Structure-function relationships in the peroxisome: Implications for human disease

BIOCHEMICAL

MEDICINE

AND

METABOLIC

Structure-Function

BIOLOGY

46,288-298

(1991)

Relationships in the Peroxisome: Implications for Human Disease GOLDER N. WILSON

Division of Pediatric Genetics and Metabolism, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9063 Received July 22, 1991 Progress relevant to human peroxisomal disorders over the past 3 years includes improved biochemical delineation of disease phenotypes and new insights into peroxisomal structure and biogenesis. Immunoblotting studies using antibodies to peroxisomal p-oxidation enzymes have defined mutations affecting each step of the pathway, some with clinical phenotypes as severe as disorders with global peroxisome deficiency. The latter disorders, typified by Zellweger syndrome, often lack matrix proteins but retain major membrane species of 150, 70,35, and 22 kDa in empty peroxisomal “ghost” structures. The hypothesis that peroxisomal deficiency disorders result from altered targeting or import of peroxisomal matrix proteins has been strengthened by the demonstration of a carboxy terminal peroxisome-targeting signal which is distinct from amino terminal signals directing proteins to mitochondria. A mutation which mistargets alanine/glyoxylate aminotransferase from peroxisomes to mitochondria in primary hyperoxaluria provides a graphic example of these signals. The structural significance of membrane function is supported by the primacy of membrane assembly in normal ontogeny or regenerating liver. The coordinate control, targeting, and striking inducibility of peroxisomal proteins suggests a potential vehicle for gene and enzyme therapy. 0 1991 Academic

Press. Inc.

The demonstration of deficient peroxisomes in Zellweger syndrome (1) gave clinical significance to an obscure organelle and was a major stimulus to peroxisome biology. Although named for oxidation reactions linked to hydrogen peroxide, peroxisomes have a diverse metabolic repertoire involving at least 40 enzymes. The recent explosion of research on peroxisomes has revealed a diversity of size, protein content, and substrate inducibility exhibited by no other subcellular organelle . Microbody is a general term for these protean and ubiquitous cytoplasmic structures. They host the glyoxylate cycle in plants (glyoxisomes) and range in size from 0.1 pm microperoxisomes in brain or fibroblasts to OS-l.5 pm mitochondrial-sized organelles in mammalian liver or kidney (2). In humans, peroxisomes are distinguished from mitochondria by their single membrane and ho288 08854505/91 $3.00 Copyright 6 1991 by Academic Press, Inc. AI1 rights of reproduction in any form reserved.

