bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural evidence for multiple transport mechanisms through the Golgi in the pancreatic β-cell line, HIT-T15
Authors: Marsh B. J. , Mastronarde D. N. , McIntosh J. R. , Howell K. E.
Published: January 2005

Accurate data on the three-dimensional architecture of the Golgi is prerequisite for evaluating the mechanisms of transit through this organelle. Here we detail the structure of the Golgi ribbon within part of an insulin-secreting cell in three dimensions at ~ 6 nm resolution. Rapid freezing, freeze-substitution and electron tomography were employed. The Golgi in this region is composed of seven cisternae. The cis-most element is structurally intermediate between the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) and the cis-most cisterna characterized in three dimensions at high resolution in a normal rat kidney cell [Ladinsky, Mastronarde, McIntosh, Howell and Staehelin (1999) J. Cell Biol. 144, 1135–1149]. There are three trans-cisternae that demonstrate morphological and functional variation. The membrane surface areas and volumes of these elements decrease from cis to trans. The two trans-most cisternae are dissociated from the stack and are fragmented by tubulation. ER closely adheres to and inserts between individual trans-cisternae. Many of the 2119 small, clathrin-negative vesicles that are in close proximity to the Golgi fill the region where trans-cisternae have moved out of register with the ribbon. These data provide evidence that cisternal progression/maturation, trafficking via membrane tubules and vesicle-mediated transport act in concert in the same region of the Golgi ribbon, and suggest an important role for the ER in regulating membrane dynamics at the trans-Golgi.
Control of membrane fusion dynamics during regulated exocytosis
Authors: Burgoyne R. D. , Fisher R. J. , Graham M. E. , Haynes L. P. , Morgan A.
Published: January 2005

The study of regulated exocytosis uniquely allows the direct measurement of intracellular membrane fusion events in real time. We have exploited this to examine factors that regulate not only the extent but also the dynamics of single fusion/release events. The general strategy used has been to assess exocytosis in transiently transfected PC12 or adrenal chromaffin cells. We aimed to design mutant constructs based on in vitro biochemistry, in some cases informed by knowledge of protein structure. Using this approach we have demonstrated an inhibitory role for the putative Rab3 effector Noc2 that requires interaction with Rab3. Using carbon-fibre amperometry on adrenal chromaffin cells, we have demonstrated regulation of the kinetics of single granule release events consistent with changes in fusion pore dynamics and switches between full fusion and ‘kiss-and-run’ fusion. These studies have demonstrated a late role for cysteine string protein in exocytosis. In addition, they have focused attention on a key role for Munc18 in the regulation of post-fusion events that affect fusion pore dynamics.
Internalization of the epidermal growth factor receptor: role in signalling
Authors: Sorkin A.
Published: January 2005

The interaction of the activated epidermal growth factor (EGF) receptor (EGFR) with the Src homology 2 (SH2) domain of Grb2 (growth-factor-receptor-bound protein 2) initiates signalling through Ras and mitogen-activated protein kinase. Grb2 can bind EGFR directly or through another SH2-containing protein, She. Activation of EGFRs by ligand also triggers rapid endocytosis of EGF-receptor complexes. To analyse the spatial and temporal regulation of EGFR interactions with SH2 domains in living cells, we have combined imaging microscopy with a modified method of measuring fluorescence resonance energy transfer (FRET) on a pixel-by-pixel basis using EGFR fused to cyan fluorescent protein (CFP) in pair with Grb2 or She fused to yellow fluorescent protein (YFP). Stimulation by EGF resulted in the recruitment of Grb2-YFP and YFP-Shc to cellular compartments that contained EGFR-CFP, and a large increase in the FRET signal. In particular, FRET measurements indicated that activated EGFR-CFP interacted with YFP-Shc and Grb2-YFP in membrane ruffles and endosomes. These results demonstrate that signalling via EGFRs can occur in the endosomal compartment. Moreover, in contrast with previous biochemical studies, FRET experiments show that a large pool of Grb2 and Shc is associated with EGFRs for a prolonged period after EGF stimulation.
Proteins involved in the synaptic organization of AMPA (α-amino-3-hydroxy-5-methylisoxazolepropionate) receptors
Authors: Henley J.
Published: January 2005

As well mediating most synaptic transmission in the mammalian central nervous system, α-amino-3-hydroxy-5-methylisoxazolepropionate (AMPA) receptors are involved in the processes of synapse formation, stabilization and plasticity. Thus the mechanisms that control the developmental and activity-dependent changes in the functional synaptic expression of AMPA receptors are of fundamental importance. In the last few years dramatic advances have been achieved towards elucidating some of the molecular events involved, and a previously unsuspected complexity of proteins that selectively interact directly or indirectly with individual AMPA receptor subunits have been identified. Here I present an overview of some of the main interacting proteins, and describe what we know about how these may fit into cellular pathways that control AMPA receptor targeting, trafficking and functional expression.
Caveolin-I, a putative tumour suppressor gene
Authors: Razani B. , Schlegel A. , Liu J. , Lisanti M. P.
Published: January 2005

Caveolae (‘little caves’) are plasma membrane specializations of 50–100 nm in diameter, and the caveolins are structural proteins used by cells to form caveolae. We and other investigators have discovered that caveolae organelles may be important both in normal signal transduction and in the pathogenesis of a number of human diseases, such as cancer. Here we describe the functional roles of the caveolin gene family and summarize the evidence that supports a role for caveolae as mediators of a number of cellular signalling processes, including apoptosis.
Role of endocytosis in signalling and regulation of G-protein-coupled receptors
Authors: von Zastrow M.
Published: January 2005

