bluuebunny/crossref_metadata_2025 huggingface dataset:
Slow delayed rectifier K+ current block by HMR 1556 increases dispersion of repolarization and promotes Torsades de Pointes in rabbit ventricles
Authors: So P P‐S , Backx P H , Dorian P
Published: October 2008

Background and purpose:

The slow delayed rectifier K+ current (IKs) contributes to ventricular repolarization when the action potential (AP) is prolonged. IKs block during drug‐induced AP prolongation may promote Torsades de Pointes (TdP), but whether this is due to additional AP prolongation is uncertain.

Experimental approach:

In bradycardic perfused rabbit ventricles, the incidence of spontaneous TdP, monophasic AP duration at 90% repolarization (MAPD90) and ECG interval between the peak and the end of T wave (Tpeak−end) (index of dispersion of repolarization) were measured after the administration of veratridine (125 nM, slows Na+ channel inactivation), dofetilide (7.5 or 10 nM, a rapid delayed rectifier blocker) and HMR 1556 (HMR, 100 nM, an IKs blocker), alone or in combinations (n=6 each).

Key results:

HMR did not prolong MAPD90, whereas veratridine or 7.5 nM dofetilide prolonged MAPD90 (P<0.01) without inducing TdP. Veratridine+7.5 nM dofetilide additively prolonged MAPD90 (P<0.05), induced 4±6 TdP per heart and prolonged Tpeak−end by 12±10 ms. Subsequent addition of HMR did not further prolonged MAPD90, but increased the number of TdP to 22±18 per heart and increased Tpeak−end by 39±21 ms (P<0.05). Increasing dofetilide concentration from 7.5 to 10 nM (added to veratridine) produced a longer MAPD90, but fewer TdP (5±5 per heart) and less Tpeak−end prolongation (17±8 ms) compared to the veratridine+7.5 nM dofetilide+HMR group (P<0.05).

Conclusions and implications:

Adding IKs block markedly increases TdP incidence in hearts predisposed to TdP development by increasing the dispersion of repolarization, but without additional AP prolongation.

British Journal of Pharmacology (2008) 155, 1185–1194; doi:10.1038/bjp.2008.354; published online 6 October 2008
Neural Mechanisms of Selective Auditory Attention in Rats (Dissertation)
Authors: Tai Lung-Hao , Zador Anthony
Published: October 2008

How does attention modulate sensory representations? In order to probe the underlying neural mechanisms, we established a simple rodent model of modality-specific attention. Rats were trained to perform distinct auditory two-tone discrimination and olfactory odor discrimination in a two alternative choice (2AC) paradigm. To determine auditory cortex’s role in this frequency discrimination task, we used GABA-A receptor agonist muscimol to transiently and reversibly inactivate auditory cortexes bilaterally in rats performing simple interleaved auditory and olfactory discrimination. With olfactory discrimination performance serving as internal control for motivation and decision making capability, we found only auditory two-tone discrimination was selectively impaired in these rats. This shows the auditory cortex is involved in this two-tone discrimination task.To investigate the neural correlate of modality-specific attention in the auditory cortex, we trained rats to perform interleaved auditory and olfactory blocks (of 50~70 trials each) in a single session. In auditory blocks, pure tones were either presented with or without a neutral odor (caproic acid, n=2 and 3 respectively), and subjects were rewarded for discriminating auditory stimuli. In olfactory blocks, both task odors and pure tones were presented simultaneously, and subjects were rewarded for discriminating olfactory stimuli. We recorded neural responses in primary auditory cortex (area A1) in freely moving rats while subjects performed this behavior. Single unit responses to tones were heterogeneous, and included transient, sustained, and suppressed. We found 205 of 802 units recorded responsive to the stimuli we used. Of these 205 units, 18.5% showed modality-specific attentional modulation of the anticipatory activity before tone onset. In addition, we also observed in smaller proportion of units (11.2%) modality-specific attentional modulation of the tone-evoked responses; in most cases, the responses to a particular auditory stimulus was enhanced in the auditory block (or, equivalently, suppressed in the olfactory block). Attention increased choice probability of the population in the auditory block. We have also observed significant behavior choice probability in small proportions of units. Our results suggest that shifting attention between audition to olfaction tasks can modulate the activity of single neurons in primary auditory cortex.
Characterization of the ubinuclein protein as a new member of the nuclear and adhesion complex components (NACos)
Authors: Aho Sirpa , Lupo Julien , Coly Pierre‐Alain , Sabine Amélie , Castellazzi Marc , Morand Patrice , Sergeant Alain , Manet Evelyne , Boyer Véronique , Gruffat Henri
Published: October 2008

Background information. We characterized previously a cellular protein through its interaction with cellular and viral transcription factors from the bZip family. The corresponding mRNA was detected in a wide range of cell types and the protein was highly expressed in the nucleus of human keratinocytes. On the basis of these observations, we named this protein ubinuclein.

Results. Using a specific monoclonal antibody, we have shown in the present study that, although endogenous ubinuclein was mainly nuclear in sparse MDCK (Madin—Darby canine kidney) cells, it was exclusively present in the cell—cell junctions in confluent MDCK cultures or in polarized HT29 cells, where it co‐localized with the tight junction marker ZO‐1 (zonula occludens 1). In accordance with this, we have shown that ubinuclein interacted with ZO‐1 in vitro and in vivo. In cultures of undifferentiated human keratinocytes, ubinuclein was essentially nuclear, but in differentiated cells, in which involucrin and periplakin reside at the apical cell membrane and at the cell—cell junctions, ubinuclein staining was observed at the lateral cell—cell borders. In human skin, ubinuclein appeared as a thread‐like pattern between the upper granular cell layer and the cornified cell layer. In mouse epithelia, including bile canaliculi, bronchioli, salivary gland ducts, and oral and olfactory epithelium, ubinuclein co‐localized with tight junction markers. Ubinuclein was, however, not present in endothelial cell—cell junctions. In addition, when overexpressed, ubinuclein localized to the nucleus and prevented MDCK cells from entering cytokinesis, resulting in multinucleated giant cells after several cycles of endoreplication.

