Showing papers in "EMBO Reports in 2004"
TL;DR: A new in vitro isothermal DNA amplification method by mimicking this in vivo mechanism, Helicase‐dependent amplification (HDA), which offers several advantages over other isothermalDNA amplification methods by having a simple reaction scheme and being a true isothermal reaction that can be performed at one temperature for the entire process.
Abstract: Polymerase chain reaction is the most widely used method for in vitro DNA amplification. However, it requires thermocycling to separate two DNA strands. In vivo, DNA is replicated by DNA polymerases with various accessory proteins, including a DNA helicase that acts to separate duplex DNA. We have devised a new in vitro isothermal DNA amplification method by mimicking this in vivo mechanism. Helicase-dependent amplification (HDA) utilizes a DNA helicase to generate single-stranded templates for primer hybridization and subsequent primer extension by a DNA polymerase. HDA does not require thermocycling. In addition, it offers several advantages over other isothermal DNA amplification methods by having a simple reaction scheme and being a true isothermal reaction that can be performed at one temperature for the entire process. These properties offer a great potential for the development of simple portable DNA diagnostic devices to be used in the field and at the point-of-care.
864 citations
TL;DR: Results clearly showed thatDJ‐1 has a role in the antioxidative stress reaction and that mutations of DJ‐1 lead to cell death, which is observed in PD.
Abstract: Deletion and point (L166P) mutations of DJ-1 have recently been shown to be responsible for the onset of familial Parkinson's disease (PD, PARK7). The aim of this study was to determine the role of DJ-1 in PD. We first found that DJ-1 eliminated hydrogen peroxide in vitro by oxidizing itself. We then found that DJ-1 knockdown by short interfering RNA rendered SH-SY5Y neuroblastoma cells susceptible to hydrogen peroxide-, MPP+- or 6-hydroxydopamine-induced cell death and that cells harbouring mutant forms of DJ-1, including L166P, became susceptible to death in parallel with the loss of oxidized forms of DJ-1. These results clearly showed that DJ-1 has a role in the antioxidative stress reaction and that mutations of DJ-1 lead to cell death, which is observed in PD.
841 citations
TL;DR: The concept of dimerization could be important in the development and screening of drugs that act through this receptor class, and the changes in ligand‐binding and signalling properties that accompany heterodimerization could give rise to an unexpected pharmacological diversity that would need to be considered.
Abstract: The classical idea that G-protein-coupled receptors (GPCRs) function as monomeric entities has been unsettled by the emerging concept of GPCR dimerization. Recent findings have indicated not only that many GPCRs exist as homodimers and heterodimers, but also that their oligomeric assembly could have important functional roles. Several studies have shown that dimerization occurs early after biosynthesis, suggesting that it has a primary role in receptor maturation. G-protein coupling, downstream signalling and regulatory processes such as internalization have also been shown to be influenced by the dimeric nature of the receptors. In addition to raising fundamental questions about GPCR function, the concept of dimerization could be important in the development and screening of drugs that act through this receptor class. In particular, the changes in ligand-binding and signalling properties that accompany heterodimerization could give rise to an unexpected pharmacological diversity that would need to be considered.
686 citations
TL;DR: This work outlines exciting progress in the biochemistry and cell biology of sphingolipids and focuses on their functional diversity, which should set the conceptual and experimental framework that will eventually lead to a fully integrated and comprehensive model of the functions of specific sphingosine‐1‐phosphate in regulating defined aspects of cell physiology.
Abstract: The extensive diversity of membrane lipids is rarely appreciated by cell and molecular biologists. Although most researchers are familiar with the three main classes of lipids in animal cell membranes, few realize the enormous combinatorial structural diversity that exists within each lipid class, a diversity that enables functional specialization of lipids. In this brief review, we focus on one class of membrane lipids, the sphingolipids, which until not long ago were thought by many to be little more than structural components of biological membranes. Recent studies have placed sphingolipids—including ceramide, sphingosine and sphingosine-1-phosphate—at the centre of a number of important biological processes, specifically in signal transduction pathways, in which their levels change in a highly regulated temporal and spatial manner. We outline exciting progress in the biochemistry and cell biology of sphingolipids and focus on their functional diversity. This should set the conceptual and experimental framework that will eventually lead to a fully integrated and comprehensive model of the functions of specific sphingolipids in regulating defined aspects of cell physiology.
