Journal of Molecular Biology

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  • Bicistronic mRNAs to Enhance Membrane Protein Overexpression
    [Feb 2015]

    Publication date: 27 February 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 4

    Author(s): Jacopo Marino , Michael Hohl , Markus A. Seeger , Oliver Zerbe , Eric R. Geertsma

    Functional overexpression of membrane proteins is essential for their structural and functional characterization. However, functional overexpression is often difficult to achieve, and frequently either no expression or expression as misfolded aggregates is observed. We present an approach for improving the functional overexpression of membrane proteins in Escherichia coli using transcriptional fusions. The method involves the use of a small additional RNA sequence upstream to the RNA sequence of the target membrane protein and results in the production of a bicistronic mRNA. In contrast to the common approach of translational fusions to enhance protein expression, transcriptional fusions do not require protease treatment and subsequent removal of the fusion protein. Using this strategy, we observed improvements in the quantity and/or the quality of the produced material for several membrane proteins to levels compatible with structural studies. Our analysis revealed that translation of the upstream RNA sequence was not essential for increased expression. Rather, the sequence itself had a large impact on protein yields, suggesting that alternative folding of the transcript was responsible for the observed effect.
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  • Novel Essential Gene Involved in 16S rRNA Processing in Escherichia coli
    [Feb 2015]

    Publication date: 27 February 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 4

    Author(s): Tatsuaki Kurata , Shinobu Nakanishi , Masayuki Hashimoto , Masato Taoka , Yukiko Yamazaki , Toshiaki Isobe , Jun-ichi Kato

    Biogenesis of ribosomes is a complex process mediated by many factors. While its transcription proceeds, ribosomal RNA (rRNA) folds itself into a characteristic three-dimensional structure through interaction with ribosomal proteins, during which its ends are processed. Here, we show that the essential protein YqgF, a RuvC family protein with an RNase-H-like motif, is involved in the processing of pre-16S rRNA during ribosome maturation. Indeed, pre-16S rRNA accumulated in cells of a temperature-sensitive yqgF mutant (yqgF ts) cultured at a non-permissive temperature. In addition, purified YqgF was shown to process the 5′ end of pre-16S rRNA within 70S ribosomes in vitro. Mass spectrometry analysis of the total proteins in the yqgF ts mutant cells showed that the expression of genes containing multiple Shine–Dalgarno-like sequences was observed to be lower than in wild type. These results are interpreted to indicate that YqgF is involved in a novel enzymic activity necessary for the processing of pre-16S rRNA, thereby affecting elongation of translation.
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  • Structural Basis of Dynamic Membrane Recognition by trans-Golgi Network Specific FAPP Proteins
    [Feb 2015]

    Publication date: 27 February 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 4

    Author(s): Marc Lenoir , Michał Grzybek , Michał Majkowski , Sandya Rajesh , Jaswant Kaur , Sara B.-M. Whittaker , Ünal Coskun , Michael Overduin

    Glycosphingolipid metabolism relies on selective recruitment of the pleckstrin homology (PH) domains of FAPP proteins to the trans-Golgi network. The mechanism involved is unclear but requires recognition of phosphatidylinositol-4-phosphate (PI4P) within the Golgi membrane. We investigated the molecular basis of FAPP1-PH domain interactions with PI4P bilayers in liposome sedimentation and membrane partitioning assays. Our data reveals a mechanism in which FAPP-PH proteins preferentially target PI4P-containing liquid disordered membranes, while liquid ordered membranes were disfavored. Additionally, NMR spectroscopy was used to identify the binding determinants responsible for recognizing trans-Golgi network-like bicelles including phosphoinositide and neighboring lipid molecules. Membrane penetration by the FAPP1-PH domain was mediated by an exposed, conserved hydrophobic wedge next to the PI4P recognition site and ringed by a network of complementary polar residues and basic charges. Our data illuminates how insertion of a structured loop provides selectivity for sensing membrane fluidity and targeting to defined membrane zones and organelles. The determinants of this membrane sensing process are conserved across the CERT, OSBP and FAPP family. Hence, lipid gradients not only result in differential membrane ordering along the secretory pathway but also specifically localize diverse proteins through recognition of ensembles of lipid ligands in dynamic and deformable bilayers in order to promote anterograde trafficking.
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  • The s2D Method: Simultaneous Sequence-Based Prediction of the Statistical Populations of Ordered and Disordered Regions in Proteins
    [Feb 2015]

    Publication date: 27 February 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 4

