Expression of the human TIMM23 and TIMM23B genes is regulated by the GABP transcription factor.

The TIM23 protein is a key component of the mitochondrial import machinery in yeast and mammals. TIM23 is the channel-forming subunit of the translocase of the inner mitochondrial membrane (TIM23) complex, which mediates preprotein translocation across the mitochondrial inner membrane. In this paper, we aimed to characterize the promoter region of the highly similar human TIM23 orthologs: TIMM23 and TIMM23B. Bioinformatic analysis revealed putative sites for the GA-binding protein (GABP) and the recombination signal binding protein for immunoglobulin kappa J (RBPJ) transcription factors in both promoters. Luciferase reporter assays, electrophoretic mobility shift assays, and chromatin immunoprecipitation experiments showed three functional sites for GABP and one functional site for RBPJ in both promoters. Moreover, silencing of GABPA, the gene encoding the DNA-binding subunit of the GABP transcription factor, resulted in reduced expression of TIMM23 and TIMM23B. Our results show an essential role of GABP in activating TIMM23 expression. More broadly, they suggest that physiological signals involved in activating mitochondrial biogenesis and oxidative function also enhance the transcription but not the protein level of TIMM23, which is essential for maintaining mitochondrial function and homeostasis.

In vitro methylation of nuclear respiratory factor-2 binding sites suppresses the promoter activity of the human TOMM70 gene.

TOMM70 is a subunit of the outer mitochondrial membrane translocase that plays a major role as a receptor of hydrophobic preproteins targeted to mitochondria. We have previously reported that two binding sites for transcription factor NRF-2 in the promoter region of the human TOMM70 gene are essential in activating transcription (Blesa et al., Mitochondrion 2004; 3:251-59. Blesa et al., Biochem Cell Biol 2006; 84:813-22). This region contains thirteen CpG methylation sites, three of which occur in the sequence 5'-CCGG-3' that is specifically recognized by HpaII methylase which modifies the internal cytosine residue. Interestingly, each NRF-2 site contains one CCGG sequence, allowing specific methylation of the NRF-2 sites and, therefore, providing an ideal model to study how methylation of these sites affects promoter activity. In this paper we report that site-specific methylation of the NRF-2 binding sites in the TOMM70 promoter down-regulated expression of a luciferase reporter in HeLa S3 cells. Electrophoretic mobility shift assays confirmed abrogation of NRF-2 binding at the methylated sites. These results suggest that methylation of the TOMM70 promoter in mammalian cells may silence TOMM70 expression. However, studies of methylation degree on DNAs from different sources found no methylation in the promoter regions of TOMM70 and other TOMM/TIMM family genes. Thus, although in vitro methylation inactivates the expression of TOMM70, our results suggest that this is not the mechanism modulating its expression in vivo. Since a number of nuclear genes encoding mitochondrial translocases have NRF-2 binding sequences containing CpG methylation sites, a possible role of methylation as a regulatory mechanism of mitochondrial biogenesis can be ruled out.

NRF-1 is the major transcription factor regulating the expression of the human TOMM34 gene.

The human TOMM34 gene encodes a cytosolic protein with chaperone-like activity that helps import some preproteins to the mitochondria by keeping them in an unfolded, import-compatible state. TOMM34 was found to be upregulated frequently in colorectal tumors, suggesting that it also has a role in the growth of cancer cells. In this context, TOMM34 is a potential target for novel anticancer drugs, and it might also be used in the diagnosis of colorectal cancer. Nuclear respiratory factors (NRFs) play an important role in governing the nuclear-mitochondrial interactions implicated in mitochondrial biogenesis. Our previous studies revealed that NRFs promote the expression of the major members of the mitochondrial transport machinery, TOMM70 and TOMM20. Here we report the existence of binding sites for NRF-1, Sp1, and NRF-2 in the 5' region of the human TOMM34 gene. We determined the effects of mutations at these sites on promoter activity in HeLa S3 and A204 cells, in conjunction with chromatin immunoprecipitation experiments, electrophoretic mobility shift assays, and in vivo methylation analysis of the promoter region. We conclude that NRF-1 is the main transcription factor regulating the expression of TOMM34. Sp1 interacts with NRF-1 to stimulate the promoter's full activity.

Molecular genetics of a patient with Mohr-Tranebjaerg Syndrome due to a new mutation in the DDP1 gene.

