synthetase(VLCS) and adrenoleukodystrophy protein (ALDP) or ALDP fragmentsdetermined by yeast two-hybrid system screening of a human cDNAlibrary

The values reported were obtained from nD 2 experiments.

�-Galactosidase (U/ml)

�-Galactosidase (U/ml)

pCLl (control) 1520 20.61568 26.6

pGADT7-T + pGBKT7-53 (positive control)

115 5.6145 7.7

pGADT7-T + pGBKT7-Lam (negative control)

0 00 0

VLCS-pGADT7 + ALDf-pGBKT7(encoding residues 1–745)

34.8 5.343.2 7.3

VLCS-pGADT7 + ALDn-pGBKT7 (encoding residues 1–74)

1.9 1.72.1 1.2

VLCS-pGADT7 + ALDm-pGBKT7 (encoding residues 75–290)

24.4 4.932.6 4.8

VLCS-pGADT7 + ALDc-pGBKT7 (encoding residues 291–745)

8.5 1.47.5 1.2

SCAH109 (yeast cells) 0 00 0

N�-terminal sequence (residues 1–74) or the peptide con-taining the C�-terminal sequence (residues 291–745) ofALDP (Table 1). These interactions are represented by anincrease in the activities of �- and �-galactosidases and sug-gest that VLCS and ALDP physically interact, possiblythrough the domain of ALDP localized on the luminal sideof the peroxisomal membrane. The absence of interactionsbetween VLCS and the N� or C�-terminal regions of ALDPindicate that the observed interactions between ALDP andVLCS or between VLCS and the ALDP domain (residues75–290) might represent a true protein–protein interactionof VLCS and ALDP on the luminal surface of the peroxi-somal membrane.

VLCS and ALDP interactions determined by surface plasmon resonance (SPR)

ALDP is a membrane protein whereas VLCS is a mem-brane-associated protein in the lumen (matrix) of peroxi-somes. Therefore, the observed interactions between ALDPand VLCS in Table 1 indicate that VLCS interact withALDP peptides at the luminal surface of the peroxisomalmembrane. To further assess the interaction between VLCSand ALDP, an ALDP peptide that projects as a loop at theluminal surface of the peroxisomal membrane was customsynthesized for this study (L1: 104RTFLSVYVARLDGRLARCIVRKDPRA). In parallel, a second synthetic peptidewith the same amino acid sequence but carrying a singleamino acid substitution (R113P) was used for the sameanalysis (L1m: 104RTFLSVYVAPLDGRLARCIVRKDPRA). These peptides were immobilized to the Biacore chipsas described in the methods section. PuriWed rat liver VLCSwas passed over a chip containing either L1 or L1m boundpeptides. Binding and dissociation kinetics were recorded.The dissociation constant (KD) for the peptide L1m carry-ing a mutation was 3.4-fold greater than the one obtainedfor the normal peptide L1 (1.35£ 10¡7 vs. 4.75£ 10¡8),when VLCS was used at concentrations of 6–200 nM(Table 2). The higher aYnity of VLCS to the normal pep-tide as compared to the peptide containing one amino acidsubstitution indicates that this interaction may representtrue ALDP and VLCS interactions. Taken together theresults from studies utilizing two complementary

Table 2Surface plasmon resonance analysis of very long chain acyl-CoA synthe-tase binding to a synthetic peptide containing the transmembranesequence of adrenoleukodystrophy protein

The K dissociation (KD; n D 2) was obtained from the regression analysisof the association–dissociation curve obtained from the binding of VLCSto peptides (L1 and L1m) over a range of concentrations (6–200 nM) ofthe puriWed VLCS. The sequence of the synthetic peptides L1 and L1mwere similar (104RTFLSVYVARLDGRLARCIVRKDPRA), but L1mcarry a R113P mutation (Arg to Pro at position 113).

