bluuebunny/crossref_metadata_2025 huggingface dataset:
Hopping, jumping and looping by restriction enzymes
Authors: Halford S. E.
Published: January 2005

Type II restriction endonucleases recognize specific DNA sequences and cleave both strands of the DNA at fixed locations at or near their recognition sites. Many of these enzymes are dimeric proteins that recognize, in symmetrical fashion, palindromic DNA sequences. They generally catalyse independent reactions at each recognition site on the DNA, although in some cases they act processively; cutting the DNA first at one site, then translocating along the DNA to another site and cutting that before leaving the DNA. The way in which the degree of processivity varies with the length of DNA between the sites can reveal the mechanism of translocation. In contrast with the common view that proteins move along DNA by ‘sliding’, the principal mode of transfer of the EcoRV endonuclease is by ‘hopping’ and ‘jumping’, i.e. the dissociation of the protein from one site followed by its re-association with another site in the same DNA molecule, either close to or distant from the original site. Other type II restriction enzymes require two copies of their recognition sites for their DNA cleavage reactions. Many of these enzymes, such as SfiI, are tetramers with two DNA-binding surfaces. SfiI has no activity when bound to just one recognition site, and instead both DNA-binding surfaces have to be filled before it becomes active. Although the two sites can be on separate DNA molecules, SfiI acts optimally with two sites on the same DNA, where it traps the DNA between the sites in a loop. SfiI thus constitutes a test system for the analysis of DNA looping.
The mechanism of protein kinase regulation by protein phosphatases
Authors: Barford D.
Published: January 2005

Protein kinases are an important class of substrate of the protein phosphatases. We have examined the mechanism of dephosphorylation of the activation segments of the insulin receptor kinase and cyclin-dependent kinase 2 by their respective phosphatases, namely the tyrosine specific phosphatase PTP1B and the dual specificity phosphatase KAP. These studies reveal that PTP1B and KAP utilize contrasting mechanisms in order to dephosphorylate their substrates specifically.
Basal and regulated transcription in Archaea
Authors: Bell S. D. , Magill C. P. , Jackson S. P.
Published: January 2005

The basal transcription machinery of Archaea is fundamentally related to the eucaryal RNA polymerase (RNAP) II apparatus. In addition to a 12-subunit RNAP, Archaea possess two general transcription factors, the activities of which are required for accurate and efficient in vitro transcription. These factors, TBP and TFB, are homologues of the eucaryal TATA-box binding protein and TFIIB respectively. Archaea also possess TFE, a homologue of the eucaryal RNAP II general transcription factor TFIIE. Although not absolutely required for transcription in vitro, TFE nonetheless plays a stimulatory role under conditions where promoter recognition by TBP is sub-optimal. The basal transcription apparatus of Archaea is closely related to that of Eucarya but archaeal transcriptional regulators resemble those of bacteria. The mode of action of two such regulators has been characterized to determine how these ‘bacterial-like’ regulators impinge on the ‘eucaryal-like’ basal machinery.
Incorporation of iron-sulphur clusters in membrane-bound proteins
Authors: Seidler A. , Jaschkowitz K. , Wollenberg M.
Published: January 2005

The completely sequenced genome of the cyano-bacterium Synechocystis PCC 6803 contains several open reading frames, of which the deduced amino acid sequences show similarities to proteins known to be involved in FeS cluster synthesis of nitrogenase (Nif proteins) and other FeS proteins (Isc proteins). In this article, the results of our studies on these proteins are summarized and discussed with respect to their relevance in FeS cluster incorporation in chloroplasts. In cyanobacteria, there appears to exist several pathways for FeS cluster synthesis.
Assembly-controlled regulation of chloroplast gene translation
Authors: Choquet Y. , Wostrikoff K. , Rimbault B. , Zito F. , Girard-Bascou J. , Drapier D. , Wollman F.-A.
Published: January 2005

Studies of the biogenesis of the photosynthetic protein complexes in the unicellular green alga Chlamydomonas reinhardtii have pointed to the importance of the concerted expression of nuclear and chloroplast genomes. The accumulation of chloroplast- and nuclear-encoded subunits is concerted, most often as a result of the rapid proteolytic disposal of unassembled subunits, but the rate of synthesis of some chloroplast-encoded subunits from photosynthetic protein complexes, designed as CES proteins (Controlled by Epistasy of Synthesis), is regulated by the availability of their assembly partners from the same complex. Cytochrome f, a major subunit of the cytochrome b6f complex is a model protein for the study of the CES process. In the absence of subunit IV, another subunit of the cytochrome b6f complex, its synthesis is decreased by 90%. This results from a negative autoregulation of cytochrome f translation initiation, mediated by a regulatory motif carried by the C-terminal domain of the unassembled protein [Choquet, Stern, Wostrikoff, Kuras, Girard-Bascou and Wollman (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 4380–4385]. Using site-directed mutagenesis, we have characterized this regulatory motif. We discuss the possible implications regarding the mechanism of the CES process for cytochrome f expression. We have studied the possible generalization of this mechanism to other CES proteins.
Structure and function of Pet100p, a molecular chaperone required for the assembly of cytochrome c oxidase in Saccharomyces cerevisiae
Authors: Forsha D. , Church C. , Wazny P. , Poyton R. O.
Published: January 2005