    Author(s): Pietro Sormanni , Carlo Camilloni , Piero Fariselli , Michele Vendruscolo

    Extensive amounts of information about protein sequences are becoming available, as demonstrated by the over 79 million entries in the UniProt database. Yet, it is still challenging to obtain proteome-wide experimental information on the structural properties associated with these sequences. Fast computational predictors of secondary structure and of intrinsic disorder of proteins have been developed in order to bridge this gap. These two types of predictions, however, have remained largely separated, often preventing a clear characterization of the structure and dynamics of proteins. Here, we introduce a computational method to predict secondary-structure populations from amino acid sequences, which simultaneously characterizes structure and disorder in a unified statistical mechanics framework. To develop this method, called s2D, we exploited recent advances made in the analysis of NMR chemical shifts that provide quantitative information about the probability distributions of secondary-structure elements in disordered states. The results that we discuss show that the s2D method predicts secondary-structure populations with an average error of about 14%. A validation on three datasets of mostly disordered, mostly structured and partly structured proteins, respectively, shows that its performance is comparable to or better than that of existing predictors of intrinsic disorder and of secondary structure. These results indicate that it is possible to perform rapid and quantitative sequence-based characterizations of the structure and dynamics of proteins through the predictions of the statistical distributions of their ordered and disordered regions.
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  • Editorial Board
    [Feb 2015]

    Publication date: 13 February 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 3









    Categories: Journal Articles
  • Content List
    [Feb 2015]

    Publication date: 13 February 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 3









    Categories: Journal Articles
  • Why Is Nuclear Organization Dynamic, Hierarchical and Intricate? Implications for Gene Regulation, Cellular Differentiation and Disease
    [Feb 2015]

    Publication date: 13 February 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 3

    Author(s): Marina Ostankovitch , Evi Soutoglou







    Categories: Journal Articles
  • “Breaking Up Is Hard to Do”: The Formation and Resolution of Sister Chromatid Intertwines
    [Feb 2015]

    Publication date: 13 February 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 3

    Author(s): Jonathan Baxter

    The absolute necessity to resolve every intertwine between the two strands of the DNA double helix provides a massive challenge to the cellular processes that duplicate and segregate chromosomes. Although the overwhelming majority of intertwines between the parental DNA strands are resolved during DNA replication, there are numerous chromosomal contexts where some intertwining is maintained into mitosis. These mitotic sister chromatid intertwines (SCIs) can be found as; short regions of unreplicated DNA, fully replicated and intertwined sister chromatids—commonly referred to as DNA catenation—and as sister chromatid linkages generated by homologous recombination-associated processes. Several overlapping mechanisms, including intra-chromosomal compaction, topoisomerase action and Holliday junction resolvases, ensure that all SCIs are removed before they can prevent normal chromosome segregation. Here, I discuss why some DNA intertwines persist into mitosis and review our current knowledge of the SCI resolution mechanisms that are employed in both prokaryotes and eukaryotes, including how deregulating SCI formation during DNA replication or disrupting the resolution processes may contribute to aneuploidy in cancer.
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  • Chromatin-Driven Behavior of Topologically Associating Domains
    [Feb 2015]

    Publication date: 13 February 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 3

    Author(s): Filippo Ciabrelli , Giacomo Cavalli

    Metazoan genomes are highly organized inside the cell nucleus. Topologically associating domains (TADs) represent the building blocks of genome organization, but their linear modularity does not explain alone their spatial organization. Indeed, the chromatin type adorning a TAD can shape its structure and drives its nuclear positioning and its function. Genome-wide association studies revealed mainly four chromatin types: active chromatin, Polycomb-repressed chromatin, null chromatin and constitutive heterochromatin. In this review, we will describe the main three-dimensional features of each chromatin type and finally their relationships with TAD organization and epigenetic memory
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  • Reshaping Chromatin after DNA Damage: The Choreography of Histone Proteins
    [Feb 2015]

    Publication date: 13 February 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 3

    Author(s): Sophie E. Polo

    DNA damage signaling and repair machineries operate in a nuclear environment where DNA is wrapped around histone proteins and packaged into chromatin. Understanding how chromatin structure is restored together with the DNA sequence during DNA damage repair has been a topic of intense research. Indeed, chromatin integrity is central to cell functions and identity. However, chromatin shows remarkable plasticity in response to DNA damage. This review presents our current knowledge of chromatin dynamics in the mammalian cell nucleus in response to DNA double strand breaks and UV lesions. I provide an overview of the key players involved in regulating histone dynamics in damaged chromatin regions, focusing on histone chaperones and their concerted action with histone modifiers, chromatin remodelers and repair factors. I also discuss how these dynamics contribute to reshaping chromatin and, by altering the chromatin landscape, may affect the maintenance of epigenetic information.
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  • INO80-C and SWR-C: Guardians of the Genome
    [Feb 2015]

