Peroxisome-Mediated Metabolism Is Required for Immune Response to Microbial Infection.

The innate immune response is critical for animal homeostasis and is conserved from invertebrates to vertebrates. This response depends on specialized cells that recognize, internalize, and destroy microbial invaders through phagocytosis. This is coupled to autonomous or non-autonomous cellular signaling via reactive oxygen species (ROS) and cytokine production. Lipids are known signaling factors in this process, as the acute phase response of macrophages is accompanied by systemic lipid changes that help resolve inflammation. We found that peroxisomes, membrane-enclosed organelles central to lipid metabolism and ROS turnover, were necessary for the engulfment of bacteria by Drosophila and mouse macrophages. Peroxisomes were also required for resolution of bacterial infection through canonical innate immune signaling. Reduced peroxisome function impaired the turnover of the oxidative burst necessary to fight infection. This impaired response to bacterial challenge affected cell and organism survival and revealed a previously unknown requirement for peroxisomes in phagocytosis and innate immunity.

Different contributions of the peroxisomal import protein Pex5 and Pex7 to development, stress response and virulence of insect fungal pathogen Beauveria bassiana.

AIMS: Peroxins Pex5 and Pex7 belong to the peroxisomal import machinery and recognize proteins containing peroxisomal targeting signal (PTS) type 1 and type 2, respectively. This study seeks to characterize these two peroxins in the entomopathogenic fungus Beauveria bassiana. METHODS AND RESULTS: The orthologs of Pex5 and Pex7 in B. bassiana (BbPex5 and BbPex7) were functionally analyzed via protein localization and gene disruption. BbPex5 and BbPex7 were associated with peroxisome and specifically required for PTS1 and PTS2 pathways, respectively, which were demonstrated to be involved in development, tolerance to oxidative stress and virulence. ΔBbPex5 mutant displayed additionally defectives that were undetected in ΔBbPex7 in vegetative growth and resistance to osmotic and cell wall-perturbing stresses. Notably, Woronin body major protein Hex1 with PTS1 linked this organelle to the development and virulence of B. bassiana, which indicates that Woronin body is associated with the roles of PTS1 pathway. CONCLUSION: Both PTS1 and PTS2 pathways are involved in broad physiological process, and the PTS1 pathway acts as a main peroxisomal import pathway. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows the functional divergence of different peroxins and improves our understanding of organellar physiology involved in biocontrol potential of the entomopathogenic fungi.

Fusarium verticillioides Pex7/20 mediates peroxisomal PTS2 pathway import, pathogenicity, and fumonisin B1 biosynthesis.

Fusarium verticillioides, a well-known fungal pathogen that causes severe disease in maize and contaminates the grains with fumonisin B1 (FB1) mycotoxin, affects the yield and quality of maize worldwide. The intrinsic roles of peroxisome targeting signal (PTS)-containing proteins in phytopathogens remain elusive. We therefore explored the regulatory role and other biological functions of the components of PTS2 receptor complex, FvPex7 and FvPex20, in F. verticillioides. We found that FvPex7 directly interacts with the carboxyl terminus of FvPex20 in F. verticillioides. PTS2-containing proteins are recognized and bound by the FvPex7 receptor or the FvPex7-Pex20 receptor complex in the cytoplasm, but the peroxisome localization of the PTS2-Pex7-Pex20 complex is only determined by Pex20 in F. verticillioides. However, we observed that some putative PTS2 proteins that interact with Pex7 are not transported into the peroxisomes, but a PTS1 protein that interacts with Pex5 was detected in the peroxisomes. Furthermore, ΔFvpex7pex20 as well as ΔFvpex7pex5 double mutants exhibited reduced pathogenicity and FB1 biosynthesis, along with defects in conidiation. The PTS2 receptor complex mutants (ΔFvpex7pex20) grew slowly on minimal media and showed reduced sensitivity to cell wall and cell membrane stress-inducing agents compared to the wild type. Taken together, we conclude that the PTS2 receptor complex mediates peroxisome matrix proteins import and contributes to pathogenicity and FB1 biosynthesis in F. verticillioides. KEY POINTS: • FvPex7 directly interacts with FvPex20 in F. verticillioides. • vThe PTS2 receptor complex is essential for the importation of PTS2-containing matrix protein into peroxisomes in F. verticillioides. • Fvpex7/pex20 is involved in pathogenicity and FB1 biosynthesis in F. verticillioides.

Pex7 selectively imports PTS2 target proteins to peroxisomes and is required for anthracnose disease development in Colletotrichum scovillei.

