Adenine nucleotide transporters in organelles: novel genes and functions

In eukaryotes, cellular energy in the form of ATP is produced in the cytosol via glycolysis or in the mitochondria via oxidative phosphorylation and, in photosynthetic organisms, in the chloroplast via photophosphorylation. Transport of adenine nucleotides among cell compartments is essential and is performed mainly by members of the mitochondrial carrier family, among which the ADP/ATP carriers are the best known. This work reviews the carriers that transport adenine nucleotides into the organelles of eukaryotic cells together with their possible functions. We focus on novel mechanisms of adenine nucleotide transport, including mitochondrial carriers found in organelles such as peroxisomes, plastids, or endoplasmic reticulum and also mitochondrial carriers found in the mitochondrial remnants of many eukaryotic parasites of interest. The extensive repertoire of adenine nucleotide carriers highlights an amazing variety of new possible functions of adenine nucleotide transport across eukaryotic organelles.     

Plant mitochondrial carriers: an overview

In the two last decades, biochemical studies using mitochondrial swelling experiments or direct solute uptake in isolated mitochondria have lead to the identification of different transport systems at the level of the plant mitochondrial inner membrane. Although most of them have been found to have similar features to those identified in animal mitochondria, some differences have been observed between plant and animal transporters. More recently, molecular biology studies have revealed that most of the mitochondrial exchanges are performed by nuclear encoded proteins, which form a superfamily. Members of this family have been reported in animals, yeast as well as plants. This review attempts to give an overview of the present knowledge concerning the biochemical and molecular characterisation of plant members of the mitochondrial carrier family and, when possible, a comparison with carriers from other organisms.     

Single-nucleotide evolution quantifies the importance of each site along the structure of mitochondrial carriers

Mitochondrial carriers are membrane-embedded proteins consisting of a tripartite structure, a three-fold pseudo-symmetry, related sequences, and similar folding whose main function is to catalyze the transport of various metabolites, nucleotides, and coenzymes across the inner mitochondrial membrane. In this study, the evolutionary rate in vertebrates was screened at each of the approximately 50,000 nucleotides corresponding to the amino acids of the 53 human mitochondrial carriers. Using this information as a starting point, a scoring system was developed to quantify the evolutionary pressure acting on each site of the common mitochondrial carrier structure and estimate its functional or structural relevance. The degree of evolutionary selection varied greatly among all sites, but it was highly similar among the three symmetric positions in the tripartite structure, known as symmetry-related sites or triplets, suggesting that each triplet constitutes an evolutionary unit. Based on evolutionary selection, 111 structural sites (37 triplets) were found to be important. These sites play a key role in structure/function of mitochondrial carriers and are involved in either conformational changes (sites of the gates, proline–glycine levels, and aromatic belts) or in binding and specificity of the transported substrates (sites of the substrate-binding area in between the two gates). Furthermore, the evolutionary pressure analysis revealed that the matrix short helix sites underwent different degrees of selection with high inter-paralog variability. Evidence is presented that these sites form a new sequence motif in a subset of mitochondrial carriers, including the ADP/ATP translocator, and play a regulatory function by interacting with ligands and/or proteins of the mitochondrial matrix.     

Divergent effects of the major mast cell products histamine, tryptase and TNF-alpha on human fibroblast behaviour

Fibroblast proliferation is a key process in tissue remodeling and mast cells (MCs) are thought to play a crucial role. Having established that the three major MC products, tryptase, histamine and TNF-alpha (TNF) are normally present in human skin MCs, which are in close proximity to dermal fibroblasts, we studied their individual effects on cell cycle-controlled human dermal fibroblasts (HFFF2). These cells express receptors (H1, PAR2, TNFR1/2) for the major MC mediators, but only tryptase or a PAR2 agonist peptide stimulated proliferation and gene expression. TNF was antimitotic, and histamine, while elevating intracellular Ca²⁺ levels at high concentrations, did not affect proliferation. We conclude that MC products but also composition and numbers of respective receptors on fibroblasts are crucially responsible for fibroproliferative events. Received: 28 June 2005; received after revision 28 September 2005; accepted 6 October 2005     

