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Kinetoplastid parasites have essential organelles called glycosomes that are analogous to peroxisomes present in other eukaryotes. While many of the processes that regulate glycosomes are conserved, there are several unique aspects of their biology that are divergent from other systems and may be leveraged as therapeutic targets for the treatment of kinetoplastid diseases. Glycosomes are heterogeneous organelles that likely exist as sub-populations with different protein composition and function in a given cell, between individual cells, and between species. However, the limitations posed by the small size of these organelles makes the study of this heterogeneity difficult. Recent advances in the analysis of small vesicles by flow-cytometry provide an opportunity to overcome these limitations. In this review, we describe studies that document the diverse nature of glycosomes and propose an approach to using flow cytometry and organelle sorting to study the diverse composition and function of these organelles. Because the cellular machinery that regulates glycosome protein import and biogenesis is likely to contribute, at least in part, to glycosome heterogeneity we highlight some ways in which the glycosome protein import machinery differs from that of peroxisomes in other eukaryotes., (© 2021 The Author(s).)

Importance of the Field: Parasitic diseases that pose a threat to human life include leishmaniasis - caused by protozoa of Leishmania species. Existing drugs have limitations due to deleterious side effects like teratogenicity and factors like cost and drug resistance, thus furthering the need to develop this area of research., Areas Covered in This Review: We came across drug targets, very recently characterised, cloned and validated by genomics and bioinformatics. We bring these promising drug targets into focus so that they can be explored to their fullest., What the Reader Will Gain: In an effort to bridge the gaps between existing knowledge and future prospects of drug discovery, we found interesting studies validating drug targets and paving the way for better experiments to be designed. In a few cases, novel pathways have been characterized, while in others, well established pathways when probed further, led to the discovery of new drug targets., Take Home Message: The review constitutes a comprehensive report on upcoming drug targets, with emphasis on glycosylphosphatidylinositol (GPI)-anchored glycoconjugates along with related biochemistry of enolase, glycosome and purine salvage pathways, as we strive to bring ourselves a step closer to being able to combat this deadly disease.

Protozoan Kinetoplastida, a group that comprises the pathogenic Trypanosoma brucei, compartmentalize several metabolic systems such as the major part of the glycolytic pathway, in multiple peroxisome-like organelles, designated glycosomes. Trypanosomes have a complicated life cycle, involving two major, distinct stages living in the mammalian bloodstream and several stages inhabiting different body parts of the tsetse fly. Previous studies on non-differentiating trypanosomes have shown that the metabolism and enzymatic contents of glycosomes in bloodstream-form and cultured procyclic cells, representative of the stage living in the insect's midgut, differ considerably. In this study, the morphology of glycosomes and their position relative to the lysosome were followed, as were the levels of some glycosomal enzymes and markers for other subcellular compartments, during the differentiation from bloodstream-form to procyclic trypanosomes. Our studies revealed a small tendency of glycosomes to associate with the lysosome when a population of long-slender bloodstream forms differentiated into short-stumpy forms which are pre-adapted to live in the fly. The same phenomenon was observed during the short-stumpy to procyclic transformation, but then the process was fast and many more glycosomes were associated with the dramatically enlarged degradation organelle. The observations suggested an efficient glycosome turnover involving autophagy. Changes observed in the levels of marker enzymes are consistent with the notion that, during differentiation, glycosomes with enzymatic contents specific for the old life-cycle stage are degraded and new glycosomes with different contents are synthesized, causing that the metabolic repertoire of trypanosomes is, at each stage, optimally adapted to the environmental conditions encountered.

Microparticles (MPs) are small fragments generated from the plasma membrane after cell stimulation. Among the candidate proteins harbored by MPs, we recently showed that sonic hedgehog (Shh) is present in MPs generated from activated/apoptotic human T lymphocytes [Martínez et al., Blood (2006) vol. 108, 3012-3020]. We show here that Shh carried by MPs induces nitric oxide (NO) release from endothelial cells, triggers changes in the expression and phosphorylation of enzymes related to the NO pathway, and decreases production of reactive oxygen species. When PI3-kinase and ERK signaling were specifically inhibited, the effects of MPs were reversed. In vivo injection of MPs in mice was also able to improve endothelial function by increasing NO release, and it reversed endothelial dysfunction after ischemia/reperfusion. Silencing the effects of Shh with cyclopamine, a specific inhibitor of Shh, or siRNA, an inhibitor of the Shh receptor Patched, strongly reduced production of NO elicited by MPs. Taken together, we propose that the biological message carried by MPs harboring Shh may represent a new therapeutic approach against endothelial dysfunction during acute severe endothelial injury.

Peroxisomes are present in nearly every eukaryotic cell and compartmentalize a wide range of important metabolic processes. Glycosomes of Kinetoplastid parasites are peroxisome-like organelles, characterized by the presence of the glycolytic pathway. The two replicating stages of Trypanosoma brucei brucei, the mammalian bloodstream form (BSF) and the insect (procyclic) form (PCF), undergo considerable adaptations in metabolism when switching between the two different hosts. These adaptations involve also substantial changes in the proteome of the glycosome. Comparative (non-quantitative) analysis of BSF and PCF glycosomes by nano LC-ESI-Q-TOF-MS resulted in the validation of known functional aspects of glycosomes and the identification of novel glycosomal constituents.

Background: In multicellular organisms, apoptosis and subsequent microparticle shedding play a key role in homeostasis. Having long been considered as << cellular dust >>, microparticles released in biological fluids upon cell activation or apoptosis appear as multifunctional bioeffectors involved in the modulation of key functions including immunity, inflammation, hemostasis and thrombosis, angiogenesis. MP constitute reliable markers of vascular damage, accessible to biological detection whilst the cells they originate from remain sequestered in tissues or are promptly submitted to phagocytosis., Recent Findings: MP modulate biological functions of target cells through the transfer of cytoplasmic content, lipids and membrane receptors. The pharmacological modulation of circulating levels of microparticles could be of particular interest in thrombotic or inflammatory diseases, cancer or hemophilia., Conclusion: MP can now be viewed not only as a hallmark of cell damage but also as a true biological tool.

In trypanosomatids (Trypanosoma and Leishmania), protozoa responsible for serious diseases of mankind in tropical and subtropical countries, core carbohydrate metabolism including glycolysis is compartmentalized in peculiar peroxisomes called glycosomes. Proper biogenesis of these organelles and the correct sequestering of glycolytic enzymes are essential to these parasites. Biogenesis of glycosomes in trypanosomatids and that of peroxisomes in other eukaryotes, including the human host, occur via homologous processes involving proteins called peroxins, which exert their function through multiple, transient interactions with each other. Decreased expression of peroxins leads to death of trypanosomes. Peroxins show only a low level of sequence conservation. Therefore, it seems feasible to design compounds that will prevent interactions of proteins involved in biogenesis of trypanosomatid glycosomes without interfering with peroxisome formation in the human host cells. Such compounds would be suitable as lead drugs against trypanosomatid-borne diseases.