Conclusions. Ubinuclein mRNA and its corresponding protein are expressed in almost all cell types. Analyses have revealed that in most cells ubinuclein occurred in the nucleoplasm, but in cells forming tight junctions it is recruited to the plaque structure of the zonula occludens. This recruitment appeared to be dependent on cell density. Therefore ubinuclein is a new NACos (nuclear and adhesion complex component) protein.
Discovery of a new RNA‐containing nuclear structure in UVC‐induced apoptotic cells by integrated laser electron microscopy
Authors: Karreman Matthia A. , Agronskaia Alexandra V. , Verkleij Arie J. , Cremers Fons F.M. , Gerritsen Hans C. , Humbel Bruno M.
Published: October 2008

Background information. Treatment of cells with UVC radiation leads to the formation of DNA cross‐links which, if not repaired, can lead to apoptosis. γ‐H2AX and cleaved caspase 3 are proteins formed during UVC‐induced DNA damage and apoptosis respectively. The present study sets out to identify early morphological markers of apoptosis using a new method of correlative microscopy, ILEM (integrated laser electron microscopy). Cleaved caspase 3 and γ‐H2AX were immunofluorescently labelled to mark the cells of interest. These cells were subsequently searched in the fluorescence mode of the ILEM and further analysed at high resolution with TEM (transmission electron microscopy).

Results. Following the treatment of HUVECs (human umbilical vein endothelial cells) with UVC radiation, in the majority of the cells γ‐H2AX was formed, whereas only in a subset of cells caspase 3 was activated. In severely damaged cells with high levels of γ‐H2AX a round, electron‐dense nuclear structure was found, which was hitherto not identified in UV‐stressed cells. This structure exists only in nuclei of cells containing cleaved caspase 3 and is present during all stages of the apoptotic process. Energy‐loss imaging showed that the nuclear structure accumulates phosphorus, indicating that it is rich in nucleic acids. Because the nuclear structure did not label for DNA and was not affected by regressive EDTA treatment, it is suggested that the UV‐induced nuclear structure contains a high amount of RNA.

Conclusions. Because the UV‐induced nuclear structure was only found in cells labelled for cleaved caspase 3 it is proposed as an electron microscopic marker for all stages of apoptosis. Such a marker will especially facilitate the screening for early apoptotic cells, which lack the well‐known hallmarks of apoptosis within a cell population. It also raises new questions on the mechanisms involved in the UV‐induced apoptotic pathway.
NFAT directly regulates Nkx2‐5 transcription during cardiac cell differentiation
Authors: Chen Yong , Cao Xinmin
Published: October 2008

Background information. The transcription factor NFAT (nuclear factor of activated T‐cell) family comprises important regulators in immuno‐responses and mouse embryonic development, including early cardiovascular and heart valve development. The mechanism involved, however, is not fully understood. Nkx2‐5 (NK2 transcription factor related, locus 5) is one of the earliest genes expressed in early cardiac progenitor cells and is essential for heart tube development by control of a subset of cardiac muscle‐specific genes. Previously we found that downregulation of mitochondrial respiratory chain complex I caused severe cardiac deficiencies during heart tube development in Xenopus embryos associated with compromised Nkx2‐5 expression. However, the heart defects and Nkx2‐5 expression could be rescued by a constitutively activated NFAT, suggesting a possible link between NFAT and Nkx2‐5 during early heart development.

Results. In the present study, we demonstrate that NFAT regulates Nkx2‐5 expression in both mouse ES (embryonic stem) cells and P19 cells, a mouse model for embryonic differentiation. We found that there are six core NFAT‐binding elements in the 5′ regulatory region of the Nkx2‐5 gene. Although NFAT is able to bind directly to all but one of these elements, it activates Nkx2‐5 transcription only via a specific binding site in the distal enhancer region. Interestingly, the transcriptional activity of NFAT is largely dependent on the co‐factor GATA (GATA‐binding transcription factor), which binds to an element adjacent to this key NFAT‐binding site. Furthermore, binding of the endogenous NFAT to this particular site was observed during cardiac differentiation in mouse ES and P19 cells.

Conclusions. The results suggest that Nkx2‐5 is a direct target of NFAT that co‐ordinates with other transcription factors such as GATA4 to regulate Nkx2‐5 during cardiogenesis.
Synergistic induction of nuclear factor-κB by transforming growth factor-β and tumour necrosis factor-α is mediated by protein kinase A-dependent RelA acetylation
Authors: Ishinaga Hajime , Jono Hirofumi , Lim Jae Hyang , Komatsu Kensei , Xu Xiangbin , Lee Jiyun , Woo Chang-Hoon , Xu Haidong , Feng Xin-Hua , Chen Lin-Feng , Yan Chen , Li Jian-Dong
Published: October 2008

