Journal of Molecular Biology

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  • Chromatin Structure and Replication Origins: Determinants of Chromosome Replication and Nuclear Organization
    [Oct 2014]

    Publication date: 9 October 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 20

    Author(s): Owen K. Smith , Mirit I. Aladjem

    The DNA replication program is, in part, determined by the epigenetic landscape that governs local chromosome architecture and directs chromosome duplication. Replication must coordinate with other biochemical processes occurring concomitantly on chromatin, such as transcription and remodeling, to insure accurate duplication of both genetic and epigenetic features and to preserve genomic stability. The importance of genome architecture and chromatin looping in coordinating cellular processes on chromatin is illustrated by two recent sets of discoveries. First, chromatin-associated proteins that are not part of the core replication machinery were shown to affect the timing of DNA replication. These chromatin-associated proteins could be working in concert, or perhaps in competition, with the transcriptional machinery and with chromatin modifiers to determine the spatial and temporal organization of replication initiation events. Second, epigenetic interactions are mediated by DNA sequences that determine chromosomal replication. In this review, we summarize recent findings and current models linking spatial and temporal regulation of the replication program with epigenetic signaling. We discuss these issues in the context of the genome's three-dimensional structure with an emphasis on events occurring during the initiation of DNA replication.
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  • Genome-Scale Acetylation-Dependent Histone Eviction during Spermatogenesis
    [Oct 2014]

    Publication date: 9 October 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 20

    Author(s): Afsaneh Goudarzi , Hitoshi Shiota , Sophie Rousseaux , Saadi Khochbin

    A genome-wide histone hyperacetylation is known to occur in the absence of transcription in haploid male germ cells, spermatids, before and during the global histone eviction and their replacement by non-histone DNA-packaging proteins. Although the occurrence of this histone hyperacetylation has been correlated with histone removal for a long time, the underlying mechanisms have remained largely obscure. Important recent discoveries have not only shed light on how histone acetylation could drive a subsequent transformation in genome organization but also revealed that the associated nucleosome dismantlement is a multi-step process, requiring the contribution of histone variants, critical destabilizing histone modifications and chromatin readers, including Brdt, working together to achieve the full packaging of the male genome, indispensable for the propagation of life.
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  • Emerging Technologies to Map the Protein Methylome
    [Oct 2014]

    Publication date: 9 October 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 20

    Author(s): Scott M. Carlson , Or Gozani

    Protein methylation plays an integral role in cellular signaling, most notably by modulating proteins bound at chromatin and increasingly through regulation of non-histone proteins. One central challenge in understanding how methylation acts in signaling is identifying and measuring protein methylation. This includes locus-specific modification of histones, on individual non-histone proteins, and globally across the proteome. Protein methylation has been studied traditionally using candidate approaches such as methylation-specific antibodies, mapping of post-translational modifications by mass spectrometry, and radioactive labeling to characterize methylation on target proteins. Recent developments have provided new approaches to identify methylated proteins, measure methylation levels, identify substrates of methyltransferase enzymes, and match methylated proteins to methyl-specific reader domains. Methyl-binding protein domains and improved antibodies with broad specificity for methylated proteins are being used to characterize the “protein methylome”. They also have the potential to be used in high-throughput assays for inhibitor screens and drug development. These tools are often coupled to improvements in mass spectrometry to quickly identify methylated residues, as well as to protein microarrays, where they can be used to screen for methylated proteins. Finally, new chemical biology strategies are being used to probe the function of methyltransferases, demethylases, and methyl-binding “reader” domains. These tools create a “system-level” understanding of protein methylation and integrate protein methylation into broader signaling processes.
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  • Keeping Them All Together: β-Propeller Domains in Histone Methyltransferase Complexes
    [Oct 2014]

    Publication date: 9 October 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 20

    Author(s): Elisa Bergamin , Alexandre Blais , Jean-François Couture

    Histone methyltransferases (HKMTs) residing in multi-subunit protein complexes frequently require the presence of β-propeller proteins to achieve their biological functions. Recent biochemical studies have highlighted the functional diversity of these scaffolding proteins in maintaining the integrity of the complexes, allosterically regulating HKMT enzymatic activity and acting as “histone tethering devices” to facilitate the interaction between HKMTs and their substrates. Structural studies have revealed that, while β-propeller domain proteins share structural similarity, they employ divergent mechanisms to achieve their functions. This review focuses on the progress made in the last decade to identify the biochemical determinants underlying the functions of these important proteins.
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  • Chromatin Regulation of DNA Damage Repair and Genome Integrity in the Central Nervous System
    [Oct 2014]