Peroxisomes are membrane-bounded organelles that compartmentalize a variety of metabolic functions. Perhaps the most divergent peroxisomes known are the glycosomes of trypanosomes and their relatives. The glycolytic pathway of these organisms resides within the glycosome. The development of robust molecular genetic and proteomic approaches coupled with the completion of the genome sequence of the pathogens Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major provides an opportunity to determine the complement of proteins within the glycosome and the function of compartmentation. Studies now suggest that regulation of glycolysis is a strong driving force for maintenance of the glycosome., (Copyright 2004 Blackwell Publishing Ltd)

Trypanosomatid parasites are disease agents with an extraordinarily broad host range including humans, livestock and plants. Recent work has revealed that trypanosomatids harbour numerous genes sharing apparent common ancestry with plants and/or bacteria. Although there is no evidence of a plastid (chloroplast-like organelle) in trypanosomatids, the presence of such genes suggests lateral gene transfer from some photosynthetic organism(s) during trypanosomatid evolution. Remarkably, many products of these horizontally acquired genes now function in the glycosome, a highly modified peroxisome unique to trypanosomatids and their near relatives.

Using a combination of in vivo and in vitro assays, we characterized the sorting pathway and molecular targeting signal for the Arabidopsis 22-kD peroxisome membrane protein (PMP22), an integral component of the membrane of all peroxisomes in the mature plant. We show that nascent PMP22 is sorted directly from the cytosol to peroxisomes and that it is inserted into the peroxisomal boundary membrane with its N- and C-termini facing the cytosol. This direct sorting of PMP22 to peroxisomes contrasts with the indirect sorting reported previously for cottonseed (Gossypium hirsutum) ascorbate peroxidase, an integral PMP that sorts to peroxisomes via a subdomain of the endoplasmic reticulum. Thus, at least two different sorting pathways for PMPs exist in plant cells. At least four distinct regions within the N-terminal one-half of PMP22, including a positively charged domain present in most peroxisomal integral membrane-destined proteins, functions in a cooperative manner in efficient peroxisomal targeting and insertion. In addition, targeting with high fidelity to peroxisomes requires all four membrane-spanning domains in PMP22. Together, these results illustrate that the PMP22 membrane peroxisomal targeting signal is complex and that different elements within the signal may be responsible for mediating unique aspects of PMP22 biogenesis, including maintaining the solubility before membrane insertion, targeting to peroxisomes, and ensuring proper assembly in the peroxisomal boundary membrane.

In subgroups of a New Zealand obese mouse-derived backcross population with defined aberrations of glucose homeostasis, a comprehensive study of the hepatic expression of cytochrome P450 and glutathione S-transferase was performed. Three patterns of alterations in response to insulin resistance (normoglycemia/hyperinsulinemia) or diabetes (hyperglycemia/hypoinsulinemia) were observed: mRNA levels of Cyp2b9, Cyp3a16, Cyp4a14, and Gstt2 as assessed by Northern- and dot-blot analysis were increased markedly in liver from diabetic mice with no or only a slight increase in insulin resistant mice. Western-blot analysis detected the corresponding changes of the CYP2B and CYP4A proteins. In contrast, expression of Cyp2c22, Cyp2c29, and Cyp2c40 was reduced in diabetic, but normal in insulin resistant mice. These alterations were correlated with changes in serum free fatty acid levels and, therefore, seem to be mediated by the peroxisome proliferator activated receptor-alpha. Furthermore, expression of Cyp1a2, Cyp7b1, Gstm3, and Gstm6 was reduced in both diabetic and insulin resistant mice. Because this third pattern was not correlated with the alterations of serum free fatty acid levels, it seems to reflect an early alteration in the course of the disease, and may be related to the progression of the syndrome from insulin resistance to the type 2-like diabetes.

We previously isolated and characterized a Chinese hamster ovary (CHO) cell mutant, ZPG207, that is defective in import of proteins carrying a peroxisome-targeting signal type 2 (PTS2) nonapeptide. Herein we have cloned Chinese hamster (Cl) PEX7 encoding the PTS2 receptor. ClPex7p consists of 318 amino acids, shorter than human Pex7p by 5 residues, showing 91 and 30% identity with Pex7p from humans and the yeast Saccharomyces cerevisiae, respectively. Expression of ClPEX7 rescued the impaired PTS2 import in pex7 ZPG207. Mutation in ZPG207 PEX7 was determined by reverse transcription PCR; a G-to-A transition caused a 1-amino acid substitution, W221ter. We investigated the molecular dysfunction of Pex7p variants in mammals, including Pex7p-W221ter and Pex7p with one site mutation at G217R, A218V, or L292ter, which frequently occurs in the human fatal genetic peroxisomal disease rhizomelic chondrodysplasia punctata, showing a cell phenotype of PTS2 import defect. All types of the mutations affected Pex7p in binding to both PTS2 cargo protein and the longer isoform of PTS1 receptor Pex5pL that is responsible for transport of the Pex7p-PTS2 complex. Subcellular fractionation and protease protection studies demonstrated bimodal distribution of Pex7p between the cytoplasm and peroxisomes in CHO and human cells. Moreover, expression of Pex5pL, but not of the shorter isoform Pex5pS, enhanced translocation of Pex7p-PTS2 proteins into peroxisomes, thereby implying that both PTS receptors shuttle between peroxisomes and the cytosol. Furthermore, a ClPex7p mutant with a deletion of 7 amino acids from the N terminus retained peroxisome-restoring activity, whereas an 11-amino acid truncation abrogated the activity. ClPex7p with a C-terminal 9- amino acid truncation, comprising residues 1--309, maintained the activity, whereas a 14-amino acid shorter form lacking several amino acids of the sixth WD motif lost the activity. Therefore, nearly the full length of Pex7p, including all WD motifs, is required for its function.

This review summarizes materials on the mechanisms of intracellular degradation of proteins whose topogenesis is disturbed at one stage or another. Chaperone and proteolytic systems involved in this process in the endoplasmic reticulum, mitochondria, and chloroplasts of eucaryotic cells as well as those in distinct subcellular compartments of procaryotic cells are considered. The available data suggest that living cells contain numerous systems keeping under control both folding of newly synthesized and newly imported polypeptide chains and their incorporation into heterooligomeric complexes. The point of view is elaborated that organelle formation is controlled not only at the level of individual protein molecules but also at the supermolecular level when whole organelles incapable of carrying out their integral key functions become targets for partial or total elimination. This type of control is realized through an autophagic mechanism involving lysosomes/vacuoles.