623 citations
TL;DR: In vivo, HMGB1 enhances the primary antibody responses to soluble antigens and transforms poorly immunogenic apoptotic lymphoma cells into efficient vaccines and reduces the activation induced by necrotic wild‐type cell supernatants.
Abstract: Immune responses against pathogens require that microbial components promote the activation of antigen-presenting cells (APCs). Autoimmune diseases and graft rejections occur in the absence of pathogens; in these conditions, endogenous molecules, the so-called 'innate adjuvants', activate APCs. Necrotic cells contain and release innate adjuvants; necrotic cells also release high-mobility group B1 protein (HMGB1), an abundant and conserved constituent of vertebrate nuclei. Here, we show that necrotic HMGB1(-/-) cells have a reduced ability to activate APCs, and HMGB1 blockade reduces the activation induced by necrotic wild-type cell supernatants. In vivo, HMGB1 enhances the primary antibody responses to soluble antigens and transforms poorly immunogenic apoptotic lymphoma cells into efficient vaccines.
584 citations
TL;DR: A previously unknown function of Oct4 in maintaining viability of mammalian germline is suggested using the conditional Cre/loxP gene targeting strategy to assess Oct4 function in primordial germ cells (PGCs).
Abstract: Previous studies have shown that Oct4 has an essential role in maintaining pluripotency of cells of the inner cell mass (ICM) and embryonic stem cells. However, Oct4 null homozygous embryos die around the time of implantation, thus precluding further analysis of gene function during development. We have used the conditional Cre/loxP gene targeting strategy to assess Oct4 function in primordial germ cells (PGCs). Loss of Oct4 function leads to apoptosis of PGCs rather than to differentiation into a trophectodermal lineage, as has been described for Oct4-deficient ICM cells. These new results suggest a previously unknown function of Oct4 in maintaining viability of mammalian germline.
554 citations
TL;DR: It is shown that elicited peritoneal murine macrophages do not produce tumour necrosis factor‐α or interleukin‐6 in response to purified PGs, suggesting that PG detection is more likely to occur intracellularly (through Nod1/Nod2) rather than from the extracellular compartment.
Abstract: Toll-like receptor 2 (TLR2) has been shown to recognize several classes of pathogen-associated molecular patterns including peptidoglycan (PG). However, studies linking PG with TLR2 recognition have relied mainly on the use of commercial Staphylococcus aureus PG and have not addressed TLR2 recognition of other PG types. Using highly purified PGs from eight bacteria (Escherichia coli, Pseudomonas aeruginosa, Yersinia pseudotuberculosis, Helicobacter pylori, Bacillus subtilis, Listeria monocytogenes, Streptococcus pneumoniae and S. aureus), we show that these PGs are not sensed through TLR2, TLR2/1 or TLR2/6. PG sensing is lost after removal of lipoproteins or lipoteichoic acids (LTAs) from Gram-negative and Gram-positive cell walls, respectively. Accordingly, purified LTAs are sensed synergistically through TLR2/1. Finally, we show that elicited peritoneal murine macrophages do not produce tumour necrosis factor-alpha or interleukin-6 in response to purified PGs, suggesting that PG detection is more likely to occur intracellularly (through Nod1/Nod2) rather than from the extracellular compartment.
512 citations
TL;DR: Evidence is focused on that supports a role for transcriptional dysfunction and impaired protein folding and degradation as early events in disease pathogenesis in Huntington's disease.
Abstract: Huntington's disease (HD) is a late‐onset neurodegenerative disorder that is caused by a CAG repeat expansion in the IT15 gene, which results in a long stretch of polyglutamine close to the amino‐terminus of the HD protein huntingtin (htt). The normal function of htt, and the molecular mechanisms that contribute to the disease pathogenesis, are in the process of being elucidated. In this review, we outline the potential functions of htt as defined by the proteins with which it has been found to interact. We then focus on evidence that supports a role for transcriptional dysfunction and impaired protein folding and degradation as early events in disease pathogenesis.
