bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanisms of initiation and termination of signalling by neuropeptide receptors: a comparison with the proteinase-activated receptors
Authors: DeFea K. , Schmidlin F. , Déry O. , Grady E. F. , Bunnett N. W.
Published: August 2015

Biological responses to neuropeptides are rapidly attenuated by overlapping mechanisms that include peptide degradation by cell-surface proteases, receptor uncoupling from heterotrimeric G-proteins and receptor endocytosis. We have investigated the mechanisms that terminate the proinflammatory effects of the neuropeptide substance P (SP), which are mediated by the neurokinin 1 receptor (NK1R). Neutral endopeptidase degrades SP in the extracellular fluid and is one of the first mechanisms to terminate signalling. G-protein receptor kinases and second-messenger kinases phosphorylate the NK1R to permit interaction with β-arrestins, which uncouple the receptor from G-proteins to terminate the signal. SP-induces NK1R endocytosis by a β-arrestin-dependent mechanism, which also involves the GTPases dynamin and Rab5a. Endocytosis contributes to desensitization by depleting receptors from the cell surface. Disruption of these mechanisms results in uncontrolled stimulation and disease. Thus the deletion of neutral endopeptidase in mice exacerbates inflammation of many tissues. There are similarities and distinct differences in the mechanisms that regulate signalling by neuropeptide receptors and other G-protein-coupled receptors, in particular those that are activated irreversibly by proteolysis.
Neuropeptidases regulating gonadal function
Authors: Smith A. I. , Shrimpton C. N. , Norman U. M. , Clarke I. J. , Wolfson A. J. , Lew R. A.
Published: August 2015

The generation and metabolism of bioactive peptides involves a series of highly ordered proteolytic events. This post-translational processing can occur either within the cell, at the cell surface or after secretion. In the central nervous system a number of extracellular peptidases have been implicated in the regulated processing of peptides, particularly in the regulation of neuroendocrine function. The aim of this study has been to identify the peptidases involved in the metabolism of gonadotropin-releasing hormone (GnRH) and to characterize the factors and the mechanisms by which the activity of these peptidases are regulated. We have shown that both prolylendo-peptidase and the thimet oligopeptidase EC 3.4.24.15 are involved in GnRH metabolism and that both oestrogen and thiol-based reductants could be involved in the physiological regulation of their activities.
Protein processing mechanisms: from angiotensin-converting enzyme to Alzheimer's disease
Authors: Hooper N. M. , Turner A. J.
Published: August 2015

Angiotensin-converting enzyme (ACE) and the Alzheimer's disease amyloid precursor protein are two examples of membrane-bound proteins that are released in a soluble form by a post-trans-lational proteolytic cleavage event involving a secretase. Site-specific antibodies and matrix-assisted laser desorption ionization-time-of-flight (‘MALDI-TOF’) MS have been used to map the secretase cleavage site in somatic ACE to Arg-1203/Ser-1204, 24 residues proximal to the membrane-anchoring domain. Trypsin, which can solubilize ACE from the membrane, cleaves the protein at the same site. The use of structurally related hydroxamic acid-based zinc metalloproteinase inhibitors indicate that tumour necrosis factor-α convertase, a member of the ADAMs (‘a disintegrin and metalloproteinase’) family of proteins, is not involved in the proteolytic release of ACE, or in the constitutive or regulated α-secretase release of the amyloid precursor protein from a human neuronal cell line.
Biochemical and molecular approaches to understanding protein import into peroxisomes
Authors: Baker A. , Charlton W. , Johnson B. , Lopez-Huertas E. , Oh J. , Sparkes I. , Thomas J.
Published: August 2015

Peroxisomes are eukaryotic organelles that perform diverse and variable functions. Although genetic studies in yeasts and mammals have identified approximately 20 genes (PEX genes) required for the biogenesis of this important organelle, biochemical studies of protein targeting and import have lagged behind and in many cases we have no idea of the function of the PEX gene products (peroxins). Using an import assay in vitro derived from sunflower cotyledon cells and recombinant proteins, we have obtained translocation intermediates on the peroxisome import pathway and are using cross-linking to identify interacting partners. We have also used antibodies raised against human PEX14 to inhibit the import of matrix proteins in this system. To obtain homologous antibodies for inhibition experiments, to immunoprecipitate cross-linked products and to enable us to study the import pathways of peroxins we have cloned and characterized plant orthologues of three PEX genes, PEX6, PEX10 and PEX14.
Structure-function relationships in an anion-translocating ATPase
Authors: Bhattacharjee H. , Zhou T. , Li J. , Gatti D. L. , Walmsley A. R. , Rosen B. P.
Published: August 2015

