Peroxisomes during postnatal development of mouse endocrine and exocrine pancreas display cell-type- and stage-specific protein composition.

Peroxisomal dysfunction unhinges cellular metabolism by causing the accumulation of toxic metabolic intermediates (e.g. reactive oxygen species, very -chain fatty acids, phytanic acid or eicosanoids) and the depletion of important lipid products (e.g. plasmalogens, polyunsaturated fatty acids), leading to various proinflammatory and devastating pathophysiological conditions like metabolic syndrome and age-related diseases including diabetes. Because the peroxisomal antioxidative marker enzyme catalase is low abundant in Langerhans islet cells, peroxisomes were considered scarcely present in the endocrine pancreas. Recently, studies demonstrated that the peroxisomal metabolism is relevant for pancreatic cell functionality. During the postnatal period, significant changes occur in the cell structure and the metabolism to trigger the final maturation of the pancreas, including cell proliferation, regulation of energy metabolism, and activation of signalling pathways. Our aim in this study was to (i) morphometrically analyse the density of peroxisomes in mouse endocrine versus exocrine pancreas and (ii) investigate how the distribution and the abundance of peroxisomal proteins involved in biogenesis, antioxidative defence and fatty acid metabolism change during pancreatic maturation in the postnatal period. Our results prove that endocrine and exocrine pancreatic cells contain high amounts of peroxisomes with heterogeneous protein content indicating that distinct endocrine and exocrine cell types require a specific set of peroxisomal proteins depending on their individual physiological functions. We further show that significant postnatal changes occur in the peroxisomal compartment of different pancreatic cells that are most probably relevant for the metabolic maturation and differentiation of the pancreas during the development from birth to adulthood.

Sequential lipidomic, metabolomic, and proteomic analyses of serum, liver, and heart tissue specimens from peroxisomal biogenesis factor 11α knockout mice.

Peroxisomes are versatile single membrane-enclosed cytoplasmic organelles, involved in reactive oxygen species (ROS) and lipid metabolism and diverse other metabolic processes. Peroxisomal disorders result from mutations in Pex genes-encoded proteins named peroxins (PEX proteins) and single peroxisomal enzyme deficiencies. The PEX11 protein family (α, ß, and γ isoforms) plays an important role in peroxisomal proliferation and fission. However, their specific functions and the metabolic impact caused by their deficiencies have not been precisely characterized. To understand the systemic molecular alterations caused by peroxisomal defects, here we utilized untreated peroxisomal biogenesis factor 11α knockout (Pex11α KO) mouse model and performed serial relative-quantitative lipidomic, metabolomic, and proteomic analyses of serum, liver, and heart tissue homogenates. We demonstrated significant specific changes in the abundances of multiple lipid species, polar metabolites, and proteins and dysregulated metabolic pathways in distinct biological specimens of the Pex11α KO adult mice in comparison to the wild type (WT) controls. Overall, the present study reports comprehensive semi-quantitative molecular omics information of the Pex11α KO mice, which might serve in the future as a reference for a better understanding of the roles of Pex11α and underlying pathophysiological mechanisms of peroxisomal biogenesis disorders.

The mitochondrial phosphate carrier TbMCP11 is essential for mitochondrial function in the procyclic form of Trypanosoma brucei.

Conserved amongst all eukaryotes is a family of mitochondrial carrier proteins (SLC25A) responsible for the import of various solutes across the inner mitochondrial membrane. We previously reported that the human parasite Trypanosoma brucei possesses 26 SLC25A proteins (TbMCPs) amongst which two, TbMCP11 and TbMCP8, were predicted to function as phosphate importers. The transport of inorganic phosphate into the mitochondrion is a prerequisite to drive ATP synthesis by substrate level and oxidative phosphorylation and thus crucial for cell viability. In this paper we describe the functional characterization of TbMCP11. In procyclic form T. brucei, the RNAi of TbMCP11 blocked ATP synthesis on mitochondrial substrates, caused a drop of the mitochondrial oxygen consumption and drastically reduced cell viability. The functional complementation in yeast and mitochondrial swelling experiments suggested a role for TbMCP11 as inorganic phosphate carrier. Interestingly, procyclic form T. brucei cells in which TbMCP11 was depleted displayed an inability to either replicate or divide the kinetoplast DNA, which resulted in a severe cytokinesis defect.

Analysis of the Level of Plasmid-Derived mRNA in the Presence of Residual Plasmid DNA by Two-Step Quantitative RT-PCR.

