Article

Peroxisome Dynamics in Cultured Mammalian Cells

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Abstract

Despite the identification and characterization of various proteins that are essential for peroxisome biogenesis, the origin and the turnover of peroxisomes are still unresolved critical issues. In this study, we used the HaloTag technology as a new approach to examine peroxisome dynamics in cultured mammalian cells. This technology is based on the formation of a covalent bond between the HaloTag protein--a mutated bacterial dehalogenase which is fused to the protein of interest--and a synthetic haloalkane ligand that contains a fluorophore or affinity tag. By using cell-permeable ligands of distinct fluorescence, it is possible to image distinct pools of newly synthesized proteins, generated from a single genetic HaloTag-containing construct, at different wavelengths. Here, we show that peroxisomes display an age-related heterogeneity with respect to their capacity to incorporate newly synthesized proteins. We also demonstrate that these organelles do not exchange their protein content. In addition, we present evidence that the matrix protein content of pre-existing peroxisomes is not evenly distributed over new organelles. Finally, we show that peroxisomes in cultured mammalian cells, under basal growth conditions, have a half-life of approximately 2 days and are mainly degraded by an autophagy-related mechanism. The implications of these findings are discussed.

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... Interestingly, both, de novo formation of peroxisomes from the ER via pre-peroxisomal vesicles or from pre-existing organelles via membrane growth and division, lead to the formation of membrane compartments which mature by subsequent import of matrix proteins (Hoepfner et al. 2005;Delille et al. 2010). The matrix protein content of pre-existing peroxisomes is therefore not evenly distributed over new organelles indicating that peroxisome formation by division is an asymmetric process (Huybrechts et al. 2009;Delille et al. 2010). Peroxisomes display an age-related heterogeneity with respect to their capacity to incorporate newly synthesized proteins (Huybrechts et al. 2009) and segregation during cell division (Kumar et al. 2018). ...
... The matrix protein content of pre-existing peroxisomes is therefore not evenly distributed over new organelles indicating that peroxisome formation by division is an asymmetric process (Huybrechts et al. 2009;Delille et al. 2010). Peroxisomes display an age-related heterogeneity with respect to their capacity to incorporate newly synthesized proteins (Huybrechts et al. 2009) and segregation during cell division (Kumar et al. 2018). This also applies to peroxisomal membrane proteins (PMPs), which reorganize in the peroxisomal membrane during membrane growth and division (Delille et al. 2010;Cepińska et al. 2011). ...
... Adapted from (Schrader and Fahimi 2008). See legend Fig. 1 and text for further details synthesized matrix proteins (Huybrechts et al. 2009;Delille et al. 2010). The membrane peroxin Pex11β is a key factor in the regulation of peroxisome number in mammals, which has now been associated with all steps of peroxisomal growth and division (Fig. 1, 2). ...
Article
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Peroxisomes are key metabolic organelles, which contribute to cellular lipid metabolism, e.g. the β-oxidation of fatty acids and the synthesis of myelin sheath lipids, as well as cellular redox balance. Peroxisomal dysfunction has been linked to severe metabolic disorders in man, but peroxisomes are now also recognized as protective organelles with a wider significance in human health and potential impact on a large number of globally important human diseases such as neurodegeneration, obesity, cancer, and age-related disorders. Therefore, the interest in peroxisomes and their physiological functions has significantly increased in recent years. In this review, we intend to highlight recent discoveries, advancements and trends in peroxisome research, and present an update as well as a continuation of two former review articles addressing the unsolved mysteries of this astonishing organelle. We summarize novel findings on the biological functions of peroxisomes, their biogenesis, formation, membrane dynamics and division, as well as on peroxisome–organelle contacts and cooperation. Furthermore, novel peroxisomal proteins and machineries at the peroxisomal membrane are discussed. Finally, we address recent findings on the role of peroxisomes in the brain, in neurological disorders, and in the development of cancer.
... Fluctuations in organelle abundance can be expected to have significant effects on their functional output, and-to adjust organelle quantity in response to changing environmental and developmental stimuli-cells are equipped with mechanisms that coordinate the formation of new organelles and their subsequent degradation once they are excessive or non-functional ( Figure 1). In mammals, new peroxisomes are formed de novo (e.g., from the ER [16,17] or by fusion of mitochondria-derived vesicles and ER-derived pre-peroxisomes (Figure 1b) [18]) or by asymmetric growth and division of pre-existing organelles [19,20]. Damaged or superfluous peroxisomes are mainly degraded by the autophagy-lysosome pathway, in a process known as pexophagy [21,22]. ...
... Redundant or dysfunctional mitochondria are selectively removed by autophagy through a process called mitophagy [24]. Unlike mitochondria, mature peroxisomes cannot fuse with one another [19,25]. In the following sections, we further discuss the mechanisms underlying co-regulation of peroxisomal and mitochondrial abundance and activity in mammalian cells. ...
Article
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Over the past decades, peroxisomes have emerged as key regulators in overall cellular lipid and reactive oxygen species metabolism. In mammals, these organelles have also been recognized as important hubs in redox-, lipid-, inflammatory-, and innate immune-signaling networks. To exert these activities, peroxisomes must interact both functionally and physically with other cell organelles. This review provides a comprehensive look of what is currently known about the interconnectivity between peroxisomes and mitochondria within mammalian cells. We first outline how peroxisomal and mitochondrial abundance are controlled by common sets of cis- and trans-acting factors. Next, we discuss how peroxisomes and mitochondria may communicate with each other at the molecular level. In addition, we reflect on how these organelles cooperate in various metabolic and signaling pathways. Finally, we address why peroxisomes and mitochondria have to maintain a healthy relationship and why defects in one organelle may cause dysfunction in the other. Gaining a better insight into these issues is pivotal to understanding how these organelles function in their environment, both in health and disease.
... Since the formation of the UGP/PEPCK heterodimer may occur mainly during UGP import into the organelle, the analysis of the UGP/PEPCK interactions using the PLA approach provides new insights regarding glycosomal import of proteins and multiplication of the organelles. In mammalian cells, peroxisomes multiply by growth and division using an asymmetric process generating new peroxisomes via formation of a membrane compartment and subsequent import of newly synthesised matrix proteins (Huybrechts et al., 2009;Delille et al., 2010;Costello and Schrader, 2018). In mammalian cells, overexpression of the membrane peroxin Pex11p resulted in the formation of pre-peroxisomal membrane structures composed of mature globular domains and tubular extensions, the latter being maturated by import of matrix proteins (Delille et al., 2010). ...
... cells, while the very few red dots observed within the control ∆pepck/ EXP UGP-MYC.i and EXP PEPCK-TY cells represent background signals.Staining with an immune serum against the glycosomal PPDK showed that the PLA signals are found in very close proximity to the PPDK-containing organelles without showing clear co-localisation with them(Figure 2.35C). This suggests the existence of different pools of glycosomes with different import capacities as observed for peroxisomes in mammalian cells(Huybrechts et al., 2009;Delille et al., 2010; Schrader, 2018, Gualdrón-Lopez et al., 2013b) (see the discussion section). ...
Thesis
Trypanosoma brucei, un protiste responsable de la Trypanosomose Humaine Africaine, également connue sous le nom de la maladie du sommeil, est transmis par la mouche tsé-tsé (Glossina sp.). La découverte d'organites de type peroxysome spécialisés dans la glycolyse, appelés glycosomes, a soulevé un certain nombre de questions sur le rôle de cet organite dans la biologie des trypanosomes. Plusieurs voies métaboliques présentes dans le cytosol d'autres eucaryotes, comme la glycolyse et la biosynthèse des sucres nucléotidiques, sont compartimentées dans les glycosomes. Les raisons et les avantages de la présence des enzymes glycolytiques dans l'organite ont été largement discutés, mais la fonctionnalité et le rôle des voies de biosynthèse des sucres nucléotidiques glycosomales ne sont pas connus. Notre étude s'est focalisée sur l'UDP-glucose pyrophosphorylase (UGP), une enzyme impliquée dans la synthèse de l'UDP-glucose (UDP-Glc). Sur la base de la double localisation glycosomale et cytosolique de l'UGP mise en évidence ici à l'aide de plusieurs techniques de localisation subcellulaire, nous avons abordé deux questions en utilisant comme modèle les formes procycliques de T. brucei présentes dans l'insecte vecteur. La première est liée au mécanisme d'import de l'UGP dans les glycosomes, car cette protéine ne possède aucun signal d'adressage aux peroxysomes de type PTS1 ou PTS2. Nous avons montré que l'UGP est importée dans les glycosomes par "piggybacking" en s'associant à la phosphoénolpyruvate décarboxylase (PEPCK) possédant un signal d’adressage PTS1. Les interactions entre l'UGP et la PEPCK ont été montrées in situ et l'identification les régions impliquées dans ces interactions ont été identifiées. Nos résultats suggèrent que le complexe UGP-PEPCK est formé de manière transitoire lors de son import dans les glycosomes nouvellement produits et compétents pour l'import des protéines. La seconde question concerne le rôle de l'UGP dans les glycosomes. Nous avons montré que l'UGP est essentielle à la croissance des trypanosomes et que les voies métaboliques glycosomales et cytosoliques dont l'UGP fait partie sont fonctionnelles. En effet, des mutants viables contenant l'UGP exclusivement dans les glycosomes ou dans le cytosol sont viables et produisent des quantités similaires d'UDP-Glc. La raison d'être de la production glycosomale d'UDP-Glc par l'UGP reste inconnue, mais n'est probablement pas liée aux réactions de glycosylation, étant donné qu'aucune glycosyltransférase n'a été détectée dans l'organite.Un autre aspect de ce travail concerne le rôle des intermédiaires du cycle de l'acide tricarboxylique (TCA) dans le métabolisme mitochondrial des formes procycliques. Dans le tractus digestif de son insecte vecteur, les trypanosomes dépendent de la proline pour alimenter leur métabolisme énergétique. Cependant, la disponibilité d'éventuelles autres sources de carbone pouvant être utilisées par le parasite est actuellement inconnue. Nous avons montré que les intermédiaires du cycle TCA, i.e. succinate, malate et a-cétoglutarate, stimulent la croissance des formes procycliques incubées dans un milieu contenant 2 mM de proline, concentration se situant dans la gamme des quantités mesurées dans l'intestin de la mouche. De plus, le développement de nouvelles approches ont permis d'étudier une branche peu explorée du cycle TCA convertissant le malate en a-cétoglutarate, précédemment décrite comme peu ou pas utilisée par le parasite, quellles que soient les quantités de glucose disponibles. L'activité de cette branche suggère qu'un cycle TCA complet peut être mis en œuvre dans les formes procycliques et probablement dans les autres formes parasitaires de l'insecte. Nos données élargissent le potentiel métabolique des trypanosomes et ouvrent la voie vers une meilleure compréhension du métabolisme de ce parasite dans divers organes de la mouche tsé-tsé, où il évolue.
... The estimated half-life of mammalian peroxisomes is 1.5~2 days, suggesting that peroxisome homeostasis is a dynamic process [7]. More importantly, the abundance and activity of peroxisomes can be rapidly adjusted to meet the metabolic needs induced by a changing environment. ...
... By using HaloTag technology to examine peroxisome dynamics, it was found that peroxisomes in cultured mammalian cells have a half-life of 1.5~2 days under basal growth conditions and treatment with 3-methyladenine, an inhibitor of the class III phosphoinositide 3-kinase (PI3K) complex, prevents the degradation of peroxisomes, suggesting that autophagy is involved in peroxisome turnover [7]. In addition to basal turnover, pexophagy can be triggered by various stimuli, as described below. ...
