Purification of a Recombinant Human PEX1/PEX6 AAA+ ATPase Complex from HEK293TT Cells.

The heteromeric complex of the two AAA+ ATPases PEX1 and PEX6 is involved in the export of the monoubiquitinated import receptor PEX5 from the peroxisomal membrane. Mutations in this complex make up for over 60% of the patients with Peroxisomal Biogenesis Disorders. To have better options for the treatment of the milder mutations we purified the human PEX1/PEX6 complex after overexpression of plasmids encoding tagged proteins from HEK293TT cells. We used a combination of a HisTrap Column (Ni-NTA chromatography) and a Strep-Tactin®XT cartridge for small-scale purification of the complex using the His-tag of PEX1 and the Strep-tagII of PEX6.

Chemically monoubiquitinated PEX5 binds to the components of the peroxisomal docking and export machinery.

Peroxisomal matrix proteins contain either a peroxisomal targeting sequence 1 (PTS1) or a PTS2 that are recognized by the import receptors PEX5 and PEX7, respectively. PEX5 transports the PTS1 proteins and the PEX7/PTS2 complex to the docking translocation module (DTM) at the peroxisomal membrane. After cargo release PEX5 is monoubiquitinated and extracted from the peroxisomal membrane by the receptor export machinery (REM) comprising PEX26 and the AAA ATPases PEX1 and PEX6. Here, we investigated the protein interactions of monoubiquitinated PEX5 with the docking proteins PEX13, PEX14 and the REM. "Click" chemistry was used to synthesise monoubiquitinated recombinant PEX5. We found that monoubiquitinated PEX5 binds the PEX7/PTS2 complex and restores PTS2 protein import in vivo in ΔPEX5 fibroblasts. In vitro pull-down assays revealed an interaction of recombinant PEX5 and monoubiquitinated PEX5 with PEX13, PEX14 and with the REM components PEX1, PEX6 and PEX26. The interactions with the docking proteins were independent of the PEX5 ubiquitination status whereas the interactions with the REM components were increased when PEX5 is ubiquitinated.

Peroxisomal monoubiquitinated PEX5 interacts with the AAA ATPases PEX1 and PEX6 and is unfolded during its dislocation into the cytosol.

PEX1 and PEX6 are two members of the ATPases associated with diverse cellular activities (AAA) family and the core components of the receptor export module of the peroxisomal matrix protein import machinery. Their role is to extract monoubiquitinated PEX5, the peroxisomal protein-shuttling receptor, from the peroxisomal membrane docking/translocation module (DTM), so that a new cycle of protein transportation can start. Recent data have shown that PEX1 and PEX6 form a heterohexameric complex that unfolds substrates by processive threading. However, whether the natural substrate of the PEX1-PEX6 complex is monoubiquitinated PEX5 (Ub-PEX5) itself or some Ub-PEX5-interacting component(s) of the DTM remains unknown. In this work, we used an established cell-free in vitro system coupled with photoaffinity cross-linking and protein PEGylation assays to address this problem. We provide evidence suggesting that DTM-embedded Ub-PEX5 interacts directly with both PEX1 and PEX6 through its ubiquitin moiety and that the PEX5 polypeptide chain is globally unfolded during the ATP-dependent extraction event. These findings strongly suggest that DTM-embedded Ub-PEX5 is a bona fide substrate of the PEX1-PEX6 complex.

Towards the molecular mechanism of the integration of peroxisomal membrane proteins.

The correct topogenesis of peroxisomal membrane proteins is a crucial step for the formation of functioning peroxisomes. Although this process has been widely studied, the exact mechanism with which it occurs has not yet been fully characterized. Nevertheless, it is generally accepted that peroxisomes employ three proteins - Pex3, Pex19 and Pex16 in mammals - for the insertion of peroxisomal membrane proteins into the peroxisomal membrane. Structural biology approaches have been utilized for the elucidation of the mechanistic questions of peroxisome biogenesis, mainly by providing information on the architecture of the proteins significant for this process. This review aims to summarize, compare and put into perspective the structural knowledge that has been generated mainly for Pex3 and Pex19 and their interaction partners in recent years.

Progesterone rapidly down-regulates the biosynthesis of 5-lipoxygenase products in human primary monocytes.