The ArsAB ATPase is an efflux pump located in the inner membrane of Escherichia coli. This transport ATPase confers resistance to arsenite and antimonite by their extrusion from the cells. The pump is composed of two subunits, the catalytic ArsA subunit and the membrane subunit ArsB. The complex is similar in many ways to ATP-binding cassette (‘ABC’) transporters, which typically have two groups of six transmembrane-spanning helical segments and two nucleotide-binding domains (NBDs). The 45 kDa ArsB protein has 12 transmembrane-spanning segments. ArsB contains the substrate translocation pathway and is capable of functioning as an anion uniporter. The 63 kDa ArsA protein is a substrate-activated ATPase. It has two homologous halves, A1 and A2, which are clearly the result of an ancestral gene duplication and fusion. Each half has a consensus NBD. The mechanism of allosteric activation of the ArsA ATPase has been elucidated by a combination of molecular genetics and biochemical, structural and kinetic analyses. Conformational changes produced by binding of substrates, activator and/or products could be revealed by stopped-flow fluorescence measurements with single-tryptophan derivatives of ArsA. The results demonstrate that the rate-limiting step in the overall reaction is a slow isomerization between two conformations of the enzyme. Allosteric activation increases the rate of this isomerization such that product release becomes rate-limiting, thus accelerating catalysis. ABC transporters, which exhibit similar substrate activation of ATPase activity, can undergo similar conformational changes to overcome a rate-limiting step. Thus the ArsAB pump is a useful model for elucidating mechanistic aspects of the ABC superfamily of transport ATPases.
Life-cycle differentiation in Trypanosoma brucei: molecules and mutants
Authors: Hendriks E. , van Deursen F. J. , Wilson J. , Sarkar M. , Timms M. , Matthews K. R.
Published: August 2015

Differentiation between bloodstream and tsetse midgut procyclic forms during the life cycle of the African trypanosome is an attractive model for the analysis of stage-regulated events. In particular, this transformation occurs synchronously, there are well-defined markers for stage-regulated processes and cell lines with specific defects in differentiation have been identified. This combination of tools, combined with the developing Trypanosoma brucei genome database is allowing its underlying controls to be investigated at the molecular and cytological levels. This paper examines some recent discoveries that illuminate some of the key events during trypanosome life-cycle progression.
Toll-like receptors: lessons from knockout mice
Authors: Akira S.
Published: August 2015

The Toll signalling pathway, which is required for establishment of dorsoventral polarity in Drosophila embryos, plays an important role in the response to microbial infections. Recently, Tolllike receptors (TLRs) have also been identified in mammals. TLR4 has been shown to function as the transmembrane component of the lipopolysaccharide receptor, while TLR2 recognizes peptidoglycans from Gram-positive bacteria, lipoproteins and yeast. Although various microbial cell-wall components are recognized by different receptors, all of these responses are abrogated in MyD88-deficient cells. These results show that different TLRs recognize different microbial cell-wall components, and that MyD88 is an essential signalling molecule shared among interleukin-1 receptor/Toll family members.
The Toll/interleukin-1 receptor domain: a molecular switch for inflammation and host defence
Authors: O'neill L.
Published: August 2015

The pro-inflammatory cytokine interleukin-1 (IL-1) signals via the Type-1 IL-1 receptor (IL-1RI), inducing an increase in the expression of many genes with roles in immunity and inflammation. The signalling pathways involve two adapter proteins, MyD88 and Tollip, which via two IL-1 receptor-associated kinases (IRAK and IRAK-2) activate transcription factors such as nuclear factor-κB and protein kinases such as p38 mitogen-activated protein kinase. A role for the low-molecular-mass G-proteins Rac, Ras and Rap in these processes has also been indicated. IL-1RI is the founder of a diverse superfamily of receptors, which all share a cytosolic domain, termed the Toll/IL-1 receptor (TIR) domain. The superfamily can be divided broadly into three subgroups. The first of these is most similar to IL-1RI and includes the receptor for IL-18 and the Th2 cell regulator T1/ST2. The second subgroup is most similar to the Drosophila melanagaster protein Toll and includes Toll-like receptor 2 (TLR2), which is required for host defence against Gram-positive bacteria and fungi, and TLR4, which is required for lipopolysaccharide responsiveness, and thus is involved in host defence against Gram-negative bacteria. There are also a number of TLRs in plants and insects, all involved in host defence. The third subgroup contains nonreceptor proteins which possess a TIR domain and are cytosolic. MyD88 is a member, and it presumably complexes with IL-1RI via a TIR-TIR interaction. The other two members are proteins encoded by the vaccinia virus, A46R and A52R, which block TIR-dependent signalling. This receptor superfamily therefore appears to play a central role in inflammation and host defence against infection, pointing to the TIR domain as a critical molecular player in the innate immune response.
Toll-like receptor family and signalling pathway
Authors: Muzio M. , Polentarutti N. , Bosisio D. , Kumar P. P. Manoj , Mantovani A.
Published: August 2015

Toll is a Drosophila gene essential for ontogenesis and anti-microbial resistance. Several orthologues of Toll have been identified and cloned in vertebrates, namely Toll-like receptors (TLRs). Human TLRs are a growing family of molecules involved in innate immunity. TLRs are characterized structurally by a cytoplasmic Toll/inter-leukin-1 receptor (TIR) domain and by extracellular leucine-rich repeats. TLRs characterized so far activate the MyD88/interleukin-l receptor-associated kinase (IRAK) signalling pathway. Genetic, gene-transfer and dominant-negative approaches have involved TLR family members (TLR2 and TLR4) in Gram-positive and Gram-negative bacteria recognition and signalling. Accumulating evidence suggests that TLR2 is also involved in signalling-receptor complexes that recognize components of yeast and mycobacteria. However, the definitive roles of other TLRs are still lacking. A systematic approach has been used to determine whether different human leucocyte populations selectively or specifically express TLR mRNA. Based on expression pattern, TLR can be classified as ubiquitous (TLR1), restricted (TLR2, TLR4 and TLR5) and specific (TLR3). Expression and regulation of distinct but overlapping ligand-recognition patterns may underlie the existence of a large, seemingly redundant TLR family. Alternatively, the expression of a TLR in a single cell type may indicate a specific role for this molecule in a restricted setting.
Recent developments in cartilage research: matrix biology of the collagen II/IX/XI heterofibril network
Authors: Eyre D. R. , Wu J.-J. , Fernandes R. J. , Pietka T. A. , Weis M. A.
Published: August 2015

