bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation and characterization of a polymerized prion protein
Authors: LU Bao-Yuan , CHANG Jui-Yoa
Published: August 2015

A polymerized form of recombinant mouse prion protein (mPrP) domain 23–231 [mPrP-(23–231)], designated mPrP-z, was generated at acidic pH (pH 2–5) in the presence of selected concentrations of denaturant (2M guanidinium chloride or 5M urea). This isoform of mPrP is stable in acidic solution after removal of denaturant. It can be isolated and purified using reversed-phase HPLC or size-exclusion HPLC. mPrP-z bears structural properties that partially resemble those of scrapie prion. Unlike the native mPrP-(23–231) (mPrP-N), mPrP-z exhibits a high content of β-sheet structure, as shown by CD spectroscopy, and exists as an oligomer with an approximate molecular mass of 340000Da, as measured by light scattering. However, similarly to mPrP-N, mPrP-z contains the intact disulphide bond and is sensitive to digestion by proteinase K.
l-Lactate transport into rat heart mitochondria and reconstruction of the l-lactate/pyruvate shuttle
Authors: VALENTI Daniela , de BARI Lidia , ATLANTE Anna , PASSARELLA Salvatore
Published: August 2015

In vitro reconstruction of the l-lactate/pyruvate shuttle has been performed, which allows NADH oxidation outside rat heart mitochondria. Such a shuttle occurs due to the combined action of the novel mitochondrial l-lactate/pyruvate antiporter, which differs from the monocarboxylate carrier, and the mitochondrial l-lactate dehydrogenase. The rate of l-lactate/pyruvate antiport proved to regulate the shuttle in vitro.
Plant-derived phenolic compounds prevent the DNA single-strand breakage and cytotoxicity induced by tert-butylhydroperoxide via an iron-chelating mechanism
Authors: SESTILI Piero , DIAMANTINI Giuseppe , BEDINI Annalida , CERIONI Liana , TOMMASINI Ilaria , TARZIA Giorgio , CANTONI Orazio
Published: August 2015

The protective effects of selected members from a series of caffeic acid esters and flavonoids were tested in various toxicity paradigms using U937 cells, previously shown to be sensitive to either iron chelators or bona fide radical scavengers or to both classes of compounds. It was found that all the protective polyphenols were active at very low concentrations and that their effects were observed only under those conditions in which iron chelators also afforded protection. Consistently, active polyphenolic compounds, unlike the inactive ones, effectively chelated iron in an in vitro system. It follows that, at least under the experimental conditions utilized in the present study, the most prominent activity of these polyphenolic compounds resides in their ability to chelate iron. Further studies revealed that the protective effects afforded by the caffeic acid esters and flavonoids were largely mediated by the catechol moiety and that the relative biological potency of these compounds was a direct function of their lipophilicity.
Novel disease-causing mutations in the dihydropyrimidine dehydrogenase gene interpreted by analysis of the three-dimensional protein structure
Authors: van KUILENBURG André B.P. , DOBRITZSCH Doreen , MEINSMA Rutger , HAASJES Janet , WATERHAM Hans R. , NOWACZYK Malgorzata J.M. , MAROPOULOS George D. , HEIN Guido , KALHOFF Hermann , KIRK Jean M. , BAASKE Holger , AUKETT Anne , DULEY John A. , WARD Kate P. , LINDQVIST Ylva , van GENNIP Albert H.
Published: August 2015

Dihydropyrimidine dehydrogenase (DPD) deficiency is an autosomal recessive disease characterized by thymine-uraciluria in homozygous deficient patients. Cancer patients with a partial deficiency of DPD are at risk of developing severe life-threatening toxicities after the administration of 5-fluorouracil. Thus, identification of novel disease-causing mutations is of the utmost importance to allow screening of patients at risk. In eight patients presenting with a complete DPD deficiency, a considerable variation in the clinical presentation was noted. Whereas motor retardation was observed in all patients, no patients presented with convulsive disorders. In this group of patients, nine novel mutations were identified including one deletion of two nucleotides [1039-1042delTG] and eight missense mutations. Analysis of the crystal structure of pig DPD suggested that five out of eight amino acid exchanges present in these patients with a complete DPD deficiency, Pro86Leu, Ser201Arg, Ser492Leu, Asp949Val and His978Arg, interfered directly or indirectly with cofactor binding or electron transport. Furthermore, the mutations Ile560Ser and Tyr211Cys most likely affected the structural integrity of the DPD protein. Only the effect of the Ile370Val and a previously identified Cys29Arg mutation could not be readily explained by analysis of the three-dimensional structure of the DPD enzyme, suggesting that at least the latter might be a common polymorphism. Our data demonstrate for the first time the possible consequences of missense mutations in the DPD gene on the function and stability of the DPD enzyme.
Structural basis for the unusual carbohydrate-binding specificity of jacalin towards galactose and mannose
Authors: BOURNE Yves , ASTOUL Corinne Houlès , ZAMBONI Véronique , PEUMANS Willy J. , MENU-BOUAOUICHE Laurence , Van DAMME Els J.M. , BARRE Annick , ROUGÉ Pierre
Published: August 2015

