Journal of Molecular Biology

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  • Integrating –omics: systems biology as explored through C. elegans research
    [Apr 2015]

    Publication date: Available online 1 April 2015
    Source:Journal of Molecular Biology

    Author(s): Roel Van Assche , Valérie Broeckx , Kurt Boonen , Evelyne Maes , Wouter De Haes , Liliane Schoofs , Liesbet Temmerman

    -Omics data have become indispensable to systems biology, which aims to describe the full complexity of functional cells, tissues, organs and organisms. Generating vast amounts of data via such methods, researchers have invested in ways of handling and interpreting these. From the large volumes of -omics data that have been gathered over the years, it is clear that the information derived from one -ome is usually far from complete. Now, individual techniques and methods for integration are maturing to the point that researchers can focus on network-based integration, rather than simply interpreting single-ome studies. This review evaluates the application of integrated -omics approaches with a focus on C. elegans studies, intending to direct researchers in this field to useful databases and inspiring examples.
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    Categories: Journal Articles
  • Unwinding the Mechanisms of a DEAD-Box RNA Helicase in Cancer
    [Apr 2015]

    Publication date: Available online 30 March 2015
    Source:Journal of Molecular Biology

    Author(s): Rick Russell







    Categories: Journal Articles
  • Functional Characterization of a Conserved Archaeal Viral Operon Revealing Single-Stranded DNA Binding, Annealing and Nuclease Activities
    [Apr 2015]

    Publication date: Available online 29 March 2015
    Source:Journal of Molecular Biology

    Author(s): Yang Guo , Birthe B. Kragelund , Malcolm F. White , Xu Peng

    The majority of archaeal viral genes are of unknown function hindering our understanding of the virus life cycle and viral interactions with their host. Here, we first describe functional characterization of ORF131b (gp17) and ORF436 (gp18) of Sulfolobus islandicus rod-shaped virus 2 (SIRV2), both encoding proteins of unknown function and forming an operon with ORF207 (gp19). SIRV2 gp17 was found to be a single-stranded DNA (ssDNA) binding protein different in structure from all previously characterized ssDNA binding proteins. Mutagenesis of a few conserved basic residues suggested a U-shaped binding path for ssDNA. The recombinant gp18 showed an ssDNA annealing activity often associated with helicases and recombinases. To gain insight into the biological role of the entire operon, we characterized SIRV2 gp19 and showed it to possess a 5′→3′ ssDNA exonuclease activity, in addition to the previously demonstrated ssDNA endonuclease activity. Further, in vitro pull-down assay demonstrated interactions between gp17 and gp18 and between gp18 and gp19 with the former being mediated by the intrinsically disordered C-terminus of gp17. The strand-displacement replication mode proposed previously for rudiviruses and the close interaction among the ssDNA binding, annealing and nuclease proteins strongly point to a role of the gene operon in genome maturation and/or DNA recombination that may function in viral DNA replication/repair.
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    Categories: Journal Articles
  • An ATPase-Deficient Variant of the SNF2 Family Member HELLS Shows Altered Dynamics at Pericentromeric Heterochromatin
    [Apr 2015]

    Publication date: Available online 28 March 2015
    Source:Journal of Molecular Biology

    Author(s): Cristiana Lungu , Kathrin Muegge , Albert Jeltsch , Renata Z. Jurkowska

    The HELLS (helicase, lymphoid specific, also known as lymphoid-specific helicase) protein is related to the SNF2 (sucrose non-fermentable 2) family of chromatin remodeling ATPases. It is required for efficient DNA methylation in mammals, particularly at heterochromatin-located repetitive sequences. In this study, we investigated the interaction of HELLS with chromatin and used an ATPase-deficient HELLS variant to address the role of ATP hydrolysis in this process. Chromatin fractionation experiments demonstrated that, in the absence of the ATPase activity, HELLS is retained at the nuclear matrix compartment, defined in part by lamin B1. Microscopy studies revealed a stronger association of the ATPase-deficient mutant with heterochromatin. These results were further supported by fluorescence recovery after photobleaching measurements, which showed that, at heterochromatic sites, wild-type HELLS is very dynamic, with a recovery half-time of 0.8s and a mobile protein fraction of 61%. In contrast, the ATPase-deficient mutant displayed 4.5-s recovery half-time and a reduction in the mobile fraction to 30%. We also present evidence suggesting that, in addition to the ATPase activity, a functional H3K9me3 signaling pathway contributes to an efficient release of HELLS from pericentromeric chromatin. Overall, our results show that a functional ATPase activity is not required for the recruitment of HELLS to heterochromatin, but it is important for the release of the enzyme from these sites.
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    Categories: Journal Articles
  • Editorial Board
    [Apr 2015]