Research on cartilage is intensifying as efforts expand to discover disease-modifying drugs to treat or prevent osteoarthritis. Proteolytic damage to the collagen fabric of cartilage is a critical, and probably early, component of the pathogenesis of degenerative joint disease. Here we summarize recent findings on the unique heteromeric structure of cartilage collagen fibrils, including the key role of collagen IX, a covalently bonded fibril-adapter molecule. A highly specific pattern of cross-linking sites that involves all three component gene products strongly suggests that collagen IX has evolved to function as an inter-fibrillar network-bonding agent. This is supported from the genetic evidence that mutations in all three collagen IX genes can produce a phenotype in which cartilage matrix integrity and early-onset osteoarthritis are a feature. From the structure of the cartilage collagen heteropolymer we also predict a pivotal role for telopeptide (non-triplehelical) proteolytic cleavages in the remodelling and degradation of collagen fibrils.
Role played by BRCA1 in regulating the cellular response to stress
Authors: Gilmore P.M. , Quinn J.E. , Mullan P.B. , Andrews H.N. , McCabe N. , Carty M. , Kennedy R.D. , Harkin D.P.
Published: August 2015

BRCA1 (breast-cancer susceptibility gene 1) is a tumour suppressor gene that is mutated in the germline of women with a genetic predisposition to breast and ovarian cancer. In this review, we examine the role played by BRCA1 in mediating the cellular response to stress. We review the role played by BRCA1 in detecting and signalling the presence of DNA damage, particularly double-strand DNA breaks, and look at the evidence to support a role for BRCA1 in regulating stress response pathways such as the c-Jun N-terminal kinase/stress-activated protein kinase pathway. In addition, we examine the role played by BRCA1 in mediating both cell-cycle arrest and apoptosis following different types of cellular insult, and how this may be modulated by the presence or absence of associated proteins such as p53. Finally, we explore the possibility that many of the functions associated with BRCA1 may be based on transcriptional regulation of key downstream genes that have been implicated in the regulation of these specific cellular pathways.
The co-ordination of central plant metabolism by the circadian clock
Authors: Hartwell J.
Published: August 2015

A circadian clock optimizes many aspects of plant biology relative to the light/dark cycle. One example is the circadian control of primary metabolism and CO2 fixation in plants that carry out a metabolic adaptation of photosynthesis called CAM (crassulacean acid metabolism). These plants perform primary CO2 fixation at night using the enzyme phosphoenolpyruvate carboxylase and exhibit a robust rhythm of CO2 fixation under constant conditions. Transcriptomic analysis has revealed that many genes encoding enzymes in primary metabolic pathways such as glycolysis and starch metabolism are under the control of the circadian clock in CAM plants. These transcript changes are accompanied by changes in metabolite levels associated with flux through these pathways. The molecular basis for the circadian control of CAM remains to be elucidated. Current research is focusing on the identity of the CAM central oscillator and the output pathway that links the central oscillator to the control of plant metabolism.
Implications of a genomic search for autophagy-related genes in trypanosomatids
Authors: Rigden D.J. , Herman M. , Gillies S. , Michels P.A.M.
Published: August 2015

Autophagy is the process by which cellular components are directed to and degraded in the vacuole or lysosome and has been studied largely in yeasts. We present here an in silico genomic analysis of trypanosomatid autophagy aimed at highlighting similarities and differences with autophagy in other organisms. Less than half of the yeast autophagy-related proteins examined have certain putative orthologues in trypanosomatids. A cytosol-to-vacuole transport system is clearly lacking in these organisms. Other absences are even more unexpected and have implications for our understanding of the molecular mechanisms of autophagy. The results are consistent with taxon-specific addition of components to a core autophagy machinery during evolution.
Aβ-degrading enzymes: modulators of Alzheimer's disease pathogenesis and targets for therapeutic intervention
Authors: Eckman E.A. , Eckman C.B.
Published: August 2015

The accumulation of Aβ (amyloid β-protein) peptides in the brain is a pathological hallmark of all forms of AD (Alzheimer's disease) and reducing Aβ levels can prevent or reverse cognitive deficits in mouse models of the disease. Aβ is produced continuously and its concentration is determined in part by the activities ofseveral degradative enzymes, including NEP (neprilysin), IDE (insulin-degrading enzyme), ECE-1 (endothelinconverting enzyme 1) and ECE-2, and probably plasmin. Decreased activity of any of these enzymes due to genetic mutation, or age- or disease-related alterations in gene expression or proteolytic activity, may increase the risk for AD. Conversely, increased expression of these enzymes may confer a protective effect. Increasing Aβ degradation through gene therapy, transcriptional activation or even pharmacological activation of the Aβ-degrading enzymes represents a novel therapeutic strategy for the treatment of AD that is currently being evaluated in cell-culture and animal models. In this paper, we will review the roles of NEP, IDE, ECE and plasmin in determining endogenous Aβ concentration, highlighting recent results concerning the regulation of these enzymes and their potential as therapeutic targets.
Compartmentalization of adenylate cyclase and cAMP signalling
Authors: Cooper D.M.F.
Published: August 2015

