Showing papers in "Biochemical Journal in 2010"
TL;DR: The molecular mechanisms underlying p38MAPK functions are dissected, with special emphasis on the activation and regulation of the core kinases, the interplay with other signalling pathways and the nature of p38 MAPK substrates as a source of functional diversity.
Abstract: The p38 MAPK (mitogen-activated protein kinase) signalling pathway allows cells to interpret a wide range of external signals and respond appropriately by generating a plethora of different biological effects. The diversity and specificity in cellular outcomes is achieved with an apparently simple linear architecture of the pathway, consisting of a core of three protein kinases acting sequentially. In the present review, we dissect the molecular mechanisms underlying p38 MAPK functions, with special emphasis on the activation and regulation of the core kinases, the interplay with other signalling pathways and the nature of p38 MAPK substrates as a source of functional diversity. Finally, we discuss how genetic mouse models are facilitating the identification of physiological functions for p38 MAPKs, which may impinge on their eventual use as therapeutic targets.
1,375 citations
TL;DR: The SUMOylation pathway is briefly described and an overview of the recent findings that are beginning to identify some of the mechanisms that regulate protein SUMoylation are presented.
Abstract: The post-translational modification SUMOylation is a major regulator of protein function that plays an important role in a wide range of cellular processes. SUMOylation involves the covalent attachment of a member of the SUMO (small ubiquitin-like modifier) family of proteins to lysine residues in specific target proteins via an enzymatic cascade analogous to, but distinct from, the ubiquitination pathway. There are four SUMO paralogues and an increasing number of proteins are being identified as SUMO substrates. However, in many cases little is known about how SUMOylation of these targets is regulated. Compared with the ubiquitination pathway, relatively few components of the conjugation machinery have been described and the processes that specify individual SUMO paralogue conjugation to defined substrate proteins are an active area of research. In the present review, we briefly describe the SUMOylation pathway and present an overview of the recent findings that are beginning to identify some of the mechanisms that regulate protein SUMOylation.
604 citations
TL;DR: The current literature is reviewed to examine hnRNP diversity, and how this impacts upon approaches to the classification of RNA-binding proteins in general is discussed.
Abstract: The hnRNPs (heterogeneous nuclear ribonucleoproteins) are RNA-binding proteins with important roles in multiple aspects of nucleic acid metabolism, including the packaging of nascent transcripts, alternative splicing and translational regulation. Although they share some general characteristics, they vary greatly in terms of their domain composition and functional properties. Although the traditional grouping of the hnRNPs as a collection of proteins provided a practical framework, which has guided much of the research on them, this approach is becoming increasingly incompatible with current knowledge about their structural and functional divergence. Hence, we review the current literature to examine hnRNP diversity, and discuss how this impacts upon approaches to the classification of RNA-binding proteins in general.
473 citations
TL;DR: Mitochondria are a major source of hydrogen peroxide, and this oxidant is implicated in the damage associated with aging and a number of pathologies, and the extent of this role and the mechanisms involved are currently unclear.
Abstract: Prxs (peroxiredoxins) are a family of proteins that are extremely effective at scavenging peroxides. The Prxs exhibit a number of intriguing properties that distinguish them from conventional antioxidants, including a susceptibility to inactivation by hyperoxidation in the presence of excess peroxide and the ability to form complex oligomeric structures. These properties, combined with a high cellular abundance and reactivity with hydrogen peroxide, have led to speculation that the Prxs function as redox sensors that transmit signals as part of the cellular response to oxidative stress. Multicellular organisms express several different Prxs that can be categorized by their subcellular distribution. In mammals, Prx 3 and Prx 5 are targeted to the mitochondrial matrix. Mitochondria are a major source of hydrogen peroxide, and this oxidant is implicated in the damage associated with aging and a number of pathologies. Hydrogen peroxide can also act as a second messenger, and is linked with signalling events in mitochondria, including the induction of apoptosis. A simple kinetic competition analysis estimates that Prx 3 will be the target for up to 90% of hydrogen peroxide generated in the matrix. Therefore, mitochondrial Prxs have the potential to play a major role in mitochondrial redox signalling, but the extent of this role and the mechanisms involved are currently unclear.
441 citations
TL;DR: Recent advances in research on CaMs and CMLs, CDPKs and CBLs are discussed, and the current knowledge on the different sensor classes into common physiological themes are integrated.
Abstract: Ca2+ ions play a vital role as second messengers in plant cells during various developmental processes and in response to environmental stimuli. Plants have evolved a diversity of unique proteins that bind Ca2+ using the evolutionarily conserved EF-hand motif. The currently held hypothesis is that these proteins function as Ca2+ sensors by undergoing conformational changes in response to Ca2+-binding that facilitate their regulation of target proteins and thereby co-ordinate various signalling pathways. The three main classes of these EF-hand Ca2+sensors in plants are CaMs [calmodulins; including CMLs (CaM-like proteins)], CDPKs (calcium-dependent protein kinases) and CBLs (calcineurin B-like proteins). In the plant species examined to date, each of these classes is represented by a large family of proteins, most of which have not been characterized biochemically and whose physiological roles remain unclear. In the present review, we discuss recent advances in research on CaMs and CMLs, CDPKs and CBLs, and we attempt to integrate the current knowledge on the different sensor classes into common physiological themes.
