Peroxisomal Metabolic Pathways Research Articles

Characterization and engineering of peroxisome targeting sequences for compartmentalization engineering in Pichia pastoris.

Peroxisomal compartmentalization has emerged as a highly promising strategy for reconstituting intricate metabolic pathways. In recent years, significant progress has been made in the peroxisomes through harnessing precursor pools, circumventing metabolic crosstalk, and minimizing the cytotoxicity of exogenous pathways. However, it is important to note that in methylotrophic yeasts (e.g. Pichia pastoris), the abundance and protein composition of peroxisomes are highly variable, particularly when peroxisome proliferation is induced by specific carbon sources. The intricate subcellular localization of native proteins, the variability of peroxisomal metabolic pathways, and the lack of systematic characterization of peroxisome targeting signals have limited the applications of peroxisomal compartmentalization in P. pastoris. Accordingly, this study established a high-throughput screening method based on β-carotene biosynthetic pathway to evaluate the targeting efficiency of PTS1s (Peroxisome Targeting Signal Type 1) in P. pastoris. First, 25 putative endogenous PTS1s were characterized and 3 PTS1s with high targeting efficiency were identified. Then, directed evolution of PTS1s was performed by constructing two PTS1 mutant libraries, and a total of 51 PTS1s (29 classical and 22 noncanonical PTS1s) with presumably higher peroxisomal targeting efficiency were identified, part of which were further characterized via confocal microscope. Finally, the newly identified PTS1s were employed for peroxisomal compartmentalization of the geraniol biosynthetic pathway, resulting in more than 30% increase in the titer of monoterpene compared with when the pathway was localized to the cytosol. The present study expands the synthetic biology toolkit and lays a solid foundation for peroxisomal compartmentalization in P. pastoris.

Protein ubiquitination in plant peroxisomes.

Protein ubiquitination regulates diverse cellular processes in eukaryotic organisms, from growth and development to stress response. Proteins subjected to ubiquitination can be found in virtually all subcellular locations and organelles, including peroxisomes, single-membrane and highly dynamic organelles ubiquitous in eukaryotes. Peroxisomes contain metabolic functions essential to plants and animals such as lipid catabolism, detoxification of reactive oxygen species (ROS), biosynthesis of vital hormones and cofactors, and photorespiration. Plant peroxisomes possess a complex proteome with functions varying among different tissue types and developmental stages, and during plant response to distinct environmental cues. However, how these diverse functions are regulated at the post-translational level is poorly understood, especially in plants. In this review, we summarized current knowledge of the involvement of protein ubiquitination in peroxisome protein import, remodeling, pexophagy, and metabolism, focusing on plants, and referencing discoveries from other eukaryotic systems when relevant. Based on previous ubiquitinomics studies, we compiled a list of 56 ubiquitinated Arabidopsis peroxisomal proteins whose functions are associated with all the major plant peroxisomal metabolic pathways. This discovery suggests a broad impact of protein ubiquitination on plant peroxisome functions, therefore substantiating the need to investigate this significant regulatory mechanism in peroxisomes at more depths.

Peroxisome-Mediated Reactive Oxygen Species Signals Modulate Programmed Cell Death in Plants.

Peroxisomes are a class of simple organelles that play an important role in plant reactive oxygen species (ROS) metabolism. Experimental evidence reveals the involvement of ROS in programmed cell death (PCD) in plants. Plant PCD is crucial for the regulation of plant growth, development and environmental stress resistance. However, it is unclear whether the ROS originated from peroxisomes participated in cellular PCD. Enzymes involved in the peroxisomal ROS metabolic pathways are key mediators to figure out the relationship between peroxisome-derived ROS and PCD. Here, we summarize the peroxisomal ROS generation and scavenging pathways and explain how peroxisome-derived ROS participate in PCD based on recent progress in the functional study of enzymes related to peroxisomal ROS generation or scavenging. We aimed to elucidate the role of the peroxisomal ROS regulatory system in cellular PCD to show its potential in terms of accurate PCD regulation, which contribute to environmental stress resistance.

Regulatory Roles of Peroxisomal Metabolic Pathways Involved in Musk Secretion in Muskrats.