Many G-protein-coupled receptors (GPCRs) undergo agonist-induced endocytosis. Endocytosis contributes to distinct processes that regulate the number and functional activity of receptors present in the plasma membrane, contributing to the well described processes of receptor sequestration and down-regulation. Emerging evidence suggests additional functions of endocytosis in mediating GPCR signalling via certain effector pathways, such as mitogen-activated protein kinase modules. The diverse functions of endocytosis raise fundamental questions about the nature of the vesicular carriers and membrane pathways that mediate the endocytic trafficking of specific GPCRs. Insights into the biochemical and functional properties of endocytic vesicles containing internalized opioid and adrenergic receptors will be discussed. Progress towards understanding the mechanisms that control the specificity with which distinct GPCRs are sorted to specialized sub-populations of endocytic vesicles will be highlighted.
Specificity in ligand binding and intracellular signalling by insulin and insulin-like growth factor receptors
Authors: Siddle K. , Ursø B. , Niesler C. A. , Cope D. L. , Molina L. , Surinya K. H. , Soos M. A.
Published: January 2005

The physiological roles of insulin and insulin-like growth factors (IGFs) are distinct, with insulin acting to regulate cellular uptake and metabolism of fuels, whereas IGFs promote cell growth, survival and differentiation. The only components of signalling pathways known to be unique to insulin and IGFs are their respective receptors, and even these display substantial structural and functional similarity. Specificity of action in vivo must in part reflect relative levels of receptor expression in different tissues. The extent to which the receptors differ in intrinsic signalling capacity remains unclear, but specificity might in principle arise from differences in ligand-binding mechanism or properties of intracellular domains. To identify ligand binding determinants we expressed receptor fragments as soluble proteins. Both N-terminal domains and a C-terminal peptide sequence from the α-subunit are essential for ligand binding with moderate affinity. However, binding of ligand with high affinity and specificity requires higher-order structure. To compare signalling capacities, we constructed chimaeras containing intracellular domains of insulin or IGF receptors fused to the extracellular portion of TrkC. Expression and activation of these chimaeras in cell lines reveals subtle differences in signalling and end-point responses, which may depend on cell background.
Functional effects of APS and SH2-B on insulin receptor signalling
Authors: Ahmed Z. , Pillay T. S.
Published: January 2005

APS [for ‘adapter protein with a pleckstrin homology (PH) and Src homology 2 (SH2) domain’] belongs to a family of adapter proteins involved in signalling by the receptors for insulin, insulin-like growth factor 1, platelet-derived growth factor and nerve growth factor. Other members include alternatively spliced SH2-B isoforms (SH2Bα. SH2-Byβ and SH2-Bγ) and Lnk. These have a C-terminal SH2 domain, a central PH domain and an N-terminal proline-rich region. SH2Bα, APS and Lnk have a conserved C-terminal tyrosine phosphorylation site, whereas the alternatively spliced SH2-Bβ and SH2-Bγ have distinct C-termini. There is considerable sequence similarity between APS, SH2-B and Lnk, particularly in the SH2 domain. Both APS and SH2-Bα interact with the insulin-receptor activation loop phosphorylation sites and undergo insulin-stimulated tyrosine phosphorylation, although the phosphorylation of SH2-B is considerably weaker. APS couples c-Cbl to the insulin receptor, resulting in ubiquitination of the insulin receptor. We established cell lines [Chinese hamster ovary (CHO). T-APS and CHO. T-SH2-B cells] overexpressing APS and SH2-Bα to study their roles in insulin receptor signalling. Either adapter protein enhances insulin receptor and ERK (extracellular-signal-regulated kinase) phosphorylation. In CHO. T-APS cells, Akt phosphorylation is observed earlier than in CHO.T-SH2-B cells. Both enhance insulin-stimulated Akt activation but APS seems to cause greater activation. Thus APS and SH2-B have similar effects on insulin receptor signalling, although the effects of SH2-B are independent of its phosphorylation.
Regulation of glycogen synthesis in human muscle cells
Authors: Yeaman S. J. , Armstrong J. L. , Bonavaud S. M. , Poinasamy D. , Pickersgill L. , Halse R.
Published: January 2005

Glucose uptake into muscle and its subsequent storage as glycogen is a crucial factor in energy homeostasis in skeletal muscle. This process is stimulated acutely by insulin and is impaired in both insulin-resistant states and in type 2 diabetes mellitus. A signalling pathway involving protein kinase B and glycogen synthase kinase 3 seems certain to have a key role in stimulating glycogen synthesis but other signalling pathways also contribute, including a rapamycin-sensitive pathway stimulated by amino acids. Although glycogen synthesis is one of the classical insulin-regulated pathways, it is also regulated in an insulin-independent manner; for example glycogen synthesis in muscle is stimulated significantly after strenuous exercise, with much of this stimulation being independent of the involvement of insulin. Evidence suggests that glucose and the glycogen content of the muscle have a key role in this stimulation but the molecular mechanism has yet to be fully explained.
Sterol-regulatory-element-binding protein I c mediates insulin action on hepatic gene expression
Authors: Ferré P. , Foretz M. , Azzout-Marniche D. , Bécard D. , Foufelle F.
Published: January 2005