The assembly of cytochrome c oxidase in the inner mitochondrial membranes of eukaryotic cells requires the protein products of a large number of nuclear genes. In yeast, some of these act globally and affect the assembly of several respiratory-chain protein complexes, whereas others act in a cytochrome c oxidase-specific fashion. Many of these yeast proteins have human counterparts, which when mutated lead to energy-related diseases. One of these proteins, Pet100p, is a novel molecular chaperone that functions to incorporate a subcomplex containing cytochrome c oxidase subunits VII, VIIa and VIII into holo-(cytochrome c oxidase). Here we report the topological disposition of Pet100p in the inner mitochondrial membrane and show that its C-terminal domain is essential for its function as a cytochrome c oxidase-specific ‘assembly facilitator’.
Assembly of cytochrome c oxidase: what can we learn from patients with cytochrome c oxidase deficiency?
Authors: Taanman J.-W. , Williams S. L.
Published: January 2005

Cytochrome c oxidase is an intricate metalloprotein that transfers electrons from cytochrome c to oxygen in the last step of the mitochondrial respiratory chain. It uses the free energy of this reaction to sustain a transmembrane electrochemical gradient of protons. Site-directed mutagenesis studies of bacterial terminal oxidases and the recent availability of refined crystal structures of the enzyme are rapidly expanding the understanding of the coupling mechanism between electron transfer and proton translocation. In contrast, relatively little is known about the assembly pathway of cytochrome c oxidase. Studies in yeast have indicated that assembly is dependent on numerous proteins in addition to the structural subunits and prosthetic groups. Human homologues of a number of these assembly factors have been identified and some are now known to be involved in disease. To dissect the assembly pathway of cytochrome c oxidase, we are characterizing tissues and cell cultures derived from patients with genetically defined cytochrome c oxidase deficiency, using biochemical, biophysical and immunological techniques. These studies have allowed us to identify some of the steps of the assembly process.
Chlamydomonas nuclear mutants that fail to assemble respiratory or photosynthetic electron transfer complexes
Authors: Lown F. J. , Watson A. T. , Purton S.
Published: January 2005

We are using a molecular-genetic approach to investigate the role of nuclear genes in the biogenesis of the electron transfer complexes of mitochondria and chloroplasts. Our analysis of nuclear mutants of the green alga Chlamydomonas that are defective in respiration or photosynthesis has led to the identification of genes encoding factors required for the expression of specific organellar genes, and genes encoding structural components of the complexes.
Function of Hrs in endocytic trafficking and signalling
Authors: Raiborg C. , Bache K. G. , Mehlum A. , Stenmark H.
Published: January 2005

The hepatocyte growth factor-regulated tyrosine kinase substrate, Hrs, becomes tyrosine-phosphorylated upon the binding of various growth factors and cytokines to their receptors. This protein is essential for ventral folding morphogenesis, and it shares structural similarity with Vps27p, which is involved in vacuolar protein sorting in yeast. Since Hrs is localized to endosomes and has been implicated in the regulation of signal transduction as well as membrane trafficking, it has been regarded as a potential co-ordinator of endosomal receptor sorting and signalling. Here we discuss the possible functions of Hrs in light of its interactions with phosphatidylinositol 3-phosphate and multiple proteins.
Relationship between endosomes and lysosomes
Authors: Luzio J. P. , Mullock B. M. , Pryor P. R. , Lindsay M. R. , James D. E. , Piper R. C.
Published: January 2005

Delivery of endocytosed macromolecules to lysosomes occurs by means of direct fusion of late endosomes with lysosomes. This has been formally demonstrated in a cell-free content mixing assay using late endosomes and lysosomes from rat liver. There is evidence from electron microscopy studies that the same process occurs in intact cells. The fusion process results in the formation of hybrid organelles from which lysosomes are reformed. The discovery of the hybrid organelle has opened up three areas of investigation: (i) the mechanism of direct fusion of late endosomes and lysosomes, (ii) the mechanism of re-formation of lysosomes from the hybrid organelle, and (iii) the function of the hybrid organelle. Fusion has analogies with homotypic vacuole fusion in yeast. It requires syntaxin 7 as part of the functional trans-SNARE [SNAP receptor, where SNAP is soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein] complex and the release of lumenal calcium to achieve membrane fusion. Reformation of lysosomes from the hybrid organelle occurs by a maturation process involving condensation of lumenal content and probably removal of some membrane proteins by vesicular traffic. Lysosomes may thus be regarded as a type of secretory granule, storing acid hydrolases in between fusion events with late endosomes. The hybrid organelle is predicted to function as a ‘cell stomach’, acting as a major site of hydrolysis of endocytosed macromolecules.
Signalling and non-caveolar rafts
Authors: Waugh M. G. , Minogue S. , Anderson J. S. , dos Santos M. , Hsuan J. J.
Published: January 2005