Article
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Peroxisomes play essential roles in diverse cellular metabolism functions, and their dynamic homeostasis is maintained through the coordination of peroxisome biogenesis and turnover. Pexophagy, selective autophagic degradation of peroxisomes, is a major mechanism for removing damaged and/or superfluous peroxisomes. Dysregulation of pexophagy impairs the physiological functions of peroxisomes and contributes to the progression of many human diseases. However, the mechanisms and functions of pexophagy in mammalian cells remain largely unknown compared to those in yeast. This review focuses on mammalian pexophagy and aims to advance the understanding of the roles of pexophagy in human health and diseases. Increasing evidence shows that ubiquitination can serve as a signal for pexophagy, and ubiquitin-binding receptors, substrates, and E3 ligases/deubiquitinases involved in pexophagy have been described. Alternatively, pexophagy can be achieved in a ubiquitin-independent manner. We discuss the mechanisms of these ubiquitin-dependent and ubiquitin-independent pexophagy pathways and summarize several inducible conditions currently used to study pexophagy. We highlight several roles of pexophagy in human health and how its dysregulation may contribute to diseases.
... Taking a value of 3 days for this, and estimating A as 4π r 2 + f πw L ≈ 10 5 nm 2 , we conclude that τ ∼ 1.5 × 10 5 s. This corresponds to a mean peroxisome lifetime of just under 2 days, which agrees well with previously measured values [3]. ...
... Wild-type value Section Lipid flow rate, α 75 nm 2 /s §1. 3 ...
... In contrast to other organelles, peroxisome are constantly recycled in healthy cell populations, and degraded to remove old or damaged peroxisomes [51,52]. Defining the predominant process of peroxisome production is a current topic of debate [53,54,55,56]. ...
... While yeast cells only have 5-20 peroxisomes per cell, humans and other mammals need larger number of peroxisomes (100-500). It is therefore possible that mammals evolved to use fission as a primary mechanism for peroxisome proliferation [51,14]. ...
Chapter
The regulation of organelle abundance sustains critical biological processes, such as metabolism and energy production. Biochemical models mathematically express these temporal changes in terms of reactions, and their rates. The rate parameters are critical components of the models, and must be experimentally inferred. However, the existing methods for rate inference are limited, and not directly applicable to organelle dynamics.
... To maintain a functional peroxisome population, excess or dysfunctional organelles need to be selectively removed. Half-life studies of peroxisomes indicate that peroxisomes have a half-life of around 1.5 -2 days (Huybrechts et al., 2009). Peroxisomes are mainly degraded by the autophagy-lysosome pathway, in a process called pexophagy (Nordgren et al., 2013). ...
... Similar to endoplasmatic reticulum associated degradation (ERAD), misfolded or dysfunctional peroxisomal proteins can be targeted to the cytosol for proteasomal degradation in a process called peroxisome-associated matrix protein degradation (PexAD) (Williams, 2014). Since the turnover rate of some PMPs (t½(PEX3) = 2 -6 h) is much faster than that of matrix proteins (t½ = 2 days) (Fransen, 2012) and furthermore dependent on the proteasomal degradation system (Huybrechts et al., 2009), different degradation pathways for matrix and membrane proteins can be assumed. The existence of a protease within the peroxisomal matrix might allow for intra-peroxisomal degradation of matrix proteins (Okumoto et al., 2011). ...
... Mammalian pexophagy plays a critical role in peroxisome quality control. Mammalian peroxisomes have half-lives of 1.5-2 days, highlighting the dynamicity of their basal turnover [141][142][143]. One common theme that has emerged in yeasts and mammals is the necessity of the matrix import machinery to maintain peroxisome quality, wherein defects in matrix import machinery act as a signal to designate peroxisomes for pexophagy. ...
Article
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The removal of damaged or superfluous organelles from the cytosol by selective autophagy is required to maintain organelle function, quality control and overall cellular homeostasis. Precisely how substrate selectivity is achieved, and how individual substrates are degraded during selective autophagy in response to both extracellular and intracellular cues is not well understood. The aim of this review is to highlight pexophagy, the autophagic degradation of peroxisomes, as a model for selective autophagy. Peroxisomes are dynamic organelles whose abundance is rapidly modulated in response to metabolic demands. Peroxisomes are routinely turned over by pexophagy for organelle quality control yet can also be degraded by pexophagy in response to external stimuli such as amino acid starvation or hypoxia. This review discusses the molecular machinery and regulatory mechanisms governing substrate selectivity during both quality-control pexophagy and pexophagy in response to external stimuli, in yeast and mammalian systems. We draw lessons from pexophagy to infer how the cell may coordinate the degradation of individual substrates by selective autophagy across different cellular cues.
... Thus, peroxisomal biogenesis and degradation must be tightly controlled to prevent dysregulated metabolism and oxidative stresses [1,3]. The estimated half-life of a peroxisome is approximately few days in CHO cells, implying that peroxisome biogenesis and degradation are very dynamic processes [4,5]. Various peroxisomal proteins, known as peroxins (PEXs), regulate peroxisome biogenesis [6,7]. ...
Article
Quality control of peroxisomes is essential for cellular homeostasis. However, the mechanism underlying pexophagy is largely unknown. In this study, we identified HSPA9 as a novel pexophagy regulator. Downregulation of HSPA9 increased macroautophagy/autophagy but decreased the number of peroxisomes in vitro and in vivo. The loss of peroxisomes by HSPA9 depletion was attenuated in SQSTM1-deficient cells. In HSPA9-deficient cells, the level of peroxisomal reactive oxygen species (ROS) increased, while inhibition of ROS blocked pexophagy in HeLa and SH-SY5Y cells. Importantly, reconstitution of HSPA9 mutants found in Parkinson disease failed to rescue the loss of peroxisomes, whereas reconstitution with wild type inhibited pexophagy in HSPA9-depleted cells. Knockdown of Hsc70-5 decreased peroxisomes in Drosophila, and the HSPA9 mutants failed to rescue the loss of peroxisomes in Hsc70-5-depleted flies. Taken together, our findings suggest that the loss of HSPA9 enhances peroxisomal degradation by pexophagy.
... However, we saw no evidence for rapid removal of peroxisome material. It is likely that peroxisome removal and replacement constitute a relatively slow process, as pulse-chase studies of fluorescently tagged or isotopelabeled peroxisomal proteins indicated a half-life of 1-3 days for liver peroxisomes (33,61). The observations of a steep decline in peroxisome numbers 1-2 h after light onset track a peak in numbers of phagosomes in this study, suggesting the possibility of false negatives or misclassification of structures, when phagosomes accumulate and traffic more basally. ...
Article
The retinal pigment epithelium (RPE) supports the outer retina through essential roles in the retinoid cycle, nutrient supply, ion exchange and waste removal. Each day the RPE removes the oldest ~10% of photoreceptor outer segment (OS) disk membranes through phagocytic uptake, which peaks following light onset. Impaired degradation of phagocytosed OS material by the RPE can lead to toxic accumulation of lipids, oxidative tissue damage, inflammation and cell death. OSs are rich in very long chain fatty acids which are preferentially catabolized in peroxisomes. Despite the importance of lipid degradation in RPE function, the regulation of peroxisome number and activity relative to diurnal OS ingestion is relatively unexplored. Using immunohistochemistry, immunoblotting and catalase activity assays, we investigated peroxisome abundance and activity at 6 am, 7 am (light onset), 8 am, and 3 pm, in WT mice and mice lacking microtubule-associated protein 1 light chain 3B ( Lc3b), that have impaired phagosome degradation. We found that catalase activity, but not the amount of catalase protein, is 50% higher in the morning compared with 3 pm, in RPE of WT but not Lc3b -/- mice. Surprisingly, we found that peroxisome abundance was stable during the day in RPE of WT mice, however numbers were elevated overall in Lc3b -/- mice, implicating LC3B in autophagic organelle turnover in RPE. Our data suggest that RPE peroxisome function is regulated in coordination with phagocytosis, possibly through direct enzyme regulation, and may serve to prepare RPE peroxisomes for daily surges in ingested lipid-rich OS.
... Cells were then treated either with a vehicle or with doxorubicin, and tracked longitudinally (Fig. 2C, D). We discovered that neurons treated with a vehicle had the half-life of the peroxisomal pool being~3 days, which is longer than the half-life of the peroxisomal pool in cell lines and the liver (1.5-2.2 days) (Fransen, 2014;Huybrechts et al., 2009;Leighton et al., 1969;Poole et al., 1969;Price et al., 1962). Neurons exposed to doxorubicin had the half-life of the photoswitched Dendra2-per fluorescence increased, indicating that pexophagy is impaired by doxorubicin. ...
... The peroxisomal lifespan in mammalian cells lasts about 2 to 3 days (Poole et al., 1969;Huybrechts et al., 2009;Moruno-Manchon et al., 2018b). Peroxisomes are then degraded by a selective form of macroautophagy: macropexophagy, which specifically targets peroxisomes (Yang and Klionsky, 2010;Bartoszewska et al., 2012;Cho et al., 2018). ...
Article
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Peroxisomes exist in most cells, where they participate in lipid metabolism, as well as scavenging the reactive oxygen species (ROS) that are produced as by-products of their metabolic functions. In certain tissues such as the liver and kidneys, peroxisomes have more specific roles, such as bile acid synthesis in the liver and steroidogenesis in the adrenal glands. In the brain, peroxisomes are critically involved in creating and maintaining the lipid content of cell membranes and the myelin sheath, highlighting their importance in the central nervous system (CNS). This review summarizes the peroxisomal lifecycle, then examines the literature that establishes a link between peroxisomal dysfunction, cellular aging, and age-related disorders that affect the CNS. This review also discusses the gap of knowledge in research on peroxisomes in the CNS.
... The formation of peroxisomes by growth and division from pre-existing organelles requires remodelling and expansion of the peroxisomal membrane through the formation of tubular membrane extensions which then constrict and divide into new peroxisomes [33] (Figure 2). Multiplication by growth and division is an asymmetric process, which generates new peroxisomes via formation of a membrane compartment and subsequent import of newly synthesised matrix proteins [34,35] (Figure 2). Several key proteins involved in peroxisome dynamics and multiplication have been identified (Figure 1), but their coordinated interplay, and how these processes are regulated is not well understood. ...
Article
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Peroxisome biogenesis is governed by molecular machineries, which are either unique to peroxisomes or are partially shared with mitochondria. As peroxisomes have important protective functions in the cell, modulation of their number is important for human health and disease. Significant progress has been made towards our understanding of the mechanisms of peroxisome formation, revealing a remarkable plasticity of the peroxisome biogenesis pathway. Here we discuss most recent findings with particular focus on peroxisome formation in mammalian cells.
... Both the HaloTag 1,4,16-20 and the SNAP tag 7,21,22 systems have been used with spectrally distinct dyes to visualize newly synthesized proteins, to differentiate between populations of newly formed versus aged proteins, to follow proteins at different subcellular locations and to measure protein half-lifetimes [23][24][25][26][27][28][29][30][31] . In such experiments, however, incomplete labeling -that is, binding sites that remain dye-free, as well as fluorescence from residual unbound ligands -represent significant confounds. ...
