Journal of Molecular Biology

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  • O-GlcNAc Modification of tau Directly Inhibits Its Aggregation without Perturbing the Conformational Properties of tau Monomers
    [Apr 2014]

    Publication date: 17 April 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 8

    Author(s): Scott A. Yuzwa , Adrienne H. Cheung , Mark Okon , Lawrence P. McIntosh , David J. Vocadlo

    The aggregation of the microtubule-associated protein tau into paired helical filaments to form neurofibrillary tangles constitutes one of the pathological hallmarks of Alzheimer's disease. Tau is post-translationally modified by the addition of N-acetyl-d-glucosamine O-linked to several serine and threonine residues (O-GlcNAc). Previously, increased O-GlcNAcylation of tau has been shown to block the accumulation of tau aggregates within a tauopathy mouse model. Here we show that O-GlcNAc modification of full-length human tau impairs the rate and extent of its heparin-induced aggregation without perturbing its activity toward microtubule polymerization. O-GlcNAcylation, however, does not impact the “global-fold” of tau as measured by a Förster resonance energy transfer assay. Similarly, nuclear magnetic resonance studies demonstrated that O-GlcNAcylation only minimally perturbs the local structural and dynamic features of a tau fragment (residues 353–408) spanning the last microtubule binding repeat to the major GlcNAc-acceptor Ser400. These data indicate that the inhibitory effects of O-GlcNAc on tau aggregation may result from enhanced monomer solubility or the destabilization of fibrils or soluble aggregates, rather than by altering the conformational properties of the monomeric protein. This work further underscores the potential of targeting the O-GlcNAc pathway for potential Alzheimer's disease therapeutics.
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  • The C-Terminal Domain of SRA1p Has a Fold More Similar to PRP18 than to an RRM and Does Not Directly Bind to the SRA1 RNA STR7 Region
    [Apr 2014]

    Publication date: 17 April 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 8

    Author(s): Stephanie M. Bilinovich , Caroline M. Davis , Daniel L. Morris , Louis A. Ray , Jeremy W. Prokop , Gregory J. Buchan , Thomas C. Leeper

    Steroid receptor activator RNA protein (SRA1p) is the translation product of the bi-functional long non-coding RNA steroid receptor activator RNA 1 (SRA1) that is part of the steroid receptor coactivator-1 acetyltransferase complex and is indicated to be an epigenetic regulatory component. Previously, the SRA1p protein was suggested to contain an RNA recognition motif (RRM) domain. We have determined the solution structure of the C-terminal domain of human SRA1p by NMR spectroscopy. Our structure along with sequence comparisons among SRA1p orthologs and against authentic RRM proteins indicates that it is not an RRM domain but rather an all-helical protein with a fold more similar to the PRP18 splicing factor. NMR spectroscopy on the full SRA1p protein suggests that this structure is relevant to the native full-length context. Furthermore, molecular modeling indicates that this fold is well conserved among vertebrates. Amino acid variations in this protein seen across sequenced human genomes, including those in tumor cells, indicate that mutations that disrupt the fold occur vary rarely and highlight that its function is well conserved. SRA1p had previously been suggested to bind to the SRA1 RNA, but NMR spectra of SRA1p in the presence of its 80-nt RNA target suggest otherwise and indicate that this protein must be part of a multi-protein complex in order to recognize its proposed RNA recognition element.
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  • Structure and Function of Steroid Receptor RNA Activator Protein, the Proposed Partner of SRA Noncoding RNA
    [Apr 2014]

    Publication date: 17 April 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 8