    Publication date: 9 October 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 20

    Author(s): Ling Pan , Jay Penney , Li-Huei Tsai

    With the continued extension of lifespan, aging and age-related diseases have become a major medical challenge to our society. Aging is accompanied by changes in multiple systems. Among these, the aging process in the central nervous system is critically important but very poorly understood. Neurons, as post-mitotic cells, are devoid of replicative associated aging processes, such as senescence and telomere shortening. However, because of the inability to self-replenish, neurons have to withstand challenge from numerous stressors over their lifetime. Many of these stressors can lead to damage of the neurons' DNA. When the accumulation of DNA damage exceeds a neuron's capacity for repair, or when there are deficiencies in DNA repair machinery, genome instability can manifest. The increased mutation load associated with genome instability can lead to neuronal dysfunction and ultimately to neuron degeneration. In this review, we first briefly introduce the sources and types of DNA damage and the relevant repair pathways in the nervous system (summarized in Fig. 1). We then discuss the chromatin regulation of these processes and summarize our understanding of the contribution of genomic instability to neurodegenerative diseases.
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  • Epigenetic Signaling in Psychiatric Disorders
    [Oct 2014]

    Publication date: 9 October 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 20

    Author(s): Catherine J. Peña , Rosemary C. Bagot , Benoit Labonté , Eric J. Nestler

    Psychiatric disorders are complex multifactorial illnesses involving chronic alterations in neural circuit structure and function. While genetic factors are important in the etiology of disorders such as depression and addiction, relatively high rates of discordance among identical twins clearly indicate the importance of additional mechanisms. Environmental factors such as stress or prior drug exposure are known to play a role in the onset of these illnesses. Such exposure to environmental insults induces stable changes in gene expression, neural circuit function, and ultimately behavior, and these maladaptations appear distinct between developmental and adult exposures. Increasing evidence indicates that these sustained abnormalities are maintained by epigenetic modifications in specific brain regions. Indeed, transcriptional dysregulation and associated aberrant epigenetic regulation is a unifying theme in psychiatric disorders. Aspects of depression and addiction can be modeled in animals by inducing disease-like states through environmental manipulations (e.g., chronic stress, drug administration). Understanding how environmental factors recruit the epigenetic machinery in animal models reveals new insight into disease mechanisms in humans.
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  • Structural Insights into Estrogen Receptor α Methylation by Histone Methyltransferase SMYD2, a Cellular Event Implicated in Estrogen Signaling Regulation
    [Oct 2014]

    Publication date: 9 October 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 20

    Author(s): Yuanyuan Jiang , Laura Trescott , Joshua Holcomb , Xi Zhang , Joseph Brunzelle , Nualpun Sirinupong , Xiaobing Shi , Zhe Yang

    Estrogen receptor (ER) signaling plays a pivotal role in many developmental processes and has been implicated in numerous diseases including cancers. We recently showed that direct ERα methylation by the multi-specificity histone lysine methyltransferase SMYD2 regulates estrogen signaling through repressing ERα-dependent transactivation. However, the mechanism controlling the specificity of the SMYD2–ERα interaction and the structural basis of SMYD2 substrate binding diversity are unknown. Here we present the crystal structure of SMYD2 in complex with a target lysine (Lys266)-containing ERα peptide. The structure reveals that ERα binds SMYD2 in a U-shaped conformation with the binding specificity determined mainly by residues C-terminal to the target lysine. The structure also reveals numerous intrapeptide contacts that ensure shape complementarity between the substrate and the active site of the enzyme, thereby likely serving as an additional structural determinant of substrate specificity. In addition, comparison of the SMYD2–ERα and SMYD2–p53 structures provides the first structural insight into the diverse nature of SMYD2 substrate recognition and suggests that the broad specificity of SMYD2 is achieved by multiple molecular mechanisms such as distinct peptide binding modes and the intrinsic dynamics of peptide ligands. Strikingly, a novel potentially SMYD2-specific polyethylene glycol binding site is identified in the CTD domain, implicating possible functions in extended substrate binding or protein–protein interactions. Our study thus provides the structural basis for the SMYD2-mediated ERα methylation, and the resulting knowledge of SMYD2 substrate specificity and target binding diversity could have important implications in selective drug design against a wide range of ERα-related diseases.
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  • Histone Methyltransferase EZH2 Is Transcriptionally Induced by Estradiol as Well as Estrogenic Endocrine Disruptors Bisphenol-A and Diethylstilbestrol
    [Oct 2014]