Platelet monoamine oxidase B: use and misuse

The human platelet in addition to having serotonin (5-HT) receptors, uptake carriers (receptor) and transmitter storage vesicles, primarily possesses mitochondrial monoamine oxidase (MAO) type B. Similar to the major form of MAO in the human brain, this enzyme actively oxidizes A-B and B substrates (tyramine, dopamine, phenylethylamine) as well as the novel secondary amine anticonvulsant, milacemide and dopaminergic neurotoxin, MPTP. 5-HT oxidation is hardly affected by the platelet enzyme and MAO inhibitors have no net effect on its accumulation. MAO-B is selectively inhibited by 1-deprenyl and thus the platelet enzyme may be useful to monitor the anti-Parkinson activity of such drugs, as related to their ability to inhibit brain MAO-B. The oxidation of the anticonvulsant, milacemide, to glycine in vitro and in vivo by MAO-B, may herald new prospects for the development of inert prodrugs capable of being metabolized to neuroactive substances by MAO-B. The plasma levels of their metabolites may be an index of MAO-B activity found in the platelet and brain.     

Platelet monoamine oxidase B: Use and misuse

The human platelet in addition to having serotonin (5-HT) receptors, uptake carriers (receptor) and transmitter storage vesicles, primarily possesses mitochondrial monoamine oxidase (MAO) type B. Similar to the major form of MAO in the human brain, this enzyme actively oxidizes A-B and B substrates (tyramine, dopamine, phenylethylamine) as well as the novel secondary amine anticonvulsant, milacemide and dopaminergic neurotoxin, MPTP. 5-HT oxidation is hardly affected by the platelet enzyme and MAO inhibitors have no net effect on its accumulation. MAO-B is selectively inhibited by 1-deprenyl and thus the platelet enzyme may be useful to monitor the anti-Parkinson activity of such drugs, as related to their ability to inhibit brain MAO-B. The oxidation of the anticonvulsant, milacemide, to glycine in vitro and in vivo by MAO-B, may herald new prospects for the development of inert prodrugs capable of being metabolized to neuroactive substances by MAO-B. The plasma levels of their metabolites may be an index of MAO-B activity found in the platelet and brain.     

DING proteins: numerous functions, elusive genes, a potential for health

DING proteins, named after their conserved N-terminus, form an overlooked protein family whose members were generally discovered through serendipity. It is characterized by an unusually high sequence conservation, even between distantly related species, and by an outstanding diversity of activities and ligands. They all share a demonstrated capacity to bind phosphate with high affinity or at least a predicted phosphate-binding site. However, DING protein genes are conspicuously absent from databases. The many novel family members identified in recent years have confirmed that DING proteins are ubiquitous not only in animals and plants but probably also in prokaryotes. At the functional level, there is increasing evidence that they participate in many health-related processes such as cancers as well as bacterial (Pseudomonas) and viral (HIV) infections, by mechanisms that are now beginning to be understood. They thus represent potent targets for the development of novel therapeutic approaches, especially against HIV. The few genomic sequences that are now available are starting to give some clues on why DING protein genes and mRNAs are well conserved and difficult to clone. This could open a new era of research, of both fundamental and applied importance.     

Snake venom thrombin-like enzymes: from reptilase to now

The snake venom thrombin-like enzymes (SVTLEs) comprise a number of serine proteases functionally and structurally related to thrombin. Until recently, only nine complete sequences of this subgroup of the serine protease family were known. Over the past 5 years, the primary structure of several SVTLEs has been characterized, and now this family includes several members. Of particular interest is their possible use in pathologies such as thrombosis. The aim of the present review is to summarize the state of the art concerning the evolutionary, structural and biological features of the SVTLEs.Received 16 August 2003; received after revision 26 September 2003; accepted 1 October 2003     

Proteolysis in protein import and export: signal peptide processing in eu- and prokaryotes.