Pex7 is a shuttling receptor that imports matrix proteins with a type 2 peroxisomal targeting signal (PTS2) to peroxisomes. The Pex7-mediated PTS2 protein import contributes to crucial metabolic processes such as the fatty acid ß-oxidation and glucose metabolism in a number of fungi, but cellular roles of Pex7 between the import of PTS2 target proteins and metabolic processes have not been fully understood. In this study, we investigated the functional roles of CsPex7, a homolog of the yeast Pex7, by targeted gene deletion in the pepper anthracnose fungus Colletotrichum scovillei. CsPex7 was required for carbon source utilization, scavenging of reactive oxygen species, conidial production, and disease development in C. scovillei. The expression of fluorescently tagged PTS2 signal of hexokinases and 3-ketoacyl-CoA thiolases showed that peroxisomal localization of the hexokinase CsGlk1 PTS2 is dependent on CsPex7, but those of the 3-ketoacyl-CoA thiolases are independent on CsPex7. In addition, GFP-tagged CsPex7 proteins were intensely localized to the peroxisomes on glucose-containing media, indicating a role of CsPex7 in glucose utilization. Collectively, these findings indicate that CsPex7 selectively recognizes specific PTS2 signal for import of PTS2-containing proteins to peroxisomes, thereby mediating peroxisomal targeting efficiency of PTS2-containing proteins in C. scovillei. On pepper fruits, the ΔCspex7 mutant exhibited significantly reduced virulence, in which excessive accumulation of hydrogen peroxide was observed in the pepper cells. We think the reduced virulence results from the abnormality in hydrogen peroxide metabolism of the ΔCspex7 mutant. Our findings provide insight into the cellular roles of CsPex7 in PTS2 protein import system.

Twins with PEX7 related intellectual disability and cataract: Highlighting phenotypes of peroxisome biogenesis disorder 9B.

Peroxisome biogenesis disorders (PBDs) are a group of autosomal recessive disorders caused due to impaired peroxisome assembly affecting the formation of functional peroxisomes. PBDs are caused by a mutation in PEX gene family resulting in disease manifestation with extreme variability ranging from the onset of profound neurologic symptoms in newborns to progressive degenerative disease in adults. Disease causing variations in PEX7 is known to cause severe rhizomelic chondrodysplasia punctata type 1 and PBD 9B, an allelic disorder resulting in a milder phenotype, often indistinguishable from that of classic Refsum disease. This case report highlights the variability of PEX7 related phenotypes and suggests that other than RCDP1 and late onset phenotype similar to Refsum disease, some cases present with cataract and neurodevelopmetal abnormalities during childhood without chondrodysplasia or rhizomelia. This report also underlines the importance of considering PBD 9B in children presenting with neurodevelopmental abnormalities especially if they have congenital cataract.

The role of Peroxin 7 during Drosophila embryonic development.

Peroxisomes are organelles in eukaryotic cells responsible for processing several types of lipids and management of reactive oxygen species. A conserved family of peroxisome biogenesis (Peroxin, Pex) genes encode proteins essential to peroxisome biogenesis or function. In yeast and mammals, PEROXIN7 (PEX7) acts as a cytosolic receptor protein that targets enzymes containing a peroxisome targeting signal 2 (PTS2) motif for peroxisome matrix import. The PTS2 motif is not present in the Drosophila melanogaster homologs of these enzymes. However, the fly genome contains a Pex7 gene (CG6486) that is very similar to yeast and human PEX7. We find that Pex7 is expressed in tissue-specific patterns analogous to differentiating neuroblasts in D. melanogaster embryos. This is correlated with a requirement for Pex7 in this cell lineage as targeted somatic Pex7 knockout in embryonic neuroblasts reduced survival. We also found that Pex7 over-expression in the same cell lineages caused lethality during the larval stage. Targeted somatic over-expression of a Pex7 transgene in neuroblasts of Pex7 homozygous null mutants resulted in a semi-lethal phenotype similar to targeted Pex7 knockout. These findings suggest that D. melanogaster has tissue-specific requirements for Pex7 during embryo development.

Protein transport into peroxisomes: Knowns and unknowns.

Peroxisomal matrix proteins are synthesized on cytosolic ribosomes and rapidly transported into the organelle by a complex machinery. The data gathered in recent years suggest that this machinery operates through a syringe-like mechanism, in which the shuttling receptor PEX5 - the "plunger" - pushes a newly synthesized protein all the way through a peroxisomal transmembrane protein complex - the "barrel" - into the matrix of the organelle. Notably, insertion of cargo-loaded receptor into the "barrel" is an ATP-independent process, whereas extraction of the receptor back into the cytosol requires its monoubiquitination and the action of ATP-dependent mechanoenzymes. Here, we review the main data behind this model.

