Showing papers in "Biochemical Journal in 2005"

The ADAMTS metalloproteinases.

Abstract: The ADAMTSs (a disintegrin and metalloproteinase with thrombospondin motifs) are a group of proteases that are found both in mammals and invertebrates. Since the prototype ADAMTS-1 was first described in 1997, there has been a rapidly expanding body of literature describing this gene family and the proteins they encode. The complete human family has 19 ADAMTS genes, together with three members of a newly identified subgroup, the ADAMTSL (ADAMTS-like) proteins, which have several domains in common with the ADAMTSs. The ADAMTSs are extracellular, multidomain enzymes whose known functions include: (i) collagen processing as procollagen N-proteinase; (ii) cleavage of the matrix proteoglycans aggrecan, versican and brevican; (iii) inhibition of angiogenesis; and (iv) blood coagulation homoeostasis as the von Willebrand factor cleaving protease. Roles in organogenesis, inflammation and fertility are also apparent. Recently, some ADAMTS genes have been found to show altered expression in arthritis and various cancers. This review highlights progress in understanding the structural organization and functional roles of the ADAMTSs in normal and pathological conditions.

LEA proteins prevent protein aggregation due to water stress.

Abstract: LEA (late embryogenesis abundant) proteins in both plants and animals are associated with tolerance to water stress resulting from desiccation and cold shock. However, although various functions of LEA proteins have been proposed, their precise role has not been defined. Recent bioinformatics studies suggest that LEA proteins might behave as molecular chaperones, and the current study was undertaken to test this hypothesis. Recombinant forms of AavLEA1, a group 3 LEA protein from the anhydrobiotic nematode Aphelenchus avenae, and Em, a group 1 LEA protein from wheat, have been subjected to functional analysis. Heat-stress experiments with citrate synthase, which is susceptible to aggregation at high temperatures, suggest that LEA proteins do not behave as classical molecular chaperones, but they do exhibit a protective, synergistic effect in the presence of the so-called chemical chaperone, trehalose. In contrast, both LEA proteins can independently protect citrate synthase from aggregation due to desiccation and freezing, in keeping with a role in water-stress tolerance; similar results were obtained with lactate dehydrogenase. This is the first evidence of anti-aggregation activity of LEA proteins due to water stress. Again, a synergistic effect of LEA and trehalose was observed, which is significant given that non-reducing disaccharides are known to accumulate during dehydration in plants and nematodes. A model is proposed whereby LEA proteins might act as a novel form of molecular chaperone, or 'molecular shield', to help prevent the formation of damaging protein aggregates during water stress.

Activation and assembly of the NADPH oxidase: a structural perspective.

Abstract: The NADPH oxidase of professional phagocytes is a crucial component of the innate immune response due to its fundamental role in the production of reactive oxygen species that act as powerful microbicidal agents. The activity of this multi-protein enzyme is dependent on the regulated assembly of the six enzyme subunits at the membrane where oxygen is reduced to superoxide anions. In the resting state, four of the enzyme subunits are maintained in the cytosol, either through auto-inhibitory interactions or through complex formation with accessory proteins that are not part of the active enzyme complex. Multiple inputs are required to disrupt these inhibitory interactions and allow translocation to the membrane and association with the integral membrane components. Protein interaction modules are key regulators of NADPH oxidase assembly, and the protein–protein interactions mediated via these domains have been the target of numerous studies. Many models have been put forward to describe the intricate network of reversible protein interactions that regulate the activity of this enzyme, but an all-encompassing model has so far been elusive. An important step towards an understanding of the molecular basis of NADPH oxidase assembly and activity has been the recent solution of the three-dimensional structures of some of the oxidase components. We will discuss these structures in the present review and attempt to reconcile some of the conflicting models on the basis of the structural information available.

The WNK1 and WNK4 protein kinases that are mutated in Gordon's hypertension syndrome phosphorylate and activate SPAK and OSR1 protein kinases

Abstract: Mutations in the human genes encoding WNK1 [with no K (lysine) protein kinase-1] and the related protein kinase WNK4 are the cause of Gordon's hypertension syndrome. Little is known about the molecular mechanism by which WNK isoforms regulate cellular processes. We immunoprecipitated WNK1 from extracts of rat testis and found that it was specifically associated with a protein kinase of the STE20 family termed 'STE20/SPS1-related proline/alanine-rich kinase' (SPAK). We demonstrated that WNK1 and WNK4 both interacted with SPAK as well as a closely related kinase, termed 'oxidative stress response kinase-1' (OSR1). Wildtype (wt) but not catalytically inactive WNK1 and WNK4 phosphorylated SPAK and OSR1 to a much greater extent than with other substrates utilized previously, such as myelin basic protein and claudin-4. Phosphorylation by WNK1 or WNK4 markedly increased SPAK and OSR1 activity. Phosphopeptide mapping studies demonstrated that WNK1 phosphorylated kinase-inactive SPAK and OSR1 at an equivalent residue located within the T-loop of the catalytic domain (Thr233 in SPAK, Thr185 in OSR1) and a serine residue located within a C-terminal non-catalytic region (Ser373 in SPAK, Ser325 in OSR1). Mutation of Thr185 to alanine prevented the activation of OSR1 by WNK1, whereas mutation of Thr185 to glutamic acid (to mimic phosphorylation) increased the basal activity of OSR1 over 20-fold and prevented further activation by WNK1. Mutation of Ser325 in OSR1 to alanine or glutamic acid did not affect the basal activity of OSR1 or its ability to be activated by WNK1. These findings suggest that WNK isoforms operate as protein kinases that activate SPAK and OSR1 by phosphorylating the T-loops of these enzymes, resulting in their activation. Our analysis also describes the first facile assay that can be employed to quantitatively assess WNK1 and WNK4 activity.