In this study, we analyzed the main components of muskrat musk by gas chromatography-mass spectrometry, the results showed that muskrat musk contained fatty acids (29.32%), esters (31.89%), cholesterol (4.38%), cyclic ketones (16.31%), alcohols (6.42%) and other compounds, among which 9-octadecenoic acid accounted for 4.89%. We also analyzed the genes of the metabolic pathway in the scent gland at the transcriptomic level during musk-secreting and non-secreting seasons by RNA-seq (RNA sequencing). We detected 21 genes in the peroxisomal metabolic pathways, including PEX14(peroxin-14) and ACOX3(acyl-CoA oxidase), which exhibited significant differential expression between the musk-secreting season and the non-secreting season (p < 0.05). The RNA-seq results for these genes were validated by reverse transcription PCR(RT-PCR) for both seasons. In addition, we examined changes in the composition of muskrat musk from the glandular cells of scent glands cultured in vitro after RNA interference-mediated silencing of 2 differentially expressed genes, ACOX3 and HSD17B4(D-bifunctional protein, DBP). The 9-Octadecenoic acid content in muskrat musk decreased significantly following the silencing of ACOX3 and HSD17B4(D-bifunctional protein, DBP). These results suggest that peroxisomal metabolic pathways play important roles in the regulation of musk secretion in scent glands in the muskrat.

Peroxysomal Carnitine Acetyl Transferase Influences Host Colonization Capacity in Sclerotinia sclerotiorum

In lower eukaryotes, the glyoxylate cycle allows cells to utilize two-carbon compounds when simple sugars are not available. In filamentous fungi, glyoxylate metabolism is coupled with β-oxidation of fatty acids, and both are localized to ubiquitous eukaryotic organelles called peroxisomes. Acetyl coenzyme A (acetyl-CoA) produced during β-oxidation is transported via the cytosol into mitochondria for further metabolism. A peroxisomal-specific pathway for acetyl-CoA transport requiring peroxisomal carnitine acetyl transferase (CAT) activity has been identified in Magnaporthe grisea peroxisomes. Here, we report that a Sclerotinia sclerotiorum ortholog of the M. grisea peroxisomal CAT-encoding gene Pth2 (herein designated Ss-pth2) is required for virulence-associated host colonization. Null (ss-pth2) mutants, obtained by in vitro transposon mutagenesis, failed to utilize fatty acids, acetate, or glycerol as sole carbon sources for growth. Gene expression analysis of these mutants showed altered levels of transcript accumulation for glyoxylate cycle enzymes. Ss-pth2 disruption also affected sclerotial, apothecial, and appressorial development and morphology, as well as oxalic acid accumulation when cultured with acetate or oleic acid as sole carbon nutrient sources. Although mutants were able to penetrate and initially colonize host tissue, subsequent colonization was impaired. Genetic complementation with the wild-type Ss-pth2 restored wild-type virulence phenotypes. These findings suggest an essential role in S. sclerotiorum for the peroxisomal metabolic pathways for oxalic acid synthesis and host colonization.

Germ cells of male mice express genes for peroxisomal metabolic pathways implicated in the regulation of spermatogenesis and the protection against oxidative stress

Peroxisomes are organelles with main functions in the metabolism of lipids and of reactive oxygen species. Within the testis, they have different functional profiles depending on the cell types. A dysfunction of peroxisomes interferes with regular spermatogenesis and can lead to infertility due to spermatogenic arrest. However, so far only very little is known about the functions of peroxisomes in germ cells. We have therefore analyzed the peroxisomal compartment in germ cells and its alterations during spermatogenesis by fluorescence and electron microscopy as well as by expression profiling of peroxisome-related genes in purified cell populations isolated from mouse testis. We could show that peroxisomes are present in all germ cells of the germinal epithelium. During late spermiogenesis, the peroxisomes form large clusters that are segregated from the spermatozoa into the residual bodies upon release from the germinal epithelium. Germ cells express genes for proteins involved in numerous metabolic pathways of peroxisomes. Based on the expression profile, we conclude that newly identified functions of germ cell peroxisomes are the synthesis of plasmalogens as well as the metabolism of retinoids, polyunsaturated fatty acids and polyamines. Thus, germ cell peroxisomes are involved in the regulation of the homeostasis of signaling molecules regulating spermatogenesis and they contribute to the protection of germ cells against oxidative stress.

