A meta-analysis of cancer risk associated with the TP53 intron 3 duplication polymorphism (rs17878362): geographic and tumor-specific effects.

We have performed a meta-analysis of cancer risk associated with the rs17878362 polymorphism of the TP53 suppressor gene (PIN3, (polymorphism in intron 3), 16 bp sequence insertion/duplication in intron 3), using a compilation of a total of 25 published studies with 10 786 cases and 11 760 controls. Homozygote carriers of the duplicated allele (A2A2) had a significantly increased cancer risk compared with A1A1 carriers (aggregated odds ratio (OR) = 1.45, 95% confidence interval (CI) = 1.22-1.74). However, there was no significant effect for the A1A2 heterozygotes (A1A2 versus A1A1 aggregated OR = 1.08, 95% CI = 0.99-1.18). No significant heterogeneity or publication bias was detected in the data set analysed. When comparing populations groups, increased cancer risk was associated with A2A2 carriage in Indian, Mediterranean and Northern Europe populations but not in the Caucasian population of the United States. Analysis by cancer site showed an increased risk for A2A2 carriers for breast and colorectal, but not for lung cancers. These results support that the A2A2 genotype of rs17878362 is associated with increased cancer risk, with population and tumour-specific effects.

Biological functions of p53 isoforms through evolution: lessons from animal and cellular models.

The TP53 tumour-suppressor gene is expressed as several protein isoforms generated by different mechanisms, including use of alternative promoters, splicing sites and translational initiation sites, that are conserved through evolution and within the TP53 homologues, TP63 and TP73. Although first described in the eighties, the importance of p53 isoforms in regulating the suppressive functions of p53 has only become evident in the last 10 years, by analogy with observations that p63 and p73 isoforms appeared indispensable to fully understand the biological functions of TP63 and TP73. This review summarizes recent advances in the field of 'p53 isoforms', including new data on p63 and p73 isoforms. Details of the alternative mechanisms that produce p53 isoforms and cis- and trans-regulators identified are provided. The main focus is on their biological functions (apoptosis, cell cycle, aging and so on) in cellular and animal models, including mouse, zebrafish and Drosophila. Finally, the deregulation of p53 isoform expression in human cancers is reviewed. Based on these latest results, several developments are expected in the future: the identification of drugs modulating p53 isoform expression; the generation of animal models and the evaluation of the use of p53 isoform as biomarkers in human cancers.

Expression and lysosomal targeting of CLN7, a major facilitator superfamily transporter associated with variant late-infantile neuronal ceroid lipofuscinosis.

Neuronal ceroid lipofuscinoses (NCLs) constitute a group of progressive neurodegenerative disorders resulting from mutations in at least eight different genes. Mutations in the most recently identified NCL gene, MFSD8/CLN7, underlie a variant of late-infantile NCL (vLINCL). The MFSD8/CLN7 gene encodes a polytopic protein with unknown function, which shares homology with ion-coupled membrane transporters. In this study, we confirmed the lysosomal localization of the native CLN7 protein. This localization of CLN7 is not impaired by the presence of pathogenic missense mutations or after genetic ablation of the N-glycans. Expression of chimeric and full-length constructs showed that lysosomal targeting of CLN7 is mainly determined by an N-terminal dileucine motif, which specifically binds to the heterotetrameric adaptor AP-1 in vitro. We also show that CLN7 mRNA is more abundant in neurons than astrocytes and microglia, and that it is expressed throughout rat brain, with increased levels in the granular layer of cerebellum and hippocampal pyramidal cells. Interestingly, this cellular and regional distribution is in good agreement with the autofluorescent lysosomal storage and cell loss patterns found in brains from CLN7-defective patients. Overall, these data highlight lysosomes as the primary site of action for CLN7, and suggest that the pathophysiology underpinning CLN7-associated vLINCL is a cell-autonomous process.

p53 regulates the transcription of its Delta133p53 isoform through specific response elements contained within the TP53 P2 internal promoter.