    Publication date: 13 February 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 3

    Author(s): Christian-Benedikt Gerhold , Michael H. Hauer , Susan M. Gasser

    The double membrane of the eukaryotic nucleus surrounds the genome, constraining it to a nuclear sphere. Proteins, RNA protein particles and artificial chromosome rings diffuse rapidly and freely throughout the nucleoplasm, while chromosomal loci show subdiffusive movement with varying degrees of constraint. In situ biochemical approaches and live imaging studies have revealed the existence of nuclear subcompartments that are enriched for specific chromatin states and/or enzymatic activities. This sequestration is thought to enhance the formation of heterochromatin, particularly when factors of limited abundance are involved. Implicit in the concept of compartmentation is the idea that chromatin is able to move from one compartment to another. Indeed, in budding yeast, gene activation, repression and the presence of persistent DNA double-strand breaks each has been shown to provoke subnuclear relocalization of chromatin. In some cases, movement has been linked to the action of ATP-dependent chromatin remodeling complexes, more specifically to the Snf2-related ATPase-containing complexes, SWR-C and INO80-C. Here we examine how these multi-subunit remodelers contribute to chromatin-based processes linked to the DNA damage response. We review recent evidence that supports a role for yeast SWR-C and INO80-C in determining the subnuclear position of damaged domains and finally, we recap the multiple ways in which these remodelers contribute to genomic integrity.
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  • DSB (Im)mobility and DNA Repair Compartmentalization in Mammalian Cells
    [Feb 2015]

    Publication date: 13 February 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 3

    Author(s): Charlène Lemaître , Evi Soutoglou

    Chromosomal translocations are considered as causal in approximately 20% of cancers. Therefore, understanding their mechanisms of formation is crucial in the prevention of carcinogenesis. The first step of translocation formation is the concomitant occurrence of double-strand DNA breaks (DSBs) in two different chromosomes. DSBs can be repaired by different repair mechanisms, including error-free homologous recombination (HR), potentially error-prone non-homologous end joining (NHEJ) and the highly mutagenic alternative end joining (alt-EJ) pathways. Regulation of DNA repair pathway choice is crucial to avoid genomic instability. In yeast, DSBs are mobile and can scan the entire nucleus to be repaired in specialized DNA repair centers or if they are persistent, in order to associate with the nuclear pores or the nuclear envelope where they can be repaired by specialized repair pathways. DSB mobility is limited in mammals; therefore, raising the question of whether the position at which a DSB occurs influences its repair. Here, we review the recent literature addressing this question. We first present the reports describing the extent of DSB mobility in mammalian cells. In a second part, we discuss the consequences of non-random gene positioning on chromosomal translocations formation. In the third part, we discuss the mobility of heterochromatic DSBs in light of our recent data on DSB repair at the nuclear lamina, and finally, we show that DSB repair compartmentalization at the nuclear periphery is conserved from yeast to mammals, further pointing to a role for gene positioning in the outcome of DSB repair. When regarded as a whole, the different studies reviewed here demonstrate the importance of nuclear architecture on DSB repair and reveal gene positioning as an important parameter in the study of tumorigenesis.





    Categories: Journal Articles
  • Nuclear Organization Changes and the Epigenetic Silencing of FLC during Vernalization
    [Feb 2015]

    Publication date: 13 February 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 3

    Author(s): Danling Zhu , Stefanie Rosa , Caroline Dean

    Changes in nuclear organization are considered an important complement to trans-acting factors, histone modifications and non-coding RNAs in robust and stable epigenetic silencing. However, how these multiple layers interconnect mechanistically to reinforce each other's activity is still unclear. A system providing long timescales facilitating analysis of these interconnections is vernalization. This involves the Polycomb-mediated epigenetic silencing of flowering locus C (FLC) that occurs as Arabidopsis plants are exposed to prolonged cold. Analysis of changes in nuclear organization during vernalization has revealed that disruption of a gene loop and physical clustering of FLC loci are part of the vernalization mechanism. These events occur at different times and thus contribute to distinct aspects of the silencing mechanism. The physical clustering of FLC loci is tightly correlated with the accumulation of specific Polycomb complexes/H3K27me3 at a localized intragenic site during the cold. Since the quantitative nature of vernalization is a reflection of a bistable cell autonomous switch in an increasing number of cells, this correlation suggests a tight connection between the switching mechanism and changes in nuclear organization. This integrated picture is likely to be informative for many epigenetic mechanisms.
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  • Chromatin Dynamics during Differentiation of Myeloid Cells
    [Feb 2015]