5-Lipoxygenase (5-LO), the key enzyme in the biosynthesis of pro-inflammatory leukotrienes (LTs) from arachidonic acid, is regulated by androgens in human neutrophils and monocytes accounting for sex differences in LT formation. Here we show that progesterone suppresses the synthesis of 5-LO metabolites in human primary monocytes. 5-LO product formation in monocytes stimulated with Ca(2+)-ionophore A23187 or with lipopolysaccharide/formyl peptide was suppressed by progesterone at concentrations of 10-100 nM in cells from females and at 1 µM in cells from males. Progesterone down-regulated 5-LO product formation in a rapid and reversible manner, but did not significantly inhibit 5-LO activity in cell-free assays using monocyte homogenates. Also, arachidonic acid release and its metabolism to other eicosanoids in monocytes were not significantly reduced by progesterone. The inhibitory effect of progesterone on LTs was still observed when mitogen-activated protein kinases were pharmacologically blocked, stimulatory 1-oleoyl-2-acetyl-sn-glycerol was exogenously supplied, or extracellular Ca(2+) was removed by chelation. Instead, suppression of PKA by means of two different pharmacological approaches (i.e. H89 and a cell-permeable PKA inhibitor peptide) prevented inhibition of 5-LO product generation by progesterone, to a similar extent as observed for the PKA activators prostaglandin E2 and 8-Br-cAMP, suggesting the involvement of PKA. In summary, progesterone affects the capacity of human primary monocytes to generate 5-LO products and, in addition to androgens, may account for sex-specific effects on pro-inflammatory LTs.

PEX5, the shuttling import receptor for peroxisomal matrix proteins, is a redox-sensitive protein.

Peroxisome maintenance depends on the import of nuclear-encoded proteins from the cytosol. The vast majority of these proteins is destined for the peroxisomal lumen and contains a C-terminal peroxisomal targeting signal, called PTS1. This targeting signal is recognized in the cytosol by the receptor PEX5. After docking at the peroxisomal membrane and release of the cargo into the organelle matrix, PEX5 is recycled to the cytosol through a process requiring monoubiquitination of an N-terminal, cytosolically exposed cysteine residue (Cys11 in the human protein). At present, the reason why a cysteine, and not a lysine residue, is the target of ubiquitination remains unclear. Here, we provide evidence that PTS1 protein import into human fibroblasts is a redox-sensitive process. We also demonstrate that Cys11 in human PEX5 functions as a redox switch that regulates PEX5 activity in response to intracellular oxidative stress. Finally, we show that exposure of human PEX5 to oxidized glutathione results in a ubiquitination-deficient PEX5 molecule, and that substitution of Cys11 by a lysine can counteract this effect. In summary, these findings reveal that the activity of PEX5, and hence PTS1 import, is controlled by the redox state of the cytosol. The potential physiological implications of these findings are discussed.

Identification of the kinesin KifC3 as a new player for positioning of peroxisomes and other organelles in mammalian cells.

The attachment of organelles to the cytoskeleton and directed organelle transport is essential for cellular morphology and function. In contrast to other cell organelles like the endoplasmic reticulum or the Golgi apparatus, peroxisomes are evenly distributed in the cytoplasm, which is achieved by binding of peroxisomes to microtubules and their bidirectional transport by the microtubule motor proteins kinesin-1 (Kif5) and cytoplasmic dynein. KifC3, belonging to the group of C-terminal kinesins, has been identified to interact with the human peroxin PEX1 in a yeast two-hybrid screen. We investigated the potential involvement of KifC3 in peroxisomal transport. Interaction of KifC3 and the AAA-protein (ATPase associated with various cellular activities) PEX1 was confirmed by in vivo colocalization and by coimmunoprecipitation from cell lysates. Furthermore, knockdown of KifC3 using RNAi resulted in an increase of cells with perinuclear-clustered peroxisomes, indicating enhanced minus-end directed motility of peroxisomes. The occurrence of this peroxisomal phenotype was cell cycle phase independent, while microtubules were essential for phenotype formation. We conclude that KifC3 may play a regulatory role in minus-end directed peroxisomal transport for example by blocking the motor function of dynein at peroxisomes. Knockdown of KifC3 would then lead to increased minus-end directed peroxisomal transport and cause the observed peroxisomal clustering at the microtubule-organizing center.

