Publications by authors named "Özlem Demir"

67 Publications

Markov state models and NMR uncover an overlooked allosteric loop in p53.

Chem Sci 2020 Dec 16;12(5):1891-1900. Epub 2020 Dec 16.

Department of Chemistry and Biochemistry, University of California San Diego La Jolla CA 92093 USA +1-858-534-9645 +1-858-534-9629.

The tumor suppressor p53 is the most frequently mutated gene in human cancer, and thus reactivation of mutated p53 is a promising avenue for cancer therapy. Analysis of wildtype p53 and the Y220C cancer mutant long-timescale molecular dynamics simulations with Markov state models and validation by NMR relaxation studies has uncovered the involvement of loop L6 in the slowest motions of the protein. Due to its distant location from the DNA-binding surface, the conformational dynamics of this loop has so far remained largely unexplored. We observe mutation-induced stabilization of alternate L6 conformations, distinct from all experimentally-determined structures, in which the loop is both extended and located further away from the DNA-interacting surface. Additionally, the effect of the L6-adjacent Y220C mutation on the conformational landscape of the functionally-important loop L1 suggests an allosteric role to this dynamic loop and the inactivation mechanism of the mutation. Finally, the simulations reveal a novel Y220C cryptic pocket that can be targeted for p53 rescue efforts. Our approach exemplifies the power of the MSM methodology for uncovering intrinsic dynamic and kinetic differences among distinct protein ensembles, such as for the investigation of mutation effects on protein function.
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http://dx.doi.org/10.1039/d0sc05053aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8179107PMC
December 2020

Structural Characterization of a Minimal Antibody against Human APOBEC3B.

Viruses 2021 04 12;13(4). Epub 2021 Apr 12.

Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA.

APOBEC3B (A3B) is one of seven human APOBEC3 DNA cytosine deaminases that restrict viral infections as part of the overall innate immune response, but it also plays a major role in tumor evolution by mutating genomic DNA. Given the importance of A3B as a restriction factor of viral infections and as a driver of multiple human cancers, selective antibodies against A3B are highly desirable for its specific detection in various research and possibly diagnostic applications. Here, we describe a high-affinity minimal antibody, designated 5G7, obtained via a phage display screening against the C-terminal catalytic domain (ctd) of A3B. 5G7 also binds APOBEC3A that is highly homologous to A3Bctd but does not bind the catalytic domain of APOBEC3G, another Z1-type deaminase domain. The crystal structure of 5G7 shows a canonical arrangement of the heavy and light chain variable domains, with their complementarity-determining region (CDR) loops lining an antigen-binding cleft that accommodates a pair of α-helices. To understand the mechanism of A3Bctd recognition by 5G7, we used the crystal structures of A3Bctd and 5G7 as templates and computationally predicted the A3B-5G7 complex structure. Stable binding poses obtained by the simulation were further tested by site-directed mutagenesis and in vitro binding analyses. These studies mapped the epitope for 5G7 to a portion of C-terminal α6 helix of A3Bctd, with Arg374 playing an essential role. The same region of A3Bctd was used previously as a peptide antigen for generating a rabbit monoclonal antibody (mAb 5210-87-13), suggesting that this region is particularly immunogenic and that these antibodies from very different origins may share similar binding modes. Our studies provide a platform for the development of selective antibodies against A3B and other APOBEC3 family enzymes.
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http://dx.doi.org/10.3390/v13040663DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8070380PMC
April 2021

Structure and dynamics of SARS-CoV-2 proofreading exoribonuclease ExoN.

bioRxiv 2021 Apr 4. Epub 2021 Apr 4.

High-fidelity replication of the large RNA genome of coronaviruses (CoVs) is mediated by a 3'-to-5' exoribonuclease (ExoN) in non-structural protein 14 (nsp14), which excises nucleotides including antiviral drugs mis-incorporated by the low-fidelity viral RNA-dependent RNA polymerase (RdRp) and has also been implicated in viral RNA recombination and resistance to innate immunity. Here we determined a 1.6-Å resolution crystal structure of SARS-CoV-2 ExoN in complex with its essential co-factor, nsp10. The structure shows a highly basic and concave surface flanking the active site, comprising several Lys residues of nsp14 and the N-terminal amino group of nsp10. Modeling suggests that this basic patch binds to the template strand of double-stranded RNA substrates to position the 3' end of the nascent strand in the ExoN active site, which is corroborated by mutational and computational analyses. Molecular dynamics simulations further show remarkable flexibility of multi-domain nsp14 and suggest that nsp10 stabilizes ExoN for substrate RNA-binding to support its exoribonuclease activity. Our high-resolution structure of the SARS-CoV-2 ExoN-nsp10 complex serves as a platform for future development of anti-coronaviral drugs or strategies to attenuate the viral virulence.
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http://dx.doi.org/10.1101/2021.04.02.438274DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8020977PMC
April 2021

RNA Metabolism Guided by RNA Modifications: The Role of SMUG1 in rRNA Quality Control.

Biomolecules 2021 01 8;11(1). Epub 2021 Jan 8.

Department of Clinical Molecular Biology, University of Oslo, 0318 Oslo, Norway.

