bluuebunny/crossref_metadata_2025 huggingface dataset:
Use of a purified and functional recombinant calcium-channel β4 subunit in surface-plasmon resonance studies
Authors: GEIB Sandrine , SANDOZ Guillaume , MABROUK Kamel , MATAVEL Alessandra , MARCHOT Pascale , HOSHI Toshinori , VILLAZ Michel , RONJAT Michel , MIQUELIS Raymond , LÉVÊQUE Christian , de WAARD Michel
Published: August 2015

Native high-voltage-gated calcium channels are multi-subunit complexes comprising a pore-forming subunit Cav and at least two auxiliary subunits α2δ and β. The β subunit facilitates cell-surface expression of the channel and contributes significantly to its biophysical properties. In spite of its importance, detailed structural and functional studies are hampered by the limited availability of native β subunit. Here, we report the purification of a recombinant calcium-channel β4 subunit from bacterial extracts by using a polyhistidine tag. The purified protein is fully functional since it binds on the alpha1 interaction domain, its main Cav-binding site, and regulates the activity of P/Q calcium channel expressed in Xenopus oocytes in a similar way to the β4 subunit produced by cRNA injection. We took advantage of the functionality of the purified material to (i) develop an efficient surface-plasmon resonance assay of the interaction between two calcium channel subunits and (ii) measure, for the first time, the affinity of the recombinant His-β4 subunit for the full-length Cav2.1 channel. The availability of this purified material and the development of a surface-plasmon resonance assay opens two immediate research perspectives: (i) drug screening programmes applied to the Cav/β interaction and (ii) crystallographic studies of the calcium-channel β4 subunit.
Characteristics and composition of the vitamin K-dependent γ-glutamyl carboxylase-binding domain on osteocalcin
Authors: HOUBEN Roger J.T.J. , RIJKERS Dirk T.S. , STANLEY Thomas B. , ACHER Francine , AZERAD Robert , KÄKÖNEN Sanna-Maria , VERMEER Cees , SOUTE Berry A.M.
Published: August 2015

Two different sites on vitamin K-dependent γ-glutamyl carboxylase (VKC) are involved in enzyme—substrate interaction: the propeptide-binding site required for high-affinity substrate binding and the active site for glutamate carboxylation. Synthetic descarboxy osteocalcin (d-OC) is a low-Km substrate for the VKC, but unique since it possesses a high-affinity recognition site for the VKC, distinct from the propeptide which is essential as a binding site for VKC. However, the exact location and composition of this VKC-recognition domain on d-OC has remained unclear until now. Using a stereospecific substrate analogue [t-butyloxycarbonyl-(2S,4S)-4-methylglutamic acid-Glu-Val (S-MeTPT)] we demonstrate in this paper that the high affinity of d-OC for VKC cannot be explained by a direct interaction with either the active site or with the propeptide-binding site on VKC. It is shown using various synthetic peptides derived from d-OC that there are two domains on d-OC necessary for recognition: one located between residues 1 and 12 and a second between residues 26 and 39, i.e. at the C-terminal side of the γ-carboxyglutamate (Gla) domain. Both internal sequences contribute substantially to the efficiency of carboxylation. On the basis of these data we postulate the presence of a second high-affinity substrate-binding site on VKC capable of specifically binding d-OC, which is the first vitamin K-dependent substrate of which the VKC binding domain is interrupted by the Gla domain.
Proteases: a primer
Authors: Hooper Nigel M
Published: August 2015

A protease can be defined as an enzyme that hydrolyses peptide bonds. Proteases can be divided into endopeptidases, which cleave internal peptide bonds in substrates, and exopeptidases, which cleave the terminal peptide bonds. Exopeptidases can be further subdivided into aminopeptidases and carboxypeptidases. The Schechter and Berger nomenclature provides a model for describing the interactions between the peptide substrate and the active site of a protease. Proteases can also be classified as aspartic proteases, cysteine proteases, metalloproteases, serine proteases and threonine proteases, depending on the nature of the active site. Different inhibitors can be used experimentally to distinguish between these classes of protease. The MEROPs database groups proteases into families on the basis of similarities in sequence and structure. Protease activity can be regulated in vivo by endogenous inhibitors, by the activation of zymogens and by altering the rate of their synthesis and degradation.
Therapeutic approaches to the modulation of apoptosis
Authors: Murphy Finbarr J , Seery Liam T , Hayes Ian
Published: August 2015

The appreciation of the role of apoptosis in the vast majority of diseases affecting humans has revolutionized the discovery and development of drugs targeting inflammation and oncology. Novel therapeutic approaches to modulate disease by regulating apoptosis are currently being tested in preclinical and clinical settings. Enthusiasm for some of these therapies is reflected in the fact that they have received U.S. Food and Drug Administration approval in record time. Approaches include the traditional use of small molecules to target specific players in the apoptosis cascade. They also include radical new approaches such as using antisense molecules to inhibit production of the Bcl-2 protein or antibodies that ligate either death receptors, such as TRAIL (tumour necrosis factor-related apoptosis-inducing ligand), or the MHC (HLA-DR), resulting in the initiation of apoptosis of target cells. Antibodies targeting cell-specific antigens are being used in conjunction with radioactive isotopes to deliver a more specific chemotherapy, particularly in the case of B-cell lymphomas. Other therapies target mitochondria, a key organelle in the apoptosis cascade. This diverse range of therapies includes photodynamic therapy, retinoic acid and arsenic trioxide, all of which induce apoptosis by generating reactive oxygen species. As our understanding of apoptosis increases, further opportunities will arise for tailor-made therapies that will result in improved clinical outcome without the devastating side effects of current interventions.
Apoptosis: future perspectives
Authors: Ward Carol , Rossi Adriano G , Haslett Christopher , Dransfield Ian
Published: August 2015