    Publication date: 27 March 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 6, Part A









    Categories: Journal Articles
  • Editorial Board
    [Apr 2015]

    Publication date: 27 March 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 6, Part B









    Categories: Journal Articles
  • Contents List
    [Apr 2015]

    Publication date: 27 March 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 6, Part B









    Categories: Journal Articles
  • Contents List
    [Apr 2015]

    Publication date: 27 March 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 6, Part A









    Categories: Journal Articles
  • By Any Means Necessary: Discovering a Bcl-xL Specific Domain
    [Apr 2015]

    Publication date: 27 March 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 6, Part B

    Author(s): Kurt D. Deshayes







    Categories: Journal Articles
  • Editorial Overview: Elucidation of Protein Translocation Pathways, Part II
    [Apr 2015]

    Publication date: 27 March 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 6, Part A

    Author(s): Dejana Mokranjac , Stephen High







    Categories: Journal Articles
  • Potent and Specific Peptide Inhibitors of Human Pro-Survival Protein Bcl-xL
    [Apr 2015]

    Publication date: 27 March 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 6, Part B

    Author(s): Sanjib Dutta , Jeremy Ryan , T. Scott Chen , Christos Kougentakis , Anthony Letai , Amy E. Keating

    The Bcl-2 family of proteins plays a critical role regulating apoptosis, and pro-survival Bcl-2 family members are important therapeutic targets due to their overexpression in different cancers. Pro-apoptotic Bcl-2 homology 3 (BH3)-only proteins antagonize pro-survival Bcl-2 protein functions by binding directly to them, and a sub-class of BH3-only proteins termed sensitizers can initiate apoptosis via this mechanism in response to diverse signals. The five pro-survival proteins Bcl-xL, Mcl-1, Bcl-2, Bcl-w and Bfl-1 differ in their binding preferences, with Bcl-xL, Bcl-2 and Bcl-w sharing similar interaction profiles for many natural sensitizers and small molecules. Peptides that bind selectively to just one or a subset of family members have shown utility in assays that diagnose apoptotic blockades in cancer cells and as reagents for dissecting apoptotic mechanism. Combining computational design, combinatorial library screening and rational mutagenesis, we designed a series of BH3 sensitizer peptides that bind Bcl-xL with sub-nanomolar affinity and selectivity up to 1000-fold over each of the four competing pro-survival proteins. We demonstrate the efficacy of our designed BH3 peptides in assays that differentiate between cancer cells that are dependent on different pro-survival proteins.
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    Categories: Journal Articles
  • A Perspective on Transport of Proteins into Mitochondria: A Myriad of Open Questions
    [Apr 2015]

    Publication date: 27 March 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 6, Part A

    Author(s): Walter Neupert

    Mitochondria are the central hub of key cellular processes such as energy conversion, cell signaling, cell cycle regulation and cell differentiation. Therefore, in particular, mitochondrial biogenesis and protein translocation have been the focus of intense research for now nearly half a century. In spite of remarkable progress the field has made, many of the proposed mechanisms remain controversial and none of the translocation pathways is yet understood at the high-resolution level. In this context, the present article is intended to identify and discuss current major open questions and unresolved issues in the field in hope that it will stimulate and engage the pursuit of current efforts and expose new directions.
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    Categories: Journal Articles
  • Structure of Salmonella FlhE, Conserved Member of a Flagellar Type III Secretion Operon
    [Apr 2015]

    Publication date: 27 March 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 6, Part B

    Author(s): Jaemin Lee , Arthur F. Monzingo , Adrian T. Keatinge-Clay , Rasika M. Harshey

    The bacterial flagellum is assembled by a multicomponent transport apparatus categorized as a type III secretion system. The secretion of proteins that assemble into the flagellum is driven by the proton motive force. The periplasmic protein FlhE is a member of the flhBAE operon in the majority of bacteria where FlhE is found. FlhA and FlhB are established components of the flagellar type III secretion system. The absence of FlhE results in a proton leak through the flagellar system, inappropriate secretion patterns, and cell death, indicating that FlhE regulates an important aspect of proper flagellar biosynthesis. We isolated FlhE from the periplasm of Salmonella and solved its structure to 1.5Å resolution. The structure reveals a β-sandwich fold, with no close structural homologs. Possible roles of FlhE, including that of a chaperone, are discussed.
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    Categories: Journal Articles
  • Protein Transport into the Human Endoplasmic Reticulum
    [Apr 2015]

    Publication date: 27 March 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 6, Part A

    Author(s): Johanna Dudek , Stefan Pfeffer , Po-Hsien Lee , Martin Jung , Adolfo Cavalié , Volkhard Helms , Friedrich Förster , Richard Zimmermann