    Publication date: 13 February 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 3

    Author(s): Jörg Schönheit , Achim Leutz , Frank Rosenbauer

    Cellular commitment to differentiation requires a tightly synchronized, spatial–temporal interaction of regulatory proteins with the basic DNA and chromatin. A complex network of mechanisms involving induction of lineage instructive transcription factors, installation or removal of histone modifications and changes in the DNA methylation pattern locally orchestrate the three-dimensional chromatin structure and determine cell fate. Maturation of myeloid lineages from hematopoietic stem cells has emerged as a powerful model to study those principles of chromatin mechanisms in cellular differentiation and lineage fate selection. This review summarizes recent knowledge and puts forward novel ideas on how dynamics in the epigenetic landscape of myeloid cells shape the development, immune activation and leukemic transformation outcome.
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  • Regulation of HIV-1 Latency by Chromatin Structure and Nuclear Architecture
    [Feb 2015]

    Publication date: 13 February 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 3

    Author(s): Marina Lusic , Mauro Giacca

    Current antiretroviral therapies fail to cure HIV-1 (human immunodeficiency virus type 1) infection because HIV-1 persists as a transcriptionally inactive provirus in resting memory CD4+ T cells. Multiple molecular events are known to regulate HIV-1 gene expression, yet the mechanisms governing the establishment and maintenance of latency remain incompletely understood. Here we summarize different molecular aspects of viral latency, from its establishment in resting CD4+ T cells to the mechanisms involved in the reactivation of latent viral reservoirs. We focus on the relevance of chromatin structure and nuclear architecture in determining the transcriptional fate of integrated HIV-1 genomes, in light of recent findings indicating that proximity to specific subnuclear neighborhoods regulates HIV-1 gene expression.
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  • Cytoskeletal Control of Nuclear Morphology and Chromatin Organization
    [Feb 2015]

    Publication date: 13 February 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 3

    Author(s): Nisha M. Ramdas , G.V. Shivashankar

    The nucleus is sculpted toward various morphologies during cellular differentiation and development. Alterations in nuclear shape often result in changes to chromatin organization and genome function. This is thought to be reflective of its role as a cellular mechanotransducer. Recent evidence has highlighted the importance of cytoskeletal organization in defining how nuclear morphology regulates chromatin dynamics. However, the mechanisms underlying cytoskeletal control of chromatin remodeling are not well understood. We demonstrate here the differential influence of perinuclear actin- and microtubule-driven assemblies on nuclear architecture using pharmacological inhibitors and targeted RNA interference knockdown of cytoskeleton components in Drosophila cells. We find evidence that the loss of perinuclear actin assembly results in basolateral enhancement of microtubule organization and this is reflected functionally by enhanced nuclear dynamics. Cytoskeleton reorganization leads to nuclear lamina deformation that influences heterochromatin localization and core histone protein mobility. We also show that modulations in actin–microtubule assembly result in differential gene expression patterns. Taken together, we suggest that perinuclear actin and basolateral microtubule organization exerts mechanical control on nuclear morphology and chromatin dynamics.
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  • Synonymous Codon Usage Affects the Expression of Wild Type and F508del CFTR
    [Feb 2015]

    Publication date: Available online 10 February 2015
    Source:Journal of Molecular Biology

    Author(s): Kalpit Shah , Yi Cheng , Brian Hahn , Robert Bridges , Neil Bradbury , David M. Mueller