MtDNA depletion confers specific gene expression profiles in human cells grown in culture and in xenograft

BackgroundInteractions between the gene products encoded by the mitochondrial and nuclear genomes play critical roles in eukaryotic cellular function. However, the effects mitochondrial DNA (mtDNA) levels have on the nuclear transcriptome have not been defined under physiological conditions. In order to address this issue, we characterized the gene expression profiles of A549 lung cancer cells and their mtDNA-depleted ρ0 counterparts grown in culture and as tumor xenografts in immune-deficient mice.ResultsCultured A549 ρ0 cells were respiration-deficient and showed enhanced levels of transcripts relevant to metal homeostasis, initiation of the epithelial-mesenchymal transition, and glucuronidation pathways. Several well-established HIF-regulated transcripts showed increased or decreased abundance relative to the parental cell line. Furthermore, growth in culture versus xenograft has a significantly greater influence on expression profiles, including transcripts involved in mitochondrial structure and both aerobic and anaerobic energy metabolism. However, both in vitro and in vivo, mtDNA levels explained the majority of the variance observed in the expression of transcripts in glucuronidation, tRNA synthetase, and immune surveillance related pathways. mtDNA levels in A549 xenografts also affected the expression of genes, such as AMACR and PHYH, involved in peroxisomal lipid metabolic pathways.ConclusionWe have identified mtDNA-dependent gene expression profiles that are shared in cultured cells and in xenografts. These profiles indicate that mtDNA-depleted cells could provide informative model systems for the testing the efficacy of select classes of therapeutics, such as anti-angiogenesis agents. Furthermore, mtDNA-depleted cells grown culture and in xenografts provide a powerful means to investigate possible relationships between mitochondrial activity and gene expression profiles in normal and pathological cells.

Dr Brian Gibberd (1931–2006): a pioneering clinician in Refsum's disease

Branched-chain fatty acids are common components of the human diet (phytanic acid) or are produced endogenously (bile acids), and are also used as medicines (ibuprofen). Owing to their branched-chain structure, they are metabolized in peroxisomes. In the case of phytanic acid, the presence of a 3-methyl group prevents beta-oxidation, and instead it undergoes one round of alpha-oxidation to allow further metabolism. Defects in this process give rise to neurological diseases and cancer. Dr Brian F. Gibberd was one of the first U.K. physicians to recognize the importance of these peroxisomal metabolic pathways in clinical medicine, and pioneered their study. This obituary recognizes his many achievements in neurology and especially in the treatment of peroxisomal disorders. The following four papers from this mini-symposium entitled 'Advances in peroxisomal alpha-, beta- and omega-oxidation' describe work done in this area as part of a collaborative study in which Dr Gibberd played a key role. This work was presented as part of the Cardiovascular Bioscience focused topic at the Life Sciences 2007 conference, and this mini-symposium was dedicated to Dr Gibberd and his important contributions to this field.

Multiple contributions of peroxisomal metabolic function to fungal pathogenicity in Colletotrichum lagenarium.

In Colletotrichum lagenarium, which is the causal agent of cucumber anthracnose, PEX6 is required for peroxisome biogenesis and appressorium-mediated infection. To verify the roles of peroxisome-associated metabolism in fungal pathogenicity, we isolated and functionally characterized ICL1 of C. lagenarium, which encodes isocitrate lyase involved in the glyoxylate cycle in peroxisomes. The icl1 mutants failed to utilize fatty acids and acetate for growth. Although Icl1 has no typical peroxisomal targeting signals, expression analysis of the GFP-Icl1 fusion protein indicated that Icl1 localizes in peroxisomes. These results indicate that the glyoxylate cycle that occurs inside the peroxisome is required for fatty acid and acetate metabolism for growth. Importantly, in contrast with the pex6 mutants that form nonmelanized appressoria, the icl1 mutants formed appressoria that were highly pigmented with melanin, suggesting that the glyoxylate cycle is not essential for melanin biosynthesis in appressoria. However, the icl1 mutants exhibited a severe reduction in virulence. Appressoria of the icl1 mutants failed to develop penetration hyphae in the host plant, suggesting that ICL1 is involved in host invasion. The addition of glucose partially restored virulence of the icl1 mutant. Heat shock treatment of the host plant also enabled the icl1 mutants to develop lesions, implying that the infection defect of the icl1 mutant is associated with plant defense. Together with the requirement of PEX6 for appressorial melanization, our findings suggest that peroxisomal metabolic pathways play functional roles in appressorial melanization and subsequent host invasion steps, and the latter step requires the glyoxylate cycle.

Role of ALDP (ABCD1) and mitochondria in X-linked adrenoleukodystrophy.