The tumor suppressor p53 protein is activated by genotoxic stress and regulates genes involved in senescence, apoptosis and cell-cycle arrest. Nine p53 isoforms have been described that may modulate suppressive functions of the canonical p53 protein. Among them, Delta133p53 lacks the 132 proximal residues and has been shown to modulate p53-induced apoptosis and cell-cycle arrest. Delta133p53 is expressed from a specific mRNA, p53I4, driven by an alternative promoter P2 located between intron 1 and exon 5 of TP53 gene. Here, we report that the P2 promoter is regulated in a p53-dependent manner. Delta133p53 expression is increased in response to DNA damage by doxorubicin in p53 wild-type cell lines, but not in p53-mutated cells. Chromatin immunoprecipitation and luciferase assays using P2 promoter deletion constructs indicate that p53 binds functional response elements located within the P2 promoter. We also show that Delta133p53 does not bind specifically to p53 consensus DNA sequence in vitro, but competes with wild-type p53 in specific DNA-binding assays. Finally, we report that Delta133p53 counteracts p53-dependent growth suppression in clonogenic assays. These observations indicate that Delta133p53 is a novel target of p53 that may participate in a negative feedback loop controlling p53 function.

Molecular physiology and pathophysiology of lysosomal membrane transporters.

In contrast to lysosomal hydrolytic enzymes, the lysosomal membrane remains poorly characterized. In particular, although the genetic study of cystinosis and sialic acid storage disorders led to the identification of two lysosomal transporters for cystine and sialic acids, respectively, ten years ago, most transporters responsible for exporting lysosomal hydrolysis products to the cytosol are still unknown at the molecular level. However, two lines of investigation recently started to fill this gap in the knowledge of lysosomal biology. First, novel proteomic approaches are now able to provide a reliable inventory of lysosomal membrane proteins. On the other hand, a novel functional approach based on intracellular trafficking mechanisms allows direct transport measurement in whole cells by redirecting recombinant lysosomal transporters to the cell surface. After surveying the current state of knowledge in this field, the review focuses on the sialic acid transporter sialin and shows how recent functional data using the above whole-cell approach shed new light on the pathogenesis of sialic acid storage disorders by revealing the existence of a residual transport activity associated with Salla disease.

A Na(+)/Cl(-)-dependent transporter for catecholamines, identified as a norepinephrine transporter, is expressed in the brain of the teleost fish medaka (Oryzias latipes).

We report the isolation, functional characterization, and localization of a Na(+)/Cl(-)-dependent catecholamine transporter (meNET) present in the brain of the teleost fish medaka. This carrier is very similar to the human neuronal norepinephrine transporter (NET) and the human neuronal dopamine transporter (DAT), showing 70 and 64% amino acid identity, respectively. When expressed in COS-7 cells, this transporter mediates the high-affinity uptake of dopamine (K(M) = 290 nM) and norepinephrine (K(M) = 640 nM). Its pharmacological profile reveals more similarities with NET, including a high affinity for the tricyclic antidepressants desipramine (IC(50) = 0.92 nM) and nortriptyline (IC(50) = 16 nM). In situ hybridization on the medaka brain shows that meNET mRNA is present only in a subset of tyrosine hydroxylase-positive neurons found in the noradrenergic areas of the hindbrain, such as the locus ceruleus and area postrema. None of the dopaminergic areas anterior to the isthmus contains any labeled neurons. Neither reverse transcriptase-polymerase chain reaction with degenerate primers specific for gamma-aminobutyric acid transporter/NET nor autoradiographic experiments with [(125)I]3b-(4-iodophenyl)-tropane-2b-carboxylic acid methyl ester revealed an additional catecholamine transporter in the medaka brain. Uptake experiments with medaka brain synaptosomes show an endogenous transport with a pharmacological profile identical to that of the recombinant meNET. Thus, meNET is probably the predominant--if not the only--catecholamine transporter in the medaka fish brain. In view of the highly conserved primary structures and pharmacological properties of meNET, it is tempting to speculate that a specific dopamine transport developed later in vertebrate evolution and probably accompanied the tremendous enlargement of the meso-telencephalic dopaminergic pathways in amniotes.

Identification and characterization of a lysosomal transporter for small neutral amino acids.