    Author(s): David B. McKay , Linghe Xi , Kristen K.B. Barthel , Thomas R. Cech

    In a widely accepted model, the steroid receptor RNA activator protein (SRA protein; SRAP) modulates the transcriptional regulatory activity of SRA RNA by binding a specific stem–loop of SRA. We first confirmed that SRAP is present in the nucleus as well as the cytoplasm of MCF-7 breast cancer cells, where it is expressed at the level of about 105 molecules per cell. However, our SRAP–RNA binding experiments, both in vitro with recombinant protein and in cultured cells with plasmid-expressed protein and RNA, did not reveal a specific interaction between SRAP and SRA. We determined the crystal structure of the carboxy-terminal domain of human SRAP and found that it does not have the postulated RRM (RNA recognition motif). The structure is a five-helix bundle that is distinct from known RNA-binding motifs and instead is similar to the carboxy-terminal domain of the yeast spliceosome protein PRP18, which stabilizes specific protein–protein interactions within a multisubunit mRNA splicing complex. SRA binding experiments with this domain gave negative results. Transcriptional regulation by SRA/SRAP was examined with siRNA knockdown. Effects on both specific estrogen-responsive genes and genes identified by RNA-seq as candidates for regulation were examined in MCF-7 cells. Only a small effect (~20% change) on one gene resulting from depletion of SRA/SRAP could be confirmed. We conclude that the current model for SRAP function must be reevaluated; we suggest that SRAP may function in a different context to stabilize specific intermolecular interactions in the nucleus.
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  • A Novel C-Terminal Homologue of Aha1 Co-Chaperone Binds to Heat Shock Protein 90 and Stimulates Its ATPase Activity in Entamoeba histolytica
    [Apr 2014]

    Publication date: 17 April 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 8

    Author(s): Meetali Singh , Varun Shah , Utpal Tatu

    Cytosolic heat shock protein 90 (Hsp90) has been shown to be essential for many infectious pathogens and is considered a potential target for drug development. In this study, we have carried out biochemical characterization of Hsp90 from a poorly studied protozoan parasite of clinical importance, Entamoeba histolytica. We have shown that Entamoeba Hsp90 can bind to both ATP and its pharmacological inhibitor, 17-AAG (17-allylamino-17-demethoxygeldanamycin), with K d values of 365.2 and 10.77μM, respectively, and it has a weak ATPase activity with a catalytic efficiency of 4.12×10−4 min−1 μM−1. Using inhibitor 17-AAG, we have shown dependence of Entamoeba on Hsp90 for its growth and survival. Hsp90 function is regulated by various co-chaperones. Previous studies suggest a lack of several important co-chaperones in E. histolytica. In this study, we describe the presence of a novel homologue of co-chaperone Aha1 (activator of Hsp90 ATPase), EhAha1c, lacking a canonical Aha1 N-terminal domain. We also show that EhAha1c is capable of binding and stimulating ATPase activity of EhHsp90. In addition to highlighting the potential of Hsp90 inhibitors as drugs against amoebiasis, our study highlights the importance of E. histolytica in understanding the evolution of Hsp90 and its co-chaperone repertoire.
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  • Immunoglobulin G1 Fc Domain Motions: Implications for Fc Engineering
    [Apr 2014]

    Publication date: 17 April 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 8

    Author(s): Martin Frank , Ross C. Walker , William N. Lanzilotta , James H. Prestegard , Adam W. Barb

    The fragment crystallizable (Fc) region links the key pathogen identification and destruction properties of immunoglobulin G (IgG). Pathogen opsonization positions Fcs to activate pro-inflammatory Fcγ receptors (FcγRs) on immune cells. The cellular response and committal to a damaging, though protective, immune response are tightly controlled at multiple levels. Control mechanisms are diverse and in many cases unclear, but one frequently suggested contribution originates in FcγR affinity being modulated through shifts in Fc conformational sampling. Here, we report a previously unseen IgG1 Fc conformation. This observation motivated an extensive molecular dynamics investigation of polypeptide and glycan motions that revealed greater amplitude of motion for the N-terminal Cγ2 domains and N-glycan than previously observed. Residues in the Cγ2/Cγ3 interface and disulfide-bonded hinge were identified as influencing the Cγ2 motion. Our results are consistent with a model of Fc that is structurally dynamic. Conformational states that are competent to bind immune-stimulating FcγRs interconverted with Fc conformations distinct from those observed in FcγR complexes, which may represent a transient, nonbinding population.
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  • Relative Domain Folding and Stability of a Membrane Transport Protein
    [Apr 2014]

    Publication date: 17 April 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 8

    Author(s): Nicola J. Harris , Heather E. Findlay , John Simms , Xia Liu , Paula J. Booth