Peptide KD

L1 3.3 £ 10¡8

6.0 £ 10¡8

L1m (R113P) 1.1 £ 10¡7

1.6 £ 10¡7

134 R.S. Makkar et al. / Archives of Biochemistry and Biophysics 451 (2006) 128–140

techniques (yeast two-hybrid system and SPR) indicate thatALDP and VLCS physically interact in the peroxisomemembrane.

Protein–protein interaction in the peroxisome matrix

Catalase interacting proteins determined by yeast two-hybrid system

Screening of human liver cDNA library for catalaseinteracting proteins using Matchmaker Two-Hybrid Sys-tem 3 (Fig. 1C) at high stringent conditions revealed 32 col-onies positive for X-Gal. Further, plasmid rescuing andDNA sequencing revealed that 5 out of 32 positive colonieshad the L-bifunctional enzyme (L-BFE) gene. The remain-ing 28 colonies encoded for other peroxisomal enzymes andcytoplasmic proteins (data not shown). L-BFE positiveyeast cell colonies were used to determine �- and �-galacto-sidase activities. It conWrmed that catalase interactsstrongly with L-BFE protein (Table 3), compared with suit-able positive and negative controls. Moreover, yeast cellstransformed with plasmid pCL1 had high activity for both�- and �-galactosidase due to the presence of completeGAL4 gene.

Catalase-binding protein in cytoplasm and in matrix of peroxisomes of rat liver

To evaluate the signiWcance of the interaction deter-mined by the yeast two-hybrid system, catalase wasimmobilized to a resin and used for Wshing proteins fromcytosol by aYnity chromatography. Incubating rat livercytosolic fraction with immobilized catalase (catalase-linked to resin) resulted in the binding of a major proteinwith apparent molecular weight of 74-kDa and 2 minorproteins of 19 and 12-kDa (data not shown), in SDS–PAGE gel electrophoresis (Fig. 2A). The intensity of theband increased with incubation time and increased mark-edly after 48 h. As expected, the incubation of resin alonewith cytosolic fraction did not retain any protein(Fig. 2A).

To further conWrm the speciWcity/selectivity of the inter-action between catalase and the 74-kDa protein, catalase-

Table 3Molecular interaction between catalase and L-bifunctional enzyme deter-mined by yeast two-hybrid system screening of human cDNA library

The values reported were obtained from n D 2 experiments.

�-Galactosidase (U/ml)

�-Galactosidase (U/ml)

PCLl (control) 1520 20.61568 26.6

pGADT7-T + pGBKT7-53 (positive control)

115 5.6145 7.7

pGADT7-T + pGBKT7-Lam (negative control)

0 00 0

CAT-pGBKT7 + L-BFE-pGADT7

50 5.668 7.7

SCAH109 (yeast cells) 0 00 0

linked to resin was incubated with puriWed peroxisome pro-teins. Incubation of peroxisomal proteins with catalase-linked to resin also bound a 74-kDa protein, identiWedwhen the complex was analyzed by electrophoresis(Fig. 2B).

We assessed whether the interaction between the 74-kDaprotein and catalase depends exclusively on the nativestructure of catalase or it also recognizes the unfolded(denatured) catalase. PuriWed catalase transferred fromSDS-gel to nitrocellulose membrane after electrophoresis(denatured protein) was incubated with rat liver cytosolicfraction, and analysis of the protein retained on the mem-branes was performed using SDS–PAGE. Denatured cata-lase retained a protein of 74-kDa, whereas, a control ofmembrane alone incubated with cytosolic fraction did notretain any protein (Fig. 3).