427 citations
TL;DR: The results suggest that mutation of Ser910 and/or Ser935 to disrupt 14-3-3 binding does not affect intrinsic protein kinase activity, but induces LRRK2 to accumulate within discrete cytoplasmic pools, perhaps resembling inclusion bodies.
Abstract: LRRK2 (leucine-rich repeat protein kinase 2) is mutated in a significant number of Parkinson's disease patients, but still little is understood about how it is regulated or functions. In the present study we have demonstrated that 14-3-3 protein isoforms interact with LRRK2. Consistent with this, endogenous LRRK2 isolated from Swiss 3T3 cells or various mouse tissues is associated with endogenous 14-3-3 isoforms. We have established that 14-3-3 binding is mediated by phosphorylation of LRRK2 at two conserved residues (Ser910 and Ser935) located before the leucine-rich repeat domain. Our results suggests that mutation of Ser910 and/or Ser935 to disrupt 14-3-3 binding does not affect intrinsic protein kinase activity, but induces LRRK2 to accumulate within discrete cytoplasmic pools, perhaps resembling inclusion bodies. To investigate links between 14-3-3 binding and Parkinson's disease, we studied how 41 reported mutations of LRRK2 affected 14-3-3 binding and cellular localization. Strikingly, we found that five of the six most common pathogenic mutations (R1441C, R1441G, R1441H, Y1699C and I2020T) display markedly reduced phosphorylation of Ser910/Ser935 thereby disrupting interaction with 14-3-3. We have also demonstrated that Ser910/Ser935 phosphorylation and 14-3-3 binding to endogenous LRRK2 is significantly reduced in tissues of homozygous LRRK2(R1441C) knock-in mice. Consistent with 14-3-3 regulating localization, all of the common pathogenic mutations displaying reduced 14-3-3-binding accumulated within inclusion bodies. We also found that three of the 41 LRRK2 mutations analysed displayed elevated protein kinase activity (R1728H, ~2-fold; G2019S, ~3-fold; and T2031S, ~4-fold). These results provide the first evidence suggesting that 14-3-3 regulates LRRK2 and that disruption of the interaction of LRRK2 with 14-3-3 may be linked to Parkinson's disease.
367 citations
TL;DR: The function of cell-death modifiers have considerably broadened over the last few years, and these molecules are increasingly recognized as arbiters of cellular homoeostasis, from cell division, to intracellular signalling to cellular adaptation.
Abstract: From the realization that cell number homoeostasis is fundamental to the biology of all metazoans, and that deregulation of this process leads to human diseases, enormous interest has been devoted over the last two decades to map the requirements of cell death and cell survival. This effort has led to tangible progress, and we can now chart with reasonable accuracy complex signalling circuitries controlling cell-fate decisions. Some of this knowledge has translated into novel therapeutics, and the outcome of these strategies, especially in cancer, is eagerly awaited. However, the function of cell-death modifiers have considerably broadened over the last few years, and these molecules are increasingly recognized as arbiters of cellular homoeostasis, from cell division, to intracellular signalling to cellular adaptation. This panoply of functions is best exemplified by members of the IAP (inhibitor of apoptosis) gene family, molecules originally narrowly defined as endogenous caspase inhibitors, but now firmly positioned at the crossroads of multiple normal and transformed cellular responses.
361 citations
TL;DR: Treatment of Swiss 3T3 cells or lymphoblastoid cells derived from control or a Parkinson's disease patient harbouring a homozygous LRRK2(G2019S) mutation with two structurally unrelated inhibitors of L RRK2 induced dephosphorylation of endogenous LRB2 at Ser910 and Ser935, thereby disrupting 14-3-3 interaction.
Abstract: LRRK2 (leucine-rich repeat protein kinase 2) is mutated in a significant number of Parkinson's disease patients. Since a common mutation that replaces Gly2019 with a serine residue enhances kinase catalytic activity, small-molecule LRRK2 inhibitors might have utility in treating Parkinson's disease. However, the effectiveness of inhibitors is difficult to assess, as no physiological substrates or downstream effectors have been identified that could be exploited to develop a robust cell-based assay. We recently established that LRRK2 bound 14-3-3 protein isoforms via its phosphorylation of Ser910 and Ser935. In the present study we show that treatment of Swiss 3T3 cells or lymphoblastoid cells derived from control or a Parkinson's disease patient harbouring a homozygous LRRK2(G2019S) mutation with two structurally unrelated inhibitors of LRRK2 (H-1152 or sunitinib) induced dephosphorylation of endogenous LRRK2 at Ser910 and Ser935, thereby disrupting 14-3-3 interaction. Our results suggest that H-1152 and sunitinib induce dephosphorylation of Ser910 and Ser935 by inhibiting LRRK2 kinase activity, as these compounds failed to induce significant dephosphorylation of a drug-resistant LRRK2(A2016T) mutant. Moreover, consistent with the finding that non-14-3-3-binding mutants of LRRK2 accumulated within discrete cytoplasmic pools resembling inclusion bodies, we observed that H-1152 causes LRRK2 to accumulate within inclusion bodies. These findings indicate that dephosphorylation of Ser910/Ser935, disruption of 14-3-3 binding and/or monitoring LRRK2 cytoplasmic localization can be used as an assay to assess the relative activity of LRRK2 inhibitors in vivo. These results will aid the elaboration and evaluation of LRRK2 inhibitors. They will also stimulate further research to understand how phosphorylation of Ser910 and Ser935 is controlled by LRRK2, and establish any relationship to development of Parkinson's disease.