In eukaryotic cells, lysosomes represent a major site for macromolecule degradation. Hydrolysis products are eventually exported from this acidic organelle into the cytosol through specific transporters. Impairment of this process at either the hydrolysis or the efflux step is responsible of several lysosomal storage diseases. However, most lysosomal transporters, although biochemically characterized, remain unknown at the molecular level. In this study, we report the molecular and functional characterization of a lysosomal amino acid transporter (LYAAT-1), remotely related to a family of H+-coupled plasma membrane and synaptic vesicle amino acid transporters. LYAAT-1 is expressed in most rat tissues, with highest levels in the brain where it is present in neurons. Upon overexpression in COS-7 cells, the recombinant protein mediates the accumulation of neutral amino acids, such as gamma-aminobutyric acid, l-alanine, and l-proline, through an H+/amino acid symport. Confocal microscopy on brain sections revealed that this transporter colocalizes with cathepsin D, an established lysosomal marker. LYAAT-1 thus appears as a lysosomal transporter that actively exports neutral amino acids from lysosomes by chemiosmotic coupling to the H+-ATPase of these organelles. Homology searching in eukaryotic genomes suggests that LYAAT-1 defines a subgroup of lysosomal transporters in the amino acid/auxin permease family.

Differential regulation of tyrosine hydroxylase in the basal ganglia of mice lacking the dopamine transporter.

Mice lacking the dopamine transporter (DAT) display biochemical and behavioural dopaminergic hyperactivity despite dramatic alteration in dopamine homeostasis. In order to determine the anatomical and functional integrity of the dopaminergic system, we examined the expression of tyrosine hydroxylase (TH), the rate-limiting enzyme of dopamine synthesis as well as DOPA decarboxylase and vesicular monoamine transporter. TH-positive neurons in the substantia nigra were only slightly decreased (-27.6 +/- 4.5%), which can not account for the dramatic decreases in the levels of TH and dopamine that we previously observed in the striatum. TH mRNA levels were decreased by 25% in the ventral midbrain with no modification in the ratio of TH mRNA levels per cell. However, TH protein levels were decreased by 90% in the striatum and 35% in the ventral midbrain. In the striatum, many dopaminergic projections had no detectable TH, while few projections maintained regular labelling as demonstrated using electron microscopy. DOPA decarboxylase levels were not modified and vesicular transporter levels were decreased by only 28.7% which suggests that the loss of TH labelling in the striatum is not due to loss of TH projections. Interestingly, we also observed sporadic TH-positive cell bodies using immunohistochemistry and in situ hybridization in the striatum of homozygote mice, and to some extent that of wild-type animals, which raises interesting possibilities as to their potential contribution to the dopamine hyperactivity and volume transmission previously reported in these animals. In conjunction with our previous findings, these results highlight the complex regulatory mechanisms controlling TH expression at the level of mRNA, protein, activity and distribution. The paradoxical hyperdopaminergia in the DAT KO mice despite a marked decrease in TH and dopamine levels suggests a parallel to Parkinson's disease implying that blockade of DAT may be beneficial in this condition.

The vesicular monoamine transporter: from chromaffin granule to brain.

All characterized monoaminergic cells utilize the same transport system for the vesicular accumulation of monoamines prior to their release. This system operates in neuronal (catecholaminergic, serotoninergic or histaminergic) as well as in endocrine or neuroendocrine cells. For several decades, chromaffin granules from bovine adrenal medulla have been used as a model system, allowing progress in the understanding of the biophysics, the biochemistry and the pharmacology of the monoamine vesicular transporter. The transporters from rat, bovine and man have been cloned. Surprisingly, two genes encode different isoforms of the protein which are differentially expressed in monoaminergic systems. The conjunction of recombinant DNA techniques and expression in secretory or non-secretory cells with the large body of data obtained on the chromaffin granule transporter has allowed rapid progress in the study of the protein. But interestingly enough, this progress has open new possibilities in the study of biological problems, especially in the brain. The transporter is useful for the determination of the relationship between small and large dense core vesicles, for the understanding of the mechanism of the drugs such as 1-methyl-4-phenylpyridinium (MPP+), tetrabenazine or amphetamines, and as a marker in brain development. The possibility of regulations at the vesicular transporter level and of their effect on the quantum size has to be investigated. The vesicular monoamine transporter is also an important target for brain imaging.