Fig. 2. Catalase-binding protein is present in cytosol and peroxisomesfrom rat liver. AYnity puriWcation of a 74-kDa protein from cytosolic (A)and peroxisomal (B) fraction using catalase-linked to resin was achievedas described in methods section. In (A): resin alone incubated with cyto-solic fraction for 144 h (lane 1) and catalase-linked resin incubated inbatch with PBS for 144 h (lane 2), or rat liver cytosol for diVerent periodsof time: 24 h (lane 3), 48 h (lane 4), 72 h (lane 5), and 144 h (lane 6) at 4 °C.In (B): Catalase-linked resin incubated in batch with PBS for 144 h (lane1), or rat liver peroxisomal fraction for diVerent periods of time: 24 h (lane2), 48 h (lane 3), 72 h (lane 4), and 144 h (lane 5) at 4 °C. After incubation,the resin was washed with PBS, resuspended and heated in sample buVerand subjected to SDS–PAGE. After electrophoresis, the gel was stainedwith Coomassie blue to visualize the protein bands. Protein pattern wasacquired by scanning the gel and the Wgure rendered by Adobe Photo-shop. Lane 7 in (A) shows protein standards. Small arrowhead indicatesthe 74-kDa protein band; big arrowhead indicates the protein band corre-sponding to catalase.

R.S. Makkar et al. / Archives of Biochemistry and Biophysics 451 (2006) 128–140 135

Catalase-binding protein co-immunoprecipitates with catalase

To assess whether the molecular interaction observedbetween catalase and the 74-kDa protein occurs in thematrix of peroxisomes or whether it is a transient interac-tion in the cytosol, matrix proteins from puriWed peroxi-somes were immunoprecipitated using anti-catalaseantibodies. Interestingly, the analysis of the proteins of theimmunoprecipitated complex by SDS–PAGE and stainingof the gel with Coomassie blue demonstrated the presenceof 74-kDa protein (Fig. 4).

Immunological identiWcation of the 74-kDa catalase binding protein

The identity of the 74-kDa protein was evaluated byWestern blot analysis of the proteins transferred to nitro-cellulose membranes after SDS–PAGE of the immobilizedcatalase experiments; from catalase immobilized to resin(Fig. 5A), and catalase immobilized to nitrocellulose mem-branes (Fig. 5B). Antibodies against L-BFE recognized asingle band of 74-kDa (Fig. 5A and B).

Proteomic analysis of the catalase-binding proteinThe 74-kDa bands excised from the stained gels after

electrophoresis from the immobilized catalase and the co-immunoprecipitation experiments were subjected to pro-teomics analysis for identiWcation. Mass spectrometry (MS)analyses of the peptides generated by trypsin digestion wereanalyzed by MALDI-MS (Fig. 6), and the peptide frag-ments (peptide Wngerprinting) identiWed the 74-kDa proteinas enoyl-Coenzyme A hydratase/3-hydroxyacyl CoenzymeA dehydrogenase (Table 4), also known as L-multifunc-

Fig. 3. Denatured catalase is recognized by the catalase-binding proteinpresent in cytosolic fraction. PuriWed catalase was electrophoresed andtransferred to nitrocellulose membrane as described in methods section.The nitrocellulose membrane (NC) alone or nitrocellulose membrane con-taining catalase (catalase-NC) were blocked in PBS-milk powder for 1 h atroom temperature. The membranes were washed with PBS and incubatedwith cytosolic fraction for 144 h (lane 1), with PBS for 144 h (lane 2), orrat liver cytosol for diVerent periods of time: 24 h (lane 3), 48 h (lane 4),72 h (lane 5), and 144 h (lane 6) at 4 °C. After the incubation, the mem-branes were washed with PBS, heated in sample buVer and subjected toSDS–PAGE. After electrophoresis, the gel was stained with Coomassieblue. Protein pattern was acquired as described in legend for Fig. 2. Smallarrowhead indicates the 74-kDa protein band; big arrowhead indicatesthe protein band corresponding to catalase.

tional enzyme or L-bifunctional enzyme (L-BFE), a enzymelocalized in the peroxisome matrix.