The TGF-β (transforming growth factor-β) pathway represents an important signalling pathway involved in regulating diverse biological processes, including cell proliferation, differentiation and inflammation. Despite the critical role for TGF-β in inflammatory responses, its role in regulating NF-κB (nuclear factor-κB)-dependent inflammatory responses still remains unknown. In the present study we show that TGF-β1 synergizes with proinflammatory cytokine TNF-α (tumour necrosis factor-α) to induce NF-κB activation and the resultant inflammatory response in vitro and in vivo. TGF-β1 synergistically enhances TNF-α-induced NF-κB DNA binding activity via induction of RelA acetylation. Moreover, synergistic enhancement of TNF-α-induced RelA acetylation and DNA-binding activity by TGF-β1 is mediated by PKA (protein kinase A). Thus the present study reveals a novel role for TGF-β in inflammatory responses and provides new insight into the regulation of NF-κB by TGF-β signalling.
Vascular endothelial growth factor acts through novel, pregnancy-enhanced receptor signalling pathways to stimulate endothelial nitric oxide synthase activity in uterine artery endothelial cells
Authors: Grummer Mary A. , Sullivan Jeremy A. , Magness Ronald R. , Bird Ian M.
Published: October 2008

During pregnancy, VEGF (vascular endothelial growth factor) regulates in part endothelial angiogenesis and vasodilation. In the present study we examine the relative roles of VEGFRs (VEGF receptors) and associated signalling pathways mediating the effects of VEGF165 on eNOS (endothelial nitric oxide synthase) activation. Despite equal expression levels of VEGFR-1 and VEGFR-2 in UAECs (uterine artery endothelial cells) from NP (non-pregnant) and P (pregnant) sheep, VEGF165 activates eNOS at a greater level in P- compared with NP-UAEC, independently of Akt activation. The selective VEGFR-1 agonist PlGF (placental growth factor)-1 elicits only a modest activation of eNOS in P-UAECs compared with VEGF165, whereas the VEGFR-2 kinase inhibitor blocks VEGF165-stimulated eNOS activation, suggesting VEGF165 predominantly activates eNOS via VEGFR-2. Although VEGF165 also activates ERK (extracellular-signal-regulated kinase)-1/2, this is not necessary for eNOS activation since U0126 blocks ERK-1/2 phosphorylation, but not eNOS activation, and the VEGFR-2 kinase inhibitor inhibits eNOS activation, but not ERK-1/2 phosphorylation. Furthermore, the inability of PlGF to activate ERK-1/2 and the ability of the VEGFR-2 selective agonist VEGF-E to activate ERK-1/2 and eNOS suggests again that both eNOS and ERK-1/2 activation occur predominately via VEGFR-2. The lack of VEGF165-stimulated Akt phosphorylation is consistent with a lack of robust phosphorylation of Ser1179-eNOS. Although VEGF165-stimulated eNOS phosphorylation is observed at Ser617 and Ser635, pregnancy does not significantly alter this response. Our finding that VEGF165 activation of eNOS is completely inhibited by wortmannin but not LY294002 implies a downstream kinase, possibly a wortmannin-selective PI3K (phosphoinositide 3-kinase), is acting between the VEGFR-2 and eNOS independently of Akt.
WITHDRAWN: Curcumin, a dietary polyphenol, emerges as a novel inhibitor of DNA topoisomerase I of kinetoplastid parasite Leishmania donovani
Authors: Das Rakhee , Roy Amit , Ganguly Agneyo , Datta Neeta , Majumder Hemanta K
Published: October 2008

This paper published as an Accepted Manuscript on 29 September 2008 was withdrawn by the Editorial Board of the Biochemical Journal on 12 December 2008.
Isolation at physiological temperature of detergent-resistant membranes with properties expected of lipid rafts: the influence of buffer composition
Authors: Chen Xi , Jen Angela , Warley Alice , Lawrence M. Jayne , Quinn Peter J. , Morris Roger J.
Published: October 2008

The failure of most non-ionic detergents to release patches of DRM (detergent-resistant membrane) at 37 °C undermines the claim that DRMs consist of lipid nanodomains that exist in an Lo (liquid ordered) phase on the living cell surface. In the present study, we have shown that inclusion of cations (Mg2+, K+) to mimic the intracellular environment stabilizes membranes during solubilization sufficiently to allow the isolation of DRMs at 37 °C, using either Triton X-100 or Brij 96. These DRMs are sensitive to chelation of cholesterol, maintain outside-out orientation of membrane glycoproteins, have prolonged (18 h) stability at 37 °C, and are vesicles or sheets up to 150–200 nm diameter. DRMs containing GPI (glycosylphosphatidylinositol)-anchored proteins PrP (prion protein) and Thy-1 can be separated by immunoaffinity isolation, in keeping with their separate organization and trafficking on the neuronal surface. Thy-1, but not PrP, DRMs are associated with actin. EM (electron microscopy) immunohistochemistry shows most PrP, and some Thy-1, to be clustered on DRMs, again maintaining their organization on the neuronal surface. For DRMs labelled for either protein, the bulk of the surface of the DRM is not labelled, indicating that the GPI-anchored protein is a minor component of its lipid domain. These 37 °C DRMs thus have properties expected of raft membrane, yet pose more questions about how proteins are organized within these nanodomains.
Glucose induces an autocrine activation of the Wnt/β-catenin pathway in macrophage cell lines
Authors: Anagnostou Sasha H. , Shepherd Peter R.
Published: October 2008

