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Biochimica et Biophysica Acta 1822 (2012) 1326–1336

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Biochimica et Biophysica Acta

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Review Molecular basis of peroxisomal biogenesis disorders caused by defects in peroxisomal matrix protein import☆

Shirisha Nagotu 1, Vishal C. Kalel 1, Ralf Erdmann ⁎, Harald W. Platta ⁎

Abteilung für Systembiochemie, Medizinische Fakultät der Ruhr-Universität Bochum, D-44780 Bochum, Germany

article info abstract

Article history: Peroxisomal biogenesis disorders (PBDs) represent a spectrum of autosomal recessive metabolic disorders Received 19 December 2011 that are collectively characterized by abnormal peroxisome assembly and impaired peroxisomal function. Received in Revised form 26 March 2012 The importance of this ubiquitous organelle for human health is highlighted by the fact that PBDs are mul- Accepted 9 May 2012 tisystemic disorders that often cause death in early infancy. Peroxisomes contribute to central metabolic Available online 19 May 2012 pathways. Most enzymes in the peroxisomal matrix are linked to lipid metabolism and detoxification of reactive oxygen species. Proper assembly of peroxisomes and thus also import of their enzymes relies on Keywords: fi Peroxisome biogenesis disorders speci c peroxisomal biogenesis factors, so called peroxins with PEX being the gene acronym. To date, 13 Zellweger syndrome spectrum PEX genes are known to cause PBDs when mutated. Studies of the cellular and molecular defects in cells PEX derived from PBD patients have significantly contributed to the understanding of the functional role of the Peroxin corresponding peroxins in peroxisome assembly. In this review, we discuss recent data derived from both Ubiquitination human cell culture as well as model organisms like yeasts and present an overview on the molecular mech- anism underlying peroxisomal biogenesis disorders with emphasis on disorders caused by defects in the per- oxisomal matrix protein import machinery. This article is part of a Special Issue entitled: Metabolic Functions and Biogenesis of Peroxisomes in Health and Disease. © 2012 Elsevier B.V. All rights reserved.

1. Peroxisomes — general introduction Unlike in yeast and plants, β-oxidation of fatty acids occurs in both per- oxisomes and mitochondria of mammalian cells. Very long chain fatty acids, long chain dicarboxylic acids, some unsaturated fatty acids, Peroxisomes are single membrane bound, dynamic organelles of pristanoic acids, di and tri-hydroxycholestanoic acids are metabolized eukaryotic cells. They were first identified in the electron microscopy in peroxisomes. In humans, peroxisomes are also involved in synthesis images of mouse kidney cells [1]. They are mostly spherical, 0.1 to of cholesterol, bile acids and ether lipids such as plasmalogens, which ac- 1 μm in diameter and surrounded by a single lipid bilayer membrane count for the major portion of the ethanolamine glycerophospholipids in [2]. They do not contain DNA or a protein translation machinery and the adult human brain, notably 80–90% of these lipids in the white mat- hence import nuclear coded proteins synthesized in the cytosol post- ter of the brain [5–10]. Apart from the above mentioned functions, re- translationally [3]. The number, size and function of peroxisomes de- cently a role for peroxisomes in the antiviral innate immunity has been pend on the cell type or organism and the environmental conditions. described. In mouse embryonic fibroblasts and human hepatocytes, the This is also reflected by their unique variability in enzyme content antiviral signaling protein MAVS (mitochondrial antiviral-signaling pro- and thus metabolic functions, which marks them as “multi-purpose or- tein) has been localized to both peroxisomes and mitochondria. The ganelles” that adjust their metabolic capabilities according to the cellu- data demonstrate that peroxisomes provide a significant site of antiviral lar needs [4]. Until now 50 different enzymes have been identified in signal transduction and that they promote a rapid response to viral infec- the peroxisomal matrix, which are linked to different biochemical path- tion [11]. ways. However, the central function of these organelles in all instances After their initial description in 1954 [1], peroxisomes were first is β-oxidation of fatty acids and hydrogen peroxide detoxification. associated with human disease in 1973, when it was discovered that kidney and liver tissue from Zellweger syndrome patients were devoid of peroxisomes [12]. In 1984, a specific biomarker was identified [13] ☆ This article is part of a Special Issue entitled: Metabolic Functions and Biogenesis of Peroxisomes in Health and Disease. that could be used for the screening of patients and the first gene defect ⁎ Corresponding authors at: Institut für Physiologische Chemie, Ruhr-Universität associated with a peroxisomal biogenesis defect was identified in 1992 Bochum, Universitätsstr, 150, D-44780 Bochum, Germany. Tel.: +49 234 322 4943; [14]. fax: +49 234 321 4266. The finding that peroxisomes constitute the sole site for breakdown E-mail addresses: [email protected] (R. Erdmann), [email protected] (H.W. Platta). of fatty acids in fungi and the synthesis of plasmalogens in mammalian 1 Joint first authors. cells, has been used for the screening of yeast [15] and Chinese hamster