359 citations
TL;DR: The present review discusses the molecular mechanisms by which insulin signalling regulates metabolism in Drosophila, comparing and contrasting with the mammalian system.
Abstract: The insulin signalling pathway is highly conserved from mammals to Drosophila. Insulin signalling in the fly, as in mammals, regulates a number of physiological functions, including carbohydrate and lipid metabolism, tissue growth and longevity. In the present review, I discuss the molecular mechanisms by which insulin signalling regulates metabolism in Drosophila, comparing and contrasting with the mammalian system. I discuss both the intracellular signalling network, as well as the communication between organs in the fly.
342 citations
TL;DR: In silico analyses show that the NAC TRDs contain group-specific sequence motifs and are characterized by a high degree of intrinsic disorder, and demonstrate that the biochemical and functional specificity of NAC TFs is associated with both the DBDs and the TRDs.
Abstract: TFs (transcription factors) are modular proteins minimally containing a DBD (DNA-binding domain) and a TRD (transcription regulatory domain). NAC [for NAM (no apical meristem), ATAF, CUC (cup-shaped cotyledon)] proteins comprise one of the largest plant TF families. They are key regulators of stress perception and developmental programmes, and most share an N-terminal NAC domain. On the basis of analyses of gene expression data and the phylogeny of Arabidopsis thaliana NAC TFs we systematically decipher structural and functional specificities of the conserved NAC domains and the divergent C-termini. Nine of the ten NAC domains analysed bind a previously identified conserved DNA target sequence with a CGT[GA] core, although with different affinities. Likewise, all but one of the NAC proteins analysed is dependent on the C-terminal region for transactivational activity. In silico analyses show that the NAC TRDs contain group-specific sequence motifs and are characterized by a high degree of intrinsic disorder. Furthermore, ANAC019 was identified as a new positive regulator of ABA (abscisic acid) signalling, conferring ABA hypersensitivity when ectopically expressed in plants. Interestingly, ectopic expression of the ANAC019 DBD or TRD alone also resulted in ABA hypersensitivity. Expression of stress-responsive marker genes [COR47 (cold-responsive 47), RD29b (responsive-to-desiccation 29b) and ERD11 (early-responsive-to-dehydration 11)] were also induced by full-length and truncated ANAC019. Domain-swapping experiments were used to analyse the specificity of this function. Chimaeric proteins, where the NAC domain of ANAC019 was replaced with the analogous regions from other NAC TFs, also have the ability to positively regulate ABA signalling. In contrast, replacing the ANAC019 TRD with other TRDs abolished ANAC019-mediated ABA hypersensitivity. In conclusion, our results demonstrate that the biochemical and functional specificity of NAC TFs is associated with both the DBDs and the TRDs.
340 citations
TL;DR: How developmental plasticity, although beneficial for short-term survival, can subsequently programme glucose intolerance and insulin resistance in adult life by eliciting changes in key organ structures and the epigenome is highlighted.
Abstract: There are many instances in life when the environment plays a critical role in the health outcomes of an individual, yet none more so than those experienced in fetal and neonatal life. One of the most detrimental environmental problems encountered during this critical growth period are changes in nutrition to the growing fetus and newborn. Disturbances in the supply of nutrients and oxygen to the fetus can not only lead to adverse fetal growth patterns, but they have also been associated with the development of features of metabolic syndrome in adult life. This fetal response has been termed developmental programming or the developmental origins of health and disease. The present review focuses on the epidemiological studies that identified this association and the importance that animal models have played in studying this concept. We also address the potential mechanisms that may underpin the developmental programming of future disease. It also highlights (i) how developmental plasticity, although beneficial for short-term survival, can subsequently programme glucose intolerance and insulin resistance in adult life by eliciting changes in key organ structures and the epigenome, and (ii) how aberrant mitochondrial function can potentially lead to the development of Type 2 diabetes and other features of metabolic syndrome.
TL;DR: It is discovered that 14-3-3 interacts with two residues that are phosphorylated by basophilic kinases and located in the DISC1 (disrupted-in-schizophrenia 1)-interacting region of Ndel1 that is implicated in cognitive disorders.
