bluuebunny/crossref_metadata_2025 huggingface dataset:
The solution structure of ribosomal protein L18 from Thermus thermophilus reveals a conserved RNA-binding fold
Authors: WOESTENENK Esmeralda A. , GONGADZE George M. , SHCHERBAKOV Dmitry V. , RAK Alexey V. , GARBER Maria B. , HÄRD Torleif , BERGLUND Helena
Published: August 2015

We have determined the solution structure of ribosomal protein L18 from Thermus thermophilus. L18 is a 12.5kDa protein of the large subunit of the ribosome and binds to both 5S and 23S rRNA. In the uncomplexed state L18 folds to a mixed α/β globular structure with a long disordered N-terminal region. We compared our high-resolution structure with RNA-complexed L18 from Haloarcula marismortui and T. thermophilus to examine RNA-induced as well as species-dependent structural differences. We also identified T. thermophilus S11 as a structural homologue and found that the structures of the RNA-recognition sites are conserved. Important features, for instance a bulge in the RNA-contacting β-sheet, are conserved in both proteins. We suggest that the L18 fold recognizes a specific RNA motif and that the resulting RNA—protein-recognition module is tolerant to variations in sequence.
Chicken avidin-related proteins show altered biotin-binding and physico-chemical properties as compared with avidin
Authors: LAITINEN Olli H. , HYTÖNEN Vesa P. , AHLROTH Mervi K. , PENTIKÄINEN Olli T. , GALLAGHER Ciara , NORDLUND Henri R. , OVOD Vladimir , MARTTILA Ari T. , PORKKA Eevaleena , HEINO Sanna , JOHNSON Mark S. , AIRENNE Kari J. , KULOMAA Markku S.
Published: August 2015

Chicken avidin and bacterial streptavidin are proteins familiar from their use in various (strept)avidin—biotin technological applications. Avidin binds the vitamin biotin with the highest affinity known for non-covalent interactions found in nature. The gene encoding avidin (AVD) has homologues in chicken, named avidin-related genes (AVRs). In the present study we used the AVR genes to produce recombinant AVR proteins (AVRs 1, 2, 3, 4/5, 6 and 7) in insect cell cultures and characterized their biotin-binding affinity and biochemical properties. Amino acid sequence analysis and molecular modelling were also used to predict and explain the properties of the AVRs. We found that the AVR proteins are very similar to avidin, both structurally and functionally. Despite the numerous amino acid substitutions in the subunit interface regions, the AVRs form extremely stable tetramers similar to those of avidin. Differences were found in some physico-chemical properties of the AVRs as compared with avidin, including lowered pI, increased glycosylation and, most notably, reversible biotin binding for two AVRs (AVR1 and AVR2). Molecular modelling showed how the replacement Lys111→isoleucine in AVR2 alters the shape of the biotin-binding pocket and thus results in reversible binding. Both modelling and biochemical analyses showed that disulphide bonds can form and link monomers in AVR4/5, a property not found in avidin. These, together with the other properties of the AVRs described in the present paper, may offer advantages over avidin and streptavidin, making the AVRs applicable for improved avidin—biotin technological applications.
Overloading and removal of N-glycosylation targets on human acetylcholinesterase: effects on glycan composition and circulatory residence time
Authors: CHITLARU Theodor , KRONMAN Chanoch , VELAN Baruch , SHAFFERMAN Avigdor
Published: August 2015

Optimization of post-translational modifications was shown to affect the ability of recombinant human acetylcholinesterase (rHuAChE) produced in HEK-293 cells to be retained in the circulation for prolonged periods of time [Kronman, Velan, Marcus, Ordentlich, Reuveny and Shafferman (1995) Biochem. J. 311, 959–967; Chitlaru, Kronman, Zeevi, Kam, Harel, Ordentlich, Velan and Shafferman (1998) Biochem. J. 336, 647–658; Chitlaru, Kronman, Velan and Shafferman (2001) Biochem. J. 354, 613–625]. To evaluate the possible contribution of the number of appended N-glycans in determining the pharmacokinetic behaviour of AChE, a series of sixteen recombinant human AChE glycoforms, differing in their number of appended N-glycans (2, 3, 4 or 5 glycans), state of assembly (dimeric or tetrameric) and terminal glycan sialylation (partially or fully sialylated) were generated. Extensive structural analysis of N-glycans demonstrated that the various glycan types associated with all the different rHuAChE glycoforms are essentially similar both in structure and abundance, and that production of the various glycoforms in the sialyltransferase-overexpressing 293ST-2D6 cell line resulted in the generation of enzyme species that carry glycans sialylated to the same extent. Pharmacokinetic profiling of the rHuAChE glycoforms in their fully tetramerized and sialylated state clearly demonstrated that circulatory longevity correlated directly with the number of attached N-glycans (mean residence times for rHuAChE glycoforms harbouring 2, 3, and 4 glycans = 200, 740, and 1055min respectively). This study provides evidence that glycan loading, together with N-glycan terminal processing and enzyme subunit oligomerization, operate in a hierarchical and concerted manner in determining the pharmacokinetic characteristics of AChE.
Metabolic labelling of membrane microdomains/rafts in Jurkat cells indicates the presence of glycerophospholipids implicated in signal transduction by the CD3 T-cell receptor
Authors: ROUQUETTE-JAZDANIAN Alexandre K. , PELASSY Claudette , BREITTMAYER Jean-Philippe , COUSIN Jean-Louis , AUSSEL Claude
Published: August 2015

