review article abc transporter subfamily d: distinct differences...

Review ArticleABC Transporter Subfamily D Distinct Differences inBehavior between ABCD1ndash3 and ABCD4 in SubcellularLocalization Function and Human Disease

Kosuke Kawaguchi and Masashi Morita

Department of Biological Chemistry Graduate School of Medicine and Pharmaceutical Sciences University of Toyama2630 Sugitani Toyama 930-0194 Japan

Correspondence should be addressed to Kosuke Kawaguchi kkawaphau-toyamaacjp

Received 24 June 2016 Accepted 29 August 2016

Academic Editor Hiroshi Nakagawa

Copyright copy 2016 K Kawaguchi and M MoritaThis is an open access article distributed under the Creative CommonsAttributionLicense which permits unrestricted use distribution and reproduction in anymedium provided the originalwork is properly cited

ATP-binding cassette (ABC) transporters are one of the largest families of membrane-bound proteins and transport a wide varietyof substrates across both extra- and intracellular membranes They play a critical role in maintaining cellular homeostasis To datefour ABC transporters belonging to subfamily D have been identified ABCD1ndash3 and ABCD4 are localized to peroxisomes andlysosomes respectively ABCD1 and ABCD2 are involved in the transport of long and very long chain fatty acids (VLCFA) or theirCoA-derivatives into peroxisomes with different substrate specificities while ABCD3 is involved in the transport of branched chainacyl-CoA into peroxisomes On the other hand ABCD4 is deduced to take part in the transport of vitamin B

12from lysosomes into

the cytosol It is well known that the dysfunction of ABCD1 results in X-linked adrenoleukodystrophy a severe neurodegenerativedisease Recently it is reported that ABCD3 and ABCD4 are responsible for hepatosplenomegaly and vitamin B

12deficiency

respectively In this review the targeting mechanism and physiological functions of the ABCD transporters are summarized alongwith the related disease

1 Introduction

There are 48 ATP-binding cassette (ABC) transporters inhumans that are classified into seven subfamilies A to Gbased on structural organization and amino acid homology[1 2] ABC transporters exist on plasma membranes andintracellular compartments such as the mitochondria per-oxisomes endoplasmic reticulum (ER) Golgi apparatus andlysosomes and play important roles in transporting a widevariety of substrates across membranes in order to sustaincellular homeostasis Defects in their functions are related tovarious diseases [3]

To date four ABCD transporters have been identi-fied adrenoleukodystrophy protein (ALDPABCD1) ALDP-related protein (ALDRPABCD2) the 70-kDa peroxiso-mal membrane protein (PMP70ABCD3) and the PMP70-related protein (P70RABCD4) [4ndash7] The ABCD trans-porters have a predicted ABC half-transporter structure withone transmembrane domain (TMD) and one nucleotide-binding domain (NBD) (Figure 1) ABCD1ndash3 are known to

be peroxisomal proteins and predominantly function as ahomodimer [8] but a heterodimeric structure has also beensuggested [9] It is reported that ABCD1 and ABCD2 areinvolved in the transport of long and very long chain fattyacids (VLCFA) or their CoA-derivatives into peroxisomes[10 11] ABCD3 is thought to play an important role inthe transport of branched chain acyl-CoA and the bile acidintermediates di- and tri-hydroxycholestanoyl-CoA (DHCAand THCA) [12] It is known that ABCD1 andABCD3 defectsare the cause of X-linked adrenoleukodystrophy (X-ALD) aneurodegenerative disease and hepatosplenomegaly a liverdisease respectively [5 13]

ABCD4 was identified by a homology search for ABCD1-and ABCD3-related sequences in a database of expressedsequence tags and is thus considered to be a peroxisomalprotein [7] We reported that ABCD4 is not a peroxisomeresident protein but rather an ER resident protein How-ever the function of ABCD4 still remained unknown atthat time [14] In 2012 it was reported that mutations inABCD4 cause a newly identified inborn error of vitamin B

12

Hindawi Publishing CorporationBioMed Research InternationalVolume 2016 Article ID 6786245 11 pageshttpdxdoiorg10115520166786245

2 BioMed Research International

COOH

Membrane

Matrix

Cytosol

ABC signature

TMD

NBD

Walker A

Walker B NH2

Figure 1 Hypothesized structure of the ABCD transporter The ABCD transporters are comprised of a half-size ABC transporter with onetransmembrane domain (TMD) and one nucleotide-binding domain (NBD) Six transmembrane domains are located in the NH

2-terminal

half of the transporter and Walker A Walker B and ABC signature are located in the COOH-terminal half of the transporter

(cobalamin) metabolism [15] The dysfunction of ABCD4results in a failure in lysosomal cobalamin release whichmimics the cobalamin deficiency caused by the defect oflysosomal membrane protein LMBD1 [16] It is indicatedthat ABCD4 has a function on the lysosomal membraneRecently we showed that ABCD4 is translocated fromthe ER to lysosomes through an interaction with LMBD1[17]

As mentioned above dysfunction of any of the ABCDtransporters other than ABCD2 may be the cause of diseaseIn this review we will focus on the targeting mechanismof ABCD transporters to peroxisomes or lysosomes theirfunctions and related diseases

2 Role of Human Peroxisomes and Lysosomes

21 Peroxisomes Peroxisomes are ubiquitous organelles in alleukaryotic cells and participate in various activities relatedto cellular homeostasis especially lipid metabolism [18] Theindispensable role of the peroxisome in human health anddevelopment is evidenced by the large number of geneticdiseases termed peroxisomal disorders [19]

The peroxisomes contain more than 50 different enzymesthat catalyze a variety of metabolic pathways including fattyacid 120573-oxidation the biosynthesis of ether phospholipids andbile acids and the metabolism of reactive oxygen species Inaddition peroxisomes fulfill essential roles in the synthesisof the ether phospholipid such as plasmalogen and the 120572-oxidation of phytanic acid Among these various functionsthe 120573-oxidation of fatty acid is generally considered to be themain one [20]

In peroxisomes 120573-oxidation is performed on VLCFA(gtC220) branched chain fatty acids (eg pristanic acid)bile acid intermediates such as DHCA and THCA [21]poly-unsaturated fatty acid (eg tetracosahexaenoic acid)long chain dicarboxylic acids [22] 2-hydroxy fatty acidsand a number of prostanoids The 120573-oxidation of thesesubstrates is restricted to peroxisomes and does not occur inmitochondria where long chain medium chain and shortchain fatty acids (C18 and shorter) are 120573-oxidized

These fatty acids are imported into peroxisomes viaspecific transporters as mentioned above where they aredegraded by four 120573-oxidation cycles Like the activity ofmitochondrial 120573-oxidation the 120573-oxidation performed inperoxisomes is conducted by oxidation hydration dehydro-genation and thiolytic cleavage [23] In the first step theacyl-CoA oxidase ACOX1 or ACOX2 catalyze the reactionACOX1 preferentially degrades saturated and unsaturatedstraight chain fatty acids while ACOX2 has a high affinityfor 2-methyl branched fatty acids such as pristanoyl-CoADHCA-CoA and THCA-CoA In the second and thirdsteps D- and L-bifunctional enzymes function as an enoyl-CoA hydratase and a 3-hydroxyacyl CoA dehydrogenaserespectively Two thiolases 3-ketoacyl-CoA-thiolase 1 (pTH1)and SCPx (pTH2) are involved in the last step pTH1metabolizes only straight chain fatty acids The branchedchain fatty acids and bile acid precursors (pristanic acidDHCA and THCA) are solely cleaved by pTH2 Once thefatty acid chains are shortened to medium chain fatty acyl-CoA via peroxisomal 120573-oxidation they are conjugated tocarnitine exit the peroxisomes and undergo further 120573-oxidation in mitochondria On the other hand choloyl-CoAand deoxycholoyl-CoA are converted to taurine- or glycine-conjugated cholic acid or deoxycholic acid by bile acids-CoA amino acid N-acyltransferase and then exported intothe cytosol

When functional peroxisomes or enzymes are absent theundegraded molecules such as VLCFA DHCA THCA andphytanic acid accumulate in the cell while physiologicallyessential molecules such as bile acids and plasmalogenbecome deficient In addition to lipid metabolism peroxi-somes play a role in several nonlipid metabolic pathwaysincluding purine polyamine glyoxylate and amino acidmetabolism

