PeroxisomeDB 2.0: an integrative view of the global peroxisomal metabolome

Peroxisomes are essential organelles that play a key role in redox signalling and lipid homeostasis. They contain a highly diverse enzymatic network among different species, mirroring the varied metabolic needs of the organisms. The previous PeroxisomeDB version organized the peroxisomal proteome of humans and Saccharomyces cerevisiae based on genetic and functional information into metabolic categories with a special focus on peroxisomal disease. The new release (http://www.peroxisomeDB.org) adds peroxisomal proteins from 35 newly sequenced eukaryotic genomes including fungi, yeasts, plants and lower eukaryotes. We searched these genomes for a core ensemble of 139 peroxisomal protein families and identified 2706 putative peroxisomal protein homologs. Approximately 37% of the identified homologs contained putative peroxisome targeting signals (PTS). To help develop understanding of the evolutionary relationships among peroxisomal proteins, the new database includes phylogenetic trees for 2386 of the peroxisomal proteins. Additional new features are provided, such as a tool to capture kinetic information from Brenda, CheBI and Sabio-RK databases and more than 1400 selected bibliographic references. PeroxisomeDB 2.0 is a freely available, highly interactive functional genomics platform that offers an extensive view on the peroxisomal metabolome across lineages, thus facilitating comparative genomics and systems analysis of the organelle

The PEX7-Mediated Peroxisomal Import System Is Required for Fungal Development and Pathogenicity in Magnaporthe oryzae

In eukaryotes, microbodies called peroxisomes play important roles in cellular activities during the life cycle. Previous studies indicate that peroxisomal functions are important for plant infection in many phytopathogenic fungi, but detailed relationships between fungal pathogenicity and peroxisomal function still remain unclear. Here we report the importance of peroxisomal protein import through PTS2 (Peroxisomal Targeting Signal 2) in fungal development and pathogenicity of Magnaporthe oryzae. Using an Agrobacterium tumefaciens-mediated transformation library, a pathogenicity-defective mutant was isolated from M. oryzae and identified as a T-DNA insert in the PTS2 receptor gene, MoPEX7. Gene disruption of MoPEX7 abolished peroxisomal localization of a thiolase (MoTHL1) containing PTS2, supporting its role in the peroxisomal protein import machinery. ΔMopex7 showed significantly reduced mycelial growth on media containing short-chain fatty acids as a sole carbon source. ΔMopex7 produced fewer conidiophores and conidia, but conidial germination was normal. Conidia of ΔMopex7 were able to develop appressoria, but failed to cause disease in plant cells, except after wound inoculation. Appressoria formed by ΔMopex7 showed a defect in turgor generation due to a delay in lipid degradation and increased cell wall porosity during maturation. Taken together, our results suggest that the MoPEX7-mediated peroxisomal matrix protein import system is required for fungal development and pathogenicity M. oryzae

PhylomeDB v3.0: an expanding repository of genome-wide collections of trees, alignments and phylogeny-based orthology and paralogy predictions

The growing availability of complete genomic sequences from diverse species has brought about the need to scale up phylogenomic analyses, including the reconstruction of large collections of phylogenetic trees. Here, we present the third version of PhylomeDB (http://phylomeDB.org), a public database for genome-wide collections of gene phylogenies (phylomes). Currently, PhylomeDB is the largest phylogenetic repository and hosts 17 phylomes, comprising 416 093 trees and 165 840 alignments. It is also a major source for phylogeny-based orthology and paralogy predictions, covering about 5 million proteins in 717 fully-sequenced genomes. For each protein-coding gene in a seed genome, the database provides original and processed alignments, phylogenetic trees derived from various methods and phylogeny-based predictions of orthology and paralogy relationships. The new version of phylomeDB has been extended with novel data access and visualization features, including the possibility of programmatic access. Available seed species include model organisms such as human, yeast, Escherichia coli or Arabidopsis thaliana, but also alternative model species such as the human pathogen Candida albicans, or the pea aphid Acyrtosiphon pisum. Finally, PhylomeDB is currently being used by several genome sequencing projects that couple the genome annotation process with the reconstruction of the corresponding phylome, a strategy that provides relevant evolutionary insights

A bacteria-derived tail anchor localizes to peroxisomes in yeast and mammalian cells