The field of apoptosis has expanded to include research in almost all subject areas of cell biology. Despite this growth, there are still many unanswered questions, some of which are fundamental to the development of therapeutic strategies. Most importantly, the underlying mechanisms involved in conferring specificity of apoptotic signals must be determined. This will allow the development of treatments to delete injured or transformed cells and minimize damage to other tissues. Such information is also necessary to combat degenerative diseases, to prevent critical loss of cells. In addition, it would be of therapeutic benefit to selectively induce apoptosis in specific cells where removal of unwanted cells is required (e.g. tumour cells in malignant tissue or inflammatory cells responsible for tissue damage in chronic inflammatory disease). Therefore we have to understand how the full complement of signalling pathways, and survival and apoptotic proteins determine the end effect of an apoptotic signal. Apoptotic cells are known to functionally influence phagocytes that ingest them, but little is known of the effect of apoptosis on neighbouring cells or of signals generated during the apoptotic process itself. Although the processes of mitosis and apoptosis would appear to be diametrically opposed, cell-cycle proteins are involved in apoptosis, and may be able to influence cell-death mechanisms; conversely, proliferation may be caspase-dependent in some cells. Further study is necessary to discover how these pathways are interlinked since such knowledge would be particularly useful in cancer treatment. The link between pathogen-survival strategies and apoptotic mechanisms will also reward further work. Viruses, in particular, exploit cellular survival and death pathways for replication purposes and may represent a targeting approach for specific treatments.
Nuclear receptors and disease: androgen receptor
Authors: Gottlieb Bruce , Beitel Lenore K , Wu Jianhui , Elhaji Youssef A , Trifiro Mark
Published: August 2015

The androgen receptor (AR) protein regulates transcription of certain genes. Usually this depends upon a central DNA-binding domain that permits the binding of androgen–AR complexes to regulatory DNA sequences near or in a target gene. The AR also has a C-terminal ligand-binding domain and an Nterminal transcription modulatory domain. These N- and C-terminal domains interact directly, and with co-regulatory, non-receptor proteins, to exert precise control over a gene’s transcription rate. The precise roles of these proteins are active research areas. Severe X-linked AR gene (AR) mutations cause complete androgen insensitivity, mild ones impair virilization with or without infertility, and moderate ones yield a wide phenotypic spectrum sometimes among siblings. Different phenotype expressivity may reflect variability of ARinteractive proteins. Mutations occur throughout the AR but are concentrated in specific areas of the gene known as hot spots. A number of these mutations of somatic origin are associated with prostate cancer. N-terminal polyglutamine (polyGln) tract expansion reduces AR transactivation, and when there are more than 38 glutamine residues it causes spinobulbar muscular atrophy, a motor neuron disease, due to a gain of function. Variations in polyGln tract length have been associated as risk factors with prostate, breast, uterine, endometrial and colorectal cancer, as well as male infertility.
Ultraviolet radiation and free radical damage to skin
Authors: Tyrrell Rex M.
Published: August 2015

Solar UVB (290-320 nm) and particularly UVA (320-380 nm) radiations have a capacity to generate reactive chemical species, including free radicals, in cells. These intermediates have been shown to be involved in various biological effects in cultured human skin cells (e.g. cell death) and skin (e.g. erythema). Endogenous glutathione is a critical molecule in protection against the cytotoxic effects of both wavelength ranges. Although there is evidence from cellular studies for the involvement of an oxidative component of UVC/UVB radiations in activation of several genes, the doses used are generally extremely cytotoxic and could cause aberrant signalling. Genes activated by sublethal doses of UVA radiations (e.g. haem oxygenase 1 and the CL100 phosphatase) are clearly redox regulated. The strong induction of haem oxygenase 1 in human fibroblasts has been implicated in an adaptive response to oxidative membrane damage that involves increased synthesis of the iron storage protein, ferritin.
Thiyl radicals in biological systems: significant or trivial?
Authors: Kalyanaraman B.
Published: August 2015

Thiyl radicals are formed from one-electron oxidation of thiols. Thiyl radicals participate in a number of reactions including electron transfer, hydrogen abstraction and addition reactions with several biological constituents and xenobiotics. Thiyl radicals can be detected by optical spectroscopy or by electron spin resonance (ESR) spectroscopy. Thiyl radicals appear to play a role in the nitrosylation of thiols and protein thiols. The exact mechanism of thiol-induced enhancement of oxidative modification of low-density lipoprotein remains questionable. The proposed role of thiyl radicals in lipid peroxidation needs to be re-examined. It has been proposed that thiyl radicals are detoxified by superoxide dismutase in mammalian cells and by a thiol-specific enzyme in bacterial systems. We propose that thiols or protein thiols act as potent antioxidants in radical-induced damage via formation of thiyl radicals.
In vivo measurements of mitochondrial function and cell death following hypoxic/ischaemic damage to the new-born brain
Authors: Cooper Chris E.
Published: August 2015