Recently new functions for peroxisome have been report-edly demonstrated It is reported that peroxisomes as wellas mitochondria act as intracellular signaling platforms ininnate immunity and are important for early stage antiviralsignaling [24] Moreover it has been shown that perox-isomes have a role in the transport of free cholesterol

BioMed Research International 3

from lysosomes to the ER through lysosome-peroxisomemembrane contact [25] It is thus evident that peroxi-somes are multifunctional organelles that interact with otherorganelles such as mitochondria lysosomes the ER andlipid droplets while performing a diverse array of biologicalfunctions

22 Lysosomes Lysosomes are membrane-bound intracellu-lar organelles present in all eukaryotic cells that internallyhave an acidic pH (sim50) that is maintained by an ATP-dependent proton pump the V-type H+-ATPase Lysosomesare dynamic organelles crucially involved in many physio-logical processes such as the degradation andor recycling ofmacromolecules cholesterol homeostasis plasmamembranerepair pathogen defense cell death and cell signaling as wellas being core regulators of cell homeostasis [26] Two classesof proteins are essential for these lysosomal activities Oneis comprised of soluble lysosomal hydrolases referred to asacid hydrolases Acid hydrolases function at a low pH in thisorganelle and possess a wide variety of substrate specificitiesMore than 60 hydrolases (including proteases peptidasesphosphatases nucleases glycosidases sulfatases and lipases)have been identified to date [27] The other class is madeup of integral lysosomal membrane proteins These havea variety of functions including the transport of substrateand metabolic products establishment of the pH gradientsvesicular transport and maintenance of lysosomal structuralintegrity There are 25 lysosomal membrane proteins iden-tified thus far but the existence of additional membraneproteins is expected [28ndash30]

The primary cellular function of the lysosome is thedegradation and recycling of macromolecules During theseprocesses cytoplasmic components are delivered to lyso-somes by endocytosis phagocytosis or autophagy Sev-eral classes of macromolecules are hydrolyzed by solublelysosomal hydrolases including proteins polysaccharideslipids and nucleic acids The end products of lysosomaldigestion are recycled in the cell after diffusion or undergocarrier-mediated transport across the lysosomal membraneThe process of autophagy is classified into macroautophagymicroautophagy and chaperone-mediated autophagy [31]Macroautophagy is a process whereby cytoplasmic organellesand substances are sequestered within autophagosomes thatin turn fuse with lysosomes and their contents are degradedMicroautophagy is the direct sequestration of cytoplasmiccargo at the boundary of the lysosomal membrane Duringchaperone-mediated autophagy cytosolic proteins possess-ing specific recognition motifs are delivered to lysosomes viaa chaperone in a lysosomal receptor LAMP-2A dependentmanner

Cholesterol is an essential structural element of cellularmembranes as well as a precursor for the synthesis of steroidhormones bile acids and lipoproteins Cellular cholesterolhomeostasis is controlled by lysosomal cholesterol effluxthrough the Niemann-Pick type C1 (NPC1) and NPC2 pro-teins [32 33] NPC1 is a large protein with 13 transmembranedomainswhich localizes to themembranes of endosomes andlysosomes NPC2 exists in endosomes and lysosomes as asoluble protein

Lysosomes are also involved in a secretory pathwayknown as ldquolysosomal exocytosisrdquo that plays a major rolein important processes such as the immune response cellsignaling and plasma membrane repair Plasma membraneinjury is a common event in mammalian cells especiallyin cells that operate under conditions of mechanical stressPlasma membrane damage results in calcium influx afterwhich lysosomal exocytosis and plasma membrane repairare initiated by calcium binding to synaptotagmin 7 at thelysosomal membrane [34] Lysosomal cell death signaling istriggered by a release of lysosomal cathepsins through an asyet unknown membrane protein

The lysosomal system is of considerable biomedicalimportance Lysosomal dysfunction causes and contributesto numerous diseases the majority of which are classified aslysosomal storage diseases (LSDs) LSDs result from defectsin soluble lysosomal hydrolases or lysosomal membraneproteins Over 50 LSDs have been identified to date [30]

3 Targeting of ABCD Transporters tothe Peroxisome or Lysosome

The subcellular localization of proteins is strictly controlledin cells as it is inherently important for their vital func-tioning For the trafficking of ABCD transporters an NH

2-

terminal hydrophilic region containing an H0 motif plays animportant role [35] The H0 motif is a hydrophobic segmentadjacent to the NH

2-terminal portion of TMD1

The ABCD transporters are translated on free polysomesThe ABCD1ndash3 forms possessing the H0 motif are selectivelycaptured by Pex19p which is essential for the early stepsof peroxisome biogenesis and most likely also involvedin peroxisomal membrane synthesis and then destined forperoxisomes [8] In contrast ABCD4 hardly interacts withPex19p because of its lack of the NH

2-terminal H0 motif

and as a result ABCD4 is recognized by certain signalrecognition particles and integrated into the ER membrane[14] Subsequently ABCD4 is translocated to lysosomes(Figure 2) [17]

Concerning the trafficking of the newly synthesizedperoxisomal ABCD transporters ABCD3 has been studied ingreatest detail [36ndash39] As ABCD3 is a hydrophobic integralmembrane protein binding with Pex19p is indispensable forABCD3 to retain its soluble form and proper conformationfor targeting to peroxisomes In order to investigate theregion of ABCD3 (AA1ndash659) that is critical for targeting toperoxisomes Kashiwayama et al prepared various truncatedor mutated ABCD3 fused with GFP The COOH termi-nally truncatedABCD3 (AA1ndash144)-GFP containing deducedTMD1 and TMD2 along with GFP-ABCD3 (AA263ndash375)containing TMD5 and TMD6 was found to be localizedto peroxisomes Further analysis using mutated ABCD3revealed that ABCD3 is recognized and binds to Pex19p atthe NH

2-terminal hydrophobic motif constituted by Leu21-

Leu22-Leu23 and the region of TMD5-TMD6 Subsequentlythey prepared various mutated forms of ABCD3 to iden-tify the peroxisomal membrane protein targeting signals(mPTSs) It was suggested that the hydrophobic aminoacid pairs Ile70-Leu71 and Ile307-Leu308 adjacent to TMD1


Peroxisome

ABCD1ndash3

ABCD4

COOH

NBD

TMD

H0 motif

Pex19p

COOH

NBD

TMDPex19p

SRP

Peroxisome ER Lysosome

ABCD4-HA LMBD1-GFP Merge120572-LAMP1

ABCD1-GFP Merge120572-catalase

HA-ABCD2 Merge120572-catalase


NH2

NH2

Figure 2 Targeting and localization of the ABCD transporters For the trafficking of theABCD transporters anNH2-terminalH0motif plays

an important roleTheABCD1ndash3 forms possessing this H0motif are selectively captured by Pex19p and destined for peroxisomes In contrastABCD4 hardly interacts with Pex19p because of its lack of the H0motif and as a result ABCD4 is recognized by a signal recognition particle(SRP) and integrated into the ER membrane Subsequently ABCD4 is translocated to lysosomes through the association with LMBD1 GFP-tagged ABCD1 and ABCD3 were expressed in CHO cells The distribution of ABCD1 and ABCD3 was compared with that of peroxisomesstained with anti-catalase [36] The localization of ABCD2 was cited from [6] with some modification HA-tagged ABCD2 was expressedin COS cells The distribution of HA-ABCD2 was compared with that of catalase HA-tagged ABCD4 was expressed in CHO cells stablyexpressing LMBD1-GFP [17] The subcellular localization of ABCD4-HA and LMBD1-GFP was compared with that of lysosomes labeledwith anti-LAMP1 Bar 10120583m

and TMD5 respectively are essential for the targeting toperoxisomesThusABCD3 forms a complexwith Pex19p thatis transported to peroxisomes by mPTSs Finally ABCD3 isinserted into the peroxisomal membranes through a putativeproteinaceous receptor on the peroxisomal membrane Inthis process at least two TMDs (TMD1 and TMD2) arerequired for proper insertion Furthermore it was shownthat the TMD1 segment of ABCD3 possesses a potent ERtargeting function and a certain cis-acting element in orderfor TMD1 to suppress ER targeting [40] For this suppressioncertain NH