Rafts are small membrane domains containing discrete subsets of lipids and proteins. Although microscopic raft structures termed ‘caveolae’ were described nearly 50 years ago, the importance of rafts, particularly signalling within rafts, is only beginning to be understood. Our studies focus on receptor-dependent phosphoinositide signalling. Using their characteristic buoyancy in density gradients, we and others found that the epidermal growth factor (EGF) receptor, phosphatidyl-inositol 4-kinase and phosphoinositides are localized within a caveolin-rich fraction of A431 carcinoma cells. We subsequently found that membrane fragments containing the EGF receptor and most cellular phosphoinositides can be separated from caveolae. Consequently, components of EGF-dependent phosphoinositide signalling localize to one or more novel types of raft, the composition of which we are currently determining. A key component is the type II phosphatidylinositol 4-kinase, which, for many years, has proven difficult to purify and clone. We describe our recent purification from rafts and cloning of this elusive enzyme, and discuss how the structure sheds light on the rafting of this enzyme.
Insulin receptor substrate proteins and neuroendocrine function
Authors: Withers D. J.
Published: January 2005

A family of insulin receptor substrate (IRS) proteins mediates the pleiotropic effects of insulin and insulin-like growth factor 1 (IGF-1) on cellular function by recruiting several intracellular signalling networks. Conventional murine knockout strategies have started to reveal distinct physiological roles for the IRS proteins. Deletion of Irsl produces a mild metabolic phenotype with compensated insulin resistance but also causes marked growth retardation. In contrast, mice lacking IRS-2 display nearly normal growth but develop diabetes owing to a combination of peripheral insulin resistance and β-cell failure. As well as the classical metabolic events regulated by insulin signalling pathways, studies in lower organisms have implicated insulin/IGF-1 signalling pathways in the control of food intake and reproductive function. Our analysis of IRS-2 knockout mice shows that female mice are infertile owing to defects in the hypothalamus, pituitary and gonad. IRS-2−1 mice have small, anovulatory ovaries with reduced numbers of follicles. Levels of the pituitary hormones luteinizing hormone and prolactin and gonadal steroids are low in these animals. Pituitaries of IRS-2−1 animals are decreased in size and contain reduced numbers of gonadotrophs. Additionally, IRS-2−1 females display increased food intake and develop obesity, despite elevated leptin levels, suggesting abnormalities in hypothalamic function. Coupled with recent observations that brain-specific deletion of the insulin receptor causes a similar phenotype, these findings implicate IRS signalling pathways in the neuroendocrine regulation of reproduction and energy homeostasis.
Growth factor regulation of the novel class II phosphoinositide 3-kinases
Authors: Brown R. A. , Shepherd P. R.
Published: January 2005

It is well established that the class-I phosphoinositide (PI) 3-kinases play a crucial role in growth factor signalling pathways. However, evidence has recently emerged that the alpha isoform of the class-II PI 3-kinase (PI 3K-C2α) is activated by growth factors, although the consequences of this are poorly understood. Here we demonstrate that the activation of PI 3K-C2aL is not associated with a change in subcellular localization. Furthermore, we provide the first evidence that PI 3K-C2β is activated by insulin, albeit with slower kinetics than activation of PI 3K-C2α. These findings suggest that both these class-II PI 3-kinase isoforms are likely to participate in insulin-signalling pathways in the cell.
Insulin-regulated gene expression
Authors: O'Brien R. M. , Streeper R. S. , Ayala J. E. , Stadelmaier B. T. , Hornbuckle L. A.
Published: January 2005

Insulin regulates the expression of more than 150 genes, indicating that this is a major action of this hormone. At least eight distinct consensus insulin response sequence (IRSs) have been defined through which insulin can regulate gene transcription. These include the serum response element, the activator protein 1 (‘AP-1’) motif, the Ets motif, the E-box motif and the thyroid transcription factor 2 (‘TTF-2’) motif. All of these IRSs mediate stimulatory effects of insulin on gene transcription. In contrast, an element with the consensus sequence T(G/A)TTT(T/G)-(G/T), which we refer to as the phosphoenol-pyruvate carboxykinase (PEPCK)-like motif, mediates the inhibitory effect of insulin on transcription of the genes encoding PEPCK, insulinlike-growth-factor-binding protein 1 (IGFBP-1), tyrosine aminotransferase and the glucose-6-phos-phatase (G6Pase) catalytic subunit. The forkhead transcription factor FKHR has recently been shown to bind this PEPCK-like IRS motif and a model has been proposed in which insulin inhibits gene transcription by stimulating the phosphorylation and nuclear export of FKHR. Our results suggest that this model is consistent with the action of insulin on transcription of the gene encoding IGFBP-1 but not that of the G6Pase catalytic subunit. Thus, even though the IRSs in both promoters seem identical, they are functionally distinct. In addition, in the G6Pase catalytic subunit promoter, hepatocyte nuclear factor 1 (‘HNF-1’), acts as an accessory factor to enhance the effect of insulin mediated through the IRS.
Structural implications for the transformation of the Bacillus thuringiensis δ-endotoxins from water-soluble to membrane-inserted forms
Authors: Li J. , Derbyshire D. J. , Promdonkoy B. , Ellar D. J.
Published: January 2005

Crystal structures combined with biochemical data show that the δ-endotoxins from Bacillus thuringiensis are structurally poised towards large-scale, irreversible conformational changes that transform them from the soluble protein bound at the cell surface into a membrane-embedded form causing lysis of susceptible insect cells. Cry δ-endotoxins are made of a helix bundle, a β-prism and a β-sandwich. The conformational change involves an umbrella-like opening between the helix-4,5-hairpin and the remaining helices, and between the helical domain and the two sheet domains. Comparison of Cry1Ac structures with and without the bound receptor ligand GalNAc associates occupation of the high-affinity site on the β-sandwich with an increase of temperature factors in the helical, pore-forming domain, which may indicate how receptor binding could trigger the required major conformational change. The structure of Cyt δ-endotoxins indicates that the surface helix hairpins must peel away to expose the β-strands for membrane attack. Single amino acid substitutions in hinge residues or the core can restore activity following an inhibitory mutation.
Assembly of pore proteins on gold electrodes
Authors: Hong Q. , Terrettaz S. , Ulrich W.-P. , Vogel H. , Lakey J. H.
Published: January 2005