Huntington's disease (HD) is an autosomal‐dominant disorder characterized by irrepressible motor dysfunction, cognitive decline and psychiatric disturbances, which lead to progressive dementia and death approximately 15–20 years after disease onset (Bates et al , 2002). It belongs to a family of neurodegenerative diseases caused by mutations in which an expanded CAG repeat tract results in long stretches of polyglutamine (polyQ) in the encoded protein. This family also includes dentatorubral‐pallidoluysian atrophy (DRPLA), spinal and bulbar muscular atrophy (SBMA) and the spinocerebellar ataxias (SCAs) 1–3, 6, 7 and 17. Apart from their polyQ repeats, the proteins involved are unrelated, and although they are all widely expressed in the central nervous system and peripheral tissues, they lead to characteristic patterns of neurodegeneration. In HD, the selective neurodegeneration of the γ‐aminobutyric acid‐releasing spiny‐projection neurons of the striatum is predominant, although loss of neurons in many other brain regions has also been reported. In the unaffected population, the number of CAG repeats in the IT15 gene that encodes the HD protein huntingtin (htt) varies from 6 to 35; repeats of 36 or more define an HD allele. The length of the CAG expansion is inversely correlated with age of …
486 citations
TL;DR: It is suggested that J‐like proteins might function to regulate the activity of bona fide J proteins during protein translocation, assembly and disassembly.
Abstract: DnaJ is a molecular chaperone and the prototypical member of the J-protein family. J proteins are defined by the presence of a J domain that can regulate the activity of 70-kDa heat-shock proteins. Sequence analysis on the genome of Saccharomyces cerevisiae has revealed 22 proteins that establish four distinguishing structural features of the J domain: predicted helicity in segments I-IV, precisely placed interhelical contact residues, a lysine-rich surface on helix II and placement of the diagnostic sequence HPD between the predicted helices II and III. We suggest that this definition of the J-protein family could be used for other genome-wide studies. In addition, three J-like proteins were identified in yeast that contain regions closely resembling a J domain, but in which the HPD motif is non-conservatively replaced. We suggest that J-like proteins might function to regulate the activity of bona fide J proteins during protein translocation, assembly and disassembly.
484 citations
TL;DR: By undergoing a cAMP‐induced conformational change, CFP–Epac–YFP serves as a highly sensitive cAMP indicator in vivo, and when compared with a protein kinase A (PKA)‐based sensor, Epac‐based cAMP probes show an extended dynamic range and a better signal‐to‐noise ratio.
Abstract: Epac1 is a guanine nucleotide exchange factor for Rap1 that is activated by direct binding of cAMP. In vitro studies suggest that cAMP relieves the interaction between the regulatory and catalytic domains of Epac. Here, we monitor Epac1 activation in vivo by using a CFP–Epac–YFP fusion construct. When expressed in mammalian cells, CFP–Epac–YFP shows significant fluorescence resonance energy transfer (FRET). FRET rapidly decreases in response to the cAMP-raising agents, whereas it fully recovers after addition of cAMP-lowering agonists. Thus, by undergoing a cAMP-induced conformational change, CFP–Epac–YFP serves as a highly sensitive cAMP indicator in vivo. When compared with a protein kinase A (PKA)-based sensor, Epac-based cAMP probes show an extended dynamic range and a better signal-to-noise ratio; furthermore, as a single polypeptide, CFP–Epac–YFP does not suffer from the technical problems encountered with multisubunit PKA-based sensors. These properties make Epac-based FRET probes the preferred indicators for monitoring cAMP levels in vivo.
464 citations
TL;DR: How are human and veterinary medicines in soils and water bodies affecting human and environmental health?
Abstract: How are human and veterinary medicines in soils and water bodies affecting human and environmental health?
TL;DR: The structure of the DNA‐binding NAC domain of Arabidopsis ANAC (abscisic‐acid‐responsive NAC) has been determined by X‐ray crystallography to 1.9 Å resolution, the first structure determined for a member of the NAC family of plant‐specific transcriptional regulators.