Article
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Background: HaloTag is a modified bacterial enzyme that binds rapidly and irreversibly to an array of synthetic ligands, including chemical dyes. When expressed in live cells in conjunction with a protein of interest, HaloTag can be used to study protein trafficking, synthesis, and degradation. For instance, sequential HaloTag labeling with spectrally separable dyes can be used to separate preexisting protein pools from proteins newly synthesized following experimental manipulations or the passage of time. Unfortunately, incomplete labeling by the first dye, or labeling by residual, trapped dye pools can confound interpretation. Methods : Labeling specificity of newly synthesized proteins could be improved by blocking residual binding sites. To that end, we synthesized a non-fluorescent, cell permeable blocker (1-chloro-6-(2-propoxyethoxy)hexane; CPXH), essentially the HaloTag ligand backbone without the reactive amine used to attach fluorescent groups. Results : High-content imaging was used to quantify the ability of CPXH to block HaloTag ligand binding in live HEK cells expressing a fusion protein of mTurquoise2 and HaloTag. Full saturation was observed at CPXH concentrations of 5-10 µM at 30 min. No overt effects on cell viability were observed at any concentration or treatment duration. The ability of CPXH to improve the reliability of newly synthesized protein detection was then demonstrated in live cortical neurons expressing the mTurquoise2-HaloTag fusion protein, in both single and dual labeling time lapse experiments. Practically no labeling was observed after blocking HaloTag binding sites with CPXH when protein synthesis was suppressed with cycloheximide, confirming the identification of newly synthesized protein copies as such, while providing estimates of protein synthesis suppression in these experiments. Conclusions: CPXH is a reliable (and inexpensive) non-fluorescent ligand for improving assessment of protein-of-interest metabolism in live cells using HaloTag technology.
... Peroxisomes exhibited typical Brownian and saltatory movements as described in olfactory neural stem cells and other mammalian cells (Figures 4A-C and Supplementary Videos S1, S2) (Huybrechts et al., 2009;Wali et al., 2016). The frequency distribution of peroxisome speeds from patient and control axons had similar positively skewed non-normal distributions (skewness > 1) (Figures 4E,F), indicating that most peroxisomes traveled at slow speeds with few peroxisomes traveling at fast speeds. ...
Article
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Hereditary spastic paraplegia (HSP) is a group of inherited disorders characterized by progressive spasticity and paralysis of the lower limbs. Autosomal dominant mutations in SPAST gene account for ∼40% of adult-onset patients. We have previously shown that SPAST patient cells have reduced organelle transport and are therefore more sensitive to oxidative stress. To test whether these effects are present in neuronal cells, we first generated 11 induced pluripotent stem (iPS) cell lines from fibroblasts of three healthy controls and three HSP patients with different SPAST mutations. These cells were differentiated into FOXG1-positive forebrain neurons and then evaluated for multiple aspects of axonal transport and fragmentation. Patient neurons exhibited reduced levels of SPAST encoded spastin, as well as a range of axonal deficits, including reduced levels of stabilized microtubules, lower peroxisome transport speed as a consequence of reduced microtubule-dependent transport, reduced number of peroxisomes, and higher density of axon swellings. Patient axons fragmented significantly more than controls following hydrogen peroxide exposure, suggesting for the first time that the SPAST patient axons are more sensitive than controls to the deleterious effects of oxidative stress. Treatment of patient neurons with tubulin-binding drugs epothilone D and noscapine rescued axon peroxisome transport and protected them against axon fragmentation induced by oxidative stress, showing that SPAST patient axons are vulnerable to oxidative stress-induced degeneration as a consequence of reduced axonal transport.
... Loss of peroxisomes can be followed enzymatically or by immunoblot, monitoring enzymes such as fatty acyl-CoA oxidase (note that this enzyme is sometimes abbreviated "AOX," but should not be confused with the enzyme alcohol oxidase that is frequently used in assays for yeast pexophagy) or catalase, and also by EM. 551,552 Finally, a HaloTag 1 -PTS1 marker that is targeted to peroxisomes has been used to fluorescently label the organelle. 553 Cautionary notes: There are many assays that can be used to monitor specific types of autophagy, but caution must be used in choosing an appropriate marker(s). It is best to monitor more than one protein, and to include an inner membrane or matrix component in the analysis. ...
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... The number, morphology, and size of peroxisomes are dynamically regulated in response to environmental and developmental cues (Heiland and Erdmann, 2005;Huybrechts et al., 2009). The biogenesis of peroxisomes is very complicated and regulated by more than 30 different PEX proteins (Kiel et al., 2006;Mayerhofer, 2016;Schlüter et al., 2006). ...
Article
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Autophagy is an intracellular degradation pathway for large protein aggregates and damaged organelles. Recent studies have indicated that autophagy targets cargoes through a selective degradation pathway called selective autophagy. Peroxisomes are dynamic organelles that are crucial for health and development. Pexophagy is selective autophagy that targets peroxisomes and is essential for the maintenance of homeostasis of peroxisomes, which is necessary in the prevention of various peroxisome-related disorders. However, the mechanisms by which pexophagy is regulated and the key players that induce and modulate pexophagy are largely unknown. In this review, we focus on our current understanding of how pexophagy is induced and regulated, and the selective adaptors involved in mediating pexophagy. Furthermore, we discuss current findings on the roles of pexophagy in physiological and pathological responses, which provide insight into the clinical relevance of pexophagy regulation. Understanding how pexophagy interacts with various biological functions will provide fundamental insights into the function of pexophagy and facilitate the development of novel therapeutics against peroxisomal dysfunction-related diseases.
... Once again, the fluorescence intensity of 3 was higher than that of 2 in both cases ( Figure 3I and Figure S12). Extra fusion proteins for peroxisomes 87 were examined as well, with equally good results (HsPex3p (1−230) -HaloTag p8, Figure 4 and Figure S15). ...
Article
Tools to image membrane tension in response to mechanical stimuli are badly needed in mechanobiology. We have recently introduced mechanosensitive flipper probes to report quantitatively global membrane tension changes in fluorescence lifetime imaging microscopy (FLIM) images of living cells. However, to address specific questions on physical forces in biology, the probes need to be localized precisely in the membrane of interest (MOI). Herein we present a general strategy to image the tension of the MOI by tagging our newly introduced HaloFlippers to self-labeling HaloTags fused to proteins in this membrane. The critical challenge in the construction of operational HaloFlippers is the tether linking the flipper and the HaloTag: It must be neither too taut nor too loose, be hydrophilic but lipophilic enough to passively diffuse across membranes to reach the HaloTags, and allow partitioning of flippers into the MOI after the reaction. HaloFlippers with the best tether show localized and selective fluorescence after reacting with HaloTags that are close enough to the MOI but remain nonemissive if the MOI cannot be reached. Their fluorescence lifetime in FLIM images varies depending on the nature of the MOI and responds to myriocin-mediated sphingomyelin depletion as well as to osmotic stress. The response to changes in such precisely localized membrane tension follows the validated principles, thus confirming intact mechanosensitivity. Examples covered include HaloTags in the Golgi apparatus, peroxisomes, endolysosomes, and the ER, all thus becoming accessible to the selective fluorescence imaging of membrane tension.
... In addition, mitochondria and peroxisomes have an equilibrium between oxidant and antioxidant agents to avoid an oxidative stress that could ultimately alter their function. A misregulation of this balance could induce an uncontrolled proliferation, eventually leading to cancer [57,63,64,[66][67][68]. ...
Article
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The immune synapse (IS) is a well-known intercellular communication platform, organized at the interphase between the antigen presenting cell (APC) and the T cell. After T cell receptor (TCR) stimulation, signaling from plasma membrane proteins and lipids is amplified by molecules and downstream pathways for full synapse formation and maintenance. This secondary signaling event relies on intracellular reorganization at the IS, involving the cytoskeleton and components of the secretory/recycling machinery, such as the Golgi apparatus and the endolysosomal system (ELS). T cell activation triggers a metabolic reprogramming that involves the synthesis of lipids, which act as signaling mediators, and an increase of mitochondrial activity. Then, this mitochondrial activity results in elevated reactive oxygen species (ROS) production that may lead to cytotoxicity. The regulation of ROS levels requires the concerted action of mitochondria and peroxisomes. In this review, we analyze this reprogramming and the signaling implications of endolysosomal, mitochondrial, peroxisomal, and lipidic systems in T cell activation.
... In each cell the biogenesis of peroxisomes is balanced by removal of old/excess peroxisomes. In mammalian cells, the population of peroxisomes is turned over approximately every 2 days (Huybrechts et al., 2009). In yeast or mammalian cells, defective or excess peroxisomes are removed by autophagy (pexophagy, reviewed in Germain and Kim, 2020). ...
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Research using the fruit fly Drosophila melanogaster has traditionally focused on understanding how mutations affecting gene regulation or function affect processes linked to animal development. Accordingly, flies have become an essential foundation of modern medical research through repeated contributions to our fundamental understanding of how their homologs of human genes function. Peroxisomes are organelles that metabolize lipids and reactive oxygen species like peroxides. However, despite clear linkage of mutations in human genes affecting peroxisomes to developmental defects, for many years fly models were conspicuously absent from the study of peroxisomes. Now, the few early studies linking the Rosy eye color phenotype to peroxisomes in flies have been joined by a growing body of research establishing novel roles for peroxisomes during the development or function of specific tissues or cell types. Similarly, unique properties of cultured fly Schneider 2 cells have advanced our understanding of how peroxisomes move on the cytoskeleton. Here, we profile how those past and more recent Drosophila studies started to link specific effects of peroxisome dysfunction to organ development and highlight the utility of flies as a model for human peroxisomal diseases. We also identify key differences in the function and proliferation of fly peroxisomes compared to yeast or mammals. Finally, we discuss the future of the fly model system for peroxisome research including new techniques that should support identification of additional tissue specific regulation of and roles for peroxisomes.
... The estimated half-life of mammalian peroxisomes is approximately 1.5-2 days, suggesting that biogenesis and degradation of peroxisomes are dynamic processes (Huybrechts et al. 2009;Poole et al. 1969). Little was known about the regulation of peroxisome degradation in mammalian cells, but recently several studies uncovered mechanisms for regulating mammalian pexophagy. ...
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Peroxisomes are ubiquitous and highly dynamic organelles that play a central role in the metabolism of lipids and reactive oxygen species. The importance of peroxisomal metabolism is illustrated by severe peroxisome biogenesis disorders in which functional peroxisomes are absent or disorders caused by single peroxisomal enzyme deficiencies. These multisystemic diseases manifest specific clinical and biochemical disturbances that originate from the affected peroxisomal pathways. An emerging role of the peroxisome has been identified in many types of diseases, including cancer, neurodegenerative disorders, aging, obesity, and diabetes. Peroxisome homeostasis is achieved via a tightly regulated interplay between peroxisome biogenesis and degradation via selective autophagy, which is commonly known as “pexophagy”. Dysregulation of either peroxisome biogenesis or pexophagy may be detrimental to the health of cells and contribute to the pathophysiology of these diseases. Autophagy is an evolutionary conserved catabolic process for non-selective degradation of macromolecules and organelles in response to various stressors. In selective autophagy, specific cargo-recognizing receptors connect the cargo to the core autophagic machinery, and additional posttranslational modifications such as ubiquitination and phosphorylation regulate this process. Several stress conditions have been shown to stimulate pexophagy and decrease peroxisome abundance. However, our understanding of the mechanisms that particularly regulate mammalian pexophagy has been limited. In recent years considerable progress has been made uncovering signaling pathways, autophagy receptors and adaptors as well as posttranslational modifications involved in pexophagy. In this review, which is published back-to-back with a peroxisome review by Islinger et al. [(Histochem Cell Biol 137:547–574, 2018). The peroxisome: an update on mysteries 2.0], we focus on recent novel findings on the underlying molecular mechanisms of pexophagy in yeast and mammalian cells and highlight concerns and gaps in our knowledge.