AS160, the Akt substrate regulating GLUT4 translocation, has a functional Rab GTPase-activating protein domain.

Abstract: Recently, we described a 160 kDa protein (designated AS160, for Akt substrate of 160 kDa) with a predicted Rab GAP (GTPase-activating protein) domain that is phosphorylated on multiple sites by the protein kinase Akt. Phosphorylation of AS160 in adipocytes is required for insulin-stimulated translocation of the glucose transporter GLUT4 to the plasma membrane. The aim of the present study was to determine whether AS160 is in fact a GAP for Rabs, and, if so, what its specificity is. We first identified a group of 16 Rabs in a preparation of intracellular vesicles containing GLUT4 by MS. We then prepared the recombinant GAP domain of AS160 and examined its activity against many of these Rabs, as well as several others. The GAP domain was active against Rabs 2A, 8A, 10 and 14. There was no significant activity against 14 other Rabs. GAP activity was further validated by the finding that the recombinant GAP domain with the predicted catalytic arginine residue replaced by lysine was inactive. Finally, it was found by immunoblotting that Rabs 2A, 8A and 14 are present in GLUT4 vesicles. These results indicate that AS160 is a Rab GAP, and suggest novel Rabs that may participate in GLUT4 translocation.

Identification and characterization of pleckstrin-homology-domain-dependent and isoenzyme-specific Akt inhibitors

Abstract: We developed a high-throughput HTRF (homogeneous time-resolved fluorescence) assay for Akt kinase activity and screened approx. 270000 compounds for their ability to inhibit the three isoforms of Akt. Two Akt inhibitors were identified that exhibited isoenzyme specificity. The first compound (Akt-I-1) inhibited only Akt1 (IC50 4.6 microM) while the second compound (Akt-I-1,2) inhibited both Akt1 and Akt2 with IC50 values of 2.7 and 21 microM respectively. Neither compound inhibited Akt3 nor mutants lacking the PH (pleckstrin homology) domain at concentrations up to 250 microM. These compounds were reversible inhibitors, and exhibited a linear mixed-type inhibition against ATP and peptide substrate. In addition to inhibiting kinase activity of individual Akt isoforms, both inhibitors blocked the phosphorylation and activation of the corresponding Akt isoforms by PDK1 (phosphoinositide-dependent kinase 1). A model is proposed in which these inhibitors bind to a site formed only in the presence of the PH domain. Binding of the inhibitor is postulated to promote the formation of an inactive conformation. In support of this model, antibodies to the Akt PH domain or hinge region blocked the inhibition of Akt by Akt-I-1 and Akt-I-1,2. These inhibitors were found to be cell-active and to block phosphorylation of Akt at Thr308 and Ser473, reduce the levels of active Akt in cells, block the phosphorylation of known Akt substrates and promote TRAIL (tumour-necrosis-factor-related apoptosis-inducing ligand)-induced apoptosis in LNCap prostate cancer cells.

Specificity and versatility of SH3 and other proline-recognition domains: structural basis and implications for cellular signal transduction.

Abstract: Protein-protein interactions occurring via the recognition of short peptide sequences by modular interaction domains play a central role in the assembly of signalling protein complexes and larger protein networks that regulate cellular behaviour. In addition to spatial and temporal factors, the specificity of signal transduction is intimately associated with the specificity of many co-operative, pairwise binding events upon which various pathways are built. Although protein interaction domains are usually identified via the recognition code, the consensus sequence motif, to which they selectively bind, they are highly versatile and play diverse roles in the cell. For example, a given interaction domain can bind to multiple sequences that exhibit no apparent identity, and, on the other hand, domains of the same class or different classes may favour a given consensus motif. This promiscuity in ligand selection is typified by the SH3 (Src homology 3) domain and several other interaction modules that commonly recognize proline-rich sequences. Furthermore, interaction domains are highly adaptable, a property that is essential for the evolution of novel pathways and modulation of signalling dynamics. The ability of certain interaction domains to perform multiple tasks, however, poses a challenge for the cell to control signalling specificity when cross-talk between pathways is undesired. Extensive structural and biochemical analysis of many interaction domains in recent years has started to shed light on the molecular basis underlying specific compared with diverse binding events that are mediated by interaction domains and the role affinity plays in affecting domain specificity and regulating cellular signal transduction.

RhoGDI: multiple functions in the regulation of Rho family GTPase activities.

Abstract: RhoGDI (Rho GDP-dissociation inhibitor) was identified as a down-regulator of Rho family GTPases typified by its ability to prevent nucleotide exchange and membrane association. Structural studies on GTPase-RhoGDI complexes, in combination with biochemical and cell biological results, have provided insight as to how RhoGDI exerts its effects on nucleotide binding, the membrane association-dissociation cycling of the GTPase and how these activities are controlled. Despite the initial negative roles attributed to RhoGDI, recent evidence has come to suggest that it may also act as a positive regulator necessary for the correct targeting and regulation of Rho activities by conferring cues for spatial restriction, guidance and availability to effectors. These potential functions are discussed in the context of RhoGDI-associated multimolecular complexes, the newly emerged shuttling capability and the importance of the particular membrane microenvironment that represents the site of action for GTPases. All these results point to a wider role for RhoGDI than initially perceived, making it a binding partner that can tightly control Rho GTPases, but which also allows them to reach their full spectrum of activities.