The role of conserved PEX3 regions in PEX19-binding and peroxisome biogenesis.

The human peroxins PEX3 and PEX19 are essential for peroxisome biogenesis. They mediate the import of membrane proteins as well as the de novo formation of peroxisomes. PEX19 binds newly synthesized peroxisomal membrane proteins post-translationally and directs them to peroxisomes by engaging PEX3, a protein anchored in the peroxisomal membrane. After protein insertion into the lipid bilayer, PEX19 is released back to the cytosol. Crystallographic analysis provided detailed insights into the PEX3-PEX19 interaction and identified three highly conserved regions, the PEX19-binding region, a hydrophobic groove and an acidic cluster, on the surface of PEX3. Here, we used site-directed mutagenesis and biochemical and functional assays to determine the role of these regions in PEX19-binding and peroxisome biogenesis. Mutations in the PEX19-binding region reduce the affinity for PEX19 and destabilize PEX3. Furthermore, we provide evidence for a crucial function of the PEX3-PEX19 complex during de novo formation of peroxisomes in peroxisome-deficient cells, pointing to a dual function of the PEX3-PEX19 interaction in peroxisome biogenesis. The maturation of preperoxisomes appears to require the hydrophobic groove near the base of PEX3, presumably by its involvement in peroxisomal membrane protein insertion, while the acidic cluster does not appear to be functionally relevant.

Testosterone suppresses phospholipase D, causing sex differences in leukotriene biosynthesis in human monocytes.

Sex disparities in inflammation have been reported, but the cellular and molecular basis for these discrepancies is unknown. Monocytes are central effector cells in immunity and possess high capacities to produce proinflammatory leukotrienes (LTs). Here, we investigated sex differences in the activation of 5-lipoxygenase (5-LO), the key enzyme in LT biosynthesis, in human peripheral monocytes. In cells from females, 5-LO product formation was 1.8-fold higher than in cells from males, as evaluated by HPLC. When female monocytes were resuspended in plasma from males, 5-LO products were significantly lower than in female plasma. Interestingly, 5α-dihydrotestosterone (5α-DHT, 10 nM) repressed LT synthesis in female cells down to the levels observed in males, while estradiol (100 nM) was without effect, and progesterone (100 nM) caused only a slight inhibition. 5α-DHT (10 nM) caused ERK phosphorylation and inhibition of phospholipase D (PLD), as evaluated by Western blot and measurement of PLD activity via radioenzymatic diacylglyceride (DAG) and nonradioactive choline assays. Accordingly, PLD activity and DAG formation were 1.4- to 1.8-fold lower in male vs. female monocytes connected to increased ERK phosphorylation. Our data indicate that ERK activation by androgens in monocytes represses PLD activity, resulting in impaired 5-LO product formation due to lack of activating DAGs.

Pex11pß-mediated maturation of peroxisomes.

Peroxisomes are highly dynamic, multifunctional organelles that display remarkable changes in morphology, number and enzyme content. Peroxisomes multiply by growth and division of pre-existing organelles, but they can also form de novo from the ER. Growth and division of peroxisomes in mammalian cells involves elongation, membrane constriction and final fission and requires the peroxisome biogenesis Pex11 proteins as well as the recruitment of Dynamin-like protein DLP1/Drp1. We recently exploited the division-inhibiting properties of a unique Pex11pß-YFP fusion protein to further dissect the process of peroxisomal growth and division. By applying life cell imaging and the HaloTag technology, our study revealed that Pex11pß-mediated growth (elongation) and division of peroxisomes follows a multistep maturation pathway, which is initiated by the formation of an early peroxisomal membrane compartment from a pre-existing peroxisome and its stepwise conversion into a mature, metabolically active peroxisome compartment. Our observations support the view that peroxisomes formed by growth and division of pre-existing ones contain new membrane and matrix components. Peroxisome division is an asymmetric process, which is more complex than simple (symmetric) division of a preexisting organelle and equal distribution of the protein content. Our findings are in favor of Pex11pß acting as a peroxisomal membrane shaping protein.

Insights into peroxisome function from the structure of PEX3 in complex with a soluble fragment of PEX19.

