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The paper is the fundamental beginning of research series aimed at understanding the processes associated with high performance in higher animals. The research aim is to study correlation of dicarboxylic acid cycle with tricarboxylic acid cycle with establishment of activity and dislocation of enzymes, confirming the hypothesis of availability and active metabolic participation of peroxisome in highly productive animals. Research was conducted on the basis of the VNIIFBiP animal vivarium in 2019 with a group of piglets of the Irish Landrace breed (n = 10). After slaughter at the age of 210 days, the nuclear (with large tissue particles), mitochondrial and postmitochondrial fractions of the liver were studied with assessment of succinate dehydrogenase and activity of other dehydrogenes of the Krebs cycle. It was found that peroxisomes act as universal agents of communication and cooperation, and microtelets are able to generate various chemical signals that carry information, to control and arrange a number of mechanisms in the metabolic processes in the body. Despite the fact that the Krebs cycle dehydrogenases are considered mitochondrial enzymes, the experiment showed an increase in activity of priruvate dehydrogenase (P > 0.1), isocitrate dehydrogenase (0.1 > P > 0.05) and malate dehydrogenase (0.1 > P > 0.05), which, when comparing the mitochondrial and postmitochondrial fractions, indicates a higher activity of peroxisomal fractions. The peroxisome localization place is the postmitochondrial fraction, and the lower layer contains larger peroxisomes to a greater extent, while the upper layer contains smaller ones. It was found that indicator enzymes of glyoxylate cycle isocitratliase and malate synthase exhibit catalytic activity in the peroxisomal fraction of liver of highly productive pigs. The obtained data on functioning of key glyoxylate cycle enzymes and their intracellular compartmentalization in highly productive pigs allow learning more about the specifics of metabolism and its regulation processes. Application of this knowledge in practice opens up prospects for rationalizing the production of livestock products of increased quantity, improved quality with less feed, labor and financial resources spent.

Numerous studies have used genome-editing modules such as CRISPR-Cas9 for site-directed mutagenesis; however, evaluation of the efficiency of these modules remains a time-consuming process. Here, we report the development of SKL-mediated Peroxisome Targeting Imaging (SKLPT imaging), an efficient in vivo pre-evaluation method based on the change in subcellular localization of a fluorescent protein. In this method, frameshifts resulting from successful editing cause the fusion of green fluorescent protein to the peroxisome localization signal Serine-Lysine-Leucine (SKL). Using SKLPT imaging, we pre-evaluated three CRISPR-Cas9 modules in vivo at the single-cell level, and then efficiently mutagenized the liverwort (Marchantia polymorpha) genome using a high-efficiency module.

In an in silico search for correlated gene loss with fungal peroxisomal uric acid oxidase (UOX), we identified PMP22-like proteins, some of which function as promiscuous channels in organellar membranes. To investigate whether PMP22 channels have a role in peroxisomal uric acid transport and catabolism, we functionally analyzed the closest homologue in Aspergillus nidulans, named SspA. We confirmed that SspA is a peroxisomal membrane protein that co-localizes significantly with PTS1-tagged mRFP, UOX or HexA, the latter considered a protein of Woronin bodies (WB), organelles originating from peroxisomes that dynamically plug septal pores in ascomycetes. Our results suggest that in A. nidulans, unlike some other ascomycetes, there is no strict protein segregation of peroxisomal and WB-specific proteins. Importantly, genetic deletion of sspA, but not of hexA, led to lack of peroxisomal localization at septal pores, suggesting that SspA is a key factor for septal pore functioning. Additionally, ΔsspA resulted in increased sensitivity to oxidative stress, apparently as a consequence of not only the inability to plug septal pores, but also a recorded reduction in peroxisome biogenesis. However, deleting sspA had no effect on uric acid or purine utilization, as we hypothesized, a result also in line with the observation that expression of SspA was not affected by regulatory mutants and conditions known to control purine catabolic enzymes. Our results are discussed within the framework of previous studies of SspA homologues in other fungi, as well as, the observed gene losses of PMP22 and peroxisomal uric acid oxidase.