    Publication date: 9 October 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 20

    Author(s): Arunoday Bhan , Imran Hussain , Khairul I. Ansari , Samara A.M. Bobzean , Linda I. Perrotti , Subhrangsu S. Mandal

    Enhancer of Zeste homolog 2 (EZH2), a methyltransferase specific to histone 3 lysine 27, is a critical player in gene silencing and is overexpressed in breast cancer. Our studies demonstrate that EZH2 is transcriptionally induced by estradiol in cultured breast cancer cells and in the mammary glands of ovariectomized rats. EZH2 promoter contains multiple functional estrogen-response elements. Estrogen receptors (ERs) and ER coregulators such as mixed lineage leukemia (MLL) histone methylases (MLL2 and MLL3) and histone acetyltransferase CBP/P300 bind to the EZH2 promoter in the presence of estradiol and regulate estradiol-induced EZH2 expression. EZH2 expression is also increased upon exposure to estrogenic endocrine disrupting chemicals (EDCs) such as bisphenol-A (BPA) and diethylstilbestrol (DES). Similar to estradiol, BPA and DES-induced EZH2 expression is coordinated by ERs, MLLs and CBP/P300. In summary, we demonstrate that EZH2 is transcriptionally regulated by estradiol in vitro and in vivo, and its expression is potentially dysregulated upon exposure to estrogenic EDCs.
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  • Molecular Basis for the Antiparasitic Activity of a Mercaptoacetamide Derivative That Inhibits Histone Deacetylase 8 (HDAC8) from the Human Pathogen Schistosoma mansoni
    [Oct 2014]

    Publication date: 9 October 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 20

    Author(s): Diana A. Stolfa , Martin Marek , Julien Lancelot , Alexander-Thomas Hauser , Alexandra Walter , Emeline Leproult , Jelena Melesina , Tobias Rumpf , Jean-Marie Wurtz , Jean Cavarelli , Wolfgang Sippl , Raymond J. Pierce , Christophe Romier , Manfred Jung

    Schistosomiasis, caused by the parasitic flatworm Schistosoma mansoni and related species, is a tropical disease that affects over 200 million people worldwide. A new approach for targeting eukaryotic parasites is to tackle their dynamic epigenetic machinery that is necessary for the extensive phenotypic changes during the life cycle of the parasite. Recently, we identified S. mansoni histone deacetylase 8 (smHDAC8) as a potential target for antiparasitic therapy. Here, we present results on the investigations of a focused set of HDAC (histone deacetylase) inhibitors on smHDAC8. Besides several active hydroxamates, we identified a thiol-based inhibitor that inhibited smHDAC8 activity in the micromolar range with unexpected selectivity over the human isotype, which has not been observed so far. The crystal structure of smHDAC8 complexed with the thiol derivative revealed that the inhibitor is accommodated in the catalytic pocket, where it interacts with both the catalytic zinc ion and the essential catalytic tyrosine (Y341) residue via its mercaptoacetamide warhead. To our knowledge, this is the first complex crystal structure of any HDAC inhibited by a mercaptoacetamide inhibitor, and therefore, this finding offers a rationale for further improvement. Finally, an ester prodrug of the thiol HDAC inhibitor exhibited antiparasitic activity on cultured schistosomes in a dose-dependent manner.
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  • The Gene Silencing Transcription Factor REST Represses miR-132 Expression in Hippocampal Neurons Destined to Die
    [Oct 2014]

    Publication date: 9 October 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 20

    Author(s): Jee-Yeon Hwang , Naoki Kaneko , Kyung-Min Noh , Fabrizio Pontarelli , R. Suzanne Zukin