    Protein transport into the endoplasmic reticulum (ER) is essential for all eukaryotic cells and evolutionary related to protein transport into and across the cytoplasmic membrane of eubacteria and archaea. It is based on amino-terminal signal peptides in the precursor polypeptides plus various transport components in cytosol plus ER and can occur either cotranslationally or posttranslationally. The two mechanisms merge at the heterotrimeric Sec61 complex in the ER membrane, which forms an aqueous polypeptide-conducting channel. Since the mammalian ER is also the main intracellular calcium storage organelle, the Sec61 complex is tightly regulated in its dynamics between the open and closed conformations by various ligands, such as precursor polypeptides at the cytosolic face and the Hsp70-type molecular chaperone BiP at the ER lumenal face (Hsp, heat shock protein). Furthermore, BiP binding to the incoming precursor polypeptide contributes to unidirectionality and efficiency of transport. Recent insights into the structural dynamics of the Sec61 complex and related complexes in eubacteria and archaea have various mechanistic and functional implications.
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    Categories: Journal Articles
  • Multiple Pathways for Protein Transport to Peroxisomes
    [Apr 2015]

    Publication date: 27 March 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 6, Part A

    Author(s): P.K. Kim , E.H. Hettema

    Peroxisomes are unique among the organelles of the endomembrane system. Unlike other organelles that derive most if not all of their proteins from the ER (endoplasmic reticulum), peroxisomes contain dedicated machineries for import of matrix proteins and insertion of membrane proteins. However, peroxisomes are also able to import a subset of their membrane proteins from the ER. One aspect of peroxisome biology that has remained ill defined is the role the various import pathways play in peroxisome maintenance. In this review, we discuss the available data on matrix and membrane protein import into peroxisomes.
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    Categories: Journal Articles
  • Oligosaccharide and Substrate Binding in the Starch Debranching Enzyme Barley Limit Dextrinase
    [Apr 2015]

    Publication date: 27 March 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 6, Part B

    Author(s): Marie S. Møller , Michael S. Windahl , Lyann Sim , Marie Bøjstrup , Maher Abou Hachem , Ole Hindsgaul , Monica Palcic , Birte Svensson , Anette Henriksen

    Complete hydrolytic degradation of starch requires hydrolysis of both the α-1,4- and α-1,6-glucosidic bonds in amylopectin. Limit dextrinase (LD) is the only endogenous barley enzyme capable of hydrolyzing the α-1,6-glucosidic bond during seed germination, and impaired LD activity inevitably reduces the maltose and glucose yields from starch degradation. Crystal structures of barley LD and active-site mutants with natural substrates, products and substrate analogues were sought to better understand the facets of LD–substrate interactions that confine high activity of LD to branched maltooligosaccharides. For the first time, an intact α-1,6-glucosidically linked substrate spanning the active site of a LD or pullulanase has been trapped and characterized by crystallography. The crystal structure reveals both the branch and main-chain binding sites and is used to suggest a mechanism for nucleophilicity enhancement in the active site. The substrate, product and analogue complexes were further used to outline substrate binding subsites and substrate binding restraints and to suggest a mechanism for avoidance of dual α-1,6- and α-1,4-hydrolytic activity likely to be a biological necessity during starch synthesis.
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    Categories: Journal Articles
  • The Code for Directing Proteins for Translocation across ER Membrane: SRP Cotranslationally Recognizes Specific Features of a Signal Sequence
    [Apr 2015]

    Publication date: 27 March 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 6, Part A

    Author(s): IngMarie Nilsson , Patricia Lara , Tara Hessa , Arthur E. Johnson , Gunnar von Heijne , Andrey L. Karamyshev

    The signal recognition particle (SRP) cotranslationally recognizes signal sequences of secretory proteins and targets ribosome-nascent chain complexes to the SRP receptor in the endoplasmic reticulum membrane, initiating translocation of the nascent chain through the Sec61 translocon. Although signal sequences do not have homology, they have similar structural regions: a positively charged N-terminus, a hydrophobic core and a more polar C-terminal region that contains the cleavage site for the signal peptidase. Here, we have used site-specific photocrosslinking to study SRP–signal sequence interactions. A photoreactive probe was incorporated into the middle of wild-type or mutated signal sequences of the secretory protein preprolactin by in vitro translation of mRNAs containing an amber-stop codon in the signal peptide in the presence of the Nε-(5-azido-2 nitrobenzoyl)-Lys-tRNAamb amber suppressor. A homogeneous population of SRP–ribosome-nascent chain complexes was obtained by the use of truncated mRNAs in translations performed in the presence of purified canine SRP. Quantitative analysis of the photoadducts revealed that charged residues at the N-terminus of the signal sequence or in the early part of the mature protein have only a mild effect on the SRP–signal sequence association. However, deletions of amino acid residues in the hydrophobic portion of the signal sequence severely affect SRP binding. The photocrosslinking data correlate with targeting efficiency and translocation across the membrane. Thus, the hydrophobic core of the signal sequence is primarily responsible for its recognition and binding by SRP, while positive charges fine-tune the SRP–signal sequence affinity and targeting to the translocon.
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    Categories: Journal Articles
  • Functional Consequences of the Oligomeric Assembly of Proteorhodopsin
    [Apr 2015]

