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Berlin Brandenburg

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  • 1
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 23 October 2018, Vol.115(43), pp.E10022-E10031
    Description: SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase (dNTPase) that depletes cellular dNTPs in noncycling cells to promote genome stability and to inhibit retroviral and herpes viral replication. In addition to being substrates, cellular nucleotides also allosterically regulate SAMHD1 activity. Recently, it was shown that high expression levels of SAMHD1 are also correlated with significantly worse patient responses to nucleotide analog drugs important for treating a variety of cancers, including acute myeloid leukemia (AML). In this study, we used biochemical, structural, and cellular methods to examine the interactions of various cancer drugs with SAMHD1. We found that both the catalytic and the allosteric sites of SAMHD1 are sensitive to sugar modifications of the nucleotide analogs, with the allosteric site being significantly more restrictive. We crystallized cladribine-TP, clofarabine-TP, fludarabine-TP, vidarabine-TP, cytarabine-TP, and gemcitabine-TP in the catalytic pocket of SAMHD1. We found that all of these drugs are substrates of SAMHD1 and that the efficacy of most of these drugs is affected by SAMHD1 activity. Of the nucleotide analogs tested, only cladribine-TP with a deoxyribose sugar efficiently induced the catalytically active SAMHD1 tetramer. Together, these results establish a detailed framework for understanding the substrate specificity and allosteric activation of SAMHD1 with regard to nucleotide analogs, which can be used to improve current cancer and antiviral therapies.
    Keywords: Samhd1 ; Allosteric Regulation ; Dntpase ; Nucleotide Analog Drugs ; Substrate Selection ; Allosteric Site -- Drug Effects ; Catalytic Domain -- Drug Effects ; Drug Interactions -- Physiology ; Leukemia, Myeloid, Acute -- Metabolism ; SAM Domain and HD Domain-Containing Protein 1 -- Metabolism
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 2
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 17 April 2018, Vol.115(16), pp.E3798-E3807
    Description: Sterile alpha motif and HD-domain-containing protein 1 (SAMHD1) blocks replication of retroviruses and certain DNA viruses by reducing the intracellular dNTP pool. SAMHD1 has been suggested to down-regulate IFN and inflammatory responses to viral infections, although the functions and mechanisms of SAMHD1 in modulating innate immunity remain unclear. Here, we show that SAMHD1 suppresses the innate immune responses to viral infections and inflammatory stimuli by inhibiting nuclear factor-κB (NF-κB) activation and type I interferon (IFN-I) induction. Compared with control cells, infection of SAMHD1-silenced human monocytic cells or primary macrophages with Sendai virus (SeV) or HIV-1, or treatment with inflammatory stimuli, induces significantly higher levels of NF-κB activation and IFN-I induction. Exogenous SAMHD1 expression in cells or SAMHD1 reconstitution in knockout cells suppresses NF-κB activation and IFN-I induction by SeV infection or inflammatory stimuli. Mechanistically, SAMHD1 inhibits NF-κB activation by interacting with NF-κB1/2 and reducing phosphorylation of the NF-κB inhibitory protein IκBα. SAMHD1 also interacts with the inhibitor-κB kinase ε (IKKε) and IFN regulatory factor 7 (IRF7), leading to the suppression of the IFN-I induction pathway by reducing IKKε-mediated IRF7 phosphorylation. Interactions of endogenous SAMHD1 with NF-κB and IFN-I pathway proteins were validated in human monocytic cells and primary macrophages. Comparing splenocytes from knockout and heterozygous mice, we further confirmed SAMHD1-mediated suppression of NF-κB activation, suggesting an evolutionarily conserved property of SAMHD1. Our findings reveal functions of SAMHD1 in down-regulating innate immune responses to viral infections and inflammatory stimuli, highlighting the importance of SAMHD1 in modulating antiviral immunity.
    Keywords: Nf-kB ; Samhd1 ; Inflammatory Stimuli ; Type I Interferon ; Viral Infection ; Immunity, Innate ; Inflammation -- Immunology ; Interferon-Alpha -- Biosynthesis ; Nf-Kappa B -- Metabolism ; SAM Domain and HD Domain-Containing Protein 1 -- Physiology ; Virus Diseases -- Immunology
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 3
    Language: English
    In: Nature Communications, 01 January 2018, Vol.9(1), pp.1-9
    Description: The sterile alpha-motif and histidine-aspartate domain-containing protein 1 (SAMHD1) is a dNTP phosphohydrolase that blocks HIV-1 infection by depleting cellular dNTPs. Here the authors present the structures of full-length mouse SAMHD1 in different nucleotide bound states and give insights into SAMHD1 activity regulation.