Prokaryotes can provide new genetic information to eukaryotes by horizontal gene transfer (HGT), and such transfers are likely to have been particularly consequential in the era of eukaryogenesis. Since eukaryotes are highly compartmentalized, it is worthwhile to consider the mechanisms by which newly transferred proteins might reach diverse organellar destinations. Toward this goal, we have focused our attention upon the behavior of bacteria-derived tail anchors (TAs) expressed in the eukaryote Saccharomyces cerevisiae. In this study, we report that a predicted membrane-associated domain of the Escherichia coli YgiM protein is specifically trafficked to peroxisomes in budding yeast, can be found at a pre-peroxisomal compartment (PPC) upon disruption of peroxisomal biogenesis, and can functionally replace an endogenous, peroxisome-directed TA. Furthermore, the YgiM(TA) can localize to peroxisomes in mammalian cells. Since the YgiM(TA) plays no endogenous role in peroxisomal function or assembly, this domain is likely to serve as an excellent tool allowing further illumination of the mechanisms by which TAs can travel to peroxisomes. Moreover, our findings emphasize the ease with which bacteria-derived sequences might target to organelles in eukaryotic cells following HGT, and we discuss the importance of flexible recognition of organelle targeting information during and after eukaryogenesis.Peer reviewe

New insights into the peroxisomal protein inventory: Acyl-CoA oxidases and -dehydrogenases are an ancient feature of peroxisomes

Journal ArticleCopyright © 2014 Elsevier B.V. All rights reserved.Peroxisomes are ubiquitous organelles which participate in a variety of essential biochemical pathways. An intimate interrelationship between peroxisomes and mitochondria is emerging in mammals, where both organelles cooperate in fatty acid β-oxidation and cellular lipid homeostasis. As mitochondrial fatty acid β-oxidation is lacking in yeast and plants, suitable genetically accessible model systems to study this interrelationship are scarce. Here, we propose the filamentous fungus Ustilago maydis as a suitable model for those studies. We combined molecular cell biology, bioinformatics and phylogenetic analyses and provide the first comprehensive inventory of U. maydis peroxisomal proteins and pathways. Studies with a peroxisome-deficient Δpex3 mutant revealed the existence of parallel and complex, cooperative β-oxidation pathways in peroxisomes and mitochondria, mimicking the situation in mammals. Furthermore, we provide evidence that acyl-CoA dehydrogenases (ACADs) are bona fide peroxisomal proteins in fungi and mammals and together with acyl-CoA oxidases (ACOX) belong to the basic enzymatic repertoire of peroxisomes. A genome comparison with baker's yeast and human gained new insights into the basic peroxisomal protein inventory shared by humans and fungi and revealed novel peroxisomal proteins and functions in U. maydis. The importance of our findings for the evolution and function of the complex interrelationship between peroxisomes and mitochondria in fatty acid β-oxidation is discussed.Portuguese Foundation for Science and Technology (FCT)FEDER/COMPETEBBSRCCRUP/Treaty of Windso

Peroxisome membrane proteins: Multiple trafficking routes and multiple functions?

PMPs (peroxisome membrane proteins) play essential roles in organelle biogenesis and in co-ordinating peroxisomal metabolism with pathways in other subcellular compartments through transport of metabolites and the operation of redox shuttles. Although the import of soluble proteins into the peroxisome matrix has been well studied, much less is known about the trafficking of PMPs. Pex3 and Pex19 (and Pex16 in mammals) were identified over a decade ago as critical components of PMP import; however, it has proved surprisingly difficult to produce a unified model for their function in PMP import and peroxisome biogenesis. It has become apparent that each of these peroxins has multiple functions and in the present review we focus on both the classical and the more recently identified roles of Pex19 and Pex3 as informed by structural, biochemical and live cell imaging studies. We consider the different models proposed for peroxisome biogenesis and the role of PMP import within them, and propose that the differences may be more perceived than real and may reflect the highly dynamic nature of peroxisomes

The cycling peroxisomal targeting signal type 1 - receptor Pex5p: reaching the circle’s end with ubiquitin

Peroxisomes are single-membrane bound organelles that are found nearly ubiquitiously in eukaryotic cells. Their main task is the breakdown of fatty acids by beta-oxidation and the detoxification of hydrogen peroxide. However, these so called “multi-purpose organelles” also display several other metabolic functions, which can differ between species, tissues or growth conditions of the cells. This high plasticity of peroxisomal functions is enabled by an adjustment of the protein composition, which in turn is regulated by the dynamically operating protein import receptors. Subsequent to their synthesis on free ribosomes in the cytosol, peroxisomal matrix proteins are recognizes by import receptors by means of a peroxisomal targeting sequence (PTS). Most peroxisomal matrix proteins harbor a PTS-type 1 (PTS1) signal, which is bound by the PTS1-receptor Pex5p in the cytosol. The PTS1-receptor/cargo-complex reaches a docking complex at the peroxisome, where Pex5p is thought to become a building block of a transiently opened translocation pore. After the translocation of the folded cargo proteins over the membrane into the peroxisomal matrix, Pex5p is exported back to the cytosol for further rounds of matrix protein import. This dislocation step comprises the only energy-consuming reactions of the entire receptor cycle, because Pex5p has to be monoubiquitinated at a conserved cysteine before it can be extracted from the membrane by the AAA-type ATPases Pex1p and Pex6p. In case this recycling pathway is hampered, Pex5p gets polyubiquitinated on lysine residues and degraded by the proteasome. This review focuses on the PTS1-receptor Pex5p and discusses recent data and concepts regarding the molecular mechanism of cargo recognition, pore formation, cargo release and ubiquitination-dependent export and highlights the clinical relevance of Pex5p in health and disease