Identification of Peroxisomal Protein Complexes with PTS Receptors, Pex5 and Pex7, in Mammalian Cells.

Pex5 and Pex7 are cytosolic receptors for peroxisome targeting signal type-1 (PTS1) and type-2 (PTS2), respectively, and play a pivotal role in import of peroxisomal matrix proteins. Recent advance in mass spectrometry analysis has facilitated comprehensive analysis of protein-protein interaction network by a combination with immunoprecipitation or biochemical purification. In this chapter, we introduce several findings obtained by these methods applied to mammalian cells. Exploring Pex5-binding partners in mammalian cells revealed core components comprising the import machinery complex of matrix proteins and a number of PTS1-type cargo proteins. Biochemical purification of the Pex5-export stimulating factor from rat liver cytosol fraction identified Awp1, providing further insight into molecular mechanisms of the export step of mono-ubiquitinated Pex5. Identification of DDB1 (damage-specific DNA-binding protein 1), a component of CRL4 (Cullin4A-RING ubiquitin ligase) E3 complex, as a Pex7-interacting protein revealed that quality control of Pex7 by CRL4A is important for PTS2 protein import by preventing the accumulation of dysfunctional Pex7. Furthermore, analysis of binding partners of an intraperoxisomal processing enzyme, trypsin-domain containing 1 (Tysnd1), showed a protein network regulating peroxisomal fatty acid ß-oxidation.

Stress exposure results in increased peroxisomal levels of yeast Pnc1 and Gpd1, which are imported via a piggy-backing mechanism.

Saccharomyces cerevisiae glycerol phosphate dehydrogenase 1 (Gpd1) and nicotinamidase (Pnc1) are two stress-induced enzymes. Both enzymes are predominantly localised to peroxisomes at normal growth conditions, but were reported to localise to the cytosol and nucleus upon exposure of cells to stress. Import of both proteins into peroxisomes depends on the peroxisomal targeting signal 2 (PTS2) receptor Pex7. Gpd1 contains a PTS2, however, Pnc1 lacks this sequence. Here we show that Pnc1 physically interacts with Gpd1, which is required for piggy-back import of Pnc1 into peroxisomes. Quantitative fluorescence microscopy analyses revealed that the levels of both proteins increased in peroxisomes and in the cytosol upon exposure of cells to stress. However, upon exposure of cells to stress we also observed enhanced cytosolic levels of the control PTS2 protein thiolase, when produced under control of the GPD1 promoter. This suggests that these conditions cause a partial defect in PTS2 protein import, probably because the PTS2 import pathway is easily saturated.

Pexophagy and peroxisomal protein turnover in plants.

Peroxisomes are dynamic, vital organelles that sequester a variety of oxidative reactions and their toxic byproducts from the remainder of the cell. The oxidative nature of peroxisomal metabolism predisposes the organelle to self-inflicted damage, highlighting the need for a mechanism to dispose of damaged peroxisomes. In addition, the metabolic requirements of plant peroxisomes change during development, and obsolete peroxisomal proteins are degraded. Although pexophagy, the selective autophagy of peroxisomes, is an obvious mechanism for executing such degradation, pexophagy has only recently been described in plants. Several recent studies in the reference plant Arabidopsis thaliana implicate pexophagy in the turnover of peroxisomal proteins, both for quality control and during functional transitions of peroxisomal content. In this review, we describe our current understanding of the occurrence, roles, and mechanisms of pexophagy in plants.

Structural biology of the import pathways of peroxisomal matrix proteins.

The peroxisomal proteins (peroxins) that mediate the import of peroxisomal matrix proteins have been identified. Recently, the purification of a functional peroxisomal translocon has been reported. However, the molecular details of the import pathways and the mechanisms by which the cargo is translocated into the lumen of the organelle are still poorly understood. Structural studies have begun to provide insight into molecular mechanisms of peroxisomal import pathways for cargo proteins that harbor peroxisomal targeting signals, PTS1 and PTS2, at their C- and N-termini, respectively. So far structures have been reported for binary or tertiary protein-protein interfaces, and highlight the role of intrinsically disordered regions for these interactions. Here, we provide an overview of the currently available structural biology of peroxisomal import pathways. Current challenges and future perspectives of the structural biology of peroxisomal protein translocation are discussed.

The first minutes in the life of a peroxisomal matrix protein.

