M-LP,Mpv17-like protein,has a peroxisomal membrane targeting signal comprising a transmembrane domain and a positively charged loop and up-regulates expression of the manganese superoxide dismutase gene

M-LP (Mpv17-like protein) has been identified as a new protein that has high sequence homology with Mpv17 protein, a peroxisomal membrane protein involved in the development of early onset glomerulosclerosis. In this study, we verified the peroxisomal localization of M-LP by performing dual-color confocal analysis of COS-7 cells cotransfected with green fluorescent protein-tagged M-LP and DsRED2-PTS1, a red fluorescent peroxisomal marker. To characterize the peroxisomal membrane targeting signal, we examined the intracellular localizations of several green fluorescent protein-tagged deletion mutants and demonstrated that, of the three transmembrane segments predicted, the first near the NH(2) terminus and NH(2)-terminal half of the following loop region, which is abundant in positively charged amino acids, were necessary and sufficient for peroxisomal targeting. To elucidate the function of M-LP, we examined the activities of several enzymes involved in reactive oxygen species metabolism in COS-7 cells and found that transfection with M-LP increased the superoxide dismutase activity significantly. Quantitative real-time PCR analysis revealed that the manganese SOD (SOD2) mRNA level of COS-7 cells transfected with M-LP was elevated. These results indicate that M-LP participates in reactive oxygen species metabolism.     

A novel alternative spliced Mpv17-like protein isoform localizes in cytosol and is expressed in a kidney- and adult-specific manner

Mpv17-like protein (M-LP) has been identified as a new protein that shows high sequence homology with Mpv17 protein, a peroxisomal membrane protein involved in the development of early onset glomerulosclerosis. We previously showed that the originally identified M-LP isoform, designated M-LPL, is, like Mpv17, localized in peroxisomes, and that transfection with M-LPL up-regulates expression of the manganese superoxide dismutase (SOD2) gene [R. Iida, T. Yasuda, E. Tsubota, H. Takatsuka, M. Masuyama, T. Matsuki, K. Kishi, M-LP, Mpv17-like protein, has a peroxisomal membrane targeting signal comprising a transmembrane domain and a positively charged loop and up-regulates expression of the manganese superoxide dismutase gene. J. Biol. Chem. 278 (2003) 6301-6306.]. We report here the identification of a novel alternative splicing product of the M-LP gene, designated M-LPS. A comparison of the genomic sequence with the cDNA sequences and an analysis of 5'-flanking regions revealed that the two isoforms are generated by alternative usage of two promoters. M-LPS consists of the C-terminal half of M-LPL (90 amino acids) and therefore lacks the peroxisome targeting signal of membrane protein that exists near the N-terminus of M-LPL. Expression of green fluorescent protein-tagged M-LPS in COS-7 cells demonstrated that M-LPS localizes in the cytosol. In mice, M-LPS is expressed exclusively in kidneys after the age of 6 weeks. Moreover, quantitative real-time PCR analysis revealed that transfection with M-LPS up-regulates expression of the SOD2 gene and down-regulates expression of the cellular glutathione peroxidase (Gpx1) and plasma glutathione peroxidase (Gpx3) genes. Taken together, these results suggest different functional attributes of the two M-LP isoforms during aging and development.     

The glomerulosclerosis gene Mpv17 encodes a peroxisomal protein producing reactive oxygen species.

The mutant mouse strain Mpv17 carries a retroviral insert in its genome which inactivates the Mpv17 gene. At a young age these mice develop glomerulosclerosis and nephrotic syndrome which resembles human disease. We show here that the Mpv17 gene product is highly conserved and encodes a peroxisomal protein. Loss of the Mpv17 protein does not impair peroxisome biogenesis but instead leads to a reduced ability to produce reactive oxygen species (ROS). In turn, overproduction of the Mpv17 gene in transfected cells results in dramatically enhanced levels of intracellular ROS indicating a direct involvement of Mpv17 in ROS production. These data reveal a role for the Mpv17 protein in peroxisomal reactive oxygen metabolism and establish a novel link between peroxisomal ROS production and glomerulosclerosis.     