Evidence is presented that the specificity of jacalin, the seed lectin from jack fruit (Artocarpus integrifolia), is not directed exclusively against the T-antigen disaccharide Galβ1,3GalNAc, lactose and galactose, but also against mannose and oligomannosides. Biochemical analyses based on surface-plasmon-resonance measurements, combined with the X-ray-crystallographic determination of the structure of a jacalin—α-methyl-mannose complex at 2Å resolution, demonstrated clearly that jacalin is fully capable of binding mannose. Besides mannose, jacalin also interacts readily with glucose, N-acetylneuraminic acid and N-acetylmuramic acid. Structural analyses demonstrated that the relatively large size of the carbohydrate-binding site enables jacalin to accommodate monosaccharides with different hydroxyl conformations and provided unambiguous evidence that the β-prism structure of jacalin is a sufficiently flexible structural scaffold to confer different carbohydrate-binding specificities to a single lectin.
Engineering N-terminal domain of tissue inhibitor of metalloproteinase (TIMP)-3 to be a better inhibitor against tumour necrosis factor-α-converting enzyme
Authors: LEE Meng-Huee , VERMA Vandana , MASKOS Klaus , NATH Deepa , KNÄUPER Vera , DODDS Philippa , AMOUR Augustin , MURPHY Gillian
Published: August 2015

We previously reported that full-length tissue inhibitor of metalloproteinase-3 (TIMP-3) and its N-terminal domain form (N-TIMP-3) displayed equal binding affinity for tissue necrosis factor-α (TNF-α)-converting enzyme (TACE). Based on the computer graphic of TACE docked with a TIMP-3 model, we created a number of N-TIMP-3 mutants that showed significant improvement in TACE inhibition. Our strategy was to select those N-TIMP-3 residues that were believed to be in actual contact with the active-site pockets of TACE and mutate them to amino acids of a better-fitting nature. The activities of these mutants were examined by measuring their binding affinities (Kappi) and association rates (kon) against TACE. Nearly all mutants at position Thr-2 exhibited slightly impaired affinity as well as association rate constants. On the other hand, some Ser-4 mutants displayed a remarkable increase in their binding tightness with TACE. In fact, the binding affinities of several mutants were less than 60pM, beyond the sensitivity limits of fluorimetric assays. Further studies on cell-based processing of pro-TNF-α demonstrated that wild-type N-TIMP-3 and one of its tight-binding mutants, Ser-4Met, were capable of inhibiting the proteolytic shedding of TNF-α. Furthermore, the Ser-4Met mutant was also significantly more active (P<0.05) than the wild-type N-TIMP-3 in its cellular inhibition. Comparison of N-TIMP-3 and full-length TIMP-3 revealed that, despite their identical TACE-interaction kinetics, the latter was nearly 10 times more efficient in the inhibition of TNF-α shedding, with concomitant implications for the importance of the TIMP-3 C-terminal domain in vivo.
Caspases and apoptosis
Authors: Salvesen Guy S
Published: August 2015

The ability of metazoan cells to undergo programmed cell death is vital to both the precise development and long-term survival of the mature adult. Cell deaths that result from engagement of this programme end in apoptosis, the ordered dismantling of the cell that results in its 'silent' demise, in which packaged cell fragments are removed by phagocytosis. This co-ordinated demise is mediated by members of a family of cysteine proteases known as caspases, whose activation follows characteristic apoptotic stimuli, and whose substrates include many proteins, the limited cleavage of which causes the characteristic morphology of apoptosis. In vertebrates, a subset of caspases has evolved to participate in the activation of pro-inflammatory cytokines, and thus members of the caspase family participate in one of two very distinct intracellular signalling pathways.
Guardians of cell death: the Bcl-2 family proteins
Authors: Daniel Peter T , Schulze-Osthoff Klaus , Belka Claus , Güner Dilek
Published: August 2015

Apoptosis is mediated through at least three major pathways that are regulated by (i) the death receptors, (ii) the mitochondria and (iii) the ER (endoplasmic reticulum). In most cells, these pathways are controlled by the Bcl-2 family of proteins that can be divided into anti-apoptotic and pro-apoptotic members. Although the overall amino acid sequence homology between the family members is relatively low, they contain highly conserved domains, referred to as BH (Bcl-2 homology) domains (BH1-4), that are essential for homo- and hetero-complex formation, as well as for their cell-death-inducing capacity. Structural and functional analyses revealed that the pro-apoptotic homologues can be subdivided into the Bax subfamily and the growing BH3-only subfamily. Recent data indicate that BH3-only proteins act as mediators that link various upstream signals, including death receptors and DNA damage signalling, to the mitochondrial and the ER pathway. This review discusses recent structural and functional insights into how these subfamilies promote or inhibit cell-death signals, and how these properties may be utilized for development of apoptosis-promoting small molecules, e.g. in cancer therapy.
Role of molecular chaperones in steroid receptor action
Authors: Pratt William B , Galigniana Mario D , Morishima Yoshihiro , Murphy Patrick J M
Published: August 2015

Unliganded steroid receptors are assembled into heterocomplexes with heat-shock protein (hsp) 90 by a multiprotein chaperone machinery. In addition to binding the receptors at the chaperone site, hsp90 binds cofactors at other sites that are part of the assembly machinery, as well as immunophilins that connect the assembled receptor-hsp90 heterocomplexes to a protein trafficking pathway. The hsp90-/hsp70-based chaperone machinery interacts with the unliganded glucocorticoid receptor to open the steroid-binding cleft to access by a steroid, and the machinery interacts in very dynamic fashion with the liganded, transformed receptor to facilitate its translocation along microtubular highways to the nucleus. In the nucleus, the chaperone machinery interacts with the receptor in transcriptional regulatory complexes after hormone dissociation to release the receptor and terminate transcriptional activation. By forming heterocomplexes with hsp90, the chaperone machinery stabilizes the receptor to degradation by the ubiquitin-proteasome pathway of proteolysis.
Glucocorticoid and mineralocorticoid receptors and associated diseases
Authors: Kino Tomoshige , Chrousos George P
Published: August 2015