The deafness-dystonia syndrome (DDS) or Mohr-Tranebjaerg syndrome (MTS, MIM 304700) is a rare X-linked recessive neurological disorder resulting from loss-of-function mutations in the nuclear DDP1/TIMM8A gene, involved in the transport and sorting of proteins to the mitochondrial inner membrane. A Mohr-Tranebjaerg patient and his mother were subjected to clinical and molecular studies. Screening of mutations were performed in TIMM8A, TIMM13, and other mitochondrial protein transport genes by conformation sensitive gel electrophoresis (CSGE), followed by direct DNA sequencing of tissue samples from the patient. Mitochondrial DNA of the patient was also sequenced at the genes for COX subunits and some mitochondrial tRNAs. Respiratory chain activities in a muscle biopsy and cultured fibroblasts from the patient were assessed using biochemical methods. mRNA expression of TIMM8A and TIMM13 was determined by RT-PCR in cultured fibroblasts. We identified a new case of Mohr-Tranebjaerg syndrome and report the characteristics of a new pathogenic de novo mutation (c.112C>T, pGln38X) in the TIMM8A gene. Biochemical measures of respiratory chain complex activities in muscle biopsy and fibroblasts did not show a major deficiency or alteration. mRNA expression studies demonstrated increased TIMM8A mRNA levels in cultured fibroblasts from the patient. Phenotypic differences among published cases seem not to be related with the mutation location or type. Our results support the idea that dysfunctions of mitochondrial protein transport, in addition to OXPHOS deficiency, can be the basis of important mitochondrial pathologies.

NRF-2 transcription factor is required for human TOMM20 gene expression.

TOMM20 is a subunit of the outer mitochondrial membrane translocase that plays a major role as a receptor of precursor proteins with N-terminal cleavable presequences targeted to the mitochondria. Nuclear respiratory factors 1 and 2 (NRF-1 and NRF-2) play an important role in governing nucleo-mitochondrial interactions implicated in mitochondrial biogenesis. It was recently reported that NRF-2 is critical for maintaining normal transcriptional levels of the TOMM20 gene, but the promoter of this gene is uncharacterized. We report the presence of a NRF-2 and two NRF-1 binding motifs in the 5'-flanking region of the human TOMM20 gene and provide insight into their roles for promoter activity by using chromatin immunoprecipitation experiments and reporter assays in HeLa S3 and A204 cells. We show that only NRF-2 and the proximal NRF-1 motifs are involved in the expression of the gene. The NRF-2 binding site is required to activate transcription. The proximal NRF-1 site cooperates with NRF-2 in regulating the expression of the gene. The distal NRF-1 binding site is not functional.

Distinct functional contributions of 2 GABP-NRF-2 recognition sites within the context of the human TOMM70 promoter.

TOMM70 is a subunit of the outer mitochondrial membrane translocase that plays a major role as a receptor of hydrophobic preproteins targeted to mitochondria. We have previously reported 2 binding sites for the transcription factor GABP-NRF-2 in the promoter region of the human TOMM70 gene that are important in activating transcription. To assess the functionality and actual role of these sites, chromatin immunoprecipitation, site-directed mutagenesis, and electrophoretic mobility shift assays were carried out. We conclude that GABP-NRF-2 binds in vivo to the TOMM70 promoter, and that the 2 GABP-NRF-2 binding sites of the promoter have different functional contributions in promoting TOMM70 expression. Evidence is provided that they work in an additive manner as single sites.

Conformation-sensitive gel electrophoresis as an ideal high-throughput strategy for accurate detection of sequence variations in DNA: screening hTomm and hTimm genes.

Conformation-sensitive gel electrophoresis is a heteroduplex-based method that is particularly well suited to high-throughput analyses. Its simplicity makes it amenable to various adaptations and modifications to enhance its applicability to genome-wide mutation scans. Technical aspects that markedly improve the conformation-sensitive gel electrophoresis performance by combining high throughput and high resolution of the bands facilitating the interpretation of the results are described here. The authors report some of the results they have obtained in the screening of the exon 1 of human Timm8A gene as an example of the suitability of the conformation-sensitive gel electrophoresis-based protocol that has been adapted to optimize its throughput, speed, and simplicity in the recognition of both heterozygous and homozygous DNA mutations. The higher throughput is achieved by using 12 batches per gel. The length of the gel is sufficient for an adequate well-to-read distance for each batch that allows a clear distinction and resolution of the conformation-sensitive gel electrophoresis bands. Standardization of the procedure using multichannel pipettes reduces the preparation time of the 96-well PCRs to 10 min and also accelerates the gel loading. The resulting bands give high-quality images, allowing easy detection of known as well as novel mutations.

NRF-2 transcription factor is essential in promoting human Tomm70 gene expression.

TOMM70 is a subunit of the outer mitochondrial membrane translocase that plays a major role as a receptor of hydrophobic pre-proteins targeted to the mitochondria. We report the presence of two nuclear respiratory factor 2 (NRF-2) binding motifs in the 5'-flanking region of the human Tomm70 gene and establish their essential role for promoter activity by using reporter assays in HeLa cells. We show that both NRF-2 binding sites are functional and present evidence that interactions between these sites and a CpG island contribute to expression. Mobility shift assays show that these NRF-2 sites are specifically recognized by NRF-2 present in HeLa nuclear extracts.

Report of hereditary persistence of alpha-fetoprotein in a Spanish family: molecular basis and clinical concerns.