Abstract: The structure of the DNA-binding NAC domain of Arabidopsis ANAC (abscisic-acid-responsive NAC) has been determined by X-ray crystallography to 1.9 A resolution (Protein Data Bank codes 1UT4 and 1UT7). This is the first structure determined for a member of the NAC family of plant-specific transcriptional regulators. NAC proteins are characterized by their conserved N-terminal NAC domains that can bind both DNA and other proteins. NAC proteins are involved in developmental processes, including formation of the shoot apical meristem, floral organs and lateral shoots, as well as in plant hormonal control and defence. The NAC domain does not possess a classical helix–turn–helix motif; instead it reveals a new transcription factor fold consisting of a twisted β-sheet surrounded by a few helical elements. The functional dimer formed by the NAC domain was identified in the structure, which will serve as a structural template for understanding NAC protein function at the molecular level.
TL;DR: It is demonstrated that lentiviral vectors can be used to generate transgenic lines with an efficiency in the order of 100‐fold higher than any previously published method, with no detectable silencing of transgene expression between generations.
Abstract: An effective method for genetic modification of chickens has yet to be developed. An efficient technology, enabling production of transgenic birds at high frequency and with reliable expression of transgenes, will have many applications, both in basic research and in biotechnology. We investigated the efficiency with which lentiviral vectors could transduce the chicken germ line and examined the expression of introduced reporter transgenes. Ten founder cockerels transmitted the vector to between 4% and 45% of their offspring and stable transmission to the G2 generation was demonstrated. Analysis of expression of reporter gene constructs in several transgenic lines showed a conserved expression profile between individuals that was maintained after transmission through the germ line. These data demonstrate that lentiviral vectors can be used to generate transgenic lines with an efficiency in the order of 100-fold higher than any previously published method, with no detectable silencing of transgene expression between generations.
TL;DR: Understanding the biology of AT will lead to a greater understanding of the fundamental processes that underpin cancer and neurodegeneration, as well as inspire new ideas on how to prevent cancer.
Abstract: Ataxia telangiectasia (AT) has long intrigued the biomedical research community owing to the spectrum of defects that are characteristic of the disease, including neurodegeneration, immune dysfunction, radiosensitivity and cancer predisposition Following the identification of mutations in ATM (ataxia telangiectasia, mutated) as the underlying cause of the disease, biochemical analysis of this protein kinase has shown that it is a crucial nexus for the cellular response to DNA double-stranded breaks Many ATM kinase substrates are important players in the cellular responses that prevent cancer Accordingly, AT is a disease that results from defects in the response to specific types of DNA damage Thus, although it is a rare neurodegenerative disease, understanding the biology of AT will lead to a greater understanding of the fundamental processes that underpin cancer and neurodegeneration
TL;DR: The existence of an atypical molecular phenotype among cattle diagnosed with BSE in France suggests either some phenotypic modifications of PrPres following infection by the BSE agent or the existence of alternative origins of such diseases in cattle.
Abstract: Bovine spongiform encephalopathy (BSE) in cattle, the most likely cause of variant Creutzfeldt–Jakob disease in humans, is thought to be caused by a unique infectious agent, with stable features, even when transmitted to other species. Here, we show the existence of an atypical molecular phenotype among cattle diagnosed with BSE in France. Following western blot analysis, three cases showed unusual features of the electrophoretic profiles of the protease-resistant prion protein (PrPres) accumulating in the brain. The PrPres patterns were similar in these three atypical cases, showing a higher molecular mass of unglycosylated PrPres and strong labelling by P4 monoclonal antibody compared to 55 typical BSE cases. This finding suggests either some phenotypic modifications of PrPres following infection by the BSE agent or the existence of alternative origins of such diseases in cattle.
TL;DR: Duchenne muscular dystrophy is caused by mutations in the gene that encodes the 427‐kDa cytoskeletal protein dystrophin, and upregulation of other proteins can also prevent the dystrophic process.
Abstract: Duchenne muscular dystrophy (DMD) is caused by mutations in the gene that encodes the 427‐kDa cytoskeletal protein dystrophin. Increased knowledge of the function of dystrophin and its role in muscle has led to a greater understanding of the pathogenesis of DMD. This, together with advances in the genetic toolkit of the molecular biologist, are leading to many different approaches to treatment. Gene therapy can be achieved using plasmids or viruses, mutations can be corrected using chimaeraplasts and short DNA fragments, exon skipping of mutations can be induced using oligonucleotides and readthrough of nonsense mutations can be achieved using aminoglycoside antibiotics. Blocking the proteasome degradation pathway can stabilize any truncated dystrophin protein, and upregulation of other proteins can also prevent the dystrophic process. Muscle can be repopulated with myoblasts or stem cells. All, or a combination, of these approaches hold great promise for the treatment of this devastating disease.