Expression of the Recessive Glomerulosclerosis Gene Mpv17 Regulates MMP-2 Expression in Fibroblasts, the Kidney, and the Inner Ear of Mice

The recessive mouse mutant Mpv17 is characterized by the development of early-onset glomerulosclerosis, concomitant hypertension, and structural alterations of the inner ear. The primary cause of the disease is the loss of function of the Mpv17 protein, a peroxisomal gene product involved in reactive oxygen metabolism. In our search of a common mediator exerting effects on several aspects of the phenotype, we discovered that the absence of the Mpv17 gene product causes a strong increase in matrix metalloproteinase 2 (MMP-2) expression. This was seen in the kidney and cochlea of Mpv17-negative mice as well as in tissue culture cells derived from these animals. When these cells were transfected with the human Mpv17 homolog, an inverse causal relationship between Mpv17 and MMP-2 expression was established. These results indicate that the Mpv17 protein plays a crucial role in the regulation of MMP-2 and suggest that enhanced MMP-2 expression might mediate the mechanisms leading to glomerulosclerosis, inner ear disease, and hypertension in this model.     

Enhanced gamma-glutamyl transpeptidase expression and superoxide production in Mpv17-/- glomerulosclerosis mice.

Recently, gamma-glutamyl transpeptidase, which initiates cleavage of extracellular glutathione, has been shown to promote oxidative damage to cells. Here we examined a murine disease model of glomerulosclerosis, involving loss of the Mpv17 gene coding for a peroxisomal protein. In Mpv17-/- cells, enzyme activity and mRNA expression (examined by quantitative RT-PCR) of membrane-bound gamma-glutamyl transpeptidase were increased, while plasma glutathione peroxidase and superoxide dismutase levels were lowered. Superoxide anion production in these cells was increased as documented by electron spin resonance spectroscopy. In the presence of Mn(III)tetrakis(4-benzoic acid)porphyrin, the activities of gamma-glutamyl transpeptidase and plasma glutathione peroxidase were unchanged, suggesting a relationship between enzyme expression and the amount of reactive oxygen species. Inhibition of gamma-glutamyl transpeptidase by acivicin reverted the lowered plasma glutathione peroxidase and superoxide dismutase activities, indicating reciprocal control of gene expression for these enzymes.     

Abnormal basement membrane in the inner ear and the kidney of the Mpv17-/- mouse strain: ultrastructural and immunohistochemical investigations

The loss of the function of the peroxisomal Mpv17-protein and associated imbalanced radical oxygen species (ROS) homeostasis leads to an early onset of focal segmental glomerulosclerosis and sensorineural deafness associated with severe degeneration of cochlear structures. An excessive enlargement of basal laminae of the stria vascularis capillaries and glomeruli indicates numerous changes in their molecular composition. The basement membrane (BM) of the glomeruli and the stria vascularis are simultaneously affected in early stages of the disease and the lamination, splitting of the membrane and formation of the “basket weaving” seen at the onset of the disease in the kidney are similar to the ultrastructural alterations characteristic for Alportȁ9s syndrome. The progressive alteration of the BMs is accompanied by irregularity in the distribution of the collagen IV subunits and by an accumulation of the laminin B2(γ1) in the inner ear and B(β1) in the kidney. Since Mpv17 protein contributes to ROS homeostasis, further studies are necessary to elucidate downstream signaling molecules activated by ROS. These studies explain the cellular responses to missing Mpv17-protein, such as accumulation of the extracellular matrix, degeneration, and apoptosis in the inner ear.     

The human PEX3 gene encoding a peroxisomal assembly protein: genomic organization, positional mapping, and mutation analysis in candidate phenotypes

In yeasts, the peroxin Pex3p was identified as a peroxisomal integral membrane protein that presumably plays a role in the early steps of peroxisomal assembly. In humans, defects of peroxins cause peroxisomal biogenesis disorders such as Zellweger syndrome. We previously reported data on the human PEX3 cDNA and its protein, which in addition to the peroxisomal targeting sequence contains a putative endoplasmic reticulum targeting signal. Here we report the genomic organization, sequencing of the putative promoter region, chromosomal localization, and physical mapping of the human PEX3 gene. The gene is composed of 12 exons and 11 introns spanning a region of approximately 40 kb. The highly conserved putative promoter region is very GC rich, lacks typical TATA and CCAAT boxes, and contains potential Sp1, AP1, and AP2 binding sites. The gene was localized to chromosome 6q23-24 and D6S279 was identified to be the closest positional marker. As yeast mutants deficient in PEX3 have been shown to lack peroxisomes as well as any peroxisomal remnant structures, human PEX3 is a candidate gene for peroxisomal assembly disorders. Mutation analysis of the human PEX3 gene was therefore performed in fibroblasts from patients suffering from peroxisome biogenesis disorders. Complementation groups 1, 4, 7, 8, and 9 according to the numbering system of Kennedy Krieger Institute were analyzed but no difference to the wild-type sequence was detected. PEX3 mutations were therefore excluded as the molecular basis of the peroxisomal defect in these complementation groups.     