    There is a limited understanding of the folding of multidomain membrane proteins. Lactose permease (LacY) of Escherichia coli is an archetypal member of the major facilitator superfamily of membrane transport proteins, which contain two domains of six transmembrane helices each. We exploit chemical denaturation to determine the unfolding free energy of LacY and employ Trp residues as site-specific thermodynamic probes. Single Trp LacY mutants are created with the individual Trps situated at mirror image positions on the two LacY domains. The changes in Trp fluorescence induced by urea denaturation are used to construct denaturation curves from which unfolding free energies can be determined. The majority of the single Trp tracers report the same stability and an unfolding free energy of approximately +2kcal mol−1. There is one exception; the fluorescence of W33 at the cytoplasmic end of helix I on the N domain is unaffected by urea. In contrast, the equivalent position on the first helix, VII, of the C-terminal domain exhibits wild-type stability, with the single Trp tracer at position 243 on helix VII reporting an unfolding free energy of +2kcal mol−1. This indicates that the region of the N domain of LacY at position 33 on helix I has enhanced stability to urea, when compared the corresponding location at the start of the C domain. We also find evidence for a potential network of stabilising interactions across the domain interface, which reduces accessibility to the hydrophilic substrate binding pocket between the two domains.
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  • Determination of the Individual Roles of the Linker Residues in the Interdomain Motions of Calmodulin Using NMR Chemical Shifts
    [Apr 2014]

    Publication date: 17 April 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 8

    Author(s): Predrag Kukic , Carlo Camilloni , Andrea Cavalli , Michele Vendruscolo

    Many protein molecules are formed by two or more domains whose structures and dynamics are closely related to their biological functions. It is thus important to develop methods to determine the structural properties of these multidomain proteins. Here, we characterize the interdomain motions in the calcium-bound state of calmodulin (Ca2+-CaM) using NMR chemical shifts as replica-averaged structural restraints in molecular dynamics simulations. We find that the conformational fluctuations of the interdomain linker, which are largely responsible for the overall interdomain motions of CaM, can be well described by exploiting the information provided by chemical shifts. We thus identify 10 residues in the interdomain linker region that change their conformations upon substrate binding. Five of these residues (Met76, Lys77, Thr79, Asp80 and Ser81) are highly flexible and cover the range of conformations observed in the substrate-bound state, while the remaining five (Arg74, Lys75, Asp78, Glu82 and Glu83) are much more rigid and do not populate conformations typical of the substrate-bound form. The ensemble of conformations representing the Ca2+-CaM state obtained in this study is in good agreement with residual dipolar coupling, paramagnetic resonance enhancement, small-angle X-ray scattering and fluorescence resonance energy transfer measurements, which were not used as restraints in the calculations. These results provide initial evidence that chemical shifts can be used to characterize the conformational fluctuations of multidomain proteins.
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  • Structural Analysis of a Fungal Methionine Synthase with Substrates and Inhibitors
    [Apr 2014]

    Publication date: 17 April 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 8

    Author(s): Devinder Ubhi , Grace Kago , Arthur F. Monzingo , Jon D. Robertus

    The cobalamin-independent methionine synthase from Candida albicans, known as Met6p, is a 90-kDa enzyme that consists of two (βα)8 barrels. The active site is located between the two domains and has binding sites for a zinc ion and substrates l-homocysteine and 5-methyl-tetrahydrofolate-glutamate3. Met6p catalyzes transfer of the methyl group of 5-methyl-tetrahydrofolate-glutamate3 to the l-homocysteine thiolate to generate methionine. Met6p is essential for fungal growth, and we currently pursue it as an antifungal drug design target. Here we report the binding of l-homocysteine, methionine, and several folate analogs. We show that binding of l-homocysteine or methionine results in conformational rearrangements at the amino acid binding pocket, moving the catalytic zinc into position to activate the thiol group. We also map the folate binding pocket and identify specific binding residues, like Asn126, whose mutation eliminates catalytic activity. We also report the development of a robust fluorescence-based activity assay suitable for high-throughput screening. We use this assay and an X-ray structure to characterize methotrexate as a weak inhibitor of fungal Met6p.
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  • The Novel Microtubule-Destabilizing Drug BAL27862 Binds to the Colchicine Site of Tubulin with Distinct Effects on Microtubule Organization
    [Apr 2014]

    Publication date: 17 April 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 8