Critically impaired gas exchange to the brain due to decreased oxygen (hypoxia) or reduced blood flow (ischaemia) is a major cause of brain injury in the perinatal period. There is an accumulating body of evidence suggesting that the irreversible cellular damage in the neonatal brain that occurs subsequent to an hypoxic/ischaemic insult is at the level of the mitochondria. Much of this evidence has been obtained by novel non-invasive measurements of mitochondrial function in vivo. This review focuses on four techniques: near-infrared spectroscopy, magnetic resonance spectroscopy, magnetic resonance imaging and electron paramagnetic resonance spectroscopy. The advantages and disadvantages of these in vivo methods are described in patients and animal models. The picture that emerges is of a slow (1-2 day) energy failure, occurring at the level of the brain mitochondria and leading to a primarily apoptotic cell death. Moderate post-insult hypothermia prevents this damage by an unknown mechanism. It is stressed that isolated cell studies alone are not sufficient to understand the processes occurring at the biochemical and physiological levels. The use of the non-invasive techniques described to understand the biochemistry occurring in vivo is therefore an invaluable aid in integrating cellular and organismal studies of the role of mitochondria in cell death.
The mitochondrial permeability transition pore
Authors: Crompton Martin , Virji Sukaina , Doyle Veronica , Johnson Nicholas , Ward John M.
Published: August 2015

This chapter reviews recent advances in the identification of the structural elements of the permeability transition pore. The discovery that cyclosporin A (CsA) inhibits the pore proved instrumental. Various approaches indicate that CsA blocks the pore by binding to cyclophilin (CyP)-D. In particular, covalent labelling of CyP-D in situ by a photoactive CsA derivative has shown that pore ligands have the same effects on the degree to which CsA both blocks the pore and binds to CyP-D. The recognition that CyP-D is a key component has enabled the other constituents to be resolved. Use of a CyP-D fusion protein as affinity matrix has revealed that CyP-D binds very strongly to 1:1 complexes of the voltage-dependent anion channel (from the outer membrane) and adenine nucleotide translocase (inner membrane). Our current model envisages that the pore arises as a complex between these three components at contact sites between the mitochondrial inner and outer membranes. This is in line with recent reconstitutions of pore activity from protein fractions containing these proteins. The strength of interaction between these proteins suggests that it may be a permanent feature rather than assembled only under pathological conditions. Calcium, the key activator of the pore, does not appear to affect pore assembly; rather, an allosteric action allowing pore flicker into an open state is indicated. CsA inhibits pore flicker and lowers the binding affinity for calcium. Whether adenine nucleotide translocase or the voltage-dependent anion channel (via inner membrane insertion) provides the inner membrane pore has not been settled, and data relevant to this issue are also documented.
The role of post-translational modification in ϐ-amyloid precursor protein processing
Authors: Georgopoulou Niki , McLaughlin Mark , McFarlane Ian , Breen Kieran C.
Published: August 2015

The ϐ-amyloid precursor protein (APP) plays a pivotal role in the early stages of neurodegeneration associated with Alzheimer's disease. An alteration in the processing pattern of the protein results in an increase in the generation of the 40-42-amino-acid ϐ-amyloid (Aϐ) peptide, which coalesces to form insoluble, extracellular amyloid deposits. A greater understanding of the factors that influence APP processing may assist in the design of effective therapeutic agents to halt progression of Alzheimer's disease. APP is a sialoglycoprotein with two potential N-linked glycosylation sites, one of which may contain a complex oligosaccharide chain. An alteration in the glycosylation state of APP by the generation of oligomannosyl oligosaccharides results in a decrease in the secretion of the neuroprotective, soluble form of the protein and a parallel increase in the deposition of the cellular protein within the perinuclear region of the cell. Conversely, the attachment of additional terminal sialic acid residues on to the oligosaccharide chain results in an increase in secretion of soluble APP (sAPPα). One factor that has been widely reported to alter APP processing is the activation of protein kinase C (PKC). This process has been characterized using synaptosomal preparations, which suggests that the PKC action is occurring at the level of the plasma membrane. Furthermore, when cells are transfected with the sialyltransferase enzyme, there is a direct relationship between the sialylation potential of APP and the fold stimulation of sAPPα, after PKC activation. These results suggest that the post-translational modification of APP by glycosylation is a key event in determining the processing of the protein.
Dysfunctional intracellular calcium homoeostasis: a central cause of neurodegeneration in Alzheimer's disease
Authors: O'Neill Cora , Cowburn Richard F. , Bonkale Willy L. , Ohm Thomas G. , Fastbom Johan , Carmody Mark , Kelliher Mary
Published: August 2015

The clinical symptoms of all forms of Alzheimer's disease (AD) result from a slowly progressive neurodegeneration that is associated with the excessive deposition of ϐ-amyloid (Aϐ) in plaques and in the cerebrovasculature, and the formation of intraneuronal neurofibrillary tangles, which are composed primarily of abnormally hyperphosphorylated tau protein. The sequence of cellular events that cause this pathology and neurodegeneration is unknown. It is, however, most probably linked to neuronal signal transduction systems that become misregulated in the brains of certain individuals, causing excessive Aϐ to be formed and/or deposited, tau to become aggregated and hyperphosphorylated and neurons to degenerate. We hypothesize that a progressive alteration in the ability of neurons to regulate intracellular calcium, particularly at the level of the endoplasmic reticulum, is a crucial signal transduction event that is linked strongly to the initiation and development of AD pathology. In this chapter we will discuss the key findings that lend support to this hypothesis.
Manipulating the folding of membrane proteins: using the bilayer to our advantage
Authors: Booth Paula J. , Curran A. Rachael , Templer Richard H. , Lu Hui , Meijberg Wim
Published: August 2015