Abstract: More than 200 phosphorylated 14-3-3-binding sites in the literature were analysed to define 14-3-3 specificities, identify relevant protein kinases, and give insights into how cellular 14-3-3/phosphoprotein networks work. Mode I RXX(pS/pT)XP motifs dominate, although the +2 proline residue occurs in less than half, and LX(R/K)SX(pS/pT)XP is prominent in plant 14-3-3-binding sites. Proline at +1 is rarely reported, and such motifs did not stand up to experimental reanalysis of human Ndel1. Instead, we discovered that 14-3-3 interacts with two residues that are phosphorylated by basophilic kinases and located in the DISC1 (disrupted-in-schizophrenia 1)-interacting region of Ndel1 that is implicated in cognitive disorders. These data conform with the general findings that there are different subtypes of 14-3-3-binding sites that overlap with the specificities of different basophilic AGC (protein kinase A/protein kinase G/protein kinase C family) and CaMK (Ca2+/calmodulin-dependent protein kinase) protein kinases, and a 14-3-3 dimer often engages with two tandem phosphorylated sites, which is a configuration with special signalling, mechanical and evolutionary properties. Thus 14-3-3 dimers can be digital logic gates that integrate more than one input to generate an action, and coincidence detectors when the two binding sites are phosphorylated by different protein kinases. Paired sites are generally located within disordered regions and/or straddle either side of functional domains, indicating how 14-3-3 dimers modulate the conformations and/or interactions of their targets. Finally, 14-3-3 proteins bind to members of several multi-protein families. Two 14-3-3-binding sites are conserved across the class IIa histone deacetylases, whereas other protein families display differential regulation by 14-3-3s. We speculate that 14-3-3 dimers may have contributed to the evolution of such families, tailoring regulatory inputs to different physiological demands.
TL;DR: A hierarchical classification of polysaccharide lyases is shown thoroughly for the first time, which should help annotate relevant genes in genomic efforts and is available and constantly updated at the Carbohydrate-Active Enzymes Database.
Abstract: Carbohydrate-active enzymes face huge substrate diversity in a highly selective manner using only a limited number of available folds. They are therefore subjected to multiple divergent and convergent evolutionary events. This and their frequent modularity render their functional annotation in genomes difficult in a number of cases. In the present paper, a classification of polysaccharide lyases (the enzymes that cleave polysaccharides using an elimination instead of a hydrolytic mechanism) is shown thoroughly for the first time. Based on the analysis of a large panel of experimentally characterized polysaccharide lyases, we examined the correlation of various enzyme properties with the three levels of the classification: fold, family and subfamily. The resulting hierarchical classification, which should help annotate relevant genes in genomic efforts, is available and constantly updated at the Carbohydrate-Active Enzymes Database (http://www.cazy.org).
TL;DR: The present review discusses the state of the understanding of FA synthesis with a focus on ACP, and the structural and dynamic properties of an increasing number of acyl-ACPs have been described, leading to an improved comprehension of this central carrier protein.
Abstract: FA (fatty acid) synthesis represents a central, conserved process by which acyl chains are produced for utilization in a number of end-products such as biological membranes. Central to FA synthesis, the ACP (acyl carrier protein) represents the cofactor protein that covalently binds all fatty acyl intermediates via a phosphopantetheine linker during the synthesis process. FASs (FA synthases) can be divided into two classes, type I and II, which are primarily present in eukaryotes and bacteria/plants respectively. They are characterized by being composed of either large multifunctional polypeptides in the case of type I or consisting of discretely expressed mono-functional proteins in the type II system. Owing to this difference in architecture, the FAS system has been thought to be a good target for the discovery of novel antibacterial agents, as exemplified by the antituberculosis drug isoniazid. There have been considerable advances in this field in recent years, including the first high-resolution structural insights into the type I mega-synthases and their dynamic behaviour. Furthermore, the structural and dynamic properties of an increasing number of acyl-ACPs have been described, leading to an improved comprehension of this central carrier protein. In the present review we discuss the state of the understanding of FA synthesis with a focus on ACP. In particular, developments made over the past few years are highlighted.
TL;DR: Direct comparison of purified 20S proteasome and immunoproteasome demonstrated that the immunoproteinasome can selectively degrade oxidized proteins, which is important during transient (reversible) adaptation, to oxidative stress in murine embryonic fibroblasts.
Abstract: Oxidized cytoplasmic and nuclear proteins are normally degraded by the proteasome, but accumulate with age and disease. We demonstrate the importance of various forms of the proteasome during transient (reversible) adaptation (hormesis), to oxidative stress in murine embryonic fibroblasts. Adaptation was achieved by ‘pre-treatment’ with very low concentrations of H2O2, and tested by measuring inducible resistance to a subsequent much higher ‘challenge’ dose of H2O2. Following an initial direct physical activation of pre-existing proteasomes, the 20S proteasome, immunoproteasome and PA28αβ regulator all exhibited substantially increased de novo synthesis during adaptation over 24 h. Cellular capacity to degrade oxidatively damaged proteins increased with 20S proteasome, immunoproteasome and PA28αβ synthesis, and was mostly blocked by the 20S proteasome, immunoproteasome and PA28 siRNA (short interfering RNA) knockdown treatments. Additionally, PA28αβ-knockout mutants achieved only half of the H2O2-induced adaptive increase in proteolytic capacity of wild-type controls. Direct comparison of purified 20S proteasome and immunoproteasome demonstrated that the immunoproteasome can selectively degrade oxidized proteins. Cell proliferation and DNA replication both decreased, and oxidized proteins accumulated, during high H2O2 challenge, but prior H2O2 adaptation was protective. Importantly, siRNA knockdown of the 20S proteasome, immunoproteasome or PA28αβ regulator blocked 50–100% of these adaptive increases in cell division and DNA replication, and immunoproteasome knockdown largely abolished protection against protein oxidation.