    The gene silencing transcription factor REST [repressor element 1 silencing transcription factor]/NRSF (neuron-restrictive silencer factor) actively represses a large array of coding and noncoding neuron-specific genes important to synaptic plasticity including miR-132. miR-132 is a neuron-specific microRNA and plays a pivotal role in synaptogenesis, synaptic plasticity and structural remodeling. However, a role for miR-132 in neuronal death is not, as yet, well-delineated. Here we show that ischemic insults promote REST binding and epigenetic remodeling at the miR-132 promoter and silencing of miR-132 expression in selectively vulnerable hippocampal CA1 neurons. REST occupancy was not altered at the miR-9 or miR-124a promoters despite the presence of repressor element 1 sites, indicating REST target specificity. Ischemia induced a substantial decrease in two marks of active gene transcription, dimethylation of lysine 4 on core histone 3 (H3K4me2) and acetylation of lysine 9 on H3 (H3K9ac) at the miR-132 promoter. RNAi-mediated depletion of REST in vivo blocked ischemia-induced loss of miR-132 in insulted hippocampal neurons, consistent with a causal relation between activation of REST and silencing of miR-132. Overexpression of miR-132 in primary cultures of hippocampal neurons or delivered directly into the CA1 of living rats by means of the lentiviral expression system prior to induction of ischemia afforded robust protection against ischemia-induced neuronal death. These findings document a previously unappreciated role for REST-dependent repression of miR-132 in the neuronal death associated with global ischemia and identify a novel therapeutic target for amelioration of the neurodegeneration and cognitive deficits associated with ischemic stroke.
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  • Gene-Specific Methylation Control of H3K9 and H3K36 on Neurotrophic BDNF versus Astroglial GFAP Genes by KDM4A/C Regulates Neural Stem Cell Differentiation
    [Oct 2014]

    Publication date: 9 October 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 20

    Author(s): Anna Cascante , Susanne Klum , Moumita Biswas , Beatriz Antolin-Fontes , Fanie Barnabé-Heider , Ola Hermanson

    Neural stem cell (NSC) state and fate depend on spatially and temporally synchronized transcriptional and epigenetic regulation of the expression of extrinsic signaling factors and intrinsic cell-specific genes, but the functional roles for chromatin-modifying enzymes in neural differentiation remain poorly understood. Here we show that the histone demethylases KDM4A (JMJD2A) and KDM4C (JMJD2C) are essential for proper differentiation of NSCs in vitro and in vivo. KDM4A/C were required for neuronal differentiation, survival and expression of the neurotrophic signaling factor BDNF in association with promoter H3K9 demethylation and RNA polymerase II recruitment. Unexpectedly, KDM4A/C were essential for selective H3K36 demethylation and loss of RNA polymerase II recruitment in transcribed regions of the astrocyte-characteristic gene GFAP, thereby in parallel repressing astrocytic differentiation by control of elongation. We propose that gene- and lysine-specific KDM4A/C-mediated control of histone methylation and thereby regulation of intrinsic factors and signaling factors such as BDNF provide a novel control mechanism of lineage decision.
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  • Additional Articles published in Volume 426
    [Oct 2014]

    Publication date: 9 October 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 20









    Categories: Journal Articles
  • The Microbiota, Chemical Symbiosis, and Human Disease
    [Oct 2014]

    Publication date: Available online 8 October 2014
    Source:Journal of Molecular Biology

    Author(s): Matthew R. Redinbo

    Our understanding of mammalian-microbial mutualism has expanded by combing microbial sequencing with evolving molecular and cellular methods, and unique model systems. Here, the recent literature linking the microbiota to diseases of three of the key mammalian mucosal epithelial compartments – nasal, lung and gastrointestinal (GI) tract – is reviewed with a focus on new knowledge about the taxa, species, proteins and chemistry that promote health and impact progression toward disease. The information presented is further organized by specific diseases now associated with the microbiota:, Staphylococcus aureus infection and rhinosinusitis in the nasal-sinus mucosa; cystic fibrosis (CF), chronic obstructive pulmonary disorder (COPD), and asthma in the pulmonary tissues. For the vast and microbially dynamic GI compartment, several disorders are considered, including obesity, atherosclerosis, Crohn’s disease, ulcerative colitis, drug toxicity, and even autism. Our appreciation of the chemical symbiosis ongoing between human systems and the microbiota continues to grow, and suggest new opportunities for modulating this symbiosis using designed interventions.
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  • Structural and functional divergence of the aldolase fold in Toxoplasma gondii
    [Oct 2014]

    Publication date: Available online 2 October 2014
    Source:Journal of Molecular Biology

    Author(s): Michelle L. Tonkin , Andrei S. Halavaty , Raghavendran Ramaswamy , Jiapeng Ruan , Makoto Igarashi , Huân M. Ngô , Martin J. Boulanger