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mogenous matrix visible by routine electron microscopy. While they number up to loo0 per liver cell (2.5% of liver protein), special stains such as the diaminobenzidine substrate for catalase activity may be required for visualization of small or defective peroxisomes (3). Even in liver, their ovoid shapes represent cross sections of a complex peroxisomal reticulum which evades unidimensional characterization (4). The deficiency of plasmalogens (5) and of enzymes for plasmalogen synthesis (6) in Zellweger syndrome initiated a productive period of correlation between altered metabolites and peroxisomal function. Elevated levels of very long chain fatty acids, abnormal bile acids, phytanic acid, and pipecolic acid in serum or urine and decreased levels of myelin, plasmalogens, and cholesterol have been related to peroxisomal enzyme deficiencies (7,8). Certain abnormalities of Zellweger syndrome such as elevated serum iron or urine dicarboxylic acids have yet to be explained, as do the clinical correlates of peroxisomal involvement in cholesterol, dolichol, and prostaglandin metabolism (summarized in Ref. (9)). With Zellweger syndrome as the prototype disorder, other diseases have been added to the peroxisomal disease category based on related clinical and metabolic alterations or a peroxisomal location of the responsible enzyme. Excitement over the novel insights which peroxisomal disorders may provide into dysmorphology, metabolism, and protein topogenesis has stimulated several recent reviews (2,712). This paper will summarize advances in peroxisomal medicine and biology over the past 3 years with an eye toward future research. Significant findings concern the nature of human peroxisomal deficiency diseases, mechanisms by which proteins are targeted to peroxisomes, and insights into peroxisomal proliferation and assembly. PEROXISOMAL DISEASES Peroxisomal disorders can be preliminarily separated into two classes: those with normal peroxisome structure and defects in a single enzyme or pathway versus those with defective peroxisome structure and multiple enzyme deficiencies (Fig. 1). Acatalasemia, X-linked adrenoleukodystrophy, adult Refsum disease, and primary hyperoxaluria type 1 seem to involve single enzyme defects while Zellweger syndrome, neonatal adrenoleukodystrophy, and infantile Refsum disease are examples of polyenzymopathies (7-9). The peroxisomal pathways for very long-chain fatty acid (VLCFA) oxidation are diagrammed in Fig. 1, but those for ether lipid synthesis and phytanate/pipecolate/bile acid oxidation are also impacted, yielding abnormal levels of various intermediates which can serve as diagnostic assays. The contrast between single versus multiple enzyme disease is supported by studies of peroxisome biogenesis in yeast, where mutants unable to grow on oleate fall into two groups-those lacking one of the enzymes of fatty acid oxidation and those with multiple enzyme deficiencies and absent peroxisomes (13). As shown in Fig. 1, isolated defects at each step of fatty acid oxidation have now been defined although this conclusion is compromised by the limited spectrum of peroxisomal proteins analyzed in these diseases and the lack of measurements in heterozygotes. Of interest is the fact that single enzyme defects-i.e., acyl-

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1. Enzymes of the peroxisomal fatty acid oxidation pathway are shown as examples of matrix proteins;other examples would includeenzymesinvolved in ether lipid synthesisand phytanate/pipecolate oxidation. Individual enzyme defects (m) in the P-oxidation pathway are contrasted with global peroxisomal deficiencydisorders whichlack multiplematrix enzymes.The singleperoxisomal membrane with several of its integral proteins (PMPs) is shown; these proteins are present in “ghost” membrane structures in peroxisomal deficiency diseases and are synthesized prior to matrix assembly in normal ontogeny (see text). The targeting and uptake of peroxisomal matrix proteins is diagrammed at the right, along with the mistargeting which occurs in primary hyperoxaluria type 1 PHOXl). Known NH,-terminal mitochondrial (mt) and COOH-terminal peroxisomal (SKL) targeting signals are diagrammed. A second potential peroxisomal targeting signal (?p) is shown in the alanine/glyoxylate aminotransferase enzyme which is mistargeted to mitochondria from peroxisomes in PHOXl (see text). ZS, Zellwegersyndrome;IRD, infantile Refsumdisease; NALD, neonataladrenoleukodystrophy; X-ALD, X-linked adrenoleukodystrophy. FIG.

CoA oxidase (pseudo-neonatal adrenoleukodystrophy) or 3-oxoacyl-CoA thiolase (pseudo-Zellweger syndrome)*an produce clinical phenotypes indistinguishable from,, their cognate disorders with no detectable peroxisomes. Heterogeneity amon;-clinical disease categories is also demonstrated by restoration of peroxisomal functions by cell fusion; as many as five complementation groups have been demonstrated for Zellweger syndrome alone (llJ2).

Recent progress in generating antibodies and complementary DNA (cDNA) clones for the p-oxidation enzymes has allowed more discriminating analysis of single and multienzyme deficiencies. As would be expected from their lack of visible peroxisomes, cells from patients with Zellweger syndrome or neonatal

adrenoleukodystrophy usually are deficient in all P-oxidation enzyme proteins (Fig. 1). However, intermediate biochemical phenotypes have now been defined with the single and polyenzyme defects representing extremes of a spectrum. Guerroui et al. (14) have examined the levels of P-oxidation proteins in liver samples from 15 patients with elevations of plasma VLCFA. Twelve of these patients, including four diagnosed as Zellweger syndrome, five as neonatal ad-