Cloning of a functional vesicular GABA and glycine transporter by screening of genome databases.

The unc-47 locus of Caenorhabditis elegans has been suggested to encode a synaptic vesicle GABA transporter. Here we used hydropathy plot analysis to identify a candidate vesicular GABA transporter in genomic sequences derived from a region of the physical map comprising unc-47. A mouse homologue was identified and cloned from EST database information. In situ hybridization in rat brain revealed codistribution with both GABAergic and glycinergic neuronal markers. Moreover, expression in COS-7 and PC12 cells induced an intracellular, glycine-sensitive GABA uptake activity. These observations, consistent with previous data on GABA and glycine uptake by synaptic vesicles, demonstrate that the mouse clone encodes a vesicular inhibitory amino acid transporter.

The photoactivatable inhibitor 7-azido-8-iodoketanserin labels the N terminus of the vesicular monoamine transporter from bovine chromaffin granules.

In monoaminergic cells, the hormone or neurotransmitter is concentrated into secretory vesicles by a tetrabenazine- and reserpine-sensitive vesicular monoamine transporter (VMAT), catalyzing a H+/monoamine antiport. Ketanserin is another powerful inhibitor of VMAT that binds to the tetrabenazine binding site. A photoactivatable derivative, 7-azido-8-iodoketanserin (AZIK), labels covalently the transporter from bovine chromaffin granules, VMAT-2. Digestion with endoproteinases V8 or Lys-C, which cleave peptide bonds at acidic or lysine residues, respectively, revealed that the AZIK label is located in a 7 kDa segment of the VMAT-2 polypeptide. The photolabeled chromaffin granule transporter was purified by DEAE and WGA chromatography followed by selective aggregation and size-exclusion HPLC. After treatment by V8 or Lys-C, digestion products were separated by electrophoresis in SDS and sequenced. For both enzymes, the material comigrating with the labeled peptide produced a sequence matching the N terminus of VMAT-2. A K55E mutant of the bovine VMAT-2 cDNA was constructed and expressed in COS-7 cells. The mutant protein exhibited a full VMAT activity and could be labeled by AZIK. However, the formation of the 7 kDa labeled peptide upon Lys-C proteolysis was prevented in the mutant, with a redistribution of the label in higher-molecular mass digestion products. The localization of the label upstream of lysine 55 was confirmed by an immunological and enzymatic analysis. We conclude that the segment 2-55 of bovine VMAT-2, which encompasses the cytosolic N terminus and the first transmembrane segment in the current topological model of the transporter, contains residues involved in the binding of ketanserin and, possibly, tetrabenazine.

SDS-resistant aggregation of membrane proteins: application to the purification of the vesicular monoamine transporter.

The vesicular monoamine transporter, which catalyses a H+/ monoamine antiport in monoaminergic vesicle membrane, is a very hydrophobic intrinsic membrane protein. After solubilization, this protein was found to have a high tendency to aggregate, as shown by SDS/PAGE, especially when samples were boiled in the classical Laemmli buffer before electrophoresis. This behavior was analysed in some detail. The aggregation was promoted by high temperatures, organic solvents and acidic pH, suggesting that it resulted from the unfolding of structure remaining in SDS. The aggregates were very stable and could be dissociated only by suspension in anhydrous trifluoroacetic acid. This SDS-resistant aggregation behaviour was shared by very few intrinsic proteins of the chromaffin granule membrane. Consequently, a purification procedure was based on this property. A detergent extract of chromaffin granule membranes enriched in monoamine transporter was heated and the aggregates were isolated by size-exclusion HPLC in SDS. The aggregates, containing the transporter, were dissociated in the presence of trifluoroacetic acid and analysed on the same HPLC column. This strategy might be of general interest for the purification of membrane proteins that exhibit SDS-resistant aggregation.

Involvement of the peptide sensitive channel in the translocation of basic peptides into mitochondria.

