Denis I. Crane

Localization of a Portion of Extranuclear ATM to Peroxisomes

The gene mutated in the human genetic disorder ataxia-telangiectasia codes for a protein, ATM, the known functions of which include response to DNA damage, cell cycle control, and meiotic recombination. Consistent with these functions, ATM is predominantly present in the nucleus of proliferating cells; however, a significant proportion of the protein has also been detected outside the nucleus in cytoplasmic vesicles. To understand the possible role of extra-nuclear ATM, we initially investigated the nature of these vesicles. In this report we demonstrate that a portion of ATM co-localizes with catalase, that ATM is present in purified mouse peroxisomes, and that there are reduced levels of ATM in the post-mitochondrial membrane fraction of cells from a patient with a peroxisome biogenesis disorder. Furthermore the use of the yeast two-hybrid system demonstrated that ATM interacts directly with a protein involved in the import of proteins into the peroxisome matrix. Because peroxisomes are major sites of oxidative metabolism, we investigated catalase activity and lipid hydroperoxide levels in normal and A-T fibroblasts. Significantly decreased catalase activity and increased lipid peroxidation was observed in several A-T cell lines. The localization of ATM to peroxisomes may contribute to the pleiotropic nature of A-T.

Interaction of Pex5p, the Type 1 Peroxisome Targeting Signal Receptor, with the Peroxisomal Membrane Proteins Pex14p and Pex13p

Pex5p, a receptor for peroxisomal matrix proteins with a type 1 peroxisome targeting signal (PTS1), has been proposed to cycle from the cytoplasm to the peroxisomal membrane where it docks with Pex14p and Pex13p, the latter an SH3 domain-containing protein. Using in vitro binding assays we have demonstrated that binding of Pex5p to Pex14p is enhanced when Pex5p is loaded with a PTS1-containing peptide. In contrast, Pex5p binding to Pex13p, which involves only the SH3 domain, occurs at 20–40-fold lower levels and is reduced when Pex5p is preloaded with a PTS1 peptide. Pex14p was also shown to bind weakly to the Pex13p SH3 domain. Site-directed mutagenesis of the Pex13p SH3 domain attenuated binding to Pex5p and Pex14p, consistent with both of these proteins being binding partners for this domain. The SH3 binding site in Pex5p was determined to lie within a 114-residue peptide (Trp100-Glu213) in the amino-terminal region of the protein. The interaction between this peptide and the SH3 domain was competitively inhibited by Pex14p. We interpret these data as suggesting that docking of the Pex5p-PTS1 protein complex at the peroxisome membrane occurs at Pex14p and that the Pex13p SH3 domain functions as an associated component possibly involved in sequestering Pex5p after relinquishment of the PTS1 protein cargo to components of the translocation machinery.

Expression of the α1, α2 and α3 isoforms of the GABAA receptor in human alcoholic brain

The expression of the α1, α2 and α3 isoforms of the GABAA receptor was studied in the superior frontal and motor cortices of 10 control, 10 uncomplicated alcoholic and 7 cirrhotic alcoholic cases matched for age and post-mortem delay. The assay was based on competitive RT/PCR using a single set of primers specific to the α class of isoform mRNA species, and was normalized against a synthetic cRNA internal standard. The assay was shown to be quantitative for all three isoform mRNA species. Neither the patients age nor the post-mortem interval significantly affected the expression of any isoform in either cortical area. The profile of expression was shown to be significantly different between the case groups, particularly because α1 expression was raised in both groups of alcoholics cf controls. The two groups of alcoholics could be differentiated on the basis of regional variations in α1 expression. In frontal cortex, α1 mRNA expression was significantly increased when uncomplicated alcoholics were compared with control cases whereas alcoholic-cirrhotic cases were not significantly different from either controls or uncomplicated alcoholic cases. In the motor cortex, α1 expression was elevated only when alcoholic-cirrhotic cases were compared with control cases. There was no significant difference between case groups or areas for any other isoform.