The human peroxins PEX3 and PEX19 play a central role in peroxisomal membrane biogenesis. The membrane-anchored PEX3 serves as the receptor for cytosolic PEX19, which in turn recognizes newly synthesized peroxisomal membrane proteins. After delivering these proteins to the peroxisomal membrane, PEX19 is recycled to the cytosol. The molecular mechanisms underlying these processes are not well understood. Here, we report the crystal structure of the cytosolic domain of PEX3 in complex with a PEX19-derived peptide. PEX3 adopts a novel fold that is best described as a large helical bundle. A hydrophobic groove at the membrane-distal end of PEX3 engages the PEX19 peptide with nanomolar affinity. Mutagenesis experiments identify phenylalanine 29 in PEX19 as critical for this interaction. Because key PEX3 residues involved in complex formation are highly conserved across species, the observed binding mechanism is of general biological relevance.

Hyperforin is a novel type of 5-lipoxygenase inhibitor with high efficacy in vivo.

We previously showed that, in vitro, hyperforin from St. John's wort (Hypericum perforatum) inhibits 5-lipoxygenase (5-LO), the key enzyme in leukotriene biosynthesis. Here, we demonstrate that hyperforin possesses a novel and unique molecular pharmacological profile as a 5-LO inhibitor with remarkable efficacy in vivo. Hyperforin (4 mg/kg, i.p.) significantly suppressed leukotriene B(4) formation in pleural exudates of carrageenan-treated rats associated with potent anti-inflammatory effectiveness. Inhibition of 5-LO by hyperforin, but not by the iron-ligand type 5-LO inhibitor BWA4C or the nonredox-type inhibitor ZM230487, was abolished in the presence of phosphatidylcholine and strongly reduced by mutation (W13A-W75A-W102A) of the 5-LO C2-like domain. Moreover, hyperforin impaired the interaction of 5-LO with coactosin-like protein and abrogated 5-LO nuclear membrane translocation in ionomycin-stimulated neutrophils, processes that are typically mediated via the regulatory 5-LO C2-like domain. Together, hyperforin is a novel type of 5-LO inhibitor apparently acting by interference with the C2-like domain, with high effectiveness in vivo.

ERK-mediated regulation of leukotriene biosynthesis by androgens: a molecular basis for gender differences in inflammation and asthma.

5-Lipoxygenase initiates the biosynthesis of leukotrienes, lipid mediators involved in normal host defense and in inflammatory and allergic disorders. Despite an obvious gender bias in leukotriene-related diseases (e.g., asthma), gender aspects have been neglected in studies on leukotrienes and 5-lipoxygenase. Here, we show that leukotriene formation in stimulated whole blood or neutrophils from males is substantially lower compared with females, accompanied by changed 5-lipoxygenase trafficking. This is due to gender-specific differential activation of extracellular signal-regulated kinases (ERKs). The differences are directly related to variant male/female testosterone plus 5alpha-dihydrotestosterone levels, and addition of 5alpha-dihydrotestosterone to female blood or neutrophils reduced the high (female) LT biosynthesis capacity to low (male) levels. In conclusion, regulation of ERKs and leukotriene formation by androgens constitutes a molecular basis for gender differences in the inflammatory response, and in inflammatory diseases such as asthma.

The role of diacylglyceride generation by phospholipase D and phosphatidic acid phosphatase in the activation of 5-lipoxygenase in polymorphonuclear leukocytes.