Cell membranes contain sphingolipids and cholesterol, which cluster together in distinct domains called rafts. The outer-membrane leaflet of these peculiar membrane domains contains glycosylphosphatidylinositol-anchored proteins, while the inner leaflet contains proteins implicated in signalling, such as the acylated protein kinase p56lck and the palmitoylated adaptator LAT (linker for activation of T-cells). We present here an approach to study the lipid composition of rafts and its change upon T-cell activation. Our method is based on metabolic labelling of Jurkat T-cells with different precursors of glycerophospholipid synthesis, including glycerol and fatty acids with different lengths and degrees of saturation as well as phospholipid polar head groups. The results obtained indicate that lipid rafts isolated by the use of sucrose density-gradient centrifugation after Triton X-100 extraction in the cold, besides sphingolipids and cholesterol, contain unambiguously all classes of glycerophospholipids: phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine and phosphatidylcholine. Fatty acid labelling shows that lipid rafts are labelled preferentially with saturated fatty acids while the rest of the plasma membrane incorporates mostly long-chained polyunsaturated fatty acids. To see whether the raft composition as measured by metabolic labelling of phospholipids is involved in T-cell activation, we investigated the production of sn-1,2-diacylglycerol (DAG) in CD3-activated cells. DAG production occurs within rafts, confirming previous demonstration of protein kinase C translocation into membrane microdomains. Our data demonstrate that raft disorganization by methyl-β-cyclodextrin impairs both CD3-induced DAG production and changes in cytosolic Ca2+ concentration. These lines of evidence support the conclusion that the major events in T-cell activation occur within or due to lipid rafts.
Mutation of conserved active-site threonine residues in creatine kinase affects autophosphorylation and enzyme kinetics
Authors: STOLZ Martin , HORNEMANN Thorsten , SCHLATTNER Uwe , WALLIMANN Theo
Published: August 2015

Muscle-type creatine kinase (MM-CK) is a member of an isoenzyme family with key functions in cellular energetics. It has become a matter of debate whether the enzyme is autophosphorylated, as reported earlier [Hemmer, Furter-Graves, Frank, Wallimann and Furter (1995) Biochim. Biophys. Acta 1251, 81–90], or exclusively nucleotidylated. In the present paper, we demonstrate unambiguously that CK is indeed autophosphorylated. However, this autophosphorylation is not solely responsible for the observed microheterogeneity of MM-CK on two-dimensional isoelectric focusing gels. Using phosphoamino-acid analysis of 32P-labelled CK isoforms, phosphothreonine (P-Thr) residues were identified as the only product of autophosphorylation for all CK isoenzymes. The phosphorylated residues in chicken MM-CK were allocated to a region in the vicinity of the active site, where five putative phosphorylation sites were identified. Site-directed threonine—valine-replacement mutants reveal that autophosphorylation is not specific for one particular residue but occurs at all examined threonine residues. The enzyme kinetic parameters indicate that the autophosphorylation of CK exerts a modulatory effect on substrate binding and the equilibrium constant, rather than on the catalytic mechanism itself.
Glutathione synthetase from Plasmodium falciparum
Authors: MEIERJOHANN Svenja , WALTER Rolf D. , MÜLLER Sylke
Published: August 2015

GSH is the major low-molecular-mass thiol in most organisms. The tripeptide maintains a reduced intracellular environment and protects cellular components from damaging oxidation. GSH is synthesized by the action of two ATP-dependent enzymic steps, in which γ-glutamylcysteine synthetase (γ-GCS) catalyses the ligation of glutamate and cysteine and subsequently glutathione synthetase (GS) adds glycine to the dipeptide. Recently it was shown that the synthesis of γ-glutamylcysteine is crucial for the survival of the erythrocytic stages of the malaria parasite Plasmodium falciparum by using the specific γ-GCS inhibitor buthionine sulphoximine. In order to investigate further the synthetic pathway of the tripeptide in the parasite, GS was cloned and expressed recombinantly. The deduced amino acid sequence of P. falciparum GS shares only a moderate degree of identity with other known GSs, but the residues responsible for substrate and co-factor binding are almost all conserved, with the exception of the ones involved in γ-glutamylcysteine binding. The protein is active as a dimer, with a subunit molecular mass of 77kDa, and the addition of reducing reagents such as dithiothreitol is essential in maintaining enzymic activity, indicating that thiol groups are important for stability and enzymic activity. The Kappm values for γ-glutamyl-α-aminobutyrate, ATP and glycine were determined to be 107.1μM, 59.1μM and 5.04mM, respectively, and the Vmax of 5.24±0.7μmol·min−1·mg−1 was in the same range as that of the mammalian enzymes. However, the negative co-operativity observed for γ-glutamylcysteine binding to the rat enzyme was not found for the parasite protein. This may be due to the alteration of several amino acids in the γ-glutamylcysteine-binding site.
The 45 kDa collagen-binding fragment of fibronectin induces matrix metalloproteinase-13 synthesis by chondrocytes and aggrecan degradation by aggrecanases
Authors: STANTON Heather , UNG Linh , FOSANG Amanda J.
Published: August 2015