The folding mechanisms of integral membrane proteins have largely eluded detailed study. This is owing to the inherent difficulties in folding these hydrophobic proteins in vitro, which, in turn, reflects the often apparently insurmountable problem of mimicking the natural membrane bilayer with lipid or detergent mixtures. There is, however, a large body of information on lipid properties and, in particular, on phosphatidylcholine and phosphatidylethanolamine lipids, which are common to many biological membranes. We have exploited this knowledge to develop efficient in vitro lipid-bilayer folding systems for the membrane protein, bacteriorhodopsin. Furthermore, we have shown that a rate-limiting apoprotein folding step and the overall folding efficiency appear to be controlled by particular properties of the lipid bilayer. The properties of interest are the stored curvature elastic energy within the bilayer, and the lateral pressure that the lipid chains exert on the their neighbouring folding proteins. These are generic properties of the bilayer that can be achieved with simple mixtures of biological lipids, and are not specific to the lipids studied here. These bilayer properties also seem to be important in modulating the function of several membrane proteins, as well as the function of membranes in vivo. Thus, it seems likely that careful manipulations of lipid properties will shed light on the forces that drive membrane protein folding, and will aid the development of bilayer folding systems for other membrane proteins.
Neoglycoconjugates: trade and art
Authors: Bovin Nicolai V.
Published: August 2015

This chapter deals with the tendencies in design of multivalent neoglycoconjugates for glycobiology research and high-throughput profiling technologies, including cellular phenotyping. Soluble polyacrylamide (PAA) conjugates are remarkable owing to a variety of possibilities for synthesis and application. PAA is soluble and stable, and the molecule is flexible, PAA-tethered ligands are capable of adjusting to a receptor during multiple-point interactions and PAA does not bind to the cell surface. Synthesis provides unlimited diversity of the probe types (biotin, fluorescein, allyl, digoxygenin, 3H, radiolabelled I), glyco-particles, glyco-surfaces, multiarrays, immunogens etc. Several examples illustrate the most advanced applications. (i) Dynamic systems: the selectin ligands immobilized on the surface as sugar–PAA conjugates made the study of the kinetics of rolling in the model system possible. Carbohydrate ligands that are covalently attached to the chip as sugar–PAA conjugates are of use with the surface plasmon resonance method. (ii) Pseudoglycoprotein: some questions arise regarding the biologically active glycoproteins, e.g.: is a carbohydrate or peptide fragment responsible for the activity? We have proposed the approach that promotes to answer this and other questions. The pool of oligosaccharides that were spitted off a glycoprotein is attached to PAA resulting in a pseudoglycoprotein. (iii) Virtual (dynamic) glycotope: receptor-ligand recognition, such as that of P-selectin with its receptor P-selectin glycoprotein ligand 1, frequently involves molecular interactions at two distinct sites. Using P-selectin as a model, we developed an approach to discover novel ligands. PAA was synthesized with multiple ligands; a marked synergistic inhibitory effect was observed.
Proteases as drug targets
Authors: Docherty Andy J. , Crabbe Tom , O'Connell James P. , Groom Colin R.
Published: August 2015

The effective management of AIDS with HIV protease inhibitors, or the use of angiotensin-converting enzyme inhibitors to treat hypertension, indicates that proteases do make good drug targets. On the other hand, matrix metalloproteinase (MMP) inhibitors from several companies have failed in both cancer and rheumatoid arthritis clinical trials. Mindful of the MMP inhibitor experience, this chapter explores how tractable proteases are as drug targets from a chemistry perspective. It examines the recent success of other classes of drug for the treatment of rheumatoid arthritis, and highlights the need to consider where putative targets lie on pathophysiological pathways--regardless of what kind of therapeutic entity would be required to target them. With genome research yielding many possible new drug targets, it explores the likelihood of discovering proteolytic enzymes that are causally responsible for disease processes and that might therefore make better targets, especially if they lead to the development of drugs that can be administered orally. It also considers the impact that biologics are having on drug discovery, and in particular whether biologically derived therapeutics such as antibodies are likely to significantly alter the way we view proteases as targets and the methods used to discover therapeutic inhibitors.
Radical reactions of nitric oxide synthases
Authors: Stuehr Dennis J. , Wei Chin-Chuan , Santolini Jerome , Wang Zhi- Qiang , Aoyagi Mika , Getzoff Elizabeth D.
Published: August 2015

NOSs (nitric oxide synthases) are flavohaem enzymes that function broadly in human health and disease. We are combining mutagenesis, crystallographic and rapid kinetic methods to understand their mechanism and regulation. The NOSs create a transient tetrahydrobiopterin radical within the enzyme to generate their free radical product (NO). Recent work is revealing how critically important this process is at all levels of catalysis. This article will synthesize four seemingly disparate but related aspects of NOS tetrahydrobiopterin radical formation: (i) how it enables productive O2 activation by providing an electron to the enzyme haem, (ii) what structural features help to regulate this electron transfer, (iii) how it enables NOS to synthesize NO from its diamagnetic substrate and (iv) how it allows NOS to release NO after each catalytic cycle instead of other nitorgen oxide-containing products.
Reversible - through calmodulin - electrostatic interactions between basic residues on proteins and acidic lipids in the plasma membrane.
Authors: McLaughlin Stuart , Hangyás-Mihályné Gyöngyi , Zaitseva Irina , Golebiewska Urszula
Published: August 2015