TL;DR: Scientists now have the tools to unravel biological complexity and overcome the limitations of reductionism, according to research published in Science magazine.
Abstract: The reductionist method of dissecting biological systems into their constituent parts has been effective in explaining the chemical basis of numerous living processes. However, many biologists now realize that this approach has reached its limit. Biological systems are extremely complex and have emergent properties that cannot be explained, or even predicted, by studying their individual parts. The reductionist approach—although successful in the early days of molecular biology—underestimates this complexity and therefore has an increasingly detrimental influence on many areas of biomedical research, including drug discovery and vaccine development.
TL;DR: The results of a database search for the sequence of the BAR domain of Amphiphysin and Arfaptin 2 identified a variety of related proteins, most of which are involved in intracellular transport and especially in endocytosis.
Abstract: BAR-domains recently took centre stage in science through a report on the crystal structure of this domain in Drosophila Amphiphysin. Though only weakly conserved at the sequence level, the structure of the BAR domain shows striking similarity to the GTPase-binding domain of Arfaptin 2, an effector of Rho- and Arf- GTPases. On the basis of this sequence and structural similarity, these two proteins have been classified as belonging to the same family, the BAR-domain family, and they probably also have similar functional characteristics. Presented here are the results of a database search for the sequence of the BAR domain of Amphiphysin and Arfaptin 2. This search identified a variety of related proteins, most of which are involved in intracellular transport and especially in endocytosis. For example, the BAR-domain family includes Endophilins, GTPase-activating proteins of the Centaurinβ family and Oligophrenins, the adaptor proteins APPL1 and APPL2 that were recently shown to interact with the small GTPase Rab5, as well as members of the Sorting nexin family. On the basis of the structures of Amphiphysin and Arfaptin 2 and the cellular role of Amphiphysins in the early steps of endocytosis, the functions of the BAR domain have been defined as a dimerization motif and as sensing and inducing membrane curvature. However, data on Arfaptin 2 and now also on the Adaptor proteins APPL1 and 2 suggest that another function of the BAR domain is to bind to small GTPases.
TL;DR: The results suggest that the Ash1 and Trx HMTases are not ‘coactivators’ required for transcriptional activation of HOX genes, but function specifically as anti‐repressors.
Abstract: Transcriptional on and off states of HOX genes and other developmental control genes are maintained by antagonistic regulators encoded by trithorax group (trxG) and Polycomb group (PcG) genes. The trxG proteins Ash1 and hTRX and the PcG repressor E(z) are histone methyltransferases (HMTases) that methylate distinct lysine residues in the N‐terminal tail of histone H3. trxG proteins are generally thought to function as activators of HOX genes, but how histone methylation by Ash1 and Trx promotes HOX gene transcription is not clear. Here, we show that in ash1 and trx mutants expression of HOX genes is lost within their normal expression domains, but we find that, contrary to expectation, this expression is restored in ash1 and trx mutants that also lack PcG gene function. Moreover, such trxG PcG double mutants show severe misexpression of HOX genes and, hence, ectopic activation of HOX genes caused by the removal of PcG gene function also occurs in the absence of ash1 and trx function. Together, these results suggest that the Ash1 and Trx HMTases are not ‘coactivators’ required for transcriptional activation of HOX genes, but function specifically as anti‐repressors. We propose that histone methylation by Ash1 and Trx is required continuously throughout development to prevent inappropriate PcG silencing of HOX genes in cells in which they must stay transcriptionally active.
TL;DR: The receptor for activated C‐kinase (RACK1) is a scaffold protein that is able to interact simultaneously with several signalling molecules and binds to protein kinases and membrane‐bound receptors in a regulated fashion, which allows translation to be regulated in response to cell stimuli.