Protein-protein interactions at the cell surface are important in the activity of bacterial toxins such as colicins. We have developed methods to study these events using tethered lipid bilayers, which can be probed by impedance spectroscopy and surface plasmon resonance. Recently we have attached the receptor proteins directly to gold electrodes and this offers new possibilities for measuring protein-protein interactions on solid supports.
The energetics of binding of a signal peptide to lipid bilayers: the role of bilayer properties
Authors: McIntosh T. J. , Vidal A. , Simon S. A.
Published: January 2005

The interactions between a signal peptide and a variety of lipid bilayers have been studied with direct binding assays, CD and isothermal titration calorimetry. We find that the binding of this peptide is influenced by charges and dipoles located in the bilayer interfacial region, as well as by the presence of cholesterol in the bilayer. These studies show that bilayer compositional and mechanical variations found in different biological membranes can affect the partitioning of peptides into the bilayer.
Connexin mimetic peptides: specific inhibitors of gap-junctional intercellular communication
Authors: Evans W. H. , Boitano S.
Published: January 2005

Intercellular co-operation is a fundamental and widespread feature in tissues and organs. An important mechanism ensuring multicellular homoeostasis involves signalling between cells via gap junctions that directly connect the cytosolic contents of adjacent cells. Cell proliferation and intercellular communication across gap junctions are closely linked, and a number of pathologies in which communication is disrupted are known where connexins, the gap-junctional proteins, are modified. The proteins of gap junctions thus emerge as therapeutic targets inviting the development and exploitation of chemical tools and drugs that specifically influence intercellular communication. Connexin mimetic peptides that correspond to short specific sequences in the two extracellular loops of connexins are a class of benign, specific and reversible inhibitors of gap-junctional communication that have been studied recently in a broad range of cells, tissues and organs. This review summarizes the properties and uses of these short synthetic peptides, and compares their probable mechanism of action with those of a wide range of other less specific traditional gap-junction inhibitors.
Influenza fusion peptides
Authors: Skehel J. J. , Cross K. , Steinhauer D. , Wiley D. C.
Published: January 2005

The ‘fusion peptides’ of a group of enveloped viruses that includes influenza, paramyxo-, retro-and filo-viruses are N-terminal regions of their membrane fusion proteins generated by cleavage of non-functional precursors. For the influenza membrane fusion protein, haemagglutinin (HA), the three-dimensional structures of precursor HA, cleaved HA and fusion-activated HA show that the fusion peptides are located in different positions in all three forms and adopt different structures. Analyses of mutant HAs with changes in fusion peptide sequence indicate the importance of specific residues for membrane-fusion activity and suggest a structure for the fusion peptide in a fusion-active molecule.
Choosing between growth arrest and apoptosis through the retinoblastoma tumour suppressor protein, Abl and p73
Authors: Wang J. Y. J. , Ki S. W.
Published: January 2005

The choice between growth arrest and apoptosis is made during differentiation, leading to survival with permanent arrest (e.g. neurons), or to death (e.g. epithelium). Genotoxic stress can also cause growth arrest or apoptosis, in addition to the activation of cell cycle checkpoint pathways. The p53 tumour suppressor can simulate growth arrest and apoptosis in response to DNA damage. Thus, p53 alone is not sufficient to specify these two mutually exclusive fates in damaged cells. The retinoblastoma tumour suppressor protein (RB) is a necessary downstream effector in p53-mediated growth arrest. RB inhibits E2F and the nuclear c-Abl tyrosine kinase. Interestingly, E2F activates the transcription of p73 mRNA and c-Abl stabilizes the p73 protein and activates its pro-apoptotic function. Because of RB, the c-Abl/p73 apoptosis pathway is activated in S/G2 cells but not in G1 cells. Taken together, the current data suggests RB to be an important player in directing the choice between permanent arrest and apoptosis. The antagonism between RB and c-Abl/p73 may modulate the function of p53 to direct the choice between growth arrest and apoptosis in DNA damaged cells.
DNA damage signalling and NF-κB: implications for survival and death in mammalian cells
Authors: Boland M. P.
Published: January 2005

Nuclear factor κB (NF-κB), p53 and signal transducer and activator of transcription (STAT) proteins are transcription factors that are known to regulate cell fate in response to apoptotic stimuli. They may, therefore, represent components of drug responses that determine drug efficacy. This review will describe data illustrating some recent progress in our understanding of the mechanism of NF-κB activation after treatment of cells with DNA-damaging anti-cancer drugs. Furthermore, it will discuss how this and other transcriptional events, including STAT transcription factor activation, may effect the promotion or attenuation of apoptosis in target cells. In this regard, certain STAT family members may lie downstream of signalling pathways that are activated after DNA damage. Finally, it will be discussed how proteins normally linked with DNA damage signalling such as p53 may be regulated in response to physiological signals, revealing their role in developmental checkpoints such as differentiation commitment.
Caspases: cellular demolition experts
Authors: Creagh E. M. , Martin S. J.
Published: January 2005