The canonical Wnt signalling pathway acts by slowing the rate of ubiquitin-mediated β-catenin degradation. This results in the accumulation and subsequent nuclear translocation of β-catenin, which induces the expression of a number of genes involved in growth, differentiation and metabolism. The mechanisms regulating the Wnt signalling pathway in the physiological context is still not fully understood. In the present study we provide evidence that changes in glucose levels within the physiological range can acutely regulate the levels of β-catenin in two macrophage cell lines (J774.2 and RAW264.7 cells). In particular we find that glucose induces these effects by promoting an autocrine activation of Wnt signalling that is mediated by the hexosamine pathway and changes in N-linked glycosylation of proteins. These studies reveal that the Wnt/β-catenin system is a glucose-responsive signalling system and as such is likely to play a role in pathways involved in sensing changes in metabolic status.
Histidine-regulated activity of M-ficolin
Authors: Tanio Michikazu , Kohno Toshiyuki
Published: October 2008

Human M-ficolin is a pathogen-associated molecular recognition molecule in the innate immune system, and it binds to some sugars, such as GlcNAc (N-acetylglucosamine), on pathogen surfaces. From previous structural and functional studies of the FD1 (M-ficolin fibrinogen-like domain), we proposed that the ligand-binding region of FD1 exists in a conformational equilibrium between active and non-active states depending on three groups with a pKa of 6.2, which are probably histidine residues, and suggested that the 2-state conformational equilibrium as well as the trimer formation contributes to the discrimination mechanism between self and non-self of FD1 [Tanio, M., Kondo, S., Sugio, S. and Kohno, T. (2007) J. Biol. Chem. 282, 3889–3895]. To investigate the origins of the pH dependency, mutational analyses were performed on FD1 expressed by Brevibacillus choshinensis. The GlcNAc binding study of a series of single histidine mutants of FD1 demonstrated that His251, His284 and His297 are required for the activity, and thus we concluded that the three histidines are the origins of the pH dependency of FD1. Monomeric mutants of FD1 show weaker affinity for the ligand than the trimeric wild-type, indicating that trimer formation confers high avidity for the ligand. In addition, analyses of the GlcNAc association and dissociation of FD1 provided evidence that FD1 always exchanges between the active and non-active states with the pH-dependent populations in solution. The biological roles of the histidine-regulated conformational equilibrium of M-ficolin are discussed in terms of the self and non-self discrimination mechanism.
Oleoyl-oestrone inhibits lipogenic, but maintains thermogenic, gene expression of brown adipose tissue in overweight rats
Authors: Romero María del Mar , Fernández-López José A. , Esteve Montserrat , Alemany Marià
Published: October 2008

In the present study we intended to determine how BAT (brown adipose tissue) maintained thermogenesis under treatment with OE (oleoyl-oestrone), a powerful slimming hormone that sheds off body lipid but maintains the metabolic rate. Overweight male rats were subjected to daily gavages of 10 nmol/g of OE or vehicle (control) for 10 days. A PF (pair-fed) vehicle-receiving group was used to discount the effects attributable to energy availability limitation. Interscapular BAT mass, lipid, DNA, mRNA and the RT-PCR (real-time PCR) expression of lipid and energy metabolism genes for enzymes and regulatory proteins were measured. BAT mass and lipid were decreased in OE and PF, with the latter showing a marked reduction in tissue mRNA. Maintenance of perilipin gene expression in PF and OE rats despite the loss of lipid suggests the preservation of the vacuolar interactive surface, a critical factor for thermogenic responsiveness. OE and, to a lesser extent, PF maintained the expression of genes controlling lipolysis and fatty acid oxidation, but markedly decreased the expression of those genes involved in lipogenic and acyl-glycerol synthesis. OE did not affect UCP1 (uncoupling protein 1) (decreased in PF), β3 adrenergic receptors or hormone-sensitive lipase gene mRNAs, which may translate in maintaining a full thermogenic system potential. OE rats were able to maintain a less energetically stressed BAT (probably through glucose utilization) than PF rats. These changes were not paralleled in PF rats, in which lower thermogenesis and glucose preservation resulted in a heavier toll on internal fat stores. Thus the mechanism of action of OE is more complex and tissue-specific than previously assumed.
Cross-reactivity of pollen and food allergens: soybean Gly m 4 is a member of the Bet v 1 superfamily and closely resembles yellow lupine proteins
Authors: Berkner Hanna , Neudecker Philipp , Mittag Diana , Ballmer-Weber Barbara K. , Schweimer Kristian , Vieths Stefan , Rösch Paul
Published: October 2008

In many cases, patients allergic to birch pollen also show allergic reactions after ingestion of certain fruits or vegetables. This observation is explained at the molecular level by cross-reactivity of IgE antibodies induced by sensitization to the major birch pollen allergen Bet v 1 with homologous food allergens. As IgE antibodies recognize conformational epitopes, a precise structural characterization of the allergens involved is necessary to understand cross-reactivity and thus to develop new methods of allergen-specific immunotherapy for allergic patients. Here, we report the three-dimensional solution structure of the soybean allergen Gly m 4, a member of the superfamily of Bet v 1 homologous proteins and a cross-reactant with IgE antibodies originally raised against Bet v 1 as shown by immunoblot inhibition and histamine release assays. Although the overall fold of Gly m 4 is very similar to that of Bet v 1, the three-dimensional structures of these proteins differ in detail. The Gly m 4 local structures that display those differences are also found in proteins from yellow lupine with known physiological function. The three-dimensional structure of Gly m 4 may thus shed some light on the physiological function of this subgroup of PR10 proteins (class 10 of pathogenesis-related proteins) and, in combination with immunological data, allow us to propose surface patches that might represent cross-reactive epitopes.
Much to know about proteolysis: intricate proteolytic machineries compromise essential cellular functions
Authors: Marfany Gemma , Farràs Rosa , Salido Eduardo , Xirodimas Dimitris P. , Rodríguez Manuel S.
Published: October 2008