0925-4439/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.bbadis.2012.05.010 S. Nagotu et al. / Biochimica et Biophysica Acta 1822 (2012) 1326–1336 1327 ovary (CHO)-cell mutants [16] affected in peroxisome function. These PTS2). Notable exceptions are C. elegans and diatoms, which have lost mutants were instrumental for the identification of the corresponding the genes for targeting PTS2 proteins during evolution and import all genes by functional complementation which led to the discovery of matrix proteins via PTS1 signals [36,37]. the first proteins essential for peroxisome formation [17,18].Mostof PTS1 was initially discovered as the carboxyterminal tripeptide the currently known peroxisome biogenesis factors, collectively called SKL in the firefly luciferase protein [38]. Sequence comparisons be- peroxins (PEX), were identified by these genetic approaches, some tween species led to a consensus sequence (S/A/C)-(K/R/H)-(L/M) [39]. later on by proteomic approaches [19] or as in the case of the recently In recent years, it became clear that additional amino acid residues with- discovered PEX34, by their interaction with known peroxins [20]. in the cargo protein are of relevance for the interaction with the PTS1- Apart from the study of their metabolic functions another widely receptor, which led to a refinement of the definition of the PTS1 [40]. studied research area is peroxisome biogenesis. Essential aspects of ThePTS1asatargetingsignalisusedbymajorityoftheperoxisomal peroxisome biogenesis concern the cellular origin of the organelle proteins. Mammalian catalase and alanine-glyoxylate aminotransferase and the matrix and membrane protein import, which are completely are examples of two proteins that need interactions in addition to the independent processes. A role for the endoplasmic reticulum (ER) in binding of the PTS1 for proper targeting to the peroxisomes. A KANL the origin of peroxisomal membrane was hypothesized initially sequence at the C-terminus is required for the targeting of mammalian based on the close proximity of ER and peroxisomes in electron mi- catalase [41] whereas two signals are required for the targeting of crographs [21]. However the post-translational import of peroxisomal alanine-glyoxylate aminotransferase, a C-terminal KKKL and an internal proteins developed the idea of peroxisomes as an autonomous organ- eight amino acid sequence [42]. The PTS2 is located near the N-terminus elle which multiply by growth and division [22]. This concept of per- of the proteins and conforms to the motif R-(L/V/I/Q)-xx-(L/V/I/H)-(L/S/ oxisomes being autonomous organelles was challenged with the G/A)-x-(H/Q)-(L/A) [43]. Very few proteins are targeted to the peroxi- discovery that peroxisomes can be formed from the endoplasmic re- somes by PTS2 in mammals. However, in plants approximately one ticulum upon the reintroduction of Pex3 in peroxisome-deficient third of the peroxisomal proteins are targeted via the PTS2 pathway cells. [23–25]. While, the possibility of de novo formation of peroxi- [44].InSaccharomyces cerevisiae, thiolase (Fox3) and glycerol phosphate somes is now well accepted, the significance of this contribution to dehydrogenase (Gpd1) are the only proteins known to use the PTS2 the peroxisome content is still under debate. While one view is that [45,46]. all peroxisomal membrane proteins traffic via the ER [26], other Proteins that harbor a peroxisomal targeting signal are recognized work demonstrates that under wild type conditions peroxisomes by their respective receptors in the cytosol — which is Pex5 for the are generally formed by growth and division and only under circum- PTS1 [47] and Pex7 for the PTS2 import [48]. The C-terminal domain stances when there are no peroxisomes in a cell, they can be formed of Pex5 binds to the PTS1 sequence of the cargo and is characterized from the ER after reintroduction of the corresponding gene [27]. Also by seven tetratricopeptide repeats. Pex7 is a WD40 protein which recog- in higher eukaryotes, it is shown that peroxisomal membrane pro- nizes the PTS2 signal. A major difference between Pex5 and Pex7 is that teins are directly targeted to peroxisomes [28]. Several recent data, Pex5 functions independently whereas Pex7 requires co-receptors such however, point towards a more semi-autonomous nature of peroxi- as Pex18 and Pex21 in S. cerevisiae or Pex20 in Yarrowia lipolytica, Pichia somes, where certain peroxisomal membrane proteins might traffic pastoris, Hansenula polymorpha and Neurospora [49–51]. Humans and to peroxisomes via the ER [29–31]. plants lack these co-receptors but express a longer splice variant of In contrast to peroxisomal membrane protein targeting, the basic Pex5 which contains a Pex7 binding site [52–54]. Two isoforms of principles of matrix protein import are widely accepted. The ability Pex5 are present in mammalian cells, namely a short (Pex5S) and a lon- to import folded, co-factor bound and oligomeric proteins distinguishes ger species (Pex5L). Both of these isoforms function as PTS1 receptors peroxisomes from other organelles like mitochondria [32–34]. whereas only Pex5L is also required for PTS2 import due to its additional The importance of peroxisomes for human health is highlighted Pex7-binding domain [53]. Thus, while PTS1 and PTS2 import pathways by severe inborn peroxisomal diseases. These can be caused by defects function independently in yeasts and fungi, they converge in higher in peroxisome biogenesis or can be due to peroxisomal single enzyme eukaryotes such as mammals and plants already at the level of cargo deficiencies. Peroxisome biogenesis disorders are caused by defects recognition. in PEX genes. At present, 33 different PEX genes are known and 13 While most peroxisomal proteins use either the PTS1 or PTS2 sig- orthologous human PEX genes have been described (Table 1). nal for targeting, there are some proteins which do not contain such In this review, we focus on the import of peroxisomal matrix pro- a signal [55]. Interestingly, the import of these non-PTS proteins still teins and the importance of this process for humans. We highlight the depends on the presence of Pex5, mostly via an interaction to the increased understanding of the molecular mechanism of this trans- N‐terminal portion of Pex5. Some examples include acyl-CoA oxidase port process and explain the manifestation of the severe peroxisomal in S. cerevisiae and Y. lipolytica, alcohol oxidase in H. polymorpha [56,57]. biogenesis disorders (PBD), which occur when this import process is Moreover, some proteins that lack a PTS might hijack the PTS-pathway defective. Recent developments of various animal models and thera- by piggy-backing onto proteins that do contain a PTS. Enoyl-CoA peutic strategies for mild PBDs associated with a defective matrix im- isomerases Eci1 and Dci1 in S. cerevisiae and the five acyl-CoA oxi- port will also be discussed here. dase isoforms in Y. lipolytica are examples for this mechanism [58,59]. Also in mammals, the copper–zinc superoxide dismutase 1 (SOD1), a 2. Matrix protein import non-PTS protein, is targeted to peroxisomes with the aid of its inter- acting partner, the copper chaperone of SOD1 [60].