Apoptosis is co-ordinated by a family of cysteine proteases, the caspases, that dismantle the cell by targeting a panoply of proteins for limited proteolysis. The mammalian caspase family contains 14 members, a subset of which participates in apoptosis, with the remainder likely to be involved in the processing of pro-inflammatory cytokines. Apical caspase activation events are typically initiated by adaptor molecules that promote caspase aggregation and facilitate caspase auto-activation. In contrast, distal caspase activation events are controlled by caspases activated earlier in the cascade. Many cellular stresses provoke apoptosis by damaging mitochondria which results in the release of factors [such as cytochrome c and SMAC (second mitochondrial-derived activator of caspase)/Diablo] that trigger caspase activation and cell death. Here, we discuss the hierarchical nature of the caspase cascade that is triggered upon the release of mitochondrial cytochrome c into the cytoplasm, and the role of specific caspases within this cascade in targeting proteins for degradation. Finally, feedback amplification loops and important control points within the caspase cascade will be discussed.
Regulation of the serotonin transporter by interacting proteins
Authors: Haase J. , Killian A.-M. , Magnani F. , Williams C.
Published: January 2005

The serotonin transporter (SERT) plays a critical role in the maintenance of normal neurotransmission by serotonin [5-hydroxytryptamine (5-HT)]. Recent evidence suggests that SERT and other neurotransmitter transporters are tightly regulated. Activation of protein kinase C results in a decrease in SERT-mediated 5-HT uptake, which is due to an internalization of the transporter. However, to date little is known about the mechanism and proteins involved in the down-regulation of the transporter. One candidate SERT-regulatory protein is the SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor) protein, syntaxin 1A (Syn1A), which has recently been implicated in the regulation of ion channels as well as the SERT-related γ-aminobutyric acid- and glycine-transporters. Using 5-HT uptake assays, confocal microscopy and glutathione S-transferase (GST) pull-down assays we showed that Syn1A also interacts with SERT and alters the subcellular localization of the transporter, resulting in a reduction of 5-HT transport. In addition, we have used the yeast two-hybrid system to search for novel regulatory proteins that interact with the cytoplasmic N-terminal domain of SERT. By screening rat brain cDNA library we have identified six potential SERT-binding proteins. Here we also present progress towards the elucidation of the biological relevance of these proteins and their potential role for the regulation of the serotonin transporter.
Life without UCPI: mitochondrial, cellular and organismal characteristics of the UCPI-ablated mice
Authors: Nedergaard J. , Golozoubova V. , Matthias A. , Shabalina I. , Ohba K.-i. , Ohlson K. , Jacobsson A. , Cannon B.
Published: January 2005

Mice devoid of the original uncoupling protein UCP1 have provided opportunities to delineate UCP1 function in a series of biochemical and physiological contexts. The isolated brown-fat mitochondria from such mice are fully coupled (without the addition of GDP), but still exhibit a depressed capacity for ATP synthesis. However, they only show a 2-fold decrease in sensitivity to the de-energizing effect of free fatty acids, compared with UCP1-containing mitochondria, whereas they possess a (UCP1-independent) 50-fold higher sensitivity than liver mitochondria; the fatty acid sensitivities in wild-type and UCP1-deficient mitochondria may, however, be of different natures. Despite the fact that brown-fat cells from UCP1-ablated mice cannot produce heat when stimulated by noradrenaline (‘norepinephrine’) or fatty acids, UCP1-ablated mice can be induced to tolerate extended cold exposure, but the heat then fully results from shivering thermogenesis. Recruitable or adaptive (by cold acclimation or adaptation to a cafeteria diet) adrenergically-stimulated thermogenesis does not exist in the UCP1-ablated animals, demonstrating the unique ability of UCP1 to mediate recruitable non-shivering thermogenesis. In addition to information on the function of UCP1, the UCP1 -ablated mice can be used to gain information concerning the function of the UCP1 homologues. Thus whereas an uncoupling function of the UCP1 homologues cannot be excluded, UCP1-ablated animals clearly lack any ability to recruit any UCP1 homologue to functionally replace the loss of thermogenesis resulting from UCP1. UCP1 (thermogenin) thus remains the only protein the activity of which can be recruited for the purpose of facultative thermogenesis.
UCP3 and its putative function: consistencies and controversies
Authors: Harper M.-E. , Dent R. M. , Bezaire V. , Antoniou A. , Gauthier A. , Monemdjou S. , McPherson R.
Published: January 2005