Fragments of fibronectin occur naturally in vivo and are increased in the synovial fluid of arthritis patients. We have studied the 45kDa fragment (Fn-f 45), representing the N-terminal collagen-binding domain of fibronectin, for its ability to modulate the expression of metalloproteinases by porcine articular chondrocytes in vitro. We report that stimulation of cultured chondrocytes, or cartilage explants, with Fn-f 45 increased the levels of matrix metalloproteinase-13 (MMP-13; collagenase-3) released into the conditioned medium in a dose-dependent manner. Increased levels of MMP-13 were due to stimulation of MMP-13 synthesis, rather than release of MMP-13 from accumulated matrix stores. Fn-f 45 also stimulated the synthesis of MMP-3 (stromelysin-1) from cultured chondrocytes and cartilage cultures. The Fn-f 45-induced increase in MMP-3 and MMP-13 synthesis occurred via an interleukin 1-independent mechanism, since the receptor antagonist of interleukin-1 was unable to block the increased synthesis. The gelatinases, MMP-2 and MMP-9, were not modulated by Fn-f 45 in these culture systems. Fn-f 45 also stimulated the release of aggrecan from cartilage explants into conditioned medium. Neoepitope antibodies specific for aggrecan fragments generated by MMPs or aggrecanases showed that the Fn-f 45-induced aggrecan loss was mediated by aggrecanases, and not by MMPs. Extracts of cultured cartilage contained elevated levels of the aggrecanase-derived ITEGE373-G1 domain, whereas levels of the matrix metalloproteinase-derived DIPEN341-G1 domain were unchanged. These studies show that Fn-f 45 can induce a catabolic phenotype in articular chondrocytes by up-regulating the expression of metalloproteinases specific for the degradation of collagen and aggrecan.
Characterization of a novel intracellular heparanase that has a FERM domain
Authors: BAME Karen J. , VENKATESAN Indumati , DEHDASHTI Jean , McFARLANE Jeffrey , BURFEIND Rebecca
Published: August 2015

The catabolism of cell-surface heparan sulphate proteoglycans is initiated by endosomal heparanases, which are endoglycosidases that cleave the glycosaminoglycans off core proteins and degrade them to shorter oligosaccharides. We have purified previously four intracellular heparanase activities from Chinese hamster ovary (CHO) cells [Bame, Hassall, Sanderson, Venkatesan and Sun (1998) Biochem. J. 336, 191–200], and in the present study we characterize further the most abundant activity (C1A heparanase). This enzyme purifies as a family of 37–48kDa proteins from both CHO cells and the rat liver, with the major species being 37 and 40kDa. Amino acid sequence analysis shows the purified C1A heparanase protein is highly homologous with the N-terminal domain, or FERM domain, of the ≈80kDa proteins ezrin, radixin and moesin (ERM proteins, after ezrin-radixin-moesin). This domain, which is also found in erythrocyte protein 4.1, links cytoplasmic proteins to membranes. Antibodies against the FERM domain recognize all the C1A heparanase proteins on Western blots, suggesting that the smaller species are derived from a larger protein. Activity binds to, and is affected by, molecules known to interact with FERM domains, supporting the hypothesis that the intracellular C1A heparanase is the purified FERM domain protein. Since bacterially expressed FERM domains of radixin and moesin lack heparanase activity, and some tryptic peptides generated from the enzyme do not have a match in any ERM protein, it appears that, rather than being derived from ezrin, radixin or moesin, C1A heparanase may be a new member of the FERM domain family.
Proteolytic processing of the amyloid-beta protein precursor of Alzheimer's disease
Authors: Nunan Janelle , Small David H
Published: August 2015

The proteolytic processing of the amyloid-beta protein precursor plays a key role in the development of Alzheimer's disease. Cleavage of the amyloid-beta protein precursor may occur via two pathways, both of which involve the action of proteases called secretases. One pathway, involving beta- and gamma-secretase, liberates amyloid-beta protein, a protein associated with the neurodegeneration seen in Alzheimer's disease. The alternative pathway, involving alpha-secretase, precludes amyloid-beta protein formation. In this review, we describe the progress that has been made in identifying the secretases and their potential as therapeutic targets in the treatment or prevention of Alzheimer's disease.
The ubiquitin-proteasome pathway of intracellular proteolysis
Authors: Doherty Fergus J , Dawson Simon , Mayer R John
Published: August 2015

Intracellular proteins are targeted for degradation by the covalent attachment of chains of the small protein ubiquitin; a process known as ubiquitylation. Many proteins are phosphorylated prior to ubiquitylation, and therefore ubiquitylation and degradation of these proteins is regulated by kinase activity and signalling cascades. Many ubiquitylated proteins are degraded by the 26 S proteasome complex, which is found in the cytosol and nucleus. The 26 S proteasome consists of a 20 S core with proteolytic activity and 18 S regulatory complexes containing ATPases and ubiquitin-chain-binding proteins. Proteins degraded by the ubiquitin-proteasome pathway include cyclins and other regulators of the cell cycle, and transcription factors. Abnormal polypeptides are also degraded by the ubiquitin pathway, including abnormal polypeptides in the endoplasmic reticulum, which are translocated back out of the endoplasmic reticulum prior to ubiquitylation and degradation by the proteasome. The ubiquitin-proteasome pathway is implicated in numerous diseases including cancer and neurodegenerative diseases.
The final step in programmed cell death: phagocytes carry apoptotic cells to the grave
Authors: deCathelineau Aimee M , Henson Peter M
Published: August 2015