... Peroxisomes have a half-life of 1.3~2.2 days [249], so efficient removal and generation of peroxisomes is necessary. There are three main ways to eliminate peroxisomes: (1) peroxisomal matrix proteins can be degraded by Lon protease (LONP) [250]; (2) peroxisomes can undergo autolysis mediated by 15-lipoxygenase (15-LOX) [251,252]; and (3) peroxisomes can be selectively degraded by autophagy, a process known as pexophagy [253,254]. ...
Article
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Macroautophagy (hereafter called autophagy) is a highly conserved physiological process that degrades over-abundant or damaged organelles, large protein aggregates and invading pathogens via the lysosomal system (the vacuole in plants and yeast). Autophagy is generally induced by stress, such as oxygen-, energy- or amino acid-deprivation, irradiation, drugs, etc. In addition to non-selective bulk degradation, autophagy also occurs in a selective manner, recycling specific organelles, such as mitochondria, peroxisomes, ribosomes, endoplasmic reticulum (ER), lysosomes, nuclei, proteasomes and lipid droplets (LDs). This capability makes selective autophagy a major process in maintaining cellular homeostasis. The dysfunction of selective autophagy is implicated in neurodegenerative diseases (NDDs), tumorigenesis, metabolic disorders, heart failure, etc. Considering the importance of selective autophagy in cell biology, we systemically review the recent advances in our understanding of this process and its regulatory mechanisms. We emphasize the ‘cargo-ligand-receptor’ model in selective autophagy for specific organelles or cellular components in yeast and mammals, with a focus on mitophagy and ER-phagy, which are finely described as types of selective autophagy. Additionally, we highlight unanswered questions in the field, helping readers focus on the research blind spots that need to be broken.
... Studies in yeast show that deficiencies in exportomer components Pex1, Pex6, or Pex15 (the yeast ortholog of PEX26) increase pexophagy more than other peroxin deficiencies [161]. The constitutive turnover of peroxisomes is estimated to be around 30% per day [162]. ...
... Samples for immunofluorescence microscopy were fixed and processed as described before (23). Cells for live-cell imaging were seeded and imaged in FluoroDish cell culture dishes (FD35; World Precision Instruments). ...
Article
Aims: Peroxisomes are ubiquitous, single-membrane-bounded organelles that contain considerable amounts of enzymes involved in the production or breakdown of hydrogen peroxide (H2O2), a key signaling molecule in multiple biological processes and disease states. Despite this, the role of this organelle in cross-compartmental H2O2 signaling remains largely unclear, mainly because of the difficulty to modulate peroxisomal H2O2 production in a selective manner. This study aimed at establishing and validating a cellular model suitable to decipher the complex signaling processes associated with peroxisomal H2O2 release. Results: Here, we report the development of a human cell line that can be used to selectively generate H2O2 inside peroxisomes in a time- and dose-controlled manner. In addition, we provide evidence that peroxisome-derived H2O2 can oxidize redox-sensitive cysteine residues in multiple proteins within (e.g., peroxiredoxin-5 [PRDX5]) and outside (e.g., nuclear factor kappa B subunit 1 [NFKB1] and subunit RELA proto-oncogene [RELA], phosphatase and tensin homolog [PTEN], forkhead box O3 [FOXO3], and peroxin 5 [PEX5]) the peroxisomal compartment. Furthermore, we show that the extent of protein oxidation depends on the subcellular location of the target protein and is inversely correlated to catalase activity and cellular glutathione content. Finally, we demonstrate that excessive H2O2 production inside peroxisomes does not induce their selective degradation, at least not under the conditions examined. Innovation: This study describes for the first time a powerful model system that can be used to examine the role of peroxisome-derived H2O2 in redox-regulated (patho)physiological processes, a research area in need of further investigation and innovative approaches. Conclusion: Our results provide unambiguous evidence that peroxisomes can serve as regulatory hubs in thiol-based signaling networks. Antioxid. Redox Signal. 00, 000-000.
... The number of peroxisomes can range from 100 to more than 1000 depending on the type of tissue and metabolic state in human cells. 1 Failure to assemble functional peroxisomes causes severe multisystem diseases of the Zellweger Spectrum Disorder (ZSD). 2 PEX genes express proteins called peroxins, which include matrix proteins, cytosolic receptors, and peroxisome membrane proteins. Up to now, more than 70 different proteins have been found in association with mammalian peroxisomes. ...
Article
The peroxisome is responsible for a variety of vital pathways in primary metabolism, including the very long-chain fatty-acid oxidation and plasmalogen lipid biosynthesis. Autosomal recessive disorder of the Zellweger spectrum (ZSD) is a major subset of peroxisome biogenesis disorders (PBDs) that can be caused by mutations in any of the 14 PEX genes. Zellweger syndrome (ZS) is the foremost common and severe phenotype within the heterogeneous ZSD. However, missense mutations encode proteins with residual functions, which are associated with phenotypes that are milder than ZS. Mutations in the PEX1 gene are among the most prevalent. PEX1 and PEX6 proteins, belonging to the AAA family of ATPases, form a hexameric complex, which is associated with peroxisome membranes and essential for peroxisome biology. In this study, a two-month-old Iranian boy with hypotonia, poor feeding, and difficulty in breathing was diagnosed with Zellweger syndrome. The parents of the patient were second cousins and healthy and no similar cases were observed in the parents' family. The PEX1 gene was sequenced in the patient and his parents. The compound heterozygous mutations, p. Arg949Trp and p. Gly970Ala, were identified in the patient, while the parents were heterozygous for these alleles. Sequence analysis of the mutant PEX1 D2 domain revealed that mutation p. Arg949Trp precisely occurred in a conserved arginine residue (P4 Arg), which hinders the substrate processing of the complex. Several database records have reported mutation p. Arg949Trp(R949W) but its clinical significance is given as uncertain. We report here a novel mutation, p. Gly970Ala, which is not recorded before and may prevent proper interaction of PEX1 and PEX6 proteins. In summary, the clinical findings and peroxisome profile of the patient suggested that compound heterozygosity for these two missense mutations resulted in a nonfunctional PEX1/PEX6 complex causing the severe ZS phenotype.
... In mammalian cells, peroxisomes multiply by the de novo ER route and by growth and division. The latter case involves an asymmetric process generating new peroxisomes via formation of a membrane compartment and subsequent import of newly synthesized matrix proteins (55)(56)(57). Indeed, overexpression of the membrane peroxin Pex11pβ resulted in the formation in mammalian cells of preperoxisomal membrane structures composed of mature globular domains and tubular extensions, the latter being maturated by import of matrix proteins (56). Equivalent clusters of tubular glycosomal membranes were also observed by overexpressing Pex11 in T. brucei (58), and clusters of elongated glycosomes have more recently been observed in BSF trypanosomes by whole-cell reconstruction using threedimensional (3D) electron microscopy (59). ...
Article
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Glycosomes are peroxisome-related organelles of trypanosomatid parasites containing metabolic pathways, such as glycolysis and biosynthesis of sugar nucleotides, usually present in the cytosol of other eukaryotes. UDP-glucose pyrophosphorylase (UGP), the enzyme responsible for the synthesis of the sugar nucleotide UDP-glucose, is localized in the cytosol and glycosomes of the bloodstream and procyclic trypanosomes, despite the absence of any known peroxisome-targeting signal (PTS1 and PTS2). The questions that we address here are (i) is the unusual glycosomal biosynthetic pathway of sugar nucleotides functional and (ii) how is the PTS-free UGP imported into glycosomes? We showed that UGP is imported into glycosomes by piggybacking on the glycosomal PTS1-containing phosphoenolpyruvate carboxykinase (PEPCK) and identified the domains involved in the UGP/PEPCK interaction. Proximity ligation assays revealed that this interaction occurs in 3 to 10% of glycosomes, suggesting that these correspond to organelles competent for protein import. We also showed that UGP is essential for the growth of trypanosomes and that both the glycosomal and cytosolic metabolic pathways involving UGP are functional, since the lethality of the knockdown UGP mutant cell line ( RNAi UGP, where RNAi indicates RNA interference) was rescued by expressing a recoded UGP (rUGP) in the organelle ( RNAi UGP/ EXP rUGP-GPDH, where GPDH is glycerol-3-phosphate dehydrogenase). Our conclusion was supported by targeted metabolomic analyses (ion chromatography-high-resolution mass spectrometry [IC-HRMS]) showing that UDP-glucose is no longer detectable in the RNAi UGP mutant, while it is still produced in cells expressing UGP exclusively in the cytosol (PEPCK null mutant) or glycosomes ( RNAi UGP/ EXP rUGP-GPDH). Trypanosomatids are the only known organisms to have selected functional peroxisomal (glycosomal) sugar nucleotide biosynthetic pathways in addition to the canonical cytosolic ones. IMPORTANCE Unusual compartmentalization of metabolic pathways within organelles is one of the most enigmatic features of trypanosomatids. These unicellular eukaryotes are the only organisms that sequestered glycolysis inside peroxisomes (glycosomes), although the selective advantage of this compartmentalization is still not clear. Trypanosomatids are also unique for the glycosomal localization of enzymes of the sugar nucleotide biosynthetic pathways, which are also present in the cytosol. Here, we showed that the cytosolic and glycosomal pathways are functional. As in all other eukaryotes, the cytosolic pathways feed glycosylation reactions; however, the role of the duplicated glycosomal pathways is currently unknown. We also showed that one of these enzymes (UGP) is imported into glycosomes by piggybacking on another glycosomal enzyme (PEPCK); they are not functionally related. The UGP/PEPCK association is unique since all piggybacking examples reported to date involve functionally related interacting partners, which broadens the possible combinations of carrier-cargo proteins being imported as hetero-oligomers.
... Peroxisomes are small organelles that degrade lipids in the cytoplasm. Because the estimated half-life of these structures is approximately 2 days, both biogenesis and degradation of peroxisomes are probably dynamic processes [57]. Degradation of peroxisomes by autophagy called 'pexophagy', requires the participation of specific autophagy receptors. ...
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Autophagy is a process of self-degradation that plays an important role in removing damaged proteins, organelles or cellular fragments from the cell. Under stressful conditions such as hypoxia, nutrient deficiency or chemotherapy, this process can also become the strategy for cell survival. Autophagy can be nonselective or selective in removing specific organelles, ribosomes, and protein aggregates, although the complete mechanisms that regulate aspects of selective autophagy are not fully understood. This review summarizes the most recent research into understanding the different types and mechanisms of autophagy. The relationship between apoptosis and autophagy on the level of molecular regulation of the expression of selected proteins such as p53, Bcl-2/Beclin 1, p62, Atg proteins, and caspases was discussed. Intensive studies have revealed a whole range of novel compounds with an anticancer activity that inhibit or activate regulatory pathways involved in autophagy. We focused on the presentation of compounds strongly affecting the autophagy process, with particular emphasis on those that are undergoing clinical and preclinical cancer research. Moreover, the target points, adverse effects and therapeutic schemes of autophagy inhibitors and activators are presented.
... As another example, the peroxisomal membrane protein PEX14 delivers the organelle to autophagosomes via direct LC3 interaction [107,108]. Although pexophagy can be triggered by many diverse stimuli [109], under basal conditions it determines the renewal rate of peroxisomes with an approximate half-life of 2 days [110]. Impaired peroxisomal homeostasis is associated with Zellweger syndrome, a disease that, similar to DEE, includes craniofacial dysmorphism and infantile epileptic seizures as characteristic features [111,112]. ...