The inner leaflet of a typical mammalian plasma membrane contains 20-30% univalent PS (phosphatidylserine) and 1% multivalent PtdIns(4,5)P2. Numerous proteins have clusters of basic (or basic/hydrophobic) residues that bind to these acidic lipids. The intracellular effector CaM (calmodulin) can reverse this binding on a wide variety of proteins, including MARCKS (myristoylated alanine-rich C kinase substrate), GAP43 (growth-associated protein 43, also known as neuromodulin), gravin, GRK5 (G-protein-coupled receptor kinase 5), the NMDA (N-methyl-d-aspartate) receptor and the ErbB family. We used the first principles of physics, incorporating atomic models and the Poisson-Boltzmann equation, to describe how the basic effector domain of MARCKS binds electrostatically to acidic lipids on the plasma membrane. The theoretical calculations show the basic cluster produces a local positive electrostatic potential that should laterally sequester PtdInsP2, even when univalent acidic lipids are present at a physiologically relevant 100-fold excess; four independent experimental measurements confirm this prediction. Ca2+/CaM binds with high affinity (Kd approximately 10nM) to this domain and releases the PtdIns(4,5)P2. MARCKS, a major PKC (protein kinase C) substrate, is present at concentrations comparable with those of PtdIns(4,5)P2 (approx. 10 μM) in many cell types. Thus MARCKS can act as a reversible PtdIns(4,5)P2 buffer, binding PtdIns(4,5)P2 in a quiescent cell, and releasing it locally when the intracellular Ca2+ concentration increases. This reversible sequestration is important because PtdIns(4,5)P2 plays many roles in cell biology. Less is known about the role of CaM-mediated reversible membrane binding of basic/hydrophobic clusters for the other proteins.
Specialized transcription factories
Authors: Bartlett Jon , Blagojevic Jelena , Carter David , Eskiw Christopher , Fromaget Maud , Job Christy , Shamsher Monee , Trindade Inês Faro , Xu Meng , Cook Peter R.
Published: August 2015

We have previously suggested a model for the eukaryotic genome based on the structure of the bacterial nucleoid where active RNA polymerases cluster to loop the intervening DNA. This organization of polymerases into clusters – which we call transcription ‘factories’ – has important consequences. For example, in the nucleus of a HeLa cell the concentration of soluble RNA polymerase II is ∼1 mM, but the local concentration in a factory is 1000-fold higher. Because a promoter can diffuse ∼100 nm in 15 s, one lying near a factory is likely to initiate; moreover, when released at termination, it will still lie near a factory, and the movement and modifications (e.g. acetylation) accompanying elongation will leave it in an ‘open’ conformation. Another promoter out in a long loop is less likely to initiate, because the promoter concentration falls off with the cube of the distance from the factory. Moreover, a long tether will buffer it from transcription-induced movement, making it prone to deacetylation, deposition of HP1 (heterochromatin protein 1), and incorporation into heterochromatin. The context around a promoter will then be self-sustaining: productive collisions of an active promoter with the factory will attract factors increasing the frequency of initiation, and the longer an inactive promoter remains inactive, the more it becomes embedded in heterochromatin. We review here the evidence that different factories may specialize in the transcription of different groups of genes.
Non-coding RNA in transcription initiation
Authors: O'Gorman William , Kwek Kon Yew , Thomas Benjamin , Akoulitchev Alexandre
Published: August 2015

Diverse classes of non-coding RNAs, including snRNAs (small nuclear RNAs), play fundamental regulatory roles in gene expression. For example, 7SK RNA and the components of the splicing apparatus U1–U6 snRNAs are implicated in the regulation of transcriptional elongation. The first evidence for the involvement of RNA in the regulation of transcriptional initiation is now emerging. TFIIH (transcription factor IIH), a general transcription initiation factor, appears to associate specifically with U1 snRNA, a core splicing component. Reconstituted transcription in vitro demonstrates an increase in the rate of formation of the first phosphodiester bond by RNA polymerase II in presence of U1 snRNA. Reconstituted re-initiation is also stimulated by U1 snRNA. These results suggest that U1 snRNA functions in the regulation of transcription by RNA polymerase II in addition to its role in RNA processing. The implications of these data extend to the development of new technologies that will allow the identification and analysis of diverse RNA species present as regulatory components in transcription-related ribonucleoprotein complexes.
Inositol trisphosphate and calcium oscillations
Authors: Berridge Michael J.
Published: August 2015