A remarkable feature of peroxisomes is the import of fully folded 2.2. Docking of the receptor/cargo complex at the membrane even oligomeric and co-factor bound proteins [35]. This import pro- cess can be divided into five steps as (i) cargo recognition, (ii) receptor Subsequent to the formation of the receptor–cargo complex in the docking, (iii) cargo translocation, (iv) cargo release and (v) receptor cytosol, the receptor ferries the cargo protein to the peroxisomal release and recycling (Fig. 1). membrane. The membrane-bound peroxins Pex13 and Pex14 are re- quired for the initial binding step. Lack of one of these docking com- 2.1. Cargo recognition by the import receptors in the cytosol plex peroxins significantly affects the import of both PTS1 and PTS2 targeted proteins into the peroxisome [61–66]. Pex13 is an integral Proteins that are to be imported into the peroxisomal matrix typ- membrane protein and binds to Pex14 by its SH3 domain which con- ically contain one of the two peroxisome-targeting signals (PTS1 and tains a proline rich SH3 ligand motif. However, a second binding site 1328 S. Nagotu et al. / Biochimica et Biophysica Acta 1822 (2012) 1326–1336

Table 1 Peroxisomal biogenesis factors and associated biogenesis disorders. The table lists the known human and S. cerevisiae proteins required for peroxisomal biogenesis. Abbreviations used are IRD (infantile Refsum disease), NALD (neonatal adrenoleukodystrophy), RCDP1 (Rhizomelic Chondrodysplasia Punctata type 1) and ZS (Zellweger syndrome). The asterisk * points out that the protein is absent in S. cerevisiae and other yeast species with an exception of Y. lipolytica. The ✓ marks the corresponding matrix protein import defect. The (✓) indicates a stress-related matrix protein import defect. For detailed information we refer to the text and for cross-references to [20,35,102,103,113,116–118,127,129,133].

Role H. sapiens S. cerevisiae Human disease Gene deletion phenotype in S. cerevisiae — mislocalization of matrix proteins

PTS1 PTS2

Matrix protein import PTS1 receptor Pex5 Pex5 ZS, NALD ✓ PTS2 receptor Pex7 Pex7 RCDP1, Adult Refsum disease ✓ PTS2 co-receptor Pex5L Pex18 ✓ Pex21 ✓ Docking complex Pex13 Pex13 ZS, NALD ✓✓ Pex14 Pex14 ZS ✓✓ Pex17 ✓✓ Importomer assembly Pex8 ✓✓ RING finger ligase complex Pex2 Pex2 ZS, IRD ✓✓ Pex10 Pex10 ZS, NALD ✓✓ Pex12 Pex12 ZS, NALD, IRD ✓✓ Receptor ubiquitin conjugation UbcH5a/b/c Pex4 Unknown ✓✓ (E2D1/2/3) Pex22 ✓✓ Receptor deubiquitination Ubp15 (✓) Unknown USP9X Unknown AAA export complex Pex1 Pex1 ZS, NALD, IRD ✓✓ Pex6 Pex6 ZS, NALD, IRD ✓✓ Pex26 Pex15 ZS, NALD, IRD ✓✓ AWP1 Unknown Membrane biogenesis and regulatory peroxins Gene deletion phenotype in S. cerevisiae Membrane biogenesis Pex3 Pex3 ZS Absence of peroxisomes Pex19 Pex19 ZS Absence of peroxisomes Pex16 (Pex16)* ZS Peroxisome proliferation Pex11α,β,γ Pex11,25,27 Unknown Reduced number of peroxisomes Regulation of size, number and distribution Pex28–32, Pex34 Altered peroxisome number and/or morphology