In transfection experiments with mammalian cells aiming to overexpress a specific protein, it is often necessary to correctly quantify the level of the recombinant and the corresponding endogenous mRNA. In our case, mouse calvarial osteoblasts were transfected with a vector containing the complete Pex11ß cDNA (plasmid DNA). The Pex11ß mRNA level, as calculated using the RT-qPCR product, was unrealistically higher (>1000-fold) in transfected compared to non-transfected cells, and we assumed that there were large amounts of contaminating plasmid DNA in the RNA sample. Thus, we searched for a simple way to distinguish between plasmid-derived mRNA, endogenous genome-derived mRNA and plasmid DNA, with minimal changes to standard RT-PCR techniques. We succeeded by performing a plasmid mRNA-specific reverse transcription, and the plasmid cDNA was additionally tagged with a nonsense tail. A subsequent standard qPCR was conducted using appropriate PCR primers annealing to the plasmid cDNA and to the nonsense tail. Using this method, we were able to determine the specific amount of mRNA derived from the transfected plasmid DNA in comparison to the endogenous genome-derived mRNA, and thus the transfection and transcription efficiency.

Characterisation of a mitochondrial iron transporter of the pathogen Trypanosoma brucei.

Similar to higher eukaryotes, the protist parasite T. brucei harbours several iron-containing proteins that regulate DNA and protein processing, oxidative stress defence and mitochondrial respiration. The synthesis of these proteins occurs either in the cytoplasm or within the mitochondrion. For mitochondrial iron cluster protein synthesis, iron needs to be transported across the solute impermeable mitochondrial membrane. In T. brucei we previously identified 24 mitochondrial carrier proteins (TbMCPs) sharing conserved structural and functional features with those from higher eukaryotes. One of these carriers (TbMCP17) displayed high similarity with the iron carriers MRS3, MRS4 from yeast and mitoferrin from mammals, insects and plants. In the present study we demonstrated that TbMCP17 functions as an iron carrier by complementation studies using MRS3/4-deficient yeast. Depletion of TbMCP17 in procyclic form T. brucei resulted in growth deficiency, increased sensitivity to iron deprivation, and lowered mitochondrial iron content. Taken together our results suggest that TbMCP17 functions as a mitochondrial iron transporter in the parasite T. brucei.

PPARα-mediated peroxisome induction compensates PPARγ-deficiency in bronchiolar club cells.

Despite the important functions of PPARγ in various cell types of the lung, PPARγ-deficiency in club cells induces only mild emphysema. Peroxisomes are distributed in a similar way as PPARγ in the lung and are mainly enriched in club and AECII cells. To date, the effects of PPARγ-deficiency on the overall peroxisomal compartment and its metabolic alterations in pulmonary club cells are unknown. Therefore, we characterized wild-type and club cell-specific PPARγ knockout-mice lungs and used C22 cells to investigate the peroxisomal compartment and its metabolic roles in the distal airway epithelium by means of 1) double-immunofluorescence labelling for peroxisomal proteins, 2) laser-assisted microdissection of the bronchiolar epithelium and subsequent qRT-PCR, 3) siRNA-transfection of PPARγand PPRE dual-luciferase reporter activity in C22 cells, 4) PPARg inhibition by GW9662, 5) GC-MS based lipid analysis. Our results reveal elevated levels of fatty acids, increased expression of PPARα and PPRE activity, a strong overall upregulation of the peroxisomal compartment and its associated gene expression (biogenesis, α-oxidation, ß-oxidation, and plasmalogens) in PPARγ-deficient club cells. Interestingly, catalase was significantly increased and mistargeted into the cytoplasm, suggestive for oxidative stress by the PPARγ-deficiency in club cells. Taken together, PPARα-mediated metabolic induction and proliferation of peroxisomes via a PPRE-dependent mechanism could compensate PPARγ-deficiency in club cells.

A plant-like mitochondrial carrier family protein facilitates mitochondrial transport of di- and tricarboxylates in Trypanosoma brucei.