In the field of intracellular protein sorting, peroxisomes are most famous by their capacity to import oligomeric proteins. The data supporting this remarkable property are abundant and, understandably, have inspired a variety of hypothetical models on how newly synthesized (cytosolic) proteins reach the peroxisome matrix. However, there is also accumulating evidence suggesting that many peroxisomal oligomeric proteins actually arrive at the peroxisome still as monomers. In support of this idea, recent data suggest that PEX5, the shuttling receptor for peroxisomal matrix proteins, is also a chaperone/holdase, binding newly synthesized peroxisomal proteins in the cytosol and blocking their oligomerization. Here we review the data behind these two different perspectives and discuss their mechanistic implications on this protein sorting pathway.

Peroxisomal protein import pores.

Peroxisomal protein import is essentially different to the translocation of proteins into other organelles. The molecular mechanisms by which completely folded or even oligomerized proteins cross the peroxisomal membrane remain to be disclosed. The identification of a water-filled pore that is mainly constituted by Pex5 and Pex14 led to the assumption that proteins are translocated through a large, probably transient, protein-conducting channel. Here, we will review the work that led to the identification of this translocation pore. In addition, we will discuss the main biophysical features of the pore and compare it with other protein­translocation channels.

Regulation of peroxisome dynamics by phosphorylation.

Peroxisomes are highly dynamic organelles that can rapidly change in size, abundance, and protein content in response to alterations in nutritional and other environmental conditions. These dynamic changes in peroxisome features, referred to as peroxisome dynamics, rely on the coordinated action of several processes of peroxisome biogenesis. Revealing the regulatory mechanisms of peroxisome dynamics is an emerging theme in cell biology. These mechanisms are inevitably linked to and synchronized with the biogenesis and degradation of peroxisomes. To date, the key players and basic principles of virtually all steps in the peroxisomal life cycle are known, but regulatory mechanisms remained largely elusive. A number of recent studies put the spotlight on reversible protein phosphorylation for the control of peroxisome dynamics and highlighted peroxisomes as hubs for cellular signal integration and regulation. Here, we will present and discuss the results of several studies performed using yeast and mammalian cells that convey a sense of the impact protein phosphorylation may have on the modulation of peroxisome dynamics by regulating peroxisomal matrix and membrane protein import, proliferation, inheritance, and degradation. We further put forward the idea to make use of current data on phosphorylation sites of peroxisomal and peroxisome-associated proteins reported in advanced large-scale phosphoproteomic studies.

Characterization, prediction and evolution of plant peroxisomal targeting signals type 1 (PTS1s).

Our knowledge of the proteome of plant peroxisomes and their functional plasticity is far from being complete, primarily due to major technical challenges in experimental proteome research of the fragile cell organelle. Several unexpected novel plant peroxisome functions, for instance in biotin and phylloquinone biosynthesis, have been uncovered recently. Nevertheless, very few regulatory and membrane proteins of plant peroxisomes have been identified and functionally described up to now. To define the matrix proteome of plant peroxisomes, computational methods have emerged as important powerful tools. Novel prediction approaches of high sensitivity and specificity have been developed for peroxisome targeting signals type 1 (PTS1) and have been validated by in vivo subcellular targeting analyses and thermodynamic binding studies with the cytosolic receptor, PEX5. Accordingly, the algorithms allow the correct prediction of many novel peroxisome-targeted proteins from plant genome sequences and the discovery of additional organelle functions. In this review, we provide an overview of methodologies, capabilities and accuracies of available prediction algorithms for PTS1 carrying proteins. We also summarize and discuss recent quantitative, structural and mechanistic information of the interaction of PEX5 with PTS1 carrying proteins in relation to in vivo import efficiency. With this knowledge, we develop a model of how proteins likely evolved peroxisomal targeting signals in the past and still nowadays, in which order the two import pathways might have evolved in the ancient eukaryotic cell, and how the secondary loss of the PTS2 pathway probably happened in specific organismal groups.

Peroxisome biogenesis, protein targeting mechanisms and PEX gene functions in plants.

Peroxisomes play diverse and important roles in plants. The functions of peroxisomes are dependent upon their steady state protein composition which in turn reflects the balance of formation and turnover of the organelle. Protein import and turnover of constituent peroxisomal proteins are controlled by the state of cell growth and environment. The evolutionary origin of the peroxisome and the role of the endoplasmic reticulum in peroxisome biogenesis are discussed, as informed by studies of the trafficking of peroxisome membrane proteins. The process of matrix protein import in plants and its similarities and differences with peroxisomes in other organisms is presented and discussed in the context of peroxin distribution across the green plants.

Peroxisomal abnormalities in the immortalized human hepatocyte (IHH) cell line.