2-terminal nine amino acids especially Ser5 are

indispensableThe targeting of ABCD1 to peroxisomes has also been

characterized [41] It was shown that the 14-amino-acid

motif (F(FL)X(RQK)(LF)(LIK)XLLKIL(FIV)P) adja-cent to TMD1 functions as an mPTS of ABCD1 and itwas demonstrated that the substitution or deletion of thesehydrophobic residues significantly reduced the targetingefficiency In particular the three amino acids Leu78-Leu79-Arg80 were shown to be critical for peroxisomal target-ing of ABCD1 This region corresponds to Ile70-Leu71-Lys72 in ABCD3 which was identified as an mPTS inABCD3 In ABCD2 a potential Pex19p binding site wasalso identified as ABCD1 [41] This corresponds to AA84ndash97 which are localized in proximity to the putative TMD1However no experimental data are available as yet tosupport the functionality of this putative Pex19p bindingsite


Membrane

CytosolABCD1 ABCD2 ABCD3

LCFA-CoATHCA-CoADHCA-CoAPristanoyl-CoAPhytanoyl-CoA

C240-CoAC260-CoA

C200-CoAC220-CoA

C201-CoAC221-CoA

C181-CoA

C246-CoAC226-CoA

Matrix

Membrane

CytosolABCD4

MatrixPeroxisome

Lysosome

Vitamin B12

Figure 3 Substrate specificity of the ABCD transporters ABCD1ndash3 predominately exist as a homodimer ABCD1 and ABCD2 haveoverlapping substrate specificities toward saturated and monounsaturated VLCFA-CoAs However ABCD1 has a higher specificity to C240-CoA and C260-CoA than ABCD2 In contrast ABCD2 has a higher specificity for polyunsaturated C226-CoA and C246-CoA ABCD3 isthought to be involved in the transport of LCFA-CoA branched chain acyl-CoA and the bile acid intermediates THCA-CoA and DHCA-CoA ABCD4 is deduced to be involved in the transport of cobalamin from lysosomes to the cytosol

Newly synthesized ABCD4 is inserted into ER mem-branes and then translocated to lysosomes through aninteraction with the lysosomal membrane protein LMBD1Very recently we demonstrated the underlying mechanismof this translocation [17] When ABCD4 is exogenouslyexpressed alone in HuH7 cells ABCD4 is localized in ERmembrane and exists as homodimer However coexpressionof LMBD1 in the cells drastically changes the localization ofABCD4 to lysosomes During this translocation the ABCD4dimer forms a complex with LMBD1 To identify the regionsof ABCD4 critical for the translocation to lysosomes weprepared chimeric ABCD4 proteins in which each TMD ofABCD4 was exchanged to the corresponding putative trans-membrane helix with ABCD1 which does not interact withLMBD1 The distribution pattern of the ABCD4 chimeras 1ndash6 coexpressed with LMBD1 revealed that the regions aroundtransmembrane helices 2 and 5 of ABCD4 are criticallyimportant for the translocation of ABCD4 to lysosomesalong with LMBD1 As mentioned above wild-type ABCD4exists as a homodimer on the ER membrane However theABCD4 chimeras 2 and 5 exist as a bigger complex indicatinghigher-order oligomer formation and this impaired ABCD4dimer formation disturbs the targeting to lysosomes withLMBD1 Concerning the targeting of LMBD1 to lysosomesLMBD1 possesses a putative AP-2 bindingmotif [42] MutantLMBD1 with this motif modified does not localize tolysosomes but to the cell surface When this mutant LMBD1and ABCD4 are coexpressed the distribution pattern ofABCD4 is superimposable on the mutant LMBD1 on theplasma membrane of the cells Hence the translocation ofABCD4 from the ER to lysosomes depends on the lysosomaltargeting ability of LMBD1 Furthermore it was confirmedthat endogenous ABCD4 was localized to both lysosomesand ER in HEK293 cells by equilibrium iodixanol density

gradient centrifugation Lysosomal localization of ABCD4was reduced to sim40 in LMBD1 knockout HEK293 cellsThese results show that LMBD1 is responsible for the local-ization of endogenous ABCD4 The subcellular localizationof ABCD4 is determined through its association with anadaptor protein

4 Function of the ABCD Proteins

41 ABCD1 A defect in the ABCD1 protein causes X-ALDwhich is characterized by the accumulation of VLCFA intissues Thus it is thought that ABCD1 transports VLCFAacross the peroxisomalmembrane for120573-oxidation of VLCFAThis is supported by the result that exogenous expressionof ABCD1 in X-ALD skin fibroblasts recovered the VLCFA120573-oxidation and consequently the VLCFA content wasdecreased to normal in the fibroblasts [43] Furthermoreit has been demonstrated that ABCD1 is involved in thetransport of saturated monounsaturated and polyunsatu-rated VLCFA-CoA such as C180- C220- C240- C260-C181- and C246-CoA across the peroxisomal membraneThis was shown by expressing human ABCD1 in the yeastpxa1pxa2Δ mutant which lacks peroxisomal half-size ABCtransporters (Figure 3) [10 11] However the mechanism offatty acid transport by ABCD1 is controversial Two modelsare generally utilized In the first model esterified fatty acidsare delivered directly to the peroxisomal matrix [44] In theother model acyl-CoA is hydrolyzed prior to its entry intothe peroxisomal matrix and reesterified by a peroxisomalacyl-CoA synthetase [45] The first model depends on thefact that 120573-oxidation of VLCFA-CoA directly depends onABCD1 without the need for any of the additional CoA thatis required for reesterification of VLCFA by the acyl-CoAsynthetase in the peroxisomes isolated from fibroblasts Onthe other hand it was confirmed that the CoA moiety is


cleaved during the transport cycle using isotopic labeling ofyeast cells with 18O Furthermore it was shown that CTSa homolog of human ABCD1 in Arabidopsis thaliana itselfpossesses intrinsic acyl-CoA thioesterase activity [46] Wealso confirmed that the human ABCD1 expressed in themethylotrophic yeast also hydrolyzes acyl-CoAHowever theprecise transport mechanism of VLCFA-CoA as yet is stillunclear

42 ABCD2 It is known that ABCD2 shares functionalredundancy with ABCD1 [47] In fact overexpression ofABCD2 in X-ALD fibroblasts fully restores the 120573-oxidationdefect However the phenotype of fatty acid abnormalitiesin Abcd2minusminus mice is different from that in Abcd1minusminus mice[48] Abcd1minusminus mice exhibit a higher accumulation of C240and especially C260 In contrast Abcd2minusminus mice displayfatty acid abnormalities especially at the level of mono- andpolyunsaturated fatty acids indicating the different substratespecificities of ABCD1 and ABCD2 This was shown inexperiments using the yeast pxa1pxa2Δ mutant expressingABCD1 andor ABCD2 [11] ABCD2 shows overlappingsubstrate specificities with ABCD1 toward saturated andmonounsaturated fatty acids ABCD1 has a higher specificityfor saturated VLCFA-CoA In contrast ABCD2 has an affin-ity for polyunsaturated fatty acids such as C226-CoA andC246-CoA but ABCD1 does not (Figure 3)These results areconsistent with the results fromH4IIEC3 cells overexpressingABCD2 [49] However the actual function of ABCD2 in vivois still unclear because the endogenous expression level ofABCD2 is quite low in human cells and there is no reporteddisease caused by a mutation of ABCD2

43 ABCD3 ABCD3 is one of the most abundant per-oxisomal membrane proteins at least in hepatocytes [50]and has been reported to be involved in the transportof various fatty acids It has been confirmed that ABCD3possesses substrate specificity that overlaps with ABCD1 andABCD2 but ABCD3 has been shown to have roles in thetransport of more hydrophilic substrates such as long chainsaturated unsaturated long-branched chain and long chaindicarboxylic fatty acids using the yeast pxa1pxa2Δ mutantexpressing human ABCD3 (Figure 3) [12] In Abcd3minusminus micethere are clearly evident bile acid abnormalities In theliver bile and intestine there was a significant reductionof chenodeoxycholic acid and cholic acid conjugated withtaurine or glycine On the other hand a remarkable increaseof bile acid intermediates with C27 was observed in the liverbile and intestine Furthermore for animals on a phytol dietthere was a marked accumulation of phytanic and pristanicacid in the plasma of Abcd3minusminus mice [13] Thus ABCD3is involved in the transport of branched chain fatty acidsand C27 bile acid intermediates into peroxisomes WhetherABCD3possesses intrinsic acyl-CoA thioesterase activity likeABCD1 has yet to be elucidated