The procyclic form of the human parasite Trypanosoma brucei harbors one single, large mitochondrion containing all tricarboxylic acid (TCA) cycle enzymes and respiratory chain complexes present also in higher eukaryotes. Metabolite exchange among subcellular compartments such as the cytoplasm, the mitochondrion, and the peroxisomes is crucial for redox homeostasis and for metabolic pathways whose enzymes are dispersed among different organelles. In higher eukaryotes, mitochondrial carrier family (MCF) proteins transport TCA-cycle intermediates across the inner mitochondrial membrane. Previously, we identified several MCF members that are essential for T. brucei survival. Among these, only one MCF protein, TbMCP12, potentially could transport dicarboxylates and tricarboxylates. Here, we conducted phylogenetic and sequence analyses and functionally characterised TbMCP12 in vivo. Our results suggested that similarly to its homologues in plants, TbMCP12 transports both dicarboxylates and tricarboxylates across the mitochondrial inner membrane. Deleting this carrier in T. brucei was not lethal, while its overexpression was deleterious. Our results suggest that the intracellular abundance of TbMCP12 is an important regulatory element for the NADPH balance and mitochondrial ATP-production.

New insights into the distribution, protein abundance and subcellular localisation of the endogenous peroxisomal biogenesis proteins PEX3 and PEX19 in different organs and cell types of the adult mouse.

Peroxisomes are ubiquitous organelles mainly involved in ROS and lipid metabolism. Their abundance, protein composition and metabolic function vary depending on the cell type and adjust to different intracellular and environmental factors such as oxidative stress or nutrition. The biogenesis and proliferation of these important organelles are regulated by proteins belonging to the peroxin (PEX) family. PEX3, an integral peroxisomal membrane protein, and the cytosolic shuttling receptor PEX19 are thought to be responsible for the early steps of peroxisome biogenesis and assembly of their matrix protein import machinery. Recently, both peroxins were suggested to be also involved in the autophagy of peroxisomes (pexophagy). Despite the fact that distribution and intracellular abundance of these proteins might regulate the turnover of the peroxisomal compartment in a cell type-specific manner, a comprehensive analysis of the endogenous PEX3 and PEX19 distribution in different organs is still missing. In this study, we have therefore generated antibodies against endogenous mouse PEX3 and PEX19 and analysed their abundance and subcellular localisation in various mouse organs, tissues and cell types and compared it to the one of three commonly used peroxisomal markers (PEX14, ABCD3 and catalase). Our results revealed that the abundance of PEX3, PEX19, PEX14, ABCD3 and catalase strongly varies in the analysed organs and cell types, suggesting that peroxisome abundance, biogenesis and matrix protein import are independently regulated. We further found that in some organs, such as heart and skeletal muscle, the majority of the shuttling receptor PEX19 is bound to the peroxisomal membrane and that a strong variability exists in the cell type-specific ratio of cytosol- and peroxisome-associated PEX19. In conclusion, our results indicate that peroxisomes in various cell types are heterogeneous with regards to their matrix, membrane and biogenesis proteins.

Peroxisomes in cardiomyocytes and the peroxisome / peroxisome proliferator-activated receptor-loop.

It is well established that the heart is strongly dependent on fatty acid metabolism. In cardiomyocytes there are two distinct sites for the ß-oxidisation of fatty acids: the mitochondrion and the peroxisome. Although the metabolism of these two organelles is believed to be tightly coupled, the nature of this relationship has not been fully investigated. Recent research has established the significant contribution of mitochondrial function to cardiac ATP production under normal and pathological conditions. In contrast, limited information is available on peroxisomal function in the heart. This is despite these organelles harbouring metabolic pathways that are potentially cardio-protective, and findings that patients with peroxisomal diseases, such as adult Refsum´s disease, can develop heart failure. In this article, we provide a comprehensive overview on the current knowledge of peroxisomes and the regulation of lipid metabolism by PPARs in cardiomyocytes. We also present new experimental evidence on the differential expression of peroxisome-related genes in the heart chambers and demonstrate that even a mild peroxisomal biogenesis defect (Pex11α-/-) can induce profound alterations in the cardiomyocyte´s peroxisomal compartment and related gene expression, including the concomitant deregulation of specific PPARs. The possible impact of peroxisomal dysfunction in the heart is discussed and a model for the modulation of myocardial metabolism via a peroxisome/PPAR-loop is proposed.

Proteins and lipids of glycosomal membranes from Leishmania tarentolae and Trypanosoma brucei.