Peroxisomes of higher eukaryotes, glycosomes of kinetoplastids, and glyoxysomes of plants are related microbody organelles that perform differing metabolic functions tailored to their cellular environments. The close evolutionary relationship of these organelles is most clearly evidenced by the conservation of proteins involved in matrix protein import and biogenesis. The glycosome can be viewed as an offshoot of the peroxisomal lineage with additional metabolic functions, specifically glycolysis and purine salvage. Within the parasitic protozoa, only kinetoplastids have been conclusively demonstrated to possess glycosomes or indeed any peroxisome-like organelle. The importance of glycosomal pathways and their compartmentation emphasizes the potential of the glycosome and glycosomal proteins as drug targets.

Desensitization of macrophages is important during the development of sepsis. It was our intention to identify mechanisms that promote macrophage deactivation upon contact with endotoxin (LPS) and interferon-gamma (IFN-gamma) in vitro. Macrophage activation was achieved with 12-O-tetradecanoylphorbol 13-acetate (TPA), and the oxidative burst (i.e., oxygen radical formation) was followed by oxidation of the redox-sensitive dyes hydroethidine and dichlorodihydrofluorescein diacetate. Prestimulation of macrophages for 15 h with a combination of LPS/IFN-gamma attenuated oxygen radical formation in response to TPA. Taking the anti-inflammatory properties of the peroxisome proliferator-activating receptorgamma (PPARgamma) into consideration, we established activation of PPARgamma in response to LPS/IFN-gamma by an electrophoretic mobility shift, supershift, and a reporter gene assay. The reporter contains a triple PPAR-responsive element (PPRE) in front of a thymidine kinase minimal promoter driving the luciferase gene. We demonstrated that PPRE decoy oligonucleotides, supplied in front of LPS/IFN-gamma, allowed a full oxidative burst to recover upon TPA addition. Furthermore, we suppressed the oxidative burst by using the PPARgamma agonists 15-deoxy-Delta12,14-prostaglandin J2, BRL 49653, or ciglitazone. No effect was observed with WY 14643, a PPARalpha agonist. We conclude that activation of PPARs, most likely PPARgamma, promotes macrophage desensitization, thus attenuating the oxidative burst. This process appears important during development of sepsis.

High expression of the peroxisome proliferator-activated receptor alpha (PPARalpha) differentiates brown fat from white, and is related to its high capacity of lipid oxidation. We analyzed the effects of PPARalpha activation on expression of the brown fat-specific uncoupling protein-1 (ucp-1) gene. Activators of PPARalpha increased UCP-1 mRNA levels severalfold both in primary brown adipocytes and in brown fat in vivo. Transient transfection assays indicated that the (-4551)UCP1-CAT construct, containing the 5'-regulatory region of the rat ucp-1 gene, was activated by PPARalpha co-transfection in a dose-dependent manner and this activation was potentiated by Wy 14,643 and retinoid X receptor alpha. The coactivators CBP and PPARgamma-coactivator-1 (PGC-1), which is highly expressed in brown fat, also enhanced the PPARalpha-dependent regulation of the ucp-1 gene. Deletion and point-mutation mapping analysis indicated that the PPARalpha-responsive element was located in the upstream enhancer region of the ucp-1 gene. This -2485/-2458 element bound PPARalpha and PPARgamma from brown fat nuclei. Moreover, this element behaved as a promiscuous responsive site to either PPARalpha or PPARgamma activation, and we propose that it mediates ucp-1 gene up-regulation associated with adipogenic differentiation (via PPARgamma) or in coordination with gene expression for the fatty acid oxidation machinery required for active thermogenesis (via PPARalpha).

The expression of uncoupling protein (UCP)-3 mRNA in skeletal muscle is dramatically reduced during lactation in mice. The reduction in UCP-3 mRNA levels lowers the amount of the UCP-3 protein in skeletal muscle mitochondria during lactation. Spontaneous or abrupt weaning reverses the downregulation of the UCP-3 mRNA but not the reduction in UCP-3 protein levels. In lactating and virgin mice, however, fasting increases UCP-3 mRNA levels. Changes in UCP-3 mRNA occur in parallel with modifications in the levels of free fatty acids, which are reduced in lactation and are upregulated due to weaning or fasting. Modifications in the energy nutritional stress of lactating dams achieved by manipulating litter sizes do not influence UCP-3 mRNA levels in skeletal muscle. Conversely, when mice are fed a high-fat diet after parturition, the downregulation of UCP-3 mRNA and UCP-3 protein levels due to lactation is partially reversed, as is the reduction in serum free fatty acid levels. Treatment of lactating mice with a single injection of bezafibrate, an activator of the peroxisome proliferator-activated receptor (PPAR), raises UCP-3 mRNA in skeletal muscle to levels similar to those in virgin mice. 4-chloro-6-[(2,3-xylidine)-pirimidinylthio] acetic acid (WY-14,643), a specific ligand of the PPAR-alpha subtype, causes the most dramatic increase in UCP-3 mRNA, whereas troglitazone, a specific activator of PPAR-gamma, also significantly increases UCP-3 mRNA abundance in skeletal muscle of lactating mice. However, in virgin mice, bezafibrate and WY-14,643 do not significantly affect UCP-3 mRNA expression, whereas troglitazone is at least as effective as it is in lactating dams. It is proposed that the UCP-3 gene is regulated in skeletal muscle during lactation in response to changes in circulating free fatty acids by mechanisms involving activation of PPARs. The impaired expression of the UCP-3 gene is consistent with the involvement of UCP-3 gene regulation in the reduction of the use of fatty acids as fuel by the skeletal muscle and in impaired adaptative thermogenesis, both of which are major metabolic adaptations that occur during lactation.

The peroxisome proliferator-activated receptors (PPARs) are a family of transcription factors belonging to the nuclear receptor superfamily. Until recently, the genes regulated by PPARs were those believed to be predominantly associated with lipid metabolism. Recently, an immunomodulatory role for PPAR gamma has been described in cells critical to the innate immune system, the monocyte/macrophage. In addition, evidence for an antiinflammatory role of the PPAR gamma ligand, 15-deoxy-Delta 12,14-PGJ2 (15d-PGJ2) has been found. In the present studies, we demonstrate, for the first time, that murine helper T cell clones and freshly isolated splenocytes express PPAR gamma 1. The PPAR gamma expressed is of functional significance in that two ligands for PPAR gamma, 15d-PGJ2 and a thiazolidinedione, ciglitazone, mediate significant inhibition of proliferative responses of both the T cell clones and the freshly isolated splenocytes. This inhibition is mediated directly at the level of the T cell and not at the level of the macrophage/APC. Finally, we demonstrate that the two ligands for PPAR gamma mediate inhibition of IL-2 secretion by the T cell clones while not inhibiting IL-2-induced proliferation of such clones. The demonstration of the expression and function of PPAR gamma in T cells reveals a new level of immunoregulatory control for PPARs and significantly increases the role and importance of PPAR gamma in immunoregulation.