Adrenal corticosteroids, i.e. glucocorticoids and mineralocorticoids, play important physiological roles in humans. Their actions are mediated by intracellular receptor molecules, the glucocorticoid receptor (GR) and mineralocorticoid receptor (MR), which function as hormone-dependent transcription factors. Ligand-activated receptors modulate the transcription rates of responsive genes by interacting with responsive elements in the promoters of these genes or by influencing the activities of other transcription factors, via protein-protein interactions. Natural inactivating mutations of the GR or MR genes have been reported in humans with significant clinical phenotypes. The former causes sporadic or familial glucocorticoid resistance characterized by generalized partial insensitivity of tissues to glucocorticoids and subsequent activation of the hypothalamic/pituitary/adrenal axis with resultant hyperandrogenism in children and women and/or mineralocorticoid excess symptoms in both sexes. The latter develop pseudohypoaldosteronism type 1, i.e. hypotension and hyperkalaemic acidosis, as a result of reduced aldosterone actions in the kidney. An activating mutation in the MR gene causing early-onset, periodic hypertension was reported recently. The biological relevance of the GR and MR receptors was also addressed in mice whose GR or MR genes were inactivated or modified by gene targeting. The results were generally confirmatory of the concepts obtained by the human studies. Similarly, natural, compensated glucocorticoid and/or mineralocorticoid 'resistance' were described in several mammalian species, including non-human primates and rodents. Here we discuss the actions of GR and MR and the molecular defects of naturally occuring mutations in these receptors with associated pathophysiological changes.
Mitochondria, NO and neurodegeneration
Authors: Beal M.Flint
Published: August 2015

A role for mitochondrial dysfunction in neurodegenerative disease is gaining increasing support. Mitochondrial dysfunction may be linked to neurodegenerative diseases through a variety of different pathways, including free-radical generation, impaired calcium buffering and the mitochondrial permeability transition. This can lead to both apoptotic and necrotic cell death. Recent evidence has shown that there is a mitochondrial defect in Friedreich's ataxia, which leads to increased mitochondrial iron content, that appears to be linked to increased free-radical generation. There is evidence that the point mutations in superoxide dismutase which are associated with amyotrophic lateral sclerosis may contribute to mitochondrial dysfunction. There is also evidence for bioenergetic defects in Huntington's disease. Studies of cybrid cell lines have implicated mitochondrial defects in both Parkinson's disease and Alzheimer's disease. If mitochondrial dysfunction plays a role in neurodegenerative diseases then therapeutic strategies such as coenzyme Q10 and creatine may be useful in attempting to slow the disease process.
Mitochondrial dysfunction in Parkinson's disease
Authors: Greenamyre J.Timothy , MacKenzie Gillian , Peng Tsung-I , Stephans Stacy E.
Published: August 2015

The cause of Parkinson's disease (PD) is unknown, but reduced activity of complex I of the electron-transport chain has been implicated in the pathogenesis of both mitochondrial permeability transition pore-induced Parkinsonism and idiopathic PD. We developed a novel model of PD in which chronic, systemic infusion of rotenone, a complex-I inhibitor, selectively kills dopaminergic nerve terminals and causes retrograde degeneration of substantia nigra neurons over a period of months. The distribution of dopaminergic pathology replicates that seen in PD, and the slow time course of neurodegeneration mimics PD more accurately than current models. Our model should enhance our understanding of neurodegeneration in PD. Metabolic impairment depletes ATP, depresses Na+/K(+)-ATPase activity, and causes graded neuronal depolarization. This relieves the voltage-dependent Mg2+ block of the N-methyl-d-aspartate (NMDA) subtype of the glutamate receptor, which is highly permeable to Ca2+. Consequently, innocuous levels of glutamate become lethal via secondary excitotoxicity. Mitochondrial impairment also disrupts cellular Ca2+ homoeostasis. Moreover, the facilitation of NMDA-receptor function leads to further mitochondrial dysfunction. To a large part, this occurs because Ca2+ entering neurons through NMDA receptors has 'privileged' access to mitochondria, where it causes free-radical production and mitochondrial depolarization. Thus there may be a feed-forward cycle wherein mitochondrial dysfunction causes NMDA-receptor activation, which leads to further mitochondrial impairment. In this scenario, NMDA-receptor antagonists may be neuroprotective.
Mitochondria in heart ischaemia and aging
Authors: Hansford Richard G. , Tsuchiya Naotaka , Pepe Salvatore
Published: August 2015