Diacylglycerides (DAGs) such as 1-oleoyl-2-acetyl-sn-glycerol (OAG) stimulate 5-lipoxygenase (5-LO) enzyme activity and function as agonists for human polymorphonuclear leukocytes (PMNL) to induce 5-LO product synthesis. Here, we addressed the role of endogenous DAG generation in agonist-induced 5-LO activation in human PMNL. Preincubation of PMNL with the phospholipase D (PLD) inhibitor 1-butanol potently suppressed 5-LO product synthesis induced by the Ca(2)(+) ionophore A23187 or thapsigargin (TG) and blocked A23187-evoked translocation of 5-LO from the cytosol to the nuclear membrane, analyzed by subcellular fractionation as well as by indirect immunofluorescence microscopy. Tertiary-butanol, a rather poor inhibitor of PLD, caused only moderate suppression of 5-LO and hardly inhibited 5-LO translocation. Interestingly, 1-butanol failed to inhibit 5-LO product formation when PMNL were stimulated with OAG (30 microM). Moreover, coincubation of A23187- or TG-stimulated PMNL with OAG reversed inhibition of 5-LO product formation by 1-butanol in a concentration-dependent manner (EC(50), approximately 1 muM) and also restored 5-LO translocation. In addition, inhibition of phosphatidic acid phosphatase (PA-P) by propranolol or bromoenol lactone caused suppression of 5-LO product formation and of translocation, which could be reversed by addition of exogenous OAG. Together, our data suggest that in agonist-stimulated PMNL, the endogenous formation of DAGs via the PLD/PA-P pathway determines 5-LO activation.

Phytotoxicity and innate immune responses induced by Nep1-like proteins.

We show that oomycete-derived Nep1 (for necrosis and ethylene-inducing peptide1)-like proteins (NLPs) trigger a comprehensive immune response in Arabidopsis thaliana, comprising posttranslational activation of mitogen-activated protein kinase activity, deposition of callose, production of nitric oxide, reactive oxygen intermediates, ethylene, and the phytoalexin camalexin, as well as cell death. Transcript profiling experiments revealed that NLPs trigger extensive reprogramming of the Arabidopsis transcriptome closely resembling that evoked by bacteria-derived flagellin. NLP-induced cell death is an active, light-dependent process requiring HSP90 but not caspase activity, salicylic acid, jasmonic acid, ethylene, or functional SGT1a/SGT1b. Studies on animal, yeast, moss, and plant cells revealed that sensitivity to NLPs is not a general characteristic of phospholipid bilayer systems but appears to be restricted to dicot plants. NLP-induced cell death does not require an intact plant cell wall, and ectopic expression of NLP in dicot plants resulted in cell death only when the protein was delivered to the apoplast. Our findings strongly suggest that NLP-induced necrosis requires interaction with a target site that is unique to the extracytoplasmic side of dicot plant plasma membranes. We propose that NLPs play dual roles in plant pathogen interactions as toxin-like virulence factors and as triggers of plant innate immune responses.

Peroxisome biogenesis disorders.

Defects in PEX genes impair peroxisome assembly and multiple metabolic pathways confined to this organelle, thus providing the biochemical and molecular bases of the peroxisome biogenesis disorders (PBD). PBD are divided into two types--Zellweger syndrome spectrum (ZSS) and rhizomelic chondrodysplasia punctata (RCDP). Biochemical studies performed in blood and urine are used to screen for the PBD. DNA testing is possible for all of the disorders, but is more challenging for the ZSS since 12 PEX genes are known to be associated with this spectrum of PBD. In contrast, PBD-RCDP is associated with defects in the PEX7 gene alone. Studies of the cellular and molecular defects in PBD patients have contributed significantly to our understanding of the role of each PEX gene in peroxisome assembly.

IGF-independent effects of IGFBP-2 on the human breast cancer cell line Hs578T.

There is evidence that insulin-like growth factor-binding protein (IGFBP-2), a modulator of the actions of IGFs, also has IGF-independent effects in human tumor cell lines. These involve specific binding of IGFBP-2 to alpha5beta1-integrin, followed by alterations in the phosphorylation status of downstream signaling molecules. Previously, IGFBP-2 has also been shown to be associated with cell proliferation, adhesion and migration. Here, we investigated direct effects of IGFBP-2 on apoptosis and alterations in the expression of related proteins. The breast cancer cell line Hs578T, which shows no IGFBP-2 production of its own and is independent of the IGF-I receptor, was treated with human recombinant IGFBP-2 in order to study the changes in gene expression induced by IGFBP-2. The methods employed for this purpose were oligonucleotide microarrays, real-time RT-PCR, western blotting, and immunoassays. Out of the 440 genes covered by the Oligo GEArray Human Cancer Microarray OHS-802, the expression of 77 genes was directly influenced by IGFBP-2. By the use of real-time quantitative RT-PCR, the gene expression of Nuclear Factor (NF)kappaB, p53, transforming growth factor beta (TGF beta-1), LAMB1 (Laminin, Beta 1), Bcl-2, and IIp45 was found to be significantly upregulated (by 1.2- to 3.05-fold; all P < 0.001). Accordingly, NFkappaB, p53, and TGF beta-1 proteins, as measured by Western blotting and immunoassay, were upregulated > 1.5-fold. By using an ELISA-based and a flow cytometry-based apoptosis assay, IGFBP-2 was found to have a pro-apoptotic effect on Hs578T cells. Our results suggest that IGFBP-2-induced gene expressions are of functional significance for proliferation, cell adhesion, cell migration and apoptosis, and showed that IGFBP-2 can promote apoptosis in tumor cells independent of IGF.