44 ABCD4 Recently it was shown that ABCD4 is involvedin a newly discovered inherited defect affecting vitaminB12

(cobalamin) metabolism [15] In humans cobalamin

forms a complex with transcobalamin in the blood streamThis complex is taken up into lysosomes by endocytosisCobalamin is then released into the cytosol and convertedinto the two active cofactors methylcobalamin (MeCbl) andadenosylcobalamin (AdoCbl) [51] A defect inABCD4 resultsin the accumulation of cobalamin in lysosomes In patientfibroblasts the intracellular enzyme-bound cobalamin levelis significantly lower than control fibroblasts The expressionof wild-type ABCD4 in patient fibroblasts was shown tonormalize the intracellular enzyme-bound cobalamin leveland remarkably increase the synthesis of MeCbl and AdoCblOn the other hand the expression of ABCD4 mutated atthe putative ATP-binding site leads to a reduced synthesisof both cofactors This suggests that the ATPase activity ofABCD4 may be involved in the intracellular processing ofcobalamin As mentioned above mutations of ABCD4 andLMBD1 result in a quite similar phenotypeThis suggests thatthese two proteins function as a complex in the process oftransporting cobalamin from lysosomes to the cytosol Veryrecently we revealed that LMBD1 associates with ABCD4on the ER and supports the translocation of ABCD4 tolysosomes [17] The bacterial ABC transporter BtuCD isinvolved in the import of cobalamin into the cytoplasmacross the inner membrane and possesses ATPase activity[52] ABCC1 a multidrug resistance-associated protein onthe plasma membranes of mammalian cells is suggested tobe involved in the export of cobalamin out of cells [53]ABCC1 itself also possesses ATPase activity Therefore wepostulate that ABCD4 is composed of a transporter unit andthat LMBD1 is an accessory protein

ABCD1ndash3 transport substrates from the cytosol to perox-isomes In contrast ABCD4 reportedly transports cobalaminfrom lysosomes to the cytosol although it has not yet beendemonstrated that ABCD4 directly transports cobalamin Itis of special interest to determine the mechanism underlyingthe difference in the direction of this transport

5 Mutation and Disease

51 ABCD1 X-ALD is the most frequent peroxisomal dis-order caused by mutations or deletions of the ABCD1 genewith an average incidence in males of 1 20000 [54] TheABCD1 gene is composed of 10 exons and encodes theABCD1protein with length of 745 amino acids [5] At present717 nonrecurrent mutations are described in the X-ALDdatabase (httpwwwx-aldnl) The mutation types consistof missense (61) frame shift (22) nonsense (10) aminoacid insertiondeletion (4) and one or more exons deleted(3) Deletion frame shift and nonsense mutations generatetruncated ABCD1 proteins In contrast missense mutationsfrequently result in the instability and thereby a decreasein the amount of the ABCD1 protein It seems likely thatan ABCD1 protein with a missense mutation undergoesincorrect folding miss-targeting and a failure to form adimer all of which result in the instability of the mutantABCD1 protein

The missense mutations that occur in X-ALD patientshave been found throughout the entire gene and there are 209different amino acids with single amino acid substitutions


R401Q R518Q

P560L

R617H

E609K R660W

H0 motif

TMD NBD

EEA-like motif

G266R

Walker A Walker BABC signature

0 100 200 300 400 500 600 700

R554H

R401Q R518Q

P560L

R617H

E609K999 R660W

G266R R554H

Num

ber o

f pat

ient

s

60

50

40

30

20

10

0

Figure 4 Distribution of the missense mutations reported in X-ALD patients 209 different amino acids with single amino acid substitutionshave been identified Based on the X-ALDdatabase G266R R401Q R518Q R554H P560L E609K R617H and R660Ware themost frequentThe mutant ABCD1 proteins such as G266R R401Q and R554H are unaffected in terms of protein stability In contrast mutant ABCD1proteinswithmissensemutations (R518Q P560L E609K R617H andR660W) inCOOH-terminal nucleotide-binding domains are decreasedor not detected in the corresponding patient fibroblasts

that have been identified as disease-causing missense muta-tions [55] Based on the X-ALD database G266R R401QR518Q R554H P560L E609K R617H and R660W arethe most frequently identified (Figure 4) The mutationsG266R (loop 4) R401Q (downstream of TMD6) and R554H(between Walker A and Walker B) did not exert any effecton protein stability indicating that these amino acid residuesare critical for their function In contrast mutant ABCD1proteins with missense mutations (R518Q P560L E609KR617H and R660W) in COOH-terminal nucleotide-bindingdomains are either decreased or not detected It is thuslikely that the dysfunction of the ABCD1 protein is mainlycaused by the instability of the mutant ABCD1 proteinWe previously reported that mutant ABCD1 protein with amissense mutation in the COOH-terminal was frequentlydegraded by proteasomes or additional proteases [56]

ABCD protein is a half-sized ABC transporter that needsto form a dimer for its proper function [57] The NBDforms a sandwich dimer with two composite ATP-bindingsites comprising Walker A Walker B and H-loop of oneNBD and the signature motif of the other NBD [58] Themissense mutation for R518Q E609K and R617H is locatedin the region of Walker A the signature motif and WalkerB respectively suggesting that these mutant proteins mayfail to form dimer In contrast it was shown that the 87COOH-terminal amino acids (AA658ndash745) are involved inthe dimer formation [57] In addition no missense mutationhas been found in the COOH-terminal region after AA693These results suggest that the COOH-terminal region ofAA658ndash693 but not AA694ndash745 is required for dimer-ization Accordingly X-ALD fibroblasts having truncatedABCD1 protein (AA1ndash693) reportedly retain VLCFA 120573-oxidation activity [59] In X-ALD fibroblasts with a nonsensemutation (Q672X) the translation product lacking the 74COOH-terminal amino acids (AA1ndash671) was not detectable

indicating that AA672ndash745 is essential for its correct folding[60] Taken together the region between AA672 and 693appears to be critically important for dimerization Thisis supported by the recent report that showed that theCOOH-terminal region of the yeast peroxisomal ABCDprotein Pxa2p corresponding to W679 and L684 in ABCD1is involved in the heterodimerization that occurs with Pxa1p[61]

52 ABCD3 Recently a patient with an ABCD3 defect wasidentified [13]Thepatient exhibited hepatosplenomegaly andsevere liver disease along with a significant accumulationof the peroxisomal C27 bile acid intermediates DHCA andTHCA in plasmaThe peroxisomal 120573-oxidation of C260 and120573-oxidation enzyme activities were normal whereas the 120573-oxidation of pristanic acid was decreased The ABCD3 geneis composed of 24 exons and the normal length of ABCD3cDNA is 1980 bp In this patient there was a deletion ofexon 24 and part of the 31015840UTR (c1903-573 lowast1108) Thisdeletion results in a truncated form of ABCD3 lacking 24COOH-terminal amino acids but the truncated ABCD3 isnevertheless still present in the fibroblasts of the patient Asshown in our targeting experiment in ABCD3 the deletionof theNBD (AA375ndash660) does not exert any influence on thesorting to peroxisomes [36] Therefore it is assumed that thetruncated ABCD3 loses the ability to transport substrates Inthe case of ABCD1 it is deduced that the region of AA672ndash693 is critically important for dimerization as describedabove The 24 COOH-terminal amino acids of ABCD3 lostin the patient overlap with this region Hence it seems thatthe truncated ABCD3 is unable to form dimer and this is thereason for the lack of functionality