The basal proton conductance of mitochondria depends on adenine nucleotide translocase content

Abstract: The basal proton conductance of mitochondria causes mild uncoupling and may be an important contributor to metabolic rate. The molecular nature of the proton-conductance pathway is unknown. We show that the proton conductance of muscle mitochondria from mice in which isoform 1 of the adenine nucleotide translocase has been ablated is half that of wild-type controls. Overexpression of the adenine nucleotide translocase encoded by the stress-sensitive B gene in Drosophila mitochondria increases proton conductance, and underexpression decreases it, even when the carrier is fully inhibited using carboxyatractylate. We conclude that half to two-thirds of the basal proton conductance of mitochondria is catalysed by the adenine nucleotide carrier, independently of its ATP/ADP exchange or fatty-acid-dependent proton-leak functions.

Computational modelling of the receptor-tyrosine-kinase-activated MAPK pathway

Abstract: The MAPK (mitogen-activated protein kinase) pathway is one of the most important and intensively studied signalling pathways It is at the heart of a molecular-signalling network that governs the growth, proliferation, differentiation and survival of many, if not all, cell types It is de-regulated in various diseases, ranging from cancer to immunological, inflammatory and degenerative syndromes, and thus represents an important drug target Over recent years, the computational or mathematical modelling of biological systems has become increasingly valuable, and there is now a wide variety of mathematical models of the MAPK pathway which have led to some novel insights and predictions as to how this system functions In the present review we give an overview of the processes involved in modelling a biological system using the popular approach of ordinary differential equations Focusing on the MAPK pathway, we introduce the features and functions of the pathway itself before comparing the available models and describing what new biological insights they have led to

Mammalian Lass6 and its related family members regulate synthesis of specific ceramides

Abstract: The Lass (longevity-assurance homologue) family members, which are highly conserved among eukaryotes, function in ceramide synthesis. In the mouse, there are at least five Lass family members, Lass1, Lass2, Lass4, Lass5 and the hitherto uncharacterized Lass6. To investigate specific roles for each Lass member in ceramide synthesis, we cloned these five mouse proteins. Overproduction of any Lass protein in cultured cells resulted in an increase in cellular ceramide, but the ceramide species produced varied. Overproduction of Lass1 increased C18:0-ceramide levels preferentially, and overproduction of Lass2 and Lass4 increased levels of longer ceramides such as C22:0- and C24:0-ceramides. Lass5 and Lass6 produced shorter ceramide species (C14:0- and C16:0-ceramides); however, their substrate preferences towards saturated/unsaturated fatty acyl-CoA differed. In addition to differences in substrate preferences, we also demonstrated by Northern blotting that Lass family members are differentially expressed among tissues. Additionally, we found that Lass proteins differ with regard to glycosylation. Of the five members, only Lass2, Lass5 and Lass6 were N-glycosylated, each at their N-terminal Asn residue. The occurrence of N-glycosylation of some Lass proteins provides topological insight, indicating that the N-termini of Lass family members probably face the luminal side of the endoplasmic reticulum membrane. Furthermore, based on a proteinase K digestion assay, we demonstrated that the C-terminus of Lass6 faces the cytosolic side of the membrane. From these data we propose topology for the conserved Lag1 motif in Lass family members, namely that the N-terminal region faces the luminal side and the C-terminal region the cytosolic side of the endoplasmic reticulum membrane.

PAK and other Rho-associated kinases--effectors with surprisingly diverse mechanisms of regulation.

Abstract: The Rho GTPases are a family of molecular switches that are critical regulators of signal transduction pathways in eukaryotic cells. They are known principally for their role in regulating the cytoskeleton, and do so by recruiting a variety of downstream effector proteins. Kinases form an important class of Rho effector, and part of the biological complexity brought about by switching on a single GTPase results from downstream phosphorylation cascades. Here we focus on our current understanding of the way in which different Rho-associated serine/threonine kinases, denoted PAK (p21-activated kinase), MLK (mixed-lineage kinase), ROK (Rho-kinase), MRCK (myotonin-related Cdc42-binding kinase), CRIK (citron kinase) and PKN (protein kinase novel), interact with and are regulated by their partner GTPases. All of these kinases have in common an ability to dimerize, and in most cases interact with a variety of other proteins that are important for their function. A diversity of known structures underpin the Rho GTPase-kinase interaction, but only in the case of PAK do we have a good molecular understanding of kinase regulation. The ability of Rho GTPases to co-ordinate spatial and temporal phosphorylation events explains in part their prominent role in eukaryotic cell biology.

CLOCK is involved in the circadian transactivation of peroxisome-proliferator-activated receptor α (PPARα) in mice

Abstract: PPARalpha (peroxisome-proliferator-activated receptor alpha) is a member of the nuclear receptor superfamily of ligand-activated transcription factors that regulate the expression of genes associated with lipid metabolism. In the present study, we show that circadian expression of mouse PPARalpha mRNA requires the basic helix-loop-helix PAS (Per-Arnt-Sim) protein CLOCK, a core component of the negative-feedback loop that drives circadian oscillators in mammals. The circadian expression of PPARalpha mRNA was abolished in the liver of homozygous Clock mutant mice. Using wild-type and Clock-deficient fibroblasts derived from homozygous Clock mutant mice, we showed that the circadian expression of PPARalpha mRNA is regulated by the peripheral oscillators in a CLOCK-dependent manner. Transient transfection and EMSAs (electrophoretic mobility-shift assays) revealed that the CLOCK-BMAL1 (brain and muscle Arnt-like protein 1) heterodimer transactivates the PPARalpha gene via an E-box-rich region located in the second intron. This region contained two perfect E-boxes and four E-box-like motifs within 90 bases. ChIP (chromatin immunoprecipitation) also showed that CLOCK associates with this E-box-rich region in vivo. Circadian expression of PPARalpha mRNA was intact in the liver of insulin-dependent diabetic and of adrenalectomized mice, suggesting that endogenous insulin and glucocorticoids are not essential for the rhythmic expression of the PPARalpha gene. These results suggested that CLOCK plays an important role in lipid homoeostasis by regulating the transcription of a key protein, PPARalpha.