Numerous proteins in pro- and eukaryotes must cross cellular membranes in order to reach their site of function. Many of these proteins carry signal sequences that are removed by specific signal peptidases during, or shortly after, membrane transport. Signal peptidases have been identified in the rough endoplasmic reticulum, the matrix and inner membrane of mitochondria, the stroma and thylakoid membrane of chloroplasts, the bacterial plasma membrane and the thylakoid membrane of cyanobacteria. The composition of these peptidases varies between one and several subunits. No site-specific inhibitors are known for the majority of these enzymes. Accordingly, signal peptidases recognize structural motifs rather than linear amino acid sequences. Such motifs have become evident by employing extensive site-directed mutagenesis to investigate the anatomy of signal sequences. Analysis of the reaction specificities and the primary sequences of several signal peptidases suggests that the enzymes of the endoplasmic reticulum, the inner mitochondrial membrane and the thylakoid membrane of chloroplasts all have evolved from bacterial progenitors.     

CADM1 isoforms differentially regulate human mast cell survival and homotypic adhesion

Cell adhesion molecule 1 (CADM1), expressed by human lung mast cells (HLMCs), mediates their adhesion to airway smooth muscle (ASM), and contributes to ASM-dependent HLMC proliferation and survival. CADM1 is expressed in alternatively spliced isoforms, but those present in HLMCs and their function are not known. We cloned three functional and one cryptic non-functional isoform with alternative splicing between exons 7/11 and 1/2, respectively, from HLMCs and human MC lines (HMC-1 and LAD2). Differentiated HLMCs and LAD2 cells expressed the functional isoform SP4 containing exons 7/8/11 (~80% of clones), as well as SP1 (exons 7/8/9/11) and a novel SP6 (exons 7/8/9/10/11). In contrast, immature HMC-1 cells expressed only functional SP4. SP4 overexpression in HMC-1 cells and HLMCs augmented homotypic adhesion to a greater extent than SP1 in various conditions. In contrast, CADM1 downregulation abolished homotypic adhesion, indicating that CADM1 is the sole receptor mediating mast cell aggregation. CADM1-mediated adhesion was enhanced by the presence of cell survival factors. SP1 overexpression in HMC-1 cells compromised survival compared to SP4 overexpression or control. CADM1 downregulation resulted in reduced viability and decreased expression of the pro-survival protein Mcl-1(L), but not Blc-2 or Bcl-X(L), and increased caspase-3/7 activity in both HMC-1 cells and HLMCs. This coincided with decreased basal Kit levels in HLMCs. In summary, human MCs express multiple CADM1 isoforms which exhibit differential regulation of survival and homotypic adhesion. The most highly expressed SP4 isoform is likely to contribute to MC aggregation and longevity in mastocytosis, and augment the pathophysiology of allergic diseases.     

Proteolysis in protein import and export: Signal peptide processing in eu-and prokaryotes

Numerous proteins in pro-and eukaryotes must cross cellular membranes in order to reach their site of function. Many of these proteins carry signal sequences that are removed by specific signal peptidases during, or shortly after, membrane transport. Signal peptidases have been identified in the rough endoplasmic reticulum, the matrix and inner membrane of mitochondria, the stroma and thylakoid membrane of chloroplasts, the bacterial plasma membrane and the thylakoid membrane of cyanobacteria. The composition of these peptidases varies between one and several subunits. No site-specific inhibitors are known for the majority of these enzymes. Accordingly, signal peptidases recognize structural motifs rather than linear amino acid sequences. Such motifs have become evident by employing extensive site-directed mutagenesis to investigate the anatomy of signal sequences. Analysis of the reaction specificities and the primary sequences of several signal peptidases suggests that the enzymes of the endoplasmic reticulum, the inner mitochondrial membrane and the thylakoid membrane of chloroplasts all have evolved from bacterial progenitors.     