We have examined the effect of dietary polyunsaturated fatty acids (PUFAs) upon mitochondrial Ca2+ content and dehydrogenase activation in the rat heart. Diets were either a conventional low-fat chow (Ref) or were rich in n-3 PUFAs from fish oils (n-3) or n-6 PUFAs from animal fat (n-6). We found that the n-3 diet minimized the rise in mitochondrial Ca2+ seen in response to positive inotropic intervention with noradrenaline, and also minimized the activation of pyruvate dehydrogenase, which is Ca2+ dependent. As the work output of all three groups of hearts was the same, this observation may explain the previous finding of increased thermodynamic efficiency of the n-3 heart relative to the n-6 heart. When hearts were subjected to low-flow ischaemia (15 min), followed by 5 min of reperfusion, increases of mitochondrial Ca2+ were less in the n-3 group than in the n-6 group. In more prolonged ischaemia and reperfusion, n-3 feeding may confer protection against mitochondrial Ca2+ overload, opening of the permeability transition pore and cell death. Notably, the effects of n-3 feeding on mitochondrial functioning were most apparent in hearts from senescent rats (23 months). This is consistent with our finding that the decrease in mitochondrial membrane cardiolipin content, and increase in phosphatidylcholine, which occurred with aging in the Ref and n-6 groups, was totally prevented by n-3 feeding. Thus there are a number of reasons to regard an n-3-rich diet as being protective of the heart in aging mammals.
Mitochondrial dysfunction in sepsis
Authors: Singer Mervyn , Brealey David
Published: August 2015

The current mainstream view of organ failure induced by sepsis revolves around inflammation and loss of vascular control. However, there has been a resurgence in interest in bioenergetic failure due to mitochondrial dysfunction. This concept is not new--studies date back 30 years; however, the data have been highly conflicting with findings of either decreased, increased or unchanged mitochondrial activity and/or nucleotide levels. These studies are virtually all based on non-human cells, isolated perfused organs or in vivo animal models that have received a variety of insults ranging from mild to severe, and monitored for different durations ranging from minutes to weeks. As a generalization, there does appear to be depression of mitochondrial function with longer-duration models of greater severity. This is confirmed by the scanty human data currently available. This chapter provides an overview, and attempts to relate the biochemical changes to the clinical condition. The potential roles of nitric oxide, intracellular calcium and reactive oxygen species are highlighted.
The mitochondrial permeability transition: its molecular mechanism and role in reperfusion injury
Authors: Halestrap Andrew P.
Published: August 2015

The mitochondrial permeability transition (mPT) involves the opening of a non-specific pore in the inner membrane of mitochondria, converting them from organelles whose production of ATP sustains the cell, to instruments of death. Here, I first summarize the evidence in favour of our model for the molecular mechanism of the mPT. It is proposed that the adenine nucleotide translocase (ANT) is converted into a non-specific pore through a calcium-mediated conformational change. This requires the binding of a unique cyclophilin (cyclophilin-D, CyP-D) to the ANT, except when matrix [Ca2+] is very high. Binding of CyP-D is increased in response to oxidative stress and some thiol reagents which sensitize the mPT to [Ca2+]. Matrix adenine nucleotides decrease the sensitivity of the mPT to [Ca2+] by binding to the ANT. This is antagonized by carboxyatractyloside (an inhibitor of the ANT) and by modification of specific thiol groups on the ANT by oxidative stress or thiol reagents; such treatments thus enhance the mPT. In contrast, decreasing intracellular pH below 7.0 greatly desensitizes the mPT to [Ca2+]. Conditions which sensitize the mPT towards [Ca2+] are found in hearts reperfused after a period of ischaemia, a process that may irreversibly damage the heart (reperfusion injury). We have demonstrated directly that mPT pores open during reperfusion (but not ischaemia) using a technique that involves entrapment of [3H]deoxyglucose in mitochondria that have undergone the mPT. The mPT may subsequently reverse in hearts that recover from ischaemia/reperfusion, the extent of resealing correlating with recovery of heart function. A variety of agents that antagonize the mPT protect the heart from reperfusion injury, including cyclosporin A, pyruvate and propofol. Mitochondria that undergo the mPT and then reseal may cause cytochrome c release and thus initiate apoptosis in cells subjected to stresses less severe than those causing necrosis. An example is the apoptotic cell death in the hippocampus that occurs several days after insulin-induced hypoglycaemia, and can be prevented by prior treatment with cyclosporin A.
Sites of phosphorylation in tau and factors affecting their regulation
Authors: Anderton Brian H. , Betts Joanna , Blackstock Walter P. , Brion Jean-Pierre , Chapman Sara , Connell James , Dayanandan Rejith , Gallo Jean-Marc , Gibb Graham , Hanger Diane P. , Hutton Mike , Kardalinou Efterpi , Leroy Karell , Lovestone Simon , Mack Till , Reynolds C.Hugh , Van Slegtenhorst M.
Published: August 2015

The microtubule-associated protein, tau, is the principal component of paired helical filaments (PHFs) in Alzheimer's disease. PHF-tau is highly phosphorylated and a total of 25 sites of phosphorylation have so far been identified. Many of these sites are serine or threonine residues that are immediately followed in the sequence by proline residues, and hence are candidate phosphorylation sites for proline-directed kinases. In vitro, glycogen synthase kinase-3 (GSK-3), extracellular signal-related kinase-1 and -2, and mitogen-activated protein kinases, p38 kinase and c-jun N-terminal kinase, all phosphorylate many of these sites, although with different efficiencies for particular sites. Phosphorylation studies in transfected cells and neurons show that GSK-3 phosphorylates tau more extensively than do these other proline-directed kinases. Mutations in tau have been shown to affect in vitro phosphorylation of tau by GSK-3. The Arg406-->Trp (R406W) tau mutation also affects tau phosphorylation in cells.
Receptor-G-protein signalling in Alzheimer's disease
Authors: Cowburn Richard F. , O'Neill Cora , Bonkale Willy L. , Ohm Thomas G. , Fastbom Johan
Published: August 2015