Concepts of cAMP signalling have changed dramatically from the linear cascades of just a few years ago, with the realization that numerous cellular processes affect this motif. These influences include other signalling pathways – most significantly Ca2+, scaffolding proteins (which are themselves variously regulated) to organize the elements of the pathway, and subcellular targeting of components. An obvious implication of this organization is that global measurements of cAMP may trivialize the complexity of the cAMP signals and obscure the regulation of targets. In this presentation, current developments on the targeting and assembly of ACs (adenylate cyclases) and their delivery to selected raft or non-raft domains of the plasma membrane will be discussed, along with the susceptibility of raft-targeted ACs to very discrete modes of increases in the intracellular Ca2+ concentration. Single-cell explorations of cAMP dynamics, as measured with cyclic nucleotide-gated channels, are also described in this paper, particularly as applied to cells in which the composition of AKAP (A-kinase anchoring protein)–PKA (protein kinase A)–PDE (phosphodiesterase) assemblies is probed by RNA interference ablation of defined AKAPs.
Lipid phosphate phosphatases and lipid phosphate signalling
Authors: Pyne S. , Long J.S. , Ktistakis N.T. , Pyne N.J.
Published: August 2015

Mammalian LPPs (lipid phosphate phosphatases) are integral membrane proteins that belong to a superfamily of lipid phosphatases/phosphotransferases. They have broad substrate specificity in vitro, dephosphorylating PA (phosphatidic acid), S1P (sphingosine 1-phosphate), LPA (lysophosphatidic acid) etc. Their physiological role may include the attenuation of S1P- and LPA-stimulated signalling by virtue of an ecto-activity (i.e. dephosphorylation of extracellular S1P and LPA), thereby limiting the activation of LPA- and S1P-specific G-protein-coupled receptors at the cell surface. However, our recent work suggests that an intracellular action of LPP2 and LPP3 may account for the reduced agonist-stimulated p42/p44 mitogen-activated protein kinase activation of HEK-293 (human embryonic kidney 293) cells. This may involve a reduction in the basal levels of PA and S1P respectively and the presence of an early apoptotic phenotype under conditions of stress (serum deprivation). Additionally, we describe a model whereby LPP2, but not LPP3, may be functionally linked to the phospholipase D1-derived PA-dependent recruitment of sphingosine kinase 1 to the perinuclear compartment. We also consider the potential regulatory mechanisms for LPPs, which may involve oligomerization. Lastly, we highlight many aspects of the LPP biology that remain to be fully defined.
Role of GSH in the modulation of NOS-2 expression in the weaned mammary gland
Authors: Zaragozá R. , García-Trevijano E.R. , Miralles V.J. , Mata M. , García C. , Carmena R. , Barber T. , Pallardó F.V. , Torres L. , Viña J.R.
Published: August 2015

GSH delivery to the lactating mammary gland is essential for the maintenance of lactation as its decrease leads to apoptosis and involution of the mammary gland. In fact, it has already been demonstrated that some of the changes in gene expression found in the lactating mammary gland after forced weaning are reproduced in rats treated with buthionine sulphoximine to deplete GSH levels. An oligonucleotide microarray experiment would give us a better knowledge of the mRNA expression patterns during lactation and after weaning and the possible functions of GSH in the modulation of these events.
Functional mapping of Toll/interleukin-1 signalling networks by expression cloning
Authors: Kiss-Toth E. , Wyllie D.H. , Holland K. , Marsden L. , Jozsa V. , Oxley K.M. , Polgar T. , Qwarnstrom E.E. , Dower S.K.
Published: August 2015

Multiple cellular proteins have been identified as participating in Toll/interleukin-1 receptor-mediated inflammatory gene expression. The continuing isolation of novel components, based on sequence similarities, protein–protein interactions and protein purification, suggests that many elements of this signalling network remain to be identified. We report here the development of a high-throughput functional screening platform and its application for the identification of components of inflammatory signalling networks. Our results enable us to estimate that 100–150 gene products are involved in controlling the transcription of the human interleukin 8 gene. The approach, which is simple and robust, constitutes a general method for mapping signal transduction systems and for rapid isolation of a large number of signalling components based on the control of pathways leading to regulation of gene expression.
Ramifications of impaired PRPP synthesis in Saccharomyces cerevisiae
Authors: Vavassori S. , Wang K. , Schweizer L.M. , Schweizer M.
Published: August 2015

The model eukaryote Saccharomyces cerevisiae is well suited to investigate the causes of metabolic disturbance. PRPP [5-phospho-D-ribosyl-1(α)-pyrophosphate] may be regarded as a junction of carbon and nitrogen metabolism. As a result of this central position, perturbations in its synthesis can give rise to many unexpected cellular events, such as impaired cell integrity. We have taken advantage of S. cerevisiae's genetic tractability to investigate the metabolic links responsible for connecting the biochemical intermediate PRPP to apparently unrelated cellular functions. This approach provides insight into the co-ordination of different biological processes.
Nucleolar biogenesis: the first small steps
Authors: Prieto J.-L. , McStay B.
Published: August 2015