53 ABCD4 It was shown that ABCD4 is involved in a newlyidentified inherited defect affecting cobalamin metabolism


[15] To date nine inherited defects in the intracellularprocessing of cobalamin are known designated cblA to cblGcblJ andmut [15 62]These defects result in the accumulationof methylmalonic acid homocysteine or both which in turnleads tomethylmalonic aciduria andor isolated homocystin-uria The mutation of ABCD4 which is known as the cblJcomplementation group results in the failure of cobalaminrelease from lysosomes At present there are four patientswith a reported mutation in ABCD4 gene [15 63] Patient1 possesses two mutations a missense mutation c956AgtG(pY319C) and a dinucleotide insertion c1746 1747insCT(pE583LfsX9) resulting in a frameshift and the introductionof a premature stop codon that results in the removal of 14COOH-terminal amino acids Patient 2 also possesses twomutations c542+1GgtT and c1456GgtT resulting in in-framedeletions of pD143 S181del and pG443 S485del Patients 3and 4 possess a missense mutation c423CgtG (pN141K)There are no data on whether mutated ABCD4 is present ornot in these patients In the case of patient 1 N141 is locatedin the loop between the second and third transmembranehelices of the TMD based on the predicted topology ofthe ABCD4 The substitution of this asparagine to lysine ispredicted to be ldquoprobably damagingrdquo by the PolyPhen-2 pro-gram (httpgeneticsbwhharvardedupph2) In additionthe truncated region at the COOH-terminal is overlappedwith the region deduced to be important for dimerization inABCD1 described above It is therefore speculated that themutant ABCD4 in patient 1 is not able to form the properconformation In patient 2 the deletion of pD143 S181 inABCD4 is supposed to result in lacking TMD3 based on thepredicted topology Consequently the overall structure of thepassageway for the substrate across the membrane that iscomposed of twelve-transmembrane segments is completelydisrupted In addition the pN141K mutation in ABCD4identified in patients 3 and 4 is predicted to be ldquodamagingrdquoby both PolyPhen-2 and SIFT programs (httpsiftjcviorg)The region of AA141ndash144 (NPDQ) is conserved among allABCD transporters

6 Clinical Symptoms of X-ALD andthe Development of Therapeutics

There are several X-ALD phenotypes that are categorizedbased on the onset and severity of the disease the childhoodcerebral form (CCALD) the adolescent cerebral form (Ado-CALD) the adult cerebral form (ACALD) adrenomyeloneu-ropathy (AMN) and Addisonrsquos disease alone (Table 1) [54]Themost frequent forms are CCALD andAMNTheCCALDform is associated with inflammation of the cerebral whitematter cerebral demyelination and adrenocortical insuffi-ciency with the onset occurring in childhood between 3 and10 years of age In contrast AMN is characterized by a slowlyprogressive axonopathy in males with an onset of around 20to 30 years old

The type of a given ABCD1 mutation such as missensenonsense frameshift deletion or insertion does not predictthe disease phenotype Different clinical phenotypes haveeven been reported in monozygotic twins [64] It has been

Table 1 Clinical forms of X-ALD

Phenotype Ratio () Age of onsetCerebral ALDChildhood cerebral ALD sim38 3ndash10Adolescent cerebral ALD sim7 11ndash21Adult cerebral ALD sim5 21sim

Adrenomyeloneuropathy (AMN) sim50 20ndash30Addisonrsquos only

also reported that X-ALD patients with a complete absenceof ABCD1 due to large deletions exhibit a milder AMNphenotype [59] Therefore modifying influences such asgenetic epigenetic and environmental factors seem to beimportant in determining the severity of the disease [59]Matsukawa et al have reported SNPs of ABCD1 ABCD2ABCD3 and ABCD4 genes in X-ALD patients Howeverthere were no significant associations between these SNPsand phenotypes [65]

At present there is no effective treatment for preventingthe onset or the progression of the neurological pathogen-esis that occurs in X-ALD Allogeneic hematopoietic stemcell transplantation (HSCT) is the only currently availabletherapeutic procedure which halts the progress of cerebraldemyelination in patients with early stages of CCALD [66]The mechanism by which HSCT stops demyelination hasnot yet been made clear It seems likely that HSCT allowsstem cells to penetrate into the brain parenchyma throughthe brain blood barrier where the engrafted stem cells fulfilla role by acting as microglia-like cells However HSCT isstill associated with a higher risk of mortality because ofthe graft-versus-host reaction and the immunodeficiencyinduced by myeloablation Autologous HSC-gene therapyin which the ABCD1 gene is transduced into hematopoi-etic stem cell using a lentivirus vector has been testedas an alternative option It is an important advantage forCCALD patients without HLA-matched donors In 2009Cartier et al reported that lentiviral-mediated gene therapyof hematopoietic stem cells halted the progression of X-ALD[67] However caution is required because of the issue ofgenotoxicity

In addition to HSCT-based therapy pharmacologicaltherapies are still of importance in order to delay boththe onset and progression of the disease Many candidatecompounds have been reported for X-ALD therapy to dateThe targets of these compounds can be categorized into4 groups [68] pharmacological induction of ABCD2 geneexpression [69] stimulation of residual peroxisomal VLCFA120573-oxidation [70] suppression of excess fatty acid elongation[71] and reduction of oxidative stress [72] However nopromising drug has emerged so further studies are requiredfor the development of effective treatment

Competing Interests

The authors declare that they have no competing interests


Acknowledgments

The authors thank Professor Tsuneo Imanaka for his com-ments and suggestions This work was supported in partby the Sasakawa Scientific Research Grant from The JapanScience Society (27-427 to Kosuke Kawaguchi) and Grants-in-Aid for Scientific Research (C) (16K09961 to MasashiMorita) from the Ministry of Education Culture SportsScience and Technology of Japan Pacific Edit reviewed themanuscript prior to submission

References

[1] E Dassa and P Bouige ldquoThe ABC of ABCs a phylogenetic andfunctional classification of ABC systems in living organismsrdquoResearch in Microbiology vol 152 no 3-4 pp 211ndash229 2001

[2] V Vasiliou K Vasiliou and D W Nebert ldquoHuman ATP-binding cassette (ABC) transporter familyrdquo Human Genomicsvol 3 no 3 pp 281ndash290 2009

[3] K Ueda ldquoABC proteins protect the human body and maintainoptimal healthrdquo Bioscience Biotechnology and Biochemistryvol 75 no 3 pp 401ndash409 2011

[4] K Kamijo S Taketani S Yokota T Osumi and T HashimotoldquoThe 70-kDa peroxisomal membrane protein is a member ofthe Mdr (P-glycoprotein)-related ATP-binding protein super-familyrdquo The Journal of Biological Chemistry vol 265 no 8 pp4534ndash4540 1990

[5] J Mosser A-M Douart C-O Sarde et al ldquoPutative X-linkedadrenoleukodystrophy gene shares unexpected homology withABC transportersrdquoNature vol 361 no 6414 pp 726ndash730 1993

[6] G Lombard-Platet S Savary C-O Sarde J-L Mandel and GChimini ldquoA close relative of the adrenoleukodystrophy (ALD)gene codes for a peroxisomal protein with a specific expressionpatternrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 93 no 3 pp 1265ndash1269 1996

[7] N Shani G Jimenez-Sanchez G Steel M Dean and D ValleldquoIdentification of a fourth half ABC transporter in the humanperoxisomal membranerdquoHumanMolecular Genetics vol 6 no11 pp 1925ndash1931 1997

[8] M Morita and T Imanaka ldquoPeroxisomal ABC transportersstructure function and role in diseaserdquoBiochimica et BiophysicaActa (BBA)mdashMolecular Basis of Disease vol 1822 no 9 pp1387ndash1396 2012

[9] F Geillon C Gondcaille S Charbonnier et al ldquoStructure-function analysis of peroxisomal ATP-binding cassette trans-porters using chimeric dimersrdquoThe Journal of Biological Chem-istry vol 289 no 35 pp 24511ndash24520 2014

[10] C W T van Roermund W F Visser L Ijlst et al ldquoThehuman peroxisomal ABC half transporter ALDP functions asa homodimer and accepts acyl-CoA estersrdquoThe FASEB Journalvol 22 no 12 pp 4201ndash4208 2008

[11] C W T van Roermund W F Visser L Ijlst H R Waterhamand R J A Wanders ldquoDifferential substrate specificities ofhuman ABCD1 and ABCD2 in peroxisomal fatty acid 120573-oxidationrdquo Biochimica et Biophysica ActamdashMolecular and CellBiology of Lipids vol 1811 no 3 pp 148ndash152 2011