    Keywords: Biology
    E-ISSN: 2041-1723
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  • 4
    Language: English
    In: Journal of virology, 15 March 2018, Vol.92(6)
    Description: Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) regulates intracellular deoxynucleoside triphosphate (dNTP) levels and functions as a retroviral restriction factor through its dNTP triphosphohydrolase (dNTPase) activity. Human SAMHD1 interacts with cell cycle regulatory proteins cyclin A2, cyclin-dependent kinase 1 (CDK1), and CDK2. This interaction mediates phosphorylation of SAMHD1 at threonine 592 (T592), which negatively regulates HIV-1 restriction. We previously reported that the interaction is mediated, at least in part, through a cyclin-binding motif (RXL, amino acids [aa] 451 to 453). To understand the role of the RXL motif in regulating SAMHD1 activity, we performed structural and functional analyses of RXL mutants and the effect on HIV-1 restriction. We found that the RXL mutation (R451A and L453A, termed RL/AA) disrupted SAMHD1 tetramer formation and abolished its dNTPase activity and in cells. Compared to wild-type (WT) SAMHD1, the RL/AA mutant failed to restrict HIV-1 infection and had reduced binding to cyclin A2. WT SAMHD1 and RL/AA mutant proteins were degraded by Vpx from HIV-2 but were not spontaneously ubiquitinated in the absence of Vpx. Analysis of proteasomal and autophagy degradation revealed that WT and RL/AA SAMHD1 protein levels were enhanced only when both pathways of degradation were simultaneously inhibited. Our results demonstrate that the RXL motif of human SAMHD1 is required for its HIV-1 restriction, tetramer formation, dNTPase activity, and efficient phosphorylation at T592. These findings identify a new functional domain of SAMHD1 important for its structural integrity, enzyme activity, phosphorylation, and HIV-1 restriction. SAMHD1 is the first mammalian dNTPase identified as a restriction factor that inhibits HIV-1 replication by decreasing the intracellular dNTP pool in nondividing cells, although the critical mechanisms regulating SAMHD1 function remain unclear. We previously reported that mutations of a cyclin-binding RXL motif in human SAMHD1 significantly affect protein expression levels, half-life, nuclear localization, and phosphorylation, suggesting an important role of this motif in modulating SAMHD1 functions in cells. To further understand the significance and mechanisms of the RXL motif in regulating SAMHD1 activity, we performed structural and functional analyses of the RXL motif mutation and its effect on HIV-1 restriction. Our results indicate that the RXL motif is critical for tetramer formation, dNTPase activity, and HIV-1 restriction. These findings help us understand SAMHD1 interactions with other host proteins and the mechanisms regulating SAMHD1 structure and functions in cells.
    Keywords: HIV-1 ; Samhd1 ; Cyclin Binding ; Dntpase ; Infection ; Restriction ; Structure ; HIV Infections ; HIV-1 ; Mutation, Missense ; Protein Multimerization ; SAM Domain and HD Domain-Containing Protein 1
    ISSN: 0022538X
    E-ISSN: 1098-5514
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  • 5
    In: Protein Science, November 2018, Vol.27(11), pp.1969-1977
    Description: We present the structure of an engineered protein–protein interface between two beta barrel proteins, which is mediated by interactions between threonine (Thr) residues. This Thr zipper structure suggests that the protein interface is stabilized by close‐packing of the Thr residues, with only one intermonomer hydrogen bond (H‐bond) between two of the Thr residues. This Thr‐rich interface provides a unique opportunity to study the behavior of Thr in the context of many other Thr residues. In previous work, we have shown that the side chain () dihedral angles of interface and core Thr residues can be predicted with high accuracy using a hard sphere plus stereochemical constraint (HS) model. Here, we demonstrate that in the Thr‐rich local environment of the Thr zipper structure, we are able to predict the dihedral angles of most of the Thr residues. Some, however, are not well predicted by the HS model. We therefore employed explicitly solvated molecular dynamics (MD) simulations to further investigate the side chain conformations of these residues. The MD simulations illustrate the role that transient H‐bonding to water, in combination with steric constraints, plays in determining the behavior of these Thr side chains. Broader Audience Statement: Protein–protein interactions are critical to life and the search for ways to disrupt adverse protein–protein interactions involved in disease is an ongoing area of drug discovery. We must better understand protein–protein interfaces, both to be able to disrupt existing ones and to engineer new ones for a variety of biotechnological applications. We have discovered and characterized an artificial Thr‐rich protein–protein interface. This novel interface demonstrates a heretofore unknown property of Thr‐rich surfaces: mediating protein–protein interactions. PDB Code(s):