The interaction between human PEX3 and PEX19 characterized by fluorescence resonance energy transfer (FRET) analysis.

The process of peroxisome biogenesis involves several PEX genes that encode the machinery required to assemble the organelle. Among the corresponding peroxins the interaction between PEX3 and PEX19 is essential for early peroxisome biogenesis. However, the intracellular site of this protein interaction is still unclear. To address this question by fluorescence resonance energy transfer (FRET) analysis, we engineered the enhanced yellow fluorescent protein (EYFP) to the C-terminus of PEX3 and the enhanced cyan fluorescent protein (ECFP) to the N-terminus of PEX19. Functionality of the fusion proteins was shown by transfection of human PEX3- and PEX19-deficient fibroblasts from Zellweger patients with tagged versions of PEX3 and PEX19. This led to reformation of import-competent peroxisomes in both cell lines previously lacking detectable peroxisomal membrane structures. The interaction of PEX3-EYFP with ECFP-PEX19 in a PEX3-deficient cell line during peroxisome biogenesis was visualized by FRET imaging. Although PEX19 was predominantly localized to the cytoplasma, the peroxisome was identified to be the main intracellular site of the PEX3-PEX19 interaction. Results were confirmed and quantified by donor fluorescence photobleaching experiments. PEX3 deletion proteins lacking the N-terminal peroxisomal targeting sequence (PEX3 34-373-EYFP) or the PEX19-binding domain located in the C-terminal half of the protein (PEX3 1-140-EYFP) did not show the characteristic peroxisomal localization of PEX3, but were mislocalized to the cytoplasm (PEX3 34-373-EYFP) or to the mitochondria (PEX3 1-140-EYFP) and did not interact with ECFP-PEX19. We suggest that FRET is a suitable tool to gain quantitative spatial information about the interaction of peroxins during the process of peroxisome biogenesis in single cells. These findings complement and extend data from conventional in vitro protein interaction assays and support the hypothesis of PEX3 being an anchor for PEX19 at the peroxisomal membrane.

Modeling human peroxisome biogenesis disorders in the nematode Caenorhabditis elegans.

Peroxisomes are ubiquitous eukaryotic organelles. The proteins required for peroxisome biogenesis are called peroxins, and mutations in the peroxin genes cause the devastating human developmental syndromes called the peroxisome biogenesis disorders. Our interest is in elaborating the roles that peroxisomes play in Caenorhabditis elegans development, and in establishing an invertebrate model system for the human peroxisome biogenesis disorders. The genome of C. elegans encodes homologs of 11 of the 13 human peroxins. We disrupted five nematode peroxins using RNA interference (RNAi) and found that RNAi knockdown of each one causes an early larval arrest at the L1 stage. Using a green fluorescent protein reporter targeted to the peroxisome, we establish that peroxisomal import is impaired in prx-5(RNAi) nematodes. prx-5(RNAi) animals are blocked very early in the L1 stage and do not initiate normal postembryonic cell divisions, similar to starvation-arrested larvae. Cell and axonal migrations that normally occur during the L1 stage also appear blocked. We conclude that peroxisome function is required for C. elegans postembryonic development and that disruption of peroxisome assembly by prx-5(RNAi) prevents scheduled postembryonic cell divisions. Defects in the cellular localization of peroxisomal proteins and in development are shared features of human and nematode peroxisome biogenesis disorders. In setting up a C. elegans model of peroxisomal biogenesis disorders, we suggest that genetic screens for suppression of the Prx developmental block will facilitate identification of novel intervention strategies and may provide new insights into human disease pathogenesis.