in Pex13 for Pex14 was also identified and it has also been shown that deficiency of Pex17 affects import of PTS1 as well as PTS2 proteins, how- the interactions of Pex13 with Pex14 are mediated by several ever, the functional role of the protein remains enigmatic [75,76]. direct or indirect interactions [67]. The N-terminus of Pex13 binds to Pex7 and the SH3 domain interacts with the WXXXF/Y motif of the PTS1 receptor in yeast [63,65,66,68]. However, the Chinese hamster 2.3. Cargo translocation Pex5 does not interact with the SH3 domain of Pex13 but interacts with its N-terminus which also contains the Pex7 binding region The import of folded and in some cases even cofactor bound or [69]. oligomerized proteins into the matrix distinguishes peroxisomes sig- Pex14 is an integral peroxisomal membrane protein in most spe- nificantly from mitochondria and chloroplasts. However, it remained a cies but in some species it is also described to be peripherally associ- matter of speculation for a long time how the cargo proteins traverse ated with the peroxisomal membrane [61,70–72]. Several studies the membrane without affecting the permeability barrier. One hypoth- suggest that Pex14 is the initial docking protein. NMR and crystal esis was that the import receptors themselves might temporally be- structures have revealed a structural basis for the Pex14 and Pex5 in- come part of the dynamic import pore [79]. This was mainly based on teractions [73,74]. The interaction between Pex14 and Pex5 is shown to the observations that the PTS1-receptor Pex5 can bind lipids and be mediated by the Pex5 WXXXF/Y motifs. The N-terminus of Pex14 changes its topology at the peroxisomal membrane. At the membrane, comprises two hydrophobic cavities which recognize the WXXXF/Y it is partially carbonate resistant, adapts a partial protease-protected motif of Pex5. These hydrophobic cavities are separated by two aromat- state and thus behaves like an integral membrane protein [80–82].In ic residues and are flanked by several basic amino acids leading to a line with this view, Pex5 together with Pex14 turned out to represent positively charged protein surface. The Pex5 peptide adopts an amphi- the minimal unit for the import of the intraperoxisomal protein Pex8 pathic α-helical conformation in the Pex5–Pex14 complex structure in P. pastoris [83]. Furthermore, by the use of the planar lipid bilayer and binds diagonally across helices α1andα2 in Pex14 [73]. technique, it has been shown that the import receptor Pex5 and its Yeast Pex17 is a peripheral membrane protein of unknown function, docking protein Pex14 in the presence of a cargo protein form a pore which associates to peroxisomes via Pex14 [75,76]. A homolog of Pex17 with features expected for a protein-conducting channel [84].More- in higher eukaryotes has not yet been identified. In filamentous fungi, over, the dynamic behavior and physical properties of this channel a chimeric protein that consists of Pex14-like N-terminal domain and with a diameter of up to 9 nm appear to fulfill the criteria for the pas- a Pex17-like C-terminal domain has been described [77,78]. In yeast, sage of folded proteins. However, the exact composition of this pore S. Nagotu et al. / Biochimica et Biophysica Acta 1822 (2012) 1326–1336 1329 as well as the driving force and the detailed mechanism of cargo trans- 2.5. Receptor ubiquitination and recycling location is still to be disclosed. Subsequent to cargo liberation, the PTS-receptors return to the cy- tosol for further rounds of import. Early studies discovered that the 2.4. Cargo release into the matrix peroxisomal matrix protein import is an energy-dependent process requiring the hydrolysis of ATP [94]. The idea of import receptors The release of the cargo into the peroxisomal lumen after transloca- shuttling between the peroxisomal membrane and the cytosol was tion is still not well characterized. A role for the less conserved yeast first described by Marzioch et al. (1994) [95], based on the predominant Pex8 in this process has been suggested. Pex8 is an intra-peroxisomal cytosolic localization of the PTS2-receptor. Investigations in perme- peripheral membrane protein, which contains both PTS1 and PTS2 sig- abilized cell systems of human fibroblasts provided the first evidence nals for its targeting to peroxisomes [85,86]. There is some evidence for for cycling of the PTS1 receptor as the protein accumulated reversibly the role of S. cerevisiae Pex8 in the dissociation of the PTS1-receptor at the peroxisomal membrane under conditions when protein transport cargo complex [87]. Another function assigned to S. cerevisiae Pex8 was blocked [96]. Furthermore, in vitro studies revealed that the binding is that it is required to physically connect the docking complex to and translocation of Pex5 does not require ATP while the export of Pex5 the RING finger complex of the export machinery (see below) [88]. back to the cytosol is the ATP-dependent step [97]. The corresponding However, this structural function seems to be taken over in P. pastoris ATPase was identified as the peroxisomal AAA (ATPases associated by Pex3 [89]. As Pex8-like proteins are not yet identified in humans, with diverse cellular activities)-complex, consisting of Pex1 and Pex6, the general mechanism of cargo release remains elusive. both in human [98] and yeast cells [99]. The function of Pex1 and Pex6 A role for Pex14 in the release of catalase from the translocation is not redundant [86,87] and depends on the presence of their mem- machinery into the matrix has been suggested recently [90]. The au- brane anchor, Pex26 in mammalian cells and its ortholog Pex15 in thors suggest that the release of the cargo is facilitated by the interac- yeast [100,101]. The binding and consumption of ATP by the AAA pro- tion of Pex5 with the N-terminal domain of Pex14 molecules at the teins are believed to induce conformational changes that generate translocation machinery. the driving force to pull the receptor out of the membrane [102,103]. After the release into the matrix, a subset of proteins is processed The exact mechanism of substrate recognition and extraction from in peroxisomes of mammals and plants [91,92]. In mammalian cells, the membrane is not known. However, it has become increasingly the intraperoxisomal protease Tysnd1 is responsible both for the re- clear that ubiquitination of the PTS-receptor plays a major role in the re- moval of the leader peptide from PTS2 proteins and for the specific ceptor release. Ubiquitination is a highly conserved post-translational processing of PTS1 proteins. Tysnd1 turned out to be a key regulator modification that is catalyzed by a three-step enzyme cascade of the peroxisomal β-oxidation pathway. The proteolytic activity of (E1, E2, E3) and results in the covalent attachment of the 76 amino oligomeric Tysnd1 is controlled by self-cleavage and the degradation acid comprising ubiquitin to a substrate [104]. Pex5 exhibits two differ- products are removed by the peroxisomal Lon protease [91–93]. ent modes of ubiquitination, mono and polyubiquitination. For