Abbreviations: AMC, 7-amino-4-methylcoumarin; BrdU, bromodeoxyuridine; Bz-VGR-AMC, α-N-benzoyl-Val-Gly-Arg-7-amino-4-methylcoumarin; ezrinox, oxidized ezrin; Hbox, oxidized Hb; MEF, murine embryonic fibroblast; PrOxI, hypothesis, protein oxidation and immunoproteasome hypothesis of MHC class I antigen processing; siRNA, short interfering RNA; suc-LLVY-AMC, N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin; TCA, trichloroacetic acid; Z-LLE-AMC, benzyloxycarbonyl-Leu-Leu-Glu-7-amino-4-methylcoumarin
TL;DR: The results suggest that DCF-dependent fluorescence largely reflects relocation to the cytosol of lysosomal iron and/or mitochondrial cytochrome c.
Abstract: H(2)DCF-DA (dihydrodichlorofluorescein diacetate) is widely used to evaluate 'cellular oxidative stress'. After passing through the plasma membrane, this lipophilic and non-fluorescent compound is de-esterified to a hydrophilic alcohol [H(2)DCF (dihydrodichlorofluorescein)] that may be oxidized to fluorescent DCF (2',7'-dichlorofluorescein) by a process usually considered to involve ROS (reactive oxygen species). It is, however, not always recognized that, being a hydrophilic molecule, H(2)DCF does not cross membranes, except for the outer fenestrated mitochondrial ones. It is also not generally realized that oxidation of H(2)DCF is dependent either on Fenton-type reactions or on unspecific enzymatic oxidation by cytochrome c, for neither superoxide, nor H(2)O(2), directly oxidizes H(2)DCF. Consequently, oxidation of H(2)DCF requires the presence of either cytochrome c or of both redox-active transition metals and H(2)O(2). Redox-active metals exist mainly within lysosomes, whereas cytochrome c resides bound to the outer side of the inner mitochondrial membrane. Following exposure to H(2)DCF-DA, weak mitochondrial fluorescence was found in both the oxidation-resistant ARPE-19 cells and the much more sensitive J774 cells. This fluorescence was only marginally enhanced following short exposure to H(2)O(2), showing that by itself it is unable to oxidize H(2)DCF. Cells that were either exposed to the lysosomotropic detergent MSDH (O-methylserine dodecylamide hydrochloride), exposed to prolonged oxidative stress, or spontaneously apoptotic showed lysosomal permeabilization and strong DCF-induced fluorescence. The results suggest that DCF-dependent fluorescence largely reflects relocation to the cytosol of lysosomal iron and/or mitochondrial cytochrome c.
TL;DR: PF-4708671 is the first S6K1-specific inhibitor to be reported and will be a useful tool for delineating S6k1- specific roles downstream of mTOR.
Abstract: S6K1 (p70 ribosomal S6 kinase 1) is activated by insulin and growth factors via the PI3K (phosphoinositide 3-kinase) and mTOR (mammalian target of rapamycin) signalling pathways. S6K1 regulates numerous processes, such as protein synthesis, growth, proliferation and longevity, and its inhibition has been proposed as a strategy for the treatment of cancer and insulin resistance. In the present paper we describe a novel cell-permeable inhibitor of S6K1, PF-4708671, which specifically inhibits the S6K1 isoform with a Ki of 20 nM and IC50 of 160 nM. PF-4708671 prevents the S6K1-mediated phosphorylation of S6 protein in response to IGF-1 (insulin-like growth factor 1), while having no effect upon the PMA-induced phosphorylation of substrates of the highly related RSK (p90 ribosomal S6 kinase) and MSK (mitogen- and stress-activated kinase) kinases. PF-4708671 was also found to induce phosphorylation of the T-loop and hydrophobic motif of S6K1, an effect that is dependent upon mTORC1 (mTOR complex 1). PF-4708671 is the first S6K1-specific inhibitor to be reported and will be a useful tool for delineating S6K1-specific roles downstream of mTOR.
TL;DR: A high level of GSH in the nucleus may not only have an immediate effect on gene expression patterns, but also contribute to how cells retain a memory of the cellular redox environment that is transferred through generations.
Abstract: The complex antioxidant network of plant and animal cells has the thiol tripeptide GSH at its centre to buffer ROS (reactive oxygen species) and facilitate cellular redox signalling which controls growth, development and defence GSH is found in nearly every compartment of the cell, including the nucleus Transport between the different intracellular compartments is pivotal to the regulation of cell proliferation GSH co-localizes with nuclear DNA at the early stages of proliferation in plant and animal cells Moreover, GSH recruitment and sequestration in the nucleus during the G1- and S-phases of the cell cycle has a profound impact on cellular redox homoeostasis and on gene expression For example, the abundance of transcripts encoding stress and defence proteins is decreased when GSH is sequestered in the nucleus The functions of GSHn (nuclear GSH) are considered in the present review in the context of whole-cell redox homoeostasis and signalling, as well as potential mechanisms for GSH transport into the nucleus We also discuss the possible role of GSHn as a regulator of nuclear proteins such as histones and PARP [poly(ADP-ribose) polymerase] that control genetic and epigenetic events In this way, a high level of GSH in the nucleus may not only have an immediate effect on gene expression patterns, but also contribute to how cells retain a memory of the cellular redox environment that is transferred through generations
TL;DR: Recent progress made on the mechanisms regulating adiponectin gene transcription, multimerization and secretion are summarized and the potential relevance of these studies to the development of new clinical therapy for insulin resistance, Type 2 diabetes and other obesity-related metabolic disorders is discussed.