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FIG. 2. Left, normal peroxisomes with intact matrices and a normal complement proteins demonstrated by gel electrophoresis and immunobloting. Right, immunological ghost membrane structures in cells from patients with peroxisome deficiency disorders. brane structures have normal amounts of membrane proteins but lack matrices and thus density on gradients (H, heavy; L, light).

of membrane studies reveal These memhave a lighter

renoleukodystrophy, and three as infantile Refsum disease, had no detectable liver peroxisomes by electron microscopy. Eleven of them exhibited a corresponding lack of the 72-kDa acyl-CoA oxidase, the 78-kDa bifunctional enzyme, and the mature 41-kDa form of 3-oxoacyl-CoA thiolase as expected for the polyenzyme defect category. However, another patient (diagnosed as neonatal adrenoleukodystrophy) had normal amounts of all three proteins suggesting a functional defect in one of the enzyme molecules or an altered peroxisomal milieu. The last three patients (neonatal adrenoleukodystrophy) had structurally normal peroxisomes, but two of these patients had normal levels of P-oxidation enzyme proteins and the other exhibited isolated deficiency of bifunctional enzyme with a slight decrease in mature thiolase. Another intermediate biochemical phenotypes is illustrated by rhizomelic chondrodysplasia punctata. These patients have normal-appearing peroxisomes and normal VLCFA levels but exhibit impairment of plasmalogen biosynthesis and phytanate catabolism (15,16). Scrutiny of the VLCFA pathway did show an abnormality, however, in that the 44-kDa 3-oxoacyl-CoA thiolase precursor protein was not converted to its 41-kDa mature form in skin fibroblasts. A decreased rate of palmitate (16) but not lignocerate (15) oxidation could be demonstrated in vitro, but residual activity of the immature enzyme was apparently sufficient to prevent VLCFA accumulation in viva Further emphasizing the complexity of peroxisomal structure/function relationships are studies of other peroxisomal proteins by immunoblotting. A critical observation in peroxisomal biology was that by Lazarow and colleagues which demonstrated the presence of peroxisomal membrane proteins (PMPs) in the cells of Zellweger syndrome patients which lacked peroxisomes (17,18). Major membrane proteins of 145/150, 69/70, 55, 35/36, and 22 kDa can be demonstrated in cell extracts using polyclonal antibodies (Fig. 1); a novel 57-kDa PMP has also been detected with a monoclonal antibody (19). Immunoflourescent staining of intact cells revealed peroxisomal membrane “ghosts” lacking the matrix proteins necessary for visualization of peroxisomes by routine microscopy; when isolated

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TABLE 1 Peroxisomal Targeting Sequences (llJ2) Firefly luciferase Rat bifnnctional enzyme Rat acyl-CoA oxidase Pig o-amino acid oxidase Rat mate oxidase Soybean urate oxidase Rapeseed malate synthase

-Gly-Lys-Ser-Lys-Leu -His-Gly-Ser-Lys-Leu -Leu-Gin-Ser-Lys-Leu -Pro-Pro-Ser-His-Leu -Pro-Ser-Ser-Arg-Leu -Leu-Trp-Ser-Lys-Leu -Glu-Leu-Ser-Lys-Leu