Fig. 1. Composite model of peroxisomal matrix protein import. Proteins harboring a peroxisomal targeting signal type 1 (PTS1) are recognized by the soluble import receptor Pex5 in the cytosol and proteins with the PTS2 sequence are recognized by Pex7 and the cofactors Pex18 and Pex21 in S. cerevisiae, Pex5L in plants and mammals. After this step, the receptor–cargo complex is directed to the peroxisomal membrane and associates with the docking complex consisting of Pex14 and Pex13 as well as Pex17 in yeast. Assembly of the cargo-loaded Pex5 with the docking complex results in the formation of a transient pore, which mainly consists of Pex5 and Pex14. The exact architecture and components of the pore yet have to be identified. The cargo is translocated into the peroxisomal lumen in an unknown manner. The receptor–cargo complex then dissociates and the cargo is released into the peroxisomal lumen, a process which possibly involves Pex8 in yeast. The RING-complex comprises Pex2, Pex10 and Pex12 which all are ubiquitin ligases, which together with ubiquitin-conjugating enzymes Pex4 and its membrane anchor Pex22 in yeast or the Pex4-like isoforms UbcH5 a, b, c in mammals are responsible for receptor mono- ubiquitination. This modification serves as a signal for the ATP-dependent dislocation of Pex5 from the peroxisomal membrane back to the cytosol by the AAA peroxins Pex1 and Pex6. Pex1 and Pex6 are anchored to the peroxisomal membrane via Pex15 in yeast and Pex26 in mammals. In yeast the ubiquitin-conjugating enzyme Ubc4 together with the redundant proteins Ubc5 and Ubc1 polyubiquitinate Pex5 which is then degraded by the 26S proteasome. Thus, while polyubiquitination directs Pex5 to a quality control pathway, the modification by monoubiquitination primes Pex5 for new round of import. The AAA peroxin Pex6 in yeast interacts with Ubp15, a deubiquitinating enzyme acting on Pex5, while human Pex6 interacts with AWP1, an adaptor protein for ubiquitinated Pex5 in humans. In mammals, Pex5 is deubiquitinated by USP9X in the cytosol. The import machinery components present in yeast and humans are depicted in different font colors. Black — components present in both yeast and human, red — components specific to humans, blue — components specific to yeast. 1330 S. Nagotu et al. / Biochimica et Biophysica Acta 1822 (2012) 1326–1336 monoubiquitination of Pex5, a single ubiquitin protein is attached via a degradation whereas in the case of the peroxisomal import the peroxi- thioester bond to a conserved cysteine residue of the receptor whereas somal receptors are modified. Thus, the receptors in peroxisomal protein attachment of ubiquitin to conserved lysine residues and subsequent import behave like the cargos in ERAD. These considerations led to the ubiquitination of the attached ubiquitin results in polyubiquitination. export-driven import model proposing that the ERAD-like removal of The polyubiquitination cascade acting on Pex5 has been unraveled the peroxisomal import receptor is linked to protein import [124]. in S. cerevisiae. Here, the ubiquitin-conjugating enzyme (E2) Ubc4 In support of this model, the presence of a functional receptor–export and the partially redundant Ubc5 and Ubc1 are required for poly- complex is a pre-requisite for the import of matrix proteins into peroxi- ubiquitination of Pex5 [105,106]. Both Pex2 and Pex10 have been somes. This mode of protein translocation is dependent on ATP and the suggested to function as the corresponding ubiquitin-ligases (E3) AAA-dependent energy driven extraction of the receptor from the im- [107,108]. Polyubiquitinated Pex5 is degraded in the 26S proteasome port pore is supposed to be coupled to the movement and translocation and thus is considered as a quality control system for aberrant PTS1- of the cargo across the membrane [124]. Recently, work on the receptor molecules [105,106]. ubiquitination of the PTS2-co-receptor Pex18 in S. cerevisiae provided The monoubiquitination of Pex5 on a conserved cysteine is a prereq- direct evidence for this model as the cysteine-dependent mono- uisite for the export of the PTS1-receptor back to the cytosol [109–111] ubiquitination of Pex18 which is required for receptor export was and thus is essential for peroxisomal biogenesis. The E2 protein Pex4 found to be a prerequisite for translocation of cargo-loaded Pex7 across together with its membrane anchor Pex22 is required for this modifica- the peroxisomal membrane [119]. tion in yeast [111,112] while in mammals the Pex4-like isoforms UbcH5a, UbcH5b; and UbcH5c (also known as UBE2D1, UBE2D2, 3. Peroxisomal matrix protein import defects and human disorders UBE2D3) catalyze the cysteine-dependent ubiquitination [113]. Fur- thermore, the E3 ligase Pex12 is also required for this process [107]. A wide spectrum of disorders is associated with defects in peroxi- The ubiquitin moiety needs to be removed from the mon- some biogenesis affecting specific steps like matrix protein import, oubiquitinated Pex5 before it can enter a new round of import. This peroxisome membrane biogenesis or organelle division [127,128]. cleavage of ubiquitin from the substrate is generally carried out by Peroxisome biogenesis disorders (PBD) can be generally divided into ubiquitin hydrolases also called deubiquitinating enzymes [114].Re- two subsections namely Zellweger syndrome spectrum (ZSS) and clin- cent in vitro data obtained from rat suggests that the monoubiquitin ically distinct rhizomelic chondrodysplasia punctata type 1 (RCDP1). moiety of Pex5 can be cleaved in two ways, a non enzymatic release The ZSS is a genetically heterogeneous group of disorders with over- of the thioester bond between Pex5 and mono-Ub by a nucleophilic lapping clinical phenotypes, which includes the most severe Zellweger attack of glutathione or, as the major pathway, enzyme catalyzed by syndrome (ZS), less severe neonatal adrenoleukodystrophy (NALD) ubiquitin hydrolases [115]. Ubp15 was identified as such an ubiquitin and relatively milder infantile Refsum disease (IRD). Neurological ab- hydrolase in S. cerevisiae. The protein is a novel interaction partner of normalities and developmental defects are common in PBD patients Pex6 and functions as a deubiquitinating enzyme acting on Pex5 [116]. which appear early after birth. Abnormalities in fatty acid metabolism Recently, its putative mammalian ortholog, the ubiquitin-specificprote- lead to changes in the levels of various metabolites like very long ase 9X (USP9X) has been identified as a deubiquitinase acting on the chain fatty acids (VLCFA) or plasmalogens [129]. Several diseases are ubiquitin-Pex5 thioester conjugate [117]. also attributed to single peroxisomal enzyme deficiencies [130] or de- While it seems clear that the purpose of monoubiquitination is to fects in fatty acid transport across the peroxisomal membrane [131]. prime Pex5 for export mediated by the AAA peroxins as part of the This review focuses mainly on disorders caused by matrix protein im- recycling pathway, the mechanistic role of the modification remains port defects as well as on recent developments in animal models to to be investigated. In this respect, the recent discovery of a novel study such disorders and novel therapeutic strategies. adaptor protein of human Pex6, AWP1 could provide a mechanistic Experimental studies on the fibroblasts isolated from patients link [118]. AWP1, which has been described before to function as a suffering from PBDs led to the observation of distinct types of perox- ubiquitin-binding NF-kappaB modulator, is able to interact with both isomal protein import defects at a subcellular level. Various strategies Pex6 and monoubiquitinated Pex5 and thus might function as a selec- were employed to dissect the degree of these import defects. Slawecki tive linker, which enables the AAA proteins to transfer their suggested et al. [132] conducted immunofluorescence microscopy studies using pulling force to the receptor molecule intended for export. antibodies against the PTS1 (−SKL) or the PTS2 protein thiolase while Not only the PTS1-receptor Pex5, but also components of the PTS2 more recently Ebberink et al. [133] used fluorescent marker proteins pathway are ubiquitinated. The co-receptors Pex18 in S. cerevisiae GFP–PTS1 or PTS2–GFPtoelucidatetheimportdefectsinover600PBD [119,120] and Pex20 in P. pastoris [121,122] are ubiquitinated at the patient cell lines. Three types of matrix protein import defects were peroxisome membrane and this modification is crucial for PTS2-co- observed: (i) defects in PTS1 protein import, (ii) defects in PTS2 protein receptor recycling. import and (iii) defects in PTS1 as well as PTS2 protein import (Table 1).