Article
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Seizure threshold 2 (SZT2) is a component of the KICSTOR complex which, under catabolic conditions, functions as a negative regulator in the amino acid-sensing branch of mTORC1. Mutations in this gene cause a severe neurodevelopmental and epileptic encephalopathy whose main symptoms include epilepsy, intellectual disability, and macrocephaly. As SZT2 remains one of the least characterized regulators of mTORC1, in this work we performed a systematic interactome analysis under catabolic and anabolic conditions. Besides numerous mTORC1 and AMPK signaling components, we identified clusters of proteins related to autophagy, ciliogenesis regulation, neurogenesis, and neurodegenerative processes. Moreover, analysis of SZT2 ablated cells revealed increased mTORC1 signaling activation that could be reversed by Rapamycin or Torin treatments. Strikingly, SZT2 KO cells also exhibited higher levels of autophagic components, independent of the physiological conditions tested. These results are consistent with our interactome data, in which we detected an enriched pool of selective autophagy receptors/regulators. Moreover, preliminary analyses indicated that SZT2 alters ciliogenesis. Overall, the data presented form the basis to comprehensively investigate the physiological functions of SZT2 that could explain major molecular events in the pathophysiology of developmental and epileptic encephalopathy in patients with SZT2 mutations.
... Peroxisomes are maintained by proliferation of pre-existing peroxisomes or by de novo synthesis from the endoplasmic reticulum (ER) [4,5]. Both pathways contribute to the cellular pool of peroxisomes and require the import of peroxisomal membrane proteins (PMPs) [6][7][8]. Peroxisomal division that occurs through growth and division of pre-existing peroxisomes requires, besides the membrane fission machinery whose components are shared between mitochondria and peroxisomes [9][10][11][12], the PMP, Pex11, which remodels peroxisomal membranes prior to division. Pex11, one of the most abundant peroxins in the peroxisomal membrane [13], is well conserved in other eukaryotes ( Figure 1). ...
Article
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Pex11, an abundant peroxisomal membrane protein (PMP), is required for division of peroxisomes and is robustly imported to peroxisomal membranes. We present a comprehensive analysis of how the Pichia pastoris Pex11 is recognized and chaperoned by Pex19, targeted to peroxisome membranes and inserted therein. We demonstrate that Pex11 contains one Pex19-binding site (Pex19-BS) that is required for Pex11 insertion into peroxisomal membranes by Pex19, but is non-essential for peroxisomal trafficking. We provide extensive mutational analyses regarding the recognition of Pex19-BS in Pex11 by Pex19. Pex11 also has a second, Pex19-independent membrane peroxisome-targeting signal (mPTS) that is preserved among Pex11-family proteins and anchors the human HsPex11γ to the outer leaflet of the peroxisomal membrane. Thus, unlike most PMPs, Pex11 can use two mechanisms of transport to peroxisomes, where only one of them depends on its direct interaction with Pex19, but the other does not. However, Pex19 is necessary for membrane insertion of Pex11. We show that Pex11 can self-interact, using both homo- and/or heterotypic interactions involving its N-terminal helical domains. We demonstrate that Pex19 acts as a chaperone by interacting with the Pex19-BS in Pex11, thereby protecting Pex11 from spontaneous oligomerization that would otherwise cause its aggregation and subsequent degradation.
... The mother peroxisome appears to retain its matrix enzymes and certain membrane proteins indicating that peroxisomal growth and division is an asymmetric process (Huybrechts et al., 2009). How this is mediated and how the diffusion of PMPs is restricted is currently unknown. ...
Article
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Organelles within the cell are highly dynamic entities, requiring dramatic morphological changes to support their function and maintenance. As a result, organelle membranes are also highly dynamic, adapting to a range of topologies as the organelle changes shape. In particular, peroxisomes—small, ubiquitous organelles involved in lipid metabolism and reactive oxygen species homeostasis—display a striking plasticity, for example, during the growth and division process by which they proliferate. During this process, the membrane of an existing peroxisome elongates to form a tubule, which then constricts and ultimately undergoes scission to generate new peroxisomes. Dysfunction of this plasticity leads to diseases with developmental and neurological phenotypes, highlighting the importance of peroxisome dynamics for healthy cell function. What controls the dynamics of peroxisomal membranes, and how this influences the dynamics of the peroxisomes themselves, is just beginning to be understood. In this review, we consider how the composition, biophysical properties, and protein-lipid interactions of peroxisomal membranes impacts on their dynamics, and in turn on the biogenesis and function of peroxisomes. In particular, we focus on the effect of the peroxin PEX11 on the peroxisome membrane, and its function as a major regulator of growth and division. Understanding the roles and regulation of peroxisomal membrane dynamics necessitates a multidisciplinary approach, encompassing knowledge across a range of model species and a number of fields including lipid biochemistry, biophysics and computational biology. Here, we present an integrated overview of our current understanding of the determinants of peroxisome membrane dynamics, and reflect on the outstanding questions still remaining to be solved.
... These include the tail-anchored adaptor proteins FIS1 and MFF, which are dually targeted to both peroxisomes and mitochondria, where they recruit the fission GTPase DRP1 (also known as DNML1) to the organelle membrane (Costello et al., 2017b(Costello et al., , 2018Gandre-Babbe and van der Bliek, 2008;Koch et al., 2005). In contrast to mitochondria, peroxisomes do not fuse (Bonekamp et al., 2012;Huybrechts et al., 2009); elongation is instead mediated by the peroxisome-specific membrane protein and biogenesis factor PEX11β (also known as PEX11B) (Schrader et al., 1998), with lipids for membrane expansion likely provided by the endoplasmic reticulum (ER) via peroxisome-ER contacts (Costello et al., 2017a;Hua et al., 2017;Islinger et al., 2020). PEX11β is a key factor in the regulation of peroxisome abundance in mammals (Schrader et al., 2016). ...
Article
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Peroxisome membrane dynamics and division are essential to adapt the peroxisomal compartment to cellular needs. The peroxisomal membrane protein PEX11β (also known as PEX11B) and the tail-anchored adaptor proteins FIS1 (mitochondrial fission protein 1) and MFF (mitochondrial fission factor), which recruit the fission GTPase DRP1 (dynamin-related protein 1, also known as DNML1) to both peroxisomes and mitochondria, are key factors of peroxisomal division. The current model suggests that MFF is essential for peroxisome division, whereas the role of FIS1 is unclear. Here, we reveal that PEX11β can promote peroxisome division in the absence of MFF in a DRP1- and FIS1-dependent manner. We also demonstrate that MFF permits peroxisome division independently of PEX11β and restores peroxisome morphology in PEX11β-deficient patient cells. Moreover, targeting of PEX11β to mitochondria induces mitochondrial division, indicating the potential for PEX11β to modulate mitochondrial dynamics. Our findings suggest the existence of an alternative, MFF-independent pathway in peroxisome division and report a function for FIS1 in the division of peroxisomes. This article has an associated First Person interview with the first authors of the paper.
... It is now widely accepted that peroxisomes are semi-autonomous organelles, which multiply by growth and division, but depend on the ER for supply of other essential components such as lipids. [1][2][3][4][5][6][7][8][9][10][11][12] There is also general agreement that the ER contributes to the de novo formation of peroxisomes, in particular in cells without pre-existing peroxisomes. Current models derived from studies in different yeast species suppose that the core peroxisomal import machinery is first targeted to the ER (or structures close to the ER), and enriched in pre-peroxisomal vesicles, which fully assemble the peroxisomal import machinery, thereby allowing continued growth and division. ...
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A recent report from the Laboratory of Heidi McBride (McGill University) presents a role for mitochondria in the de novo biogenesis of peroxisomes in mammalian cells. Peroxisomes are essential organelles responsible for a wide variety of biochemical functions, from the generation of bile to plasmalogen synthesis, reduction of peroxides, and the oxidation of very-long-chain fatty acids . Like mitochondria, peroxisomes proliferate primarily through growth and division of pre-existing peroxisomes. However, unlike mitochondria, peroxisomes do not fuse; further, and perhaps most importantly, they can also be born de novo, a process thought to occur through the generation of pre-peroxisomal vesicles that originate from the endoplasmic reticulum. De novo peroxisome biogenesis has been extensively studied in yeast, with a major focus on the role of the ER in this process; however, in the mammalian system this field is much less explored. By exploiting patient cells lacking mature peroxisomes, the McBride laboratory now assigns a role to ER and mitochondria in de novo mammalian peroxisome biogenesis by showing that the formation of immature pre-peroxisomes occurs through the fusion of Pex3-/Pex14-containing mitochondria-derived vesicles with Pex16- containing ER-derived vesicles.
Article
Accumulating evidence indicates that peroxisome functioning, catalase localization, and cellular oxidative balance are intimately interconnected. Nevertheless, it remains largely unclear why modest increases in the cellular redox state especially interfere with the subcellular localization of catalase, the most abundant peroxisomal antioxidant enzyme. This study aimed at gaining more insight into this phenomenon. Therefore, we first established a simple and powerful approach to study peroxisomal protein import and protein-protein interactions in living cells in response to changes in redox state. By employing this approach, we confirm and extend previous observations that Cys-11 of human PEX5, the shuttling import receptor for peroxisomal matrix proteins containing a C-terminal peroxisomal targeting signal (PTS1), functions as a redox switch that modulates the protein's activity in response to intracellular oxidative stress. In addition, we show that oxidative stress affects the import of catalase, a non-canonical PTS1-containing protein, more than the import of a reporter protein containing a canonical PTS1. Furthermore, we demonstrate that changes in the local redox state do not affect PEX5-substrate binding and that human PEX5 does not oligomerize in cellulo, not even when the cells are exposed to oxidative stress. Finally, we present evidence that catalase retained in the cytosol can protect against H2O2-mediated redox changes in a manner that peroxisomally targeted catalase does not. Together, these findings lend credit to the idea that inefficient catalase import, when coupled with the role of PEX5 as a redox-regulated import receptor, constitutes a cellular defense mechanism to combat oxidative insults of extra-peroxisomal origin.
Article
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USP30 is an integral protein of the outer mitochondrial membrane that counteracts PINK1 and Parkin-dependent mitophagy following acute mitochondrial depolarisation. Here, we use two distinct mitophagy reporter systems to reveal tonic suppression by USP30, of a PINK1-dependent component of basal mitophagy in cells lacking detectable Parkin. We propose that USP30 acts upstream of PINK1 through modulation of PINK1-substrate availability and thereby determines the potential for mitophagy initiation. We further show that a fraction of endogenous USP30 is independently targeted to peroxisomes where it regulates basal pexophagy in a PINK1- and Parkin-independent manner. Thus, we reveal a critical role of USP30 in the clearance of the two major sources of ROS in mammalian cells and in the regulation of both a PINK1-dependent and a PINK1-independent selective autophagy pathway.
Article
Peroxisomes are highly dynamic intracellular organelles involved in a variety of metabolic functions essential for the metabolism of long-chain fatty acids, d-amino acids, and many polyamines. A byproduct of peroxisomal metabolism is the generation, and subsequent detoxification, of reactive oxygen and nitrogen species, particularly hydrogen peroxide (H2O2). Because of its relatively low reactivity (as a mild oxidant), H2O2 has a comparatively long intracellular half-life and a high diffusion rate, all of which makes H2O2 an efficient signaling molecule. Peroxisomes also have intricate connections to mitochondria, and both organelles appear to play important roles in regulating redox signaling pathways. Peroxisomal proteins are also subject to oxidative modification and inactivation by the reactive oxygen and nitrogen species they generate, but the peroxisomal LonP2 protease can selectively remove such oxidatively damaged proteins, thus prolonging the useful lifespan of the organelle. Peroxisomal homeostasis must adapt to the metabolic state of the cell, by a combination of peroxisome proliferation, the removal of excess or badly damaged organelles by autophagy (pexophagy), as well as by processes of peroxisome inheritance and motility. More recently the tumor suppressors ataxia telangiectasia mutate (ATM) and tuberous sclerosis complex (TSC), which regulate mTORC1 signaling, have been found to regulate pexophagy in response to variable levels of certain reactive oxygen and nitrogen species. It is now clear that any significant loss of peroxisome homeostasis can have devastating physiological consequences. Peroxisome dysregulation has been implicated in several metabolic diseases, and increasing evidence highlights the important role of diminished peroxisomal functions in aging processes.