Abstract: The receptor for activated C-kinase (RACK1) is a scaffold protein that is able to interact simultaneously with several signalling molecules. It binds to protein kinases and membrane-bound receptors in a regulated fashion. Interestingly, RACK1 is also a constituent of the eukaryotic ribosome, and a recent cryo-electron microscopy study localized it to the head region of the 40S subunit in the vicinity of the messenger RNA (mRNA) exit channel. RACK1 recruits activated protein kinase C to the ribosome, which leads to the stimulation of translation through the phosphorylation of initiation factor 6 and, potentially, of mRNA-associated proteins. RACK1 therefore links signal-transduction pathways directly to the ribosome, which allows translation to be regulated in response to cell stimuli. In addition, the fact that RACK1 associates with membrane-bound receptors indicates that it promotes the docking of ribosomes at sites where local translation is required, such as focal adhesions.
TL;DR: Data show that combinatorial interactions of SNAREs determine whether late endosomes undergo homotypic or heterotypic fusion events, and that the same Q‐SNAREs can combine with an alternative R‐ SNARE, namely VAMP7, for heterotyping fusion between late endOSomes and lysosomes.
Abstract: Both heterotypic and homotypic fusion events are required to deliver endocytosed macromolecules to lysosomes and remodel late endocytic organelles. A trans-SNARE complex consisting of Q-SNAREs syntaxin 7, Vti1b and syntaxin 8 and the R-SNARE VAMP8 has been shown by others to be responsible for homotypic fusion of late endosomes. Using antibody inhibition experiments in rat liver cell-free systems, we confirmed this result, but found that the same Q-SNAREs can combine with an alternative R-SNARE, namely VAMP7, for heterotypic fusion between late endosomes and lysosomes. Co-immunoprecipitation demonstrated separate syntaxin 7 complexes with either VAMP7 or VAMP8 in solubilized rat liver membranes. Additionally, overexpression of the N-terminal domain of VAMP7, in cultured fibroblastic cells, inhibited the mixing of a preloaded lysosomal content marker with a marker delivered to late endosomes. These data show that combinatorial interactions of SNAREs determine whether late endosomes undergo homotypic or heterotypic fusion events.
TL;DR: D4476 is the most useful CK1 inhibitor currently available for identifying physiological substrates of CK1 and is required for accelerated nuclear exclusion of FOXO1a in response to IGF‐1 and insulin.
Abstract: The protein kinase CK1 phosphorylates serine residues that are located close to another phosphoserine in the consensus pSer-Xaa-Xaa-Ser. This specificity generates regions in its target proteins containing two or more neighbouring phosphoserine residues, termed here multisite phosphorylation domains (MPDs). In this paper, we demonstrate that D4476 is a potent and rather selective inhibitor of CK1 in vitro and in cells. In H4IIE hepatoma cells, D4476 specifically inhibits the phosphorylation of endogenous forkhead box transcription factor O1a (FOXO1a) on Ser322 and Ser325 within its MPD, without affecting the phosphorylation of other sites. Our results indicate that these residues are targeted by CK1 in vivo and that the CK1-mediated phosphorylation of the MPD is required for accelerated nuclear exclusion of FOXO1a in response to IGF-1 and insulin. D4476 is much more potent and specific than IC261 or CKI-7, and is therefore the most useful CK1 inhibitor currently available for identifying physiological substrates of CK1.
TL;DR: In humans, heterotrimeric G proteins couple stimulus perception by G‐protein‐coupled receptors (GPCRs) with numerous downstream effectors to drive cell proliferation, hormone perception and ion‐channel regulation.
Abstract: In humans, heterotrimeric G proteins couple stimulus perception by G-protein-coupled receptors (GPCRs) with numerous downstream effectors By contrast, despite great complexity in their signal-transduction attributes, plants have a simpler repertoire of G-signalling components Nonetheless, recent studies on Arabidopsis thaliana have shown the importance of plant G-protein signalling in such fundamental processes as cell proliferation, hormone perception and ion-channel regulation
TL;DR: Unexpectedly, binding of PPD proteins to Dicer inhibits the RNase activity of this enzyme in vitro, indicating that interactions between these two types of proteins may occur in multiple compartments.
Abstract: PAZ PIWI domain (PPD) proteins, together with the RNA cleavage products of Dicer, form ribonucleoprotein complexes called RNA-induced silencing complexes (RISCs). RISCs mediate gene silencing through targeted messenger RNA cleavage and translational suppression. The PAZ domains of PPD and Dicer proteins were originally thought to mediate binding between PPD proteins and Dicer, although no evidence exists to support this theory. Here we show that PAZ domains are not required for PPD protein–Dicer interactions. Rather, a subregion of the PIWI domain in PPD proteins, the PIWI-box, binds directly to the Dicer RNase III domain. Stable binding between PPD proteins and Dicer was dependent on the activity of Hsp90. Unexpectedly, binding of PPD proteins to Dicer inhibits the RNase activity of this enzyme in vitro. Lastly, we show that PPD proteins and Dicer are present in soluble and membrane-associated fractions, indicating that interactions between these two types of proteins may occur in multiple compartments.