    Keywords: Protein–Protein Interaction ; Threonine ; Steric Interactions ; Molecular Dynamics Simulations ; Protein Structure ; Packing ; Hard Sphere Plus Stereochemical Constraint Model
    ISSN: 0961-8368
    E-ISSN: 1469-896X
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  • 6
    Language: English
    In: Journal of virology, 01 August 2018, Vol.92(15)
    Description: Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) restricts human immunodeficiency virus type 1 (HIV-1) replication in nondividing cells by degrading intracellular deoxynucleoside triphosphates (dNTPs). SAMHD1 is highly expressed in resting CD4 T cells, which are important for the HIV-1 reservoir and viral latency; however, whether SAMHD1 affects HIV-1 latency is unknown. Recombinant SAMHD1 binds HIV-1 DNA or RNA fragments , but the function of this binding remains unclear. Here we investigate the effect of SAMHD1 on HIV-1 gene expression and reactivation of viral latency. We found that endogenous SAMHD1 impaired HIV-1 long terminal repeat (LTR) activity in monocytic THP-1 cells and HIV-1 reactivation in latently infected primary CD4 T cells. Overexpression of wild-type (WT) SAMHD1 suppressed HIV-1 LTR-driven gene expression at a transcriptional level. Tat coexpression abrogated SAMHD1-mediated suppression of HIV-1 LTR-driven luciferase expression. SAMHD1 overexpression also suppressed the LTR activity of human T-cell leukemia virus type 1 (HTLV-1), but not that of murine leukemia virus (MLV), suggesting specific suppression of retroviral LTR-driven gene expression. WT SAMHD1 bound to proviral DNA and impaired reactivation of HIV-1 gene expression in latently infected J-Lat cells. In contrast, a nonphosphorylated mutant (T592A) and a dNTP triphosphohydrolase (dNTPase) inactive mutant (H206D R207N [HD/RN]) of SAMHD1 failed to efficiently suppress HIV-1 LTR-driven gene expression and reactivation of latent virus. Purified recombinant WT SAMHD1, but not the T592A and HD/RN mutants, bound to fragments of the HIV-1 LTR These findings suggest that SAMHD1-mediated suppression of HIV-1 LTR-driven gene expression potentially regulates viral latency in CD4 T cells. A critical barrier to developing a cure for HIV-1 infection is the long-lived viral reservoir that exists in resting CD4 T cells, the main targets of HIV-1. The viral reservoir is maintained through a variety of mechanisms, including regulation of the HIV-1 LTR promoter. The host protein SAMHD1 restricts HIV-1 replication in nondividing cells, but its role in HIV-1 latency remains unknown. Here we report a new function of SAMHD1 in regulating HIV-1 latency. We found that SAMHD1 suppressed HIV-1 LTR promoter-driven gene expression and reactivation of viral latency in cell lines and primary CD4 T cells. Furthermore, SAMHD1 bound to the HIV-1 LTR and in a latently infected CD4 T-cell line, suggesting that the binding may negatively modulate reactivation of HIV-1 latency. Our findings indicate a novel role for SAMHD1 in regulating HIV-1 latency, which enhances our understanding of the mechanisms regulating proviral gene expression in CD4 T cells.
    Keywords: HIV-1 ; Ltr ; Samhd1 ; Gene Expression ; Latency ; Reactivation ; Cd4-Positive T-Lymphocytes -- Metabolism ; Gene Expression Regulation, Viral -- Physiology ; HIV Long Terminal Repeat -- Physiology ; HIV-1 -- Physiology ; SAM Domain and HD Domain-Containing Protein 1 -- Metabolism ; Transcription, Genetic -- Physiology ; Virus Latency -- Physiology
    ISSN: 0022538X
    E-ISSN: 1098-5514
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  • 7
    Language: English
    In: eLife, 05 April 2019, Vol.8
    Description: We recently implemented a bioinformatics pipeline that can uncover novel, but rare, riboswitch candidates as well as other noncoding RNA structures in bacteria. A prominent candidate revealed by our initial search efforts was called the ' motif' because of its frequent association with a gene coding for the ThiS protein, which delivers sulfur to form the thiazole moiety of the thiamin precursor HET-P. In the current report, we describe biochemical and genetic data demonstrating that motif RNAs function as sensors of the thiamin precursor HMP-PP, which is fused with HET-P ultimately to form the final active coenzyme thiamin pyrophosphate (TPP). HMP-PP riboswitches exhibit a distinctive architecture wherein an unusually small ligand-sensing aptamer is almost entirely embedded within an otherwise classic intrinsic transcription terminator stem. This arrangement yields remarkably compact genetic switches that bacteria use to tune the levels of thiamin precursors during the biosynthesis of this universally distributed coenzyme.