Effects of insulin on the expression of liver-specific genes are part of the adaptive mechanisms aimed at maintaining energy homeostasis in mammals. When the diet is rich in carbohydrates, secreted insulin stimulates the expression of genes for enzymes involved in glucose utilization (glucokinase, L-type pyruvate kinase and lipogenic enzymes) and inhibits genes for enzymes involved in glucose production (phosphenolpyruvate carboxykinase). The mechanisms by which insulin controls the expression of these genes have been poorly understood. Recently, the transcription factor sterol-regulatory-element-binding protein 1c has been proposed as a key mediator of insulin transcriptional effects. Here we review the evidence that has led to this proposal and the consequences for our understanding of insulin effects in physiological or pathological conditions.
Peptaibols: models for ion channels
Authors: Chugh J. K. , Wallace B. A.
Published: January 2005

Peptaibols are membrane-active polypeptides isolated from fungal sources. They are characterized by the presence of an unusual amino acid, α-aminoisobutyric acid, and a C-terminal hydroxylated amino acid. Peptaibols exhibit antibiotic activity against bacteria and fungi. Their amphipathic nature allows them to self-associate into oligomeric ion-channel assemblies which span the width of lipid bilayer membranes. Over 200 peptaibol sequences have been reported to date, which are compiled in the Peptaibol Database at http://www.cryst.bbk.ac.uk/peptaibol. Alignments of these sequences have been carried out in order to define a series of related subfamilies (SFs) with common sequence features thought to be important for channel formation. Crystal structures determined for a number of peptaibols from the various SFs provide the bases both for modelling of the channel structures and for modelling structures of other members of the same SFs.
Structural model of a voltage-gated potassium channel based on spectroscopic data
Authors: Haris P. I.
Published: January 2005

It is estimated that membrane proteins comprise as much as 30% of most genomes. Yet our knowledge of membrane-protein folding is still in its infancy. Consequently, there is a great need for developing approaches that can further advance our understanding of how peptides and proteins interact with membranes and thereby attain their folded structure. An approach that we have been exploring involves dissecting voltage-gated ion channels into simple peptide domains for the purpose of determining their structure in different media using physical techniques. We have synthesized peptides corresponding to the six membrane-spanning segments, as well as the pore domain, of the Shaker channel and characterized their secondary structures. From these studies we have developed a model for the transmembrane structure of the Shaker potassium channel that is constructed from α-helices. The hard structural data obtained from these studies lends support to the recent theoretical models of this channel protein that have been developed by others.
Why and how are peptide-lipid interactions utilized for self defence?
Authors: Matsuzaki K.
Published: January 2005

Animals defend themselves against invading pathogenic micro-organisms by utilizing cationic anti-microbial peptides, which rapidly kill various micro-organisms without exerting toxicity against the host. Physicochemical peptide-lipid interactions provide attractive mechanisms for innate immunity. Many of these peptides form amphipathic secondary structures (α-helices and β-sheets) which can selectively interact with anionic bacterial membranes by electrostatic interaction. Rapid, peptide-induced membrane permeabilization is an effective mechanism of anti-microbial action. Magainin 2 from frog skin forms a dynamic peptide-lipid supramolecular-complex pore that allows mutually coupled transmembrane transport of ions and lipids. The peptide molecule is internalized upon the disintegration of the pore. Several anti-microbial peptides are known to work synergistically.
Membrane properties and amyloid fibril formation of lung surfactant protein C
Authors: Johansson J.
Published: January 2005

Pulmonary surfactant is essential for respiration and lung host defence and is composed of 80–90% lipids, mainly dipalmitoylphosphatidylcholine (DPPC). Surfactant protein C (SP-C) constitutes 1–2 % of the surfactant mass, and is one of the most hydrophobic peptides yet isolated. SP-C residues 9–34 form an α-helix with a central poly-valine segment, which perfectly matches the thickness of a fluid DPPC bilayer. The palmitoyl groups linked to Cys-5 and Cys-6 of SP-C increase the capacity of the peptide to promote lipid adsorption at an air/liquid interface, and augment the mechanical stability of SP-C/lipid mixtures. SP-C undergoes α-helix → β-sheet transition and forms amyloid fibrils. NMR and MS studies show that the poly-valine helix is kinetically stabilized, and that once it unfolds, formation of β-sheet aggregates is significantly faster than refolding. α-Helix unfolding is accelerated after removal of the palmitoyl groups. Secondary structure prediction of SP-C yields β-strand conformation of the poly-valine part. A database search revealed similar discordance between experimentally determined helices and predicted β-strands for other amyloid-forming proteins, including the prion protein associated with spongiform encephalopathies, and the amyloid-β (Aβ) peptide associated with Alzheimer's disease. For Aβ and SP-C, removal of the helix/strand discordance by residue replacements abrogates fibril formation in vitro.
Tethered-bilayer lipid membranes as a support for membrane-active peptides
Authors: Cornell B. A. , Krishna G. , Osman P. D. , Pace R. D. , Wieczorek L.
Published: January 2005

An immunosensing device, comprising a lipid membrane incorporating ion channels tethered to the surface of a gold electrode, has been reported [Cornell, Braach-Maksvytis, King, Osman, Raguse, Wieczorek and Pace (1997) Nature (London) 387, 580–583]. The present article describes key steps in the assembly of the device and provides further evidence for its proposed sensing mechanism.
Membrane-disordering effects of β-amyloid peptides
Authors: Müller W. E. , Kirsch C. , Eckert G. P.
Published: January 2005