TL;DR: In this article, the authors investigated the gene coexpression network in Saccharomyces cerevisiae and derived a new model for its evolution based on the observation that there is a positive correlation between the sequence similarity of paralogues and their probability of coexpression or sharing of transcription factor binding sites (TFBSs).
Abstract: We investigated the gene coexpression network in Saccharomyces cerevisiae, in which genes are linked when they are coregulated. This network is shown to have a scale-free, small-world architecture. Such architecture is typical of biological networks in which the nodes are connected when they are involved in the same biological process. Current models for the evolution of intracellular networks do not adequately reproduce the features that we observe in the network. We therefore derive a new model for its evolution based on the observation that there is a positive correlation between the sequence similarity of paralogues and their probability of coexpression or sharing of transcription factor binding sites (TFBSs). The simple, neutralist's model consists of (1) coduplication of genes with their TFBSs, (2) deletion and duplication of individual TFBSs and (3) gene loss. A network is constructed by connecting genes that share multiple TFBSs. Our model reproduces the scale-free, small-world architecture of the coregulation network and the homology relations between coregulated genes without the need for selection either at the level of the network structure or at the level of gene regulation.
TL;DR: Characterization of selenoproteomes allows interpretation of other UGA codons in completed genomes of prokaryotes as terminators, addressing the UGA dual‐function problem.
Abstract: In the genetic code, the UGA codon has a dual function as it encodes selenocysteine (Sec) and serves as a stop signal. However, only the translation terminator function is used in gene annotation programs, resulting in misannotation of selenoprotein genes. Here, we applied two independent bioinformatics approaches to characterize a selenoprotein set in prokaryotic genomes. One method searched for selenoprotein genes by identifying RNA stem–loop structures, selenocysteine insertion sequence elements; the second approach identified Sec/Cys pairs in homologous sequences. These analyses identified all or almost all selenoproteins in completely sequenced bacterial and archaeal genomes and provided a view on the distribution and composition of prokaryotic selenoproteomes. In addition, lineage-specific and core selenoproteins were detected, which provided insights into the mechanisms of selenoprotein evolution. Characterization of selenoproteomes allows interpretation of other UGA codons in completed genomes of prokaryotes as terminators, addressing the UGA dual-function problem.
TL;DR: It is proposed that, in certain degradation pathways, Dsk2p, Rad23p and the trimeric Cdc48 complex function together in the delivery of ubiquitinated proteins to the proteasome, avoiding malfolded protein aggregates in the cytoplasm.
Abstract: We developed a growth test to screen for yeast mutants defective in endoplasmic reticulum (ER) quality control and associated protein degradation (ERAD) using the membrane protein CTL*, a chimeric derivative of the classical ER degradation substrate CPY*. In a genomic screen of ∼5,000 viable yeast deletion mutants, we identified genes necessary for ER quality control and degradation. Among the new gene products, we identified Dsk2p and Rad23p. We show that these two proteins are probably delivery factors for ubiquitinated ER substrates to the proteasome, following their removal from the membrane via the Cdc48–Ufd1–Npl4p complex. In contrast to the ERAD substrate CTG*, proteasomal degradation of a cytosolic CPY*–GFP fusion is not dependent on Dsk2p and Rad23p, indicating pathway specificity for both proteins. We propose that, in certain degradation pathways, Dsk2p, Rad23p and the trimeric Cdc48 complex function together in the delivery of ubiquitinated proteins to the proteasome, avoiding malfolded protein aggregates in the cytoplasm.
TL;DR: An S. cerevisiae strain is generated in which glucose uptake is dependent on a chimeric hexose transporter mediating reduced sugar uptake, and shows a fully respiratory metabolism also at high glucose levels, illustrating that manipulating a single step can alter the mode of metabolism.