    Author(s): Andrea E. Prota , Franck Danel , Felix Bachmann , Katja Bargsten , Rubén M. Buey , Jens Pohlmann , Stefan Reinelt , Heidi Lane , Michel O. Steinmetz

    Microtubule-targeting agents are widely used for the treatment of cancer and as tool compounds to study the microtubule cytoskeleton. BAL27862 is a novel microtubule-destabilizing drug that is currently undergoing phase I clinical evaluation as the prodrug BAL101553. The drug is a potent inhibitor of tumor cell growth and shows a promising activity profile in a panel of human cancer models resistant to clinically relevant microtubule-targeting agents. Here, we evaluated the molecular mechanism of the tubulin–BAL27862 interaction using a combination of cell biology, biochemistry and structural biology methods. Tubulin-binding assays revealed that BAL27862 potently inhibited tubulin assembly at 37°C with an IC50 of 1.4μM and bound to unassembled tubulin with a stoichiometry of 1mol/mol tubulin and a dissociation constant of 244±30nM. BAL27862 bound to tubulin independently of vinblastine, without the formation of tubulin oligomers. The kinetics of BAL27862 binding to tubulin were distinct from those of colchicine, with evidence of competition between BAL27862 and colchicine for binding. Determination of the tubulin–BAL27862 structure by X-ray crystallography demonstrated that BAL27862 binds to the same site as colchicine at the intradimer interface. Comparison of crystal structures of tubulin–BAL27862 and tubulin–colchicine complexes shows that the binding mode of BAL27862 to tubulin is similar to that of colchicine. However, comparative analyses of the effects of BAL27862 and colchicine on the microtubule mitotic spindle and in tubulin protease-protection experiments suggest different outcomes of tubulin binding. Taken together, our data define BAL27862 as a potent, colchicine site-binding, microtubule-destabilizing agent with distinct effects on microtubule organization.
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  • NP-Sticky: A Web Server for Optimizing DNA Ligation with Non-Palindromic Sticky Ends
    [Apr 2014]

    Publication date: 17 April 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 8

    Author(s): Daphne T.W. Ng , Casim A. Sarkar

    High-efficiency DNA ligation is vital for many molecular biology experiments, and it is best achieved using reactants with non-palindromic sticky ends to maximize specificity. However, optimizing such multi-parametric ligation reactions often involves extensive trial and error. We have developed a freely available Web-based ligation calculator, NP-Sticky (http://sarkarlab.umn.edu/npsticky/), that predicts product distribution for given reactant concentrations, thus enabling straightforward computational optimization of these reactions. Built-in schemes include two-piece and three-piece linear ligation, as well as insert-vector circular ligation. The only parameters needed for the underlying thermodynamic model are the free energies of ligation for each sticky end, which can be estimated by the calculator from the overhang sequences or provided by the user from direct experimental measurement. Free energies of sticky-end mismatches are also calculated for determining the extent of byproduct formation. This ligation calculator allows rapid identification of the optimal conditions for maximizing incorporation, efficiency, and/or accuracy, based on specific needs.
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    Categories: Journal Articles
  • Editorial Board
    [Apr 2014]

    Publication date: 3 April 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 7









    Categories: Journal Articles
  • Contents List
    [Apr 2014]

    Publication date: 3 April 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 7









    Categories: Journal Articles
  • Core Structures of Ubiquitin Dictate Its Dynamics and Function
    [Apr 2014]

    Publication date: 3 April 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 7

    Author(s): Shuya Fukai







    Categories: Journal Articles
  • Transcription Factor Seeks DNA—Cognate Site Preferred
    [Apr 2014]

    Publication date: 3 April 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 7

    Author(s): Joel Mackay







    Categories: Journal Articles
  • The Ins and Outs of Viral RNA Polymerase Translocation
    [Apr 2014]

    Publication date: 3 April 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 7

    Author(s): David D. Boehr







    Categories: Journal Articles
  • Alanine Scan of Core Positions in Ubiquitin Reveals Links between Dynamics, Stability, and Function
    [Apr 2014]

    Publication date: 3 April 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 7

    Author(s): Shirley Y. Lee , Lester Pullen , Daniel J. Virgil , Carlos A. Castañeda , Dulith Abeykoon , Daniel N.A. Bolon , David Fushman