The physiological function of uncoupling protein 3 (UCP3) is as yet unknown. Based on its 57% homology to UCP1 whose physiologic function is uncoupling and thermogenesis, UCP3 was attributed with the function of mitochondrial uncoupling through proton-leak reactions. UCP3 is expressed selectively in muscle, a tissue in which it has been estimated that proton leak accounts for approx. 50% of resting energy metabolism. Genetic linkage, association and variant studies suggest a role for UCP3 in obesity and/or diabetes. Studies of the heterologous expression of UCP3 in yeast provide support for the idea that UCP3 can uncouple mitochondrial oxidative phosphorylation, but the physiological relevance of these results is questionable. In vitro studies of mitochondria from Ucp3− − mice provide support, but there are no changes in resting metabolic rate (RMR) of mice. In vivo studies demonstrate increased ATP synthesis, but estimates of substrate oxidation rate indicate no change. Mice that greatly overexpress Ucp3 in muscle have increased RMR. Inconsistent with the function of uncoupling are the observations that fasting results in increased expression of UCP3, but no change in muscle proton leak. Moreover, fasting decreases energy expenditure in muscle. Expression patterns for Ucp3 and lipid-metabolism genes support a physiological role in fatty acid oxidation. Overall, findings support a role for Ucp3 in fatty acid metabolism that may have implications for obesity and/or Type II diabetes.
Uncoupling protein 3 and fatty acid metabolism
Authors: Dulloo A. G. , Samec S. , Seydoux J.
Published: January 2005

A role for uncoupling protein (UCP) 3 in fatty acid metabolism is reviewed within the context of our proposal, first put forward in 1998, that this homologue of UCP1 may be involved in the regulation of lipids as fuel substrate rather than in the mediation of thermogenesis. Since then, the demonstrations of muscle-type differences in UCP3 gene regulation in response to dietary manipulations (starvation, high-fat feeding) or to pharmacological interferences with the flux of lipid substrates between adipose-tissue stores and skeletal-muscle mitochondrial oxidation are all in accord with this proposed role for UCP3 in regulating lipids as fuel substrate. However, given the current limitations of gene-knockout technology for evaluating/interpreting the functional importance of genes encoding mitochondrial membrane proteins, the transition from ‘associative’ to ‘cause-and-effect’ evidence for a physiological role of UCP3 in regulating fatty acid metabolism will have to await the development of assays that are sensitive to changes in UCP3 activity. Furthermore, in evaluating the physiological regulators of UCP3, the available evidence points to the existence of adipose-derived factor(s) which, independently of circulating levels of free fatty acids, initiates events leading to the transcription of genes encoding UCP3 and key enzymes of lipid oxidation in the fast glycolytic or fast oxidative-glycolytic muscles, i.e. in the bulk of the skeletal-muscle mass. It is proposed that in tissues where UCP3 co-exists with UCP2 (skeletal muscle, brown adipose tissue, heart) they may act in concert in the overall regulation of lipid oxidation, concomitant to the prevention of lipid-induced oxidative damage.
Mitochondrial uncoupling and lipid metabolism in adipocytes
Authors: Kopecký J. , Rossmeisl M. , Flachs P. , Bardová K. , Brauner P.
Published: January 2005

Metabolism of white adipose tissue is involved in the control of body fat content. In vitro experiments indicated a dependence of lipogenesis on mitochondrial ATP production, as well as a reciprocal link between hormonal effects on metabolism and energetics of adipocytes. Therefore, mitochondrial uncoupling in adipocytes that results in stimulation of energy dissipation and depression of ATP synthesis may contribute to control of lipid metabolism and adiposity. This is supported by the expression of protonophoric proteins in adipocytes, e.g. uncoupling proteins (UCPs) 2 and 5, and some anion transporters, and induction of UCP1 and UCP3 in white fat by pharmacological treatments that reduce adiposity. Negative correlation between expression of UCPs in adipocytes and accumulation of white fat was also found. Expression of UCP1 from the adipose-specific promoter in aP2-Ucp1 transgenic mice mitigated obesity induced by genetic or dietary factors. The obesity resistance, accompanied by mitochondrial uncoupling in adipocytes and increased energy expenditure, resulted from ectopic expression of UCP1 in white but not in brown fat. Probably due to depression of ATP/ADP ratio in white fat of transgenic mice, both fatty acid synthesis and lipolytic action of noradrenaline in adipocytes were relatively low. These results support the role of protonophoric proteins in adipocytes in the control of adiposity. The main function of these proteins in white fat may be modulation of lipogenesis and intracellular hormone signalling. Augmentation of energy expenditure may be of relatively small importance, in accordance with the low oxidative capacity of white adipocytes.
Perspectives on the biology of uncoupling protein (UCP) homologues
Authors: Adams S. H. , Pan G. , Yu X. X.
Published: January 2005

In mammals, it is believed that a portion of tissue metabolic rate is driven by counteraction of uncoupling, in which the energetically inefficient process of proton leak acts to diminish the mitochondrial electrochemical membrane potential. It is proposed that specific proteins associated with the mitochondrion catalyse uncoupling, and the biology of such putative uncoupling proteins (UCPs) is the subject of active research efforts. UCP4 and UCP5 are interesting in light of their abundant expression in the brain, which may signal an important metabolic function in thermogenesis or regulation of reactive oxygen species in that tissue. While each is expressed to various degrees outside of the brain, their impact on whole-animal metabolism remains to be clarified further. Transgenic mice expressing murine UCP5L, the long isoform of UCP5, using an inducible metallothionine promoter (to drive expression of the transgene in liver, testis, heart, lung, spleen, intestine, kidney and brain) did not display any overt metabolic phenotype, despite liver UCP5L mRNA expression equivalent to that of normal mouse brain. This highlights the need for further studies to examine the nature of UCP5 physiology. Evidence for uncoupling behaviour has recently emerged from studies of the human 2-oxoglutarate carrier (OGC), indicating that the possibility of physiological proton leak elicited by the OGC and other mitochondrial carriers warrants further experimental evaluation.
Mechanism of uncoupling protein action
Authors: Garlid K. D. , Jaburek M. , Jezek P.
Published: January 2005