PEX13 is mutated in complementation group 13 of the peroxisome-biogenesis disorders.

The peroxisome-biogenesis disorders (PBDs) are a genetically and phenotypically diverse group of diseases caused by defects in peroxisome assembly. One of the milder clinical variants within the PBDs is neonatal adrenoleukodystrophy (NALD), a disease that is usually associated with partial defects in the import of peroxisomal matrix proteins that carry the type 1 or type 2 peroxisomal targeting signals. Here, we characterize the sole representative of complementation group 13 of the PBDs, a patient with NALD (patient PBD222). Skin fibroblasts from patient PBD222 display defects in the import of multiple peroxisomal matrix proteins. However, residual matrix-protein import can be detected in cells from patient PBD222, consistent with the relatively mild phenotypes of the patient. PEX13 encodes a peroxisomal membrane protein with a cytoplasmically exposed SH3 domain, and we find that expression of human PEX13 restores peroxisomal matrix-protein import in cells from patient PBD222. Furthermore, these cells are homozygous for a missense mutation at a conserved position in the PEX13 SH3 domain. This mutation attenuated the activity of human PEX13, and an analogous mutation in yeast PEX13 also reduced its activity. The mutation was absent in >100 control alleles, indicating that it is not a common polymorphism. Previous studies have demonstrated extragenic suppression in the PBDs, but the phenotypes of patient PBD222 cells could not be rescued by expression of any other human PEX genes. Taken together, these results provide strong evidence that mutations in PEX13 are responsible for disease in patient PBD222 and, by extension, in complementation group 13 of the PBDs.

RhoA is highly up-regulated in the process of early heart development of the chick and important for normal embryogenesis

We have used molecular techniques, combined with classic embryological methods, to identify up‐regulated genes associated with early heart development. One of the cDNAs identified and isolated by screening a chick lambda cDNA library was the small guanosine triphosphatase RhoA. RhoA has at least three different length mRNA species, each varying in the length of the 3′ untranslated region. In situ hybridisation and immunocytochemistry analysis of RhoA expression show marked up‐regulation in the heart‐forming region. In other systems, RhoA signalling has been shown to be important for both gene expression and morphology. To investigate the function of RhoA in early heart development, we used small interfering RNAs (siRNA) in early chick embryos. Disruption of RhoA expression by siRNA treatment resulted in lack of heart tube fusion and abnormal head development. These data indicate that RhoA is important for normal embryogenesis. Developmental Dynamics 227:35–47, 2003.

Failure of microtubule-mediated peroxisome division and trafficking in disorders with reduced peroxisome abundance

In contrast to peroxisomes in normal cells, remnant peroxisomes in cultured skin fibroblasts from a subset of the clinically severe peroxisomal disorders that includes the biogenesis disorder Zellweger syndrome and the single-enzyme defect D-bifunctional protein (D-BP) deficiency, are enlarged and significantly less abundant. We tested whether these features could be related to the known role of microtubules in peroxisome trafficking in mammalian cells. We found that remnant peroxisomes in fibroblasts from patients with PEX1-null Zellweger syndrome or D-BP deficiency exhibited clustering and loss of alignment along peripheral microtubules. Similar effects were observed for both cultured embryonic fibroblasts and brain neurons from a PEX13-null mouse with a Zellweger-syndrome-like phenotype, and a less-pronounced effect was observed for fibroblasts from an infantile Refsum patient who was homozygous for a milder PEX1 mutation. By contrast, such changes were not seen for patients with peroxisomal disorders characterized by normal peroxisome abundance and size. Stable overexpression of PEX11β to induce peroxisome proliferation largely re-established the alignment of peroxisomal structures along peripheral microtubules in both PEX1-null and D-BP-deficient cells. In D-BP-deficient cells, peroxisome division was apparently driven to completion, as induced peroxisomal structures were similar to the spherical parental structures. By contrast, in PEX1-null cells the majority of induced peroxisomal structures were elongated and tubular. These structures were apparently blocked at the division step, despite having recruited DLP1, a protein necessary for peroxisome fission. These findings indicate that the increased size, reduced abundance, and disturbed cytoplasmic distribution of peroxisomal structures in PEX1-null and D-BP-deficient cells reflect defects at different stages in peroxisome proliferation and division, processes that require association of these structures with, and dispersal along, microtubules.