Chapter
The peroxisome is an organelle with diverse functions essential for eukaryotic cell function and human health. Respiration using hydrogen peroxide and β-oxidation of fatty acids are highly conserved functions. Plasmalogen, cholesterol and bile acid synthesis, catabolism of purines and polyamines, cell signaling, the glyoxylate cycle, and immune defense are other important functions. Peroxisomes collaborate metabolically with the ER, mitochondria, and chloroplasts. The peroxisome is evolutionarily ancient and functionally highly diverse. Its abundance and repertoire of enzymes are regulated in response to environmental demands. Peroxisomes, like mitochondria and chloroplasts, are formed by growth and division, with some contribution from the ER. Failures of peroxisome function or biogenesis cause grave childhood illnesses, and peroxisomes are implicated in the neurodegenerative diseases associated with aging.
Article
Peroxisomes have the intrinsic ability to produce and scavenge hydrogen peroxide (H2O2), a diffusible second messenger that controls diverse cellular processes by modulating protein activity through cysteine oxidation. Current evidence indicates that H2O2, a molecule whose physicochemical properties are similar to those of water, traverses cellular membranes through specific aquaporin channels, called peroxiporins. Until now, no peroxiporin-like proteins have been identified in the peroxisomal membrane, and it is widely assumed that small molecules such as H2O2 can freely permeate this membrane through PXMP2, a non-selective pore-forming protein with an upper molecular size limit of 300-600 Da. By employing the CRISPR-Cas9 technology in combination with a Flp-In T-REx 293 cell line that can be used to selectively generate H2O2 inside peroxisomes in a controlled manner, we provide evidence that PXMP2 is not essential for H2O2 permeation across the peroxisomal membrane, neither in control cells nor in cells lacking PEX11B, a peroxisomal membrane-shaping protein whose yeast homologue facilitates the permeation of molecules up to 400 Da. During the course of this study, we unexpectedly noted that inactivation of PEX11B leads to partial localization of both peroxisomal membrane and matrix proteins to mitochondria and a decrease in peroxisome density. These findings are discussed in terms of the formation of a functional peroxisomal matrix protein import machinery in the outer mitochondrial membrane.
Chapter
Peroxisomal disorders (PD) are genetic disorders caused by peroxisome dysfunction and are classified into two groups: genetic defects in peroxisome-localized proteins and genetic defects in peroxisomal biogenesis. The dawn of PD research came with the detailed analysis of the Zellweger syndrome, the prototype of PD. Even recently, new PD are still being identified by whole-exome sequencing analysis, which means that the concept of PD has been expanding. Furthermore, the role of peroxisome in cancer and age-related diseases has also been studied. In contrast, PD pathophysiology and treatment are not clarified yet completely and even in adrenoleukodystrophy, which is the most common PD, the prognosis of phenotype and disease in pre-symptomatic patients is a difficult task.
Chapter
Peroxisomal metabolism and its regulation play important roles in various cellular functions. The regulation of peroxisomal metabolism is controlled by modulation of peroxisome biogenesis as well as the degradation of intra-organellar components and the organelle itself. An accumulation of experimental findings demonstrate that the majority of organelle degradation is accomplished through autophagy, an important cellular process involving transport of cytoplasmic constituents into lysosomes for degradation. The first part of this chapter discusses several processes responsible for the degradation of peroxisomes in mammalian cells, including autophagy. Next, following a general description of the molecular machinery of autophagy, molecular details of selective autophagy of peroxisomes, termed pexophagy, are described based on studies conducted in yeast and mammalian cells. In the final section, expected medical applications associated with pexophagy are described along with potential future developments in this field.
Thesis
La déficience en Acyl-CoA oxydase 1 (ACOX1) est une leucodystrophie peroxysomale rare et sévère associée à un déficit dans la beta-oxydation des acides gras à très long chaine. A cause du rôle clé de ce déficit peroxysomal microglial dans la physiopathogenèse de la déficience en ACOX1, nous avons utilisé la lignée microgliale murine BV-2 comme modèle : (i) pour évaluer les propriétés antioxydantes et anti-inflammatoires des extraits de raquettes du cactus Opuntia ficus-indica ; (ii) pour caractériser une nouvelle lignée BV-2 déficiente en ACOX1 générée récemment dans notre laboratoire par édition génique grâce à la méthode CRISPR-Cas9. Dans la première partie des travaux, les cellules BV-2, activées par exposition à quatre sérotypes de lipopolysaccharides (LPS), montre un lien entre la structure du LPS et l’effet sur la -oxydation des acides gras et les enzymes antioxydantes dans le peroxysome : les LPS dérivant d’Escherichia coli diminuent l’activité ACOX1 alors que les LPS de Salmonella minnesota réduisent l’activité catalase. Remarquablement, les différents extraits de cactus stimulent l’activité catalase. Cet effet antioxydant est accompagné par un effet anti-inflammatoire attesté par la réduction de la production LPS-dépendante d’oxyde nitrique dans les BV-2. Nos résultats suggèrent que les extraits de cactus auraient une activité neuroprotectrice sur les cellules microgliales activées à travers l’induction d’activités anti-oxydantes peroxysomales et l’inhibition de la production de NO. Dans la deuxième partie des travaux, la caractérisation de la lignée BV-2 déficiente en ACOX1, portant des mutations alléliques, confirme l’absence de la protéine et de l’activité ACOX1. Bien que ces cellules aient une croissance plus faible, elles ne montrent pas de modifications morphologiques détectables. Par contre, l’activité catalase, due à l’enzyme peroxysomale dégradant l’H2O2, est augmentée. Les études par fractionnement subcellulaire et par ultracentrifugation en gradient Nycodenz révèlent une modification de la densité et de la taille de peroxysomes. De plus, ces cellules microgliales déficientes montrent une profonde modification de l’expression des gènes liés à l’inflammation (IL-1b, IL-4, TNF-alpha) et particulièrement l’expression de la protéine CCL2/MCP-1 impliquée dans la neuro-inflammation. Cette nouvelle lignée microgliale déficiente en ACOX1 révèlent les mêmes dérégulations biochimiques que celles décrites chez les patients déficients en ACOX1 et représente donc un modèle pour l’étude des conséquences du déficit de la -oxydation peroxysomale dans la microglie sur les fonctions peroxysomales, le stress oxydatif, l’inflammation et les fonctions cellulaires.
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Peroxisomes are subcellular organelles which play a central role in human physiology by catalyzing a range of unique metabolic functions. The importance of peroxisomes for human health is exemplified by the existence of a group of usually severe diseases caused by an impairment in one or more peroxisomal functions. Among others these include the Zellweger spectrum disorders, X-linked adrenoleukodystrophy and Refsum disease. In order to fulfill their role in metabolism, peroxisomes require the continued interaction with other subcellular organelles including lipid droplets, lysosomes, the endoplasmic reticulum, and mitochondria. In recent years it has become clear that the metabolic alliance between peroxisomes and other organelles requires the active participation of tethering proteins to bring the organelles physically closer together thereby achieving efficient transfer of metabolites. This review intends to describe the current state of knowledge about the metabolic role of peroxisomes in humans with particular emphasis on the metabolic partnership between peroxisomes and other organelles and the consequences of genetic defects in these processes. We will also describe the biogenesis of peroxisomes and the consequences of the multiple genetic defects therein. In addition, we will discuss the functional role of peroxisomes in different organs and tissues and will include relevant information derived from model systems notably peroxisomal mouse models. Finally, we will pay particular attention to a hitherto underrated role of peroxisomes in viral infections.
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Peroxisomes exist in nearly every cell, oxidizing fats, synthesizing lipids and maintaining redox balance. As the brain ages, multiple pathways are negatively affected, but it is currently unknown if peroxisomal proteins are affected by aging in the brain. While recent studies have investigated a PEX5 homolog in aging C. elegans models and found that it is reduced in aging, it is unclear if PEX5, a mammalian peroxisomal protein that plays a role in peroxisomal homeostasis and degradation, is affected in the aging brain. To answer this question, we first determined the amount of PEX5, in brain homogenates from young (3 months) and aged (26 through 32+ months of age) wild-type mice of both sexes. PEX5 protein was decreased in aged male brains, but this reduction was not significant in female brains. RNAScope and real-time qPCR analyses showed that Pex5 mRNA was also reduced in aged male brain cortices, but not in females. Immunohistochemistry assays of cortical neurons in young and aged male brains showed that the amount of neuronal PEX5 was reduced in aged male brains. Cortical neurons in aged female mice also had reduced PEX5 levels in comparison to younger female mice. In conclusion, total PEX5 levels and Pex5 gene expression both decrease with age in male brains, and neuronal PEX5 levels lower in an age-dependent manner in the cortices of animals of both sexes.
Chapter
Besides the major organellesOrganelles of the cell, such as nucleus, mitochondria, EREndoplasmatic Reticulum (ER)and Golgi apparatusGolgi apparatus, the lysosomesLysosomesand peroxisomesPeroxisomes may fulfill prominent functions in the response to mechanical property alterations of their surrounding environment and possible also in providing the overall cell mechanicsCell mechanics. However, their biological functions are still not less important and are presented here additionally. In specific detail, the structure and function of lysosomesLysosomesand peroxisomesPeroxisomes are presented. The effect of environmental mechanics on organellesOrganelles, such as lysosomes and peroxisomes, is discussed.
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Aims Pancreatic beta-cell lipo-dysfunction decreases insulin secretion and predisposes to the development of type 2 diabetes. Through targeted Pex11β knockdown and peroxisome depletion, our aim was to investigate the specific contribution of peroxisomes to palmitate mediated pancreatic beta-cell dysfunction. Methods MIN6 cells were transfected with probes targeted against Pex11β, a regulator of peroxisome abundance, or with scrambled control probes. Peroxisome abundance was measured by PMP-70 protein expression. 48hrs post transfection, cells were incubated with 250μM palmitate or BSA control for a further 48hrs before measurement of glucose stimulated insulin secretion and of reactive oxygen species. Results Pex11β knockdown decreased target gene expression by more than 80% compared with the scrambled control (P<0.001). This led to decreased PMP-70 expression (p<0.01) and a 22% decrease in peroxisome number (p<0.05). At 25mM glucose, palmitate treatment decreased insulin secretion by 64% in the scrambled control cells (2.54±0.25 vs 7.07±0.83 [mean±SEM] ng/hr/μg protein; Palmitate vs BSA P<0.001), but by just 37% in the Pex11β knockdown cells. Comparing responses in the presence of palmitate, insulin secretion at 25mM glucose was significantly greater in the Pex11β knockdown cells compared with the scrambled controls (4.04±0.46 vs 2.54±0.25 ng/hr/μg protein; p<0.05). Reactive oxygen species generation with palmitate was lower in the Pex11β knockdown cells compared with the scrambled controls (P<0.001). Conclusion Pex11β knockdown decreased peroxisome abundance, decreased palmitate mediated reactive oxygen species generation, and reversed the inhibitory effect of palmitate on insulin secretion. These findings reveal a distinct role of peroxisomes in palmitate mediated beta-cell dysfunction.