As cells undergo apoptosis, they are recognized and removed from the body by phagocytes. This oft-overlooked yet critical final step in the cell-death programme protects tissues from exposure to the toxic contents of dying cells and also serves to prevent further tissue damage by stimulating production of anti-inflammatory cytokines and chemokines. The clearance of apoptotic-cell corpses occurs throughout the lifespan of multicellular organisms and is important for normal development during embryogenesis, the maintenance of normal tissue integrity and function, and the resolution of inflammation. Many of the signal-transduction molecules implicated in the phagocytosis of apoptotic cells appear to have a high degree of evolutionary conservation, and therefore the engulfment of apoptotic cells is likely to represent one of the most primitive forms of phagocytosis. With the realization that the signals that govern apoptotic-cell removal also serve to attenuate inflammation and the immune response, as well as initiate signals for tissue repair and remodelling in response to cell death, the study of apoptotic cell clearance is a field experiencing a dynamic increase in interest and momentum.
E3 ubiquitin ligases
Authors: Ardley Helen C. , Robinson Philip A.
Published: August 2015

The selectivity of the ubiquitin–26 S proteasome system (UPS) for a particular substrate protein relies on the interaction between a ubiquitin-conjugating enzyme (E2, of which a cell contains relatively few) and a ubiquitin–protein ligase (E3, of which there are possibly hundreds). Post-translational modifications of the protein substrate, such as phosphorylation or hydroxylation, are often required prior to its selection. In this way, the precise spatio-temporal targeting and degradation of a given substrate can be achieved. The E3s are a large, diverse group of proteins, characterized by one of several defining motifs. These include a HECT (homologous to E6-associated protein C-terminus), RING (really interesting new gene) or U-box (a modified RING motif without the full complement of Zn2+-binding ligands) domain. Whereas HECT E3s have a direct role in catalysis during ubiquitination, RING and U-box E3s facilitate protein ubiquitination. These latter two E3 types act as adaptor-like molecules. They bring an E2 and a substrate into sufficiently close proximity to promote the substrate's ubiquitination. Although many RING-type E3s, such as MDM2 (murine double minute clone 2 oncoprotein) and c-Cbl, can apparently act alone, others are found as components of much larger multi-protein complexes, such as the anaphase-promoting complex. Taken together, these multifaceted properties and interactions enable E3s to provide a powerful, and specific, mechanism for protein clearance within all cells of eukaryotic organisms. The importance of E3s is highlighted by the number of normal cellular processes they regulate, and the number of diseases associated with their loss of function or inappropriate targeting.
Ubiquitin and control of transcription
Authors: Dhananjayan Sarath C. , Ismail Ayesha , Nawaz Zafar
Published: August 2015

Eukaryotic transcription is one of the most complex cellular processes and constitutes the first step in protein synthesis. Ubiquitination and subsequent degradation by the 26 S proteasome, on the other hand, represents the final chapter in the life of a protein. Intriguingly, ubiquitin and the ubiquitin– proteasome system play vital roles in the regulation of transcription. Ubiquitin has dual modus operandi: firstly, ubiquitin functions via the 26 S proteasome — it is tagged to components of the transcription machinery, marking them for degradation via the proteasome, which results in the proper exchange of complexes during transcription and the prompt removal of activators after each round of transcription; and secondly, ubiquitin can function independently of the proteasome — histone ubiquitination results in heterochromatin relaxation and assembly of transcription complexes on the promoter, and ubiquitination of transcription factors enhances their transcriptional-activation function. Although ubiquitin and the ubiquitin–proteasome system were initially perceived as a graveyard for proteins, recent advances in molecular biological techniques have redefined their role as a regulatory system that influences the fate of many cellular processes, such as apoptosis, transcription and cell cycle progression.
Fluorescent reporters for the ubiquitin–proteasome system
Authors: Salomons Florian A. , Verhoef Lisette G.G.C. , Dantuma Nico P.
Published: August 2015

Regulated turnover of proteins in the cytosol and nucleus of eukaryotic cells is primarily performed by the ubiquitin–proteasome system (UPS). The UPS is involved in many essential cellular processes. Alterations in this proteolytic system are associated with a variety of human pathologies, such as neurodegenerative diseases, cancer, immunological disorders and inflammation. The precise role of the UPS in the pathophysiology of these diseases, however, remains poorly understood. Detection of UPS aberrations has been a major challenge because of the complexity of the system. Most studies focus on various aspects of the UPS, such as substrate recognition, ubiquitination, deubiquitination or proteasome activity, and do not provide a complete picture of the UPS as an integral system. To monitor the efficacy of the UPS, a number of reporter substrates have been developed based on fluorescent proteins, such as the green fluorescent protein and its spectral variants. These fluorescent UPS reporters contain specific degradation signals that target them with high efficiency and accuracy for proteasomal degradation. Several studies have shown that these reporters can probe the functionality of the UPS in cellular and animal models and provide us with important information on the status of the UPS under various conditions. Moreover, these reporters can aid the identification and development of novel anti-cancer and anti-inflammatory drugs based on UPS inhibition.
Proteasome inhibitors as therapeutics
Authors: Mitsiades Constantine S. , Mitsiades Nicholas , Hideshima Teru , Richardson Paul G. , Anderson Kenneth C.
Published: August 2015