Lutein transport by Caco-2 TC-7 cells occurs partly by a facilitated process involving the scavenger receptor class B type I (SR-BI).

Abstract: The carotenoid lutein is thought to play a role in the human eye and to protect against age-related macular degeneration. Lutein transport in the human intestine has not been characterized. We examined lutein transport processes using Caco-2 TC-7 monolayers as a model for human intestinal epithelium. Purified lutein was mixed with phospholipids, lysophospholipids, cholesterol, mono-olein, oleic acid and taurocholate to obtain lutein-rich mixed micelles that mimicked those found under physiological conditions. The micelles were added to the apical side of Caco-2 TC-7 cell monolayers for 30 min or 3 h at 37 °C. Absorbed lutein, i.e. the sum of lutein recovered in the scraped cells and in the basolateral chamber, was quantified by HPLC. Transport rate was measured (i) as a function of time (from 15 to 60 min), (ii) as a function of micellar lutein concentration (from 1.5 to 15 μM), (iii) at 4 °C, (iv) in the basolateral to apical direction, (v) after trypsin pretreatment, (vi) in the presence of β-carotene and/or lycopene, (vii) in the presence of increasing concentrations of antibody against SR-BI (scavenger receptor class B type 1) and (viii) in the presence of increasing concentrations of a chemical inhibitor of the selective transfer of lipids mediated by SR-BI, i.e. BLT1 (blocks lipid transport 1). The rate of transport of lutein as a function of time and as a function of concentration was saturable. It was significantly lower at 4 °C than at 37 °C (approx. 50%), in the basal to apical direction than in the opposite direction (approx. 85%), and after trypsin pretreatment (up to 45%). Co-incubation with β-carotene, but not lycopene, decreased the lutein absorption rate (approx. 20%) significantly. Anti-SR-BI antibody and BLT1 significantly impaired the absorption rate (approx. 30% and 57% respectively). Overall, these results indicate that lutein absorption is, at least partly, protein-mediated and that some lutein is taken up through SR-BI.

c-Cbl and Cbl-b ubiquitin ligases: substrate diversity and the negative regulation of signalling responses

Abstract: The activation of signalling pathways by ligand engagement with transmembrane receptors is responsible for determining many aspects of cellular function and fate. While these outcomes are initially determined by the nature of the ligand and its receptor, it is also essential that intracellular enzymes, adaptor proteins and transcription factors are correctly assembled to convey the intended response. In recent years, it has become evident that proteins that regulate the amplitude and duration of these signalling responses are also critical in determining the function and fate of cells. Of these, the Cbl family of E3 ubiquitin ligases and adaptor proteins has emerged as key negative regulators of signals from many types of cell-surface receptors. The array of receptors and downstream signalling proteins that are regulated by Cbl proteins is diverse; however, in most cases, the receptors have a common link in that they either possess a tyrosine kinase domain or they form associations with cytoplasmic PTKs (protein tyrosine kinases). Thus Cbl proteins become involved in signalling responses at a time when PTKs are first activated and therefore provide an initial line of defence to ensure that signalling responses proceed at the desired intensity and duration.

Biochemical dysfunction in heart mitochondria exposed to ischaemia and reperfusion.

Abstract: Heart tissue is remarkably sensitive to oxygen deprivation. Although heart cells, like those of most tissues, rapidly adapt to anoxic conditions, relatively short periods of ischaemia and subsequent reperfusion lead to extensive tissue death during cardiac infarction. Heart tissue is not readily regenerated, and permanent heart damage is the result. Although mitochondria maintain normal heart function by providing virtually all of the heart9s ATP, they are also implicated in the development of ischaemic damage. While mitochondria do provide some mechanisms that protect against ischaemic damage (such as an endogenous inhibitor of the F 1 F o -ATPase and antioxidant enzymes), they also possess a range of elements that exacerbate it, including ROS (reactive oxygen species) generators, the mitochondrial permeability transition pore, and their ability to release apoptotic factors. This review considers the process of ischaemic damage from a mitochondrial viewpoint. It considers ischaemic changes in the inner membrane complexes I–V, and how this might affect formation of ROS and high-energy phosphate production/degradation. We discuss the contribution of various mitochondrial cation channels to ionic imbalances which seem to be a major cause of reperfusion injury. The different roles of the H + , Ca 2+ and the various K + channel transporters are considered, particularly the K + ATP (ATP-dependent K + ) channels. A possible role for the mitochondrial permeability transition pore in ischaemic damage is assessed. Finally, we summarize the metabolic and pharmacological interventions that have been used to alleviate the effects of ischaemic injury, highlighting the value of these or related interventions in possible therapeutics.