Polyamines (PAs) are implicated in developmental processes and stress responses of plants. Polyamine oxidases (PAOs), flavin adenine dinucleotide-dependent enzymes that function in PA catabolism, play a critical role. Even though PAO gene families of Arabidopsis and rice have been intensely characterized and their expression in response to developmental and environmental changes has been investigated, little is known about PAOs in tomato (Solanum lycopersicum). We found seven PAO genes in S. lycopersicum and named them SlPAO1∼7. Plant PAOs form four clades in phylogenetic analysis, of which SlPAO1 belongs to clade-I, SlPAO6 and SlPAO7 to clade-III, and the residual four (SlPAO2∼5) to clade-IV, while none belongs to clade-II. All the clade-IV members in tomato also retain the putative peroxisomal-targeting signals in their carboxy termini, suggesting their peroxisome localization. SlPAO1 to SlPAO5 genes consist of 10 exons and 9 introns, while SlPAO6 and SlPAO7 are intronless genes. To address the individual roles of SlPAOs, we analyzed their expression in various tissues and during flowering and fruit development. The expression of SlPAO2∼4 was constitutively high, while that of the other SlPAO members was relatively lower. We further analyzed the expressional changes of SlPAOs upon abiotic stresses, oxidative stresses, phytohormone application, and PA application. Based on the data obtained, we discuss the distinctive roles of SlPAOs.

Subcellular localization is emerging as an important mechanism for mTORC1 regulation. We report that the tuberous sclerosis complex (TSC) signalling node, TSC1, TSC2 and Rheb, localizes to peroxisomes, where it regulates mTORC1 in response to reactive oxygen species (ROS). TSC1 and TSC2 were bound by peroxisomal biogenesis factors 19 and 5 (PEX19 and PEX5), respectively, and peroxisome-localized TSC functioned as a Rheb GTPase-activating protein (GAP) to suppress mTORC1 and induce autophagy. Naturally occurring pathogenic mutations in TSC2 decreased PEX5 binding, and abrogated peroxisome localization, Rheb GAP activity and suppression of mTORC1 by ROS. Cells lacking peroxisomes were deficient in mTORC1 repression by ROS, and peroxisome-localization-deficient TSC2 mutants caused polarity defects and formation of multiple axons in neurons. These data identify a role for the TSC in responding to ROS at the peroxisome, and identify the peroxisome as a signalling organelle involved in regulation of mTORC1.

Mammalian gene expression constructs are generally prepared in a plasmid vector, in which a promoter and terminator are located upstream and downstream of a protein-coding sequence, respectively. In this study, we found that front terminator constructs-DNA constructs containing a terminator upstream of a promoter rather than downstream of a coding region-could sufficiently express proteins as a result of end joining of the introduced DNA fragment. By taking advantage of front terminator constructs, FLAG substitutions, and deletions were generated using mutagenesis primers to identify amino acids specifically recognized by commercial FLAG antibodies. A minimal epitope sequence for polyclonal FLAG antibody recognition was also identified. In addition, we analyzed the sequence of a C-terminal Ser-Lys-Leu peroxisome localization signal, and identified the key residues necessary for peroxisome targeting. Moreover, front terminator constructs of hepatitis B surface antigen were used for deletion analysis, leading to the identification of regions required for the particle formation. Collectively, these results indicate that front terminator constructs allow for easy manipulations of C-terminal protein-coding sequences, and suggest that direct gene expression with PCR-amplified DNA is useful for high-throughput protein analysis in mammalian cells.

The biological functions of human acyl-CoA thioesterase III (ACTEIII/PTE-1), initially identified as an HIV-1 Nef binding protein, have remained unclear. We report herein that the stable overexpression of ACTEIII/PTE-1 in human and murine T-cell lines resulted in an increase in both peroxisome number and lipid droplet formation in a manner dependent on the amount of the protein. Peroxisome proliferation was evidenced by immunofluorescence staining for catalase, a peroxisome marker protein, as well as by direct peroxisome enumeration on electron micrographs. Consistently, the amount of catalase was elevated as the amount of ACTEIII/PTE-1 was increased. ACTEIII/PTE-1 mutants with reduced enzymatic activity or with the defect in peroxisome localization did not induce peroxisome proliferation, indicating that peroxisome proliferation was mediated by metabolites generated by ACTEIII/PTE-1 within peroxisomes. Finally, thymocytes isolated from a T-cell-specific ACTEIII/PTE-1 transgenic mouse as well as human and murine cell lines of lymphoid and non-lymphoid origins exhibited a similar proliferation of peroxisomes. Thus, ACTEIII/PTE-1 may be involved in the metabolic regulation of peroxisome proliferation.