β-Amyloid (Aβ) protein is the major constituent of senile plaques and cerebrovascular deposits characteristic of Alzheimer's disease (AD). The causal relationship between Aβ and AD-specific lesions like neurodegeneration and atrophy is still not known. The present article summarizes our studies indicating that rather low concentrations of Aβ significantly alter the fluidity of cell membranes and subcellular fractions from different tissues and different species including humans, as a possible initial step of its biological effects. Using different fluorescent probes our data show clearly that Aβ peptides specifically disturb the acylchain layer of cell membranes in a very distinct fashion. By contrast, membrane properties at the level of the polar heads of the phospholipid bilayer at the interface with membrane proteins are much less affected.
Keilin's Cytochromes: How Bacteria Use Them, Vary Them and Make Them
Authors: Ferguson S. J.
Published: January 2005

Many proteins with one or more haem groups bound per polypeptide chain are called cytochromes. They function in electron transfer reactions and some are involved directly in the catalysis of chemical reactions, most prominently the reduction of oxygen to water in the terminal step of cell respiration. When unmodified haem is present the cytochromes are referred to as b-type, but if the haem is covalently attached to thiol groups of a Cys-Xaa-Xaa-Cys-His motif then the cytochrome is a c-type. Neither the purpose of this post-translational modification, nor the mechanisms of the machineries that are necessary for formation of the thioether bonds between protein and haem, are fully understood. In bacteria the c-type cytochromes function in the periplasm where they are involved in a range of electron transport activities, including the reactions of denitrification, in which nitrate is reduced sequentially via nitrite, nitric oxide and nitrous oxide to nitrogen gas. Other types of cytochromes have haem molecules with modifications to their porphyrin ring. These include the a-, d-, d1- and o-types. Although Keilin first described the a-, b- and c- types of cytochrome more than 60 years ago, we still do not have clear explanations as to why one type of haem moiety does not suffice for the requirements of mitochondrial, thylakoid and bacterial electron transport.
Thromboxane A2 Signalling in Humans: a ‘Tail’ of Two Receptors
Authors: Kinsella B. T.
Published: January 2005

Since its discovery in 1975, we now have a wealth of knowledge relating to the biochemical, pharmacological and physiological actions of thromboxane A2 (TXA2) and its related metabolites. These molecular insights have been greatly expedited by the molecular cloning and characterization of a cDNA for the human TXA2 receptor, now termed the T Prostanoid or TP receptor, from a megakaryocytic/placental cDNA library in 1991, and later through the discovery of a cDNA encoding a second isoform of the human TP receptor in 1994. The requirement for two TP receptors in primates, but not in other species thus far investigated, is unclear, but points to potential species-specific physiological differences. In this review, I will describe some recent advances in the research field of TXA2/TP receptor signalling, focusing particularly on studies pertaining to the human TP receptor isoforms.
Detecting, signalling and repairing DNA double-strand breaks
Authors: Jackson S. P.
Published: January 2005

DNA double-strand breaks (DSBs) can be generated by a variety of genotoxic agents, including ionizing radiation and radiomimetic chemicals. They can also occur when DNA replication complexes encounter other forms of DNA damage, and are produced as intermediates during certain site-specific recombination processes. It is crucial that cells recognize DSBs and bring about their efficient repair, because a single unrepaired cellular DSB can induce cell death, and defective DSB repair can lead to mutations or the loss of significant segments of chromosomal material. Eukaryotic cells have evolved a variety of systems to detect DNA DSBs, repair them, and signal their presence to the transcription, cell cycle and apoptotic machineries. In this review, I describe how work on mammalian cells and also on model organisms such as yeasts has revelaed that such systems are highly conserved throughout evolution, and has provided insights into the molecular mechanisms by which DNA DSBs are recognized, signalled and repaired. I also explain how defects in the proteins that function in these pathways are associated with a variety of human pathological states.
ATM (ataxia telangiectasia mutated): expanding roles in the DNA damage response and cellular homeostasis
Authors: Shiloh Y.
Published: January 2005

DNA damage is one of the most acute threats to cellular homeostasis and life. The cell responds to such damage by activating a vast array of responses, ranging from DNA repair to numerous signalling pathways, which temporarily slow down the cellular life cycle while the damage is being repaired. Sophisticated relays convey the DNA damage alarm to all these systems immediately after damage infliction. Such relays must be capable of sensing the damage and rapidly creating functional contact with many signalling networks. The ataxia telangiectasia mutated (ATM) protein is a prominent example of such a relay. It responds swiftly to a critical DNA damage – the double strand break (DSB) – by phosphorylating key proteins in numerous signalling pathways. Evidence is emerging, however, that the ATM protein might also be involved in other processes related to cellular homeostasis, which are not directly associated with the damage response. ATM is the protein product of the gene mutated in the multisystem disorder ataxia-telangiectasia (AT), which is characterized by neuronal degeneration, immunodeficiency, chromosomal instability and cancer predisposition. The AT phenotype and the functions of the ATM protein revealed to date demonstrate the exceptionally multifaceted nature of this protein.
Mechanisms of p53-dependent apoptosis
Authors: Schuler M. , Green D. R.
Published: January 2005