STAT3-mediated transcription of Bcl-2, Mcl-1 and c-IAP2 prevents apoptosis in polyamine-depleted cells

Abstract: Activation of STAT3 (signal transducer and activator of transcription 3) plays a crucial role in cell survival and proliferation. The aim of the present study was to clarify the role of STAT3 signalling in the protection of polyamine-depleted intestinal epithelial cells against TNF-α (tumour necrosis factor-α)-induced apoptosis. Polyamine depletion by DFMO (α-difluoromethylornithine) caused phosphorylation of STAT3 at Tyr-705 and Ser-727. Phospho-Tyr-705 STAT3 was immunolocalized at the cell periphery and nucleus, whereas phospho-Ser-727 STAT3 was predominantly detected in the nucleus of polyamine-depleted cells. Sustained phosphorylation of STAT3 at tyrosine residues was observed in polyamine-depleted cells after exposure to TNF-α. Inhibition of STAT3 activation by AG490 or cell-membrane-permeant inhibitory peptide (PpYLKTK; where pY represents phospho-Tyr) increased the sensitivity of polyamine-depleted cells to apoptosis. Expression of DN-STAT3 (dominant negative-STAT3) completely eliminated the protective effect of DFMO against TNF-α-induced apoptosis. Polyamine depletion increased mRNA and protein levels for Bcl-2, Mcl-1 (myeloid cell leukaemia-1) and c-IAP2 (inhibitor of apoptosis protein-2). Significantly higher levels of Bcl-2 and c-IAP2 proteins were observed in polyamine-depleted cells before and after 9 h of TNF-α treatment. Inhibition of STAT3 by AG490 and DN-STAT3 decreased Bcl-2 promoter activity. DN-STAT3 decreased mRNA and protein levels for Bcl-2, Mcl-1 and c-IAP2 in polyamine-depleted cells. siRNA (small interfering RNA)-mediated inhibition of Bcl-2, Mcl-1 and c-IAP2 protein levels increased TNF-α-induced apoptosis. DN-STAT3 induced the activation of caspase-3 and PARP [poly(ADP-ribose) polymerase] cleavage in polyamine-depleted cells. These results suggest that activation of STAT3 in response to polyamine depletion increases the transcription and subsequent expression of anti-apoptotic Bcl-2 and IAP family proteins and thereby promotes survival of cells against TNF-α-induced apoptosis.

Characterization of chito -oligosaccharides prepared by chitosanolysis with the aid of papain and Pronase, and their bactericidal action against Bacillus cereus and Escherichia coli

Abstract: Papain (from papaya latex; EC 3.4.22.2) and Pronase (from Streptomyces griseus; EC 3.4.24.31) caused optimum depolymerization of chitosan at pH 3.5 and 37 degrees C, resulting in LMMC (low molecular mass chitosan) and chito-oligomeric-monomeric mixture. The yield of the latter was 14-16% and 14-19% respectively for papain- and Pronase-catalysed reactions, depending on the reaction time (1-5 h). HPLC revealed the presence of monomer(s) and oligomers of DP (degree of polymerization) 2-6, which was also confirmed by matrix-assisted laser-desorption ionization-time-of-flight MS. Along with the chito-oligomers, the appearance of only GlcNAc (N-acetylglucosamine) in Pronase-catalysed chitosanolysis was indicative of its different action pattern compared with papain. Fourier-transform infrared, liquid-state 13C-NMR spectra and CD analyses of chito-oligomeric-monomeric mixture indicated the release of GlcNAc/GlcNAc-rich oligomers. The monomeric sequence at the non-reducing ends of chito-oligomers was elucidated using N-acetylglucosaminidase. The chito-oligomeric-monomeric mixture showed better growth inhibitory activity towards Bacillus cereus and Escherichia coli compared with native chitosan. Optimum growth inhibition was observed with chito-oligomers of higher DP having low degree of acetylation. The latter caused pore formation and permeabilization of the cell wall of B. cereus, whereas blockage of nutrient flow due to the aggregation of chito-oligomers-monomers was responsible for the growth inhibition and lysis of E. coli, which were evidenced by scanning electron microscopy analysis. The spillage of cytoplasmic enzymes and native PAGE of the cell-free supernatant of B. cereus treated with chito-oligomeric-monomeric mixture further confirmed bactericidal activity of the latter. Use of papain and Pronase, which are inexpensive and easily available, for chitosanolysis, is of commercial importance, as the products released are of considerable biomedical value.

Identification of a functional hypoxia-responsive element that regulates the expression of the egl nine homologue 3 (egln3/phd3) gene

Abstract: Low oxygen levels induce an adaptive response in cells through the activation of HIFs (hypoxia-inducible factors) These transcription factors are mainly regulated by a group of proline hydroxylases that, in the presence of oxygen, target HIF for degradation The expression of two such enzymes, EGLN1 [EGL nine homologous protein 1, where EGL stands for egg laying defective (Caenorhabditis elegans gene)] and EGLN3, is induced by hypoxia through a negative feedback loop, and we have demonstrated recently that hypoxic induction of EGLN expression is HIF-dependent In the present study, we have identified an HRE (hypoxia response element) in the region of the EGLN3 gene using a combination of bioinformatics and biological approaches Initially, we isolated a number of HRE consensus sequences in a region of 40 kb around the human EGLN3 gene and studied their evolutionary conservation Subsequently, we examined the functionality of the conserved HRE sequences in reporter and chromatin precipitation assays One of the HREs, located within a conserved region of the first intron of the EGLN3 gene 12 kb downstream of the transcription initiation site, bound HIF in vivo Furthermore, this sequence was able to drive reporter gene expression under conditions of hypoxia in an HRE-dependent manner Indeed, we were able to demonstrate that HIF was necessary and sufficient to induce gene expression from this enhancer sequence

Myricetin, quercetin and catechin-gallate inhibit glucose uptake in isolated rat adipocytes