InsP3 has two important functions in generating Ca2+ oscillations. It releases Ca2+ from the internal store and it can contribute to Ca2+ entry. A hypothesis has been developed to describe a mechanism for Ca2+ oscillations with particular emphasis on the way agonist concentration regulates oscillator frequency. The main idea is that the InsP3 receptors are sensitized to release Ca2+ periodically by cyclical fluctuations of Ca2+ within the lumen of the endoplasmic reticulum. Each time a pulse of Ca2+ is released, the luminal level of Ca2+ declines and has to be replenished before the InsP3 receptors are resensitized to deliver the next pulse of Ca2+. It is this loading of the internal store that explains why frequency is sensitive to external Ca2+ and may also account for how variations in agonist concentration are translated into changes in oscillation frequency. Variations in agonist-induced entry of external Ca2+, which can occur through different mechanisms, determine the variable rates of store loading responsible for adjusting the sensitivity of the InsP3 receptors to produce the periodic pulses of Ca2+. The Ca2+ oscillator is an effective analogue-to-digital converter in that variations in the concentration of the external stimulus are translated into a change in oscillator frequency.
Oligosaccharides in molecular recognition
Authors: FEIZI TEN
Published: August 2015

Summary

The development, organization and growth of complex organisms as well as their interactions with the environment involve an intricate array of molecular recognition events. There is an increased awareness of the involvement of oligosaccharides in many of these processes. In this article, studies of oligosaccharide antigenicity, and the way these have been interpreted with respect to oligosaccharide function will be discussed. In addition, examples of oligosaccharides as receptor, first, as receptors and determinants of susceptibility to an exogenous infective agent and secondly, as recognition structures possibly involved in endogenous interactions, will be described. This will be followed by a discussion of the recent hypothesis in which oligosaccharides arc envisaged as recognition structures and integral components of cell growth-regulating networks. Finally, an outline of new strategies for decoding the information content in glycoprotein oligosaccharides will be given.
Hansenula polymorpha as a novel yeast system for the expression of heterologous genes
Authors: SUDBERY P. E. , GLEESON M. A. , VEALE R. A. , LEDEBOER A. M. , ZOETMULDER M. C. M.
Published: August 2015

Summary

The advantages of Hansenula polymorpha as a new yeast expression system are discussed in terms of the powerful and regulatable methanol oxidase promoter and the organism's ability to grow on cheap carbon sources. The development of techniques for conventional genetic analysis is described. A total of 218 mutants have been assigned to 62 complementation groups, three genes have been found to be linked forming the first linkage group in this organism. Methods for molecular transformation have been developed allowing the expression of heterologous genes. The disruptive integration and expression of the neomycin phosphotransferase is described.
Heparan sulphate proteoglycans on rat parathyroid cells recycle in low Ca2+ medium
Authors: TAKEUCHI YASUHIRO , SAKAGUCHI KAZUSHIGE , YANAGISHITA MASAKI , HASCALL VINCENT C.
Published: August 2015

Summary

A rat parathyroid cell line, with some differentiated properties of the parathyroid gland, synthesizes predominantly a heparan sulphate proteoglycan (HS-PG) typical of cell surface HS-PGs (core protein = ∼ 70 kDa, three to four HS chains of ∼ 30 kDa). A 10 min pulse-chase protocol was used to determine the metabolic fate of the HS-PGs for cells maintained in 2.1 mM-Ca2+ (high Ca) or in 0.05 mM-Ca2+ (low Ca). In low Ca, ∼ 60% of the labelled HS-PGs reach the cell surface (t1/2= ∼ 15 min) as determined by trypsin accessibility. This population of HS-PGs recycles (t1/2= ∼ 9 min) between the cell surface and an intracellular (presumably endosome) compartment. After ∼ 2 h, this population of HS-PGs is internalized and rapidly degraded in lysosomes. In high Ca, only ∼ 10% of the HS-PGs reach the cell surface, where they do not recycle. Changing from high to low Ca any time between 30–120 min of chase, rapidly (t1/2 less than 4 min) redistributes the HS-PGs to the cell surface where they begin recycling; conversely, changing from low to high Ca leads to a rapid sequestration of the cell surface HS-PGs within the cells. Other divalent cations fail to minic the response to Ca2+. The results suggest that most of the HS-PGs in this cell line are anchored in a membrane compartment involved in a transport process between endosomes and the cell surface which is regulated by the concentration of extracellular Ca2+.
ATP-promoted interaction between Clp A and Clp P in activation of Clp protease from Escherichia coli
Authors: Maurizi Michael R.
Published: August 2015

Summary

Clp protease is a high relative molecular mass, ATP-dependent protease found in the cytoplasm of Escherichia coli. Clp protease is composed of two protein components, Clp A, which has ATPase activity, and Clp P, which has the proteolytic active site and is activated by Clp A in the presence of ATP. Clp P subunits (Mr = 21 500) are arranged in two hexagonal rings directly superimposed on each other, and under low salt conditions two dodecamers associate to form a particle with Mr ~ 440 000. Clp A (subunit Mr = 83 000) and Clp P do not associate in the absence of nucleotide, but Clp A with ATP bound associates with Clp P to form an active proteolytic complex with Mr ~ 700 000. Although adenosine 5′-[βγ-imido]triphosphate (AMPPNP) weakly promotes association between Clp A and Clp P, non-hydrolysable analogues of ATP do not activate proteolysis, indicating that association between the components is not sufficient to allow proteolysis. Association between Clp A and Clp P does not alter the basal ATPase activity of Clp A, but addition of protein substrates is accompanied by an increase in ATP hydrolysis by Clp A. Chemically-inactivated Clp P or inactive mutants of Clp P also associate with Clp A, but no increase in the ATPase activity of Clp A is observed, either in the presence or absence of protein substrates, when Clp P is inactive. Thus the increased ATP hydrolysis is dependent on active proteolysis. These data indicate that ATP has two functions in activating Clp protease: an allosteric role in promoting association between the subunits and a mechanistically undefined role in promoting continuous rounds of peptide bond cleavage. These functions may be separately fulfilled by the two ATP-binding sites identified by sequence analysis on each Clp A subunit.
Modulation of signal-transduction pathways by chemopreventive agents
Authors: Manson M. M. , Holloway K. A. , Howells L. M. , Hudson E. A. , Plummer S. M. , Squires M. S. , Prigent S. A.
Published: August 2015