RNA modifications are essential for proper RNA processing, quality control, and maturation steps. In the last decade, some eukaryotic DNA repair enzymes have been shown to have an ability to recognize and process modified RNA substrates and thereby contribute to RNA surveillance. Single-strand-selective monofunctional uracil-DNA glycosylase 1 (SMUG1) is a base excision repair enzyme that not only recognizes and removes uracil and oxidized pyrimidines from DNA but is also able to process modified RNA substrates. SMUG1 interacts with the pseudouridine synthase dyskerin (DKC1), an enzyme essential for the correct assembly of small nucleolar ribonucleoproteins (snRNPs) and ribosomal RNA (rRNA) processing. Here, we review rRNA modifications and RNA quality control mechanisms in general and discuss the specific function of SMUG1 in rRNA metabolism. Cells lacking SMUG1 have elevated levels of immature rRNA molecules and accumulation of 5-hydroxymethyluridine (5hmU) in mature rRNA. SMUG1 may be required for post-transcriptional regulation and quality control of rRNAs, partly by regulating rRNA and stability.
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http://dx.doi.org/10.3390/biom11010076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7826747PMC
January 2021

An integrated view of p53 dynamics, function, and reactivation.

Curr Opin Struct Biol 2021 04 2;67:187-194. Epub 2021 Jan 2.

Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, CA, 92093, USA. Electronic address:

The tumor suppressor p53 plays a vital role in responding to cell stressors such as DNA damage, hypoxia, and tumor formation by inducing cell-cycle arrest, senescence, or apoptosis. Expression level alterations and mutational frequency implicates p53 in most human cancers. In this review, we show how both computational and experimental methods have been used to provide an integrated view of p53 dynamics, function, and reactivation potential. We argue that p53 serves as an exceptional case study for developing methods in modeling intrinsically disordered proteins. We describe how these methods can be leveraged to improve p53 reactivation molecule design and other novel therapeutic modalities, such as PROteolysis TARgeting Chimeras (PROTACs).
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http://dx.doi.org/10.1016/j.sbi.2020.11.005DOI Listing
April 2021

Functional and Structural Insights into a Vif/PPP2R5 Complex Elucidated Using Patient HIV-1 Isolates and Computational Modeling.

J Virol 2020 10 14;94(21). Epub 2020 Oct 14.

Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota, USA

Human immunodeficiency virus type 1 (HIV-1) Vif recruits a cellular ubiquitin ligase complex to degrade antiviral APOBEC3 enzymes (APOBEC3C-H) and PP2A phosphatase regulators (PPP2R5A to PPP2R5E). While APOBEC3 antagonism is the canonical function of HIV-1 Vif, this viral accessory protein is also known to trigger G/M cell cycle arrest. Vif initiates G/M arrest by degrading multiple PPP2R5 family members, an activity prevalent among diverse HIV-1 and simian immunodeficiency virus (SIV) isolates. Here, computational protein-protein docking was used to delineate a Vif/CBF-β/PPP2R5 complex in which Vif is predicted to bind the same PPP2R5 surface as physiologic phosphatase targets. This model was tested using targeted mutagenesis of amino acid residues within or adjacent to the putative interface to show loss or retention, respectively, of Vif-induced PPP2R5 degradation activity. Additionally, expression of a peptide that mimics cellular targets of PPP2R5s robustly inhibited Vif-mediated degradation of PPP2R5A but not APOBEC3G. Moreover, live-cell imaging studies examining Vif-mediated degradation of PPP2R5A and APOBEC3G within the same cell revealed that PPP2R5A degradation kinetics are comparable to those of APOBEC3G with a half-life of roughly 6 h postinfection, demonstrating that Vif can concurrently mediate the degradation of distinct cellular substrates. Finally, experiments with a panel of patient-derived Vif isolates indicated that PPP2R5A degradation activity is common in patient-derived isolates. Taken together, these results support a model in which PPP2R5 degradation and global changes in the cellular phosphoproteome are likely to be advantageous for viral pathogenesis. A critical function of HIV-1 Vif is to counteract the family of APOBEC3 innate immune proteins. It is also widely accepted that Vif induces G/M cell cycle arrest in several different cell types. Recently, it has been shown that Vif degrades multiple PPP2R5 phosphoregulators to induce the G/M arrest phenotype. Here, computational approaches are used to test a structural model of the Vif/PPP2R5 complex. In addition, imaging studies are used to show that Vif degrades these PPP2R5 substrates in roughly the same time frame as APOBEC3 degradation and that this activity is prevalent in patient-derived Vif isolates. These studies are important by further defining PPP2R5 proteins as a bona fide substrate of HIV-1 Vif.
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http://dx.doi.org/10.1128/JVI.00631-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7565612PMC
October 2020

Active site plasticity and possible modes of chemical inhibition of the human DNA deaminase APOBEC3B.

FASEB Bioadv 2020 Jan 24;2(1):49-58. Epub 2019 Dec 24.

Department of Biochemistry, Molecular Biology and Biophysics University of Minnesota Minneapolis MN USA.