Based on radioligand binding studies, it has long been assumed that the neurochemical pathology of Alzheimer's disease (AD) does not involve widespread changes in post-synaptic neurotransmitter function. However, more recent studies suggest that receptor function in AD may be compromised due to disrupted post-receptor signal transduction, in particular that mediated by the G-protein regulated phosphoinositide hydrolysis and adenylate cyclase (AC) pathways. The phosphoinositide hydrolysis pathway has been shown to be altered at a number of levels in AD post-mortem brains, including impaired agonist and G-protein regulation of phospholipase C, decreased protein kinase C (PKC) levels and activity, and a reduced number of receptor sites for the second messenger, Ins(1,4,5)P3. Of these, loss of Ins(1,4,5)P3 receptors and PKC in the entorhinal cortex and hippocampus correlates with AD-related neurofibrillary changes, as staged according to Braak's protocol. Disregulation of the phosphoinositide hydrolysis pathway may therefore have consequences for the progression of AD pathology. In contrast to the extensive pattern of disruption seen with the phosphoinositide hydrolysis pathway, changes to AC signalling in AD appear more circumscribed. Disruptions include a lesion at the level of Gs-protein stimulation of AC and, at least in the hippocampus, reduced enzyme activities in response to forskolin stimulation. Of these, the latter change has been shown to precede neurofibrillary changes. Apart from a loss of calcium/calmodulin sensitive AC isoforms, other components of this signalling pathway, including G-protein levels, Gi-protein mediated inhibition and protein kinase A levels and activity, remain relatively preserved in the disorder.
Validation of protein-unfolding transition states identified in molecular dynamics simulations
Authors: Daggett Valerie
Published: August 2015

Experimental and simulation studies can complement each other nicely in the area of protein folding. Experiment reports on the average properties of a large ensemble (approx. 10(17)-10(19) molecules), typically over time. Molecular dynamics simulations, on the other hand, provide detailed information for a single molecule, a component of the ensemble. By combining these approaches we can obtain not only a more complete picture of folding, but we can also take advantage of the strengths of different methods. For example, experiment cannot provide molecular structures. Molecular dynamics simulations can provide such information, but the simulations are meaningless without a linked experiment. Thus, the interrelated nature of simulation in assessing experimental assumptions and in providing structures to augment energetic descriptions, and experiment in judging whether the simulations are reasonable, provides more confidence in the resulting information about folding. This combination yields tested and testable molecular models of states that evade characterization by conventional methods. Therefore, we have explored the combined use of these methods to map folding/unfolding pathways at atomic resolution, in collaboration with Alan Fersht. Here we focus on chymotrypsin inhibitor 2, a small single-domain, two-state folding protein.
Genomic analysis of C-type lectins
Authors: Drickamer Kurt , Fadden Andrew J.
Published: August 2015

Many biological effects of complex carbohydrates are mediated by lectins that contain discrete carbohydrate-recognition domains. At least seven structurally distinct families of carbohydrate-recognition domains are found in lectins that are involved in intracellular trafficking, cell adhesion, cell–cell signalling, glycoprotein turnover and innate immunity. Genome-wide analysis of potential carbohydrate-binding domains is now possible. Two classes of intracellular lectins involved in glycoprotein trafficking are present in yeast, model invertebrates and vertebrates, and two other classes are present in vertebrates only. At the cell surface, calcium-dependent (C-type) lectins and galectins are found in model invertebrates and vertebrates, but not in yeast; immunoglobulin superfamily (I-type) lectins are only found in vertebrates. The evolutionary appearance of different classes of sugar-binding protein modules parallels a development towards more complex oligosaccharides that provide increased opportunities for specific recognition phenomena. An overall picture of the lectins present in humans can now be proposed. Based on our knowledge of the structures of several of the C-type carbohydrate-recognition domains, it is possible to suggest ligand-binding activity that may be associated with novel C-type lectin-like domains identified in a systematic screen of the human genome. Further analysis of the sequences of proteins containing these domains can be used as a basis for proposing potential biological functions.
Role of TIMPs (tissue inhibitors of metalloproteinases) in pericellular proteolysis: the specificity is in the detail
Authors: Murphy Gillian , Knäuper Vera , Lee Meng-Huee , Amour Augustin , Worley Joanna R. , Hutton Mike , Atkinson Susan , Rapti Magdalene , Williamson Richard
Published: August 2015

Pericellular proteolysis represents one of the key modes by which the cell can modulate its environment, involving not only turnover of the extracellular matrix but also the regulation of cell membrane proteins, such as growth factors and their receptors. The metzincins are active players in such proteolytic events, and their mode of regulation is therefore of particular interest and importance. The TIMPs (tissue inhibitors of metalloproteinases) are established endogenous inhibitors of the matrix metalloproteinases (MMPs), and some have intriguing abilities to associate with the pericellular environment. It has been shown that TIMP-2 can bind to cell surface MT1-MMP (membrane-type 1 MMP) to act as a 'receptor' for proMMP-2 (progelatinase A), such that the latter can be activated efficiently in a localized fashion. We have examined the key structural features of TIMP-2 that determine this unique function, showing that Tyr\36 and Glu192-Asp193 are vital for specific interactions with MT1-MMP and proMMP-2 respectively, and hence activation of proMMP-2. TIMP-3 is sequestered at the cell surface by association with the glycosaminoglycan chains of proteoglycans, especially heparan sulphate, and we have shown that it may play a role in the regulation of some ADAMs (a disintegrin and metalloproteinases), including tumour necrosis factor α-converting enzyme (TACE; ADAM17). We have established that key residues in TIMP-3 determine its interaction with TACE. Further studies of the features of TIMP-3 that determine specific binding to both ADAM and glycosaminoglycan are required in order to understand these unique properties.
Cystatins
Authors: Abrahamson Magnus , Alvarez-Fernandez Marcia , Nathanson Carl-Michael
Published: August 2015