Alzheimer's disease (AD) is characterized by the extracellular deposition of beta-amyloid fibrils within the brain and the subsequent association and phenotypic activation of microglial cells associated with the amyloid plaque. The activated microglia mount a complex local proinflammatory response with the secretion of a diverse range of inflammatory products. Nonsteroidal anti-inflammatory drugs (NSAIDs) are efficacious in reducing the incidence and risk of AD and significantly delaying disease progression. A recently appreciated target of NSAIDs is the ligand-activated nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma). PPARgamma is a DNA-binding transcription factor whose transcriptional regulatory actions are activated after agonist binding. We report that NSAIDs, drugs of the thiazolidinedione class, and the natural ligand prostaglandin J2 act as agonists for PPARgamma and inhibit the beta-amyloid-stimulated secretion of proinflammatory products by microglia and monocytes responsible for neurotoxicity and astrocyte activation. The activation of PPARgamma also arrested the differentiation of monocytes into activated macrophages. PPARgamma agonists were shown to inhibit the beta-amyloid-stimulated expression of the cytokine genes interleukin-6 and tumor necrosis factor alpha. Furthermore, PPARgamma agonists inhibited the expression of cyclooxygenase-2. These data provide direct evidence that PPARgamma plays a critical role in regulating the inflammatory responses of microglia and monocytes to beta-amyloid. We argue that the efficacy of NSAIDs in the treatment of AD may be a consequence of their actions on PPARgamma rather than on their canonical targets the cyclooxygenases. Importantly, the efficacy of these agents in inhibiting a broad range of inflammatory responses suggests PPARgamma agonists may provide a novel therapeutic approach to AD.

Cilia are motile processes extending from the basal bodies, playing important roles in the mucociliary clearance in the respiratory tract and the transport of the ovum from the ovary to the uterus in mammals. Ciliogenesis is divided into four stages: (1) duplication of centrioles; (2) migration of centrioles to the apical cell surface to become basal bodies; (3) elongation of cilia containing the axoneme; and (4) formation of accessory structures of basal bodies. The orderly course of ciliogenesis appears to be disturbed by various internal and external factors and, as a result, various unusual forms of the ciliary apparatus develop in the cell. Inhibition of basal body migration results in development of intracytoplasmic axonemes, cilia within periciliary sheaths, and intracellular ciliated cysts. Swollen cilia and the bulging type of compound cilia are formed during ciliary budding and elongation. This review also discusses the origin, composition, and function of the centriolar precursor structures.

Concern exists regarding peroxisome proliferator (PP) xenobiotic exposure because many PPs are potent hepatocarcinogens in rodents. The mechanism of carcinogenicity induced by PPs is atypical compared with those of other hepatocarcinogens in that the former appears to involve alterations in expression of PP-activated receptor (PPAR) target genes rather than direct mutagenicity. To begin to identify some of these genes, we used differential display to compare mRNA expression between hepatic adenomas and adjacent non-tumor liver from rats fed the potent PP Wy-14643 (WY) for 78 wk. Here, we report increased expression of the acute-phase protein (APP) gene alpha-1 antitrypsin (AT) and decreased expression of alpha2-urinary globulin in the tumors. Similar changes were seen in hepatic adenomas induced by a diethylnitrosamine and phenobarbital protocol, indicating a lack of specificity for PP-induced tumors. Additional APP genes, including ceruloplasmin, haptoglobin, beta-fibrinogen, and alpha1-acid glycoprotein were also upregulated in WY-induced tumors but were downregulated in the livers of rats administered a different PP for 13 wk. Mice treated with either WY or di(2-ethylhexyl) phthalate for 3 wk had decreased hepatic AT expression but increased expression of ceruloplasmin and haptoglobin. PPARalpha-null mice showed no hepatic APP gene alteration after PP treatment but had higher basal expression than did wild-type controls. We conclude that PPARalpha activation by several different PPs leads to dysregulation of hepatic APP gene expression in rats and mice. This dysregulation may indicate alterations in cytokine signaling networks regulating both APP gene expression and hepatocellular proliferation., (Copyright 1999 Wiley-Liss, Inc.)

The peroxisome proliferator-activated receptor gamma (PPARgamma) quickly evolved over the last decade from a new orphan receptor to one of the best characterized nuclear receptors. This fast pace in PPARgamma research was triggered by two main discoveries. Firstly, that PPARgamma was shown to have a key role in adipogenesis and be a master controller of the "thrifty gene response" leading to efficient energy storage. Secondly, the discovery that its synthetic ligands, the thiazolidinediones, are promising insulin sensitizing drugs, which are currently being developed for the treatment of Type II (non-insulin-dependent) diabetes mellitus. More recently this nuclear receptor emerged from a role limited to metabolism (diabetes and obesity) to a power player in general transcriptional control of numerous cellular processes, with implications in cell cycle control, carcinogenesis, inflammation, atherosclerosis and immunomodulation. This widened role of PPARgamma will certainly initiate a new flurry of research, which will not only refine our current often partial knowledge of PPARgamma but more importantly also establish that this receptor has a definite role as a primary link adapting cellular, tissue and whole body homeostasis to energy stores.

We have cloned the Hansenula polymorpha PEX1 and PEX6 genes by functional complementation of the corresponding peroxisome-deficient (pex) mutants. The gene products, HpPex1p and HpPex6p, are ATPases which both belong to the AAA protein family. Cells deleted for either gene (Deltapex1 or Deltapex6) were characterized by the presence of small peroxisomal remnants which contained peroxisomal membrane proteins and minor amounts of matrix proteins. The bulk of the matrix proteins, however, resided in the cytosol. In cell fractionation studies HpPex1p and HpPex6p co-sedimented with the peroxisomal membrane protein HpPex3p in both wild-type cells and in Deltapex4, Deltapex8 or Deltapex14 cells. Both proteins are loosely membrane-bound and face the cytosol. Furthermore, HpPex1p and HpPex6p physically and functionally interact in vivo. Overexpression of PEX6 resulted in defects in peroxisomal matrix protein import. By contrast, overexpression of PEX1 was not detrimental to the cells. Interestingly, co-overproduction of HpPex1p rescued the protein import defect caused by HpPex6p overproduction. Overproduced HpPex1p and HpPex6p remained predominantly membrane-bound, but only partially co-localized with the peroxisomal membrane protein HpPex3p. Our data indicate that HpPex1p and HpPex6p function in a protein complex associated with the peroxisomal membrane and that overproduced, mislocalized HpPex6p prevents HpPex1p from reaching its site of activity., (Copyright 1999 John Wiley & Sons, Ltd.)