The single-stranded DNA cytosine deaminase APOBEC3B (A3B) functions in innate immunity against viruses, but it is also strongly implicated in eliciting mutations in cancer genomes. Because of the critical role of A3B in promoting virus and tumor evolution, small molecule inhibitors are desirable. However, there is no reported structure for any of the APOBEC3-family enzymes in complex with a small molecule bound in the active site, which hampers the development of small molecules targeting A3B. Here we report high-resolution structures of an active A3B catalytic domain chimera with loop 7 residues exchanged with those from the corresponding region of APOBEC3G (A3G). The structures reveal novel open conformations lacking the catalytically essential zinc ion, with the highly conserved active site residues extensively rearranged. These inactive conformations are stabilized by 2-pyrimidone or an iodide ion bound in the active site. Molecular dynamics simulations corroborate the remarkable plasticity of the engineered active site and identify key interactions that stabilize the native A3B active site. These data provide insights into A3B active site dynamics and suggest possible modes of its inhibition by small molecules, which would aid in rational design of selective A3B inhibitors for constraining virus and tumor evolution.
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http://dx.doi.org/10.1096/fba.2019-00068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6996314PMC
January 2020

Dynamics and Molecular Mechanisms of p53 Transcriptional Activation.

Biochemistry 2018 11 12;57(46):6528-6537. Epub 2018 Nov 12.

Department of Chemistry and Biochemistry , University of California, San Diego , 9500 Gilman Drive , La Jolla , California 92092-0340 , United States.

The "guardian of the genome", p53, functions as a tumor suppressor that responds to cell stressors such as DNA damage, hypoxia, and tumor formation by inducing cell-cycle arrest, senescence, or apoptosis. Mutation of p53 disrupts its tumor suppressor function, leading to various types of human cancers. One particular mutant, R175H, is a structural mutant that inactivates the DNA damage response pathway and acquires oncogenic functions that promotes both cancer and drug resistance. Our current work aims to understand how p53 wild-type function is disrupted due to the R175H mutation. We use a series of atomistic integrative models built previously from crystal structures of the full-length p53 tetramer bound to DNA and model the R175H mutant using in silico site-directed mutagenesis. Explicitly solvated all-atom molecular dynamics (MD) simulations on wild-type and the R175H mutant p53 reveal insights into how wild-type p53 searches and recognizes DNA, and how this mechanism is disrupted as a result of the R175H mutation. Specifically, our work reveals the optimal quaternary DNA binding mode of the DNA binding domain and shows how this binding mode is altered via symmetry loss as a result of the R175H mutation, indicating a recognition mechanism that is reminiscent of the asymmetry seen in wild type p53 binding to nonspecific genomic elements. Altogether our work sheds new light into the hitherto unseen molecular mechanisms governing transcription factor, DNA recognition.
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http://dx.doi.org/10.1021/acs.biochem.8b01005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7560988PMC
November 2018

Neolymphostin A Is a Covalent Phosphoinositide 3-Kinase (PI3K)/Mammalian Target of Rapamycin (mTOR) Dual Inhibitor That Employs an Unusual Electrophilic Vinylogous Ester.

J Med Chem 2018 12 28;61(23):10463-10472. Epub 2018 Nov 28.

Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography , University of California, San Diego , La Jolla , California 92093 , United States.

Using a novel chemistry-based assay for identifying electrophilic natural products in unprocessed extracts, we identified the PI3-kinase/mTOR dual inhibitor neolymphostin A from Salinispora arenicola CNY-486. The method further showed that the vinylogous ester substituent on the neolymphostin core was the exact site for enzyme conjugation. Tandem MS/MS experiments on PI3Kα treated with the inhibitor revealed that neolymphostin covalently modified Lys802 with a shift in mass of +306 amu, corresponding to addition of the inhibitor and elimination of methanol. The binding pose of the inhibitor bound to PI3Kα was modeled, and hydrogen-deuterium exchange mass spectrometry experiments supported this model. Against a panel of kinases, neolymphostin showed good selectivity for PI3-kinase and mTOR. In addition, the natural product blocked AKT phosphorylation in live cells with an IC of ∼3 nM. Taken together, neolymphostin is the first reported example of a covalent kinase inhibitor from the bacterial domain of life.
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http://dx.doi.org/10.1021/acs.jmedchem.8b00975DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6688905PMC
December 2018

APOBEC3H Subcellular Localization Determinants Define Zipcode for Targeting HIV-1 for Restriction.

Mol Cell Biol 2018 12 13;38(23). Epub 2018 Nov 13.

Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota, USA

APOBEC enzymes are DNA cytosine deaminases that normally serve as virus restriction factors, but several members, including APOBEC3H, also contribute to cancer mutagenesis. Despite their importance in multiple fields, little is known about cellular processes that regulate these DNA mutating enzymes. We show that APOBEC3H exists in two distinct subcellular compartments, cytoplasm and nucleolus, and that the structural determinants for each mechanism are genetically separable. First, native and fluorescently tagged APOBEC3Hs localize to these two compartments in multiple cell types. Second, a series of genetic, pharmacologic, and cell biological studies demonstrate active cytoplasmic and nucleolar retention mechanisms, whereas nuclear import and export occur through passive diffusion. Third, APOBEC3H cytoplasmic retention determinants relocalize APOBEC3A from a passive cell-wide state to the cytosol and, additionally, endow potent HIV-1 restriction activity. These results indicate that APOBEC3H has a structural zipcode for subcellular localization and selecting viral substrates for restriction.
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http://dx.doi.org/10.1128/MCB.00356-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6234287PMC
December 2018

Determinants of Oligonucleotide Selectivity of APOBEC3B.