Cellular stresses, such as growth factor deprivation, DNA damage or oncogene expression, lead to stabilization and activation of the p53 tumour suppressor protein. Depending on the cellular context, this results in one of two different outcomes: cell cycle arrest or apoptotic cell death. Cell death induced through the p53 pathway is executed by the caspase proteinases, which, by cleaving their substrates, lead to the characteristic apoptotic phenotype. Caspase activation by p53 occurs through the release of apoptogenic factors from the mitochondria, including cytochrome c and Smac/DIABLO. Released cytochrome c allows the formation of a high-molecular weight complex, the apoptosome, which consists of the adapter protein Apaf-1 and caspase 9, which is activated following recruitment into the apoptosome. Active caspase 9 then cleaves and activates the effector caspases, such as caspases-3 and -7, which execute the death program. Released Smac/DIABLO facilitates caspase activation through repression of the IAP caspase inhibitor proteins. The release of mitochondrial apoptogenic factors is regulated by the pro- and anti-apoptotic Bcl-2 family proteins, which either induce or prevent the permeabilization of the outer mitochondrial membrane. The mechanism by which p53 signals to the Bcl-2 family proteins is unclear. It was shown that some of the pro-apoptotic family members, such as Bax, Noxa or PUMA, are transcriptional targets of p53. In addition, transcription-independent, pro-apoptotic activities of p53 have been described. The elucidation of the p53-dependent pathway, resulting in mitochondrial outer membrane permeabilization through the pro-apoptotic Bcl-2 family proteins, is a key to unveiling the mechanism of stress-induced apoptosis.
Secondary DNA structures as molecular targets for cancer therapeutics
Authors: Hurley L. H.
Published: January 2005

DNA sequence information is pivotal to transcription, replication and recombination. DNA structure is dependent upon intracellular conditions such as ion concentration and the presence of proteins that may bind to DNA to facilitate the interconversion between different forms and to stabilize specific secondary structures. Dependent upon the primary DNA sequence, purine- and pyrimidine-rich strands of DNA can adopt four-stranded structures known as G-quadruplexes and i-motifs, respectively. These structures have been proposed to exist in biologically important regions of DNA, e.g. at the end of chromosomes and in the regulatory regions of oncogenes such as c-myc. Proteins such as topoisomerase I and Rap1 can facilitate the formation of G-quadruplex structures, and for transcriptional activation of c-myc, proteins such as NM23–H2 and hnRNP K are required. These proteins bind to the non-duplex forms of the nuclease hypersensitivity element III, of c-myc. The design and synthesis of small molecules that target these secondary DNA structures and the biochemical and biological effects of these compounds are of potential importance in cancer chemotherapy.
Cytotoxic signalling by inhibitors of DNA topoisomerase II
Authors: Beck W. T. , Mo Y.-Y. , Bhat U. G.
Published: January 2005

DNA topoisomerase (topo) II inhibitors either stabilize DNA–topo II complexes by blocking DNA religation (e.g. etoposide) or block the enzyme's catalytic activity (e.g. dexrazoxane). The former class of drugs causes direct DNA damage through topo II, while the latter class does not, but both classes cause apoptosis. We cloned the Fas ligand (FasL) promoter and coupled it to the luciferase gene. Treatment of cells transfected with this construct revealed that complex-stabilizing (DNA-damaging) agents induce FasL expression, but the catalytic inhibitors do not, suggesting that the FasL pathway may not be involved in all cases of topoisomerase-mediated apoptosis. Some topo II inhibitors activate a pathway involving stress-activated protein kinases, which include c-Jun N-terminal kinase-1 (JNK-1). We will discuss the effects of these agents on components of this pathway. Our earlier work revealed that topo IIα interacts with the cell cycle regulatory protein, retinoblastoma protein (Rb). This interaction and the subcellular distribution of these proteins are altered by topo II inhibitory drugs and lead to apoptosis. In addition, agents that affect Rb, such as E1A and E2F1/DP-1, when transfected into cells, also alter topo IIα-Rb localization, activate jun kinase pathways and cause apoptosis. This paper discusses current studies that are designed to determine the contributions of these signalling events to the alterations in subcellular protein distribution and apoptosis. We suggest that protein-protein interactions are important for mediation of cytotoxic signalling by anticancer drugs.
Pyrrolo-1,5-benzoxazepines: a new class of apoptotic agents
Authors: Zisterer D. M. , McGee M. M. , Campiani G. , Ramunno A. , Fattorusso C. , Nacci V. , Lawler M. , Williams D. C.
Published: January 2005

Some members of a series of novel pyrrolo-1,5-benzoxazepines (PBOXs) potently induce apoptosis in a number of human cancerous cell lines including HL-60 cells and the drug-resistant chronic myelogenous leukaemia cell line, K562. The apoptotic induction seems to be independent of the mitochondrial peripheral-type benzodiazepine receptor (PBR), which binds these PBOXs with high affinity, due to a lack of correlation between their affinities for the receptor and their apoptotic potencies and their high apoptotic activity in PBR-deficient cells. PBOX-6, a potent member of the series, induces a transient activation of c-Jun N-terminal kinase (JNK) in a dose-dependent manner, which correlates with induction of apoptosis. Expression of a cytoplasmic inhibitor of the JNK signal transduction pathway, Jip-1, prevents JNK activity and significantly reduces the extent of apoptosis induced by PBOX-6. This demonstrates the requirement for JNK in the cellular response to this apoptotic agent. In addition, PBOX-6 activates caspase-3-like proteases in K562 and HL-60 cells. The caspase-3 inhibitor, Z-Asp-Glu-Val-Asp-fluoromethylketone (z-DEVD-fmk), blocks caspase-3-like protease activity in both cell types but only prevents PBOX-6-induced apoptosis in HL-60 cells, suggesting that the requirement for caspase-3-like proteases in the apoptotic pathway is dependent on the cell type.
Molecular characterization of substrate-binding sites in the glutamate transporter family
Authors: Kanner B. I. , Kavanaugh M. P. , Bendahan A.
Published: January 2005