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Human D‐aspartate oxidase (hDASPO) is a FAD‐dependent enzyme responsible for the degradation of D‐aspartate (D‐Asp). In the mammalian central nervous system, D‐Asp behaves as a classical neurotransmitter, it is thought to be involved in neural development, brain morphology and behavior, and appears to be involved in several pathological states, such as schizophrenia and Alzheimer's disease. Apparently, the human DDO gene produces alternative transcripts encoding for three putative hDASPO isoforms, constituted by 341 (the "canonical” form), 369 and 282 amino acids. Despite the increasing interest in hDASPO and its physiological role, little is known about these different isoforms. Here, the additional N‐terminal peptide present in the hDASPO_369 isoform only, has been identified in hippocampus of Alzheimer’s disease female patients, while peptides corresponding to the remaining part of the protein were present in samples from male and female healthy controls and Alzheimer’s disease patients. The hDASPO_369 isoform was largely expressed in E. coli as insoluble protein, hampering with its biochemical characterization. Furthermore, we generated U87 human glioblastoma cell clones stably expressing hDASPO_341 and, for the first time, hDASPO_369 isoforms; the latter protein showed a lower expression compared to the canonical isoform. Both protein isoforms are active (showing similar kinetic properties), localize to the peroxisomes, are very stable (a half‐life of approximately 100 hours has been estimated) and are primarily degraded through the ubiquitin‐proteasome system. These studies shed light on the properties of hDASPO isoforms with the final aim to clarify the mechanisms controlling brain levels of the neuromodulator D‐Asp.
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The processes of peroxisome formation and proliferation are still a matter of debate. We have previously shown that peroxisomes share some components of their division machinery with mitochondria. hFis1, a tail-anchored membrane protein, regulates the membrane fission of both organelles by DLP1/Drp1 recruitment, but nothing is known about the mechanisms of the dual targeting of hFis1. Here we demonstrate for the first time that peroxisomal targeting of hFis1 depends on Pex19p, a peroxisomal membrane protein import factor. hFis1/Pex19p binding was demonstrated by expression and co-immunoprecipitation studies. Using mutated versions of hFis1 an essential binding region for Pex19p was located within the last 26 C-terminal amino acids of hFis1, which are required for proper targeting to both mitochondria and peroxisomes. The basic amino acids in the very C terminus are not essential for Pex19p binding and peroxisomal targeting, but are instead required for mitochondrial targeting. Silencing of Pex19p by small interference RNA reduced the targeting of hFis1 to peroxisomes, but not to mitochondria. In contrast, overexpression of Pex19p alone was not sufficient to shift the targeting of hFis1 to peroxisomes. Our findings indicate that targeting of hFis1 to peroxisomes and mitochondria are independent events and support a direct, Pex19p-dependent targeting of peroxisomal tail-anchored proteins.
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Human Pex19p binds a broad spectrum of peroxisomal membrane proteins (PMPs). It has been proposed that this peroxin may: (i) act as a cycling PMP receptor protein, (ii) facilitate the insertion of newly synthesized PMPs into the peroxisomal membrane, or (iii) function as a chaperone to associate and/or dissociate complexes comprising integral PMPs already in the peroxisomal membrane. We previously demonstrated that human Pex19p binds peroxisomal integral membrane proteins at regions distinct from their sorting sequences. Here we demonstrate that a mutant of Pex13p that fails to bind to Pex19p nevertheless targets to and integrates into the peroxisomal membrane. In addition, through in vitro biochemical analysis, we show that Pex19p competes with Pex5p and Pex13p for binding to Pex14p, supporting a role for this peroxin in regulating assembly/disassembly of membrane-associated protein complexes. To further examine the molecular mechanism underlying this competition, six evolutionarily conserved amino acids in the Pex5p/Pex13p/Pex19p binding domain of Pex14p were subjected to site-directed mutagenesis and the corresponding mutants functionally analyzed. Our results indicate that the physically overlapping binding sites of Pex14p for Pex5p, Pex13p, and Pex19p are functionally distinct, suggesting that competition occurs through induction of structural changes, rather than through direct competition. Importantly, we also found that amino acid substitutions resulting in a strongly reduced binding affinity for Pex13p affect the peroxisomal localization of Pex14p.
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Peroxisomes are degraded by autophagic machinery termed “pexophagy” in yeast; however, whether this is essential for peroxisome degradation in mammals remains unknown. Here we have shown that Atg7, an essential gene for autophagy, plays a pivotal role in the degradation of excess peroxisomes in mammals. Following induction of peroxisomes by a 2-week treatment with phthalate esters in control and Atg7-deficient livers, peroxisomal degradation was monitored within 1 week after discontinuation of phthalate esters. Although most of the excess peroxisomes in the control liver were selectively degraded within 1 week, this rapid removal was exclusively impaired in the mutant liver. Furthermore, morphological analysis revealed that surplus peroxisomes, but not mutant hepatocytes, were surrounded by autophagosomes in the control. Our results indicated that the autophagic machinery is essential for the selective clearance of excess peroxisomes in mammals. This is the first direct evidence for the contribution of autophagic machinery in peroxisomal degradation in mammals.
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Peroxisomes have long been viewed as semiautonomous, static, and homogenous organelles that exist outside the secretory and endocytic pathways of vesicular flow. However, growing evidence supports the view that peroxisomes actually constitute a dynamic endomembrane system that originates from the endoplasmic reticulum. This review highlights the various strategies used by evolutionarily diverse organisms for coordinating the flow of membrane-enclosed carriers through the peroxisomal endomembrane system and critically evaluates the dynamics and molecular mechanisms of this multistep process.
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Peroxisomes import virtually all of their membrane and matrix proteins post-translationally. It is presently unknown whether, in mammalian cells, their exists a pool of mature peroxisomes which have received their complement of proteins and are import-incompetent. Previous work has shown that fibroblasts are capable of importing microinjected peroxisomal proteins into peroxisomes. This report describes the import of a hybrid peroxisomal protein into virtually all peroxisomes of the microinjected cell. The peroxisomal import was uniform in both short and long incubations. Pretreatment of the cells with cycloheximide did not affect the import of the peroxisomal protein, nor was there any difference in the distribution of the imported protein. Sequential microinjection experiments demonstrated that peroxisomes that had imported luciferase were capable of importing another peroxisomal protein injected 24 hours later. These results suggest that, in fibroblasts, all peroxisomes have associated protein-import machinery; this evidence does not support the hypothesis that there exists a pool of import-incompetent peroxisomes.
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After preliminary experiments had established that the injection of Triton WR-1339 necessary for the separation of lysosomes and peroxisomes did not affect the turnover rate of catalase, the decay of ³H-leucine incorporated into peroxisomes was studied in whole particles and in protein subfractions. It was shown that peroxisomes are destroyed in a completely random way, probably as wholes since the apparent half-life was the same for all subfractions, about 3½ days. In agreement with the results of Price et al. (11), the half-life of catalase derived from the rate of recovery from aminotriazole inhibition was about 11½ days, as was the apparent half-life of the heme prosthetic groups measured with ¹⁴C-α-aminolevulinic acid. Guanidino-labeled arginine gave an apparent half-life of 2½ days with large statistical uncertainty. Either the leucine label was reutilized very extensively in our animals and the true half-life of peroxisomes is 1½ days, or the prosthetic groups of catalase turn over more rapidly than the protein part of the molecule.
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The ultrastructure of peroxisomes in partially differentiated cells of the mouse preputial gland was investigated using serial thin sections and three-dimensional reconstruction as well as the alkaline diaminobenzidine technique for visualization of the peroxidatic activity of catalase. An analysis of serial sections indicates that the different types of intensely stained peroxisomal profiles, classified according to their shape, represent random planes through highly complex peroxisomes. These organelles exceed 4 μm in length and exhibit a focal heterogeneity with respect to their size, shape and enzyme distribution. The graphical three-dimensional reconstruction demonstrates that the most intricate peroxisomes are characterized by tortuous, elongate, and branched tubular segments of varying diameter equipped with enlarged terminal hollow-spherical structures which engulf areas of cytoplasm. A close spatial relationship is established between adjacent peroxisomes and peroxisomes and mitochondria, the latter two of which synchronously develop into highly complex structures. A close association is also observed between peroxisomes and the endoplasmic reticulum, whereby membrane continuities between the two compartments cannot be demonstrated. These observations are inconsistent with traditional concepts concerning peroxisomal shape and size, the number per cell, as well as their biogenesis from the endoplasmic reticulum. The functional significance of individual highly complex peroxisomes and their assemblage forming an extensive netlike membraneous system throughout the cell is discussed with respect to intracellular energy transport and trans-membrane electron exchange.
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Peroxisomes are organelles that carry out diverse biochemical processes in eukaryotic cells, including the core pathways of beta-oxidation of lipid molecules and detoxification of reactive oxygen species. In multicellular organisms defects in peroxisome assembly result in multiple biochemical and developmental abnormalities. As peroxisomes do not contain genetic material, their protein content, and therefore function, is determined by the import of nuclearly encoded proteins from the cytosol and, presumably, removal of damaged or obsolete proteins. Import of matrix proteins can be broken down into four steps: targeting signal recognition by the cycling import receptors; receptor-cargo docking at the peroxisome membrane; translocation and cargo unloading; and receptor recycling. Import is mediated by a set of evolutionarily conserved proteins called peroxins that have been identified primarily via genetic screens, but knowledge of their biochemical activities remains largely unresolved. Recent studies have filled in some of the blanks regarding receptor recycling and the role of ubiquitination but outstanding questions remain concerning the nature of the translocon and its ability to accommodate folded, even oligomeric proteins, and the mechanism of cargo unloading and turnover of peroxisomal proteins. This review seeks to integrate recent findings from yeast, mammalian and plant systems to present an up to date account of how proteins enter the peroxisome matrix.
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Here we discuss the mechanisms for the degradation of excess peroxisomes in mammalian hepatocytes which include (a) autophagy, (b) the action of peroxisomal Lon protease and (c) the membrane disrupting effect of 15-lipoxygenase. A recent study using Atg7 conditional-knock-out mice revealed that 70-80% of excess peroxisomes are degraded by the autophagic process. The remaining 20-30% of excess peroxisomes is most probably degraded by the action of peroxisomal Lon protease. Finally, a selective disruption of the peroxisomal membrane has been shown to be mediated by 15-lipoxygenase activity which is followed by diffusion of matrix proteins into the cytoplasm and cytoplasmic proteolysis.
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Two distinct pathways have recently been proposed for the import of peroxisomal membrane proteins (PMPs): a Pex19p- and Pex3p-dependent class I pathway and a Pex19p- and Pex3p-independent class II pathway. We show here that Pex19p plays an essential role as the chaperone for full-length Pex3p in the cytosol. Pex19p forms a soluble complex with newly synthesized Pex3p in the cytosol and directly translocates it to peroxisomes. Knockdown of Pex19p inhibits peroxisomal targeting of newly synthesized full-length Pex3p and results in failure of the peroxisomal localization of Pex3p. Moreover, we demonstrate that Pex16p functions as the Pex3p-docking site and serves as the peroxisomal membrane receptor that is specific to the Pex3p-Pex19p complexes. Based on these novel findings, we suggest a model for the import of PMPs that provides new insights into the molecular mechanisms underlying the biogenesis of peroxisomes and its regulation involving Pex3p, Pex19p, and Pex16p.