Chicken egg white cystatin was first described in the late 1960s. Since then, our knowledge about a superfamily of similar proteins present in mammals, birds, fish, insects, plants and some protozoa has expanded, and their properties as potent peptidase inhibitors have been firmly established. Today, 12 functional chicken cystatin relatives are known in humans, but a few evolutionarily related gene products still remain to be characterized. The type 1 cystatins (A and B) are mainly intracellular, the type 2 cystatins (C, D, E/M, F, G, S, SN and SA) are extracellular, and the type 3 cystatins (L- and H-kininogens) are intravascular proteins. All true cystatins inhibit cysteine peptidases of the papain (C1) family, and some also inhibit legumain (C13) family enzymes. These peptidases play key roles in physiological processes, such as intracellular protein degradation (cathepsins B, H and L), are pivotal in the remodelling of bone (cathepsin K), and may be important in the control of antigen presentation (cathepsin S, mammalian legumain). Moreover, the activities of such peptidases are increased in pathophysiological conditions, such as cancer metastasis and inflammation. Additionally, such peptidases are essential for several pathogenic parasites and bacteria. Thus cystatins not only have capacity to regulate normal body processes and perhaps cause disease when down-regulated, but may also participate in the defence against microbial infections. In this chapter, we have aimed to summarize our present knowledge about the human cystatins.
Control of lipid metabolism by regulated intramembrane proteolysis of sterol regulatory element binding proteins (SREBPs)
Authors: Rawson Robert B
Published: August 2015

In mammalian cells, the supply of lipids is co-ordinated with demand through the transcriptional control of genes encoding proteins required for synthesis or uptake. The sterol regulatory element binding proteins (SREBPs) are responsible for increased transcription of these genes when lipid level fall. Mammals have three SREBPs (-1a, -1c and -2), which are the products of two distinct genes. Synthesized as approximately 120 kDa precursors, they are inserted into membranes of the endoplasmic reticulum (ER) in a hairpin fashion. Both the N-terminal transcription factor domain and the C-terminal regulatory domain face the cytoplasm. These are connected by two transmembrane helices separated by a short loop projecting into the ER lumen. The C-terminal domain of SREBP interacts with the C-terminal domain of SREBP-cleavage-activating protein (SCAP). The N-terminal half of SCAP contains eight transmembrane helices, five of which (helices 2-6) form the sterol-sensing domain. In response to cellular demand for lipid, this complex exits the ER and transits to the Golgi apparatus, where two distinct proteases cleave the SREBP precursor to release the transcriptionally active N-terminus. This process was the first example of regulated intramembrane proteolysis for which the proteases were identified. Recent work has additionally uncovered integral membrane proteins, insig-1 and insig-2, that are required to retain the SREBP-SCAP complex in the ER in the presence of sterols, thus providing a more complete understanding of the control of proteolysis in this complex regulatory pathway.
Separase regulation during mitosis
Authors: Uhlmann Frank
Published: August 2015

The final, irreversible step in the duplication and distribution of genomes to daughter cells takes place when chromosomes split at the metaphase-to-anaphase transition. A protease of the CD clan, separase (C50 family), is the key regulator of this transition. During metaphase, cohesion between sister chromatids is maintained by a chromosomal protein complex, cohesin. Anaphase is triggered when separase cleaves the Scc1 subunit of cohesin at two specific recognition sequences. As a result of this cleavage, the cohesin complex is destroyed, allowing the spindle to pull sister chromatids into opposite halves of the cell. Because of the final and irreversible nature of Scc1 cleavage, this reaction is tightly controlled. Several independent mechanisms impose regulation on separase activity, as well as on the susceptibility of the cleavage target Scc1 to cleavage by separase. This chapter provides an overview of these multiple levels of regulation.
Membrane-type 1 matrix metalloproteinase and cell migration
Authors: Seiki Motoharu , Mori Hidetoshi , Kajita Masahiro , Uekita Takamasa , Itoh Yoshifumi
Published: August 2015

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is an integral membrane proteinase that performs processing of cell surface proteins and degradation of extracellular matrix (ECM) components. Through these proteolytic events, MT1-MMP regulates various cellular functions, including ECM turnover, promotion of cell migration and invasion, and morphogenic responses to extracellular stimuli. MT1-MMP has to be regulated strictly to accomplish its function appropriately at various steps, including at the transcriptional and post-translational levels. MT1-MMP was originally identified as an invasion-promoting enzyme expressed in malignant tumour cells, and also as a specific activator of proMMP-2, which is believed to play a role in invasion of the basement membrane. Since then, it has attracted attention as a membrane-associated MMP that promotes cancer cell invasion and angiogenesis by endothelial cells. Although MT1-MMP has now become one of the best characterized enzymes in the MMP family, there remain numerous unanswered questions. In this chapter, we summarize our recent findings on how MT1-MMP is regulated during cell migration, and how cell migration is regulated by MT1-MMP.
The use of model systems to study biological functions of Nox/Duox enzymes
Authors: Ritsick Darren R. , Edens William A. , McCoy James W. , Lambeth J. David
Published: August 2015