J Chem Inf Model 2019 05 10;59(5):2264-2273. Epub 2018 Sep 10.

Department of Chemistry and Biochemistry , University of California, San Diego , La Jolla , California 92093-0340 , United States.

APOBEC3B (A3B) is a prominent source of mutation in many cancers. To date, it has been difficult to capture the native protein-DNA interactions that confer A3B's substrate specificity by crystallography due to the highly dynamic nature of wild-type A3B active site. We use computational tools to restore a recent crystal structure of a DNA-bound A3B C-terminal domain mutant construct to its wild type sequence, and run molecular dynamics simulations to study its substrate recognition mechanisms. Analysis of these simulations reveal dynamics of the native A3Bctd-oligonucleotide interactions, including the experimentally inaccessible loop 1-oligonucleotide interactions. A second series of simulations in which the target cytosine nucleotide was computationally mutated from a deoxyribose to a ribose show a change in sugar ring pucker, leading to a rearrangement of the binding site and revealing a potential intermediate in the binding pathway. Finally, apo simulations of A3B, starting from the DNA-bound open state, experience a rapid and consistent closure of the binding site, reaching conformations incompatible with substrate binding. This study reveals a more realistic and dynamic view of the wild type A3B binding site and provides novel insights for structure-guided design efforts for A3B.
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http://dx.doi.org/10.1021/acs.jcim.8b00427DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6644697PMC
May 2019

APOBEC3B Nuclear Localization Requires Two Distinct N-Terminal Domain Surfaces.

J Mol Biol 2018 08 19;430(17):2695-2708. Epub 2018 May 19.

Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA; Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA; Institute for Molecular Virology, University of Minnesota, Minneapolis, MN 55455, USA; Howard Hughes Medical Institute, University of Minnesota, Minneapolis, MN 55455, USA. Electronic address:

The APOBEC3 family of cytosine deaminases catalyzes the conversion of cytosines-to-uracils in single-stranded DNA. Traditionally, these enzymes are associated with antiviral immunity and restriction of DNA-based pathogens. However, a role for these enzymes in tumor evolution and metastatic disease has also become evident. The primary APOBEC3 candidate in cancer mutagenesis is APOBEC3B (A3B) for three reasons: (1) A3B mRNA is upregulated in several different cancers, (2) A3B expression and mutational loads correlate with poor clinical outcomes, and (3) A3B is the only family member known to be constitutively nuclear. Previous studies have mapped non-canonical A3B nuclear localization determinants to a single surface-exposed patch within the N-terminal domain (NTD). Here, we show that A3B has an additional, distinct, surface-exposed NTD region that contributes to nuclear localization. Disruption of residues within the first 30 amino acids of A3B (import surface 1) or loop 5/α-helix 3 (import surface 2) completely abolish nuclear localization. These import determinants also graft into NTDs of related family members and mediate re-localization from cell-wide-to-nucleus or cytoplasm-to-nucleus. These findings demonstrate that both sets of residues are required for non-canonical A3B nuclear localization and describe unique surfaces that may serve as novel therapeutic targets.
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http://dx.doi.org/10.1016/j.jmb.2018.04.044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6456046PMC
August 2018

The substrate-binding cap of the UDP-diacylglucosamine pyrophosphatase LpxH is highly flexible, enabling facile substrate binding and product release.

J Biol Chem 2018 05 6;293(21):7969-7981. Epub 2018 Apr 6.

Department of Biochemistry, Molecular Biology & Biophysics, University of Minnesota, Twin Cities, Minneapolis, Minnesota 55455. Electronic address:

Gram-negative bacteria are surrounded by a secondary membrane of which the outer leaflet is composed of the glycolipid lipopolysaccharide (LPS), which guards against hydrophobic toxins, including many antibiotics. Therefore, LPS synthesis in bacteria is an attractive target for antibiotic development. LpxH is a pyrophosphatase involved in LPS synthesis, and previous structures revealed that LpxH has a helical cap that binds its lipid substrates. Here, crystallography and hydrogen-deuterium exchange MS provided evidence for a highly flexible substrate-binding cap in LpxH. Furthermore, molecular dynamics simulations disclosed how the helices of the cap may open to allow substrate entry. The predicted opening mechanism was supported by activity assays of LpxH variants. Finally, we confirmed biochemically that LpxH is inhibited by a previously identified antibacterial compound, determined the potency of this inhibitor, and modeled its binding mode in the LpxH active site. In summary, our work provides evidence that the substrate-binding cap of LpxH is highly dynamic, thus allowing for facile substrate binding and product release between the capping helices. Our results also pave the way for the rational design of more potent LpxH inhibitors.
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http://dx.doi.org/10.1074/jbc.RA118.002503DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5971466PMC
May 2018

Ensemble Docking in Drug Discovery.

Biophys J 2018 05 30;114(10):2271-2278. Epub 2018 Mar 30.