The immortalized human hepatocyte (IHH) cell line is increasingly used for studies related to liver metabolism, including hepatic glucose, lipid, lipoprotein and triglyceride metabolism, and the effect of therapeutic interventions. To determine whether the IHH cell line is a good model to investigate hepatic peroxisomal metabolism, we measured several peroxisomal parameters in IHH cells and, for comparison, HepG2 cells and primary skin fibroblasts. This revealed a marked plasmalogen deficiency and a deficient fatty acid α-oxidation in the IHH cells, due to a defect of PEX7, a cytosolic receptor protein required for peroxisomal import of a subset of peroxisomal proteins. These abnormalities have consequences for the lipid homeostasis of these cells and thus should be taken into account for the interpretation of data previously generated by using this cell line and when considering using this cell line for future research.

Alternative splicing affects the targeting sequence of peroxisome proteins in Arabidopsis.

KEY MESSAGE: A systematic analysis of the Arabidopsis genome in combination with localization experiments indicates that alternative splicing affects the peroxisomal targeting sequence of at least 71 genes in Arabidopsis. Peroxisomes are ubiquitous eukaryotic cellular organelles that play a key role in diverse metabolic functions. All peroxisome proteins are encoded by nuclear genes and target to peroxisomes mainly through two types of targeting signals: peroxisomal targeting signal type 1 (PTS1) and PTS2. Alternative splicing (AS) is a process occurring in all eukaryotes by which a single pre-mRNA can generate multiple mRNA variants, often encoding proteins with functional differences. However, the effects of AS on the PTS1 or PTS2 and the targeting of the protein were rarely studied, especially in plants. Here, we systematically analyzed the genome of Arabidopsis, and found that the C-terminal targeting sequence PTS1 of 66 genes and the N-terminal targeting sequence PTS2 of 5 genes are affected by AS. Experimental determination of the targeting of selected protein isoforms further demonstrated that AS at both the 5' and 3' region of a gene can affect the inclusion of PTS2 and PTS1, respectively. This work underscores the importance of AS on the global regulation of peroxisome protein targeting.

Neonatal Rhizomelic Chondrodysplasia Punctata Type 1: Weaving Evidence Into Clinical Practice.

Rhizomelic chondrodysplasia punctata (RCDP) is a rare genetic peroxisome biogenesis disorder with a reported incidence of 1 in 100 000 live births. The 3 genetic subtypes of RCDP are acquired by an autosomal recessive inheritance pattern. RCDP type 1 accounts for greater than 90% of all aggregate cases. Differentiating between the 3 subtypes of RCDP, as well as disorders characterized by similar punctate cartilaginous changes, is essential to guide an appropriate postnatal plan of care. Management strategies are focused toward associated clinical manifestations and require an interdisciplinary approach including ophthalmology, cardiovascular, endocrine, physical and occupational therapy, and neurology. Purposeful and frequent collaboration among all members of the neonatal/pediatric interdisciplinary team is necessary to optimize outcomes for the neonate and the family unit. The purpose of this article is to anticipate the needs of both patients with known and prenatal diagnosis of RCDP type 1 and patients with suspected clinical diagnosis of RCDP type 1 in the immediate neonatal period and to guide the appropriate plan of care. This article presents a case report of type I RCDP, as well as describes genetic influences, symptoms, diagnosis, management, and prognosis.

[Peroxisomal disorder, rhizomelyc chondrodysplasia punctata type 1: case report]. / Enfermedad peroxisomal, condrodisplasia rizomelica punctata tipo 1: reporte de caso.

INTRODUCTION: Peroxisomal diseases are a group of monogenic disorders that include defects in peroxisome biogenesis or enzyme dificiencies. Rhizomelic chondrodysplasia punctata type 1 (RCDP1) belongs to the first group, caused by autosomal recessive mutations on PEX7 gene, encoding for PTS2 receptor. The aims of this report are to describe a genetic disease of low prevalence, explaining its main characteristics and the importance of the diagnostic approach and genetic counseling. CASE REPORT: 13-month-old male infant with no medical history, family or consanguinity, demonstrate at birth upper limbs shortening. Surgery intervention at seven months old for bilateral cataract. Growth retardation, psychomotor retardation, minor craniofacial anomalies, rhyzomelic shortened upper limbs and lower limbs lesser degree. Punctata calcifications in patella cartilage. Also fatty acid phytanic and pristanic increased levels. Patient dead at age of 3 years. DISCUSSION: RCDP1 is a rare disease, with a prevalence of 1/100,000. Different mutations of PEX7 gene have been described, with variations in phenotype. The treatment is basically symptomatic and depends on the severity of clinical manifestations. The rhizomelic type has poor prognosis, most patients do not survive before the first decade of live. Genetic counseling is essential because it is consider a 25% risk of recurrence.