Glutamate transporters are essential for terminating synaptic excitation and for maintaining extracellular glutamate concentrations below neurotoxic levels. These transporters also mediate a thermodynamically uncoupled chloride flux that is activated by two of the molecules that they transport – sodium and glutamate. Five eukaryotic glutamate transporters have been cloned and identified. They exhibit ~ 50% identity and this homology is even greater in the carboxyl terminal half, which is predicted to have an unusual topology. Determination of the topology shows that the carboxyl terminal part of the molecule contains several transmembrane domains that are separated by at least two re-entrant loops. In these structural elements, we have identified several conserved amino acid residues that play crucial roles in the interaction with the transporter substrates sodium, potassium and glutamate.
Molecular cloning and characterization of a murine hemicholinium-3-sensitive choline transporter
Authors: Apparsundaram S. , Ferguson S. M. , Blakely R. D.
Published: January 2005

In cholinergic neurons, a specific requirement for precursor choline in the biosynthesis of acetylcholine (ACh) is thought to be sustained by a presynaptic, hemicholinium-3 (HC-3)-sensitive choline transporter (CHT). This transporter exhibits micromolar affinity for choline and transport activity is Na+- and Cl−-dependent. Based on the sequence information available with the recent cloning of rat and human CHTs [Okuda, Haga, Kanai, Endou, Ishihara and Katsura (2000) Nat. Neurosci. 3, 120–125; Apparsundaram, Ferguson, George Jr and Blakely (2000) Biochem. Biophys. Res. Commun. 276, 862–867; Okuda and Haga (2000) FEBS Lett. 484, 92–97], we have identified a murine CHT orthologue (mCHT) by reverse transcriptase-PCR of spinal cord mRNA and confirmed this sequence using assembled mouse genomic DNA. Inferred splice junctions for mCHT exons are conserved with those of hCHT. The mCHT cDNA encodes a protein of 580 amino acids with 93 % and 98 % amino acid identity to human CHT and rat CHT1, respectively. Hydropathy analysis of the predicted amino acid sequence of mCHT indicates a protein containing 13 transmembrane domains (TMDs), with the N-terminus oriented extracellularly and the C-terminus oriented intracellularly. Northern blot analysis of mouse tissues reveals the expression of mCHT as a single transcript of ~ 5 kb with highest expression in regions that are rich in cholinergic cell bodies, e.g. the spinal cord, brainstem, mid-brain and striatum, whereas hybridization signals are absent in regions lacking cholinergic soma, e.g. the cerebellum and kidney. Expression of mCHT in COS-7 cells results in high-affinity [3H]HC-3-binding sites (Kd = 5 nM), and Na+- and Cl−-dependent HC-3-sensitive choline uptake (Km = 2 μM), assessed in resealed membrane vesicles. The availability of cloned, functional mCHT and its cognate genomic DNA should prove useful for studies of mCHT regulation and should open possibilities for evaluation of CHT dysfunction in murine models.
Molecular mechanisms of cystine transport
Authors: McBean G. J. , Flynn J.
Published: January 2005

The transport of l-cystine into cells of the mammalian brain is an essential step in the supply of cysteine for synthesis of the antioxidant glutathione. Uptake of lcystine in rat brain synaptosomes occurs by three mechanisms that are distinguishable on the basis of their ionic dependence, kinetics of transport and specificity of inhibitors. Almost 90°% of lcystine transport is by a low-affinity, sodium-dependent mechanism (Km = 473 ± 146 μM), that is mediated by the XAG-family of glutamate transporters. Both lglutamate (IC50 = 9.1 ± 0.4 μM) and lcysteine sulphinate (IC50 = 16.4 ± 3.6 μM) are non-competitive inhibitors of sodium-dependent l[14C]cystine transport, whereas ltrans-pyrrolidine-2,4-dicarboxylic acid (IC50 = 5.6 ± 2.0 μM), lserine-O-sulphate (IC50 = 13.2 ± 5.4 μM), kainate (IC50 = 215 ± 78 μM) and lcysteine (IC50 = 363 ± 63 μM) are competitive inhibitors. lCystine has no effect on the sodium-dependent uptake of D-[3H]aspartate. These results suggest that lcystine binds to a site that is different from the lglutamate recognition site on XAG-glutamate transporters. In rat brain slices, sodium-dependent transport of both lglutamate and lcystine is necessary for maintaining glutathione levels. Uptake of lcystine is sensitive to inhibition by an increased extracellular concentration of lglutamate, which has important implications for understanding conditions that may initiate oxidative stress.
Structure, function and regulation of the 5-hydroxytryptamine (serotonin) transporter
Authors: Horschitz S. , Hummerich R. , Schloss P.
Published: January 2005