Proteolysis has traditionally been considered as a radical way to terminate the function of a protein. However, protein destruction also is the starting point for many processes as they can only occur when the way has been cleared for the action of other proteins. Protein destruction can occur virtually in all compartments and organelles of the cell, associated with cell membranes or large protein complexes, it determines subcellular partitioning, association with positive or negative regulators which conditions the action of many critical cellular factors. The third intracellular proteolysis meeting held by the University La Laguna, Canary Islands, Spain, included speakers working with some of the most important proteolytic systems present in higher eukaryotes, such as the UPS (ubiquitin–proteasome system) and autophagy. Owing to the fact that these pathways directly or indirectly regulate many cell functions, this meeting brought together an audience with a wide range of research interests, including genetic, cell biological, biochemical and structural aspects of protein degradation. Some of these topics inspired interesting discussions and a significant number of these are developed in the issues reviewed herein.
Life, death and burial: multifaceted impact of autophagy
Authors: Galluzzi Lorenzo , Morselli Eugenia , Vicencio José Miguel , Kepp Oliver , Joza Nicholas , Tajeddine Nicolas , Kroemer Guido
Published: October 2008

Macroautophagy, often referred to as autophagy, designates the process by which portions of the cytoplasm, intracellular organelles and long-lived proteins are engulfed in double-membraned vacuoles (autophagosomes) and sent for lysosomal degradation. Basal levels of autophagy contribute to the maintenance of intracellular homoeostasis by ensuring the turnover of supernumerary, aged and/or damaged components. Under conditions of starvation, the autophagic pathway operates to supply cells with metabolic substrates, and hence represents an important pro-survival mechanism. Moreover, autophagy is required for normal development and for the protective response to intracellular pathogens. Conversely, uncontrolled autophagy is associated with a particular type of cell death (termed autophagic, or type II) that is characterized by the massive accumulation of autophagosomes. Regulators of apoptosis (e.g. Bcl-2 family members) also modulate autophagy, suggesting an intimate cross-talk between these two degradative pathways. It is still unclear whether autophagic vacuolization has a causative role in cell death or whether it represents the ultimate attempt of cells to cope with lethal stress. For a multicellular organism, autophagic cell death might well represent a pro-survival mechanism, by providing metabolic supplies during whole-body nutrient deprivation. Alternatively, type II cell death might contribute to the disposal of cell corpses when heterophagy is deficient. Here, we briefly review the roles of autophagy in cell death and its avoidance.
Chaperone-driven proteasome assembly
Authors: Rosenzweig Rina , Glickman Michael H.
Published: October 2008

Assembly of the 34-subunit, 2.5 MDa 26S proteasome is a carefully choreographed intricate process. It starts with formation of a seven-membered α-ring that serves as a template for assembly of the complementary β-ring-forming ‘half-proteasomes’. Dimerization results in a latent 20S core particle that can serve further as a platform for 19S regulatory particle attachment and formation of the biologically active 26S proteasome for ubiquitin-dependent proteolysis. Both general and dedicated proteasome assembly chaperones regulate the efficiency and outcome of critical steps in proteasome biogenesis, and in complex association.
Strategies for the identification of novel inhibitors of deubiquitinating enzymes
Authors: Goldenberg Seth J. , McDermott Jeffrey L. , Butt Tauseef R. , Mattern Michael R. , Nicholson Benjamin
Published: October 2008

Dysregulation of the UPS (ubiquitin–proteasome system) has been implicated in a wide range of pathologies including cancer, neurodegeneration and viral infection. Inhibiting the proteasome has been shown to be an effective therapeutic strategy in humans; yet toxicity with this target remains high. DUBs (deubiquitinating enzymes) represent an alternative target in the UPS with low predicted toxicity. Currently, there are no DUB inhibitors that have been used clinically. To address this situation, Progenra has developed a novel assay to measure the proteolytic cleavage of Ub (ubiquitin) or UBL (Ub-like protein) conjugates such as SUMO (small Ub-related modifier), NEDD8 (neural-precursor-cell-expressed, developmentally down-regulated 8) or ISG15 (interferon-stimulated gene 15) by isopeptidases. In this review, current platforms for detecting DUB inhibitors are discussed and the advantages and disadvantages of the approaches are underlined.
To ubiquitinate or to deubiquitinate: it all depends on the partners
Authors: Marfany Gemma , Denuc Amanda
Published: October 2008

Ub (ubiquitin) and Ubls (Ub-like molecules) are peptide modifiers that change the fate and function of their substrates. A plethora of enzyme activities and protein cofactors are required for either the conjugation (mainly E3 ligases) or deconjugation of Ub and Ubls. Most of the data have been gathered on describing individual enzymes and their partners, but an increasing number of reports point to the formation of multisubunit complexes regulated by cross-talk between Ub and Ubl systems and which contain opposing conjugation/deconjugation activities. This minireview focuses on these latest reports and proposes that these complexes, which are able to recruit transient partners, shift cofactors and integrate different signalling stimuli, are a common strategy to regulate highly dynamic processes, in a switch-on/switch-off type of mechanism, thus responding promptly to cellular requirements.
Is there an alternative to the proteasome in cytosolic protein degradation?
Authors: Antón Luis C. , Villasevil Eugenia M.
Published: October 2008