The filamentous fungus Alternaria alternata includes seven pathogenic variants (pathotypes) which produce different host-selective toxins and cause diseases on different plants. The Japanese pear pathotype produces the host-selective AK-toxin, an epoxy-decatrienoic acid ester, and causes black spot of Japanese pear. Previously, we identified four genes, AKT1 , AKT2 , AKT3 , and AKTR , involved in AK toxin biosynthesis. AKT1 , AKT2 , and AKT3 encode enzyme proteins with peroxisomal targeting signal type 1 (PTS1)-like tripeptides, SKI, SKL, and PKL, respectively, at the C-terminal ends. In this study, we verified the peroxisome localization of Akt1, Akt2, and Akt3 by using strains expressing N-terminal green fluorescent protein (GFP)-tagged versions of the proteins. To assess the role of peroxisome function in AK-toxin production, we isolated Aa PEX6 , which encodes a peroxin protein essential for peroxisome biogenesis, from the Japanese pear pathotype and made Aa PEX6 disruption-containing transformants from a GFP-Akt1-expressing strain. The ΔAa PEX6 mutant strains did not grow on fatty acid media because of a defect in fatty acid β oxidation. The import of GFP-Akt1 into peroxisomes was impaired in the ΔAa PEX6 mutant strains. These strains completely lost AK toxin production and pathogenicity on susceptible pear leaves. These data show that peroxisomes are essential for AK-toxin biosynthesis. The ΔAa PEX6 mutant strains showed a marked reduction in the ability to cause lesions on leaves of a resistant pear cultivar with defense responses compromised by heat shock. This result suggests that peroxisome function is also required for plant invasion and tissue colonization in A. alternata . We also observed that mutation of Aa PEX6 caused a marked reduction of conidiation.

Multimodality imaging using several reporter genes and imaging technologies has become an increasingly important tool in determining the location(s), magnitude, and time variation of reporter gene expression in small animals. We have reported construction and validation of several triple fusion genes composed of a bioluminescent, a fluorescent, and a positron emission tomography (PET) reporter gene in cell culture and in living subjects. However, the bioluminescent and fluorescent components of fusion reporter proteins encoded by these vectors possess lesser activities when compared with the bioluminescent and fluorescent components of the nonfusions. In this study, we first created a mutant (mtfl) of a thermostable firefly luciferase (tfl) bearing the peroxisome localization signal to have greater cytoplasmic localization and improved access for its substrate, d-luciferin. Comparison between the three luciferases [mtfl, tfl, and firefly luciferase (fl)] both in cell culture and in living mice revealed that mtfl possessed 6- to 10-fold (in vitro) and 2-fold (in vivo) higher activity than fl. The improved version of the triple fusion vector carrying mtfl as the bioluminescent reporter component showed significantly (P < 0.05) higher bioluminescence than the previous triple fusion vectors. Of the three different red fluorescent reporter genes (jred, hcred, and mrfp1, isolated from jellyfish chromophore, coral Heteractis crispa, and coral Discosoma, respectively) evaluated, mrfp1 was able to preserve highest expression as a component of the triple fusion reporter gene for in vivo fluorescence imaging. A truncated version of wild-type herpes simplex virus 1 (HSV1) thymidine kinase gene (wttk) retained a higher expression level than the truncated mutant HSV1-sr39 TK (ttk) as the third reporter component of this improved triple fusion vector. Multimodality imaging of tumor-bearing mice using bioluminescence and microPET showed higher luciferase activity [(2.7 ± 0.1 versus 1.9 ± 0.1) × (106 p/s/cm2/sr)] but similar level of fluorine-18–labeled 2′-fluoro-2′-deoxyarabinofuranosyl-5-ethyluracil (18F-FEAU) uptake (1.37 ± 0.15 versus 1.37 ± 0.2) percentage injected dose per gram] by mtfl-mrfp1-wttk–expressing tumors compared with the fl-mrfp1-wttk–expressing tumors. Both tumors showed 4- to 5-fold higher accumulation (P < 0.05) of 18F-FEAU than fluorine-18–labeled 9-(4-fluoro-3-hydroxymethylbutyl)guanine. This improved triple fusion reporter vector will enable high sensitivity detection of lower numbers of cells from living animals using the combined bioluminescence, fluorescence, and microPET imaging techniques. [Cancer Res 2007;67(7):3085–93]

One of the proteins targeted to the peridinin plastid of the dinoflagellate Lingulodinium polyedrum is the iron-containing superoxide dismutase (LpSOD). Like dinoflagellate plastid proteins of class II, LpSOD carries a bipartite presequence comprising a signal peptide followed by a transit peptide. Our bioinformatic studies suggest that its signal peptide is atypical, however, and that the entire presequence may function as a mitochondrial targeting signal. It is possible that LpSOD represents a new class of proteins in algae with complex plastids, which are co-targeted to the plastid and mitochondrion. In addition to the ambiguous N-terminal targeting signal, LpSOD contains a potential type-1 peroxisome-targeting signal (PTS1) located at its C-terminus. In accordance with a peroxisome localization of this dismutase, its mRNA has two in-frame AUG codons. Our bioinformatic analyses indicate that the first start codon resides in a much weaker oligonucleotide context than the second one. This suggests that synthesis of the plastid/mitochondrion-targeted and peroxisome-targeted isoforms could proceed through so-called leaky scanning. Moreover, our results show that expression of the two isoforms could be regulated by a 'hairpin' structure located between the first and second start codons.