on Nycodenz gradients, these peroxisomal ghosts were of lighter density than control (Fig. 2). The results suggest a model for multiple peroxisomal enzyme deficiencies as degradation of matrix proteins due to faulty import or integration. Certain matrix enzymes, like catalase, are less dependent upon peroxisomal organization and retain activity in the cytoplasm. Less clear are the intermediate biochemical phenotypes with microscopically “normal” peroxisomes, where deficiency of one or several enzymes leads to a clinical picture similar to that produced by disorders with microscopically absent peroxisomes. Possible explanations include defects of assembly, import, and internal milieu in addition to proteins such as xanthine oxidase in Drosophila (20) which may have both structural and catalytic roles. PEROXISOMALTARGETING AND MEMBRANE TRANSPORT In the course of expression studies with firefly luciferase as a reporter gene, Subramani and co-workers (21,22) noted that the enzyme was targeted to peroxisomes in mammalian cells. Tailoring of the luciferase gene by in vitro mutagenesis demonstrated that a carboxy terminal serine-lysine-leucine (SKL) tripeptide was both necessary and sufficient for peroxisomal targeting. Substitution experiments show that the S can be replaced by other small amino acids (A or C), that K can be replaced by other basic amino acids (R or H), but that L is absolutely required. Since luciferase was also targeted to peroxisomes of the firefly, evolutionary conservation of the topogenetic signal was demonstrated and has since been expanded to include yeast and plants. Table 1 lists targeting signals documented for several eucaryotic peroxisomal proteins, confirming the replacements allowed in in vitro mutagenesis experiments and demonstrating that some peroxisomal proteins have internal or redundant targeting signals (11,12,23). Others such as peroxisomal3-oxoacyl-CoA thiolase lack an obvious SKL homologue, suggesting that there may be additional topogenetic signals for peroxisomal proteins (llJ2). While less well studied, the mechanism for peroxisomal protein topogenesis is remarkably different from mitochondrial protein targeting. The mitochondrion, with a larger repertoire of some 700 proteins and a double membrane, recognizes a positively charged, nonacidic amino acid presequence of 10 to 70 residues which is usually cleaved during import (24). Contrast between these uptake systems is emphasized by the work of Danpure and co-workers (25,26) on primary hyper-

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oxaluria type I. Although most patients with this disorder have no alanine/glyoxalate aminotransferase (AGT) activity or immunoreactive protein, about l/3 have residual enzyme activity which is aberrantly located in liver mitochondria (mAGT) rather than peroxisomes (Fig. 1). Polymerase chain reaction (PCR) analysis of the 1600-bp complementary DNA (cDNA) from one such patient was surprising in that three separate transitions-C154T, GeJOA, and And2G-were found. Population screening demonstrated that the C&T and A,,,,G mutations cosegregated as an allele which was present in all patients with mAGT and 8-10% of normal controls. The Ge3,,A mutation appears to have arisen independently on such an allele, since the allele with three mutations was found in all patients with mAGT, in none with absent AGT, and in none of the 42 control subjects (26). The G630A mutation is probably most important in generating the phenotype of hyperoxaluria with mAGT, but whether its pathogenetic action requires the concurrent presence of Cls4T and And2 G is not known. Also unclear is the role of each mutation in enhancing mitochondrial and/or ablating peroxisomal import, since the subcellular locations of AGT in the 8-10% of normal individuals with the GAT plus An42 T allele cannot be determined without access to liver. The authors suggest that the Cls4T mutation, which causes a proline-to-leucine conversion at residue 11 of the 392-amino acid protein, is most likely to enhance mitochondrial uptake because it increases helical amphilicity of the amino-terminal domain. The Gs3,,A mutation (glycine-to-arginine change at amino acid 170) is hypothesized to disrupt an internal peroxisomal uptake signal based on homology of this domain to a region of rat AGT and firefly luciferase. Human AGT does have a carboxy terminal KKKL sequence which is similar to the luciferase KSKL, but specific mutagenesis of luciferase KSKL to KKKL abolished its peroxisomal targeting in monkey kidney cells (21). An internal peroxisomal-targeting signal around residue 170 would fit with the dual mitochondrial/peroxisomal location of rat AGT and suggest that other peroxisomal proteins, like acyl-CoA oxidase of yeast (23), will have more than one targeting signal. On the other hand, explanation of the different locations of identical serine/pyruvate aminotransferases in rat liver mitochondria and peroxisomes required primer extension studies to demonstrate RNA coding for a cleaved presequence on the mitochondrial protein (27). Transcription and expression studies of normal and mutant human AGT alleles are thus needed with attention to the 5’ mRNA regions. Additional advances regarding peroxisomal uptake concern membrane protein structure and possible mechanisms of transport. Hashimoto and colleagues (27) have cloned the 70-kDa PMP from rat and shown sequence homology to a group of ATP-binding proteins which include bacterial transport proteins, the cystic fibrosis transmembrane regulator, and proteins conferring multidrug resistance to mammalian tumor cells. Evidence for six transmembrane segments from the hydropathy profile of the derived amino acid sequence suggests that rat PMP 70 may contribute to a membrane channel involved in active transport. This observation joins with several others in suggesting the existence of transport proteins in eucaryotic peroxisomal membranes. Van Veldhoven et al. (28) observed rapid and nonsaturable equilibration of many small solutes in isolated rat peroxisomes