Peroxisome proliferator-activated receptors (PPAR) were discovered in 1990, ending 25 years of uncertainty about the molecular mechanisms of peroxisome proliferation. Subsequently, PPARs have improved our understanding of adipocyte differentiation. But there is more to PPARs than solving a puzzle about an organelle (the peroxisome) long considered an oddity, and their medical significance goes beyond obesity too. Enhanced PPAR type alpha expression protects against cardiovascular disorders though the role of enhanced PPARgamma expression seems less favourable. PPAR mechanisms, mainly via induction of more differentiated cell phenotypes, protect against some cancers. The differentiation of many cell types (hepatocyte, fibroblast, adipocyte, keratinocyte, myocyte, and monocyte/macrophage) involves PPARs, and these nuclear receptors are now attracting the attention of many medical specialties and the pharmaceutical industry.

Conditions that stress the endoplasmic reticulum (ER) in Saccharomyces cerevisiae can elicit a combination of an unfolded protein response (UPR) and an inositol response (IR). This results in increased synthesis of ER protein-folding factors and of enzymes participating in phospholipid biosynthesis. It was suggested that in cells grown on glucose or galactose medium, the UPR and the IR are linked and controlled by the ER stress sensor Ire1p. However, our studies suggest that during growth on oleate the IR is controlled both by an Ire1p-dependent pathway and by an Ire1p-independent pathway.

The postnatal mammalian heart uses mitochondrial fatty acid oxidation (FAO) as the chief source of energy to meet the high energy demands necessary for pump function. Flux through the cardiac FAO pathway is tightly controlled in accordance with energy demands dictated by diverse physiologic and dietary conditions. In this report, we demonstrate that the lipid-activated nuclear receptor, peroxisome proliferator-activated receptor alpha (PPARalpha), regulates the expression of several key enzymes involved in cardiac mitochondrial FAO. In response to the metabolic stress imposed by pharmacologic inhibition of mitochondrial long-chain fatty acid import with etomoxir, PPARa serves as a molecular 'lipostat' factor by inducing the expression of target genes involved in fatty acid utilization including enzymes involved in mitochondrial and peroxisomal beta-oxidation pathways. In mice lacking PPARalpha (PPARalpha-/- mice), etomoxir precipitates a cardiac phenotype characterized by myocyte lipid accumulation. Surprisingly, this metabolic regulatory response is influenced by gender as demonstrated by the observation that male PPARalpha-/- mice are more susceptible to the metabolic stress compared to female animals. These results identify an important role for PPARalpha in the control of cardiac lipid metabolism.

Zellweger syndrome and related disorders represent a group of lethal, genetically heterogeneous diseases. These peroxisome biogenesis disorders (PBDs) are characterized by defective peroxisomal matrix protein import and comprise at least 10 complementation groups. The genes defective in seven of these groups and more than 90% of PBD patients are now known. Here we examine the distribution of peroxisomal membrane proteins in fibroblasts from PBD patients representing the seven complementation groups for which the mutant gene is known. Peroxisomes were detected in all PBD cells, indicating that the ability to form a minimal peroxisomal structure is not blocked in these mutants. We also observed that peroxisome abundance was reduced fivefold in PBD cells that are defective in the PEX1, PEX5, PEX12, PEX6, PEX10, and PEX2 genes. These cell lines all display a defect in the import of proteins with the type-1 peroxisomal targeting signal (PTS1). In contrast, peroxisome abundance was unaffected in cells that are mutated in PEX7 and are defective only in the import of proteins with the type-2 peroxisomal targeting signal. Interestingly, a fivefold reduction in peroxisome abundance was also observed for cells lacking either of two PTS1-targeted peroxisomal beta-oxidation enzymes, acyl-CoA oxidase and 2-enoyl-CoA hydratase/D-3-hydroxyacyl-CoA dehydrogenase. These results indicate that reduced peroxisome abundance in PBD cells may be caused by their inability to import these PTS1-containing enzymes. Furthermore, the fact that peroxisome abundance is influenced by peroxisomal 105-oxidation activities suggests that there may be metabolic control of peroxisome abundance.

There is a vast body of literature on the quality control of protein folding and assembly into multisubunit complexes. Such control takes place everywhere in the cell. The correcting mechanisms involve cytosolic and organellar proteases; the result of such control is individual molecules with proper structure and individual complexes both with proper stoichiometry and proper structure. Obviously, the formation of organelles as such requires some additional criteria of correctness and some new mechanisms of their implementation. It is proposed in this article that the ability to carry out an integral (key) function may serve as a criterion of correct organelle assembly and that autophagy can be accepted as a mechanism eliminating the assembly mistakes.

Peroxisomes are subcellular organelles catalyzing a number of indispensable functions in cellular metabolism. The importance of peroxisomes in man is stressed by the existence of an expanding group of genetic diseases in which there is an impairment in one or more peroxisomal functions. Much has been learned in recent years about these functions and many of the enzymes involved have been characterized, purified and their cDNAs cloned. This has allowed resolution of the enzymatic and molecular basis of many of the single peroxisomal enzyme deficiencies. Similarly, the molecular basis of the peroxisome biogenesis disorders is also being resolved rapidly thanks to the successful use of CHO as well as yeast mutants. In this paper we will provide an overview of the peroxisomal disorders with particular emphasis on their clinical, biochemical and molecular characteristics.

Peroxisome proliferator-activated receptors (PPARs) are ligand-dependent transcription factors, and it is assumed that the biological effects of these receptors depend on interactions with recently identified coactivators, including steroid receptor coactivator-1 (SRC-1). We assessed the in vivo function of SRC-1 on the PPARalpha-regulated gene expression in liver by generating mice in which the SRC-1 gene was inactivated by gene targeting. The homozygous (SRC-1(-/-)) mice were viable and fertile and exhibited no detectable gross phenotypic defects. When challenged with a PPARalpha ligand, such as ciprofibrate or Wy-14,643, the SRC-1(-/-) mice displayed typical pleiotropic responses, including hepatomegaly, peroxisome proliferation in hepatocytes, and increased mRNA and protein levels of genes that are regulated by PPARalpha. These alterations were indistinguishable from those exhibited by SRC-1(+/+) wild-type mice fed either ciprofibrate- or Wy-14, 643-containing diets. These results indicate that SRC-1 is not essential for PPARalpha-mediated transcriptional activation in vivo and suggest redundancy in nuclear receptor coactivators.