Study of siRNA for L-BFE on subcellular distribution, speciWc activity and protein levels of catalase

The studies of catalase and L-BFE interactions describedabove indicate that catalase as tetramer or denaturedmonomer interacts with L-BFE both in cytoplasm as well asin the matrix of peroxisomes, indicating a possible role of L-BFE in assembly/function of catalase. To further assess thebiological signiWcance of this interaction in the assembly/oligomerization or the transport of catalase, we investi-gated the expression, activity, and intracellular distributionof catalase following disruption of the expression of L-BFE(siRNA) in the PC3 cells as described in the method sec-tion. After 96 h post siRNA transfection, the levels of

Fig. 4. A 74-kDa protein co-immunoprecipitate with catalase from puri-Wed rat liver peroxisomes. Immunoprecipitation of peroxisomal fractionwith antibodies against catalase was performed as indicate in methodssection. The immunocomplex was sedimented using protein A/G agaroseand washed extensively. The precipitated protein components wereresolved by electrophoresis and visualized by gel staining with Coomassieblue. Arrowhead indicates the 74-kDa protein band.

Fig. 5. Immunological identiWcation of L-bifunctional enzyme as catalase-binding protein. A polyclonal antibody against L-BFE was used toconWrm the identity of the 74-kDa protein that interacts with native A(catalase-linked to resin) or denatured B (SDS–PAGE-catalase and trans-ferred to nitrocellulose membrane) catalase protein. Catalase linked toresin or on the nitrocellulose membrane incubated with the cytosol (lanes1 and 3) or buVer (lanes 2 and 4) for 48 and 96 h, respectively.

136 R.S. Makkar et al. / Archives of Biochemistry and Biophysics 451 (2006) 128–140

mRNA for L-BFE and catalase were measured by quantita-tive PCR (Fig. 7A and B), while the GAPDH levels weremeasured by reverse transcriptase-PCR (Fig. 7C). Theanalysis demonstrated that the mixture of diVerent siRNAfragments (T2-T4) reduced signiWcantly (50%) the levels ofL-BFE (p < 0.001 for T2, T3 or T4; Fig. 7A). Transfectionreagent alone (T1) decreased the levels of mRNA for L-BFE (p < 0.01), but this decrease (10%) was minor com-pared with the 50% decrease observed with the L-BFEsiRNA (Fig. 7A). The mixture of diVerent siRNA frag-ments did not aVected catalase mRNA levels (Fig. 7B) withthe exception of the mixture 3 (siRNA 2 plus 3), which pro-duced an 18% reduction. However, this reduction seems tobe an unspeciWc eVect because such decrease was not seenwith the other two siRNA mixtures (Fig. 7B). siRNA forGAPDH reduced the levels of GAPDH mRNA determinedby reverse transcriptase-PCR (TH, positive control) asexpected (Fig. 7C), but did not aVect signiWcantly catalase

or L-BFE mRNA levels (Fig. 7A and B). These cells werefurther analyzed for catalase activity. Total catalase activitywas measured in presence of Triton X-100 detergent. Theperoxisomal (membrane bound) activity was determined asthe diVerence between the total activity and the activity ofcatalase assayed in the presence of digitonin, which repre-sents the enzyme present in the cell cytosol. The disruptionof L-BFE expression by the mixture of siRNA did not alterthe intracellular distribution of catalase activity betweencytosol and peroxisomes (Fig. 8A). However, a signiWcantdecrease in the speciWc activity (p < 0.05; Fig. 8B) and areduction of protein levels of catalase (Fig. 8C) wereobserved in cells treated for L-BFE silencing as describedunder methods section. The observed decrease in thecatalase protein but not of its mRNA following disruptionof L-BFE expression indicates that catalase-L-BFE interac-tion might play a role in the assembly/oligomerization ofcatalase or in the translational activity of catalase mRNA.

Fig. 6. Mass spectrum of the 74-kDa-protein band. MALDI TOF/TOF mass spectrometry was used to identify the catalase-binding protein puriWed byaYnity interaction. A 74-kDa protein-band obtained after SDS-gel electrophoresis and staining was excised (gel plug) and subjected to proteomic analysis.

Arrows indicates the peptide ions listed in Table 4 for the 74-kDa protein from immobilized catalase.