    Parasites of the phylum Apicomplexa are highly successful pathogens of humans and animals world-wide. As obligate intracellular parasites, they have significant energy requirements for invasion and gliding motility that are supplied by various metabolic pathways. Aldolases have emerged as key enzymes involved in these pathways, and all apicomplexans express one or both of fructose 1,6-bisphosphate (F16BP) aldolase and 2-deoxyribose 5-phosphate (dR5P) aldolase (DERA). Intriguingly, Toxoplasma gondii, a highly successful apicomplexan parasite, expresses F16BP aldolase (TgALD1), d5RP aldolase (TgDERA), and a divergent dR5P aldolase-like protein (TgDPA) exclusively in the latent bradyzoite stage. While the importance of TgALD1 in glycolysis is well established and TgDERA is also likely to be involved in parasite metabolism, the detailed function of TgDPA remains elusive. To gain mechanistic insight into the function of different T. gondii aldolases, we first determined the crystal structures of TgALD1 and TgDPA. Structural analysis revealed that both aldolases adopt a TIM barrel fold accessorized with divergent secondary structure elements. Structural comparison of TgALD1 and TgDPA with members of their respective enzyme families revealed that while the active site residues are conserved in TgALD1, key catalytic residues are absent in TgDPA. Consistent with this observation, biochemical assays showed that while TgALD1 was active on F16BP, TgDPA was inactive on dR5P. Intriguingly, both aldolases are competent to bind polymerized actin in vitro. Altogether, structural and biochemical analyses of T. gondii aldolase and aldolase-like proteins reveal diverse functionalization of the classic TIM barrel aldolase fold.
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  • Chromatin driven behavior of topologically associating domains
    [Oct 2014]

    Publication date: Available online 2 October 2014
    Source:Journal of Molecular Biology

    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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  • Cytoskeletal control of nuclear morphology and chromatin organization
    [Oct 2014]

    Publication date: Available online 2 October 2014
    Source:Journal of Molecular Biology

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

    The nucleus is sculpted towards 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 RNAi knockdown of cytoskeleton components in Drosophila cells. We find evidence that the loss of perinuclear actin assembly results in baso-lateral enhancement of microtubule organization and this is reflected functionally by enhanced nuclear dynamics. Cytoskeleton reorganization leads to nuclear lamina deformation which influences heterochromatin localization and core histone protein mobility. We also show that modulations in actin-microtubule assembly results in differential gene expression patterns. Taken together, we suggest that perinuclear actin and baso-lateral microtubule organization exerts mechanical control on nuclear morphology and chromatin dynamics.
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  • Mechanistic basis of plasmid-specific DNA binding of the F plasmid regulatory protein, TraM
    [Oct 2014]

    Publication date: Available online 2 October 2014
    Source:Journal of Molecular Biology

    Author(s): Yun Peng , Jun Lu , Joyce J.W. Wong , Ross A. Edwards , Laura S. Frost , J.N. Mark Glover

    The conjugative transfer of bacterial F plasmids relies on TraM, a plasmid-encoded protein that recognizes multiple DNA sites to recruit the plasmid to the conjugative pore. In spite of the high degree of amino acid sequence conservation between TraM proteins, many of these proteins have markedly different DNA binding specificities that ensure the selective recruitment of a plasmid to its cognate pore. Here we present the structure of F TraM RHH (ribbon-helix-helix) domain bound to its sbmA site. The structure indicates a pair of TraM tetramers cooperatively binds an underwound sbmA site that contains 12 base pairs/turn. The sbmA is composed of 4 copies of a 5 base pair motif, each of which is recognized by an RHH domain. The structure reveals that a single conservative amino acid difference in the RHH β-ribbon between F and pED208 TraM changes its specificity for its cognate 5 base pair sequence motif. Specificity is also dictated by the positioning of 2 base pair spacer elements within sbmA; in F sbmA, the spacers are positioned between motifs 1 and 2 and motifs 3 and 4, whereas in pED208 sbmA there is a single spacer between motifs 2 and 3. We also demonstrate that a pair of F TraM tetramers can cooperatively bind its sbmC site with an affinity similar to that of sbmA, in spite of a lack of sequence similarity between these DNA elements. These results provide a basis for the prediction of the DNA binding properties of the family of TraM proteins.
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  • Precise and efficient antibody epitope determination through library design, yeast display and next generation sequencing
    [Oct 2014]

    Publication date: Available online 2 October 2014
    Source:Journal of Molecular Biology