Abstract: Adiponectin is an adipose-tissue-derived hormone with anti-diabetic, anti-atherogenic and anti-inflammatory functions. Adiponectin circulates in the bloodstream in trimeric, hexameric and high-molecular-mass species, and different forms of adiponectin have been found to play distinct roles in the regulation of energy homoeostasis. The serum levels of adiponectin are negatively correlated with obesity and insulin resistance, yet the underlying mechanisms remain elusive. In the present review, we summarize recent progress made on the mechanisms regulating adiponectin gene transcription, multimerization and secretion. We also discuss the potential relevance of these studies to the development of new clinical therapy for insulin resistance, Type 2 diabetes and other obesity-related metabolic disorders.
TL;DR: Measurement of the inhibitor sensitivity of several chimaeric transporters combining different domains of MCT1 and MCT4 revealed that the binding site for AR-C155858 is contained within the C-terminal half of M CT1, and involves TM (transmembrane) domains 7–10, consistent with previous data identifying Phe360 and Asp302 plus Arg306 as key residues in substrate binding and translocation by MCT 1.
Abstract: In the present study we characterize the properties of the potent MCT1 (monocarboxylate transporter 1) inhibitor AR-C155858. Inhibitor titrations of L-lactate transport by MCT1 in rat erythrocytes were used to determine the Ki value and number of AR-C155858-binding sites (Et) on MCT1 and the turnover number of the transporter (kcat). Derived values were 2.3±1.4 nM, 1.29±0.09 nmol per ml of packed cells and 12.2±1.1 s−1 respectively. When expressed in Xenopus laevis oocytes, MCT1 and MCT2 were potently inhibited by AR-C155858, whereas MCT4 was not. Inhibition of MCT1 was shown to be time-dependent, and the compound was also active when microinjected, suggesting that AR-C155858 probably enters the cell before binding to an intracellular site on MCT1. Measurement of the inhibitor sensitivity of several chimaeric transporters combining different domains of MCT1 and MCT4 revealed that the binding site for AR-C155858 is contained within the C-terminal half of MCT1, and involves TM (transmembrane) domains 7–10. This is consistent with previous data identifying Phe360 (in TM10) and Asp302 plus Arg306 (TM8) as key residues in substrate binding and translocation by MCT1. Measurement of the Km values of the chimaeras for L-lactate and pyruvate demonstrate that both the C- and N-terminal halves of the molecule influence transport kinetics consistent with our proposed molecular model of MCT1 and its translocation mechanism that requires Lys38 in TM1 in addition to Asp302 and Arg306 in TM8 [Wilson, Meredith, Bunnun, Sessions and Halestrap (2009) J. Biol. Chem. 284, 20011–20021].
TL;DR: The present review discusses how the 7-methylguanosine cap is synthesized by cellular enzymes, the impact that the7- methylguanoine cap has on biological processes, and how the mRNA cap methylation reaction is regulated.
Abstract: The 7-methylguanosine cap added to the 5' end of mRNA is essential for efficient gene expression and cell viability. Methylation of the guanosine cap is necessary for the translation of most cellular mRNAs in all eukaryotic organisms in which it has been investigated. In some experimental systems, cap methylation has also been demonstrated to promote transcription, splicing, polyadenylation and nuclear export of mRNA. The present review discusses how the 7-methylguanosine cap is synthesized by cellular enzymes, the impact that the 7-methylguanosine cap has on biological processes, and how the mRNA cap methylation reaction is regulated.
TL;DR: The factors involved in NMD are discussed, the different models proposed for the recognition of PTCs (premature termination codons), the diverse physiological roles of NMD, the involvement of this surveillance pathway in disease and the current strategies for medical treatment of P TC-related diseases are discussed.
Abstract: NMD (nonsense-mediated mRNA decay) belongs to the best-studied mRNA surveillance systems of the cell, limiting the synthesis of truncated and potentially harmful proteins on the one hand and playing an initially unexpected role in the regulation of global gene expression on the other hand. In the present review, we briefly discuss the factors involved in NMD, the different models proposed for the recognition of PTCs (premature termination codons), the diverse physiological roles of NMD, the involvement of this surveillance pathway in disease and the current strategies for medical treatment of PTC-related diseases.
TL;DR: The regulation of the transcription of the miR-212/132 miRNA cluster and the miRNAs derived from it are regulated by the ERK1/2 (extracellular-signal-regulated kinase 1/2) pathway, via both MSK (mitogen and stress-activated kinase)-dependent and -independent mechanisms.
Abstract: Neurotrophins are growth factors that are important in neuronal development and survival as well as synapse formation and plasticity. Many of the effects of neurotrophins are mediated by changes in protein expression as a result of altered transcription or translation. To determine whether neurotrophins regulate the production of microRNAs (miRNAs), small RNA species that modulate protein translation or mRNA stability, we used deep sequencing to identify BDNF (brain-derived neurotrophic factor)-induced miRNAs in cultured primary cortical mouse neurons. This revealed that the miR-212/132 cluster contained the miRNAs most responsive to BDNF treatment. This cluster was found to produce four miRNAs: miR-132, miR-132*, miR-212 and miR-212*. Using specific inhibitors, mouse models and promoter analysis we have shown that the regulation of the transcription of the miR-212/132 miRNA cluster and the miRNAs derived from it are regulated by the ERK1/2 (extracellular-signalregulated kinase 1/2) pathway, via both MSK (mitogen and stress-activated kinase)-dependent and -independent mechanisms.