Table 4Peptides utilized for the identiWcation of catalase-binding protein

The tabulated peptides ions were obtained from a mass spectrum like the one shown in Fig. 6. The list includes only the peptide ions present in higher rel-ative abundance in the mass spectrum of the gel plugs containing the proteins obtained from the binding to immobilized catalase and from immunoprecip-itation of catalase. The intensity matched (total aminoacid residues identiWed for each protein) corresponded to 20.3% and 12.9%, respectively.

Gel plug Calculate mass Observed mass Start Sequence End sequence Peptide sequence

74 kDa immobilized catalase 1037.52 1037.49 156 164 YLSADEALR1187.60 1187.58 266 275 ALQYAFFAEK1491.88 1491.91 119 133 VGLPEVTLGILPGAR1661.93 1661.97 535 551 KGQGLTGPSLPPGTPVR1784.88 I784.94 684 698 QNPDIPQLEPSDYLR1940.98 1941.04 684 699 QNPDIPQLEPSDYLRR1988.03 1988.11 588 603 IHKPDWSTFLSQYR

74 kDa-immunoprecipitate 1037.52 1037.54 156 164 YLSADEALR1187.60 1187.60 266 275 ALQYAFFAEK1491.88 1491.96 119 133 VGLPEVTLGILPGAR1661.93 1661.97 535 551 KGQGLTGPSLPPGTPVR1784.88 1785.16 684 698 QNPDIPQLEPSDYLR1940.98 1941.46 684 699 QNPDIPQLEPSDYLRR1988.03 1988.56 588 603 IHKPDWSTFLSQYR

R.S. Makkar et al. / Archives of Biochemistry and Biophysics 451 (2006) 128–140 137

Discussion

A central question in X-ALD pathology is how themolecular defect in ALDP results in reduced peroxisomalVLCS activity. Since ALDP is a peroxisomal membraneprotein and VLCS is a membrane-associated protein, a pos-sible explanation is that VLCS and ALDP may physicallyinteract for proper activation of VLC fatty acids in peroxi-somes. Transfection studies using human Wbroblasts havedemonstrated that overexpression of VLCS does notrestore �-oxidation function in these cells [29]; however,transfer or transfection of the ALD gene restores this activ-ity [28,30]. In addition, overexpression or pharmacologicalinduction of other ABC peroxisomal transporters also sub-stitute ALDP function [31–33]. Therefore, these observa-

Fig. 7. siRNA for L-BFE does not aVect the mRNA levels of catalase orGAPDH. The mRNA levels of L-BFE (A) and catalase (B) were deter-mined by quantitative RT-PCR whereas the levels for GAPDH weredetermined by reverse transcriptase-PCR (C) as indicated in methods. L-BFE siRNA fragments used were T2 (lane 4), T3 (lane 5), and T4 (lane 6).T1: transfection agent only; T2: siRNA fragments 1 and 2; T3: siRNAfragments 1 and 3; T4: siRNA fragments 1–3. siRNA fragment againstGAPDH (TH) was used as positive control. siRNA fragments 1–3 are:siRNA IDs 146224, 112833, and 146225 (Ambion), respectively. The datarepresent the mean § SD for n D 3 experiments done in duplicate.¤p < 0.05; ¤¤¤p < 0.001.

tions indicate that ALDP or a related protein is requiredfor proper VLCS enzymatic function in peroxisomes.Indeed, the studies presented here support a molecularinteraction between VLCS and ALDP on the luminal sideof the peroxisome membrane.

Previously, we reported the topological localization ofVLCS and ALDP in peroxisomes [11,34]. VLCS is a mem-brane-associated protein with its active site localized on theluminal side of the peroxisome membrane [11,35]. On theother hand, ALDP is an integral membrane protein thatcontains two domains: a hydrophobic transmembranedomain (residues 1–361) containing six putative membranespanning segments that form three loops protruding intothe lumen of peroxisomes; and a cytoplasmic ATP bindinghydrophilic domain (residues 362–745) [34]. The studiesusing a yeast two-hybrid system indicates that a molecularinteraction might occur between VLCS and the Wrst pro-truding loop of the transmembrane domain of ALDP inperoxisomes. This result is further supported by the studiesusing surface plasmon resonance. In these studies, the inter-action of puriWed VLCS with chip-linked ALDP synthetic