Abstract: The biochemistry of most metabolic pathways is conserved from bacteria to humans, although the control mechanisms are adapted to the needs of each cell type. Oxygen depletion commonly controls the switch from respiration to fermentation. However, Saccharomyces cerevisiae also controls that switch in response to the external glucose level. We have generated an S. cerevisiae strain in which glucose uptake is dependent on a chimeric hexose transporter mediating reduced sugar uptake. This strain shows a fully respiratory metabolism also at high glucose levels as seen for aerobic organisms, and switches to fermentation only when oxygen is lacking. These observations illustrate that manipulating a single step can alter the mode of metabolism. The novel yeast strain is an excellent tool to study the mechanisms underlying glucose-induced signal transduction.
TL;DR: Experimental and theoretical work has demonstrated substantial (order‐of‐magnitude) effects of crowding on a broad range of biochemical, biophysical and physiological processes, including—but not limited to—nucleic acid and protein conformation and stability, protein–protein and protein–DNA association equilibria and kinetics.
Abstract: This workshop took place at the Palacio de Magalia (Las Navas del Marques, Avila, Spain), between 14 and 18 June 2003, and was organized by J. Ellis, A. Minton and G. Rivas. Further details on the workshop can be found at http://www.cib.csic.es/~revers/embo2003/index.htm
![][1]
Macromolecules are present as soluble species and/or structural arrays at total concentrations of up to several hundred grams per litre in essentially all physiological compartments. Although local composition varies widely between different systems, it is evident that most macromolecular reactions and processes in vivo —as opposed to typical experiments in vitro in which the total concentration of macromolecules rarely surpasses 1 g l−1 (Ralston, 1990; Ellis, 2001)—take place in environments in which macromolecules occupy a considerable fraction (between 10% and 40%) of the total volume (Fulton, 1982; Record et al , 1998). These media are termed ‘crowded’ or ‘volume‐occupied’ rather than ‘concentrated’, because no single species of macromolecule is necessarily present at a high concentration. The term ‘macromolecular crowding’ connotes the non‐specific influence of steric repulsions on specific reactions and processes that occur in highly volume‐occupied media.
During recent decades it has gradually become recognized that crowding can considerably alter the reactivity of individual macromolecules, both qualitatively and quantitatively. Crowding can be mimicked experimentally by adding high concentrations of inert synthetic or natural macromolecules, termed crowding agents or crowders, to the system in vitro (Ellis, 2001). Experimental and theoretical work has demonstrated substantial (order‐of‐magnitude) effects of crowding on a broad range of biochemical, biophysical and physiological processes, including—but not limited to—nucleic acid and protein conformation and stability, protein–protein and protein–DNA association equilibria and kinetics (including protein crystallization, protein fibre formation and bundling), catalytic activity of enzymes and cell volume regulation (Zimmerman & Minton, 1993; Minton 1997, 2001; Ellis, 2001).
The fact that biological …
[1]: /embed/graphic-1.gif
TL;DR: The data indicate that under certain conditions, folding of newly synthesized proteins in E. coli is not totally dependent on an interaction with either TF and/or DnaK, and suggest that additional chaperones may be involved in this essential process.
Abstract: Trigger factor (TF) is a ribosome-bound protein that combines catalysis of peptidyl-prolyl isomerization and chaperone-like activities in Escherichia coli. TF was shown to cooperate with the DnaK (Hsp70) chaperone machinery in the folding of newly synthesized proteins, and the double deletion of the corresponding genes (tig and dnaK) exhibited synthetic lethality. We used a detailed genetic approach to characterize various aspects of this functional cooperation in vivo. Surprisingly, we showed that under specific growth conditions, one can delete both dnaK and tig, indicating that bacterial survival can be maintained in the absence of these two major cytosolic chaperones. The strain lacking both DnaK and TF exhibits a very narrow temperature range of growth and a high level of aggregated proteins when compared to either of the single mutants. We found that, in the absence of DnaK, both the N-terminal ribosome-binding domain and the C-terminal domain of unknown function are essential for TF chaperone activity. In contrast, the central PPIase domain is dispensable. Taken together, our data indicate that under certain conditions, folding of newly synthesized proteins in E. coli is not totally dependent on an interaction with either TF and/or DnaK, and suggest that additional chaperones may be involved in this essential process.