Peroxisomes are organelles that perform a variety of functions, including the metabolism of hydrogen peroxide and the oxidation of fatty acids. Peroxisomes do not possess organellar DNA; all peroxisomal matrix proteins are post-translationally translocated into the organelle. The mechanism of peroxisomal protein translocation has been the subject of vigorous research in the past decade. Many of the proteins (peroxins, abbreviated Pex) that play critical roles in peroxisome biogenesis have been identified through functional complementation of yeast strains and of Chinese hamster ovary cell lines that are defective in peroxisome biogenesis. Researchers are now turning towards biochemical and genetic analyses of these peroxins to define their roles in peroxisome biogenesis and to discover interacting protein partners. Evidence suggests that some of the interacting partners include molecular chaperones. Several current models for peroxisomal protein import are presented.

We have isolated the Hansenula polymorpha CPY gene encoding carboxypeptidase Y (Hp-CPY). The deduced amino acid sequence revealed that Hp-CPY consists of 541 amino acids and has a calculated Mr of 60,793. The protein is highly similar to Saccharomyces cerevisiae CPY (61.8% identity). At the N-terminus of Hp-CPY signals for the entry into the secretory pathway and subsequent sorting to the vacuole were identified. Immunocytochemically, using monospecific antibodies raised against Hp-CPY, the protein was localized to the vacuole. On Western blots, a diffuse protein band was observed in extracts of H. polymorpha cells, suggesting that the protein is glycosylated. This was confirmed by endoglycosidase H treatment, which resulted in a strong reduction of the apparent Mr of the protein. We have investigated the effect of CPY deletion on the degradation of peroxisomes, an autophagous process that occurs when the organelles become redundant for growth. In deltacpy cells peroxisomal proteins were degraded in the vacuole as efficiently as in wild-type H. polymorpha cells, indicating that CPY is not a major proteinase in this pathway.

Dietary polyunsaturated fatty acids (PUFA) have profound effects on hepatic gene transcription leading to significant changes in lipid metabolism. PUFA rapidly suppress transcription of genes encoding specific lipogenic and glycolytic enzymes and induce genes encoding specific peroxisomal and cytochrome P450 (CYP) enzymes. Using the peroxisome proliferator-activated receptor alpha (PPAR alpha)-null mouse, we showed that dietary PUFA induction of acyl CoA oxidase (AOX) and CYP4A2 require PPAR alpha. However, PPAR alpha is not required for the PUFA-mediated suppression of fatty acid synthase (FAS), S14, or L-pyruvate kinase (L-PK). Studies in primary rat hepatocytes and cultured 3T3-L1 adipocytes showed that metabolites of 20:4n-6, like prostaglandin E2 (PGE2), suppress mRNA encoding FAS, S14, and L-PK through a Gi/Go-coupled signal transduction cascade. In contrast to adipocytes, 20:4n-6-mediated suppression of lipogenic gene expression in hepatic parenchymal cells does not require cyclooxygenase. Transfection analysis of S14CAT fusion genes in primary hepatocytes shows that peroxisome proliferator-activated PPAR alpha acts on the thyroid hormone response elements (-2.8/-2.5 kb). In contrast, both PGE2 and 20:4n-6 regulate factors that act on the proximal promoter (-150/-80 bp) region, respectively. In conclusion, PUFA affects hepatic gene transcription through at least three distinct mechanisms: (i) a PPAR-dependent pathway, (ii) a prostanoid pathway, and (iii) a PPAR and prostanoid-independent pathway. PUFA regulation of hepatic lipid metabolism involves an integration of these multiple pathways.

The interaction of cells with extracellular matrix components contributes to their specific differentiation. We studied hepatic peroxisomes and their changing features during embryonic development, and we immunolocalized in the same tissue the extracellular matrix components laminin and collagen IV as well as the integrin receptor subunits alpha 1, alpha 2, beta 1, and beta 4. Rat and human embryonic liver peroxisome expression were studied at the light- and electron-microscopic level by means of localizing catalase-, D-amino acid oxidase- and polyamine oxidase activities and by means of the immunocytochemistry of six peroxisomal proteins. The successive import of catalase and the peroxisomal beta-oxidation enzymes, the late appearance of the other enzymes, and the gradual increase of peroxisomal size and number to adult values occurred as asynchronous events. Although still immature, peroxisomes were recognized at every stage examined and coexisted with laminin and collagen IV in both species. The beta 1 integrin subunit was immunodetected as early as at 12.5 days in rat. It was concluded that these extracellular matrix factors may be important for the differentiation of liver parenchyma from the liverbud stage onwards. However, the stepwise maturation of the liver-specific peroxisome suggests the involvement of many other regulating factors.

Mammalian cells typically contain hundreds of peroxisomes but can increase peroxisome abundance further in response to extracellular stimuli. We report here the identification and characterization of two novel human peroxisomal membrane proteins, PEX11alpha and PEX11beta. Overexpression of the human PEX11beta gene alone was sufficient to induce peroxisome proliferation, demonstrating that proliferation can occur in the absence of extracellular stimuli and may be mediated by a single gene. Time course studies indicated that PEX11beta induces peroxisome proliferation through a multistep process involving peroxisome elongation and segregation of PEX11beta from other peroxisomal membrane proteins, followed by peroxisome division. Overexpression of PEX11alpha also induced peroxisome proliferation but at a much lower frequency than PEX11beta in our experimental system. The patterns of PEX11alpha and PEX11beta expression were examined in the rat, the animal in which peroxisome proliferation has been examined most extensively. Levels of PEX11beta mRNA were similar in all tissues examined and were unaffected by peroxisome-proliferating agents. Conversely, PEX11alpha mRNA levels varied widely among different tissues, were highest in tissues that are sensitive to peroxisome-proliferating agents, and were induced more than 10-fold in response to the peroxisome proliferators clofibrate and di(2-ethylhexyl) phthalate. Taken together, these data implicate PEX11beta in the constitutive control of peroxisome abundance and suggest that PEX11alpha may regulate peroxisome abundance in response to extracellular stimuli.

As more functional redundancy in mammalian cells is discovered, enhanced expression of genes involved in alternative pathways may become an effective form of gene therapy. X-linked adrenoleukodystrophy (X-ALD) is a peroxisomal disorder with impaired very-long-chain fatty acid metabolism. The X-ALD gene encodes a peroxisomal membrane protein (ALDP) that is part of a small family of related peroxisomal membrane proteins. We show that 4-phenylbutyrate treatment of cells from both X-ALD patients and X-ALD knockout mice results in decreased levels of and increased beta-oxidation of very-long-chain fatty acids; increased expression of the peroxisomal protein ALDRP; and induction of peroxisome proliferation. We also demonstrate that ALDP and ALDRP are functionally related, by ALDRP cDNA complementation of X-ALD fibroblasts. Finally, we demonstrate the in vivo efficacy of dietary 4-phenylbutyrate treatment through its production of a substantial reduction of very-long-chain fatty acid levels in the brain and adrenal glands of X-ALD mice.