The ubiquitin–proteasome pathway is a principle intracellular mechanism for controlled protein degradation and has recently emerged as an attractive target for anticancer therapies, because of the pleiotropic cell-cycle regulators and modulators of apoptosis that are controlled by proteasome function. In this chapter, we review the current state of the field of proteasome inhibitors and their prototypic member, bortezomib, which was recently approved by the U.S. Food and Drug Administration for the treatment of advanced multiple myeloma. Particular emphasis is placed on the pre-clinical research data that became the basis for eventual clinical applications of proteasome inhibitors, an overview of the clinical development of this exciting drug class in multiple myeloma, and a appraisal of possible uses in other haematological malignancies, such non-Hodgkin's lymphomas.
Radicals from one-electron reduction of nitro compounds, aromatic N-oxides and quinones: the kinetic basis for hypoxia-selective, bioreductive drugs
Authors: Wardman Peter , Dennis Madeleine F. , Everett Steven A. , Patel Kantilal B. , Stratford Michael R.L. , Tracy Michael
Published: August 2015

Drugs based on nitroarene, aromatic N-oxide or quinone structures are frequently reduced by cellular reductases to toxic products. Reduction often involves free radicals as intermediates which react rapidly with oxygen to form superoxide radicals, inhibiting drug reduction. The elevation of cellular oxidative stress accompanying oxygen inhibition of reduction is generally less damaging than drug reduction to toxic products, so the drugs offer selective toxicity to hypoxic cells. Since such cells are resistant to radiotherapy, these bioreductive drugs offer potential in tumour therapy. The basis for the selectivity of action entails kinetic competition involving the contesting reaction pathways. The reduction potential of the drug, radical pKa and nature of radical/radical decay kinetics all influence drug activity and selectivity, including the range of oxygen tensions over which the drug offers selective toxicity. These properties may be quantified using generation of radicals by pulse radiolysis, presenting a physicochemical basis for rational drug design.
Antioxidant agents in raw materials and processed foods
Authors: Eriksson Caj E. , Na An
Published: August 2015

Many food raw materials contain natural antioxidants which exert control of oxidative processes in the living cells. Among antioxidative agents are found enzymes such as superoxide dismutase, glutathione peroxidase and glucose oxidase-catalase. Among naturally occurring non-enzymic antioxidants are carotenoids, especially astaxanthin (e.g. in fish), tocopherols in oils and other phenolic compounds in plant material. Enzymic antioxidants are mostly inactivated in food processing but the non-enzymic ones can be active also in heat-treated food and might also be active after consumption of the food, as is claimed with ϐ-carotene, and vitamins A and E. Vitamin C is a generally reducing substance which acts synergistically with other antioxidants. Processing of food can result in the formation of antioxidative compounds, e.g. by protein hydrolysis, Maillard reaction and fermentation by lactic acid bacteria. Curing of meat yields nitrosylhaem pigments which can act as radical scavengers and protect both the meat pigment and the lipids from oxidation. Two or more antioxidants together can act synergistically, i.e. affect lipid oxidation to a higher extent than the sum of the contributions from each single antioxidant.
Apoptosis and necrosis: different execution of the same death
Authors: Nicotera Pierluigi , Leist Marcel , Ferrando-May Elisa
Published: August 2015

Regardless of whether apoptosis or necrosis are elicited by toxicants or by pathophysiological conditions, they are considered conceptually distinct forms of cell death. Nevertheless, there is increasing evidence that classical apoptosis and necrosis represent only the extreme ends of a wide range of possible morphological and biochemical deaths. The two classical types of demise can occur simultaneously in tissues or cell cultures exposed to the same stimulus and, often, the intensity of the same initial insult decides the prevalence of either apoptosis or necrosis. The execution of the death programme seems to involve a relatively limited number of pathways. In many instances, their ordered execution results in characteristic morphological and biochemical changes termed apoptosis. However, some subroutines of the degradation programme may not be active in all cases of cell death. Then, the morphological appearance of dying cells and some of their biochemical alterations differ from those of classical apoptosis. We have recently shown that intracellular energy levels and mitochondrial function are rapidly compromised in necrosis, but not in apoptosis, of neuronal cells. Then we went on to show that pre-emptying human T-cells of ATP switches the type of demise caused by two classic apoptotic triggers (staurosporine and CD95 stimulation) from apoptosis to necrosis. Conditions of controlled intracellular ATP depletion, obtained by blocking mitochondrial and/or glycolytic ATP generation, were used in combination with repletion of the cytosolic ATP pool with glucose to redirect the death programme towards apoptosis or necrosis.
Modulation of ϐ-amyloid production and fibrillization
Authors: Allsop David , Twyman Lance J. , Davies Yvonne , Moore Susan , York Amber , Swanson Linda , Soutar Ian
Published: August 2015