Uncovering the role of host peroxisomal functions in Plasmodium liver stage infection

Tese de mestrado. Biologia (Biologia Molecular e Genética). Universidade de Lisboa, Faculdade de Ciências, 2011Malaria, the world’s leading tropical parasitic disease, is caused by protozoan parasites of the genus Plasmodium. During its life cycle, Plasmodium inhabits an insect vector and a vertebrate host. Liver infection in the vertebrate host is the asymptomatic obligatory step before the onset of malaria disease. Cellular and molecular interactions between host and parasite play a key role in the establishment of susceptibility to malaria infection, and so the identification of relevant host factors is crucial for the rational development of new antimalarial strategies. We hypothesized that peroxisomes-less Plasmodium may have acquired host-dependency at the level of liver peroxisomes, and that it can take advantage of host cell peroxisomal functions and metabolites during liver stage. The myriad pathways in which peroxisomes are involved and their abundance in mammalian livers seems to place these organelles in a privileged position to be exploited in the context of intracellular parasitism. Live fluorescence microscopy and flow cytometry of DsRedlabeled peroxisomes revealed that the intracellular presence of Plasmodium can alter the dynamic properties of the host peroxisomal population. We then focused on the two major mammalian peroxisomal functions, fatty acid β-oxidation and detoxification of reactive oxygen species. Impairment of fatty acid β-oxidation by a drug inhibitor, knockdown of β- oxidation enzymes and overexpression of a key peroxisomal thiolase showed that a hostfactor dependency does exist and that it is important for both cell invasion and subsequent parasite development. This is probably tied to the parasite’s metabolic requirements for membrane biosynthesis during these processes. Catalase inhibition and knockdown of other peroxisomal peroxidases showed that this antioxidant network does not play a strong role in Plasmodium infection, but fluorescence microscopy revealed that the peroxisomal marker enzyme catalase may be recruited by the parasite to complement the functions of its own antioxidant systems in the maintenance of redox homeostasis during liver stage.A malária constitui a principal doença parasitária tropical no mundo, sendo causada por protozoários do género Plasmodium. O ciclo de vida deste parasita inclui dois hospedeiros: um insecto vector e um vertebrado. A infecção do fígado do hospedeiro vertebrado é uma etapa obrigatória e precede a manifestação clínica da doença. As interacções celulares e moleculares entre parasita e hospedeiro têm um papel determinante no estabelecimento da susceptibilidade à infecção e, portanto, a identificação de factores do hospedeiro relevantes para o desenrolar da infecção é essencial numa perspectiva de desenvolvimento de novas estratégias anti-maláricas. No âmbito deste trabalho formulámos a hipótese de que o parasita causador da malária, o qual é desprovido de peroxissomas, poderá, ao longo da evolução, ter adquirido a capacidade de subverter as funções e/ou metabolitos peroxissomais do hospedeiro vertebrado. De facto, a diversidade de vias metabólicas em que os peroxissomas estão envolvidos, bem como a sua abundância no fígado, levantam a questão da importância destes organelos num contexto de parasitismo intracelular. Começámos por mostrar que a presença de Plasmodium pode alterar as propriedades dinâmicas da população peroxissomal da célula hospedeira. Focámo-nos, então, nas duas principais funções dos peroxissomas, a β-oxidação de ácidos gordos e a degradação de espécies reactivas de oxigénio. Bloqueio da β-oxidação através de um inibidor ou por silenciamento da expressão de enzimas-chave desta via metabólica, bem como sobreexpressão de uma importante tiolase peroxissomal, permitiu-nos demonstrar que existe de facto uma dependência entre parasita e hospedeiro e que a β-oxidação peroxissomal é importante tanto para a invasão da célula hospedeira como para o subsequente desenvolvimento do parasita. Este efeito está provavelmente associado às necessidades lipídicas do parasita, nomeadamente para a síntese de membranas durante ambos os processos. Por outro lado, inibição da catalase e silenciamento da expressão de outras peroxidases peroxissomais revelou que esta rede antioxidante não tem um papel crucial na infecção por Plasmodium. Curiosamente, experiências de microscopia de fluorescência sugerem que a catalase do hospedeiro poderá ser recrutada pelo parasita, o que poderá constituir um mecanismo de homeostase durante a infecção hepática