UT/ORNL Center for Molecular Biophysics, Oak Ridge National Laboratory, Oak Ridge, Tennessee; Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, Tennessee. Electronic address:

Ensemble docking corresponds to the generation of an "ensemble" of drug target conformations in computational structure-based drug discovery, often obtained by using molecular dynamics simulation, that is used in docking candidate ligands. This approach is now well established in the field of early-stage drug discovery. This review gives a historical account of the development of ensemble docking and discusses some pertinent methodological advances in conformational sampling.
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http://dx.doi.org/10.1016/j.bpj.2018.02.038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6129458PMC
May 2018

Conformational Switch Regulates the DNA Cytosine Deaminase Activity of Human APOBEC3B.

Sci Rep 2017 12 12;7(1):17415. Epub 2017 Dec 12.

Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota, 55455, USA.

The APOBEC3B (A3B) single-stranded DNA (ssDNA) cytosine deaminase has important roles in innate immunity but is also a major endogenous source of mutations in cancer. Previous structural studies showed that the C-terminal catalytic domain of human A3B has a tightly closed active site, and rearrangement of the surrounding loops is required for binding to substrate ssDNA. Here we report structures of the A3B catalytic domain in a new crystal form that show alternative, yet still closed, conformations of active site loops. All-atom molecular dynamics simulations support the dynamic behavior of active site loops and recapitulate the distinct modes of interactions that maintain a closed active site. Replacing segments of A3B loop 1 to mimic the more potent cytoplasmic deaminase APOBEC3A leads to elevated ssDNA deaminase activity, likely by facilitating opening of the active site. These data collectively suggest that conformational equilibrium of the A3B active site loops, skewed toward being closed, controls enzymatic activity by regulating binding to ssDNA substrates.
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http://dx.doi.org/10.1038/s41598-017-17694-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5727031PMC
December 2017

Development of an AlphaLISA high throughput technique to screen for small molecule inhibitors targeting protein arginine methyltransferases.

Mol Biosyst 2017 Nov;13(12):2509-2520

Department of Pharmacology and Toxicology, Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis, IN 46202, USA.

The protein arginine methyltransferase (PRMT) family of enzymes comprises nine family members in mammals. They catalyze arginine methylation, either monomethylation or symmetric/asymmetric dimethylation of histone and non-histone proteins. PRMT methylation of its substrate proteins modulates cellular processes such as signal transduction, transcription, and mRNA splicing. Recent studies have linked overexpression of PRMT5, a member of the PRMT superfamily, to oncogenesis, making it a potential target for cancer therapy. In this study, we developed a highly sensitive (Z' score = 0.7) robotic high throughput screening (HTS) platform to discover small molecule inhibitors of PRMT5 by adapting the AlphaLISA™ technology. Using biotinylated histone H4 as a substrate, and S-adenosyl-l-methionine as a methyl donor, PRMT5 symmetrically dimethylated H4 at arginine (R) 3. Highly specific acceptor beads for symmetrically dimethylated H4R3 and streptavidin-coated donor beads bound the substrate, emitting a signal that is proportional to the methyltransferase activity. Using this powerful approach, we identified specific PRMT5 inhibitors P1608K04 and P1618J22, and further validated their efficacy and specificity for inhibiting PRMT5. Importantly, these two compounds exhibited much more potent efficacy than the commercial PRMT5 inhibitor EPZ015666 in both pancreatic and colorectal cancer cells. Overall, our work highlights a novel, powerful, and sensitive approach to identify specific PRMT5 inhibitors. The general principle of this HTS screening method can not only be applied to PRMT5 and the PRMT superfamily, but may also be extended to other epigenetic targets. This approach allows us to identify compounds that inhibit the activity of their respective targets, and screening hits like P1608K04 and P1618J22 may serve as the basis for novel drug development to treat cancer and/or other diseases.
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http://dx.doi.org/10.1039/c7mb00391aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5759323PMC
November 2017

A Comparative Study of the Structural Dynamics of Four Terminal Uridylyl Transferases.

Genes (Basel) 2017 Jun 20;8(6). Epub 2017 Jun 20.

Department of Chemistry & Biochemistry, University of California, San Diego, La Jolla, CA 92093, USA.

African trypanosomiasis occurs in 36 countries in sub-Saharan Africa with 10,000 reported cases annually. No definitive remedy is currently available and if left untreated, the disease becomes fatal. Structural and biochemical studies of trypanosomal terminal uridylyl transferases (TUTases) demonstrated their functional role in extensive uridylate insertion/deletion of RNA. RNA Editing TUTase 1 (TbRET1) is involved in guide RNA 3' end uridylation and maturation, while TbRET2 is responsible for U-insertion at RNA editing sites. Two additional TUTases called TbMEAT1 and TbTUT4 have also been reported to share similar function. TbRET1 and TbRET2 are essential enzymes for the parasite viability making them potential drug targets. For this study, we clustered molecular dynamics (MD) trajectories of four TUTases based on active site shape measured by Pocket Volume Measurer (POVME) program. Among the four TUTases, TbRET1 exhibited the largest average pocket volume, while TbMEAT1's and TbTUT4's active sites displayed the most flexibility. A side pocket was also identified within the active site in all TUTases with TbRET1 having the most pronounced. Our results indicate that TbRET1's larger side pocket can be exploited to achieve selective inhibitor design as FTMap identifies it as a druggable pocket.
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http://dx.doi.org/10.3390/genes8060166DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5485530PMC
June 2017

Adapting AlphaLISA high throughput screen to discover a novel small-molecule inhibitor targeting protein arginine methyltransferase 5 in pancreatic and colorectal cancers.