The nucleolus is the site of rRNA transcription, pre-rRNA processing and ribosome subunit assembly. The nucleolus assembles around clusters of ribosomal gene repeats during late telophase, persists throughout interphase and then disassembles as cells enter mitosis. The initial step in nucleolar formation is ribosomal gene transcription, which is mediated by Pol I (RNA polymerase I) and its associated transcription factors: UBF (upstream-binding factor), SL1 (selectivity factor) and TIF-IA (transcription initiation factor IA)/Rrn3. Ribosomal gene clusters, termed NORs (nucleolar organizer regions), are found on each of the five human acrocentric chromosomes. Though transcription is repressed during metaphase, NORs that were active in the previous interphase form prominent cytogenetic features, namely secondary constrictions. The main defining characteristic of these constrictions is under-condensation in comparison with the rest of the chromosome. Extensive binding of UBF over the ribosomal gene repeat is responsible for the formation of this chromosomal feature. During interphase, the majority of the Pol I transcription machinery, though present in nucleoli, is not actively engaged in transcription. Interaction with UBF bound across the gene repeat provides an explanation for how this non-engaged Pol I machinery is sequestered by nucleoli.
The mechanism of hedgehog signal transduction
Authors: Kalderon D.
Published: August 2015

Hedgehog (Hh) proteins are one of a small number of families of secreted signalling proteins that are responsible for cell interactions during development in many animals. As such, Hh signals produce many different responses at different times and in different cells. As for other multifunctional ligands, this requires regulated patterns of expression, special mechanisms for ligand movement between cells and ligand destruction, and mechanisms for integrating a generic signalling state (on or off) with the status of responding cells in order to produce an appropriate cell-specific response. Here I discuss what is known about the biochemical mechanisms by which an Hh signal is transduced in order to change the patterns of gene transcription.
The specificity of Sushi peptides for endotoxin and anionic phospholipids: potential application of POPG as an adjuvant for anti-LPS strategies
Authors: Li P. , Sun M. , Ho B. , Ding J.L.
Published: August 2015

Sushi peptides [S1 (Sushi 1 peptide) and S3] are derived from the LPS (lipopolysaccharide; also known as endotoxin)-binding domains of an LPS-sensitive serine protease, Factor C, from the horseshoe crab (Carcinoscorpius rotundicauda). S1 and S3 interact at high affinity with LPS. The intermolecular disulphide bonding in the S3 dimer is indispensable for its LPS binding, disruption and consequent neutralization. Simultaneously, the specific interaction between the Sushi peptides and bacterial membrane phospholipids further explains the selective propensity of these peptides for the Gram-negative bacteria. Our findings yield insights into a complex molecular paradigm in which the juxtaposition of LPS molecules and the anionic phospholipid POPG (1-palmitoyl-2-oleoyl phosphatidylglycerol) on the bacterial outer membrane confers such interfacial properties which create the optimal environment for the interaction between the peptides and bacterial membrane lipids.
Defensins and cathelicidins in inflammatory lung disease: beyond antimicrobial activity
Authors: Hiemstra P.S.
Published: August 2015

Innate immunity provides an effective first line of defence against infections. This is of particular importance in the lung, an organ that is exposed to a large number of pathogens that are inhaled. Antimicrobial peptides play an important role in the defence against these pathogens as effector molecules of innate immunity. These peptides are mainly produced by phagocytes and epithelial cells, and kill a wide range of micro-organisms: Gram-negative and Gram-positive bacteria, fungi and (enveloped) viruses. However, it is increasingly evident that these peptides not only act as endogenous antibiotics, but also display a range of other functions, including activities that are involved in regulating immune responses and inflammation, and wound repair. In this review, these activities are highlighted and their role in inflammatory lung disorders is discussed.
Progress in artificial chromosome technology
Authors: Monaco Z. Larin , Moralli D.
Published: August 2015

Artificial chromosomes is an exciting technology which has developed rapidly since the late 1990s. HACs (human artificial chromosomes) are autonomous molecules that can function and segregate as normal chromosomes in human cells. The advantages of an artificial-chromosome-based system are 2-fold. First, HACs are an excellent research tool for investigating the requirements for normal chromosome structure and function during the cell cycle. They are important in defining the sequence requirements of functional chromosomes, and investigating the organization and composition of the chromatin. Secondly, HACs are useful gene-transfer vectors for expression studies in mammalian cells, with the capacity to incorporate large DNA segments encompassing genes and their regulatory elements. As episomes, they are stably maintained, leading to more reliable and prolonged transgene expression. HACs offer the possibility of long-term gene expression in human cells and the development of future somatic gene therapy.
Endogenous ion channel complexes: the NMDA receptor
Authors: Frank René A.W.
Published: August 2015

Ionotropic receptors, including the NMDAR (N-methyl-D-aspartate receptor) mediate fast neurotransmission, neurodevelopment, neuronal excitability and learning. In the present article, the structure and function of the NMDAR is reviewed with the aim to condense our current understanding and highlight frontiers where important questions regarding the biology of this receptor remain unanswered. In the second part of the present review, new biochemical and genetic approaches for the investigation of ion channel receptor complexes will be discussed.
Surfactant-free purification of membrane proteins with intact native membrane environment
Authors: Jamshad Mohammed , Lin Yu-Pin , Knowles Timothy J. , Parslow Rosemary A. , Harris Craig , Wheatley Mark , Poyner David R. , Bill Roslyn M. , Thomas Owen R.T. , Overduin Michael , Dafforn Tim R.
Published: August 2015