    Publication date: 27 March 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 6, Part B

    Author(s): Sunyia Hussain , Maia Kinnebrew , Nicole S. Schonenbach , Emily Aye , Songi Han

    The plasma membrane is the crucial interface between the cell and its exterior, packed with embedded proteins experiencing simultaneous protein–protein and protein–membrane interactions. A prominent example of cell membrane complexity is the assembly of transmembrane proteins into oligomeric structures, with potential functional consequences that are not well understood. From the study of proteorhodopsin (PR), a prototypical seven-transmembrane light-driven bacterial proton pump, we find evidence that the inter-protein interaction modulated by self-association yields functional changes observable from the protein interior. We also demonstrate that the oligomer is likely a physiologically relevant form of PR, as crosslinking of recombinantly expressed PR reveals an oligomeric population within the Escherichia coli membrane (putatively hexameric). Upon chromatographic isolation of oligomeric and monomeric PR in surfactant micelles, the oligomer exhibits distinctly different optical absorption properties from monomeric PR, as reflected in a prominent decrease in the pK a of the primary proton acceptor residue (D97) and slowing of the light-driven conformational change. These functional effects are predominantly determined by specific PR–PR contacts over nonspecific surfactant interactions. Interestingly, varying the surfactant type alters the population of oligomeric states and the proximity of proteins within an oligomer, as determined by sparse electron paramagnetic resonance distance measurements. Nevertheless, the dynamic surfactant environment retains the key function-tuning property exerted by oligomeric contacts. A potentially general design principle for transmembrane protein function emerges from this work, one that hinges on specific oligomeric contacts that can be modulated by protein expression or membrane composition.
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    Categories: Journal Articles
  • Unconventional Secretion of Fibroblast Growth Factor 2—A Novel Type of Protein Translocation across Membranes?
    [Apr 2015]

    Publication date: 27 March 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 6, Part A

    Author(s): Julia P. Steringer , Hans-Michael Müller , Walter Nickel

    N-terminal signal peptides are a hallmark of the vast majority of soluble secretory proteins that are transported along the endoplasmic reticulum/Golgi-dependent pathway. They are recognized by signal recognition particle, a process that initiates membrane translocation into the lumen of the endoplasmic reticulum followed by vesicular transport to the cell surface and release into the extracellular space. Beyond this well-established mechanism of protein secretion from eukaryotic cells, a number of extracellular proteins with critical physiological functions in immune surveillance and tissue organization are known to be secreted in a manner independent of signal recognition particle. Such processes have collectively been termed “unconventional protein secretion” and, while known for more than two decades, their underlying mechanisms are only beginning to emerge. Different types of unconventional secretory mechanisms have been described with the best-characterized example being based on direct translocation of cytoplasmic proteins across plasma membranes. The aim of this review is to critically assess our current knowledge of this type of unconventional secretion focusing on fibroblast growth factor 2 (FGF2) as the most established example.
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    Categories: Journal Articles
  • Contributions of the TEL-patch Amino Acid Cluster on TPP1 to Telomeric DNA Synthesis by Human Telomerase
    [Apr 2015]

    Publication date: 27 March 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 6, Part B

    Author(s): Andrew B. Dalby , Ctirad Hofr , Thomas R. Cech

    Telomere maintenance is a highly coordinated process, and its misregulation is linked to cancer and telomere-shortening syndromes. Recent studies have shown that the TEL-patch—a cluster of amino acids on the surface of the shelterin component TPP1—is necessary for the recruitment of telomerase to the telomere in human cells. However, there has been only basic biochemical analysis of the role of TPP1 in the telomerase recruitment process. Here we develop an in vitro assay to quantitatively measure the contribution of the TEL-patch to telomerase recruitment—binding and extension of the first telomeric repeat. We also demonstrate that the TEL-patch contributes to the translocation step of the telomerase reaction. Finally, our quantitative observations indicate that the TEL-patch stabilizes the association between telomerase and telomeric DNA substrates, providing a molecular explanation for its contributions to telomerase recruitment and action.
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    Categories: Journal Articles