Fig. 8. Peroxisome bound enzymatic activity, speciWc activity, and proteinlevels of catalase are diVerentially aVected by siRNA fragments of L-BFE.Latency of catalase (peroxisomes associated catalase) (A) was determinedas the diVerence taken of catalase activity assayed on 6-well plates in thepresence of Triton X-100 (total activity) and Digitonin (cytosolic frac-tion); speciWc activity of catalase (B) was determined in the presence ofTriton X-100 and was calculated in reference to the amount of proteindetermined in the 6-well plates. Immunodetection (C) was performed asindicated in Materials and methods. L-BFE siRNA fragments used wereas indicated in legend to Fig. 7. The data in A, B represent the mean § SDfor n D 3 experiments done in duplicate. ¤p < 0.05.

138 R.S. Makkar et al. / Archives of Biochemistry and Biophysics 451 (2006) 128–140

peptides demonstrated higher aYnity (KD, 3-fold diVer-ence) between VLCS and the control peptide (L1) as com-pared to the peptide with an ALD mutation (L1m). Theseresults suggest that changes in the sequences of the ALDPloop region might have consequences for the molecularinteraction between these two proteins. These observationsindicate that ALDP and VLCS have a physical interactionin the peroxisomal membrane and might resemble the inter-action between fatty acid transport protein 1 and longchain acyl-CoA synthetase described in the plasma mem-brane of adipocytes [36].

Peroxisomal proteins are synthesized on free polysomesand post translationally targeted to the existing peroxi-somes via peroxisomal targeting signals. The signals for theimport of the peroxisomal matrix proteins are either a C-terminal tripeptide (PTS1) or a N-terminal cleavable prese-quence (PTS2) [16,37]. The majority of the matrix proteinscontain a tripeptide consisting of serine-lysine-leucine(-SKL) or a closely related consensus sequence (S/A/C)(K/R/H)(L/M) [37,38]. On the other hand, catalase, has anatypical PTS1 signal consisting of the four amino acidslysine-alanine-asparagine-leucine (-KANL) [18]. These dis-tinct sequences lead to the diVerences observed in theeYciency of the catalase import, due to a weak interactionwith the soluble receptor Pex5p [39,40]. Furthermore, ascatalase oligomerizes and acquires its prosthetic groupprior to its import [21,27], we can hypothesize that otherfactor(s) and/or interactions might be supporting these pro-cess to make them more eYcient. Indeed, studies in yeastindicate that certain matrix proteins that do not display thePTS1 signal are able to piggyback or hitch-hike into peroxi-somes with an unrelated protein containing the PTS1 signal[41].

Studies with human skin Wbroblasts from patients withZellweger syndrome-like clinical features and multipleenzyme deWciencies have reported that mislocalization ofcatalase to cytosol resulted in an imbalance of the oxidativestatus of the cells [12,13]. This imbalance can be restored bytreatment with the antioxidant vitamin E [14]. Theimpaired oxidative stress and peroxisomal function in thosecells was also normalized following transfection of an engi-neered catalase gene carrying the -SKL signal instead of the-KANL catalase sequence [13]. Our search for additionalfactors (proteins) that might aid catalase in its oligomeriza-tion/import resulted in the discovery that catalase interactswith a 74-kDa protein that we identiWed as L-bifunctionalenzyme. Surprisingly, the interaction occurs with both thedenatured and native form of catalase, indicating that theinteraction between the two proteins can occur at twodiVerent structural levels: primary and tertiary proteinstructure. Moreover, catalase interacted with L-BFE incytoplasm as well as in peroxisomes, suggesting that thisinteraction might be maintained beyond oligomerization/import of catalase.

