RESUMEN
In humans, sterile alpha motif (SAM) domain- and histidine-aspartic acid (HD) domain-containing protein 1 (SAMHD1) is a dNTPase enzyme that prevents HIV-1 infection in non-cycling cells, such as differentiated THP-1 cells and human primary macrophages. Although phosphorylation of threonine 592 (T592) in SAMHD1 is recognized as the primary regulator of the ability to prevent HIV-1 infection, the contributions of SAMHD1 acetylation to this ability remain unknown. Mass spectrometry analysis of SAMHD1 proteins derived from cycling and non-cycling THP-1 cells, primary cycling B cells, and primary macrophages revealed that SAMHD1 is preferentially acetylated at lysine residues 354, 494, and 580 (K354, K494, and K580). In non-cycling cells, SAMHD1 is preferentially acetylated at K580, suggesting that this post-translational modification may contribute to the ability of SAMHD1 to block HIV-1 infection. Consistent with this finding, we found that mutations in K580 disrupted the ability of SAMHD1 to block HIV-1 infection without affecting the ability of SAMHD1 to deplete cellular dNTP levels. Gene editing of SAMHD1 in macrophage-like cells revealed that an intact K580 is required for HIV-1 restriction. This finding suggests that K580 acetylation in SAMHD1 is essential for blocking HIV-1 infection. More importantly, we found that a larger proportion of SAMHD1 featuring K580 acetylation could be detected in human primary macrophages when compared to human primary monocytes. In agreement, we found that SAMHD1 is acetylated during the monocyte-to-macrophage differentiation process. This finding agrees with the idea that the blockade of HIV-1 infection in macrophages requires SAMHD1 acetylation.IMPORTANCEThe natural inhibitor of HIV-1, sterile alpha motif (SAM) domain- and histidine-aspartic acid (HD) domain-containing protein 1 (SAMHD1), plays a pivotal role in preventing HIV-1 infection of macrophages and dendritic cells, which are vital components of the immune system. This study unveils that SAMHD1 undergoes post-translational modifications, specifically acetylation at lysines 354, 494, and 580. Our research underscores the significance of these modifications, demonstrating that acetylation at residue K580 is indispensable for SAMHD1's efficacy in blocking HIV-1 infection. Notably, K580 is found in a critical regulatory domain of SAMHD1, highlighting acetylation as a novel layer of SAMHD1 regulation for HIV-1 restriction in humans. A comprehensive understanding of the regulation mechanisms governing this anti-HIV-1 protein is crucial for leveraging nature's defense mechanisms against HIV-1 and could pave the way for innovative therapeutic strategies.
RESUMEN
SAMHD1 is a dNTPase that impedes replication of HIV-1 in myeloid cells and resting T lymphocytes. Here we elucidate the substrate activation mechanism of SAMHD1 that depends on dNTP binding at allosteric sites and the concomitant tetramerization of the enzyme. The study reveals that SAMHD1 activation involves an inactive tetrameric intermediate with partial occupancy of the allosteric sites. The equilibrium between the inactive and active tetrameric states, which is coupled to cooperative binding/dissociation of at least two allosteric dNTP ligands, controls the dNTPase activity of the enzyme, which, in addition, depends on the identity of the dNTPs occupying the four allosteric sites of the active tetramer. We show how such allosteric regulation determines deoxynucleotide triphosphate levels established in the dynamic equilibria between dNTP production and SAMHD1-catalyzed depletion. Notably, the mechanism enables a distinctive functionality of SAMHD1, which we call facilitated dNTP depletion, whereby elevated biosynthesis of some dNTPs results in more efficient depletion of others. The regulatory relationship between the biosynthesis and depletion of different dNTPs sheds light on the emerging role of SAMHD1 in the biology of dNTP homeostasis with implications for HIV/AIDS, innate antiviral immunity, T cell disorders, telomere maintenance and therapeutic efficacy of nucleoside analogs.
RESUMEN
The tumor-suppressor breast cancer 1 (BRCA1) in complex with BRCA1-associated really interesting new gene (RING) domain 1 (BARD1) is a RING-type ubiquitin E3 ligase that modifies nucleosomal histone and other substrates. The importance of BRCA1-BARD1 E3 activity in tumor suppression remains highly controversial, mainly stemming from studying mutant ligase-deficient BRCA1-BARD1 species that we show here still retain significant ligase activity. Using full-length BRCA1-BARD1, we establish robust BRCA1-BARD1-mediated ubiquitylation with specificity, uncover multiple modes of activity modulation, and construct a truly ligase-null variant and a variant specifically impaired in targeting nucleosomal histones. Cells expressing either of these BRCA1-BARD1 separation-of-function alleles are hypersensitive to DNA-damaging agents. Furthermore, we demonstrate that BRCA1-BARD1 ligase is not only required for DNA resection during homology-directed repair (HDR) but also contributes to later stages for HDR completion. Altogether, our findings reveal crucial, previously unrecognized roles of BRCA1-BARD1 ligase activity in genome repair via HDR, settle prior controversies regarding BRCA1-BARD1 ligase functions, and catalyze new efforts to uncover substrates related to tumor suppression.