In order to study the structure and function of a protein, it is generally required that the protein in question is purified away from all others. For soluble proteins, this process is greatly aided by the lack of any restriction on the free and independent diffusion of individual protein particles in three dimensions. This is not the case for membrane proteins, as the membrane itself forms a continuum that joins the proteins within the membrane with one another. It is therefore essential that the membrane is disrupted in order to allow separation and hence purification of membrane proteins. In the present review, we examine recent advances in the methods employed to separate membrane proteins before purification. These approaches move away from solubilization methods based on the use of small surfactants, which have been shown to suffer from significant practical problems. Instead, the present review focuses on methods that stem from the field of nanotechnology and use a range of reagents that fragment the membrane into nanometre-scale particles containing the protein complete with the local membrane environment. In particular, we examine a method employing the amphipathic polymer poly(styrene-co-maleic acid), which is able to reversibly encapsulate the membrane protein in a 10 nm disc-like structure ideally suited to purification and further biochemical study.
Impairment of the activity of the plasma membrane Ca2+-ATPase in Alzheimer's disease
Authors: Mata Ana M. , Berrocal María , Sepúlveda M. Rosario
Published: August 2015

AD (Alzheimer's disease) is an age-associated neurodegenerative disorder where the accumulation of neurotoxic Aβ (amyloid β-peptide) in senile plaques is a typical feature. Recent studies point out a relationship between Aβ neurotoxicity and Ca2+ dyshomoeostasis, but the molecular mechanisms involved are still under discussion. The PMCAs (plasma membrane Ca2+-ATPases) are a multi-isoform family of proteins highly expressed in brain that is implicated in the maintenance of low intraneural Ca2+ concentration. Therefore the malfunction of this pump may also be responsible for Ca2+ homoeostasis failure in AD. We have found that the Ca2+-dependence of PMCA activity is affected in human brains diagnosed with AD, being related to the enrichment of Aβ. The peptide produces an inhibitory effect on the activity of PMCA which is isoform-specific, with the greatest inhibition of PMCA4. Besides, cholesterol blocked the inhibitory effect of Aβ, which is consistent with the lack of any Aβ effect on PMCA4 found in cholesterol-enriched lipid rafts isolated from pig brain. These observations suggest that PMCAs are a functional component of the machinery that leads to Ca2+ dysregulation in AD and propose cholesterol enrichment in rafts as a protector of the Aβ-mediated inhibition on PMCA.
The Plasmodium falciparum Ca2+-ATPase PfATP6: insensitive to artemisinin, but a potential drug target
Authors: Arnou Bertrand , Montigny Cédric , Morth Jens Preben , Nissen Poul , Jaxel Christine , Møller Jesper V. , Maire Marc le
Published: August 2015

The disease malaria, caused by the parasite Plasmodium falciparum, remains one of the most important causes of morbidity and mortality in sub-Saharan Africa. In the absence of an efficient vaccine, the medical treatment of malaria is dependent on the use of drugs. Since artemisinin is a powerful anti-malarial drug which has been proposed to target a particular Ca2+-ATPase (PfATP6) in the parasite, it has been important to characterize the molecular properties of this enzyme. PfATP6 is a 139 kDa protein composed of 1228 amino acids with a 39% overall identity with rabbit SERCA1a (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 1a). PfATP6 conserves all sequences and motifs that are important for the function and/or structure of a SERCA, such as two high-affinity Ca2+-binding sites, a nucleotide-binding site and a phosphorylation site. We have been successful in isolating PfATP6 after heterologous expression in yeast and affinity chromatography in a pure, active and stable detergent-solubilized form. With this preparation, we have characterized and compared with the eukaryotic SERCA1a isoform the substrate (Ca2+ and ATP) -dependency for PfATP6 activity as well as the specific inhibition/interaction of the protein with drugs. Our data fully confirm that PfATP6 is a SERCA, but with a distinct pharmacological profile: compared with SERCA1a, it has a lower affinity for thapsigargin and much higher affinity for cyclopiazonic acid. On the other hand, we were not able to demonstrate any inhibition by artemisinin and were also not able to monitor any binding of the drug to the isolated enzyme. Thus it is unlikely that PfATP6 plays an important role as a target for artemisinin in the parasite P. falciparum.
Endocannabinoid signals in the developmental programming of delayed-onset neuropsychiatric and metabolic illnesses
Authors: Keimpema Erik , Calvigioni Daniela , Harkany Tibor
Published: August 2015

It is increasingly recognized that maternal exposure to metabolic (nutritional) stimuli, infections, illicit or prescription drugs and environmental stressors during pregnancy can predispose affected offspring to developing devastating postnatal illnesses. If detrimental maternal stimuli coincide with critical periods of tissue production and organogenesis then they can permanently derail key cellular differentiation programs. Maternal programming can thus either provoke developmental failure directly (‘direct hit’) or introduce latent developmental errors that enable otherwise sub-threshold secondary stressors to manifest as disease (‘double hit’) postnatally. Accumulating evidence suggests that nervous system development is tightly controlled by maternal metabolic stimuli, and whose synaptic wiring and integrative capacity are adversely affected by dietary and hormonal challenges, infections or episodes of illicit drug use. Endocannabinoids, a family of signal lipids derived from polyunsaturated fatty acids, have been implicated in neuronal fate determination, the control of axonal growth, synaptogenesis and synaptic neurotransmission. Therefore the continuum and interdependence of endocannabinoid actions during the formation and function of synapses together with dynamic changes in focal and circulating endocannabinoid levels upon maternal nutritional imbalance suggest that endocannabinoids can execute the ‘reprogramming’ of specific neuronal networks. In the present paper, we review molecular evidence suggesting that maternal nutrition and metabolism during pregnancy can affect the formation and function of the hippocampus and hypothalamus by altering endocannabinoid signalling such that neuropsychiatric diseases and obesity respectively ensue in affected offspring. Moreover, we propose that the placenta, fetal adipose and nervous tissues interact via endocannabinoid signals. Thus endocannabinoids are hypothesized to act as a molecular substrate of maternal programming.
Motoneuronal and muscle-selective removal of ALS-related misfolded proteins
Authors: Crippa Valeria , Galbiati Mariarita , Boncoraglio Alessandra , Rusmini Paola , Onesto Elisa , Giorgetti Elisa , Cristofani Riccardo , Zito Arianna , Poletti Angela
Published: August 2015