Abstract: The facilitative glucose transporter, GLUT4, mediates insulin-stimulated glucose uptake in adipocytes and muscles, and the participation of GLUT4 in the pathogenesis of various clinical conditions associated with obesity, visceral fat accumulation and insulin resistance has been proposed. Glucose uptake by some members of the GLUT family, mainly GLUT1, is inhibited by flavonoids, the natural polyphenols present in fruits, vegetables and wine. Therefore it is of interest to establish if these polyphenolic compounds present in the diet, known to be effective antioxidants but also endowed with several other biological activities such as protein-tyrosine kinase inhibition, interfere with GLUT4 function. In the present study, we show that three flavonoids, quercetin, myricetin and catechin-gallate, inhibit the uptake of methylglucose by adipocytes over the concentration range of 10-100 microM. These three flavonoids show a competitive pattern of inhibition, with K(i)=16, 33.5 and 90 microM respectively. In contrast, neither catechin nor gallic acid inhibit methylglucose uptake. To obtain a better understanding of the interaction among GLUT4 and flavonoids, we have derived a GLUT4 three-dimensional molecular comparative model, using structural co-ordinates from a GLUT3 comparative model and a mechanosensitive ion channel [PDB (Protein Data Bank) code 1MSL] solved by X-ray diffraction. On the whole, the experimental evidence and computer simulation data favour a transport inhibition mechanism in which flavonoids and GLUT4 interact directly, rather than by a mechanism related to protein-tyrosine kinase and insulin signalling inhibition. Furthermore, the results suggest that GLUT transporters are involved in flavonoid incorporation into cells.

Post-translationally modified residues of native human osteopontin are located in clusters: identification of 36 phosphorylation and five O-glycosylation sites and their biological implications.

Abstract: OPN (osteopontin) is an integrin-binding highly phosphorylated glycoprotein, recognized as a key molecule in a multitude of biological processes such as bone mineralization, cancer metastasis, cell-mediated immune response, inflammation and cell survival. A significant regulation of OPN function is mediated through PTM (post-translational modification). Using a combination of Edman degradation and MS analyses, we have characterized the complete phosphorylation and glycosylation pattern of native human OPN. A total of 36 phosphoresidues have been localized in the sequence of OPN. There are 29 phosphorylations (Ser 8 , Ser 1 0 , Ser 1 1 , Ser 4 6 , Ser 4 7 , Thr 5 0 , Ser 6 0 , Ser 6 2 , Ser 6 5 , Ser 8 3 , Ser 8 6 , Ser 8 9 , Ser 9 2 , Ser 1 0 4 , Ser 1 1 0 , Ser 1 1 3 , Thr 1 6 9 , Ser 1 7 9 , Ser 2 0 8 , Ser 2 1 8 , Ser 2 3 8 , Ser 2 4 7 , Ser 2 5 4 , Ser 2 5 9 , Ser 2 6 4 , Ser 2 7 5 , Ser 2 8 7 , Ser 2 9 2 and Ser 2 9 4 ) located in the target sequence of MGCK (mammary gland casein kinase) also known as the Golgi kinase (S/T-X-E/S(P)/D). Six phosphorylations (Ser 1 0 1 , Ser 1 0 7 , Ser 1 7 5 , Ser 1 9 9 , Ser 2 1 2 and Ser 2 5 1 ) are located in the target sequence of CKII (casein kinase II) [S-X-X-E/S(P)/D] and a single phosphorylation, Ser 2 0 3 , is not positioned in the motif of either MGCK or CKII. The 36 phosphoresidues represent the maximal degree of modification since variability at many sites was seen. Five threonine residues are O-glycosylated (Thr 1 1 8 , Thr 1 2 2 , Thr 1 2 7 , Thr 1 3 1 and Thr 1 3 6 ) and two potential sites for N-glycosylation (Asn 6 3 and Asn 9 0 ) are not occupied in human milk OPN. The phosphorylations are arranged in clusters of three to five phosphoresidues and the regions containing the glycosylations and the RGD (Arg-Gly-Asp) integrin-binding sequence are devoid of phosphorylations. Knowledge about the positions and nature of PTMs in OPN will allow a rational experimental design of functional studies aimed at understanding the structural and functional interdependences in diverse biological processes in which OPN is a key molecule.

The G0/G1 switch gene 2 is a novel PPAR target gene

Abstract: PPARs (peroxisome-proliferator-activated receptors) alpha, beta/delta and gamma are a group of transcription factors that are involved in numerous processes, including lipid metabolism and adipogenesis By comparing liver mRNAs of wild-type and PPARalpha-null mice using microarrays, a novel putative target gene of PPARalpha, G0S2 (G0/G1 switch gene 2), was identified Hepatic expression of G0S2 was up-regulated by fasting and by the PPARalpha agonist Wy14643 in a PPARalpha-dependent manner Surprisingly, the G0S2 mRNA level was highest in brown and white adipose tissue and was greatly up-regulated during mouse 3T3-L1 and human SGBS (Simpson-Golabi-Behmel syndrome) adipogenesis Transactivation, gel shift and chromatin immunoprecipitation assays indicated that G0S2 is a direct PPARgamma and probable PPARalpha target gene with a functional PPRE (PPAR-responsive element) in its promoter Up-regulation of G0S2 mRNA seemed to be specific for adipogenesis, and was not observed during osteogenesis or myogenesis In 3T3-L1 fibroblasts, expression of G0S2 was associated with growth arrest, which is required for 3T3-L1 adipogenesis Together, these data indicate that G0S2 is a novel target gene of PPARs that may be involved in adipocyte differentiation

Phylogenetic analysis of the phytochrome superfamily reveals distinct microbial subfamilies of photoreceptors