    Keywords: E. Coli ; Biochemistry ; Chemical Biology
    ISSN: eLife
    E-ISSN: 2050-084X
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  • 8
    Language: English
    In: Journal of virology, 15 July 2019, Vol.93(14)
    Description: The pathogenesis of Epstein-Barr virus (EBV) infection, including development of lymphomas and carcinomas, is dependent on the ability of the virus to transit from latency to the lytic phase. This conversion, and ultimately disease development, depends on the molecular switch protein, ZEBRA, a viral bZIP transcription factor that initiates transcription from promoters of viral lytic genes. By binding to the origin of viral replication, ZEBRA is also an essential replication protein. Here, we identified a novel DNA-binding motif of ZEBRA, N terminal to the canonical bZIP domain. This RRTRK motif is important for high-affinity binding to DNA and is essential for recognizing the methylation state of viral promoters. Mutations in this motif lead to deficiencies in DNA binding, recognition of DNA methylation, lytic cycle DNA replication, and viral late gene expression. This work advances our understanding of ZEBRA-dependent activation of the viral lytic cascade. The binding of ZEBRA to methylated and unmethylated viral DNA triggers activation of the EBV lytic cycle, leading to viral replication and, in some patients, cancer development. Our work thoroughly examines how ZEBRA uses a previously unrecognized basic motif to bind nonmethylated and methylated DNA targets, leading to viral lytic activation. Our findings show that two different positively charged motifs, including the canonical BZIP domain and a newly identified RRTRK motif, contribute to the mechanism of DNA recognition by a viral AP-1 protein. This work contributes to the assessment of ZEBRA as a potential therapeutic target for antiviral and oncolytic treatments.
    Keywords: AP-1-Like Protein ; Bzlf1 ; DNA Methylation ; DNA-Binding Domain ; Epstein-Barr Virus ; Zebra ; Bzip Protein ; Lytic Activation ; Noncanonical DNA-Binding Domain ; Transcription Factors
    E-ISSN: 1098-5514
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  • 9
    Language: English
    In: Nature Communications, 01 August 2019, Vol.10(1), pp.1-14
    Description: In acute myeloid leukemia, hypomethylating agents decitabine and azacytidine are used interchangeably. Here, the authors show that the major metabolite of decitabine, but not azacytidine, is subject to SAMHD1 inactivation, highlighting SAMHD1 as a potential biomarker and therapeutic target
    Keywords: Biology
    E-ISSN: 2041-1723
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  • 10
    Language: English
    In: The Journal of Allergy and Clinical Immunology, February 2018, Vol.141(2), pp.529-538.e13
    Description: Allergy immunotherapy targets the immunological cause of allergic rhinoconjunctivitis and allergic asthma and has the potential to alter the natural course of allergic disease. The primary objective was to investigate the effect of the SQ grass sublingual immunotherapy tablet compared with placebo on the risk of developing asthma. A total of 812 children (5-12 years), with a clinically relevant history of grass pollen allergic rhinoconjunctivitis and no medical history or signs of asthma, were included in the randomized, double-blind, placebo-controlled trial, comprising 3 years of treatment and 2 years of follow-up. There was no difference in time to onset of asthma, defined by prespecified asthma criteria relying on documented reversible impairment of lung function (primary endpoint). Treatment with the SQ grass sublingual immunotherapy tablet significantly reduced the risk of experiencing asthma symptoms or using asthma medication at the end of trial (odds ratio = 0.66,  〈 .036), during the 2-year posttreatment follow-up, and during the entire 5-year trial period. Also, grass allergic rhinoconjunctivitis symptoms were 22% to 30% reduced (  〈 .005 for all 5 years). At the end of the trial, the use of allergic rhinoconjunctivitis pharmacotherapy was significantly less (27% relative difference to placebo,  〈 .001). Total IgE, grass pollen–specific IgE, and skin prick test reactivity to grass pollen were all reduced compared to placebo. Treatment with the SQ grass sublingual immunotherapy tablet reduced the risk of experiencing asthma symptoms and using asthma medication, and had a positive, long-term clinical effect on rhinoconjunctivitis symptoms and medication use but did not show an effect on the time to onset of asthma.
    Keywords: Allergy Immunotherapy ; Asthma Symptoms ; Children ; Disease-Modifying Treatment ; Long-Term Trial ; Medicine
    ISBN: 0004244108000
    ISSN: 0091-6749
    E-ISSN: 1097-6825
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