2.6. Possible link between receptor export and cargo release: the export 3.1. Defects in PTS1 protein import driven import model As described in Section 2.1, Pex5 is the receptor for peroxisomal The components of the peroxisomal import receptor ubiquitination targeting of PTS1-containing proteins. Human Pex5 was identified and export machinery are evolutionarily related to the proteins of by Dodt et al. based on sequence homology with P. pastoris Pex5 the Endoplasmic Reticulum Associated Degradation (ERAD) machinery and it was shown that mutations in Pex5 lead to PBDs (118). [123–125]. ERAD represents a mechanism by which misfolded and Unlike human cells, yeast cells that completely lack Pex5 still polyubiquitinated proteins are extracted from the ER for their subse- exhibit normal PTS2 import. Yeast Pex7 binds to the co-receptors quent proteasomal degradation [126]. The structural and functional Pex18 and Pex21 (S. cerevisiae) or Pex20 (Y. lipolytica, H. polymorpha) similarities of ERAD and the peroxisomal receptor cycle might provide and the peroxisomal targeting of PTS2-proteins in yeast occurs inde- a clue to how the energy-requirement of matrix protein import might pendent of the Pex5 import pathway [52]. In humans, PTS2 protein be connected to the translocation of the cargo through the import pore. import is dependent on Pex5 since Pex7 requires the longer splice The similarity between both machineries is that they use ubiquitination isoform as a co-receptor [52]. Ebberink et al. [133] performed an anal- to mark proteins for ATP-dependent release from the membrane. How- ysis of mutations in patient cell lines and found 11 different muta- ever, the target for ubiquitination is different in both cases — in ERAD tions in Pex5, out of which 4 mutations lead to a specificdefectinthe the cargo is ubiquitinated, released and directed for proteasomal import of PTS1 containing proteins. These four mutations lie in regions S. Nagotu et al. / Biochimica et Biophysica Acta 1822 (2012) 1326–1336 1331 which are not involved in PTS2 protein import like the Pex7 binding box ZSS patients exhibit severe congenital neurological abnormalities or regions required for interaction with Pex13 or Pex14. Consequently, like disturbances in neuronal migration and differentiation which PTS2 import is normal in these cell lines. In contrast, the remaining typically leads to death within the first year after birth [141]. Hepatic seven Pex5 mutations lead to the loss of both PTS1 and PTS2 protein and renal dysfunctions are also frequently observed and therefore ZSS import, resulting in the severe clinical Zellweger phenotype (see is also referred to as cerebrohepatorenal syndrome. Patients with Section 3.3) [52,132,133]. milder PBDs like NALD or IRD survive relatively longer than ZS patients, Only PTS1 protein import defects are associated with the disorder but they also display various progressive developmental defects like NALD. Elevated levels of VLCFA and pipecolic acid are accompanied by loss of vision and hearing and other symptoms like hypotonia and adeficiency in plasmalogens. Clinical symptoms include neurological seizures. Biochemical features of these PBDs include elevated plasma abnormalities like de-myelination, hypotonia, seizures, sensorineural levels of very long chain fatty acids (VLCFAs), branched chain fatty hearing loss and psychomotor retardation [134]. acids, di- and trihydroxycholestanoic acid (DHCA/THCA) and L-pipecolic acid. On the other hand, deficiency of plasmalogens and docosahexanoic 3.2. Defects in PTS2 protein import acid (DHA) is observed in the plasma and erythrocytes [142,143]. Peroxisomal disorders were originally described based on clinical Although the PTS2 signal is less commonly utilized for peroxisomal phenotypes without the knowledge of their molecular cause. Several targeting in humans [44], crucial metabolic enzymes like 3-ketoacyl peroxins involved in peroxisome biogenesis were first identified in Co-A thiolase (VLCFA metabolism), alkylglycerone phosphate synthase various yeast species or mammalian cells like CHO [15,16].Usingthis (AGPS) (plasmalogen synthesis) and phytanic acid co-A hydrolase knowledge of peroxins from different species, the orthologous human (PHYH) (phytanic acid catabolism) are targeted by this pathway in counterparts were identified based on their sequence similarity. Subse- humans. Specific defects in the PTS2 import, potentially caused by quently, mutations of the hereby identified human PEX genes were mutations in Pex7 or the Pex7-binding region in Pex5L, lead to the demonstrated to be responsible for PBDs. Thus, the genetic basis of all mislocalization of PTS2 proteins while PTS1 import is not affected. peroxisome biogenesis disorders is known. Metabolic abnormalities caused by specific PTS2 import defects are Mutational analysis of over 600 cell lines from PBD patients revealed associated with RCDP1 disorder which differs genetically as well as clin- that mutations in Pex1 are the most common (58%) followed by Pex6 ically from PBD, ZSS [135,136]. RCDP1 patients have plasmalogen defi- (16%) [133] with the Gly843Asp substitution being the most common ciency and elevated levels of phytanic acid due to the mislocalization mutation found in Pex1. This mutation reduces the Pex1–Pex6 interac- of AGPS and PHYH respectively [136]. However, normal levels of tion [144] and is associated with temperature sensitivity observed in VLCFA are observed despite of the absence of peroxisomal 3-ketoacyl cell lines that show improved PTS1 import when cells are grown at Co-A thiolase. This probably is due to the thiolase activity of Sterol 30 °C. The repertoire of known disease causing mutations in all peroxins carrier protein X which contains a PTS1 [137]. is reported in Ebberink et al. 2011 [133] or is available at www.dbpex. S. cerevisiae Pex7 mutants are characterized by PTS2 import de- org. fects. Motley et al. [138] cloned