The serotonin transporter (SERT), which terminates serotonergic neurotransmission by rapid reuptake of 5-hydroxytryptamine (5-HT) back into the nerve terminal was the subject of two studies. First, the regulatory effects of antidepressants on the expression of SERT was investigated. After chronic exposure of SERT to antidepressants, substrate transport and antidepressant binding parameters were determined. Long-term exposure of SERT to a selective 5-HT-re-uptake inhibitor, citalopram, resulted in the down-regulation of the expression level. The observed effects depend on drug concentration and exposure time and are reversible. Basal uptake levels could be regained 48 h after cessation of the treatment. Secondly, in order to gain insight into the molecular mechanism of substrate transport, potential interactions of SERT with the noradrenaline transporter (NET) have been investigated. A chimeric concatameric transporter construct consisting of rat SERT and rat NET has been cloned and characterized pharmacologically in a heterologous expression system.
Upregulation of γ-aminobutyric acid transporter expression: role of alkylated γ-aminobutyric acid derivatives
Authors: Whitworth T. L. , Quick M. W.
Published: January 2005

Pregabalin [(S)-(+)-3-isobutylgaba] and gabapentin [1-(aminomethyl)cyclohexane acetic acid] are γ-aminobutyric acid (GABA) derivatives that are effective in the treatment of behavioural disorders, convulsions, epilepsy and hyperalgesia. The mechanisms underlying the diverse actions of these compounds in the brain have not been well elucidated. To test the hypothesis that these compounds exert some of their effects on GABA-ergic systems in the brain, we examined their role in regulating the rat brain GABA transporter GAT1, a plasma membrane protein involved in regulating synaptic transmitter levels. Prolonged incubation of hippocampal cultures, which endogenously express GAT1, with gabapentin and pregabalin caused a 2-fold increase in subsequent GABA uptake, which was concentration- and time-dependent. This increase in uptake was correlated with a redistribution of GAT1 protein from intracellular locations to the plasma membrane. Further experiments also suggested that the signal transduction cascade that modulates pregabalin-mediated GAT1 redistribution may involve pathways activated by specific GAT1 substrates and antagonists but does not involve protein kinase C and tyrosine kinases, two other pathways known to regulate GAT1 redistribution. These data suggest that pregabalin and gabapentin may exert some of their actions in the brain by altering GABAergic signalling.
Molecular determinants involved in the asymmetrical distribution of glycine transporters in polarized cells
Authors: Zafra F. , Giménez C.
Published: January 2005

The asymmetrical distribution of neurotransmitter transporters on the cell surface of neurons seems to be a generalized feature of these proteins, and is thought to be important for an appropriate removal of neurotransmitters from the extracellular milieu. To study the subcellular distribution of the glycine transporter isoforms (GLYT1a, GLYT1b, GLYT2a and GLYT2b), these proteins were expressed in epithelial cells [Madin–Darby canine kidney (MDCK) cells] and in cultured hippocampal neurons, as models of polarized cells. The localization of the transporters was assessed by immunofluorescence assays. Our results indicated that the subcellular distribution of glycine transporters is dependent on both the protein isoform and the cell type. By using site-directed mutagenesis we have been able to identify signals for basolateral/somatodendritic localization in the alternative amino terminal region of GLYT1 and in two di-leucine motifs that are located in the carboxyl tail of this protein. Moreover, the N-glycosylation sites located in the large extracellular loop of GLYT2 are involved in apical localization of this protein in polarized MDCK cells. These results contribute to define the mechanisms of asymmetrical distribution of transporters on the cell surface of polarized cells.
Coenzyme Q induces GDP-sensitive proton conductance in kidney mitochondria
Authors: Echtay K. S. , Brand M. D.
Published: January 2005

Addition of coenzyme Q10 (CoQ) at low concentration (29 nmol/mg of protein) to kidney but not liver mitochondria resulted in an increase in proton conductance. This uncoupling activity required fatty acid and was completely inhibited by GDP. CoQ activated when it was likely to be reduced but not when it was likely to become oxidized. However, the redox state of endogenous CoQ did not affect mitochondrial proton conductance. Stimulation by CoQ was not inhibited by cyclosporin A, carboxyatractylate, bongkrekate and catalase but could be reversed by superoxide dismutase. We conclude that CoQ acted in mitochondria through production of superoxide, which mediated uncoupling, probably by acting through uncoupling protein 2.
Physiological effects of variants in human uncoupling proteins: UCP2 influences body-mass index
Authors: Schonfeld-Warden N. A. , Warden C. H.
Published: January 2005

The purpose of this review is to summarize the evidence from more than 40 studies that naturally occurring variants of uncoupling proteins 1–3 (UCP1–3) have detectable physiological effects in humans. Although UCP1 is known to influence mitochondrial proton leak in vitro and core body temperature in mice, genetic studies in humans have produced only weak evidence for association of naturally occurring variants with body-mass index (BMI); the best-reported P value is 0.01. In contrast, current evidence is consistent with the hypothesis that UCP2 and 3 influence BMI, since the best reported P values are better: four studies report associations of 0.001 (2 studies) to 0.002 (1 study) and 0.005 (1 study) for a UCP2 insertion/deletion variant, while the best P values for association of UCP3 with BMI are 0.003 (1 study) and 0.0037 (1 study). UCP2 and 3 are adjacent to each other on chromosome 11 and variants in each are in linkage disequilibrium. Thus, variants in UCP2 or 3 may influence results from association studies of variants in the other. Since UCP2 has a greater influence on BMI in humans than UCP3, then the two most likely hypotheses are that only UCP2 affects BMI, and positive results for UCP3 result from linkage disequilibrium to UCP2, or both UCP2 and 3 affect BMI. It is unlikely that only UCP3 influences BMI. UCP2 associations have been observed in a variety of ethnic groups, including Caucasians, African Americans, South Indians and Chinese. Consistent results from diverse ethnic groups are concordant with the hypothesis that the UCP2 insertion/deletion variant itself underlies the association with BMI.
Co-ordinate regulation of growth factor receptors and lipid phosphate phosphatase-1 controls cell activation by exogenous lysophosphatidate
Authors: Pilquil C. , Ling Z.-C. , Singh I. , Buri K. , Zhang Q.-X. , Brindley D. N.
Published: January 2005