ROS (reactive oxygen species; including superoxide and H2O2) are conventionally thought of as being broadly reactive and cytotoxic. Phagocytes utilize an NADPH oxidase to generate large amounts of ROS, and exploit their toxic properties as a host-defence mechanism to kill invading microbes. However, the recent discovery of the Nox and Duox enzymes that are expressed in many non-phagocytic cells implies that the 'deliberate' generation of ROS has additional cellular roles, which are currently incompletely understood. Functions of ROS in mammals have been inferred primarily from cell-culture experiments, and include signalling for mitogenic growth, apoptosis and angiogenesis. Nox/Duox enzymes may also provide H2O2 as a substrate for peroxidase enzymes (or, in the case of Duox, for its own peroxidase domain), thereby supporting peroxidative reactions. A broad comparison of biological functions of ROS and Nox enzymes across species and kingdoms provides insights into possible functions in mammals. To further understand novel biological roles for Nox/Duox enzymes, we are manipulating the expression of Nox/Duox enzymes in model organisms including Caenorhabditis elegans, Drosophila melanogaster and mouse. This chapter focuses on new insights into the roles of Nox enzymes gained from these approaches.
Metallic prions
Authors: Brown David R
Published: August 2015

Prion diseases, also referred to as transmissible spongiform encephalopathies, are characterized by the deposition of an abnormal isoform of the prion protein in the brain. However, this aggregated, fibrillar, amyloid protein, termed PrPSc, is an altered conformer of a normal brain glycoprotein, PrPc. Understanding the nature of the normal cellular isoform of the prion protein is considered essential to understanding the conversion process that generates PrPSc. To this end much work has focused on elucidation of the normal function and activity of PrPc. Substantial evidence supports the notion that PrPc is a copper-binding protein. In conversion to the abnormal isoform, this Cu-binding activity is lost. Instead, there are some suggestions that the protein might bind other metals such as Mn or Zn. PrPc functions currently under investigation include the possibility that the protein is involved in signal transduction, cell adhesion, Cu transport and resistance to oxidative stress. Of these possibilities, only a role in Cu transport and its action as an antioxidant take into consideration PrPc's Cu-binding capacity. There are also more published data supporting these two functions. There is strong evidence that during the course of prion disease, there is a loss of function of the prion protein. This manifests as a change in metal balance in the brain and other organs and substantial oxidative damage throughout the brain. Thus prions and metals have become tightly linked in the quest to understand the nature of transmissible spongiform encephalopathies.
Mitochondrial superoxide and aging: uncoupling-protein activity and superoxide production
Authors: Brand Martin D. , Buckingham Julie A. , Esteves Telma C. , Green Katherine , Lambert Adrian J. , Miwa Satomi , Murphy Michael P. , Pakay Julian L. , Talbot Darren A. , Echtay Karim S.
Published: August 2015

Mitochondria are a major source of superoxide, formed by the one-electron reduction of oxygen during electron transport. Superoxide initiates oxidative damage to phospholipids, proteins and nucleic acids. This damage may be a major cause of degenerative disease and aging. In isolated mitochondria, superoxide production on the matrix side of the membrane is particularly high during reversed electron transport to complex I driven by oxidation of succinate or glycerol 3-phosphate. Reversed electron transport and superoxide production from complex I are very sensitive to proton motive force, and can be strongly decreased by mild uncoupling of oxidative phosphorylation. Both matrix superoxide and the lipid peroxidation product 4-hydroxy-trans-2-nonenal can activate uncoupling through endogenous UCPs (uncoupling proteins). We suggest that superoxide releases iron from aconitase, leading to a cascade of lipid peroxidation and the release of molecules such as hydroxy-nonenal that covalently modify and activate the proton conductance of UCPs and other proteins. A function of the UCPs may be to cause mild uncoupling in response to matrix superoxide and other oxidants, leading to lowered proton motive force and decreased superoxide production. This simple feedback loop would constitute a self-limiting cycle to protect against excessive superoxide production, leading to protection against aging, but at the cost of a small elevation of respiration and basal metabolic rate.
N-glycans as apical targeting signals in polarized epithelial cells.
Authors: Urquhart Paula , Pang Susan , Hooper Nigel M.
Published: August 2015

MDCK (Madin-Darby canine kidney) cells represent a good model of polarized epithelium to investigate the signals involved in the apical targeting of proteins. As reported previously, GPI (glycosylphosphatidylinositol) anchors mediate the apical sorting of proteins in polarized epithelial cells through their interaction with lipid rafts. However, using a naturally N-glycosylated and GPI-anchored protein, we found that the GPI anchor does not influence the targeting of the protein. It is, in fact, the N-glycans that signal the protein to the apical surface. In the present review, the role of N-glycans and GPI anchors as apical signals is discussed along with the putative mechanisms involved.
Defective kidney anion-exchanger 1 (AE1, Band 3) trafficking in dominant distal renal tubular acidosis (dRTA).
Authors: Toye Ashley M.
Published: August 2015