In kinetoplastid protists, several metabolic pathways, including glycolysis and purine salvage, are located in glycosomes, which are microbodies that are evolutionarily related to peroxisomes. With the exception of some potential transporters for fatty acids, and one member of the mitochondrial carrier protein family, proteins that transport metabolites across the glycosomal membrane have yet to be identified. We show here that the phosphatidylcholine species composition of Trypanosoma brucei glycosomal membranes resembles that of other cellular membranes, which means that glycosomal membranes are expected to be impermeable to small hydrophilic molecules unless transport is facilitated by specialized membrane proteins. Further, we identified 464 proteins in a glycosomal membrane preparation from Leishmania tarentolae. The proteins included approximately 40 glycosomal matrix proteins, and homologues of peroxisomal membrane proteins - PEX11, GIM5A and GIM5B; PXMP4, PEX2 and PEX16 - as well as the transporters GAT1 and GAT3. There were 27 other proteins that could not be unambiguously assigned to other compartments, and that had predicted trans-membrane domains. However, no clear candidates for transport of the major substrates and intermediates of energy metabolism were found. We suggest that, instead, these metabolites are transported via pores formed by the known glycosomal membrane proteins.

The phosphoarginine energy-buffering system of trypanosoma brucei involves multiple arginine kinase isoforms with different subcellular locations.

Phosphagen energy-buffering systems play an essential role in regulating the cellular energy homeostasis in periods of high-energy demand or energy supply fluctuations. Here we describe the phosphoarginine/arginine kinase system of the kinetoplastid parasite Trypanosoma brucei, consisting of three highly similar arginine kinase isoforms (TbAK1-3). Immunofluorescence microscopy using myc-tagged protein versions revealed that each isoform is located in a specific subcellular compartment: TbAK1 is exclusively found in the flagellum, TbAK2 in the glycosome, and TbAK3 in the cytosol of T. brucei. The flagellar location of TbAK1 is dependent on a 22 amino acid long N-terminal sequence, which is sufficient for targeting a GFP-fusion protein to the trypanosome flagellum. The glycosomal location of TbAK2 is in agreement with the presence of a conserved peroxisomal targeting signal, the C-terminal tripeptide 'SNL'. TbAK3 lacks any apparent targeting sequences and is accordingly located in the cytosol of the parasite. Northern blot analysis indicated that each TbAK isoform is differentially expressed in bloodstream and procyclic forms of T. brucei, while the total cellular arginine kinase activity was 3-fold higher in bloodstream form trypanosomes. These results suggest a substantial change in the temporal and spatial energy requirements during parasite differentiation. Increased arginine kinase activity improved growth of procyclic form T. brucei during oxidative challenges with hydrogen peroxide. Elimination of the total cellular arginine kinase activity by RNA interference significantly decreased growth (>90%) of procyclic form T. brucei under standard culture conditions and was lethal for this life cycle stage in the presence of hydrogen peroxide. The putative physiological roles of the different TbAK isoforms in T. brucei are further discussed.

Functional characterization of TbMCP5, a conserved and essential ADP/ATP carrier present in the mitochondrion of the human pathogen Trypanosoma brucei.

Trypanosoma brucei is a kinetoplastid parasite of medical and veterinary importance. Its digenetic life cycle alternates between the bloodstream form in the mammalian host and the procyclic form (PCF) in the bloodsucking insect vector, the tsetse fly. PCF trypanosomes rely in the glucose-depleted environment of the insect vector primarily on the mitochondrial oxidative phosphorylation of proline for their cellular ATP provision. We previously identified two T. brucei mitochondrial carrier family proteins, TbMCP5 and TbMCP15, with significant sequence similarity to functionally characterized ADP/ATP carriers from other eukaryotes. Comprehensive sequence analysis confirmed that TbMCP5 contains canonical ADP/ATP carrier sequence features, whereas they are not conserved in TbMCP15. Heterologous expression in the ANC-deficient yeast strain JL1Δ2Δ3u(-) revealed that only TbMCP5 was able to restore its growth on the non-fermentable carbon source lactate. Transport studies in yeast mitochondria showed that TbMCP5 has biochemical properties and ADP/ATP exchange kinetics similar to those of Anc2p, the prototypical ADP/ATP carrier of S. cerevisiae. Immunofluorescence microscopy and Western blot analysis confirmed that TbMCP5 is exclusively mitochondrial and is differentially expressed with 4.5-fold more TbMCP5 in the procyclic form of the parasite. Silencing of TbMCP5 expression in PCF T. brucei revealed that this ADP/ATP carrier is essential for parasite growth, particularly when depending on proline for energy generation. Moreover, ADP/ATP exchange in isolated T. brucei mitochondria was eliminated upon TbMCP5 depletion. These results confirmed that TbMCP5 functions as the main ADP/ATP carrier in the trypanosome mitochondrion. The important role of TbMCP5 in the T. brucei energy metabolism is further discussed.

Mitochondrial carrier family inventory of Trypanosoma brucei brucei: Identification, expression and subcellular localisation.