The serum-derived lipid growth factors, lysophosphatidate (LPA) and sphingosine 1-phosphate (S1P), activate cells selectively through different members of a family of endothelial differentiation gene (EDG) receptors. Activation of EDG receptors by LPA and S1P provides a variety of signalling cascades depending upon the G-protein coupling of the different EDG receptors. This leads to chemotactic and mitogenic responses, which are important in wound healing. For example, LPA stimulates fibroblast division and S1P stimulates the chemotaxis and division of endothelial cells leading to angiogenesis. Counteracting these effects of LPA and S1P, are the actions of lipid phosphate phosphatases (LPP, or phosphatidate phosphohydrolases, Type 2). The isoform LPP-1 is expressed in the plasma membrane with its active site outside the cell. This enzyme is responsible for ‘ecto-phosphatase’ activity leading to the degradation of exogenous lipid phosphate mediators, particularly LPA. Expression of LPP-1 decreases cell activation by exogenous LPA. The mechanism for this is controversial and several mechanisms have been proposed. Evidence will be presented that the LPPs cross-talk with EDG and other growth factor receptors, thus, regulating the responses of the cells to lipid phosphate mediators of signal transduction.
The sphingosine-I -phosphate receptor EDG-I is essential for platelet-derived growth factor-induced cell motility
Authors: Rosenfeldt H. M. , Hobson J. P. , Milstien S. , Spiegel S.
Published: January 2005

EDG-1, encoded by the endothelial differentiation gene-1, is a heterotrimeric guanine nucleotide binding protein-coupled receptor (GPCR) for sphingosine-1-phosphate (SPP) that has been shown to stimulate angiogenesis and cell migration in cultured endothelial cells. Unexpectedly, EDG-1 knockout embryos had a normal blood vessel network, vasculogenesis and angiogenesis, but died in utero owing to massive haemorrhaging as a result of failure of smooth muscle cells and pericytes to migrate around the circumference and reinforce endothelial tubes [Liu, Wada, Yamashita, Mi, Deng, Hobson, Rosenfeldt, Nava, Chae, Lee, et al. (2000) J. Clin. Invest. 106, 951–961]. This vascular maturation defect is similar to the phenotype of mice homozygous for disrupted alleles of platelet-derived growth factor B-subunit homodimer (PDGF-BB) or its receptor PDGFR-β. We found that fibroblasts from EDG-1 null embryos did not migrate toward PDGF or SPP, and inhibition of motility correlated with defective activation of the small guanosine triphosphatase Rac, which is required for lamellipodia formation and directional locomotion [Hobson, Rosenfeldt, Barak, Olivera, Poulton, Caron, Milstien, and Spiegel (2001) Science 291, 1800–1803]. Moreover, we showed that PDGF-directed cell migration requires both sphingosine kinase activation and expression of EDG-1, suggesting a functional link between PDGF signalling and EDG-1. Indeed, treatment of wild-type cells with PDGF transactivated EDG-1 as determined by translocation of β-arrestin and phosphorylation of EDG-1. These findings reveal a new paradigm for receptor cross-communication in which activation of a GPCR by a receptor tyrosine kinase is critical for cell motility. Our observations might also clarify the role of EDG-1 in vascular maturation and angiogenesis.
FcγRIIB-mediated inhibition of T-cell receptor signal transduction involves the phosphorylation of SH2-containing inositol 5-phosphatase (SHIP), dephosphorylation of the linker of activated T-cells (LAT) and inhibition of calcium mobilization
Authors: Jensen W. A. , Marschner S. , Ott V. L. , Cambier J. C.
Published: January 2005

The low-affinity receptor for immunoglobulin G, FcγRIIB, is expressed on most B-cells and on immature and activated mature T-cells. Co-aggregation of FcγRIIB with the B-cell antigen receptor (BCR) leads to attenuation of BCR-induced blastogenesis and cell proliferation via inhibition of p21ras, phosphatidylinositol 3-kinase (PI3-K) and phospholipase Cγ (PLCγ) activation. These effects are mediated, at least in part, by the recruitment of SH2-containing protein tyrosine phosphatase-1 (SHP-1) and -2 (SHP-2) and SH2-containing inositol 5-phosphatase(SHIP). In this report, we demonstrate that FcγRIIB co-aggregation with the T-cell antigen receptor (TCR), which may occur when T-cells recognize antibody-coated target cells, leads to inhibition of TCR-induced phosphorylation of the linker of activated T-cells (LAT). When phosphorylated, LAT functions as an adapter molecule and recruits PI3-K. Additionally, we demonstrate that PI3-K is required for TCR-induced Ca2+ mobilization. Together, these data suggest that FcγRIIB may inhibit TCR-mediated Ca2+ mobilization, in part via inhibition of LAT phosphorylation and subsequent inhibition of PI3-K activation. A similar mechanism has been described in B-cells, where FcγRIIB co-aggregation with the BCR leads to inhibition of PI3-K activity via dephosphorylation of CD19. It is likely that, in both cell types, levels of PtdIns(3,4,5)P3 are additionally modulated via the enzymic activity of SHIP.