Kluyveromyces lactis Pex7 by function- al complementation in S. cerevisiae lacking the endogenous Pex7 and 4. Disorders associated with peroxisomal protein import identified the conserved residues in Pex7. Multiple sequence align- ments of the human ORFs with yeast Pex7 led to the identification 4.1. Unique case of acquired peroxisomal targeting of the human PTS2 receptor Pex7 and it was confirmed that muta- tions in human Pex7 are responsible for RCDP1 disorder [135]. Peroxisomal disorders are associated with genetic defects in per- Adult Refsum disease is mainly (90% cases) attributed to the per- oxisome biogenesis genes or single enzyme deficiencies. However, oxisomal single enzyme deficiency disorder caused by mutations in Shepard et al. [145] described a striking link between mutations in a phytanoyl-CoA hydroxylase (PHYH). The patients are affected in the gene totally unrelated to peroxisomes and a probable role of normal alpha-oxidation pathway [139]. About 10% of the cases of adult peroxisomal import in the disease pathology. Mutations in myocilin Refsum disease are caused by mutations in Pex7 without any defects (MYOC) gene are the genetic cause of primary open angle glaucoma in PHYH [140]. It should be noted that infantile Refsum disease is a (POAG), a disease that belongs to the genetically heterogeneous group distinct disorder from adult Refsum disease with a different genetic of optic neuropathies. Myocilin is a secreted glycoprotein present in cause as well as different clinical phenotypes (See Section 3.3). the trabecular extracellular matrix tissue of the eye and is responsible for the maintenance of intraocular pressure (IOP) [146,147]. Myocilin 3.3. Defects in PTS1 and PTS2 protein import bears a cryptic PTS1 signal at its C-terminus (−SKM). This signal is not accessible for Pex5 recognition in normal cells since the protein is Both PTS1 and PTS2 pathways converge at the peroxisomal mem- secreted via the ER–Golgi pathway [145]. However, mutant myocilin brane where they share the same translocon composed of the docking is retained in the ER leading to ER stress which may trigger ERAD and peroxins (Pex14, Pex13), RING-finger peroxins (Pex2, Pex10, Pex12) dislocate the mutant protein to cytosol for degradation via the ubiquitin and AAA-type ATPase complex peroxins (Pex1, Pex6, Pex26) [102]. proteasome system [148]. The presence of mutant myocilin protein in Hence a mutation in any one of these components affects the import the cytosol or the exposure of its cryptic PTS1 due to misfolding, allows of both PTS1 and PTS2 proteins. Mutations in Pex1, Pex2, Pex5L, Pex6, the PTS1 receptor Pex5 to access the PTS1 signal and to target the Pex10, Pex12, Pex13, Pex14, or Pex26 result in general matrix protein mutant myocilin to peroxisomes. The severity and early onset of IOP import defects. As a consequence, peroxisomal metabolic pathways elevation is correlated with the mutations that cause a stronger interac- are lost leading to the metabolic abnormalities and form the basis tion with Pex5. This indicates that the gain-of-function mutations in for the lethal genetic disorders collectively termed ZSS [129]. MYOC increases its peroxisomal accumulation, facilitating the progres- Cells from ZSS patients suffering from defects in peroxisomal ma- sion of IOP and pathogenesis of POAG [145]. trix protein import however can still correctly target peroxisomal membrane proteins and hence they contain remnant peroxisomal mem- 4.2. Exploitation of peroxisomal matrix protein import by pathogens brane structures, called ghosts. In contrast, mutations in the peroxins Pex3, Pex16 and Pex19 required for the topogenesis of peroxisomal Several pathogens have been shown to hijack or exploit normal membrane proteins (PMP) cause mislocalization of PTS1 and PTS2 peroxisome functions at least in part of their life cycle [149]. It has proteins due to the complete loss of peroxisomes (reviewed by Fujiki been described that rotaviruses which cause infantile gastroenteritis, et al. in this issue). encode the spike protein VP4 that bears a highly conserved PTS1 signal 1332 S. Nagotu et al. / Biochimica et Biophysica Acta 1822 (2012) 1326–1336 at its C-terminus [150]. All 153 Rotavirus VP4 sequences in genebank disease allele PEX1–Gly843Asp and treatment with a chemical library have PTS1 signal variants (SKL, CKL, GKL, CRL, and CRI) of which CRL of small molecule drugs. Recovery of peroxisomal import indicated by is the most common variant (62%). redistribution of cytosolic GFP–PTS1 reporter to peroxisomes after Rotavirus strain SA11 that encodes VP4 protein with a C-terminal drug treatment was assessed by high-content imaging. Upon screening CRL sequence was used to infect a permissive simian (monkey) cell of 2080 bioactive compounds, four molecules were found to recover line MA104. Peroxisomal localization of VP4 was demonstrated in GFP–PTS1 import into peroxisomes. Further validation of peroxisomal virus-infected cells by immunofluorescence microscopy [151]. import and metabolism recovery was done by three independent as- Transient expression of N-terminally GFP tagged VP4 in COS-7L says; import of native peroxisomal proteins, plasmalogen synthesis cells shows punctate peroxisomal localization while truncation of and processing of PTS2. This robust and high-throughput screening C-terminal PTS1 leads to diffuse cytosolic fluorescence. This confirms protocol opens up new frontiers in exploring novel drug therapy for that rotaviruses indeed can utilize the normal PTS1 import pathway for PBD treatment. the transport of at least one of its proteins to the host organelle [151]. The functional significance or requirement of peroxisomal VP4 for the virus remains to be elucidated. 