For a disease such as cancer, where a number of alterations to normal cell function accumulate over time, there are several opportunities to inhibit, slow down or even reverse the process. Many of the changes which drive the disease process occur in cell-signalling pathways that regulate proliferation and apoptosis. As our knowledge of these complicated signalling networks improves, it is becoming clear that many molecules, both drugs and naturally occurring dietary constituents, can interact beneficially with deregulated pathways. Aspirin and other non-steroidal anti-inflammatory drugs, as well as natural compounds present in plants such as green vegetables and tea, can modulate signalling by affecting kinase activity and therefore phosphorylation of key molecules. Examples of pathways which can be modulated by these agents include activation of the transcription factor nuclear factor κB by tumour promoters or cytokines, signalling by growth factors through the growth-factor receptor/extracellular-regulated protein kinase pathways and by a number of other molecules through the stress-activated c-Jun N-terminal kinase and p38 pathways. These mitogen-activated protein kinase pathways regulate a number of transcription factors including c-Fos and c-Jun. Evidence exists, at least from in vitro experiments, that by targeting such pathways, certain dietary compounds may be able to restore abnormal rates of apoptosis and proliferation to more normal levels.
Role of the BH3 (Bcl-2 homology 3) domain in the regulation of apoptosis and Bcl-2-related proteins
Authors: Lutz R. J.
Published: August 2015

The Bcl-2 family of proteins play a prominent role in the regulation of apoptosis. From the initial identification of bcl-2 as an oncogene in follicular lymphoma through genetic studies in Caenorhabditis elegans to recent functional studies focusing on the importance of mitochondrial events in cell death signalling, the members of this protein family continue to be implicated in pivotal decision points regarding the survival of the cell. The family can be divided into two classes: those such as Bcl-2 and Bcl-xL that suppress cell death, and others, such as Bak and Bax, that appear to promote apoptosis. The Bcl-2 family is characterized by specific regions of homology termed Bcl-2 homology (BH1, BH2, BH3, BH4) domains, which are critical to the function of these proteins, including their impact on cell survival and their ability to interact with other family members and regulatory proteins. The identification of the BH3 domain as a potent mediator of cell death has led to the emergence of an additional family of pro-apoptotic proteins (such as Bad, Bik, Bid and Hrk) that share identity with Bcl-2 only within this death domain. These BH3-only proteins may be part of a regulatory network serving to integrate cell survival and death signals, an assertion that is supported by the identification of a BH3-only protein, Egl-1, as part of the central core of cell death signalling in C. elegans. While the mechanism of action of the BH3-only proteins remains unclear, recent studies on the regulation of critical protein-protein interactions and activity of Bad by phosphorylation in response to growth factor signalling suggest that the active state of BH3-only proteins may be regulated by post-translational modification. Additional modes of regulation, such as transcriptional, translational and subcellular localization, are also likely to be important.
Control of the CFTR channel's gates
Authors: Vergani P. , Basso C. , Mense M. , Nairn A.C. , Gadsby D.C.
Published: August 2015

Unique among ABC (ATP-binding cassette) protein family members, CFTR (cystic fibrosis transmembrane conductance regulator), also termed ABCC7, encoded by the gene mutated in cystic fibrosis patients, functions as an ion channel. Opening and closing of its anion-selective pore are linked to ATP binding and hydrolysis at CFTR's two NBDs (nucleotide-binding domains), NBD1 and NBD2. Isolated NBDs of prokaryotic ABC proteins form homodimers upon binding ATP, but separate after hydrolysis of the ATP. By combining mutagenesis with single-channel recording and nucleotide photolabelling on intact CFTR molecules, we relate opening and closing of the channel gates to ATP-mediated events in the NBDs. In particular, we demonstrate that two CFTR residues, predicted to lie on opposite sides of its anticipated NBD1–NBD2 heterodimer interface, are energetically coupled when the channels open but are independent of each other in closed channels. This directly links ATP-driven tight dimerization of CFTR's cytoplasmic NBDs to opening of the ion channel in the transmembrane domains. Evolutionary conservation of the energetically coupled residues in a manner that preserves their ability to form a hydrogen bond argues that this molecular mechanism, involving dynamic restructuring of the NBD dimer interface, is shared by all members of the ABC protein superfamily.
Tyrosine phosphorylation of synaptophysin in synaptic vesicle recycling
Authors: Evans G.J.O. , Cousin M.A.
Published: August 2015

The integral SV (synaptic vesicle) protein synaptophysin was one of the first nerve terminal proteins identified. However its role, if any, in the SV life cycle remains undetermined. One of the most prominent features of synaptophysin is that its cytoplasmic C-terminus largely consists of pentapeptide repeats initiated by a tyrosine residue. Synaptophysin is heavily phosphorylated by tyrosine kinases in the nerve terminal, suggesting that this phosphorylation is central to its function. This review will cover the evidence for tyrosine phosphorylation of synaptophysin and how this phosphorylation may control its function in the SV life cycle.
Structural and functional studies of defensin-inspired peptides
Authors: Clarke D.J. , Campopiano D.J.
Published: August 2015