Peroxisomal disorders have been associated with malfunction of peroxisomal metabolic pathways, but the pathogenesis of these disorders is largely unknown. X-linked adrenoleukodystrophy (X-ALD) is associated with elevated levels of very-long-chain fatty acids (VLCFA; C(>22:0)) that have been attributed to reduced peroxisomal VLCFA beta-oxidation activity. Previously, our laboratory and others have reported elevated VLCFA levels and reduced peroxisomal VLCFA beta-oxidation in human and mouse X-ALD fibroblasts. In this study, we found normal levels of peroxisomal VLCFA beta-oxidation in tissues from ALD mice with elevated VLCFA levels. Treatment of ALD mice with pharmacological agents resulted in decreased VLCFA levels without a change in VLCFA beta-oxidation activity. These data indicate that ALDP does not determine the rate of VLCFA beta-oxidation and that VLCFA levels are not determined by the rate of VLCFA beta-oxidation. The rate of peroxisomal VLCFA beta-oxidation in human and mouse fibroblasts in vitro is affected by the rate of mitochondrial long-chain fatty acid beta-oxidation. We hypothesize that ALDP facilitates the interaction between peroxisomes and mitochondria, resulting, when ALDP is deficient in X-ALD, in increased VLCFA accumulation despite normal peroxisomal VLCFA beta-oxidation in ALD mouse tissues. In support of this hypothesis, mitochondrial structural abnormalities were observed in adrenal cortical cells of ALD mice.

PEX11 promotes peroxisome division independently of peroxisome metabolism.

The PEX11 peroxisomal membrane proteins are the only factors known to promote peroxisome division in multiple species. It has been proposed that PEX11 proteins have a direct role in peroxisomal fatty acid oxidation, and that they only affect peroxisome abundance indirectly. Here we show that PEX11 proteins are unique in their ability to promote peroxisome division, and that PEX11 overexpression promotes peroxisome division in the absence of peroxisomal metabolic activity. We also observed that mouse cells lacking PEX11β display reduced peroxisome abundance, even in the absence of peroxisomal metabolic substrates, and that PEX11β− / − mice are partially deficient in two distinct peroxisomal metabolic pathways, ether lipid synthesis and very long chain fatty acid oxidation. Based on these and other observations, we propose that PEX11 proteins act directly in peroxisome division, and that their loss has indirect effects on peroxisome metabolism.

Prenatal and postnatal development of peroxisomal lipid-metabolizing pathways in the mouse

The ontogeny of the following peroxisomal metabolic pathways was evaluated in mouse liver and brain: α-oxidation, β-oxidation and ether phospholipid synthesis. In mouse embryos lacking functional peroxisomes (PEX5-/- knock-out), a deficiency of plasmalogens and an accumulation of the very-long-chain fatty acid C26:0 was observed in comparison with control littermates, indicating that ether phospholipid synthesis and β-oxidation are already active at mid-gestation in the mouse. Northern analysis revealed that the enzymes required for the β-oxidation of straight-chain substrates are present in liver and brain during embryonic development but that those responsible for the degradation of branched-chain substrates are present only in liver from late gestation onwards. The expression pattern of transcripts encoding enzymes of the α-oxidation pathway suggested that α-oxidation is initiated in the liver around birth and is not active in brain throughout development. Remarkably, a strong induction of the mRNA levels of enzymes involved in α-oxidation and β-oxidation was observed around birth in the liver. In contrast, enzyme transcripts that were expressed in brain were present at rather constant levels throughout prenatal and postnatal development. These results suggest that the defective ether phospholipid synthesis and/or peroxisomal β-oxidation of straight-chain fatty acids might be involved in the pathogenesis of the prenatal organ defects in peroxisome-deficient mice and men.

Prenatal and postnatal development of peroxisomal lipid-metabolizing pathways in the mouse.

The ontogeny of the following peroxisomal metabolic pathways was evaluated in mouse liver and brain: alpha-oxidation, beta-oxidation and ether phospholipid synthesis. In mouse embryos lacking functional peroxisomes (PEX5(-/-) knock-out), a deficiency of plasmalogens and an accumulation of the very-long-chain fatty acid C(26:0) was observed in comparison with control littermates, indicating that ether phospholipid synthesis and beta-oxidation are already active at mid-gestation in the mouse. Northern analysis revealed that the enzymes required for the beta-oxidation of straight-chain substrates are present in liver and brain during embryonic development but that those responsible for the degradation of branched-chain substrates are present only in liver from late gestation onwards. The expression pattern of transcripts encoding enzymes of the alpha-oxidation pathway suggested that alpha-oxidation is initiated in the liver around birth and is not active in brain throughout development. Remarkably, a strong induction of the mRNA levels of enzymes involved in alpha-oxidation and beta-oxidation was observed around birth in the liver. In contrast, enzyme transcripts that were expressed in brain were present at rather constant levels throughout prenatal and postnatal development. These results suggest that the defective ether phospholipid synthesis and/or peroxisomal beta-oxidation of straight-chain fatty acids might be involved in the pathogenesis of the prenatal organ defects in peroxisome-deficient mice and men.