5.3. Stimulating peroxisome proliferation Similarly, the presence of functional PTS1 is also found in Poa semi- latent virus cysteine-rich γbprotein[152]. Other notable examples of ex- Sodium 4-phenylbutyrate induces peroxisome proliferation and ploitation of human peroxisomes are association of HIV's Nef protein improves biochemical function in fibroblast cell lines from patients with peroxisomal thioesterase and interaction of influenza virus NS1 with milder PBD phenotypes [155]. Recently, docosahexaenoic acid protein with peroxisomal 17b-HSD4/MFP-2. However, in both the cases (DHA, C22:6n−3) was identified as an inducer of peroxisome divi- the physiological relevance of the peroxisome association of these pro- sion [158]. Treatment of fibroblasts from patients that carry defects teins is not known [153,154]. in peroxisomal fatty acid β-oxidation with DHA induced the prolifer- ation of peroxisomes to the level seen in normal fibroblasts [158]. 5. Development of therapies and new animal models for PBDs Earlier studies demonstrated that also patients with milder PBD phe- notypes could benefit from DHA-treatment [159,160], indicating that Early onset of disease and lethality in case of the severe forms the pharmacological induction of peroxisomes in PBD patients might of PBDs has limited scope for the treatment. But patients suffering be another worth pursuing approach to improve overall peroxisomal from milder forms of PBD and those with longer survival rates have biochemical function. some scope for correcting or at least slowing down the progressive developmental defects. Several lines of research are ongoing [155] in- cluding (a) dietary therapies — limiting the intake of metabolites such 5.4. New model systems for studying PBDs as VLCFA which tend to accumulate due to peroxisomal defects or supplementing the diet with metabolites like docosahexanoic acid Our knowledge of peroxisome biogenesis is mostly obtained from (DHA), ether lipids and bile acids which are deficient in PBD patients, studies on yeast or mammalian cells which were instrumental in identi- (b) stimulating peroxisomal proliferation or peroxisome related gene fying the corresponding human peroxins and the molecular basis under- expression or (c) screening small molecule compound libraries for new lying peroxisomal disorders. However, to gain insight into how these drugs. Since PBDs are associated with abnormalities in peroxisomal mutations correlate with the pathogenesis of the disease phenotype protein import, recovering or improving import may have beneficial ef- and the developmental defects, there is a need for complex multicellular fects. Here we discuss latest efforts towards the development of such animal model systems. Mouse models of PBDs were generated by therapies. targeted deletion of Pex2, Pex5, Pex7 and Pex13 as well as conditional knockouts of Pex5 and Pex13 which allow tissue-specific inactivation 5.1. Nonsense suppressor therapies of these peroxins [161]. An interesting mouse model concerns PEX11β, a peroxisomal Nonsense mutations (point mutation leading to premature stop membrane protein which plays an important role in the peroxisome codon) are observed in ∼15% of PEX gene alleles in PBD patients. proliferation [19,162,163]. Accordingly, in this mouse model, the loss Dranchak et al., 2011 [156] evaluated whether nonsense suppressor of PEX11β resulted in a reduction of peroxisome abundance and in- therapies which promote the translation read-through can be utilized creased peroxisome clustering and elongation [162]. The Pex11β knock- to restore expression of an active peroxin [156]. Pex2- and Pex12- out mice exhibited a similar clinical phenotype as observed in human defective patient fibroblasts responded well to nonsense suppressor PBDs. However, no defects in peroxisomal protein import or changes therapy, which led to an improvement of peroxisomal VLCFA metab- in the levels of VLCFA or plasmalogens in tissues were observed, thus olism and plasmalogen biosynthesis. Immunofluorescence microscopy challenging the view of involvement of import defects or VLCFA toxicity analysis of treated cells showed recovery of peroxisomal assembly. in the disease pathogenesis [162]. On the other hand, RCDP1 related Pex7 nonsense mutations did not C. elegans as a model system to probe human PBDs is described in respond to PTC124 (ataluren) mediated read-through. Nonetheless, [164,165]. Knockdown of five peroxin homologs of Pex5, Pex6, Pex12, the positive results observed for the Pex2- and Pex12-defective cells Pex13 and Pex19 by RNA-mediated interference (RNAi) led to a devel- provide a proof of concept for using nonsense suppressor therapies for opmental arrest in early larval stage. A drawback of the C. elegans system PBDs which certainly needs further attention. is that the PTS2 pathway does not exist in this organism, hence this model is not applicable for the study of RCDP1 which displays PTS2 im- 5.2. Small molecule screening for rescuing the peroxisomal import port defects [37]. More recently, Drosophila models of PBDs have been described. Mast Peroxin mutations may lead to misfolding or instability of the pro- et al. [166] utilized cultured Drosophila cells to characterize effects of tein or reduced interaction with its binding partners. Small molecule RNAi mediated knockdown of fourteen predicted Drosophila PEX gene compounds may alleviate these defects by mechanisms such as assisting homologs. Drosophila larvae harboring inherited PEX1 mutations most proper folding. A high-content screening assay was developed by Zhang frequently found in PBD patients showed developmental defects analo- et al., 2010 for screening small molecule compounds which can recover gous to Zellweger syndrome patients. Studies on Drosophila PEX mu- peroxisomal import functions [157]. The study was based on the expres- tants also demonstrated their requirement for germ cell development sion of GFP–PTS1 reporter in patient fibroblasts carrying the common and VLCFA metabolism [167,168]. S. Nagotu et al. / Biochimica et Biophysica Acta 1822 (2012) 1326–1336 1333

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