The mitochondrial carrier family (MCF) is a group of structurally conserved proteins that mediate the transport of a wide range of metabolic intermediates across the mitochondrial inner membrane. In this paper, an overview of the mitochondrial carrier proteins (MCPs) of the early-branching kinetoplastid parasite Trypanosoma brucei brucei is presented. Sequence analysis and phylogenetic reconstruction gave insight into the evolution and conservation of the 24 identified TbMCPs; for most of these, putative transport functions could be predicted. Comparison of the kinetoplastid MCP inventory to those previously reported for other eukaryotes revealed remarkable deviations: T. b. brucei lacks genes encoding some prototypical MCF members, such as the citrate carrier and uncoupling proteins. The in vivo expression of the identified TbMCPs in the two replicating life-cycle forms of T. b. brucei, the bloodstream-form and procyclic-form, was quantitatively assessed at the mRNA level by Northern blot analysis. Immunolocalisation studies confirmed that majority of the 24 identified TbMCPs is found in the mitochondrion of procyclic-form T. b. brucei.

Regulated expression of glycosomal phosphoglycerate kinase in Trypanosoma brucei.

In Trypanosoma brucei, the PGKB and PGKC genes-encoding phosphoglycerate kinase are co-transcribed as part of a polycistronic RNA. PGKB mRNA and the cytosolic PGKB protein are much more abundant in the procyclic life-cycle stage than in bloodstream forms, whereas PGKC mRNA and glycosomal PGKC protein are specific to bloodstream forms. We here show that a sequence between nucleotides 558 and 779 in the 3'-untranslated region of the PGKC mRNA causes low expression of the chloramphenicol acetyltransferase (CAT) reporter gene in procyclic trypanosomes. In procyclics, depletion of the RRP45 component of the exosome (3'-->5' exonuclease complex) or the 5'-->3' exonuclease XRNA increased the abundance of CAT-PGKC mRNA as a consequence of effects on the degradation of precursor and/or mature mRNAs. In bloodstream forms, inhibition of both trans splicing and transcription resulted in immediate exponential decay of PGKC mRNA with a half-life of 46 min. Inhibition of transcription alone gave non-exponential kinetics and inhibition of splicing alone resulted in a longer apparent half-life. We also found that production of mRNAs using T7 polymerase can affect the apparent half-life, and that large amounts of CAT enzyme may be toxic in trypanosomes.

Characterization and developmentally regulated localization of the mitochondrial carrier protein homologue MCP6 from Trypanosoma brucei.

Proteins of the mitochondrial carrier family (MCF) are located mainly in the inner mitochondrial membrane and mediate the transport of a large range of metabolic intermediates. The genome of Trypanosoma brucei harbors 29 genes encoding different MCF proteins. We describe here the characterization of MCP6, a novel T. brucei MCF protein. Sequence comparison and phylogenetic reconstruction revealed that MCP6 is closely related to different mitochondrial ADP/ATP and calcium-dependent solute carriers, including the ATP-Mg/Pi carrier of Homo sapiens. However, MCP6 lacks essential amino acids and sequence motifs conserved in these metabolite transporters, and functional reconstitution and transport assays with E. coli suggested that this protein indeed does not function as an ADP/ATP or ATP-Mg/Pi carrier. The subcellular localization of MCP6 is developmentally regulated: in bloodstream-form trypanosomes, the protein is predominantly glycosomal, whereas in the procyclic form, it is found mainly in the mitochondria. Depletion of MCP6 in procyclic trypanosomes resulted in growth inhibition, an increased cell size, aberrant numbers of nuclei and kinetoplasts, and abnormal kinetoplast morphology, suggesting that depletion of MCP6 inhibits division of the kinetoplast.

Comparative proteomics of glycosomes from bloodstream form and procyclic culture form Trypanosoma brucei brucei.

Peroxisomes are present in nearly every eukaryotic cell and compartmentalize a wide range of important metabolic processes. Glycosomes of Kinetoplastid parasites are peroxisome-like organelles, characterized by the presence of the glycolytic pathway. The two replicating stages of Trypanosoma brucei brucei, the mammalian bloodstream form (BSF) and the insect (procyclic) form (PCF), undergo considerable adaptations in metabolism when switching between the two different hosts. These adaptations involve also substantial changes in the proteome of the glycosome. Comparative (non-quantitative) analysis of BSF and PCF glycosomes by nano LC-ESI-Q-TOF-MS resulted in the validation of known functional aspects of glycosomes and the identification of novel glycosomal constituents.