Oncotarget 2017 Jun;8(25):39963-39977

Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, IN, USA.

Pancreatic ductal adenocarcinoma (PDAC) and colorectal cancer (CRC) are notoriously challenging for treatment. Hyperactive nuclear factor κB (NF-κB) is a common culprit in both cancers. Previously, we discovered that protein arginine methyltransferase 5 (PRMT5) methylated and activated NF-κB. Here, we show that PRMT5 is highly expressed in PDAC and CRC. Overexpression of PRMT5 promoted cancer progression, while shRNA knockdown showed an opposite effect. Using an innovative AlphaLISA high throughput screen, we discovered a lead compound, PR5-LL-CM01, which exhibited robust tumor inhibition effects in both cancers. An in silico structure prediction suggested that PR5-LL-CM01 inhibits PRMT5 by binding with its active pocket. Importantly, PR5-LL-CM01 showed higher anti-tumor efficacy than the commercial PRMT5 inhibitor, EPZ015666, in both PDAC and CRC. This study clearly highlights the significant potential of PRMT5 as a therapeutic target in PDAC and CRC, and establishes PR5-LL-CM01 as a promising basis for new drug development in the future.
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http://dx.doi.org/10.18632/oncotarget.18102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5522311PMC
June 2017

Structural basis for targeted DNA cytosine deamination and mutagenesis by APOBEC3A and APOBEC3B.

Nat Struct Mol Biol 2017 02 19;24(2):131-139. Epub 2016 Dec 19.

Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota, USA.

APOBEC-catalyzed cytosine-to-uracil deamination of single-stranded DNA (ssDNA) has beneficial functions in immunity and detrimental effects in cancer. APOBEC enzymes have intrinsic dinucleotide specificities that impart hallmark mutation signatures. Although numerous structures have been solved, mechanisms for global ssDNA recognition and local target-sequence selection remain unclear. Here we report crystal structures of human APOBEC3A and a chimera of human APOBEC3B and APOBEC3A bound to ssDNA at 3.1-Å and 1.7-Å resolution, respectively. These structures reveal a U-shaped DNA conformation, with the specificity-conferring -1 thymine flipped out and the target cytosine inserted deep into the zinc-coordinating active site pocket. The -1 thymine base fits into a groove between flexible loops and makes direct hydrogen bonds with the protein, accounting for the strong 5'-TC preference. These findings explain both conserved and unique properties among APOBEC family members, and they provide a basis for the rational design of inhibitors to impede the evolvability of viruses and tumors.
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http://dx.doi.org/10.1038/nsmb.3344DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5296220PMC
February 2017

RNA Editing TUTase 1: structural foundation of substrate recognition, complex interactions and drug targeting.

Nucleic Acids Res 2016 12 15;44(22):10862-10878. Epub 2016 Oct 15.

Department of Molecular Biology, University of Geneva, 1211 Geneva, Switzerland

Terminal uridyltransferases (TUTases) execute 3' RNA uridylation across protists, fungi, metazoan and plant species. Uridylation plays a particularly prominent role in RNA processing pathways of kinetoplastid protists typified by the causative agent of African sleeping sickness, Trypanosoma brucei In mitochondria of this pathogen, most mRNAs are internally modified by U-insertion/deletion editing while guide RNAs and rRNAs are U-tailed. The founding member of TUTase family, RNA editing TUTase 1 (RET1), functions as a subunit of the 3' processome in uridylation of gRNA precursors and mature guide RNAs. Along with KPAP1 poly(A) polymerase, RET1 also participates in mRNA translational activation. RET1 is divergent from human TUTases and is essential for parasite viability in the mammalian host and the insect vector. Given its robust in vitro activity, RET1 represents an attractive target for trypanocide development. Here, we report high-resolution crystal structures of the RET1 catalytic core alone and in complex with UTP analogs. These structures reveal a tight docking of the conserved nucleotidyl transferase bi-domain module with a RET1-specific C2H2 zinc finger and RNA recognition (RRM) domains. Furthermore, we define RET1 region required for incorporation into the 3' processome, determinants for RNA binding, subunit oligomerization and processive UTP incorporation, and predict druggable pockets.
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http://dx.doi.org/10.1093/nar/gkw917DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5159558PMC
December 2016

The investigation of the sludge reduction efficiency and mechanisms in oxic-settling-anaerobic (OSA) process.

Water Sci Technol 2016 ;73(10):2311-23

Engineering Faculty, Environmental Engineering Department, Dokuz Eylul University, Tinaztepe Campus, 35160 Buca-Izmir, Turkey.