Objective: Characterization of the defect in a patient presenting a peripheral neuropathy with atypical features of distal motor involvement mimicking Werdnig-Hoffmann disease., Patient: Clinical signs included generalized hypotonia and floppiness, absence of stretch reflexes, muscle wasting, lack of head control and lingual fasciculations associated with unaffected facial muscles, and normal intellectual development., Results: Normal muscle histology ruled out Werdnig-Hoffmann disease. Elevated plasma concentrations of very long-chain fatty acids and bile acid intermediates combined with normal plasmalogen levels in erythrocytes suggested defective peroxisomal beta-oxidation directly demonstrated by deficient pristanic acid and partially deficient C26:0 was present oxidation in cultured fibroblasts. Severely impaired pipecolic acid oxidation in liver and phytanic acid oxidation in fibroblasts was present. On light and electron microscopy of the liver tissue, rare peroxisomal membrane ghosts and trilamellar inclusions but absence of peroxisomes was noted. Immunoblot analysis revealed absence of peroxisomal beta-oxidation enzymes in liver tissue but normal results in fibroblasts. Remarkably, expression of the peroxisomal defect in fibroblasts was indicated by the finding of mainly cytoplasmatic catalase, as in liver. Preliminary studies excluded classification of this patient within the large PEX1 complementation group., Conclusions: The results suggest a novel peroxisome biogenesis disorder involving peroxisomal beta-oxidation as well as phytanic and pipecolic acid oxidation rather than an isolated defect of peroxisomal beta-oxidation. The association of a clinical picture mimicking Werdnig-Hoffmann disease with a novel peroxisomal disorder raises the question of whether investigation for peroxisomal function should be considered in every patient with an enigmatic spinal muscular atrophy-like syndrome.

Two targeting signals, PTS1 and PTS2, mediate import of proteins into the peroxisomal matrix. We have cloned and sequenced the watermelon (Citrullus vulgaris) cDNA homologue to the PTS1 receptor gene (PEX5). Its gene product, CvPex5p, belongs to the family of tetratricopeptide repeat (TPR) containing proteins like the human and yeast counterparts, and exhibits 11 repeats of the sequence W-X2-(E/S)-(Y/F/Q) in its N-terminal half. According to fractionation studies the plant Pex5p is located mainly in the cytosolic fraction and therefore could function as a cycling receptor between the cytosol and glyoxysomes, as has been proposed for the Pex5p of human and some yeast peroxisomes. Transformation of the Hansenula polymorpha peroxisome deficient pex5 mutant with watermelon PEX5 resulted in restoration of peroxisome formation and the synthesis of additional membranes surrounding the peroxisomes. These structures are labeled in immunogold experiments using antibodies against the Hansenula polymorpha integral membrane protein Pex3p, confirming their peroxisomal nature. The plant Pex5p was localized by immunogold labelling mainly in the cytosol of the yeast, but also inside the newly formed peroxisomes. However, import of the PTS1 protein alcohol oxidase is only partially restored by CvPex5p.

Niemann-Pick type C disease is an inherited disorder characterized by lysosomal accumulation of cholesterol and the mutant gene has recently been identified. The predicted gene product is a transmembrane protein showing homology to proteins involved in the regulation of cholesterol homeostasis, such as 3-hydroxy-3-methylglutaryl-coenzyme A and the sterol regulatory element binding protein cleavage-activating protein. Recent investigations have established a peroxisomal deficiency, which raised the question of whether peroxisomal proliferation could affect this cholesterol-processing error. Mutant mice with Niemann-Pick type C disease were treated with the peroxisomal inducer perfluorooctanoic acid, which increased peroxisomal beta-oxidation and catalase activity to the same level as in control mice. Not only the peroxisomal, but also the lysosomal malfunctions were corrected and the cholesterol content was decreased. Clofibrate, another peroxisomal inducer, restored both peroxisomal enzyme activities and ubiquinone content. It appears that in Niemann-Pick type C disease treatment with appropriate peroxisomal inducers restores basic cellular functions, indicating a relationship between peroxisomes and cholesterol homeostasis, and thereby may effectively interfere with the development of the disease.

This article reviews the currently available data on the role of peroxisomal function in relation to the processes of cell differentiation and carcinogenesis. In regard to tumourigenesis, both genotoxic and non-genotoxic processes have been considered, and the peroxisomal relationships with these phenomena and with differentiation are described at the level of organelle characteristics, enzyme contents, and the involvement of retinoids, steroid hormones, oxygen free radicals, growth factors, apoptosis, omega-3 polyunsaturated fatty acids and the cellular signalling networks. Overall these data serve to illustrate the unique and distinctive role of the peroxisome in differentiation and carcinogenesis, and point to the advantages of considering the peroxisomal involvement in the holistic context of the differentiation dedifferentiation continuum rather than the narrower focus of non-genotoxic carcinogenesis. The review also outlines the potential for medical benefit arising from a fuller understanding of these peroxisomal affiliations.

ZR-78 and ZR-82 cells are two peroxisomal-deficient Chinese hamster ovary (CHO) cell mutants. These cells lack normal peroxisomes and show reduced levels of plasmalogen synthesis and other peroxisomal functions attributed to the deficiency of peroxisomal matrix enzymes. As we have recently identified two HMG-CoA reductase proteins in CHO cells, a 97 kDa reductase localized in the ER and a 90 kDa reductase protein localized in peroxisomes, this enabled us to study the two reductase proteins for the first time in peroxisomal-deficient CHO cells. In this study we report the results of a detailed analysis of the isoprenoid biosynthetic pathway in the peroxisomal-deficient CHO cell lines ZR-78 and ZR-82. We demonstrate that total HMG-CoA reductase activity is significantly reduced in the peroxisomal-deficient cells as compared to the wild type cells. Analysis of the two reductase proteins in permeabilized cells indicated that in the ZR-78 and ZR-82 cells the 90 kDa peroxisomal reductase protein was mainly localized to the cytosol. We further show that the rates of both sterol (cholesterol) and non-sterol (dolichols) biosynthesis were significantly lower in the peroxisomal-deficient cells, when either [3H] acetate or [3H] mevalonate was used as substrate. In contrast, the rate of dolichol biosynthesis in the peroxisomal-deficient cells was similar to that of the wild type cells when incubated with [3H] farnesol. In addition, we demonstrate that the peroxisomal-deficient cells exhibited increased rates of lanosterol biosynthesis as compared to wild type cells. The results of this study provide further evidence for the essential requirement of peroxisomes for cholesterol biosynthesis as well as for dolichol production.