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and postulated the existence of a porin in the 22- to 28-kDa molecular weight range. However, this permeability of isolated peroxisomes may not reflect the in vivo situation, since Wolvetang et al. (29) have shown latency of peroxisomal enzyme activity in fibroblasts which is decreased by the addition of ATP. The same group has also identified an ATPase in highly purified rat liver peroxisomes which is distinctive from that of other organelles (30). Imanaka et al. (31) demonstrated an ATP requirement for the uptake of acyl-CoA oxidase into yeast peroxisomes-they postulated an energy requirement for protein uptake but found no evidence for membrane potential using the uncoupler carbonylcyanide-m-chlorophenylhydrazone (CCCP). Bellion and Goodman (32) did find inhibition by CCCP of alcohol oxidase import into yeast peroxisomes, and a proton gradient has been supported by NMR studies in several fungal species (llJ2). The bulk of evidence thus suggests that in vivo peroxisomes have a nonpermeable membrane with energy requiring uptake systems for both small molecules and proteins. Peroxisomal protein import is poorly understood compared to mitochondria, but some clear differences and similarities are emerging. Peroxisomal proteins rarely contain presequences and have carboxy terminal targeting signals with rather more stringent sequence requirements. Chaperones or binding proteins for the SKL tripeptide can be expected but are not yet identified. There is a suggestion that inhibitors of protein folding delay peroxisomal uptake of catalase (33), a finding reminiscent of the conformational requirements for mitochondrial uptake (24). As summarized above, there is accumulating evidence for ATP and proton coupling for peroxisomal transport as in mitochondria. Obviously, PMPs or other structural components important in maintaining the peroxisomal milieu would be excellent candidates for primary defect(s) in peroxisome deficiency disorders. PEROXISOME ASSEMBLY AND PROLIFERATION Several mutant cell lines have now been created which mimic the peroxisome deficiency phenotype of Zellweger syndrome (13,34,35). As mentioned above for yeast peroxisome-assembly mutants (13), the Chinese hamster ovary (CHO) cell mutations (34,35) have pleiotropic effects with conversion of catalase from a sedimentable to cytosolic form, deficiency of obligately associated peroxisome enzymes such as dihydroxyacetonephosphate acyltransferase, and failure to process certain P-oxidation enzymes into their mature forms. These CHO mutants also are like fibroblasts from human peroxisomal polyenzymopathies in their normal levels of PMPs and their ability to exhibit complementation after fusion with restoration of peroxisomal structure, latency, and enzyme function. These mutant cells, like their disease homologues, have been a useful adjunct to sedimentation studies for demonstrating peroxisomal localization of proteins such as sterol carrier protein-2-a possible regulator of cholesterol metabolism (36,37). They have also been useful in suggesting a role for plasmalogens in protecting animal cells from ultraviolet radiation (38). An important consequence of these observations has been the use of complementation and UV resistance to identify a 35-kDa PMP which restores peroxisomal biogenesis and function in a CHO cell mutant (39). Capitalizing on the plasmalogen