The methylotrophic yeast Hansenula polymorpha: its use in fundamental research and as a cell factory.

During the last decade methylotrophic yeasts havegained increasing interest, both for fundamental(van der Klei and Veenhuis, 1996) and appliedpurposes (van Dijk et al., 2000; Gellissen, 2000).The special value of these yeasts (mainly Pichiapastoris and Hansenula polymorpha) in fundamentalresearch is undoubtedly related to studies on theprinciples of peroxisome homeostasis (biogenesis vs.degradation). In particular, the morphologicalfeatures of peroxisome development and selectivedegradation are more strongly pronounced than inSaccharomyces cerevisiae, which makes it possibleto induce and follow the details in mutants moreeasily. In addition, peroxisome induction in baker’syeast is restricted to growth on oleate (and thusb-oxidation), whereas in the methylotrophic yeastsvarious peroxisomal metabolic pathways mayoccur, depending on the composition of thegrowth medium. These organisms also display thestrongest induction rates that may rise to a level atwhich up to 80% of the total cell volume is occupiedby peroxisomes.The extremely high expression rates necessary toachieve this drew the attention of biotechnologists,who studied the use of these organisms as host forthe production of valuable, heterologous proteins.Initially, P. pastoris was predominantly used; nowH. polymorpha is gaining more and more attention.The H. polymorpha system was shown to provide aremarkably versatile technology platform for het-erologous gene expression. The Hansenula toolboxnow includes a variety of host strains, new integra-tion sites and several novel strong promotersbeyond those derived from methanol metabolismgenes. In the first H. polymorpha worldwide net-work (HPWN) conference, the recent developmentsin the use of this organism were discussed in a livelyfashion. This report highlights several superb pre-sentations of important, interesting and novelresearch.

The peroxisomal membrane proteins of Candida boidinii: Gene isolation and expression

Candida boidinii is a methylotrophic yeast in which several growth substrates can cause vigorous peroxisomal proliferation. While such diverse substrates as methanol, oleic acid and D-alanine induce different peroxisomal metabolic pathways, membranes seem to contain common abundant peroxisomal membrane proteins (PMPs). These proteins have been termed PMP31, PMP32 and PMP47. The gene encoding PMP47 has been previously cloned and analysed. We now report the isolation of a second PMP47 gene (or allele) as well as PMP31 and PMP32. PMP47A and PMP47B share 95% sequence identity at the amino acid level. PMP31 and PMP32 each contain 256 amino acids and are highly similar (97% identity) in protein sequence. Both PMP31 and PMP32 are predicted to span the membrane once or twice. All abundant PMPs of C. boidinii are basic in charge; they all have predicted isoelectric points above 10. RNAs corresponding to the PMP47s and to PMPs31-32 are strongly induced by methanol, oleic acid and D-alanine. While the PMP47s probably encode substrate carriers, the functions of PMP31 and PMP32 from C. boidinii are still unknown.

Preliminary studies about novel strategies to reverse chemoresistance to adriamycin regarding glutathione metabolism, peroxisomal and extraperoxisomal hydroperoxide and valproic acid metabolic pathways

The present work was aimed at defining novel strategies to reverse chemoresistance to anticancer drugs, especially by interfering with cellular glutathione metabolism, peroxisomal and/or extraperoxisomal hydroperoxide metabolic pathways. Preliminary results are presented about molecules we demonstrated to be capable of interfering with hydrogen peroxide metabolism in cells. Prior to describing these molecules, a short overview of glutathione and free radical metabolic pathways is presented as well as a rapid presentation of the characteristics of chemo-sensitivity and -resistance towards the anticancer drug adriamycin, with special emphasis on hydrogen peroxide metabolism. The strategies currently developed to reverse chemoresistance are further presented in subsequent sections, our own strategy to achieve inhibition of hydrogen peroxide breakdown and stimulation of peroxisomal hydrogen peroxide production is illustrated on the basis of molecular modelling studies and biochemical investigations on extraperoxisomal and peroxisomal metabolic pathways. Preliminary studies on cultured cells have been initiated. The perspective for future studies is presented as well as other possible models of chemoresistance as target for the design of hydrogen peroxide metabolism-interfering pharmacomolecules.