The regulation of gene expression via the peroxisome proliferator-activated receptor (PPAR) is believed to be critical in the effects of peroxisome proliferators on lipid metabolism and possibly in hepatocarcinogenesis. The involvement of PPAR in the peroxisome proliferator-mediated induction of fatty acid metabolizing genes such as acyl-CoA oxidase (ACO), fatty acid-binding protein (FABP), and cytochrome P450IVA1 (CYP4A1) has been clearly demonstrated. However, the induction by peroxisome proliferators of important growth regulatory genes such as c-myc has not been investigated extensively. In these studies we examined the dose-response relationships for the induction of mRNA for the PPAR-regulated and lipid metabolizing genes ACO, FABP, and CYP4A1 and compared them to the immediate early gene c-myc. Liver mRNA from rats fed various amounts of the peroxisome proliferator Wy14,643 for 13 weeks was utilized. The lipid metabolism and growth regulatory genes were induced by subchronic administration of Wy14,643 but to varying degrees and with different sensitivities. The lowest dose that resulted in a significant change in ACO and FABP expression was 10 ppm. The mRNA for CYP4A1 and c-myc was significantly affected at the lowest dose examined (5 ppm). Also, the maximal induction ranged from 10(5)-fold (CYP4A1) to less than 10-fold (FABP) relative to vehicle-treated animals. The accumulation of mRNA for ACO, FABP, and CYP4A1, but not c-myc, showed typical receptor-mediated dose-response relationships. The effects on gene expression were compared to rates of hepatic cell proliferation, a pertinent marker of tumor promotion and hepatocarcinogenesis. Surprisingly, ACO mRNA showed an excellent correlation (r2 = 0.9) while c-myc mRNA exhibited a poor correlation (r2 = 0.3) with cell proliferation in rat liver. Although the differences between the dose-response relationships of ACO and c-myc mRNA accumulation may suggest immediate early genes are not controlled by PPAR, evidence from PPARalpha null mice support this receptor in both lipid metabolism and growth regulatory genes. This study shows the complexity of responses mediated by peroxisome proliferators, with ACO being a good marker of PPAR-mediated events as well as cell proliferation, while c-myc, a known growth regulatory gene, was induced by Wy14,643 partially via PPAR but did not correlate well with cell proliferation., (Copyright 1998 Academic Press.)

Peroxisomes catalyze a number of essential metabolic functions especially related to lipid metabolism. There is increasing evidence suggesting that peroxisomes are also involved in the synthesis of isoprenoids via the mevalonate pathway at least in rat liver. In order to obtain independent evidence for a role of peroxisomes in isoprenoid synthesis in man, we have measured the activity of two key enzymes of the mevalonate pathway in patients suffering from certain defined defects in peroxisome biogenesis. We now report that mevalonate kinase is not only deficient in livers from Zellweger patients in which peroxisome biogenesis is defective, but also in livers from rhizomelic chondrodysplasia punctata (RCDP) Type 1 patients. In the latter group of patients there is a selective defect in peroxisome biogenesis due to a genetic defect in the PTS2-receptor, a mobile receptor-protein guiding peroxisomal proteins with a certain peroxisomal targeting signal (PTS2) to the peroxisome. Phosphomevalonate kinase was found to be strongly deficient in Zellweger patients thus suggesting that this enzyme is also peroxisomal. Taken together, our data indicate that in human liver mevalonate kinase and phosphomevalonate kinase are truly peroxisomal enzymes which strongly suggests that peroxisomes play a major role in cholesterol biosynthesis.

The peroxisome biogenesis disorders (PBDs), including Zellweger syndrome (ZS) and neonatal adrenoleukodystrophy (NALD), are autosomal recessive diseases caused by defects in peroxisome assembly, for which at least 10 complementation groups have been reported. We have isolated a human PEX1 cDNA (HsPEX1) by functional complementation of peroxisome deficiency of a mutant Chinese hamster ovary (CHO) cell line, ZP107, transformed with peroxisome targeting signal type 1-tagged "enhanced" green fluorescent protein. This cDNA encodes a hydrophilic protein (Pex1p) comprising 1,283 amino acids, with high homology to the AAA-type ATPase family. A stable transformant of ZP107 with HsPEX1 was morphologically and biochemically restored for peroxisome biogenesis. HsPEX1 expression restored peroxisomal protein import in fibroblasts from three patients with ZS and NALD of complementation group I (CG-I), which is the highest-incidence PBD. A CG-I ZS patient (PBDE-04) possessed compound heterozygous, inactivating mutations: a missense point mutation resulting in Leu-664 --> Pro and a deletion of the sequence from Gly-634 to His-690 presumably caused by missplicing (splice site mutation). Both PBDE-04 PEX1 cDNAs were defective in peroxisome-restoring activity when expressed in the patient fibroblasts as well as in ZP107 cells. These results demonstrate that PEX1 is the causative gene for CG-I peroxisomal disorders.

After our previous report on the cloning of two cDNA species in guinea pig, both encoding the same hepatic 79 kDa multifunctional protein 1 (MFP-1) [Caira, Cherkaoui-Malki, Hoefler and Latruffe (1996) FEBS Lett. 378, 57-60], here we report the cloning of a cDNA encoding a second multifunctional peroxisomal protein (MFP-2) in guinea-pig liver. This 2356 nt cDNA encodes a protein of 735 residues (79.7 kDa) whose sequence shows 83% identity with rat MFP-2 [Dieuaide-Noubhani, Novikov, Baumgart, Vanhooren, Fransen, Goethals, Vandekerckhove, Van Veldhoven and Mannaerts (1996) Eur. J. Biochem. 240, 660-666]. In parallel, we studied the effect of ciprofibrate, a hypolipaemic agent also known as peroxisome proliferator in rodent, on the expression of MFP-1 and MFP-2 (2.6 kb) in rats and guinea pigs. By Northern blotting analysis we demonstrated that three MFP-1-related mRNA species are expressed in the guinea-pig liver. The expression of two of them (3.5 and 2.6 kb) is slightly increased by ciprofibrate, whereas the 3.0 kb MFP-1 mRNA is, unlike the rat one, strongly down-regulated in guinea pigs treated with ciprofibrate. In a similar way, the hepatic expression of the guinea-pig 2.6 kb MFP-2 mRNA is also down-regulated in guinea pigs treated with ciprofibrate. These results demonstrate (1) that in contrast with the unique 3.0 kb MFP-1 rat mRNA, at least three hepatic MFP-1-related mRNA species are co-expressed in guinea pig; and (2) that, opposed to the accepted idea of non-responsiveness of the guinea pig to ciprofibrate, this drug affects MFP-1 and MFP-2 gene expression in this species. Also, the mRNA species for acyl-CoA oxidase and thiolase, two other enzymes of the peroxisomal beta-oxidation pathway that are induced severalfold in responsive species are down-regulated in guinea pig. This paper is the first, to our knowledge, reporting the down-regulation of the expression of genes encoding enzymes involved in the peroxisomal beta-oxidation of fatty acids (MFP-1) and bile acid synthesis (MFP-2) in mammals.