dRTA (distal renal tubular acidosis) results from the failure of the a-intercalated cells in the distal tubule of the nephron to acidify the urine. A truncated form of AE1 (anion-exchanger 1; Band 3), kAE1 (kidney isoform of AE1), is located in the basolateral membrane of the intercalated cell. Mutations in the AE1 gene cause autosomal dominant and recessive forms of dRTA. All the dominant dRTA mutations investigated cause aberrant trafficking of kAE1, resulting in its intracellular retention or mistargeting to the apical plasma membrane. Therefore the intracellular retention of hetero-oligomers containing wild-type and dRTA mutants, or the mistargeted protein in the apical membrane neutralizing acid secretion, explains dominant dRTA. The kAE1 (Arg901→stop) mutant has been studied in more detail, since the mistargeting kAE1 (Arg901→stop) from the basolateral to the apical membrane is consistent with the removal of a basolateral localization signal. The C-terminal amino acids deleted by the Arg901→stop mutation, contain a tyrosine motif and a type II PDZ interaction domain. The tyrosine residue (Tyr904), but not the PDZ domain, is critical for basolateral localization. In the absence of the N-terminus of kAE1, the C-terminus was not sufficient to localize kAE1 to the basolateral membrane. This suggests that a determinant within the kAE1 N-terminus co-operates with the C-terminus for kAE1 basolateral localization. Interestingly, Tyr359, in the N-terminal domain, and Tyr904 in the C-terminus of AE1 are phosphorylated in red blood cells. A potential scheme is suggested where successive phosphorylation of these residues is necessary for correct localization and recycling of kAE1 to the basolateral membrane.
The conflicting role of brain cholesterol in Alzheimer's disease: lessons from the brain plasminogen system.
Authors: Ledesma Maria Dolores , Dotti Carlos G.
Published: August 2015

Retrospective clinical studies indicate that individuals chronically treated with cholesterol synthesis inhibitors, statins, are at lower risk of developing AD (Alzheimer's disease). Moreover, treatment of guinea pigs with high doses of simvastatin or drastic reduction of cholesterol in cultured cells decrease Aβ (β-amyloid peptide) production. These data sustain the concept that high brain cholesterol is responsible for Aβ accumulation in AD, providing the scientific support for the proposed use of statins to prevent this disease. However, a number of unresolved issues raise doubts that high brain cholesterol is to blame. First, it has not been shown that higher neuronal cholesterol increases Abeta production. Secondly, it has not been demonstrated that neurons in AD have more cholesterol than control neurons. On the contrary, the brains of AD patients show a specific down-regulation of seladin-1, a protein involved in cholesterol synthesis, and low membrane cholesterol was observed in hippocampal membranes of ApoE4 (apolipoprotein E4) AD cases. This effect was also evidenced by altered cholesterol-rich membrane domains (rafts) and raft-mediated functions, such as diminished generation of the Aβ-degrading enzyme plasmin. Thirdly, numerous genetic defects that cause neurodegeneration are due to defective cholesterol metabolism. Fourthly, in female mice, the most brain-permeant statin induces neurodegeneration and high amyloid production. Altogether, this evidence makes it difficult to accept that statins are beneficial through acting as brain cholesterol-synthesis inhibitors. It appears more likely that their advantageous role arises from improved brain oxygenation.
The relationship between higher-order chromatin structure and transcription
Authors: Gilbert Nick , Bickmore Wendy A.
Published: August 2015

It has generally been assumed that transcriptionally active genes are in an ‘open’ chromatin structure and that silent genes have a ‘closed’ chromatin structure. Here we re-assess this axiom in the light of genome-wide studies of chromatin fibre structure. Using a combination of sucrose gradient sedimentation and genomic microarrays of the human genome, we argue that open chromatin fibres originate from regions of high gene density, whether or not those genes are transcriptionally active.
The role of the phosphoinositides at the Golgi complex
Authors: De Matteis Maria Antonietta , D'Angelo Giovanni
Published: August 2015

Eukaryotic cells are organized into a complex system of subcompartments, each with its distinct protein and lipid composition. A continuous flux of membranes crosses these compartments, and in some cases direct connections exist between the different organelles. It is thus surprising that they can maintain their individual identities. Small GTPases and the phosphoinositides have emerged as the key regulators in the maintenance of the identity of the Golgi complex. This property is due to their ability to act either alone or, more often, in combination, as cues directing and controlling the recruitment of proteins that possess phosphoinositide-binding domains. Among these many proteins there are the lipid transfer proteins, which can transfer ceramide, oxysterol, cholesterol and possibly glucosylceramide. By regulating these lipid transfer proteins in this way, this binomial combination of the small GTPases and the phosphoinositides acquires a further important role: control of the synthesis and/or distribution of other important integral constituents of cell organelles, such as the sphingolipids and cholesterol. This role is particularly relevant at the level of the Golgi complex, a key organelle in the biosynthesis, transport and sorting of both lipids and proteins that is located at the intersection of the secretory and endocytic pathways.
Inositol polyphosphate kinases: regulators of nuclear function
Authors: Seeds Andrew M. , York John D.
Published: August 2015

Recent work has uncovered roles for inositide signalling pathways downstream of phospholipase C activation and inositol 1,4,5-trisphosphate in the regulation of nuclear processes including gene expression, mRNA export and DNA metabolism. The identification of several IPKs (inositol polyphosphate kinases) has renewed interest in the cellular roles of inositol tetra-, penta-, hexa- and pyro-phosphates. Discoveries of inositide receptors and novel mechanisms of inositide action have provided important insights into how such messengers couple to nuclear machinery. In this chapter, we discuss the IPK family members and the nuclear processes that their inositide products regulate.