Alzheimer's disease (AD) is the most common cause of dementia in old age and presently affects an estimated 4 million people in the U.S.A. and 0.75 million people in the U.K. It is a relentless, degenerative brain disease, characterized by progressive cognitive impairment. In the final stages of the disease, patients are often bedridden, doubly incontinent and unable to speak or to recognize close relatives. Pathological changes of Alzheimer's disease include extensive neuronal loss and the presence of numerous neurofibrillary tangles and senile plaques in the brain. The senile plaques contain amyloid fibrils derived from a 39-43-amino-acid peptide referred to as ϐ-amyloid or Aϐ. The basic theory of the so-called 'amyloid hypothesis' is that the deposition of aggregated forms of Aϐ in the brain parenchyma triggers a pathological cascade of events that leads to neurofibrillary tangle formation, neuronal loss and the associated dementia [1]. Here we discuss progress towards the identification of inhibitors of Aϐ production and fibrillization.
Neurofibrillary tangles and tau phosphorylation
Authors: Brion Jean-Pierre , Anderton Brian H. , Authelet Michéle , Dayanandan Rejith , Leroy Karelle , Lovestone Simon , Octave Jean-Noël , Pradier Laurent , Touchet Nicole , Tremp Günter
Published: August 2015

Neurofibrillary tangles (NFTs) are a characteristic neuropathological lesion of Alzheimer's disease (AD). They are composed of a highly-phosphorylated form of the microtubule-associated protein tau. We are investigating the relationship between NFTs and microtubule stability and how tau phosphorylation and function is affected in transgenic models and by co-expression with ϐ-amyloid precursor protein and presenilins. In most NFT-bearing neurons, we observed a strong reduction in acetylated α-tubulin immunoreactivity (a marker of stable microtubules) and a reduction of the in situ hybridization signal for tubulin mRNA. In transfected cells, mutated tau forms (corresponding to tau mutations identified in familial forms of frontotemporal dementias linked to chromosome 17) were less efficient in their ability to sustain microtubule growth. These observations are consistent with the hypothesis that destabilization of the microtubule network is an important mechanism of cell dysfunction in Alzheimer's disease. The glycogen synthase kinase-3 ϐ (GSK-3ϐ) generates many phosphorylated sites on tau. We performed a neuroanatomical study of GSK-3ϐ distribution showing that developmental evolution of GSK-3ϐ compartmentalization in neurons paralleled that of phosphorylated tau. Studies on transfected cells and on cultured neurons showed that GSK-3 ϐ activity controls tau phosphorylation and tau functional interaction with microtubules. Tau phosphorylation was not affected in neurons overexpressing ϐ-amyloid precursor protein. Transgenic mice expressing a human tau isoform and double transgenic animals for tau and mutated presenilin 1 have been generated; a somatodendritic accumulation of phosphorylated transgenic tau proteins, as observed in the pretangle stage in AD, has been observed but NFTs were not found, suggesting that additional factors might be necessary to induce their formation.
Regulation of gene expression by muscarinic acetylcholine receptors
Authors: von der Kammer Heinz , Demiralay Cüneyt , Andresen Barbara , Albrecht Claudia , Mayhaus Manuel , Nitsch Roger M.
Published: August 2015

In the brain, muscarinic acetylcholine receptors (mAChRs) are involved in higher cognitive functions including synaptic plasticity and memory. In Alzheimer's disease (AD) patients the cholinergic nervous system is severely damaged. In order to reinforce the cholinergic system, clinical tests were started to use cholinomimetic drugs to treat AD patients. To identify the genes involved in mAChR signalling, we used a differential display approach and found 11 genes that were readily activated by mAChR with 1 hour of activation. These included the transcription factors Egr-1, Egr-2, Egr-3, c-Jun, Jun-D and Gos-3; the growth regulator hCyr61; the signalling factors NGFi-B (nerve growth factor induced gene-B) and Etr101; the unknown gene Gig-2 (for G-protein-coupled receptor induced gene 2); and the acetylcholinesterase gene (ACHE). Our data show that multiple immediate-early genes are under the control of mAChRs, and they suggest that these genes play important roles in coupling receptor stimulation to long-term neuronal responses. The results also suggest a feedback mechanism where up-regulated ACHE expression and accelerated breakdown of acetylcholine (ACh) at the cholinergic synapses limits increases in cholinergic transmission. Three hours after m1 mAChR activation a different pattern of gene expression was demonstrated. It included the novel genes Gig-3 and Gig-4, as well as the LIM-only protein LM04. Like ACHE, these genes are target genes which may be under the control of the above immediate-early genes. Together, our data show that muscarinic receptors induce a complex and sustained pattern of gene expression that may be involved in the regulation of cholinergic transmission as well as the control of cellular functions in post-synaptic cholinergic target cells. These results may contribute to a better understanding of the effects and side effects of cholinomimetic treatment in AD patients.
Transgenic mouse models of Alzheimer's disease: phenotype and mechanisms of pathogenesis
Authors: Duff Karen
Published: August 2015

A range of transgenic mice have been created to model Alzheimer's disease. These include mice expressing human forms of the amyloid precursor protein, the presenilins and, more recently, tau. Several of the models develop features of the disease including amyloid pathology, cholinergic deficits, neurodegeneration and cognitive impairment. Progress in the characterization and use of these model animals is discussed.
Structure, stability and folding of the α-helix
Authors: Doig Andrew J. , Andrew Charles D. , Cochran Duncan A. E. , Hughes Eleri , Penel Simon , Sun Jia Ke , Stapley Benjamin J. , Clarke David T. , Jones Gareth R.
Published: August 2015