Abstract: Phys (phytochromes) are a superfamily of photochromic photoreceptors that employ a bilin-type chromophore to sense red and far-red light. Although originally thought to be restricted to plants, accumulating genetic and genomic analyses now indicate that they are also prevalent among micro-organisms. By a combination of phylogenetic and biochemical studies, we have expanded the Phy superfamily and organized its members into distinct functional clades which include the phys (plant Phys), BphPs (bacteriophytochromes), Cphs (cyanobacterial Phys), Fphs (fungal Phys) and a collection of Phy-like sequences. All contain a signature GAF (cGMP phosphodiesterase/adenylate cyclase/FhlA) domain, which houses the bilin lyase activity. A PHY domain (uppercase letters are used to denote the PHY domain specifically), which helps stabilize the Pfr form (far-red-light-absorbing form of Phy), is downstream of the GAF region in all but the Phy-like sequences. The phy, Cph, BphP and Fph families also include a PLD [N-terminal PAS (Per/Arnt/Sim)-like domain] upstream of the GAF domain. Site-directed mutagenesis of conserved residues within the GAF and PLD motifs supports their importance in chromophore binding and/or spectral activity. In agreement with Lamparter, Carrascal, Michael, Martinez, Rottwinkel and Abian [(2004) Biochemistry 43, 3659-3669], a conserved cysteine within the PLD of several BphPs was found to be necessary for binding the chromophore via the C-3 vinyl side chain on the bilin A ring. Phy-type sequences were also discovered in the actinobacterium Kineococcus radiotolerans and collections of microorganisms obtained from marine and extremely acidic environments, thus expanding further the range of these photoreceptors. Based on their organization and distribution, the evolution of the Phy superfamily into distinct photoreceptor types is proposed.

Oligomeric states of the voltage-dependent anion channel and cytochrome c release from mitochondria.

Abstract: The VDAC (voltage-dependent anion channel) plays a central role in apoptosis, participating in the release of apoptogenic factors including cytochrome c. The mechanisms by which VDAC forms a protein-conducting channel for the passage of cytochrome c are not clear. The present study approaches this problem by addressing the oligomeric status of VDAC and its role in the induction of the permeability transition pore and cytochrome c release. Chemical cross-linking of isolated mitochondria or purified VDAC with five different reagents proved that VDAC exists as dimers, trimers or tetramers. Fluorescence resonance energy transfer between fluorescently labelled VDACs supports the concept of dynamic VDAC oligomerization. Mitochondrial cross-linking prevented both permeability transition pore opening and release of cytochrome c, yet had no effect on electron transport or Ca2+ uptake. Bilayer-reconstituted purified cross-linked VDAC showed decreased conductance and voltage-independent channel activity. In the dithiobis(succinimidyl propionate)-cross-linked VDAC, these channel properties could be reverted to those of the native VDAC by cleavage of the cross-linking. Cross-linking of VDAC reconstituted into liposomes inhibited the release of the proteoliposome-encapsulated cytochrome c. Moreover, encapsulated, but not soluble cytochrome c induced oligomerization of liposome-reconstituted VDAC. Thus the results indicate that VDAC exists in a dynamic equilibrium between dimers and tetramers and suggest that oligomeric VDAC may be involved in mitochondria-mediated apoptosis.

Structure and function of steroid receptor AF1 transactivation domains: induction of active conformations

Abstract: Steroid hormones are important endocrine signalling molecules controlling reproduction, development, metabolism, salt balance and specialized cellular responses, such as inflammation and immunity. They are lipophilic in character and act by binding to intracellular receptor proteins. These receptors function as ligand-activated transcription factors, switching on or off networks of genes in response to a specific hormone signal. The receptor proteins have a conserved domain organization, comprising a C-terminal LBD (ligand-binding domain), a hinge region, a central DBD (DNA-binding domain) and a highly variable NTD (N-terminal domain). The NTD is structurally flexible and contains surfaces for both activation and repression of gene transcription, and the strength of the transactivation response has been correlated with protein length. Recent evidence supports a structural and functional model for the NTD that involves induced folding, possibly involving α-helix structure, in response to protein–protein interactions and structure-stabilizing solutes.

Ras plasma membrane signalling platforms.

Abstract: The plasma membrane is a complex, dynamic structure that provides platforms for the assembly of many signal transduction pathways. These platforms have the capacity to impose an additional level of regulation on cell signalling networks. In this review, we will consider specifically how Ras proteins interact with the plasma membrane. The focus will be on recent studies that provide novel spatial and dynamic insights into the micro-environments that different Ras proteins utilize for signal transduction. We will correlate these recent studies suggesting Ras proteins might operate within a heterogeneous plasma membrane with earlier biochemical work on Ras signal transduction.

The family feud: turning off Sp1 by Sp1-like KLF proteins

Abstract: Sp1 is one of the best characterized transcriptional activators. The biological importance of Sp1 is underscored by the fact that several hundreds of genes are thought to be regulated by this protein. However, during the last 5 years, a more extended family of Sp1-like transcription factors has been identified and characterized by the presence of a conserved DNA-binding domain comprising three Kruppel-like zinc fingers. Each distinct family member differs in its ability to regulate transcription, and, as a consequence, to influence cellular processes. Specific activation and repression domains located within the N-terminal regions of these proteins are responsible for these differences by facilitating interactions with various co-activators and co-repressors. The present review primarily focuses on discussing the structural, biochemical and biological functions of the repressor members of this family of transcription factors. The existence of these transcriptional repressors provides a tightly regulated mechanism for silencing a large number of genes that are already known to be activated by Sp1.