TL;DR: Hypomorphic Brd2 shifts energy balance toward storage without causing glucose intolerance and may provide a novel model for obese metabolically healthy humans.
Abstract: Certain human subpopulations are metabolically healthy but obese, or metabolically obese but normal weight; such mutations uncouple obesity from glucose intolerance, revealing pathways implicated in Type 2 diabetes. Current searches for relevant genes consume significant effort. We have reported previously a novel double bromodomain protein called Brd2, which is a transcriptional co-activator/co-repressor with SWI/SNF (switch mating type/sucrose non-fermenting)-like functions that regulates chromatin. In the present study, we show that wholebody disruption of Brd2, an unusual MHC gene, causes lifelong severe obesity in mice with pancreatic islet expansion, hyperinsulinaemia, hepatosteatosis and elevated pro-inflammatory cytokines, but, surprisingly, enhanced glucose tolerance, elevated adiponectin, increased weight of brown adipose tissue, heat production and expression of mitochondrial uncoupling proteins in brown adipose tissue, reduced macrophage infiltration in white adipose tissue, and lowered blood glucose, leading to an improved metabolic profile and avoiding eventual Type 2 diabetes. Brd2 is highly expressed in pancreatic beta-cells, where it normally inhibits beta-cell mitosis and insulin transcription. In 3T3-L1 pre-adipocytes, Brd2 normally co-represses PPAR-gamma (peroxisome-proliferator-activated receptor-gamma) and inhibits adipogenesis. Brd2 knockdown protects 3T3-L1 adipocytes from TNF-alpha (tumour necrosis factor-alpha)-induced insulin resistance, thereby decoupling inflammation from insulin resistance. Thus hypomorphic Brd2 shifts energy balance toward storage without causing glucose intolerance and may provide a novel model for obese metabolically healthy humans.
TL;DR: The BMPs (bone morphogenetic proteins) and the GDFs (growth and differentiation factors) together form a single family of cystine-knot cytokines, sharing the characteristic fold of the TGFbeta (transforming growth factor-beta) superfamily.
Abstract: The BMPs (bone morphogenetic proteins) and the GDFs (growth and differentiation factors) together form a single family of cystine-knot cytokines, sharing the characteristic fold of the TGFbeta (transforming growth factor-beta) superfamily. Besides the ability to induce bone formation, which gave the BMPs their name, the BMP/GDFs display morphogenetic activities in the development of a wide range of tissues. BMP/GDF homo- and hetero-dimers interact with combinations of type I and type II receptor dimers to produce multiple possible signalling complexes, leading to the activation of one of two competing sets of SMAD transcription factors. BMP/GDFs have highly specific and localized functions. These are regulated in a number of ways, including the developmental restriction of BMP/GDF expression and through the secretion of several specific BMP antagonist proteins that bind with high affinity to the cytokines. Curiously, a number of these antagonists are also members of the TGF-beta superfamily. Finally a number of both the BMP/GDFs and their antagonists interact with the heparan sulphate side chains of cell-surface and extracellular-matrix proteoglycans.
TL;DR: The present results demonstrated that miR-7 regulates the IGF1R/Akt signalling pathway by post-transcriptional regulation of IGF2R and may serve as a novel therapeutic target for TSCC patients.
Abstract: miR-7 (microRNA-7) has been characterized as a tumour suppressor in several human cancers. It targets a number of proto-oncogenes that contribute to cell proliferation and survival. However, the mechanism(s) by which miR-7 suppresses tumorigenesis in TSCC (tongue squamous cell carcinoma) is unknown. The present bioinformatics analysis revealed that IGF1R (insulin-like growth factor 1 receptor) mRNA is a potential target for miR-7. Ectopic transfection of miR-7 led to a significant reduction in IGF1R at both the mRNA and protein levels in TSCC cells. Knockdown of miR-7 in TSCC cells enhanced IGF1R expression. Direct targeting of miR-7 to three candidate binding sequences located in the 3′-untranslated region of IGF1R mRNA was confirmed using luciferase-reporter-gene assays. The miR-7-mediated down-regulation of IGF1R expression attenuated the IGF1 (insulin-like growth factor 1)-induced activation of Akt (protein kinase B) in TSCC cell lines, which in turn resulted in a reduction in cell proliferation and cell-cycle arrest, and an enhanced apoptotic rate. Taken together, the present results demonstrated that miR-7 regulates the IGF1R/Akt signalling pathway by post-transcriptional regulation of IGF1R. Our results indicate that miR-7 plays an important role in TSCC and may serve as a novel therapeutic target for TSCC patients.
TL;DR: This paper shows that the tight junction protein ZO (zonula occludens)-2 uses its first PDZ domain to form a complex with YAP2, and finds that ZO-2 facilitates the nuclear localization and pro-apoptotic function of YAP1, and that this activity of Zo-2 is PDZ-domain-dependent.