Two competing models of uncoupling protein (UCP) transport mechanism agree that fatty acids (FAs) are obligatory for uncoupling, but they disagree about which ion is transported. In Klingenberg's model, UCPs conduct protons. In Garlid's model, UCPs conduct anions, like all members of this gene family. In the latter model, UCP transports the anionic FA head group from one side of the membrane to the other, and the cycle is completed by rapid flip-flop of protonated FAs across the bilayer. The head groups of the FA analogues, long-chain alkylsulphonates, are translocated by UCP, but they cannot induce uncoupling, because these strong acids cannot be protonated for the flip-flop part of the cycle. We have overcome this limitation by ion-pair transport of undecanesulphonate with propranolol, which causes the sulphonate to deliver protons across the membrane as if it were an FA. Full GDP-sensitive uncoupling is seen in the presence of propranolol and undecanesulphonate. This result confirms that the mechanism of UCP uncoupling requires transport of the anionic FA head group by UCP and that the proton transport occurs via the bilayer and not via UCP.
Uncoupling protein 2 in the brain: distribution and function
Authors: Richard D. , Clavel S. , Huang Q. , Sanchis D. , Ricquier D.
Published: January 2005

Uncoupling protein 2 (UCP2) mRNA is expressed in a panoply of tissues, including the brain, where it is widely distributed. In the mouse brain, it is expressed in the hypothalamus (suprachiasmatic, paraventricular, dorsomedial, ventromedial and arcuate nuclei), the thalamus (submedius nucleus) and the brain-stem (dorsal motor nucleus of the vagus nerve). In the rat brain, it is also expressed in the hippocampus. The presence of UCP2 mRNA in neurons expressing corticotropin-re-leasing factor and arginine-vasopressin suggests a role for UCP2 in the control of neuroendocrine and behavioural functions. We have recently demonstrated that UCP2-deficient mice can resist the lethal effect of toxoplasmosis through an enhanced production of reactive oxygen species (ROS) from the macrophages. This finding provides evidence that UCP2 can be part of a mechanism preventing ROS production. UCP2 could therefore be involved in protecting the brain against oxidative stress. The involvement of UCP2 in neuroprotection is also consistent with the recent observation that kainic acid, which promotes Ca2+ uptake in the glutamate-activated neurons in the hippocampal CA1 field, can induce the UCP2 gene in the activated CA1 cells. The role of UCP2 in neuroprotection warrants further investigation.
Sphingolipids related to apoptosis from the point of view of membrane structure and topology
Authors: van Blitterswijk W. J. , van der Luit A. H. , Caan W. , Verheij M. , Borst J.
Published: January 2005

Sphingolipids and their metabolites are implicated in signal transduction, but the mechanisms are still poorly understood. In particular, the presumed function of ceramide as a second messenger remains controversial. Here, we emphasize the importance of both ceramide and sphingomyelin for membrane structure. The effects of sphingo-lipid turnover in the induction and effector phases of apoptosis are explained by their impact on membrane microdomains that are relevant for cell signalling or changes in morphology. The topology of sphingolipid metabolism is important because of their limited transbilayer and inter-membrane movement. For instance, glycosylceramide synthase converts de novo synthesized ceramide to glycosylceramide, but it is neither a general attenuator of ceramide accumulation at the plasma membrane, nor of the apoptotic process. Synthetic alkyl-lysophospholipids modulate membrane-lipid composition and, therefore, apoptosis sensitivity.
Antagonism of PI 3-kinase-dependent signalling pathways by the tumour suppressor protein, PTEN
Authors: Downes C. P. , Bennett D. , McConnachie G. , Leslie N. R. , Pass I. , MacPhee C. , Patel L. , Gray A.
Published: January 2005

The tumour suppressor protein, PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a member of the mixed function, serine/threonine/tyrosine phosphatase subfamily of protein phosphatases. Its physiological substrates, however, are primarily 3-phosphorylated inositol phospholipids, which are products of phosphoinositide 3-kinases. PTEN thus antagonizes PI 3-kinase-dependent signalling pathways, which explains to a large extent its tumour suppressor status. We have examined the kinetic behaviour, substrate specificity and regulation of PTEN both in vitro and in a variety of cellular models. Although PTEN can utilize both phosphatidylinositol 3,4,5-trisphosphate [PtdIns-(3,4,5)P3] and its water-soluble headgroup, inositol 1,3,4,5-tetrakisphosphate, as substrates, it displays classical features of interfacial catalysis, which greatly favour the lipid substrate (by as much as 1000-fold as judged by Kcat/Km values). Expression of PTEN in U87 cells (which lack endogenous PTEN) and measuring the levels of all known 3-phosphorylated lipids suggests that phosphatidylinositol 3,4-bisphosphate and PtdIns(3,4,5)P3 are both substrates, but that phosphatidylinositol 3-phosphate and phosphatidyl-inositol 3,5-bisphosphate are not. PTEN binds to several PDZ-domain-containing proteins via a consensus sequence at its extreme C-terminus. Disruption of targeting to PDZ-domain proteins selectively blocks some PTEN functions, but not others, suggesting the existence of spatially localized, functionally dedicated pools of signalling lipids. We have also shown recently that PTEN expression is controlled at the transcriptional level and is profoundly upregulated by peroxisome proliferator activated receptor γ agonists, thereby providing possible implications for these drugs in diabetes, inflammation and cancer.
Biochemical and genetic analyses of the Tec kinases Itk and Rlk/Txk
Authors: Czar M. J. , Debnath J. , Schaeffer E. M. , Lewis C. M. , Schwartzberg P. L.
Published: January 2005