This paper aims to provide a full understanding of the sludge reduction mechanisms in the oxic-settling-anaerobic (OSA) process and presents an evaluation of the sludge reduction efficiencies and sludge characteristics in this process compared to the conventional activated sludge process. Fifty-eight percent reduction in observed yield in the OSA process was achieved compared to the control system at the end of the operational period with no deterioration of effluent quality. The settleability of sludge in the OSA process was also found to be better than that of the control system in terms of sludge volume index. In long-term operation, capillary suction time and specific resistance to filtration values confirmed that the OSA process showed good filterability characteristics. The results of batch experiments showed that higher endogenous respiration in the systems might lead to lower sludge production and that energy uncoupling had only a limited impact on sludge reduction.
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http://dx.doi.org/10.2166/wst.2016.076DOI Listing
August 2016

The Binding Interface between Human APOBEC3F and HIV-1 Vif Elucidated by Genetic and Computational Approaches.

Cell Rep 2015 Dec 25;13(9):1781-8. Epub 2015 Nov 25.

Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA; Institute for Molecular Virology, University of Minnesota, Minneapolis, MN 55455, USA; Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA; Howard Hughes Medical Institute, University of Minnesota, Minneapolis, MN 55455, USA. Electronic address:

APOBEC3 family DNA cytosine deaminases provide overlapping defenses against pathogen infections. However, most viruses have elaborate evasion mechanisms such as the HIV-1 Vif protein, which subverts cellular CBF-β and a polyubiquitin ligase complex to neutralize these enzymes. Despite advances in APOBEC3 and Vif biology, a full understanding of this direct host-pathogen conflict has been elusive. We combine virus adaptation and computational studies to interrogate the APOBEC3F-Vif interface and build a robust structural model. A recurring compensatory amino acid substitution from adaptation experiments provided an initial docking constraint, and microsecond molecular dynamic simulations optimized interface contacts. Virus infectivity experiments validated a long-lasting electrostatic interaction between APOBEC3F E289 and HIV-1 Vif R15. Taken together with mutagenesis results, we propose a wobble model to explain how HIV-1 Vif has evolved to bind different APOBEC3 enzymes and, more generally, how pathogens may evolve to escape innate host defenses.
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http://dx.doi.org/10.1016/j.celrep.2015.10.067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4684092PMC
December 2015

Parents' Talk About Letters With Their Young Children.

Child Dev 2015 Sep-Oct;86(5):1406-18. Epub 2015 May 25.

University of Chicago.

A literacy-related activity that occurs in children's homes-talk about letters in everyday conversations-was examined using data from 50 children who were visited every 4 months between 14 and 50 months. Parents talked about some letters, including those that are common in English words and the first letter of their children's names, especially often. Parents' focus on the child's initial was especially strong in families of higher socioeconomic status, and the extent to which parents talked about the child's initial during the later sessions of the study was related to the children's kindergarten reading skill. Conversations that included the child's initial were longer than those that did not, and parents presented a variety of information about this letter.
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http://dx.doi.org/10.1111/cdev.12385DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4567909PMC
June 2016

Parental socioeconomic status and the neural basis of arithmetic: differential relations to verbal and visuo-spatial representations.

Dev Sci 2015 Sep 9;18(5):799-814. Epub 2015 Feb 9.

Department of Communication Sciences and Disorders, Northwestern University, USA.

We examined the relation of parental socioeconomic status (SES) to the neural bases of subtraction in school-age children (9- to 12-year-olds). We independently localized brain regions subserving verbal versus visuo-spatial representations to determine whether the parental SES-related differences in children's reliance on these neural representations vary as a function of math skill. At higher SES levels, higher skill was associated with greater recruitment of the left temporal cortex, identified by the verbal localizer. At lower SES levels, higher skill was associated with greater recruitment of right parietal cortex, identified by the visuo-spatial localizer. This suggests that depending on parental SES, children engage different neural systems to solve subtraction problems. Furthermore, SES was related to the activation in the left temporal and frontal cortex during the independent verbal localizer task, but it was not related to activation during the independent visuo-spatial localizer task. Differences in activation during the verbal localizer task in turn were related to differences in activation during the subtraction task in right parietal cortex. The relation was stronger at lower SES levels. This result suggests that SES-related differences in the visuo-spatial regions during subtraction might be based in SES-related verbal differences.
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http://dx.doi.org/10.1111/desc.12268DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4522207PMC
September 2015

Vocabulary, syntax, and narrative development in typically developing children and children with early unilateral brain injury: early parental talk about the "there-and-then" matters.

Dev Psychol 2015 Feb;51(2):161-75

Department of Psychology, The University of Chicago.