Interaction of mitochondrial porin with cytosolic proteins.

Intracellular phosphorylation is an important step in active uptake and utilization of carbohydrates. For example glucose and glycerol enter the liver cell along the extra intracellular gradient by facilitated diffusion through specific carriers and are concentrated inside the cell by phosphorylation via hexokinase or glycerol kinase. Depending on the function of the respective tissue the uptake of carbohydrates serves different metabolic purposes. In brain and kidney medulla cells which depend on carbohydrates, glucose and glycerol are taken up according to the energy demand. However, in tissues such as muscle which synthesize glycogen or like liver which additionally produce fat from glucose, the uptake of carbohydrates has to be regulated according to the availability of glucose and glycerol. How the reversible coupling of the kinases to the outer membrane pore and the mitochondrial ATP serves to fulfil these specific requirements will be explained as well as how this regulates the carbohydrate uptake in brain according to the activity of the oxidative phosphorylation and how this allows glucose uptake in liver and muscle to persist in the presence of high glucose 6-phosphate without activating the rate of glycolysis.     

Characterization of tumor differentiation factor (TDF) and its receptor (TDF-R)

Tumor differentiation factor (TDF) is an under-investigated protein produced by the pituitary with no definitive function. TDF is secreted into the bloodstream and targets the breast and prostate, suggesting that it has an endocrine function. Initially, TDF was indirectly discovered based on the differentiation effect of alkaline pituitary extracts of the mammosomatotropic tumor MtTWlO on MTW9/PI rat mammary tumor cells. Years later, the cDNA clone responsible for this differentiation activity was isolated from a human pituitary cDNA library using expression cloning. The cDNA encoded a 108-amino-acid polypeptide that had differentiation activity on MCF7 breast cancer cells and on DU145 prostate cancer cells in vitro and in vivo. Recently, our group focused on identification of the TDF receptor (TDF-R). As potential TDF-R candidates, we identified the members of the Heat Shock 70-kDa family of proteins (HSP70) in both MCF7 and BT-549 human breast cancer cells (HBCC) and PC3, DU145, and LNCaP human prostate cancer cells (HPCC), but not in HeLa cells, NG108 neuroblastoma, or HDF-a and BLK CL.4 cells fibroblasts or fibroblast-like cells. Here we review the current advances on TDF, with particular focus on the structural investigation of its receptor and on its functional effects on breast and prostate cells.     

Interaction of mitochondrial porin with cytosolic proteins

Summary Intracellular phosphorylation is an important step in active uptake and utilization of carbohydrates. For example glucose and glycerol enter the liver, cell along the extra intracellular gradient by facilitated diffusion through specific carriers and are concentrated inside the cell by phosphorylation via hexokinase or glycerol kinase. Depending on the function of the respective tissue the uptake of carbohydrates serves different metabolic purposes. In brain and kidney medulla cells which depend on carbohydrates, glucose and glycerol are taken up according to the energy demand. However, in tissues such as muscle which synthesize glycogen or like liver which additionally produce fat from glucose, the uptake of carbohydrates has to be regulated according to the availability of glucose and glycerol. How the reversible coupling of the kinases to the outer membrane pore and the mitochondrial ATP serves to fulfil these specific requirements will be explained as well as how this regulates the carbohydrate uptake in brain according to the activity of the oxidative phosphorylation and how this allows glucose uptake in liver, and muscle to persist in the presence of high glucose 6-phosphate without activating the rate of glycolysis.     