The serum level of alpha-fetoprotein in normal adults is lower than 10 ng/ml. High levels of alpha-fetoprotein in adults are linked to cirrhosis, acute or chronic hepatitis, hepatocellular carcinomas and other pathologies, as well as to foetal malformation, and this protein is therefore used as a regular clinical marker for these diseases. We report a Spanish family in which very high levels of alpha-fetoprotein have been detected in nine members from the screening of a total of 17 relatives. These levels of alpha-fetoprotein are not accompanied by a causing pathology, are inherited as an autosomal dominant genetic trait, and are associated to a G-->A substitution at position -116 of the 5'-flanking region of the alpha-fetoprotein gene. This is an unusual benign trait of hereditary persistence of alpha-fetoprotein. This paper provides a detailed clinical report of the family including a study of the molecular basis of this trait. The desirability of a test to detect and/or rule out this benign trait in adults with abnormal levels of alpha-fetoprotein is considered.

Manifestaciones fenotípicas de la cistinuria: estudio de 20 familias en la Comunidad Valenciana / Phenotypical traits in cystinuria: a study of 20 families in the Comunidad Valenciana (Spain)

Fundamento: El objetivo de este trabajo fue realizar una caracterización fenotípica de los pacientes con cistinuria en la Comunidad Valenciana en el contexto de su genealogía y abordar desde esta perspectiva la amplia heterogeneidad de esta enfermedad. Sujetos y método: A partir de 29 individuos que habían sido diagnosticados de cistinuria con anterioridad a este estudio se logró la colaboración de 20 familias. Se recogió una muestra de orina de cada individuo, se analizó el sedimento y se cuantificaron los aminoácidos mediante cromatografía de alta resolución. El análisis genético se llevó a cabo mediante reacción en cadena de la polimerasa (PCR) y análisis de patrones de polimorfismos de restricción (RFLP). Mediante un cuestionario se obtuvieron las variables demográficas y clínicas. Resultados: De las 20 familias participantes en el estudio, se clasificaron 4 como tipo I, 10 como tipo no I, y 6 como de tipo desconocido. Los pacientes con cistinuria tipo I presentaron valores de cistina y ornitina superiores. Asimismo, se asoció la presencia de cristales de cistina y de la mutación M467T en el gen SLC3A1 con el tipo familiar I. Sin embargo, no se encontró un mayor riesgo de nefrolitiasis asociado al tipo familiar. Conclusiones: La caracterización fenotípica de los pacientes con cistinuria en el contexto de su genealogía nos ha permitido constatar la amplia variabilidad de manifestaciones fenotípicas incluso dentro del mismo patrón de transmisión. Esta variabilidad podría ser debida a la heterogeneidad genética y ambiental que ha desencadenado esta enfermedad y modulado su evolución. (AU)

Diffuse neuroendocrine system and mitochondrial diseases: molecular and cellular bases of pathogenesis, new approaches to diagnosis and therapy.

Structural and functional alterations of mitochondria have been shown to be responsible for a wide variety of clinical disorders that are referred to as "mitochondrial diseases." It is now obvious that many factors are involved in transport of mitochondrial proteins including cytokines, chaperones, chemokines, neurosteroids, ubiquitin and many others. At the same time the participation and the role of biogenic amines and peptide hormones (which are produced by the diffuse neuroendocrine system cells located in different organs) in endogenous mechanisms of mitochondrial diseases are still unknown. Taking into account the wide spectrum of biological effects of biogenic amines and peptide hormones, and especially their regulatory role for intercellular communication, it seems important to analyze the possible participation of these molecules in the pathogenesis of mitochondrial disorders as well as to draw up new ways for elaboration of new markers for lifetime diagnosis, definition of prognosis and efficiency of specific therapy in neurodegenerative diseases.

Tau-protein expression in human blood lymphocytes: a promising marker and suitable sample for life-time diagnosis of Alzheimer's disease.

OBJECTIVES: Taking into account the hypothesis that Alzheimer's disease (AD) might be a systemic disease that affects several tissues in the body, the aim of this study was to try to detect the expression of tau-protein in human peripheral blood lymphocytes (PBL) in patients with AD. MATERIAL AND METHODS: Blood samples were obtained from patients with AD (n=16, age 67-98) and from volunteers without psychoneurological pathology (n=10, age 65-78). PBL were isolated on Ficoll-Paque gradient centrifugation. For cell fixation and permeabilization we used a fixative solution (4% formaldehyde and 0.1% glutaraldehyde) and 0.03% Triton X-100. Immunocytochemical detection of tau-protein was carried out by biotin-streptavidin complex method with tau monoclonal antibody (1:100, clone TAU-2, ICN) and universal immunostaining kit IMMU-MARK (ICN). RESULTS: The expression of tau-protein was shown in PBL in absolute majority of AD patients studied. Only in two healthy volunteers a single lymphocyte from many cells (i.e. a smear) demonstrated a very weak-positive immunostaining to tau-protein CONCLUSION: This first demonstration of clear difference in localization of tau-protein in blood lymphocytes between healthy and sick people testifies to the fact that tau-protein could be considered as a promising marker and blood lymphocytes as a suitable sample for life-time diagnosis of AD.