ALS (amyotrophic lateral sclerosis), a fatal motoneuron (motor neuron) disease, occurs in clinically indistinguishable sporadic (sALS) or familial (fALS) forms. Most fALS-related mutant proteins identified so far are prone to misfolding, and must be degraded in order to protect motoneurons from their toxicity. This process, mediated by molecular chaperones, requires proteasome or autophagic systems. Motoneurons are particularly sensitive to misfolded protein toxicity, but other cell types such as the muscle cells could also be affected. Muscle-restricted expression of the fALS protein mutSOD1 (mutant superoxide dismutase 1) induces muscle atrophy and motoneuron death. We found that several genes have an altered expression in muscles of transgenic ALS mice at different stages of disease. MyoD, myogenin, atrogin-1, TGFβ1 (transforming growth factor β1) and components of the cell response to proteotoxicity [HSPB8 (heat shock 22kDa protein 8), Bag3 (Bcl-2-associated athanogene 3) and p62] are all up-regulated by mutSOD1 in skeletal muscle. When we compared the potential mutSOD1 toxicity in motoneuron (NSC34) and muscle (C2C12) cells, we found that muscle ALS models possess much higher chymotryptic proteasome activity and autophagy power than motoneuron ALS models. As a result, mutSOD1 molecular behaviour was found to be very different. MutSOD1 clearance was found to be much higher in muscle than in motoneurons. MutSOD1 aggregated and impaired proteasomes only in motoneurons, which were particularly sensitive to superoxide-induced oxidative stress. Moreover, in muscle cells, mutSOD1 was found to be soluble even after proteasome inhibition. This effect could be associated with a higher mutSOD1 autophagic clearance. Therefore muscle cells seem to manage misfolded mutSOD1 more efficiently than motoneurons, thus mutSOD1 toxicity in muscle may not directly depend on aggregation.
Understanding non-coding DNA regions in yeast
Authors: Schlackow Margarita , Gullerova Monika
Published: August 2015

Non-coding transcripts play an important role in gene expression regulation in all species, including budding and fission yeast. Such regulatory transcripts include intergenic ncRNA (non-coding RNA), 5′ and 3′ UTRs, introns and antisense transcripts. In the present review, we discuss advantages and limitations of recently developed sequencing techniques, such as ESTs, DNA microarrays, RNA-Seq (RNA sequencing), DRS (direct RNA sequencing) and TIF-Seq (transcript isoform sequencing). We provide an overview of methods applied in yeast and how each of them has contributed to our knowledge of gene expression regulation and transcription.
Genomic impact of CRISPR immunization against bacteriophages
Authors: Barrangou Rodolphe , Coûté-Monvoisin Anne-Claire , Stahl Buffy , Chavichvily Isabelle , Damange Florian , Romero Dennis A. , Boyaval Patrick , Fremaux Christophe , Horvath Philippe
Published: August 2015

CRISPR (clustered regularly interspaced short palindromic repeats) together with cas (CRISPR-associated) genes form the CRISPR–Cas immune system, which provides sequence-specific adaptive immunity against foreign genetic elements in bacteria and archaea. Immunity is acquired by the integration of short stretches of invasive DNA as novel ‘spacers’ into CRISPR loci. Subsequently, these immune markers are transcribed and generate small non-coding interfering RNAs that specifically guide nucleases for sequence-specific cleavage of complementary sequences. Among the four CRISPR–Cas systems present in Streptococcus thermophilus, CRISPR1 and CRISPR3 have the ability to readily acquire new spacers following bacteriophage or plasmid exposure. In order to investigate the impact of building CRISPR-encoded immunity on the host chromosome, we determined the genome sequence of a BIM (bacteriophage-insensitive mutant) derived from the DGCC7710 model organism, after four consecutive rounds of bacteriophage challenge. As expected, active CRISPR loci evolved via polarized addition of several novel spacers following exposure to bacteriophages. Although analysis of the draft genome sequence revealed a variety of SNPs (single nucleotide polymorphisms) and INDELs (insertions/deletions), most of the in silico differences were not validated by Sanger re-sequencing. In addition, two SNPs and two small INDELs were identified and tracked in the intermediate variants. Overall, building CRISPR-encoded immunity does not significantly affect the genome, which allows the maintenance of important functional properties in isogenic CRISPR mutants. This is critical for the development and formulation of sustainable and robust next-generation starter cultures with increased industrial lifespans.
Cas9-dependent endogenous gene regulation is required for bacterial virulence
Authors: Sampson Timothy R. , Weiss David S.
Published: August 2015