Mammals have evolved complex self-defence mechanisms to protect themselves from infection. This innate immune system comprises a large family of hundreds of peptides and proteins which have potent antibiotic activity at nanomolar concentrations. The defensins are a group of small cationic peptides which contain a high proportion of positively charged and hydrophobic amino acids. Their exact mechanism of antimicrobial action is unclear, but it is thought that the defensins bind to and disrupt the outer cell membrane which ultimately causes lysis and cell death. They are characterized by six conserved cysteine residues which oxidize to form three intramolecular disulphide (S–S) bonds. The human and mouse defensins have been subdivided into classes based on their sequence, site of expression and the S–S bond connectivity of the cysteine residues. α-Defensins are connected by cysteines 1 and 6, 2 and 4, and 3 and 5, whereas β-defensins have a 1–5, 2–4 and 3–6 cysteine S–S connectivity. We present our structural and functional studies of a novel mouse β-defensin-related peptide (Defr1) which contains only five cysteine residues. Synthetic Defr1 was more active than its six-cysteine analogue against a large panel of pathogens. High-resolution MS techniques revealed that Defr1 contains an unusual defensin structure. These studies have guided the design of novel peptides to explore the roles of defensins as antibiotics and as stimulants of the immune response.
Green oxidations with laccase–mediator systems
Authors: Wells A. , Teria M. , Eve T.
Published: August 2015

Laccases are oxidase enzymes produced by ‘white rot’ fungi as part of a complex armoury of redox enzymes used to break down lignin – part of the carbon cycle of nature. Laccases alone or in combination with redox co-catalysts have been shown to oxidize xenobiotic compounds under conditions that can be described as ‘green’. This paper describes some novel oxidations using the laccase–mediator method and some current limitations to the use of this technology.
Metabolic and cellular bases of sphingolipidoses
Authors: Sandhoff Konrad
Published: August 2015

Lysosomes are cellular stomachs. They degrade macromolecules and release their components as nutrients into the cytosol. Digestion of sphingolipids and other membrane lipids occurs at luminal intraendosomal vesicles and IMs (intraendosomal membranes). Sphingolipid and membrane digestion needs catabolic hydrolases with the help of lipid-binding proteins [SAPs (sphingolipid activator proteins)] and anionic lipids such as BMP [bis(monoacylglycero)phosphate]. Inherited defects of hydrolases or SAPs or uptake of cationic amphiphilic drugs cause lipid accumulation, eventually leading to death, especially in inherited sphingolipid storage diseases. IMs are formed during endocytosis and their lipid composition is adjusted for degradation. Their cholesterol content, which stabilizes membranes, decreases and the level of negatively charged BMP, which stimulates sphingolipid degradation, increases. At the level of late endosomes, cholesterol is transported out of the luminal vesicles preferentially by cholesterol-binding proteins, NPC (Niemann–Pick type C)-2 and NPC-1. Their defects lead to an endolysosomal accumulation of cholesterol and sphingolipids in Niemann–Pick type C disease. BMP and ceramide stimulate NPC-2-mediated cholesterol transfer, whereas sphingomyelin inhibits it. Anionic membrane lipids also activate sphingomyelin degradation by ASM (acid sphingomyelinase), facilitating cholesterol export by NPC-2. ASM is a non-specific phospholipase C and degrades more than 23 phospholipids. SAPs are membrane-perturbing proteins which solubilize lipids, facilitating glycolipid digestion by presenting them to soluble catabolic enzymes at acidic pH. High BMP and low cholesterol levels favour lipid extraction and membrane disintegration by saposin A and B. The simultaneous inherited defect of saposins A–D causes a severe membrane and sphingolipid storage disease, also disrupting the water permeability barrier of the skin.
Is the cellular and molecular machinery docile in the stationary phase of Escherichia coli?
Authors: Mehta Parul , Jovanovic Goran , Ying Liming , Buck Martin
Published: August 2015

The bacterial cell envelope retains a highly dense cytoplasm. The properties of the cytoplasm change with the metabolic state of the cell, the logarithmic phase (log) being highly active and the stationary phase metabolically much slower. Under the differing growth phases, many different types of stress mechanisms are activated in order to maintain cellular integrity. One such response in enterobacteria is the phage shock protein (Psp) response that enables adaptation to the inner membrane (IM) stress. The Psp system consists of a transcriptional activator PspF, negative regulator PspA, signal sensors PspBC, with PspA and PspG acting as effectors. The single molecule imaging of the PspF showed the existence of dynamic communication between the nucleoid-bound states of PspF and membrane via negative regulator PspA and PspBC sensors. The movement of proteins in the cytoplasm of bacterial cells is often by passive diffusion. It is plausible that the dynamics of the biomolecules differs with the state of the cytoplasm depending on the growth phase. Therefore, the Psp response proteins might encounter the densely packed glass-like properties of the cytoplasm in the stationary phase, which can influence their cellular dynamics and function. By comparing the properties of the log and stationary phases, we find that the dynamics of PspF are influenced by the growth phase and may be controlled by the changes in the cytoplasmic fluidity.