Regulation of the prolyl hydroxylase domain protein 2 (phd2/egln-1) gene: identification of a functional hypoxia-responsive element

Abstract: The HIFs (hypoxia-inducible factors) are a family of heterodimeric transcription factors essential for the adaptation of cells to reduced oxygen supply. Three human PHDs (prolyl hydroxylase domain proteins, PHD1–PHD3) initiate oxygen-dependent degradation of HIF-α-subunits in normoxia. RNA interference directed against PHD2, but not PHD1 or PHD3, is sufficient to stabilize HIF-1α in normoxia. Therefore PHD2 is regarded as the main cellular oxygen sensor. PHD2 itself is up-regulated by hypoxia and may thus limit hypoxic signalling. By sequence analysis, we predicted a promoter approx. 3.5 kb 5′ of the translation start codon and a second promoter located in a CpG island immediately upstream of the coding sequence. A consensus HIF-1-binding site that is conserved in the murine phd2 gene was detected in the CpG island. By electrophoretic mobility-shift assay, we demonstrated binding of HIF-1 to the putative HIF-1-binding site. In luciferase reporter vectors, the isolated upstream promoter was inactive in all cell lines tested unless 200 bp were deleted at the 3′-end. The downstream promoter was active and induced by hypoxia. In reporter vectors containing both promoter sequences, luciferase activity was equal to vectors containing only the downstream promoter. In cells transfected with a vector containing both promoters, a single luciferase transcript was detectable. This transcript had the same length as transcripts from a vector containing the downstream promoter only. We conclude that the phd2 gene is transcribed exclusively from the downstream promoter that contains a functional hypoxia-responsive, cis-regulatory element. Our results establish that PHD2 is a direct HIF target gene.

The transcriptional co-activator TAZ interacts differentially with transcriptional enhancer factor-1 (TEF-1) family members

Abstract: Members of the highly related TEF-1 (transcriptional enhancer factor-1) family (also known as TEAD, for TEF-1, TEC1, ABAA domain) bind to MCAT (muscle C, A and T sites) and A/T-rich sites in promoters active in cardiac, skeletal and smooth muscle, placenta, and neural crest. TEF-1 activity is regulated by interactions with transcriptional co-factors [p160, TONDU (Vgl-1, Vestigial-like protein-1), Vgl-2 and YAP65 (Yes-associated protein 65 kDa)]. The strong transcriptional co-activator YAP65 interacts with all TEF-1 family members, and, since YAP65 is related to TAZ (transcriptional co-activator with PDZ-binding motif), we wanted to determine if TAZ also interacts with members of the TEF-1 family. In the present study, we show by GST (glutathione S-transferase) pull-down assays, by co-immunoprecipitation and by modified mammalian two-hybrid assays that TEF-1 interacts with TAZ in vitro and in vivo. Electrophoretic mobility-shift assays with purified TEF-1 and GST-TAZ fusion protein showed that TAZ interacts with TEF-1 bound to MCAT DNA. TAZ can interact with endogenous TEF-1 proteins, since exogenous TAZ activated MCAT-dependent reporter promoters. Like YAP65, TAZ interacted with all four TEF-1 family members. GST pull-down assays with increasing amounts of [35S]TEF-1 and [35S]RTEF-1 (related TEF-1) showed that TAZ interacts more efficiently with TEF-1 than with RTEF-1. This differential interaction also extended to the interaction of TEF-1 and RTEF-1 with TAZ in vivo, as assayed by a modified mammalian two-hybrid experiment. These data show that differential association of TEF-1 proteins with transcriptional co-activators may regulate the activity of TEF-1 family members.

NEDD4-2 (neural precursor cell expressed, developmentally down-regulated 4-2) negatively regulates TGF-β (transforming growth factor-β) signalling by inducing ubiquitin-mediated degradation of Smad2 and TGF-β type I receptor

Abstract: Inhibitory Smad, Smad7, is a potent inhibitor of TGF-β (transforming growth factor-β) superfamily signalling. By binding to activated type I receptors, it prevents the activation of R-Smads (receptor-regulated Smads). To identify new components of the Smad pathway, we performed yeast two-hybrid screening using Smad7 as bait, and identified NEDD4-2 (neural precursor cell expressed, developmentally down-regulated 4-2) as a direct binding partner of Smad7. NEDD4-2 is structurally similar to Smurfs (Smad ubiquitin regulatory factors) 1 and 2, which were identified previously as E3 ubiquitin ligases for R-Smads and TGF-β superfamily receptors. NEDD4-2 functions like Smurfs 1 and 2 in that it associates with TGF-β type I receptor via Smad7, and induces its ubiquitin-dependent degradation. Moreover, NEDD4-2 bound to TGF-β-specific R-Smads, Smads 2 and 3, in a ligand-dependent manner, and induced degradation of Smad2, but not Smad3. However, in contrast with Smurf2, NEDD4-2 failed to induce ubiquitination of SnoN (Ski-related novel protein N), although NEDD4-2 bound to SnoN via Smad2 more strongly than Smurf2. We showed further that overexpressed NEDD4-2 prevents transcriptional activity induced by TGF-β and BMP, whereas silencing of the NEDD4-2 gene by siRNA (small interfering RNA) resulted in enhancement of the responsiveness to TGF-β superfamily cytokines. These data suggest that NEDD4-2 is a member of the Smurf-like C2-WW-HECT (WW is Trp-Trp and HECT is homologous to the E6-accessory protein) type E3 ubiquitin ligases, which negatively regulate TGF-β superfamily signalling through similar, but not identical, mechanisms to those used by Smurfs.