    The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel composed of 1480 amino acids. The major mutation responsible for cystic fibrosis results in loss of amino acid residue, F508, (F508del). Loss of F508 in CFTR alters the folding pathway resulting in endoplasmic reticulum associated degradation (ERAD). This study investigates the role of synonymous codon in the expression of CFTR and CFTR F508del in human HEK293 cells. DNA encoding the open reading frame (ORF) for CFTR containing synonymous codon replacements, were expressed using a heterologous vector integrated into the genome. The results indicate that the codon usage greatly affects the expression of CFTR. While the promoter strength driving expression of the ORFs was largely unchanged and the mRNA half-lives were unchanged, the steady state levels of the mRNA varied by as much as 30 fold. Experiments support that this apparent inconsistency is attributed to exon junction complex independent nonsense mediated decay. The ratio of CFTR/mRNA indicates that mRNA containing native codons was more efficient in expressing mature CFTR as compared to mRNA containing synonymous high expression codons. However, when F508del CFTR was expressed after codon optimization, a greater percentage of the protein escaped ERAD resulting in considerable levels of mature F508del CFTR on the plasma membrane, which showed channel activity. These results indicate that for CFTR, codon usage has an effect on mRNA levels, protein expression and likely, for F508del CFTR, chaperone assisted folding pathway.
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    Categories: Journal Articles
  • A First Line of Stress Defence: Small Heat Shock Proteins and their function in protein homeostasis
    [Feb 2015]

    Publication date: Available online 10 February 2015
    Source:Journal of Molecular Biology

    Author(s): Martin Haslbeck , Elizabeth Vierling

    Small heat shock proteins (sHsps) are virtually ubiquitous molecular chaperones that can prevent the irreversible aggregation of denaturing proteins. To maintain protein homeostasis, sHsps complex with a variety of nonnative proteins in an ATP-independent manner and, in the context of the stress response, form a first line of defense against protein aggregation. In vertebrates they act to maintain the clarity of the eye lens, and in humans sHsp mutations are linked to myopathies and neuropathies. Although found in all domains of life, sHsps are quite diverse and have evolved independently in metazoans, plants and fungi. sHsp monomers range in size from approximately 12 to 42kDa and are defined by a conserved β-sandwich α-crystallin domain, flanked by variable N- and C-terminal sequences. Most sHsps form large oligomeric ensembles with a broad distribution of different, sphere- or barrel like oligomers, with the size and structure of the oligomers dictated by features of the N- and C-termini. The activity of sHsps is regulated by mechanisms that change the equilibrium distribution in tertiary features and/or quaternary structure of the sHsp ensembles. Cooperation and/or coassembly between different sHsps in the same cellular compartment adds an underexplored level of complexity to sHsp structure and function.
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  • Translation elongation factor EF-Tu modulates filament formation of actin-like MreB protein in vitro
    [Feb 2015]

    Publication date: Available online 10 February 2015
    Source:Journal of Molecular Biology

    Author(s): Hervé Joël Defeu Soufo , Christian Reimold , Hannes Breddermann , Hans G. Mannherz , Peter L. Graumann

    EF-Tu has been shown to interact with actin-like protein MreB and to affect its localization in Escherichia coli and in Bacillus subtilis cells. We have purified YFP-MreB in an active form, which forms filaments on glass slides in vitro, and was active in dynamic light scattering assays, polymerizing in milliseconds after addition of magnesium. Purified EF-Tu enhanced the amount of MreB filaments, as seen by sedimentation assays, the speed of filament formation and the length of MreB filaments in vitro. EF-Tu had the strongest impact on MreB filaments in a 1:1 ratio, and EF-Tu co-sedimented with MreB filaments, revealing a stoichiometric interaction between both proteins. This was supported by cross-linking assays where 1:1 species were well detectable. When expressed in E. coli cells, B. subtilis MreB formed filaments and induced the formation of colocalizing B. subtilis EF-Tu structures, indicating that MreB can direct the positioning of EF-Tu structures in a heterologous cell system. FRAP analysis showed that MreB filaments have a higher turnover in B. subtilis cells than in E. coli cells, indicating different filament kinetics in homologous or heterologous cell systems. The data show that MreB can direct the localization of EF-Tu in vivo, which in turn positively affects the formation and dynamics of MreB filaments. Thus, EF-Tu is a modulator of the activity of a bacterial actin-like protein.
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  • Prolyl isomerization and its catalysis in protein folding and protein function
    [Feb 2015]

    Publication date: Available online 9 February 2015
    Source:Journal of Molecular Biology

    Author(s): Philipp A.M. Schmidpeter , Franz X. Schmid

    Prolyl isomerizations are intrinsically slow processes. They determine the rates of many protein folding reactions and control regulatory events in folded proteins. Prolyl isomerases are able to catalyze these isomerizations, and thus they have the potential to assist protein folding and to modulate protein function. Here we provide examples for how prolyl isomerizations limit protein folding and are accelerated by prolyl isomerases, and how native-state prolyl isomerizations regulate protein functions. The roles of prolines in protein folding and protein function are closely interrelated because they both depend on the coupling between cis/trans isomerization and conformational changes that can involve extended regions of a protein.
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    Categories: Journal Articles