Protein arginine deiminase 4: a target for an epigenetic cancer therapy

The recent approvals of anticancer therapeutic agents targeting the histone deacetylases and DNA methyltransferases have highlighted the important role that epigenetics plays in human diseases, and suggested that the factors controlling gene expression are novel drug targets. Protein arginine deiminase 4 (PAD4) is one such target because its effects on gene expression parallel those observed for the histone deacetylases. We demonstrated that F- and Cl-amidine, two potent PAD4 inhibitors, display micromolar cytotoxic effects towards several cancerous cell lines (HL-60, MCF7 and HT-29); no effect was observed in noncancerous lines (NIH 3T3 and HL-60 granulocytes). These compounds also induced the differentiation of HL-60 and HT29 cells. Finally, these compounds synergistically potentiated the cell killing effects of doxorubicin. Taken together, these findings suggest PAD4 inhibition as a novel epigenetic approach for the treatment of cancer, and suggest that F- and Cl-amidine are candidate therapeutic agents for this disease.     

Increased mitochondrial palmitoylcarnitine/carnitine countertransport by flavone causes oxidative stress and apoptosis in colon cancer cells

Cancer cell metabolism is characterized by limited oxidative phosphorylation in order to minimize oxidative stress. We have previously shown that the flavonoid flavone in HT-29 colon cancer cells increases the uptake of pyruvate or lactate into mitochondria, which is followed by an increase in O₂^−.. production that finally leads to apoptosis. Similarly, a supply of palmitoylcarnitine in combination with carnitine induces apoptosis in HT-29 cells by increasing the mitochondrial respiration rate. Here we show that flavone-induced apoptosis is increased more than twofold in the presence of palmitoylcarnitine due to increased mitochondrial fatty acid transport and the subsequent metabolic generation of O₂^−. in mitochondria is the initiating factor for the execution of apoptosis. Received 12 August 2005; received after revision 12 October 2005; accepted 14 October 2005     

Dps-like proteins: structural and functional insights into a versatile protein family

Dps-like proteins are key factors involved in the protection of prokaryotic cells from oxidative damage. They act by either oxidizing iron to prevent the formation of oxidative radicals or by forming Dps-DNA complexes to physically protect DNA. All Dps-like proteins are characterized by a common three-dimensional architecture and are found as spherical dodecamers with a hollow central cavity. Despite their structural similarities, recent biochemical and structural data have suggested different functions among members of the family that range from protection inside the cells in response to various stress signals to adhesion and virulence during bacterial infections. Moreover, the Dps-like proteins have lately attracted considerable interest in the field of nanotechnology owing to their ability to act as protein cages for iron and various other metals. A better understanding of their function and mechanism could therefore lead to novel applications in biotechnology and nanotechnology.     

Genetics and molecular pathogenesis of mitochondrial respiratory chain diseases

Dysfunction of the mitochondrial respiratory chain has been recognised as a cause of human disease for over 30 years. Advances in the past 10 years have led to a better understanding of the genetics and molecular pathogenesis of many of these disorders. Over 100 primary defects in mitochondrial DNA (mtDNA) are now implicated in the pathogenesis of a group of disorders which are collectively known as the mitochondrial encephalomyopathies, and which most frequently involve skeletal muscle and/or the central nervous system. Although impaired oxidative phosphorylation is likely to be the final common pathway leading to the cellular dysfunction associated with such mtDNA mutations, the complex relationship between genotype and phenotype remains largely unexplained. Most of the genes which encode the respiratory chain reside in the nucleus, yet only five nuclear genes have been implicated in human respiratory chain diseases. There is evidence that respiratory chain dysfunction is present in common neurological diseases such as Parkinson's disease and Huntington's disease. The precise cause of this respiratory chain dysfunction and its relationship to the disease process are unclear. This review focuses upon respiratory chain disorders associated with primary defects in mtDNA.     

Small proteoglycans

In this review the structure and functions of two non-related proteoglycan families are discussed. One family represents a group of extracellular matrix macromolecules characterized by core proteins with leucine-rich repeat motifs. Within this family special attention is given to those members which carry chondroitin or dermatan sulfate glycosaminoglycan chains. The second family is characterized by repeat sequences of serine and glycine. Their members are products of a single core protein gene and are characteristic constituents of secretory vesicles in cells of the haematopoietic lineage.