    Author(s): Thomas Van Blarcom , Andrea Rossi , Davide Foletti , Purnima Sundar , Steven Pitts , Christine Bee , Jody Melton Witt , Zea Melton , Adela Hasa-Moreno , Lee Shaughnessy , Dilduz Telman , Lora Zhao , Wai Ling Cheung , Jan Berka , Wenwu Zhai , Pavel Strop , Javier Chaparro-Riggers , David L. Shelton , Jaume Pons , Arvind Rajpal

    The ability of antibodies to bind an antigen with a high degree of affinity and specificity has led them to become the largest and fastest growing class of therapeutic proteins. Clearly identifying the epitope at which they bind their cognate antigen provides insight into their mechanism of action and helps differentiate antibodies that bind the same antigen. Here we describe a method to precisely and efficiently map the epitopes of a panel of antibodies in parallel over the course of several weeks. This method relies on the combination of rational library design, quantitative yeast surface display and next generation DNA sequencing and was demonstrated by mapping the epitopes of several antibodies which neutralize alpha toxin from Staphylococcus aureus. The accuracy of this method was confirmed by comparing the results to the co-crystal structure of one antibody and alpha toxin and was further refined by the inclusion of a lower affinity variant of the antibody. In addition, this method produced quantitative insight into the epitope residues most critical for the antibody-antigen interaction and enabled the relative affinities of each antibody toward alpha toxin variants to be estimated. This affinity estimate serves as a predictor of neutralizing antibody potency and was used to anticipate the ability of each antibody to effectively bind and neutralize naturally occurring alpha toxin variants secreted by strains of S. aureus, including clinically relevant strains. Ultimately this type information can be used to help select the best clinical candidate among a set of antibodies against a given antigen.
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  • Structural and functional mechanisms of CRAC channel regulation
    [Oct 2014]

    Publication date: Available online 2 October 2014
    Source:Journal of Molecular Biology

    Author(s): Ann Hye-Ryong Shim , Leidamarie Tirado-Lee , Murali Prakriya

    In many animal cells, stimulation of cell surface receptors coupled to G proteins or tyrosine kinases mobilizes Ca2+ influx through store-operated Ca2+ release-activated Ca2+ (CRAC) channels. The ensuing Ca2+ entry regulates a wide variety of effector cell responses including transcription, motility, and proliferation. The physiological importance of CRAC channels for human health is underscored by studies indicating that mutations in CRAC channel genes produce a spectrum of devastating diseases including chronic inflammation, muscle weakness, and a severe combined immunodeficiency syndrome. Moreover, from a basic science perspective, CRAC channels exhibit a unique biophysical fingerprint characterized by exquisite Ca2+-selectivity, store-operated gating, and distinct pore properties and therefore serve as fascinating ion channels for understanding the biophysical mechanisms of ion permeation and gating. Studies in the last two decades have revealed the cellular and molecular choreography of the CRAC channel activation process, and it is now established that opening of CRAC channels is governed through direct interactions between the pore-forming Orai proteins, and the ER Ca2+ sensors, STIM1 and STIM2. In this review, we summarize the functional and structural mechanisms of CRAC channel regulation, focusing on recent advances in our understanding of the conformational and structural dynamics of CRAC channel gating.
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  • Structural determinants in prion protein folding and stability
    [Oct 2014]

    Publication date: Available online 2 October 2014
    Source:Journal of Molecular Biology

    Author(s): Federico Benetti , Xevi Biarnés , Francesco Attanasio , Gabriele Giachin , Enrico Rizzarelli , Giuseppe Legname

    Prions are responsible for a heterogeneous group of fatal neurodegenerative diseases, involving post-translational modifications of the cellular prion protein (PrPC). Epidemiological studies on Creutzfeldt-Jakob disease, a prototype prion disorder, show a majority of cases being sporadic, while the remaining occurrences are either genetic or iatrogenic. The molecular mechanisms by which PrPC is converted into its pathological isoform have not yet been established. While point mutations and seeds trigger the protein to cross the energy barriers, thus causing genetic and infectious transmissible spongiform encephalopathies (TSE), respectively, the mechanism responsible for sporadic forms remains unclear. Since prion diseases are protein-misfolding disorders, we investigated prion protein folding and stability as functions of different milieus. Using spectroscopic techniques and atomistic simulations, we dissected the contribution of major structural determinants, also defining the energy landscape of prion protein. In particular, we elucidated: (i) the essential role of the octapeptide region in prion protein folding and stability; (ii) the presence of a very enthalpically stable intermediate in prion-susceptible species; (iii) the role of the disulfide bridge in prion protein folding.
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    Categories: Journal Articles