[12] C W T van Roermund L Ijlst T Wagemans R J A Wandersand H R Waterham ldquoA role for the human peroxisomal half-transporter ABCD3 in the oxidation of dicarboxylic acidsrdquoBiochimica et Biophysica ActamdashMolecular and Cell Biology ofLipids vol 1841 no 4 pp 563ndash568 2014

[13] S Ferdinandusse G Jimenez-Sanchez J Koster et al ldquoA novelbile acid biosynthesis defect due to a deficiency of peroxisomalABCD3rdquo Human Molecular Genetics vol 24 no 2 Article IDddu448 pp 361ndash370 2015

[14] Y Kashiwayama M Seki A Yasui et al ldquo70-kDa peroxisomalmembrane protein related protein (P70RABCD4) localizes toendoplasmic reticulum not peroxisomes and NH

2-terminal

hydrophobic property determines the subcellular localization ofABC subfamilyDproteinsrdquoExperimental Cell Research vol 315no 2 pp 190ndash205 2009

[15] D Coelho J C Kim I R Miousse et al ldquoMutations in ABCD4cause a new inborn error of vitamin B

12metabolismrdquo Nature

Genetics vol 44 no 10 pp 1152ndash1155 2012[16] F Rutsch S Gailus I R Miousse et al ldquoIdentification of a

putative lysosomal cobalamin exporter altered in the cblF defectof vitamin B

12metabolismrdquo Nature Genetics vol 41 no 2 pp

234ndash239 2009[17] K Kawaguchi T Okamoto M Morita and T Imanaka

ldquoTranslocation of the ABC transporter ABCD4 from the endo-plasmic reticulum to lysosomes requires the escort proteinLMBD1rdquo Scientific Reports vol 6 Article ID 30183 2016

[18] R J AWanders ldquoMetabolic functions of peroxisomes in healthand diseaserdquo Biochimie vol 98 no 1 pp 36ndash44 2014

[19] H R Waterham S Ferdinandusse and R J A WandersldquoHuman disorders of peroxisome metabolism and biogenesisrdquoBiochimica et Biophysica ActamdashMolecular Cell Research vol1863 no 5 pp 922ndash933 2016

[20] R J A Wanders and H R Waterham ldquoBiochemistry of mam-malian peroxisomes revisitedrdquo Annual Review of Biochemistryvol 75 pp 295ndash332 2006

[21] S Ferdinandusse S Denis P A W Mooyer et al ldquoClinical andbiochemical spectrum of D-bifunctional protein deficiencyrdquoAnnals of Neurology vol 59 no 1 pp 92ndash104 2006

[22] S Ferdinandusse S Denis C W T Van Roermund R J AWanders and G Dacremont ldquoIdentification of the peroxisomal120573-oxidation enzymes involved in the degradation of long-chaindicarboxylic acidsrdquoThe Journal of Lipid Research vol 45 no 6pp 1104ndash1111 2004

[23] R J Wanders H R Waterham and S FerdinandusseldquoMetabolic interplay between peroxisomes and other subcel-lular organelles including mitochondria and the endoplasmicreticulumrdquo Frontiers in Cell and Developmental Biology vol 3article 83 2016

[24] E Dixit S Boulant Y Zhang et al ldquoPeroxisomes are signalingplatforms for antiviral innate immunityrdquo Cell vol 141 no 4 pp668ndash681 2010

[25] B-B Chu Y-C Liao W Qi et al ldquoCholesterol transportthrough lysosome-peroxisome membrane contactsrdquo Cell vol161 no 2 pp 291ndash306 2015

[26] H Appelqvist P Waster K Kagedal and K Ollinger ldquoThelysosome from waste bag to potential therapeutic targetrdquoJournal of Molecular Cell Biology vol 5 no 4 pp 214ndash226 2013

[27] D F Bainton ldquoThe discovery of lysosomesrdquoThe Journal of CellBiology vol 91 no 3 pp 66sndash76s 1981

[28] B Schroder CWrocklage C Pan et al ldquoIntegral and associatedlysosomal membrane proteinsrdquo Traffic vol 8 no 12 pp 1676ndash1686 2007

[29] J W Callahan R D Bagshaw and D J Mahuran ldquoThe integralmembrane of lysosomes its proteins and their roles in diseaserdquoJournal of Proteomics vol 72 no 1 pp 23ndash33 2009


[30] T Lubke P Lobel andD E Sleat ldquoProteomics of the lysosomerdquoBiochimica et Biophysica ActamdashMolecular Cell Research vol1793 no 4 pp 625ndash635 2009

[31] NMizushima andMKomatsu ldquoAutophagy renovation of cellsand tissuesrdquo Cell vol 147 no 4 pp 728ndash741 2011

[32] E D Carstea J A Morris K G Coleman et al ldquoNiemann-Pick C1 disease gene homology to mediators of cholesterolhomeostasisrdquo Science vol 277 no 5323 pp 228ndash231 1997

[33] S Naureckiene D E Sleat H Lacklan et al ldquoIdentification ofHE1 as the second gene of Niemann-Pick C diseaserdquo Sciencevol 290 no 5500 pp 2298ndash2301 2000

[34] I Martinez S Chakrabarti T Hellevik J Morehead KFowler and NW Andrews ldquoSynaptotagmin vii regulates Ca2+-dependent exocytosis of lysosomes in fibroblastsrdquo The Journalof Cell Biology vol 148 no 6 pp 1141ndash1149 2000

[35] A Lee K Asahina T Okamoto et al ldquoRole of NH2-terminal

hydrophobic motif in the subcellular localization of ATP-binding cassette protein subfamily D common features ineukaryotic organismsrdquo Biochemical and Biophysical ResearchCommunications vol 453 no 3 pp 612ndash618 2014

[36] Y KashiwayamaKAsahinaH Shibata et al ldquoRole of Pex19p inthe targeting of PMP70 to peroxisomerdquoBiochimica et BiophysicaActamdashMolecular Cell Research vol 1746 no 2 pp 116ndash1282005

[37] K A Sacksteder J M Jones S T South X Li Y Liu and S JGould ldquoPEX19 bindsmultiple peroxisomalmembrane proteinsis predominantly cytoplasmic and is required for peroxisomemembrane synthesisrdquo Journal of Cell Biology vol 148 no 5 pp931ndash944 2000

[38] M Biermanns and J Gartner ldquoTargeting elements in the amino-terminal part direct the human 70-kDa peroxisomal integralmembrane protein (PMP70) to peroxisomesrdquo Biochemical andBiophysical Research Communications vol 285 no 3 pp 649ndash655 2001

[39] Y Kashiwayama K Asahina M Morita and T ImanakaldquoHydrophobic regions adjacent to transmembrane domains 1and 5 are important for the targeting of the 70-kDa peroxisomalmembrane proteinrdquo The Journal of Biological Chemistry vol282 no 46 pp 33831ndash33844 2007

[40] H Sakaue S Iwashita Y Yamashita Y Kida andM SakaguchildquoThe N-terminal motif of PMP70 suppresses cotranslationaltargeting to the endoplasmic reticulumrdquo Journal of Biochem-istry vol 159 no 5 pp 539ndash551 2016

[41] A Halbach S Lorenzen C Landgraf R Volkmer-EngertR Erdmann and H Rottensteiner ldquoFunction of the PEX19-binding site of human adrenoleukodystrophy protein as target-ing motif in man and yeast PMP targeting is evolutionarilyconservedrdquoThe Journal of Biological Chemistry vol 280 no 22pp 21176ndash21182 2005

[42] L T-L Tseng C-L Lin K-Y Tzen S C Chang and M-FChang ldquoLMBD1 protein serves as a specific adaptor for insulinreceptor internalizationrdquo The Journal of Biological Chemistryvol 288 no 45 pp 32424ndash32432 2013

[43] N Cartier J Lopez P Moullier et al ldquoRetroviral-mediatedgene transfer corrects very-long-chain fatty acid metabolism inadrenoleukodystrophy fibroblastsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 92 no5 pp 1674ndash1678 1995

[44] C Wiesinger M Kunze G Regelsberger S Forss-Petter andJ Berger ldquoImpaired very long-chain acyl-CoA 120573-oxidation inhuman X-linked adrenoleukodystrophy fibroblasts is a direct

consequence of ABCD1 transporter dysfunctionrdquo The Journalof Biological Chemistry vol 288 no 26 pp 19269ndash19279 2013