While it is clear that the proteasome is the major player in degradative proteolysis in the nucleus and cytosol, there is a lack of complete agreement on whether there are alternative proteolytic pathways or activities responsible for a significant degradation of cytosolic/nuclear substrates. Particularly relevant is the case of the aminopeptidase TPPII (tripeptidyl peptidase II), which has been suggested to be able to perform some of the proteasome functions. However, the current evidence seems to support only a limited role for these cytosolic alternatives. On the other hand, there is evidence of an alternative, autophagy, a pathway involving the delivery of cytosolic substrates to the lysosome for degradation.
Do F-box proteins with a C-terminal domain homologous with the tobacco lectin play a role in protein degradation in plants?
Authors: Lannoo Nausicaä , Peumans Willy J. , Van Damme Els J.M.
Published: October 2008

Protein turnover is a key post-translational event that regulates numerous cellular processes. It enables cells to respond rapidly to intracellular signals and changing environmental conditions by adjusting the levels of pivotal proteins. A major proteolytic pathway involves the ubiquitination of target proteins and subsequent targeting to the 26S proteasome for degradation. Many F-box proteins play a determining role in the substrate specificity of this degradation pathway. In most cases, selective recognition of the target proteins relies on protein–protein interactions mediated by the C-terminal domain of the F-box proteins. In mammals, the occurrence of F-box proteins with a C-terminal SBD (sugar-binding domain) that specifically interacts with high-mannose N-glycans on target glycoproteins has been documented. The identification and characterization of these sugar-binding F-box proteins demonstrated that F-box proteins do not exclusively use protein–protein interactions but also protein–carbohydrate interactions in the Ub (ubiquitin)/proteasome pathway. Recently, putative sugar-binding F-box proteins have been identified in plants. Genome analyses in Arabidopsis and rice revealed the presence of F-box proteins with a C-terminal lectin-related domain homologous with Nictaba, a jasmonate-inducible lectin from tobacco that was shown to interact with the core structure of high-mannose and complex N-glycans. Owing to the high similarity in structure and specificity between Nictaba and the SBD of the mammalian Fbs proteins, a similar role for the plant F-box proteins with a Nictaba domain in nucleocytoplasmic protein degradation in plant cells is suggested.
S-adenosylmethionine and proliferation: new pathways, new targets
Authors: Martínez-López Nuria , Varela-Rey Marta , Ariz Usue , Embade Nieves , Vazquez-Chantada Mercedes , Fernandez-Ramos David , Gomez-Santos Laura , Lu Shelly C. , Mato Jose M. , Martinez-Chantar Maria L.
Published: October 2008

SAMe (S-adenosylmethionine) is the main methyl donor group in the cell. MAT (methionine adenosyltransferase) is the unique enzyme responsible for the synthesis of SAMe from methionine and ATP, and SAMe is the common point between the three principal metabolic pathways: polyamines, transmethylation and transsulfuration that converge into the methionine cycle. SAMe is now also considered a key regulator of metabolism, proliferation, differentiation, apoptosis and cell death. Recent results show a new signalling pathway implicated in the proliferation of the hepatocyte, where AMPK (AMP-activated protein kinase) and HuR, modulated by SAMe, take place in HGF (hepatocyte growth factor)-mediated cell growth. Abnormalities in methionine metabolism occur in several animal models of alcoholic liver injury, and it is also altered in patients with liver disease. Both high and low levels of SAMe predispose to liver injury. In this regard, knockout mouse models have been developed for the enzymes responsible for SAMe synthesis and catabolism, MAT1A and GNMT (glycine N-methyltransferase) respectively. These knockout mice develop steatosis and HCC (hepatocellular carcinoma), and both models closely replicate the pathologies of human disease, which makes them extremely useful to elucidate the mechanism underlying liver disease. These new findings open a wide range of possibilities to discover novel targets for clinical applications.
Innate link between NF-κB activity and ubiquitin-like modifiers
Authors: Lang Valérie , Rodríguez Manuel S.
Published: October 2008

Among the several signalling pathways regulated by ubiquitin and ubiquitin-like proteins, the one activating NF-κB (nuclear factor κB) is certainly one of the best characterized. The regulation of the activity of this transcription factor by members of the ubiquitin family occurs at various levels, imposing overlapping controls of security of intriguing complexity. The formation of active macromolecular complexes such as the IKK [IκB (inhibitory κB) kinase] complex is tightly regulated by these post-translational modifications probably due to the fact that many signals converge on this signal's roundabout. An additional, very important level of NF-κB control occurs through the partial or total proteolysis of precursor and inhibitor molecules exerted by the ubiquitin–proteasome pathway. Regulation at this level implicates various conjugating and de-conjugating activities for ubiquitin, SUMO (small ubiquitin-related modifier) and NEDD8. Here, we summarize some of these events and underline the importance of the interconnecting ubiquitin and ubiquitin-like conjugating pathways that determine the status of the activity of this critical transcription factor.
Regulation and function of JunB in cell proliferation
Authors: Piechaczyk Marc , Farràs Rosa
Published: October 2008