    Mutations at solvent-inaccessible core positions in proteins can impact function through many biophysical mechanisms including alterations to thermodynamic stability and protein dynamics. As these properties of proteins are difficult to investigate, the impacts of core mutations on protein function are poorly understood for most systems. Here, we determined the effects of alanine mutations at all 15 core positions in ubiquitin on function in yeast. The majority (13 of 15) of alanine substitutions supported yeast growth as the sole ubiquitin. Both the two null mutants (I30A and L43A) were less stable to temperature-induced unfolding in vitro than wild type (WT) but were well folded at physiological temperatures. Heteronuclear NMR studies indicated that the L43A mutation reduces temperature stability while retaining a ground-state structure similar to WT. This structure enables L43A to bind to common ubiquitin receptors in vitro. Many of the core alanine ubiquitin mutants, including one of the null variants (I30A), exhibited an increased accumulation of high-molecular-weight species, suggesting that these mutants caused a defect in the processing of ubiquitin-substrate conjugates. In contrast, L43A exhibited a unique accumulation pattern with reduced levels of high-molecular-weight species and undetectable levels of free ubiquitin. When conjugation to other proteins was blocked, L43A ubiquitin accumulated as free ubiquitin in yeast. Based on these findings, we speculate that ubiquitin's stability to unfolding may be required for efficient recycling during proteasome-mediated substrate degradation.
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    Categories: Journal Articles
  • Steric Mechanism of Auto-Inhibitory Regulation of Specific and Non-Specific DNA Binding by the ETS Transcriptional Repressor ETV6
    [Apr 2014]

    Publication date: 3 April 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 7

    Author(s): Soumya De , Anson C.K. Chan , H. Jerome Coyne III , Niraja Bhachech , Ulrike Hermsdorf , Mark Okon , Michael E.P. Murphy , Barbara J. Graves , Lawrence P. McIntosh

    DNA binding by the ETS transcriptional repressor ETV6 (or TEL) is auto-inhibited ~50-fold due to an α-helix that sterically blocks its ETS domain binding interface. Using NMR spectroscopy, we demonstrate that this marginally stable helix is unfolded, and not displaced to a non-inhibitory position, when ETV6 is bound to DNA containing a consensus 5′GGAA3′ recognition site. Although significantly lower in affinity, binding to non-specific DNA is auto-inhibited ~5-fold and is also accompanied by helix unfolding. Based on NMR chemical shift perturbations, both specific and non-specific DNA are bound via the same canonical ETS domain interface. However, spectral perturbations are smaller for the non-specific complex, suggesting weaker and less well-defined interactions than in the specific complex. In parallel, the crystal structure of ETV6 bound to a specific DNA duplex was determined. The structure of this complex reveals that a non-conserved histidine residue in the ETS domain recognition helix helps establish the specificity of ETV6 for DNA-binding sites containing 5′GGAA3′ versus 5′GGAT3′. These studies provide a unified steric mechanism for attenuating ETV6 binding to both specific and non-specific DNA and expand the repertoire of characterized auto-inhibitory strategies utilized to regulate ETS factors.
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  • Distinct Conformations of a Putative Translocation Element in Poliovirus Polymerase
    [Apr 2014]

    Publication date: 3 April 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 7

    Author(s): Aaron J. Sholders , Olve B. Peersen

    The mechanism whereby RNA is translocated by the single subunit viral RNA-dependent RNA polymerases is not yet understood. These enzymes lack homologs of the “O-helix” structures and associated fingers domain movements thought to be responsible for translocation in many DNA-templated polymerases. The structures of multiple picornavirus polymerase elongation complexes suggest that these enzymes use a different molecular mechanism where translocation is not strongly coupled to the opening of the active site following catalysis. Here we present the 2.0- to 2.6-Å-resolution crystal structures and biochemical data for 12 poliovirus polymerase mutants that together show how proper enzyme functions and translocation activity requires conformational flexibility of a loop sequence in the palm domain B-motif. Within the loop, the Ser288-Gly289-Cys290 sequence is shown to play a major role in the catalytic cycle based on RNA binding, processive elongation activity, and single nucleotide incorporation assays. The structures show that Ser288 forms a key hydrogen bond with Asp238, the backbone flexibility of Gly289 is required for translocation competency, and Cys290 modulates the overall elongation activity of the enzyme. Some conformations of the loop represent likely intermediates on the way to forming the catalytically competent closed active site, while others are consistent with a role in promoting translocation of the nascent base pair out of the active site. The loop structure and key residues surrounding it are highly conserved, suggesting that the structural dynamics we observe in poliovirus 3Dpol are a common feature of viral RNA-dependent RNA polymerases.
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  • Crystal Structure of the F27G AIM2 PYD Mutant and Similarities of Its Self-Association to DED/DED Interactions
    [Apr 2014]