CRISPR (clustered regularly interspaced short palindromic repeats)–Cas (CRISPR-associated) systems are known to mediate bacterial defence against foreign nucleic acids. We recently demonstrated a non-canonical role for a CRISPR–Cas system in controlling endogenous gene expression, which had not previously been appreciated. In the present article, we describe the studies that led to this discovery, beginning with an unbiased genome-wide screen to identify virulence genes in the intracellular pathogen Francisella novicida. A gene annotated as encoding a hypothetical protein, but which we now know encodes the Cas protein Cas9, was identified as one of the most critical to the ability of F. novicida to replicate and survive during murine infection. Subsequent studies revealed a role for this protein in evasion of the host innate immune response. Specifically, Cas9 represses the expression of a BLP (bacterial lipoprotein) that could otherwise be recognized by TLR2 (Toll-like receptor 2), a host protein involved in initiating an antibacterial pro-inflammatory response. By repressing BLP levels, Cas9 mediates evasion of TLR2, promoting bacterial virulence. Finally, we described the molecular mechanism by which Cas9 functions in complex with two small RNAs to target the mRNA encoding the BLP for degradation. This work greatly broadened the paradigm for CRISPR–Cas function, highlighting a role in gene regulation that could be conserved in numerous bacteria, and elucidating its integral contribution to bacterial pathogenesis.
The role of TDP-43 in the pathogenesis of ALS and FTLD
Authors: Baralle Marco , Buratti Emanuele , Baralle Francisco E.
Published: August 2015

TDP-43 (TAR DNA-binding protein 43) is an hnRNP (heterogeneous nuclear ribonucleoprotein) protein whose role in cellular processes has come to the forefront of neurodegeneration research after the observation that it is the main component of brain inclusions in ALS (amyotrophic lateral sclerosis) and FTLD (frontotemporal lobar degeneration) patients. Functionally, this aberrant aggregation and mislocalization implies that, in the affected neurons, transcripts regulated by TDP-43 may be altered. Since then, a considerable amount of data has been gathered on TDP-43 interactions and on the genes that are influenced by its absence or overexpression. At present, however, most of these data come from high-throughput searches, making it problematic to separate the direct effects of TDP-43 from secondary misregulations occurring at different levels of the gene expression process. Furthermore, our knowledge of the biochemistry of TDP-43, its RNA-binding characteristics, its nuclear and cytoplasmic targets, and the details of its interactions with other proteins is still incomplete. The understanding of these features could hold the key for uncovering TDP-43′s role in ALS and FTLD pathogenesis. We describe in the present paper our work on TDP-43 RNA binding, self-regulation and aggregation processes, and attempt to relate them to the neurodegenerative pathologies.
SMV1 virus-induced CRISPR spacer acquisition from the conjugative plasmid pMGB1 in Sulfolobus solfataricus P2
Authors: Erdmann Susanne , Shah Shiraz A. , Garrett Roger A.
Published: August 2015

Organisms of the crenarchaeal order Sulfolobales carry complex CRISPR (clustered regularly interspaced short palindromic repeats) adaptive immune systems. These systems are modular and show extensive structural and functional diversity, especially in their interference complexes. The primary targets are an exceptional range of diverse viruses, many of which propagate stably within cells and follow lytic life cycles without producing cell lysis. These properties are consistent with the difficulty of activating CRISPR spacer uptake in the laboratory, but appear to conflict with the high complexity and diversity of the CRISPR immune systems that are found among the Sulfolobales. In the present article, we re-examine the first successful induction of archaeal spacer acquisition in our laboratory that occurred exclusively for the conjugative plasmid pMGB1 in Sulfolobus solfataricus P2 that was co-infected with the virus SMV1 (Sulfolobus monocaudavirus 1). Although we reaffirm that protospacer selection is essentially a random process with respect to the pMGB1 genome, we identified single spacer sequences specific for each of CRISPR loci C, D and E that, exceptionally, occurred in many sequenced clones. Moreover, the same sequence was reproducibly acquired for a given locus in independent experiments, consistent with it being the first protospacer to be selected. There was also a small protospacer bias (1.6:1) to the antisense strand of protein genes. In addition, new experiments demonstrated that spacer acquisition in the previously inactive CRISPR locus A could be induced on freeze–thawing of the infected cells, suggesting that environmental stress can facilitate activation. Coincidentally with spacer acquisition, a mobile OrfB element was deleted from pMGB1, suggesting that interplay can occur between spacer acquisition and transposition.
Probing the kinetic and thermodynamic consequences of the tetraloop/tetraloop receptor monovalent ion-binding site in P4–P6 RNA by smFRET
Authors: Bisaria Namita , Herschlag Daniel
Published: August 2015

Structured RNA molecules play roles in central biological processes and understanding the basic forces and features that govern RNA folding kinetics and thermodynamics can help elucidate principles that underlie biological function. Here we investigate one such feature, the specific interaction of monovalent cations with a structured RNA, the P4–P6 domain of the Tetrahymena ribozyme. We employ single molecule FRET (smFRET) approaches as these allow determination of folding equilibrium and rate constants over a wide range of stabilities and thus allow direct comparisons without the need for extrapolation. These experiments provide additional evidence for specific binding of monovalent cations, Na+ and K+, to the RNA tetraloop–tetraloop receptor (TL–TLR) tertiary motif. These ions facilitate both folding and unfolding, consistent with an ability to help order the TLR for binding and further stabilize the tertiary contact subsequent to attainment of the folding transition state.