Pauling first described the α-helix nearly 50 years ago, yet new features of its structure continue to be discovered, using peptide model systems, site-directed mutagenesis, advances in theory, the expansion of the Protein Data Bank and new experimental techniques. Helical peptides in solution form a vast number of structures, including fully helical, fully coiled and partly helical. To interpret peptide results quantitatively it is essential to use a helix/coil model that includes the stabilities of all these conformations. Our models now include terms for helix interiors, capping, side-chain interactions, N-termini and 310-helices. The first three amino acids in a helix (N1, N2 and N3) and the preceding N-cap are unique, as their amide NH groups do not participate in backbone hydrogen bonding. We surveyed their structures in proteins and measured their amino acid preferences. The results are predominantly rationalized by hydrogen bonding to the free NH groups. Stabilizing side-chain-side-chain energies, including hydrophobic interactions, hydrogen bonding and polar/non-polar interactions, were measured accurately in helical peptides. Helices in proteins show a preference for having approximately an integral number of turns so that their N- and C-caps lie on the same side. There are also strong periodic trends in the likelihood of terminating a helix with a Schellman or αL C-cap motif. The kinetics of α-helix folding have been studied with stopped-flow deep ultraviolet circular dichroism using synchrotron radiation as the light source; this gives a far superior signal-to-noise ratio than a conventional instrument. We find that poly(Glu), poly(Lys) and alanine-based peptides fold in milliseconds, with longer peptides showing a transient overshoot in helix content.
Papain-like lysosomal cysteine proteases and their inhibitors: drug discovery targets?
Authors: Turk Dŭsan , Turk Boris , Turk Vito
Published: August 2015

Papain-like lysosomal cysteine proteases are processive and digestive enzymes that are expressed in organisms from bacteria to humans. Increasing knowledge about the physiological and pathological roles of cysteine proteases is bringing them into the focus of drug discovery research. These proteases have rather short active-site clefts, comprising three well defined substrate-binding subsites (S2, S1 and S1') and additional broad binding areas (S4, S3, S2' and S3'). The geometry of the active site distinguishes cysteine proteases from other protease classes, such as serine and aspartic proteases, which have six and eight substrate-binding sites respectively. Exopeptidases (cathepsins B, C, H and X), in contrast with endopeptidases (such as cathepsins L, S, V and F), possess structural features that facilitate the binding of N- and C-terminal groups of substrates into the active-site cleft. Other than a clear preference for free chain termini in the case of exopeptidases, the substrate-binding sites exhibit no strict specificities. Instead, their subsite preferences arise more from the specific exclusion of substrate types. This presents a challenge for the design of inhibitors to target a specific cathepsin: only the cumulative effect of an assembly of inhibitor fragments will bring the desired result.
Memapsin 2, a drug target for Alzheimer's disease
Authors: Koelsch Gerald , Turner Robert T. , Hong Lin , Ghosh Arun K. , Tang Jordan
Published: August 2015

Mempasin 2, a ϐ-secretase, is the membrane-anchored aspartic protease that initiates the cleavage of amyloid precursor protein leading to the production of ϐ-amyloid and the onset of Alzheimer's disease. Thus memapsin 2 is a major therapeutic target for the development of inhibitor drugs for the disease. Many biochemical tools, such as the specificity and crystal structure, have been established and have led to the design of potent and relatively small transition-state inhibitors. Although developing a clinically viable mempasin 2 inhibitor remains challenging, progress to date renders hope that memapsin 2 inhibitors may ultimately be useful for therapeutic reduction of ϐ-amyloid.
Activation of cytochrome c to a peroxidase compound I-type intermediate by H2O2: relevance to redox signalling in apoptosis
Authors: Burkitt Mark , Jones Clare , Lawrence Andrew , Wardman Peter
Published: August 2015

The release of cytochrome c from mitochondria during apoptosis results in the enhanced production of superoxide radicals, which are converted to H2O2 by Mn-superoxide dismutase. We have been concerned with the role of cytochrome c/H2O2 in the induction of oxidative stress during apoptosis. Our initial studies showed that cytochrome c is a potent catalyst of 2′,7′-dichlorofluorescin oxidation, thereby explaining the increased rate of production of the fluorophore 2′,7′-dichlorofluorescein in apoptotic cells. Although it has been speculated that the oxidizing species may be a ferryl-haem intermediate, no definitive evidence for the formation of such a species has been reported. Alternatively, it is possible that the hydroxyl radical may be generated, as seen in the reaction of certain iron chelates with H2O2. By examining the effects of radical scavengers on 2′,7′-dichlorofluorescin oxidation by cytochrome c/H2O2, together with complementary EPR studies, we have demonstrated that the hydroxyl radical is not generated. Our findings point, instead, to the formation of a peroxidase compound I species, with one oxidizing equivalent present as an oxo-ferryl haem intermediate and the other as the tyrosyl radical identified by Barr and colleagues [Barr, Gunther, Deterding, Tomer and Mason (1996) J. Biol. Chem. 271, 15498-15503]. Studies with spin traps indicated that the oxo-ferryl haem is the active oxidant. These findings provide a physico-chemical basis for the redox changes that occur during apoptosis. Excessive changes (possibly catalysed by cytochrome c) may have implications for the redox regulation of cell death, including the sensitivity of tumour cells to chemotherapeutic agents.
Biophysics (and sociology) of ceramides.
Authors: Goñi Félix M. , Contreras F-Xabier , Montes L-Ruth , Sot Jesús , Alonso Alicia
Published: August 2015