    Publication date: 3 April 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 7

    Author(s): Alvin Lu , Venkataraman Kabaleeswaran , Tianmin Fu , Venkat Giri Magupalli , Hao Wu

    Absent in melanoma 2 (AIM2) is a cytoplasmic double-stranded DNA sensor involved in innate immunity. It uses its C-terminal HIN domain for recognizing double-stranded DNA and its N-terminal pyrin domain (PYD) for eliciting downstream effects through recruitment and activation of apoptosis-associated Speck-like protein containing CARD (ASC). ASC in turn recruits caspase-1 and/or caspase-11 to form the AIM2 inflammasome. The activated caspases process proinflammatory cytokines IL-1β and IL-18 and induce the inflammatory form of cell death pyroptosis. Here we show that AIM PYD (AIM2PYD) self-oligomerizes. We notice significant sequence homology of AIM2PYD with the hydrophobic patches of death effector domain (DED)-containing proteins and confirm that mutations on these residues disrupt AIM2PYD self-association. The crystal structure at 1.82Å resolution of such a mutant, F27G of AIM2PYD, shows the canonical six-helix (H1–H6) bundle fold in the death domain superfamily. In contrast to the wild-type AIM2PYD structure crystallized in fusion with the large maltose-binding protein tag, the H2–H3 region of the AIM2PYD F27G is well defined with low B-factors. Structural analysis shows that the conserved hydrophobic patches engage in a type I interaction that has been observed in DED/DED and other death domain superfamily interactions. While previous mutagenesis studies of PYDs point to the involvement of charged interactions, our results reveal the importance of hydrophobic interactions in the same interfaces. These centrally localized hydrophobic residues within fairly charged patches may form the hot spots in AIM2PYD self-association and may represent a common mode of PYD/PYD interactions in general.
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  • Identification of Novel Alternative Splicing Events in the Huntingtin Gene and Assessment of the Functional Consequences Using Structural Protein Homology Modelling
    [Apr 2014]

    Publication date: 3 April 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 7

    Author(s): Alis C. Hughes , Matthew Mort , Lyn Elliston , Rhian M. Thomas , Simon P. Brooks , Stephen B. Dunnett , Lesley Jones

    Huntington's disease (HD) is an inherited progressive neurodegenerative disorder caused by a pathological CAG trinucleotide repeat expansion in the large multi-exon gene, huntingtin (HTT). Although multiple pathogenic mechanisms have been proposed for HD, there is increasing interest in the RNA processing of the HTT gene. In mammals, most multi-exon genes are alternatively spliced; however, few alternative transcripts have been described for HTT. Given the numerous protein bands detected in mouse and human brain tissue by Western blotting using anti-huntingtin antibodies, we examined whether alternative splicing of HTT may account for some of these fragments. Using RT-PCR in mouse brain, we detected two novel splice variants of Htt that lacked the 111-bp exon 29 (Htt∆ex29) or retained a 57-bp portion of intron 28 (Htt+57in28) via use of a cryptic splice site. The alternative transcripts were present in wild-type and homozygous Hdh(Q150/Q150) mouse brain at all ages and in all brain regions and peripheral tissues studied. Differential splicing of Htt∆ex29 was found in the cerebellum of Hdh(Q150/Q150) mice with a significant reduction in transcript levels in mutant animals. In human brain, we detected similar splice variants lacking exons 28 and 29. The ability of alternatively spliced transcripts to encode different protein isoforms with individual functions in the cell, combined with the known role of splicing in disease, renders these novel transcripts of interest in the context of HD pathogenesis.
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    Categories: Journal Articles