This study examines the role of a particular kind of linguistic input--talk about the past and future, pretend, and explanations, that is, talk that is decontextualized--in the development of vocabulary, syntax, and narrative skill in typically developing (TD) children and children with pre- or perinatal brain injury (BI). Decontextualized talk has been shown to be particularly effective in predicting children's language skills, but it is not clear why. We first explored the nature of parent decontextualized talk and found it to be linguistically richer than contextualized talk in parents of both TD and BI children. We then found, again for both groups, that parent decontextualized talk at child age 30 months was a significant predictor of child vocabulary, syntax, and narrative performance at kindergarten, above and beyond the child's own early language skills, parent contextualized talk and demographic factors. Decontextualized talk played a larger role in predicting kindergarten syntax and narrative outcomes for children with lower syntax and narrative skill at age 30 months, and also a larger role in predicting kindergarten narrative outcomes for children with BI than for TD children. The difference between the 2 groups stemmed primarily from the fact that children with BI had lower narrative (but not vocabulary or syntax) scores than TD children. When the 2 groups were matched in terms of narrative skill at kindergarten, the impact that decontextualized talk had on narrative skill did not differ for children with BI and for TD children. Decontextualized talk is thus a strong predictor of later language skill for all children, but may be particularly potent for children at the lower-end of the distribution for language skill. The findings also suggest that variability in the language development of children with BI is influenced not only by the biological characteristics of their lesions, but also by the language input they receive.
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http://dx.doi.org/10.1037/a0038476DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4307606PMC
February 2015

Molecular docking to flexible targets.

Methods Mol Biol 2015 ;1215:445-69

Department of Chemistry and Biochemistry, University of California, 3234 Urey Hall, MC-0340, San Diego, La Jolla, CA, 92093-0340, USA.

It is widely accepted that protein receptors exist as an ensemble of conformations in solution. How best to incorporate receptor flexibility into virtual screening protocols used for drug discovery remains a significant challenge. Here, stepwise methodologies are described to generate and select relevant protein conformations for virtual screening in the context of the relaxed complex scheme (RCS), to design small molecule libraries for docking, and to perform statistical analyses on the virtual screening results. Methods include equidistant spacing, RMSD-based clustering, and QR factorization protocols for ensemble generation and ROC analysis for ensemble selection.
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http://dx.doi.org/10.1007/978-1-4939-1465-4_20DOI Listing
June 2015

The differential role of verbal and spatial working memory in the neural basis of arithmetic.

Dev Neuropsychol 2014 ;39(6):440-58

a Department of Communication Sciences and Disorders , Northwestern University , Evanston , Illinois.

We examine the relations of verbal and spatial working memory (WM) ability to the neural bases of arithmetic in school-age children. We independently localize brain regions subserving verbal versus spatial representations. For multiplication, higher verbal WM ability is associated with greater recruitment of the left temporal cortex, identified by the verbal localizer. For multiplication and subtraction, higher spatial WM ability is associated with greater recruitment of right parietal cortex, identified by the spatial localizer. Depending on their WM ability, children engage different neural systems that manipulate different representations to solve arithmetic problems.
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http://dx.doi.org/10.1080/87565641.2014.939182DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4142562PMC
October 2014

A tale of two hands: children's early gesture use in narrative production predicts later narrative structure in speech.

J Child Lang 2015 May 4;42(3):662-81. Epub 2014 Aug 4.

Department of Psychology,University of Chicago.

Speakers of all ages spontaneously gesture as they talk. These gestures predict children's milestones in vocabulary and sentence structure. We ask whether gesture serves a similar role in the development of narrative skill. Children were asked to retell a story conveyed in a wordless cartoon at age five and then again at six, seven, and eight. Children's narrative structure in speech improved across these ages. At age five, many of the children expressed a character's viewpoint in gesture, and these children were more likely to tell better-structured stories at the later ages than children who did not produce character-viewpoint gestures at age five. In contrast, framing narratives from a character's perspective in speech at age five did not predict later narrative structure in speech. Gesture thus continues to act as a harbinger of change even as it assumes new roles in relation to discourse.
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http://dx.doi.org/10.1017/S0305000914000415DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4317388PMC
May 2015

Pea3 transcription factor promotes neurite outgrowth.

Front Mol Neurosci 2014 26;7:59. Epub 2014 Jun 26.

Molecular Neurobiology Laboratory, Department of Genetics and Bioengineering, Yeditepe University Istanbul, Turkey.

Pea3 subfamily of E-twenty six transcription factors consist of three major -exhibit branching morphogenesis, the function of Pea3 family in nervous system development and regeneration is only beginning to unfold. In this study, we provide evidence that Pea3 can directs neurite extension and axonal outgrowth in different model systems, and that Serine 90 is important for this function. We have also identified neurofilament-L and neurofilament-M as two putative novel targets for Pea3.
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http://dx.doi.org/10.3389/fnmol.2014.00059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4072091PMC
July 2014

Computer-aided discovery of Trypanosoma brucei RNA-editing terminal uridylyl transferase 2 inhibitors.

Chem Biol Drug Des 2014 Aug 5;84(2):131-9. Epub 2014 Jun 5.

Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, 92093, USA.

Human African trypanosomiasis (HAT) is a major health problem in sub-Saharan Africa caused by Trypanosoma brucei infection. Current HAT drugs are difficult to administer and not effective against all parasite species at different stages of the disease which indicates an unmet pharmaceutical need. TbRET2 is an indispensable enzyme for the parasite and is targeted here using a computational approach that combines molecular dynamics simulations and virtual screening. The compounds prioritized are then tested in T. brucei via Alamar blue cell viability assays. This work identified 20 drug-like compounds which are candidates for further testing in the drug discovery process.
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http://dx.doi.org/10.1111/cbdd.12302DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4317284PMC
August 2014
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