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deficiency and increased UV sensitivity of the peroxisome assembly mutant, a rat liver cDNA library was screened for clones which would bestow UV resistance after transfection. The successful clones had a common open reading frame which could be translated into a 305 amino acid unique sequence which was named peroxisome assembly factor-l (PAF-1). Antibody to a synthetic peptide demonstrated that PAF-1 comigrated with other peroxisomal proteins on sedimentation gradients and was extracted with integral membrane proteins using the sodium carbonate procedure. Like other mammalian PMPs, it does not have SKL at its terminus but does have seven cysteine residues which are reminiscent of the zincfinger DNA-binding proteins (39). It will be intriguing to investigate whether this protein may have a role in the regulation of peroxisomal protein genes. Although antibody to the synthetic peptide did not inhibit acyl-CoA oxidase uptake, the restoration of peroxisome biogenesis and function by PAF-1 makes it an obvious candidate for the primary defect in one or more peroxisome deficiency disorders. The approach followed by the authors (39) should also be applicable to human cell lines, so that the complete delineation of genes which cause a peroxisome deficiency phenotype should be accomplished in the near future. Because of its tissue variability and response to inducing agents, the importance of peroxisome biogenesis extends beyond its alteration in rare human diseases. Chemicals causing hepatomegaly and peroxisomal proliferation in rodents include hypolipidemic drugs, phthalate ester plasticizers, leucotriene antagonists, and a group of long-chain fatty acid analogues with a sulfur atom in the 3 position of the carbon chain (40). Morphologic proliferation is accompanied by an increase in peroxisomal enzyme and PMP levels that, at least for two enzymes of poxidation (41,42) and PMP 70 (27) in rat liver, occurs at the level of transcription. Common to all proliferators except the leucotriene antagonists is an ability to form hydrophobic carboxylic acids which are poorly oxidized in mitochondria. Diets high in natural substrates such as C22 fatty acids (40) or phytol (41) also support this rule by inducing peroxisome proliferation. Prolonged exposure to proliferating agents causes hepatocarcinoma in rodents, an effect which was recently correlated with DNA alterations (43). It will be interesting to examine whether the increase in P-oxidation caused by proliferating agents is accompanied by a decrease in peroxisome synthesis of certain plasmalogens since the latter are suppressive of tumor growth and protect from UV damage (37). It is now clear that proliferators fed to the mother can induce peroxisomes in certain tissues of the fetus (44,45). Enzymes of p-oxidation (44) as well as certain PMPs (45) in rodent fetal liver or kidney respond to maternal administration of clofibrate; this effect accentuates the normal increase of peroxisomes which occurs during mammalian ontogeny (44-46). Contrast of the ontogeny of peroxisomal membrane versus matrix-associated proteins demonstrated the primacy of membrane protein synthesis (45). Prior synthesis of PMPs compared to matrix proteins was also demonstrated during the biogenesis of peroxisomes after partial hepatectomy; membrane loops containing PMPs without associated matrix proteins have been demonstrated in rat liver after exposure to hypolipidemic drugs (47). Such membranes may serve as a precursor to mature peroxisomes, explaining why peroxisomes have not been visualized by matrix-dependent stains early in ontogeny

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(48). The early biogenesis of membranes followed by matrix assembly also provides a parallel with the human disorders where “ghost” membranes but not mature peroxisomes with matrices can be assembled. Although most proliferators tested in rodents are not active in man, and specific trials with clofibrate have not benefited human patients (reviewed in Ref. (9)), certain derivatives are active in primates (50). The inducibility of peroxisome structure and function in diverse eucaryotes provides some hope for treatment of peroxisomal diseases and suggests the peroxisome as a potential mediator of gene and enzyme therapy. ACKNOWLEDGMENTS Support of the Biological Humanics Foundation, Dallas, TX, and of Grant HD26324 from NICHD is gratefully acknowledged.

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N. WILSON

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