The Tec kinases have been implicated as important components of signalling pathways downstream of lymphocyte antigen receptors. Activation of these kinases requires two steps: (i) phosphorylation by Src family kinases and (ii) plasma membrane localization, which is mediated by interaction between the pleckstrin homology (PH) domains of Tec kinases and the products of phosphoinositide-3 kinase (PI-3K). Itk and Rlk/Txk are Tec kinases expressed in T-lymphocytes. Despite similarity to other Tec kinases, Rlk/Txk lacks a PH domain and instead possesses a palmi-toylated cysteine-string motif. We have found that both Rlk/Txk and Itk are phosphorylated in response to T-cell receptor stimulation and can be activated by phosphorylation by Src family kinases. However, consistent with its lack of PH domain, Rlk/Txk is phosphorylated independent of PI-3K activity. Furthermore, we demonstrated that like Itk, Rlk/Txk is associated with lipid RAFTs (detergent-insoluble, cholesterol-rich regions of the membrane), but unlike Itk, Rlk/Txk's RAFT association is independent of PI-3K activity. Despite these differences, Rlk/Txk partially compensates for loss of Itk in gene-targeted animals, suggesting overlapping functions for these kinases.
Simplifying metabolic complexity
Authors: Martin D. Brand , Curtis R. Keira
Published: January 2005

A complete description of the regulation of metabolism in even a single cell will be very hard to achieve, enormous and indigestible. However, there are two powerful ways to simplify the complexity. Firstly, related processes and intermediates can be grouped into a small number of modules, and the regulation of the simplified system can be studied. Secondly, control analysis can be used. With these simplifications to illuminate the important regulatory features, even a full description could be made intellectually and experimentally accessible without distorting the essential regulatory features. Modular control analysis is powerful because it can quantify the relative importance of different flows of regulatory information through any metabolic, physiological, signalling or transcriptional network. It can answer global questions about the importance of different pathways mediating any change to a system. It has been used to analyse how cadmium, a poison with multiple effects, changes oxidative phosphorylation in isolated mitochondria, and to quantify the regulation of energy metabolism in hepatocytes. It has been used to measure how energy metabolism is regulated during mitogen stimulation of thymocytes, quantifying the relative importance of different signalling pathways and how each pathway contributes to the activation of energy metabolism. Recently, we have applied modular control analysis to modern DNA micro-array expression profiling to measure the importance of different groups of mRNA transcripts in mediating physiological responses.
Triosephosphate isomerase deficiency: a neurodegenerative misfolding disease
Authors: Oláh Judit , Orosz Ferenc , Keserü György M. , Kovári Zoltán , Kovács János , Hollán Susan , Ovádi Judit
Published: January 2005

A number of neurodegenerative diseases are mediated by mutation-induced protein misfolding. The resulting genetic defects, however, are expressed in varying phenotypes. Of the several well-established glycolytic enzyme deficiencies, triosephosphate isomerase (TPI) deficiency is the only one in which haemolytic anaemia is coupled with progressive, severe neurological disorder. In a Hungarian family with severe decrease in TPI activity, two germ line-identical but phenotypically differing compound heterozygote brothers inherited two independent (Phe240 → Leu and Glu145 → stop codon) mutations. We have demonstrated recently [Orosz, Oláh, Alvarez, Keserü, Szabó, Wágner, Kovári, Horányi, Baróti, Martial, Hollán and Ovádi (2001) Blood 98, 3106–3112] that the mutations of TPI explain in themselves neither the severe decrease in the enzyme activity characteristic of TPI deficiency nor the enhanced ability of the mutant enzyme from haemolysate of the propositus to associate with subcellular particles. Here we present kinetic (flux analysis), thermodynamic (microcalorimetry and fluorescence spectroscopy), structural (in silico) and ultrastructural (immunoelectron microscopy) data for characterization of mutant isomerase structures and for the TPI-related metabolic processes in normal and deficient cells. The relationships between mutation-induced TPI misfolding and formation of aberrant protein aggregates are discussed.
Conversion of pancreatic cells to hepatocytes
Authors: Tosh David , Shen Chia-Ning , Slack Jonathan M. W.
Published: January 2005

Transdifferentiation is the name used to describe the conversion of one differentiated cell type to another. During development, the liver and pancreas arise from the same region of the endoderm and cells from the two organs can transdifferentiate in the adult under different experimental procedures. We have produced two in vitro models for the transdifferentiation of pancreatic cells to hepatocytes. The first utilizes a pancreatic exocrine cell line AR42J-B13 and the second comprises cultures of mouse embryonic pancreas. We have analysed the pancreatic hepatocytes and they express a range of liver markers including albumin, transferrin and transthyretin. We also present evidence for (i) the molecular mechanism which regulates the conversion between pancreas and liver and (ii) the cellular basis of the switch in phenotype.