On the role of the peroxisome in ontogeny, ageing and degenerative disease.

This article reviews the available data on the role of the peroxisome in the growth, differentiation and degeneration of mammalian tissues. Developmental progressions of peroxisomes are described, along with the influence of inhibitors of peroxisomal enzymes, peroxisome proliferators and morphogenetic agents on the ontogeny of experimental animals. The role of the peroxisome in protecting tissues from damage by oxygen free radicals is also described, as is the changing role of the peroxisome in the ageing animal. Amongst the degenerative diseases which have been associated with free radical damage are cancer, atherosclerosis, muscular dystrophy, rheumatoid arthritis and the senile degeneration of brain function. In all these conditions, the major characteristics of molecular damage have been considered, along with the particular role of the peroxisome in alleviating these effects. Proposals for further research into peroxisomal function during ontogeny and the degenerative changes associated with ageing are developed, and the possibility of palliative treatments discussed.

On the multiplicity of the enzyme catalase in mammalian liver

SummaryThe literature on the complex multiplicity of mammlian catalase and the nature of the epigenetic modifications undergone by this enzyme has been reviewed, along with relevant comment on the subcellular localization and biological role of the enzyme.The epigenetic causations of multiplicity are established as being multifactorial and include oxidoreductive conversions of sulphydryl groups, the covalent attachment of carbohydrate, and partial proteolysis of the enzyme. Each of these epigenetic transformations may give rise to sets of multiple forms, and overlaps between these separate sets may give rise to extremely complex multiplicity patterns.It is concluded that any interpretation of catalase multiplicity which places emphasis on a single epigenetic causation is not compatible with the scope and variety of the available data on this enzyme. Instead, a holistic approach is urged — one giving due emphasis to the multiple causation of catalase multiplicity, and the interrelationships of these causations in the cellular situation. Rather than viewing the multiplicity of this enzyme as merely a series of interesting chemical modifications, emphasis is directed towards the fact that catalase heterogeneity povides a sensitive indication of the functional variations which occur within separate compartments of the subcellular structure, and hence becomes an essential element in any satisfactory understanding of the role of this enzyme in cellular processes.

The role of peroxisomes in lipid metabolism

Abstract Recent studies of lipid turnover in living mammals support the concept that peroxisomes have a significant physiological role in the metabolism of neutral lipids. These findings also raise intriguing questions about the influence of peroxisomes on phospholipid catabolism, the biosynthesis of complex lipids, and inter-tissue and inter-compartmental communication.

Sequential alterations in the micro-localization of catalase in mouse liver after treatment with hypolipidemic drugs.

SummaryA comparative study has been carried out on the micro-localization of catalase in mouse tissues subsequent to treatment with a representative range of hypolipidemic drugs.A commonality of effect was shown by clofibrate (ethyl-α-p-chlorophenoxyisobutyrate), Wy-14,643 (4-chloro-6-[2,3 xylidino)-2-pyrimidinylthio] acetic acid), RMI-15,414 (5-tetradecyloxy-2-furancarboxylic acid) and aspirin (acetyl salicylic acid), in that treatments with each of these drugs was associated with the release of peroxisomal catalase into the cytoplasmic compartment of liver and kidney. It was also noticeable that this increased cytosolic activity was characterized by the presence of an ‘aged’ form of the enzyme with different mobility and activity characteristics to that of the peroxisomal enzyme.Possible molecular bases for these effects and their relationship to peroxisomal biogenesis are discussed.