JunB is a member of the AP-1 (activator protein-1) family of dimeric transcription factors. It exerts a dual action on the cell cycle. It is best known as a cell proliferation inhibitor, a senescence inducer and a tumour suppressor. As for the molecular mechanisms involved, they largely involve both positive actions on genes such as the p16INK4α cyclin-dependent kinase inhibitor and negative effects on genes such as cyclin D1 during the G1-phase of the cell cycle. However, JunB is also endowed with a cell-division-promoting activity, in particular via stimulation of cyclin A2 gene expression during S-phase. Strikingly, its role in G2 and M has received little attention so far despite its possible role in the preparation of mitosis. This review addresses the known and possible mechanisms whereby JunB is implicated in the control of the different phases of the cell cycle.
Functional analysis of the SUMOylation pathway in Drosophila
Authors: Talamillo Ana , Sánchez Jonatan , Barrio Rosa
Published: October 2008

SUMOylation, a reversible process used as a ‘fine-tuning’ mechanism to regulate the role of multiple proteins, is conserved throughout evolution. This post-translational modification affects several cellular processes by the modulation of subcellular localization, activity or stability of a variety of substrates. A growing number of proteins have been identified as targets for SUMOylation, although, for many of them, the role of SUMO conjugation on their function is unknown. The use of model systems might facilitate the study of SUMOylation implications in vivo. In the present paper, we have compiled what is known about SUMOylation in Drosophila melanogaster, where the use of genetics provides new insights on SUMOylation's biological roles.
SUMO under stress
Authors: Tempé Denis , Piechaczyk Marc , Bossis Guillaume
Published: October 2008

During the last decade, SUMOylation has emerged as a central regulatory post-translational modification in the control of the fate and function of proteins. However, how SUMOylation is regulated itself has just started to be delineated. It appears now that SUMO (small ubiquitin-related modifier) conjugation/deconjugation equilibrium is affected by various environmental stresses, including osmotic, hypoxic, heat, oxidative and genotoxic stresses. This regulation occurs either at the level of individual targets, through an interplay between stress-induced phosphorylation and SUMOylation, or via modulation of the conjugation/deconjugation machinery abundance or activity. The present review gives an overview of the connections between stress and SUMOylation, the underlying molecular mechanisms and their effects on cellular functions.
Remodelling of the ubiquitin–proteasome system in response to interferons
Authors: Seifert Ulrike , Krüger Elke
Published: October 2008

Peptide generation by the UPS (ubiquitin–proteasome system) is rate-limiting in MHC class I-restricted antigen presentation in response to virus-induced IFNs (interferons). In this process, the role of IFN-induced rapid remodelling of the UPS is less defined. IFN-mediated de novo formation of different proteasome compositions as i20S (immunoproteasomes) or m20S (mixed-type proteasomes) essentially supports the rapid adjustment of the mammalian immune system to pathogens. This adjustment is of particular importance for the immune response to rapidly replicating viruses. In agreement, i20S formation has been shown to be an accelerated and transient response. Moreover, i20S and/or PA28 (proteasome activator 28) are essentially required for the generation of certain viral epitopes. In the present paper, we discuss how IFNs consecutively regulate the UPS at different levels, thereby improving the immune responsiveness of target cells.
Role of nuclear receptors in the modulation of insulin secretion in lipid-induced insulin resistance
Authors: Sugden Mary C. , Holness Mark J.
Published: October 2008

In healthy individuals, a hyperbolic relationship exists between whole-body insulin-sensitivity and insulin secretion. Thus, for any difference in insulin-sensitivity, a reciprocal proportionate change occurs in insulin secretion. Such a feedback loop is evident in healthy individuals ingesting diets high in saturated fat and in late pregnancy where, despite lipid-induced insulin resistance, glucose tolerance is maintained through augmented GSIS (glucose-stimulated insulin secretion). NRs (nuclear receptors) are members of a superfamily of ligand-regulated and orphan transcription factors. On activation by a cognate ligand, many ligand-activated NRs recruit the RXR (retinoid X receptor) for heterodimer formation. Such NRs include the PPARs (peroxisome-proliferator-activated receptors), which are involved in lipid sensing and liporegulation. PPARs exert important lipid-lowering effects in vivo, thereby opposing the development of lipid-induced insulin resistance by relieving the inhibition of insulin-stimulated glucose disposal by muscle and lowering the necessity for augmented GSIS to counter lipid-induced insulin resistance. Long-chain fatty acids are proposed as natural PPAR ligands and some specific endogenous pathways of lipid metabolism are believed to generate PPAR agonists. Other NRs, e.g. the LXR (liver X receptor), which senses expansion of the metabolically active pool of cholesterol, and the FXR (farnesoid X receptor; NR1H4), which, like the LXR, is involved in sterol metabolism, also modulate systemic lipid levels and insulin-sensitivity. In this review, we discuss how these NRs impact insulin secretion via effects on the insulin-sensitivity–insulin secretion feedback loop and, in some cases, via direct effects on the islet itself. In addition, we discuss interactions between these nutrient/metabolite-responsive NRs and NRs that are central to the action of metabolically important hormones, including (i) the glucocorticoid receptor, critical for maintaining glucose homoeostasis in stress, inflammation and during fasting, and (ii) the thyroid hormone receptors, vital for maintenance of oxidative functions. We present data indicating that the RXR occupies a key role in directly modulating islet function and that its heterodimerization with at least two of its partners modulates GSIS.
Glucolipotoxicity of the pancreatic β-cell: myth or reality?
Authors: Poitout Vincent
Published: October 2008