[45] C W T van Roermund L Ijlst W Majczak et al ldquoPeroxisomalfatty acid uptake mechanism in Saccharomyces cerevisiaerdquo TheJournal of Biological Chemistry vol 287 no 24 pp 20144ndash201532012

[46] C De Marcos Lousa C W T Van Roermund V L G Postiset al ldquoIntrinsic acyl-CoA thioesterase activity of a peroxisomalATP binding cassette transporter is required for transport andmetabolism of fatty acidsrdquo Proceedings of the National Academyof Sciences of the United States of America vol 110 no 4 pp1279ndash1284 2013

[47] A Pujol I Ferrer C Camps et al ldquoFunctional overlap betweenABCD1 (ALD) and ABCD2 (ALDR) transporters a therapeutictarget for X-adrenoleukodystrophyrdquo Human Molecular Genet-ics vol 13 no 23 pp 2997ndash3006 2004

[48] S Fourcade M Ruiz C Camps et al ldquoA key role for the peroxi-somal ABCD2 transporter in fatty acid homeostasisrdquo AmericanJournal of PhysiologymdashEndocrinology and Metabolism vol 296no 1 pp E211ndashE221 2009

[49] E CGenin FGeillon CGondcaille et al ldquoSubstrate specificityoverlap and interaction between adrenoleukodystrophy pro-tein (ALDPABCD1) and adrenoleukodystrophy-related pro-tein (ALDRPABCD2)rdquoThe Journal of Biological Chemistry vol286 no 10 pp 8075ndash8084 2011

[50] J Berger S AlbetM Bentejac et al ldquoThe fourmurine peroxiso-mal ABC-transporter genes differ in constitutive inducible anddevelopmental expressionrdquo European Journal of Biochemistryvol 265 no 2 pp 719ndash727 1999

[51] D Coelho T SuormalaM Stucki et al ldquoGene identification forthe cblD defect of vitamin B

12metabolismrdquo The New England

Journal of Medicine vol 358 no 14 pp 1454ndash1464 2008[52] E L Borths B Poolman R N Hvorup K P Locher and

D C Rees ldquoIn vitro functional characterization of BtuCD-Fthe Escherichia coli ABC transporter for vitamin B

12uptakerdquo

Biochemistry vol 44 no 49 pp 16301ndash16309 2005[53] R Beedholm-Ebsen K van de Wetering T Hardlei E Nexoslash

P Borst and S K Moestrup ldquoIdentification of multidrugresistance protein 1 (MRP1ABCC1) as a molecular gate forcellular export of cobalaminrdquo Blood vol 115 no 8 pp 1632ndash1639 2010

[54] H W Moser A Mahmood and G V Raymond ldquoX-linkedadrenoleukodystrophyrdquo Nature Clinical Practice Neurology vol3 no 3 pp 140ndash151 2007

[55] S KempA Pujol H RWaterhamet al ldquoABCD1mutations andthe X-linked adrenoleukodystrophy mutation database role indiagnosis and clinical correlationsrdquo Human Mutation vol 18no 6 pp 499ndash515 2001

[56] N Takahashi M Morita T Maeda et al ldquoAdrenoleukodystro-phy subcellular localization and degradation of adrenoleu-kodystrophy protein (ALDPABCD1) with naturally occurringmissense mutationsrdquo Journal of Neurochemistry vol 101 no 6pp 1632ndash1643 2007

[57] M Hillebrand S E Verrier A Ohlenbusch et al ldquoLivecell FRET microscopy homo- and heterodimerization of twohuman peroxisomal ABC transporters the adrenoleukodys-trophy protein (ALDP ABCD1) and PMP70 (ABCD3)rdquo TheJournal of Biological Chemistry vol 282 no 37 pp 26997ndash27005 2007

[58] P C Smith N Karpowich L Millen et al ldquoATP binding to themotor domain from an ABC transporter drives formation of


a nucleotide sandwich dimerrdquo Molecular Cell vol 10 no 1 pp139ndash149 2002

[59] K D Smith S Kemp L T Braiterman et al ldquoX-linkedadrenoleukodystrophy genes mutations and phenotypesrdquoNeurochemical Research vol 24 no 4 pp 521ndash535 1999

[60] A Holzinger E Maier S Stockler-Ipsiroglu A Braun and AA Roscher ldquoCharacterization of a novel mutation in exon 10 ofthe adrenoleukodystrophy generdquo Clinical Genetics vol 53 no6 pp 482ndash487 1998

[61] C-Y Chuang L-Y Chen R-H Fu et al ldquoInvolvement ofthe carboxyl-terminal region of the yeast peroxisomal halfABC transporter Pxa2p in its interaction with Pxa1p and intransporter functionrdquo PLoS ONE vol 9 no 8 Article IDe104892 2014

[62] D S Froese and R A Gravel ldquoGenetic disorders of vitaminB12metabolism eight complementation groupsmdasheight genesrdquo

Expert Reviews in Molecular Medicine vol 12 article e37 2010[63] T Takeichi C-K Hsu H-S Yang et al ldquoProgressive hyper-

pigmentation in a Taiwanese child due to an inborn error ofvitamin B

12metabolism (cblJ)rdquo British Journal of Dermatology

vol 172 no 4 pp 1111ndash1115 2016[64] G C Korenke S Fuchs E Krasemann et al ldquoCerebral

adrenoleukodystrophy (ALD) in only one ofmonozygotic twinswith an identical ALD genotyperdquo Annals of Neurology vol 40no 2 pp 254ndash257 1996

[65] TMatsukawaM Asheuer Y Takahashi et al ldquoIdentification ofnovel SNPs of ABCD1 ABCD2 ABCD3 and ABCD4 genes inpatients with X-linked adrenoleukodystrophy (ALD) based oncomprehensive resequencing and association studies with ALDphenotypesrdquo Neurogenetics vol 12 no 1 pp 41ndash50 2011

[66] C Peters L R Charnas Y Tan et al ldquoCerebral X-linkedadrenoleukodystrophy the international hematopoietic celltransplantation experience from 1982 to 1999rdquo Blood vol 104no 3 pp 881ndash888 2004

[67] N Cartier S Hacein-Bey-Abina C C Bartholomae et alldquoHematopoietic stem cell gene therapy with a lentiviral vectorin X-linked adrenoleukodystrophyrdquo Science vol 326 no 5954pp 818ndash823 2009

[68] M Morita N Shimozawa Y Kashiwayama Y Suzuki and TImanaka ldquoABC subfamily d proteins and very long chain fattyacid metabolism as novel targets in adrenoleukodystrophyrdquoCurrent Drug Targets vol 12 no 5 pp 694ndash706 2011

[69] E C Genin C Gondcaille D Trompier and S SavaryldquoInduction of the adrenoleukodystrophy-related gene (ABCD2)by thyromimeticsrdquo The Journal of Steroid Biochemistry andMolecular Biology vol 116 no 1-2 pp 37ndash43 2009

[70] MMoritaM Kanai SMizuno et al ldquoBaicalein 567-trimethylether activates peroxisomal but not mitochondrial fatty acid 120573-oxidationrdquo Journal of Inherited Metabolic Disease vol 31 no 3pp 442ndash449 2008

[71] M Engelen L Tran R Ofman et al ldquoBezafibrate for X-linkedadrenoleukodystrophyrdquo PLoS ONE vol 7 no 7 Article IDe41013 2012

[72] R V Kartha J Zhou L Basso H Schroder P J Orchard andJ Cloyd ldquoMechanisms of antioxidant induction with high-doseN-acetylcysteine in childhood cerebral adrenoleukodystrophyrdquoCNS Drugs vol 29 no 12 pp 1041ndash1047 2015

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of


StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of



Advances in Pharmacological Sciences

Tropical MedicineJournal of


Medicinal ChemistryInternational Journal of


AddictionJournal of



BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Autoimmune Diseases


Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of


COOH

Membrane

Matrix

Cytosol

ABC signature

TMD

NBD

Walker A

Walker B NH2

Figure 1 Hypothesized structure of the ABCD transporter The ABCD transporters are comprised of a half-size ABC transporter with onetransmembrane domain (TMD) and one nucleotide-binding domain (NBD) Six transmembrane domains are located in the NH