The Peptide Sensitive Channel (PSC), a cationic channel of the mitochondrial outer membrane, is blocked by several highly basic peptides. Among these peptides, the most active are pCOX IV (1-12)Y, a mitochondrial addressing peptide and dynorphin B (1-13), a peptide unrelated to mitochondrial physiology. The voltage-dependent characteristics of the block duration of the PSC induced by these peptides and the fact that these peptides are imported into mitochondria in an in vitro assay suggest the involvement of the PSC in peptide translocation into mitochondria. We have analyzed the interaction of Mast Cell Degranulating peptide (MCD), a disulfide rich basic peptide, with yeast and mammalian mitochondria. Electrophysiological experiments with native and reduced forms of this peptide (nMCD and rMCD) showed an interaction of both forms with the yeast PSC. On the other hand, only rMCD blocked the electrical activity of the bovine adrenal cortex PSC. Similarly, although both forms inhibited the import of dynorphin B (1-13) into yeast mitochondria, only rMCD inhibited this import in bovine mitochondria. The correlation between electrophysiological and biochemical data strongly suggest that dynorphin B is translocated across the outer membrane at the level of the PSC.

Biochemistry and molecular biology of the vesicular monoamine transporter from chromaffin granules.

Prior to secretion, monoamines (catecholamines, serotonin, histamine) are concentrated from the cytoplasm into vesicles by vesicular monoamine transporters (VMAT). These transporters also carry non-physiological compounds, e.g. the neurotoxin methyl-4-phenylpyridinium. VMAT acts as an electrogenic antiporter (exchanger) of protons and monoamines, using a proton electrochemical gradient. Vesicular transport is inhibited by specific ligands, including tetrabenazine, ketanserin and reserpine. The mechanism of transport and the biochemistry of VMAT have been analyzed with the help of these tools, using mainly the chromaffin granules from bovine adrenal glands as a source of transporter. Although biochemical studies did not suggest a multiplicity of VMATs, two homologous but distinct VMAT genes have recently been cloned from rat, bovine and human adrenal glands. The VMAT proteins are predicted to possess 12 transmembrane segments, with both extremities lying on the cytoplasmic side. They possess N-glycosylation sites in a putative luminal loop and phosphorylation sites in cytoplasmic domains. In rat, VMAT1 is expressed in the adrenal gland whereas VMAT2 is expressed in the brain. In contrast, we found that the bovine adrenal gland expressed both VMAT1 and VMAT2. VMAT2 corresponds to the major transporter of chromaffin granules, as shown by partial peptidic sequences of the purified protein and by a pharmacological analysis of the transport obtained in transfected COS cells (COS cells are monkey kidney cells possessing the ability to replicate SV-40-origin-containing plasmids). We discuss the possibility that VMAT1 may be specifically addressed to large secretory granules vesicles, whereas VMAT2 may also be addressed to small synaptic vesicles; species differences would then reflect the distinct physiological roles of the small synaptic vesicles in the adrenal gland.

Expression and regulation of the bovine vesicular monoamine transporter gene.

In monoaminergic cells, the neurotransmitter is accumulated into secretory or synaptic vesicles by a tetrabenazine- and reserpine-sensitive transporter, catalyzing an H+/monoamine antiport. The major vesicular monoamine transporter from bovine chromaffin cells was cloned, using sequences common to adrenal medulla and brain rat vesicular monoamine transporters. Its identity was confirmed by peptide sequences, determined from the purified protein. Surprisingly, the bovine adrenal medulla sequence, bVMAT2, is more related to the transporter from human and rat brain than to that from rat adrenal medulla. PCR amplification showed that bVMAT2 is expressed in both adrenal medulla and brain, in contrast with the situation reported in rats, where distinct genes appear to be expressed in brain (SVAT or MAT, now renamed rVMAT2) and in the adrenal medulla (CGAT, now renamed rVMAT1). In bovine chromaffin cells, long-term depolarization by KCl resulted in an increase in the level of bVMAT2 mRNA, in agreement with the previously observed increase in the transporter binding sites, suggesting that a coupling between stimulation, secretion and synthesis changes the composition of the secretory granule membrane.