The glucolipotoxicity hypothesis postulates that chronically elevated levels of glucose and fatty acids adversely affect pancreatic β-cell function and thereby contribute to the deterioration of insulin secretion in Type 2 diabetes. Whereas ample experimental evidence in in vitro systems supports the glucolipotoxicity hypothesis, the contribution of this phenomenon to β-cell dysfunction in human Type 2 diabetes has been questioned. The reasons for this controversy include: differences between in vitro systems and in vivo situations; time-dependent effects of fatty acids on insulin secretion (acutely stimulatory and chronically inhibitory); and the ill-defined use of the suffix ‘-toxicity’. In vitro, prolonged exposure of insulin-secreting cells or isolated islets to concomitantly elevated levels of fatty acids and glucose impairs insulin secretion, inhibits insulin gene expression and, under certain circumstances, induces β-cell death by apoptosis. Recent studies in our laboratory have shown that cyclical and alternate infusions of glucose and Intralipid in rats impair insulin gene expression, providing evidence that inhibition of the insulin gene under glucolipotoxic conditions is an early defect that might indeed contribute to β-cell failure in Type 2 diabetes, although this hypothesis remains to be tested in humans.
The diverse roles of protein kinase C in pancreatic β-cell function
Authors: Biden Trevor J. , Schmitz-Peiffer Carsten , Burchfield James G. , Gurisik Ebru , Cantley James , Mitchell Christopher J. , Carpenter Lee
Published: October 2008

Members of the serine/threonine PKC (protein kinase C) family perform diverse functions in multiple cell types. All members of the family are activated in signalling cascades triggered by occupation of cell surface receptors, but the cPKC (conventional PKC) and nPKC (novel PKC) isoforms are also responsive to fatty acid metabolites. PKC isoforms are involved in various aspects of pancreatic β-cell function, including cell proliferation, differentiation and death, as well as regulation of secretion in response to glucose and muscarinic receptor agonists. Recently, the nPKC isoform, PKCϵ, has also been implicated in the loss of insulin secretory responsiveness that underpins the development of Type 2 diabetes.
The sensitivity of pancreatic β-cells to mitochondrial injuries triggered by lipotoxicity and oxidative stress
Authors: Li Ning , Frigerio Francesca , Maechler Pierre
Published: October 2008

Pancreatic β-cells are essential for the maintenance of glucose homoeostasis, and dysfunction of these insulin-secreting cells results in the development of diabetes. In the course of events leading from obesity to Type 2 diabetes, several mechanisms are currently envisaged. Among them, lipids and oxidative stress are considered as toxic candidates for the β-cell. The cellular link between fatty acids and ROS (reactive oxygen species) is essentially the mitochondrion, a key organelle for the control of insulin secretion. Mitochondria are the main source of ROS and are also the primary target of oxidative attacks. The present review presents the current knowledge of lipotoxicity related to oxidative stress in the context of mitochondrial function in the β-cell.
Adipose tissue expandability: the metabolic problems of obesity may arise from the inability to become more obese
Authors: Tan Chong Yew , Vidal-Puig Antonio
Published: October 2008

The prevalence of obesity is increasing and with it the prevalence of associated metabolic complications. Precisely how obesity results in metabolic disturbances remains unclear. In the face of persistent positive caloric balance, it has been postulated that the capacity of adipose tissue to safely store fat may be vital. This paper explores some of the evidence suggesting that the risk of developing metabolic disturbances is not related to how much fat an individual has, but how well their fat can expand to accommodate the caloric excess. If this is true, the metabolic consequences of obesity may arise from the inability to become more obese.
Regulation and consequences of differential gene expression in diabetic kidney disease
Authors: Murphy Madeline , Crean John , Brazil Derek P. , Sadlier Denise , Martin Finian , Godson Catherine
Published: October 2008

DN (diabetic nephropathy) is the leading cause of end-stage renal disease worldwide and develops in 25–40% of patients with Type 1 or Type 2 diabetes mellitus. Elevated blood glucose over long periods together with glomerular hypertension leads to progressive glomerulosclerosis and tubulointerstitial fibrosis in susceptible individuals. Central to the pathology of DN are cytokines and growth factors such as TGF-β (transforming growth factor β) superfamily members, including BMPs (bone morphogenetic protein) and TGF-β1, which play key roles in fibrogenic responses of the kidney, including podocyte loss, mesangial cell hypertrophy, matrix accumulation and tubulointerstitial fibrosis. Many of these responses can be mimicked in in vitro models of cells cultured in high glucose. We have applied differential gene expression technologies to identify novel genes expressed in in vitro and in vivo models of DN and, importantly, in human renal tissue. By mining these datasets and probing the regulation of expression and actions of specific molecules, we have identified novel roles for molecules such as Gremlin, IHG-1 (induced in high glucose-1) and CTGF (connective tissue growth factor) in DN and potential regulators of their bioactions.
Differential regulation of the ER stress response by long-chain fatty acids in the pancreatic β-cell
Authors: Diakogiannaki Eleftheria , Morgan Noel G.
Published: October 2008

Recent evidence indicates that treatment of pancreatic β-cells with long chain fatty acids can lead to the development of an ER (endoplasmic reticulum) stress response. This is manifest as the activation of some components of the PERK [RNA-dependent protein kinase-like ER eIF2α (eukaryotic initiation factor 2α) kinase]-dependent arm of ER stress and is seen most dramatically when cells are treated with long-chain saturated fatty acids (e.g. palmitate). By contrast, the equivalent mono-unsaturates (e.g. palmitoleate) are much less effective and they can even attenuate the ER stress response to palmitate. This may be due to the regulation of eIF2α phosphorylation in cells exposed to mono-unsaturates. The present review discusses the differential effects of saturated and mono-unsaturated fatty acids on ER stress in β-cells and considers the extent to which regulation of this pathway may be involved in mediating their effects on viability.