2-terminal

half of the transporter and Walker A Walker B and ABC signature are located in the COOH-terminal half of the transporter

(cobalamin) metabolism [15] The dysfunction of ABCD4results in a failure in lysosomal cobalamin release whichmimics the cobalamin deficiency caused by the defect oflysosomal membrane protein LMBD1 [16] It is indicatedthat ABCD4 has a function on the lysosomal membraneRecently we showed that ABCD4 is translocated fromthe ER to lysosomes through an interaction with LMBD1[17]

As mentioned above dysfunction of any of the ABCDtransporters other than ABCD2 may be the cause of diseaseIn this review we will focus on the targeting mechanismof ABCD transporters to peroxisomes or lysosomes theirfunctions and related diseases

2 Role of Human Peroxisomes and Lysosomes

21 Peroxisomes Peroxisomes are ubiquitous organelles in alleukaryotic cells and participate in various activities relatedto cellular homeostasis especially lipid metabolism [18] Theindispensable role of the peroxisome in human health anddevelopment is evidenced by the large number of geneticdiseases termed peroxisomal disorders [19]

The peroxisomes contain more than 50 different enzymesthat catalyze a variety of metabolic pathways including fattyacid 120573-oxidation the biosynthesis of ether phospholipids andbile acids and the metabolism of reactive oxygen species Inaddition peroxisomes fulfill essential roles in the synthesisof the ether phospholipid such as plasmalogen and the 120572-oxidation of phytanic acid Among these various functionsthe 120573-oxidation of fatty acid is generally considered to be themain one [20]

In peroxisomes 120573-oxidation is performed on VLCFA(gtC220) branched chain fatty acids (eg pristanic acid)bile acid intermediates such as DHCA and THCA [21]poly-unsaturated fatty acid (eg tetracosahexaenoic acid)long chain dicarboxylic acids [22] 2-hydroxy fatty acidsand a number of prostanoids The 120573-oxidation of thesesubstrates is restricted to peroxisomes and does not occur inmitochondria where long chain medium chain and shortchain fatty acids (C18 and shorter) are 120573-oxidized

These fatty acids are imported into peroxisomes viaspecific transporters as mentioned above where they aredegraded by four 120573-oxidation cycles Like the activity ofmitochondrial 120573-oxidation the 120573-oxidation performed inperoxisomes is conducted by oxidation hydration dehydro-genation and thiolytic cleavage [23] In the first step theacyl-CoA oxidase ACOX1 or ACOX2 catalyze the reactionACOX1 preferentially degrades saturated and unsaturatedstraight chain fatty acids while ACOX2 has a high affinityfor 2-methyl branched fatty acids such as pristanoyl-CoADHCA-CoA and THCA-CoA In the second and thirdsteps D- and L-bifunctional enzymes function as an enoyl-CoA hydratase and a 3-hydroxyacyl CoA dehydrogenaserespectively Two thiolases 3-ketoacyl-CoA-thiolase 1 (pTH1)and SCPx (pTH2) are involved in the last step pTH1metabolizes only straight chain fatty acids The branchedchain fatty acids and bile acid precursors (pristanic acidDHCA and THCA) are solely cleaved by pTH2 Once thefatty acid chains are shortened to medium chain fatty acyl-CoA via peroxisomal 120573-oxidation they are conjugated tocarnitine exit the peroxisomes and undergo further 120573-oxidation in mitochondria On the other hand choloyl-CoAand deoxycholoyl-CoA are converted to taurine- or glycine-conjugated cholic acid or deoxycholic acid by bile acids-CoA amino acid N-acyltransferase and then exported intothe cytosol

When functional peroxisomes or enzymes are absent theundegraded molecules such as VLCFA DHCA THCA andphytanic acid accumulate in the cell while physiologicallyessential molecules such as bile acids and plasmalogenbecome deficient In addition to lipid metabolism peroxi-somes play a role in several nonlipid metabolic pathwaysincluding purine polyamine glyoxylate and amino acidmetabolism

Recently new functions for peroxisome have been report-edly demonstrated It is reported that peroxisomes as wellas mitochondria act as intracellular signaling platforms ininnate immunity and are important for early stage antiviralsignaling [24] Moreover it has been shown that perox-isomes have a role in the transport of free cholesterol










2-




2-terminal H0 motif






Peroxisome

ABCD1ndash3

ABCD4

COOH

NBD

TMD

H0 motif

Pex19p

COOH

NBD

TMDPex19p

SRP






NH2

NH2









Membrane



C240-CoAC260-CoA

C200-CoAC220-CoA

C201-CoAC221-CoA

C181-CoA

C246-CoAC226-CoA

Matrix

Membrane

CytosolABCD4

MatrixPeroxisome

Lysosome

Vitamin B12


















R401Q R518Q

P560L

R617H

E609K R660W

H0 motif

TMD NBD

EEA-like motif

G266R


0 100 200 300 400 500 600 700

R554H

R401Q R518Q

P560L

R617H

E609K999 R660W

G266R R554H

Num

ber o

f pat

ient

s

60

50

40

30

20

10

0


















Competing Interests



Acknowledgments


References















2-terminal

















































12uptakerdquo































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of










2-




2-terminal H0 motif






Peroxisome

ABCD1ndash3

ABCD4

COOH

NBD

TMD

H0 motif

Pex19p

COOH

NBD

TMDPex19p

SRP






NH2

NH2









Membrane



C240-CoAC260-CoA

C200-CoAC220-CoA

C201-CoAC221-CoA

C181-CoA

C246-CoAC226-CoA

Matrix

Membrane

CytosolABCD4

MatrixPeroxisome

Lysosome

Vitamin B12


















R401Q R518Q

P560L

R617H

E609K R660W

H0 motif

TMD NBD

EEA-like motif

G266R


0 100 200 300 400 500 600 700

R554H

R401Q R518Q

P560L

R617H

E609K999 R660W

G266R R554H

Num

ber o

f pat

ient

s

60

50

40

30

20

10

0


















Competing Interests



Acknowledgments


References















2-terminal

















































12uptakerdquo































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


Peroxisome

ABCD1ndash3

ABCD4

COOH

NBD

TMD

H0 motif

Pex19p

COOH

NBD

TMDPex19p

SRP






NH2

NH2









Membrane



C240-CoAC260-CoA

C200-CoAC220-CoA

C201-CoAC221-CoA

C181-CoA

C246-CoAC226-CoA

Matrix

Membrane

CytosolABCD4

MatrixPeroxisome

Lysosome

Vitamin B12


















R401Q R518Q

P560L

R617H

E609K R660W

H0 motif

TMD NBD

EEA-like motif

G266R


0 100 200 300 400 500 600 700

R554H

R401Q R518Q

P560L

R617H

E609K999 R660W

G266R R554H

Num

ber o

f pat

ient

s

60

50

40

30

20

10

0


















Competing Interests



Acknowledgments


References















2-terminal

















































12uptakerdquo































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


Membrane



C240-CoAC260-CoA

C200-CoAC220-CoA

C201-CoAC221-CoA

C181-CoA

C246-CoAC226-CoA

Matrix

Membrane

CytosolABCD4

MatrixPeroxisome

Lysosome

Vitamin B12


















R401Q R518Q

P560L

R617H

E609K R660W

H0 motif

TMD NBD

EEA-like motif

G266R


0 100 200 300 400 500 600 700

R554H

R401Q R518Q

P560L

R617H

E609K999 R660W

G266R R554H

Num

ber o

f pat

ient

s

60

50

40

30

20

10

0


















Competing Interests



Acknowledgments


References















2-terminal

















































12uptakerdquo































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of













R401Q R518Q

P560L

R617H

E609K R660W

H0 motif

TMD NBD

EEA-like motif

G266R


0 100 200 300 400 500 600 700

R554H

R401Q R518Q

P560L

R617H

E609K999 R660W

G266R R554H

Num

ber o

f pat

ient

s

60

50

40

30

20

10

0


















Competing Interests



Acknowledgments


References















2-terminal

















































12uptakerdquo































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of












Competing Interests



Acknowledgments


References















2-terminal

















































12uptakerdquo































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


Acknowledgments


References















2-terminal

















































12uptakerdquo































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





























12uptakerdquo































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of
























Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

review article abc transporter subfamily d: distinct differences...

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