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Mitochondria and peroxisomes are ubiquitous subcellular organelles, which fulfil an indispensable role in the cellular metabolism of higher eukaryotes. Moreover, they are highly dynamic and display large plasticity. There is growing evidence now that both organelles exhibit a closer interrelationship than previously appreciated. This connection includes metabolic cooperations and cross-talk, a novel putative mitochondria-to-peroxisome vesicular trafficking pathway, as well as an overlap in key components of their fission machinery. Thus, peroxisomal alterations in metabolism, biogenesis, dynamics and proliferation can potentially influence mitochondrial functions, and vice versa. In this review, we present the latest progress in the emerging field of peroxisomal and mitochondrial interrelationship with a particular emphasis on organelle dynamics and its implication in diseases.
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As a step toward understanding the homeostasis of peroxisomes in mammalian cells, we investigated a degradation system of peroxisomes in Chinese hamster ovary (CHO)-K1 cells in response to the nutrient-starvation. Peroxisomal proteins were degraded apparently in a preferential manner as compared to cytosolic proteins, when CHO-K1 cells were starved in Hank's solution and then re-cultured in a normal medium. We verified whether microtubule-associated protein I light chain 3 (LC3), an essential factor for autophagy, was involved in the degradation of peroxisomal proteins. In the LC3-knocked-down CHO-K1 cells, the specific degradation of peroxisomal proteins was no longer observed and proteins including peroxisomal and cytosolic proteins were rather non-selectively degraded under the starvation condition. The starvation-dependent non-selective protein degradation was inhibited with proteasome inhibitors, MG132 and Epoxomicin. The integral membrane peroxin, Pex14p interacted with membrane-bound LC3-II, the modified form of LC3, via microtubules under the starvation condition. Taken together, these results suggest that peroxisomal proteins are degraded by two degradation systems involving autophagy and proteasomes depending on various cell-culture conditions, and that Pex14p plays a pivotal role as a prerequisite factor for the degradation of peroxisomal proteins by autophagy with the aid of microtubules.
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Transformation of mitochondria in mammalian cells is now a technical challenge. In this report, we demonstrate that the standard drug resistant genes encoding neomycin and hygromycin phosphotransferases can potentially be used as selectable markers for mammalian mitochondrial transformation. We re-engineered the drug resistance genes to express proteins targeted to the mitochondrial matrix and confirmed the location of the proteins in the cells by fusing them with GFP and by Western blot and mitochondrial content mixing analyses. We found that the mitochondrially targeted-drug resistance proteins confer resistance to high levels of G418 and hygromycin without affecting the viability of cells.
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After preliminary experiments had established that the injection of Triton WR-1339 necessary for the separation of lysosomes and peroxisomes did not affect the turnover rate of catalase, the decay of (3)H-leucine incorporated into peroxisomes was studied in whole particles and in protein subfractions. It was shown that peroxisomes are destroyed in a completely random way, probably as wholes since the apparent half-life was the same for all subfractions, about 3(1/2) days. In agreement with the results of Price et al. (11), the half-life of catalase derived from the rate of recovery from aminotriazole inhibition was about 11(1/2) days, as was the apparent half-life of the heme prosthetic groups measured with (14)C-alpha-aminolevulinic acid. Guanidino-labeled arginine gave an apparent half-life of 2(1/2) days with large statistical uncertainty. Either the leucine label was reutilized very extensively in our animals and the true half-life of peroxisomes is 1(1/2) days, or the prosthetic groups of catalase turn over more rapidly than the protein part of the molecule.
Article
The ultrastructure of peroxisomes in partially differentiated cells of the mouse preputial gland was investigated using serial thin sections and three-dimensional reconstruction as well as the alkaline diaminobenzidine technique for visualization of the peroxidatic activity of catalase. An analysis of serial sections indicates that the different types of intensely stained peroxisomal profiles, classified according to their shape, represent random planes through highly complex peroxisomes. These organelles exceed 4 micron in length and exhibit a focal heterogeneity with respect to their size, shape and enzyme distribution. The graphical three-dimensional reconstruction demonstrates that the most intricate peroxisomes are characterized by tortuous, elongate, and branched tubular segments of varying diameter equipped with enlarged terminal hollow-spherical structures which engulf areas of cytoplasm. A close spatial relationship is established between adjacent peroxisomes and peroxisomes and mitochondria, the latter two of which synchronously develop into highly complex structures. A close association is also observed between peroxisomes and the endoplasmic reticulum, whereby membrane continuities between the two compartments cannot be demonstrated. These observations are inconsistent with traditional concepts concerning peroxisomal shape and size, the number per cell, as well as their biogenesis from the endoplasmic reticulum. The functional significance of individual highly complex peroxisomes and their assemblage forming an extensive net-like membraneous system throughout the cell is discussed with respect to intracellular energy transport and transmembrane electron exchange.
Article
Peroxisomes import virtually all of their membrane and matrix proteins post-translationally. It is presently unknown whether, in mammalian cells, their exists a pool of mature peroxisomes which have received their complement of proteins and are import-incompetent. Previous work has shown that fibroblasts are capable of importing microinjected peroxisomal proteins into peroxisomes. This report describes the import of a hybrid peroxisomal protein into virtually all peroxisomes of the microinjected cell. The peroxisomal import was uniform in both short and long incubations. Pretreatment of the cells with cycloheximide did not affect the import of the peroxisomal protein, nor was there any difference in the distribution of the imported protein. Sequential microinjection experiments demonstrated that peroxisomes that had imported luciferase were capable of importing another peroxisomal protein injected 24 hours later. These results suggest that, in fibroblasts, all peroxisomes have associated protein-import machinery; this evidence does not support the hypothesis that there exists a pool of import-incompetent peroxisomes.
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The cerebro-hepato-renal syndrome of Zellweger is a fatal inherited disease caused by deficient import of peroxisomal matrix proteins. The pathogenic mechanisms leading to extreme hypotonia, severe mental retardation and early death are unknown. We generated a Zellweger animal model through inactivation of the murine Pxr1 gene (formally known as Pex5) that encodes the import receptor for most peroxisomal matrix proteins. Pxr1-/- mice lacked morphologically identifiable peroxisomes and exhibited the typical biochemical abnormalities of Zellweger patients. They displayed intrauterine growth retardation, were severely hypotonic at birth and died within 72 hours. Analysis of the neocortex revealed impaired neuronal migration and maturation and extensive apoptotic death of neurons.
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The biogenesis of peroxisomes requires the interaction of several peroxins, encoded by PEX genes and is well conserved between yeast and humans. We have cloned the human cDNA of PEX3 based on its homology to different yeast PEX3 genes. The deduced peroxin HsPEX3 is a peroxisomal membrane protein with a calculated molecular mass of 42.1 kDa. We created N- and C-terminal tagged PEX3 to assay its topology at the peroxisomal membrane by immunofluorescence microscopy. Our results and the one predicted transmembrane spanning region are in line with the assumption that H sPEX3 is an integral peroxisomal membrane protein with the N-terminus inside the peroxisome and the C-terminus facing the cytoplasm. The farnesylated peroxisomal membrane protein PEX19 interacts with HsPEX3 in a mammalian two-hybrid assay in human fibroblasts. The physical interaction could be confirmed by coimmunoprecipitation of the two in vitro transcribed and translated proteins. To address the targeting of PEX3 to the peroxisomal membrane, the expression of different N- and C-terminal PEX3 truncations fused to green fluorescent protein (GFP) was investigated in human fibroblasts. The N-terminal 33 amino acids of PEX3 were necessary and sufficient to direct the reporter protein GFP to peroxisomes and seemed to be integrated into the peroxisomal membrane. The expression of a 1-16 PEX3-GFP fusion protein did not result in a peroxisomal localization, but interestingly, this and several other truncated PEX3 fusion proteins were also localized to tubular and/or vesicular structures representing mitochondria.
Article
Based on peroxin protein 5 (Pex5p) homology searches in the expressed sequence tag database and sequencing of large full-length cDNA inserts, three novel and related human cDNAs were identified. The brain-derived cDNAs coded for two related proteins that differ only slightly at their N-terminus, and exhibit 39.8% identity to human PEX5p. The shorter liver-derived cDNA coded for the C-terminal tetratricopeptide repeat-containing domain of the brain cDNA-encoded proteins. Since these three proteins specifically bind to various C-terminal peroxisome-targeting signals in a manner indistinguishable from Pex5p and effectively compete with Pex5p in an in vitro peroxisome-targeting signal 1 (PTS1)-binding assay, we refer to them as 'Pex5p-related proteins' (Pex5Rp). In contrast to Pex5p, however, human PEX5Rp did not bind to Pex14p or to the RING finger motif of Pex12p, and could not restore PTS1 protein import in Pex5(-/-) mouse fibroblasts. Immunofluorescence analysis of epitope-tagged PEX5Rp in Chinese hamster ovary cells suggested an exclusively cytosolic localization. Northern-blot analysis showed that the PEX5R gene, which is localized to chromosome 3q26.2--3q27, is expressed preferentially in brain. Mouse PEX5Rp was also delineated. In addition, experimental evidence established that the closest-related yeast homologue, YMR018wp, did not bind PTS1. Based on its subcellular localization and binding properties, Pex5Rp may function as a regulator in an early step of the PTS1 protein import process.
Article
Degradation and turnover of peroxisomes is reviewed. First, we describe the historical aspects of peroxisome degradation research and the two major concepts for breakdown of peroxisomes, i.e., autophagy and autolysis. Next, the comprehensive knowledge on autophagy of peroxisomes in mammalian and yeast cells is reviewed. It has been shown that proliferated peroxisomes are degraded by selective autophagy, and studies using yeast cells have been especially helpful in shedding light on the molecular mechanisms of this process. The degradation of extraperoxisomal urate oxidase crystalloid is noted. Overexpressed wild-type urate oxidase in cultured cells has been shown to be degraded through an unknown proteolytic pathway distinct from the lysosomal system including autophagy or the ubiquitin-proteasome system. Finally, peroxisome autolysis mediated by 15-lipoxygenase (15-LOX) is described. 15-LOX is integrated into the peroxisome membrane causing focal membrane disruptions. The content of the peroxisomes is then exposed to cytosol proteases and seems to be digested quickly. In conclusion, the number of peroxisomes appears to be regulated by two selective pathways, autophagy, including macro- and microautophagy, and 15-LOX-mediated autolysis.
Article
Peroxisomes belong to the ubiquitous organelle repertoire of eukaryotic cells. They contribute to cellular metabolism in various ways depending on species, but a consistent feature is the presence of enzymes to degrade fatty acids. Due to the pioneering work of DeDuve and coworkers, peroxisomes were in the limelight of cell biology in the sixties with a focus on their metabolic role. During the last decade, interest in peroxisomes has been growing again, this time with focus on their origin and maintenance. This has resulted in our understanding how peroxisomal proteins are targeted to the organelle and imported into the organellar matrix or recruited into the single membrane surrounding it. With respect to the formation of peroxisomes, the field is divided. The long-held view formulated in 1985 by Lazarow and Fujiki (Lazarow PB, Fujiki Y. Biogenesis of peroxisomes. Annu Rev Cell Biol 1985; 1: 489-530) is that we are dealing with autonomous organelles multiplying by growth and division. This view is being challenged by various observations that call attention to a more active contribution of the ER to peroxisome formation. Our contribution to this debate consists of recent observations using immuno-electronmicroscopy and electron tomography in mouse dendritic cells that show the peroxisomal membrane to be derived from the ER.