In recent years, the presence of two multifunctional(bifunctional) enzymes (BFE) in peroxisomes has beendescribed: L-bifunctional enzyme with dehydrogenase,

hydratase and isomerase activities; and D-bifunctionalenzyme, with dehydrogenase, hydratase and isomeraseactivities plus the sterol carrier protein-x (SCPx) sequence[42]. Both bifunctional enzymes carry a PTS1 import signal[43,44] and hence are transported into peroxisomes via thePTS1 recognition system. Alteration in D-BFE expression isassociated with a human peroxisomal disorder of fatty acid�-oxidation [45]. On the other hand L-BFE is involved inthe degradation of dicarboxylic acids and bile acids synthe-sis in humans and rodents [46–48], in mRNA physiology inthe cytoplasm of rice [49], and in the activation of peroxi-some proliferator-activated receptor alpha (PPAR-�) in ratliver [50]. Given these observations, L-BFE is a versatileprotein that plays additional roles outside the �-oxidativepathway in diVerent tissues/species.

Possible functions that can be proposed for the interac-tion between L-BFE and catalase are assisting the oligomer-ization/assembly and/or import of catalase. Currentevidence indicates that homotetrameric catalase, whichcontains a heme group as cofactor, is assembled in the cyto-plasm and then imported into peroxisomes of human cell[27,51] as well as yeast [21,52]. Chaperone molecules, pres-ent in the cytoplasm, are known to facilitate folding andoligomerization of peroxisomal proteins [53,54]. Our resultsof intracellular distribution of catalase in peroxisome vs.cytoplasm with interference of L-BFE expression (siRNA)show no change in the subcellular distribution (latency) ofcatalase, suggesting that L-BFE might not participate in theimport of catalase. However, interference in expression ofL-BFE decreased the protein and speciWc activity of cata-lase, without aVecting its mRNA, suggesting that a decreasein L-BFE possibly interferes with catalase oligomerization,which in turn might lead to its rapid degradation. Alterna-tively, L-BFE might play a role in regulating the translationof catalase mRNA [49]. Interestingly, the disruption of theL-BFE and D-BFE genes (double KO mouse) has a lethaleVect, and immunohistochemical analysis of liver of thesemice showed an increased distribution of catalase in thecytosol vs. peroxisomes when compared to the liver of wildtype animals [48].

Our Wndings describe a further example of interactionbetween peroxisomal proteins that might be of signiWcancefor the peroxisome metabolism in vivo [55]. Similarly, inter-actions of non-speciWc lipid transfer protein (SCP2) withenzymes of the �-oxidation cycle (acyl-CoA oxidase, 3ketoacyl-CoA thiolase and BFE) [56], sterol carrier protein2 and acyl-CoA oxidase [57] in peroxisomes, and malatesynthetase with isocitrate lyase and malate dehydrogenase[58,59], catalase with isocitrate lyase [60] in glyoxysomeshas been reported previously. In the last particular example,it has been suggested that catalase might confer protectionto the isocitrate lyase and its products against H2O2-medi-ated oxidation [61].

In summary, the speciWc interactions that we describein peroxisomes for the membrane (ALDP-VLCS) as wellas the matrix (L-BFE-catalase) proteins indicate thatthese pairs participate in higher order oligomeric com-

R.S. Makkar et al. / Archives of Biochemistry and Biophysics 451 (2006) 128–140 139

plexes that contribute to proper degradation of VLCfatty acids and hydrogen peroxide in peroxisomes,respectively.

Acknowledgments

The authors thank Ms. Joyce Bryan and Mrs. JeniVerBethard and Rebecca Ettling for technical assistance. Pro-teomic and surface plasmon resonance data were obtainedusing the Mass Spectrometry Research Resource and theProteogenomics facilities of the Medical University ofSouth Carolina, respectively. This work was supported byGrant Nos. C06 RR018823 and C06 RR015455 from theExtramural Research Facilities Program of the NationalCenter for Research Resources and Grant Nos. NS-22576,NS-34741, NS-37766, and AG-25307 from National Insti-tute of Neurological Disorders and Stroke, NIH.

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