In the past decade, the long-neglected ceramides (N-acylsphingosines) have become one of the most attractive lipid molecules in molecular cell biology, because of their involvement in essential structures (stratum corneum) and processes (cell signalling). Most natural ceramides have a long (16-24 C atoms) N-acyl chain, but short N-acyl chain ceramides (two to six C atoms) also exist in Nature, apart from being extensively used in experimentation, because they can be dispersed easily in water. Long-chain ceramides are among the most hydrophobic molecules in Nature, they are totally insoluble in water and they hardly mix with phospholipids in membranes, giving rise to ceramide-enriched domains. In situ enzymic generation, or external addition, of long-chain ceramides in membranes has at least three important effects: (i) the lipid monolayer tendency to adopt a negative curvature, e.g. through a transition to an inverted hexagonal structure, is increased, (ii) bilayer permeability to aqueous solutes is notoriously enhanced, and (iii) transbilayer (flip-flop) lipid motion is promoted. Short-chain ceramides mix much better with phospholipids, promote a positive curvature in lipid monolayers, and their capacities to increase bilayer permeability or transbilayer motion are very low or non-existent.
The RNA polymerase I transcription machinery
Authors: Russell Jackie , Zomerdijk Joost C.B.M.
Published: August 2015

The rRNAs constitute the catalytic and structural components of the ribosome, the protein synthesis machinery of cells. The level of rRNA synthesis, mediated by Pol I (RNA polymerase I), therefore has a major impact on the life and destiny of a cell. In order to elucidate how cells achieve the stringent control of Pol I transcription, matching the supply of rRNA to demand under different cellular growth conditions, it is essential to understand the components and mechanics of the Pol I transcription machinery. In this review, we discuss: (i) the molecular composition and functions of the Pol I enzyme complex and the two main Pol I transcription factors, SL1 (selectivity factor 1) and UBF (upstream binding factor); (ii) the interplay between these factors during pre-initiation complex formation at the rDNA promoter in mammalian cells; and (iii) the cellular control of the Pol I transcription machinery.
The role of PI3Ks in the regulation of the neutrophil NADPH oxidase
Authors: Hawkins Phillip T. , Davidson Keith , Stephens Len R.
Published: August 2015

The NADPH oxidase complex of neutrophils and macrophages is an important weapon used by these cells to kill microbial pathogens. The regulation of this enzyme complex is necessarily complicated by the diverse receptor types that are needed to trigger its activation and also the tight control that is required to deliver this activation at the appropriate time and place. As such, several signalling pathways have been established to regulate the NADPH oxidase downstream of cell surface receptors. Central amongst these are PI3K- (phosphoinositide 3-kinase)-dependent pathways, blockade of which severely limits activation of the oxidase to several soluble and particulate stimuli. The precise roles of the phosphoinositide products of PI3K activity in regulating NADPH oxidase assembly and activation are still unclear, but there is emerging evidence that they play a key role via regulation of guanine nucleotide exchange on Rac, a key component in the oxidase complex. There is also very strong evidence that the PI3K products PtdIns(3,4)P2 and PtdIns3P can bind directly to the PX (Phox homology) domains of the core oxidase components p47phox and p40phox respectively. However, the significance of these interactions in terms of membrane localization or allosteric consequences for the oxidase complex remains to be established.
Multiple functions of inositolphosphorylceramides in the formation and intracellular transport of glycosylphosphatidylinositol-anchored proteins in yeast
Authors: Bosson Régine , Conzelmann Andreas
Published: August 2015

The mature sphingolipids of yeast consist of IPCs (inositolphosphorylceramides) and glycosylated derivatives thereof. Beyond being an abundant membrane constituent in the organelles of the secretory pathway, IPCs are also used to constitute the lipid moiety of the majority of GPI (glycosylphosphatidylinositol) proteins, while a minority of GPI proteins contain PI (phosphatidylinositol). Thus all GPI anchor lipids (as well as free IPCs) typically contain C26 fatty acids. However, the primary GPI lipid that isadded to newly synthesized proteins in the endoplasmic reticulum consists of a PI with conventional C16 and C18 fatty acids. A new class of enzymes is required to replace the fatty acid in sn-2 by a C26 fatty acid. Cells lacking this activity make normal amounts of GPI proteins but accumulate GPI anchors containing lyso-PI. As a consequence, the endoplasmic reticulum to Golgi transport of the GPI protein Gas1p is slow, and mature Gas1p is lost from the plasma membrane into the medium. The GPI anchor containing C26 in sn-2 can further be remodelled by the exchange of diacylglycerol for ceramide. This process is also dependent on the presence of specific phosphorylethanolamine side-chains on the GPI anchor.
VASODILATORS AND CALCIUM-45 UPTAKE IN VASCULAR SMOOTH AND CARDIAC MUSCLE
Authors: BOSTROM STIG-LENNART , LJUNG BENGT
Published: August 2015

A variety of substances, chemically heterogenous, has been classified as calcium antagonists. They inhibit the contractile activity of vascular smooth and cardiac muscle. This has been attributed to a blockade of the so called slow channels of the cell membrane causing an inhibition of the transmembrane influx of calcium. In the experiments reported we have studied the effect of verapamil, nifedipine and felodipine (pINN) on the uptake of calcium-45 in the rat portal vein and papillary muscle.

The results show that the substances, at their IC50 values, did not decrease the cellular uptake of calcium-45, measured with the lanthanum method.