Missing mitochondrial Mpv17 gene function induces tissue-specific cell-death pathway in the degenerating inner ear

The Mpv17 gene encodes a mitochondrial inner-membrane protein that has been implicated in the metabolism of reactive oxygen species. The loss of function in Mpv17-/- mice leads to early sensorineural deafness associated with severe inner ear degeneration and late onset of kidney failure. The present study demonstrates that the onset of the degeneration of the cochlear neuroepithelia is related to the onset of auditory function and appears to be first restricted to the outer hair cells (OHC), which subsequently undergo rapid degeneration. At the age of 18 days, the OHC lateral membrane degenerates and extensive vacuolization of the cytoplasm is followed by lysis of the OHCs. Such degenerative processes have been seen for the first time in relation to auditory dysfunction. The structural degeneration pattern of the OHC appears to be similar to the described paraptotic processes (an alternative form of programmed cell death) discussed in the literature as a cause of cytoplasmic neurodegeneration. In contrast, the melanocyte-like intermediate cells that are of neural crest origin and that are located in the stria vascularis, undergo apoptosis, as documented ultrastructurally. A lack of Mpv17 protein function in mitochondria thus seems to initiate tissue-specific cell-death pathways resulting in the pathology seen during the degeneration process.     

Identification and characterization of mouse MTO1 gene related to mitochondrial tRNA modification

The nucleotide modification in tRNA plays a pivotal role in the fidelity of translational process. The defects in nucleotide modification have often been observed in the mutated mitochondrial tRNAs associated with human diseases. Recently, MTO1-like protein in bacteria and yeast has been implicated to be a component of tRNA modification pathway. Here we report the identification and characterization of mouse MTO1 homolog. The mouse MTO1 gene containing 12 exons encodes a 669-residue protein with a strong homology to the MTO1-like proteins of bacteria and yeast, related to tRNA modification. Functional conservation of this protein is supported by the observation that the isolated mouse MTO1 cDNA can complement the respiratory-deficient phenotype of yeast mto1 cells carrying P(R)(454) mutation. MTO1 is ubiquitously expressed in various tissues, but with markedly elevated expression in tissues of high metabolic rates. Furthermore, we showed that mouse Mto1 localizes in mitochondrion. These observations suggest that the mouse MTO1 is a structural and functional homolog of yeast MTO1, thereby playing a role in the mitochondrial tRNA modification and protein synthesis.     

Isolation of human retinal genes: recoverin cDNA and gene.

A human retina cDNA library enriched for retina-specific clones was prepared by subtraction with a non-retina population of cDNA in combination with polymerase chain reaction (PCR) amplifications. A highly retina-specific cDNA clone (1190 bp) was obtained through this library encoding a 200 amino acid protein with three calcium binding sites and 87% homology to the bovine photoreceptor protein, recoverin, which has been shown to mediate the recovery of the dark current after photoactivation, and 58% homology to the calcium-binding chick cone protein, visinin. Analysis of the gene indicated a 9-10 kb single-copy gene with at least three exons and two introns. The three exons contained the entire coding sequence, and all of the calcium-binding EF-hand regions were in putative exon 1. The recoverin gene was mapped to human chromosome 17 by hybridization to a panel of human-rodent hybrid DNAs.     

Osteogenic role of endosomal chloride channels in MC3T3-E1 cells

PHR protein family consists of C. elegan Rpm-1/Drosophila Highwire/Zebrafish Esrom/Mouse Phr-1/Human Pam. Esrom is required for correct neurites exiting the paused state at intermediate targets as well as pteridine synthesis. This study reports the identification and characterization of two novel Esrom splice variants, named splice variants 2 (splicing out 5′ 24 bp of exon 17) and 3 (splicing out 5′ 24 bp of exons 17 and 18). Polypeptides encoded by 5′ 24 bp of exons 17 and 18 are part of basic amino-acid-rich region inside Esrom RCC1-like domain (RLD). These two splice variants maintain the whole protein reading frame and alternative exons usage patterns are conserved with mammal. At different developmental stages and adult zebrafish tissues, abundances of these splice variants are different. Importantly, by yeast two-hybrid screen and confocal colocalization analysis, it was found that alternative splicing of exon 18 regulates Esrom RLD interaction with kinesin family member 22 and G protein beta-subunit 1. Taken together, these results suggest that Esrom RLD functions are regulated by alternative splicing at temporal and spatial-specific manner.