Abstract: The Hippo pathway regulates the size of organs by controlling two opposing processes: proliferation and apoptosis. YAP2 (Yes kinase-associated protein 2), one of the three isoforms of YAP, is a WW domain-containing transcriptional co-activator that acts as the effector of the Hippo pathway in mammalian cells. In addition to WW domains, YAP2 has a PDZ-binding motif at its C-terminus. We reported previously that this motif was necessary for YAP2 localization in the nucleus and for promoting cell detachment and apoptosis. In the present study, we show that the tight junction protein ZO (zonula occludens)-2 uses its first PDZ domain to form a complex with YAP2. The endogenous ZO-2 and YAP2 proteins co-localize in the nucleus. We also found that ZO-2 facilitates the nuclear localization and pro-apoptotic function of YAP2, and that this activity of ZO-2 is PDZ-domain-dependent. The present paper is the first report on a PDZ-based nuclear translocation mechanism. Moreover, since the Hippo pathway acts as a tumour suppressor pathway, the YAP2-ZO-2 complex could represent a target for cancer therapy.
TL;DR: The hypothesis that mitochondrially targeted therapies may be beneficial in the treatment of diabetic nephropathy is supported and a relatively unexplored aspect of mitochondrial ROS signalling in the control of fibrosis is highlighted.
Abstract: Mitochondrial production of ROS (reactive oxygen species) is thought to be associated with the cellular damage resulting from chronic exposure to high glucose in long-term diabetic patients. We hypothesized that a mitochondria-targeted antioxidant would prevent kidney damage in the Ins2+/−AkitaJ mouse model (Akita mice) of Type 1 diabetes. To test this we orally administered a mitochondria-targeted ubiquinone (MitoQ) over a 12-week period and assessed tubular and glomerular function. Fibrosis and pro-fibrotic signalling pathways were determined by immunohistochemical analysis, and mitochondria were isolated from the kidney for functional assessment. MitoQ treatment improved tubular and glomerular function in the Ins2+/−AkitaJ mice. MitoQ did not have a significant effect on plasma creatinine levels, but decreased urinary albumin levels to the same level as non-diabetic controls. Consistent with previous studies, renal mitochondrial function showed no significant change between any of the diabetic or wild-type groups. Importantly, interstitial fibrosis and glomerular damage were significantly reduced in the treated animals. The pro-fibrotic transcription factors phospho-Smad2/3 and β-catenin showed a nuclear accumulation in the Ins2+/−AkitaJ mice, which was prevented by MitoQ treatment. These results support the hypothesis that mitochondrially targeted therapies may be beneficial in the treatment of diabetic nephropathy. They also highlight a relatively unexplored aspect of mitochondrial ROS signalling in the control of fibrosis.
TL;DR: The roles of Wnt signalling is reassessed in functionally linking cellular metabolism to tissue development and function and how they integrate and regulate cellular metabolism is reass reassessed.
Abstract: At the cellular level, the biological processes of cell proliferation, growth arrest, differentiation and apoptosis are all tightly coupled to appropriate alterations in metabolic status. In the case of cell proliferation, this requires redirecting metabolic pathways to provide the fuel and basic components for new cells. Ultimately, the successful co-ordination of cell-specific biology with cellular metabolism underscores multicellular processes as diverse as embryonic development, adult tissue remodelling and cancer cell biology. The Wnt signalling network has been implicated in all of these areas. While each of the Wnt-dependent signalling pathways are being individually delineated in a range of experimental systems, our understanding of how they integrate and regulate cellular metabolism is still in its infancy. In the present review we reassess the roles of Wnt signalling in functionally linking cellular metabolism to tissue development and function.
TL;DR: Comparative genomics is probably the single most effective strategy, for it can reliably predict functions for unknown proteins and genes for orphan enzymes and is cost-efficient and increasingly straightforward to deploy owing to a proliferation of databases and associated tools.
Abstract: Like other forms of engineering, metabolic engineering requires knowledge of the components (the 'parts list') of the target system. Lack of such knowledge impairs both rational engineering design and diagnosis of the reasons for failures; it also poses problems for the related field of metabolic reconstruction, which uses a cell's parts list to recreate its metabolic activities in silico. Despite spectacular progress in genome sequencing, the parts lists for most organisms that we seek to manipulate remain highly incomplete, due to the dual problem of 'unknown' proteins and 'orphan' enzymes. The former are all the proteins deduced from genome sequence that have no known function, and the latter are all the enzymes described in the literature (and often catalogued in the EC database) for which no corresponding gene has been reported. Unknown proteins constitute up to about half of the proteins in prokaryotic genomes, and much more than this in higher plants and animals. Orphan enzymes make up more than a third of the EC database. Attacking the 'missing parts list' problem is accordingly one of the great challenges for post-genomic biology, and a tremendous opportunity to discover new facets of life's machinery. Success will require a co-ordinated community-wide attack, sustained over years. In this attack, comparative genomics is probably the single most effective strategy, for it can reliably predict functions for unknown proteins and genes for orphan enzymes. Furthermore, it is cost-efficient and increasingly straightforward to deploy owing to a proliferation of databases and associated tools.