Asunto(s)
Neoplasias , Proteínas Supresoras de Tumor , Humanos , Proteínas Supresoras de Tumor/metabolismo , Proteína BRCA1/metabolismo , Ubiquitinación , Histonas/genética , Histonas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Reparación del ADN por Recombinación , ADN , Reparación del ADNRESUMEN
TRIM5α is an E3 ubiquitin ligase of the TRIM family that binds to the capsids of primate immunodeficiency viruses and blocks viral replication after cell entry. Here we investigate how synthesis of K63-linked polyubiquitin is upregulated by transient proximity of three RING domains in honeycomb-like assemblies formed by TRIM5α on the surface of the retroviral capsid. Proximity of three RINGs creates an asymmetric arrangement, in which two RINGs form a catalytic dimer that activates E2-ubiquitin conjugates and the disordered N-terminus of the third RING acts as the substrate for N-terminal autoubiquitylation. RING dimerization is required for activation of the E2s that contribute to the antiviral function of TRIM5α, UBE2W and heterodimeric UBE2N/V2, whereas the proximity of the third RING enhances the rate of each of the two distinct steps in the autoubiquitylation process: the initial N-terminal monoubiquitylation (priming) of TRIM5α by UBE2W and the subsequent extension of the K63-linked polyubiquitin chain by UBE2N/V2. The mechanism we describe explains how recognition of infection-associated epitope patterns by TRIM proteins initiates polyubiquitin-mediated downstream events in innate immunity.
Asunto(s)
Poliubiquitina , Ubiquitina-Proteína Ligasas , Animales , Poliubiquitina/metabolismo , Proteínas de Motivos Tripartitos/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Cápside/metabolismo , Ubiquitina/metabolismoRESUMEN
Elevated intracellular levels of dNTPs have been shown to be a biochemical marker of cancer cells. Recently, a series of mutations in the multifunctional dNTP triphosphohydrolase (dNTPase), sterile alpha motif and histidine-aspartate domain-containing protein 1 (SAMHD1), have been reported in various cancers. Here, we investigated the structure and functions of SAMHD1 R366C/H mutants, found in colon cancer and leukemia. Unlike many other cancer-specific mutations, the SAMHD1 R366 mutations do not alter cellular protein levels of the enzyme. However, R366C/H mutant proteins exhibit a loss of dNTPase activity, and their X-ray structures demonstrate the absence of dGTP substrate in their active site, likely because of a loss of interaction with the γ-phosphate of the substrate. The R366C/H mutants failed to reduce intracellular dNTP levels and restrict HIV-1 replication, functions of SAMHD1 that are dependent on the ability of the enzyme to hydrolyze dNTPs. However, these mutants retain dNTPase-independent functions, including mediating dsDNA break repair, interacting with CtIP and cyclin A2, and suppressing innate immune responses. Finally, SAMHD1 degradation in human primary-activated/dividing CD4+ T cells further elevates cellular dNTP levels. This study suggests that the loss of SAMHD1 dNTPase activity induced by R366 mutations can mechanistically contribute to the elevated dNTP levels commonly found in cancer cells.
Asunto(s)
Neoplasias del Colon , Leucemia , Mutación Missense , Proteínas de Neoplasias , Proteína 1 que Contiene Dominios SAM y HD , Sustitución de Aminoácidos , Línea Celular , Neoplasias del Colon/enzimología , Neoplasias del Colon/genética , Ciclina A2/química , Ciclina A2/genética , Ciclina A2/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN , Endodesoxirribonucleasas/química , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Humanos , Leucemia/enzimología , Leucemia/genética , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína 1 que Contiene Dominios SAM y HD/química , Proteína 1 que Contiene Dominios SAM y HD/genética , Proteína 1 que Contiene Dominios SAM y HD/metabolismo , Relación Estructura-ActividadRESUMEN
SAMHD1 impedes infection of myeloid cells and resting T lymphocytes by retroviruses, and the enzymatic activity of the protein-dephosphorylation of deoxynucleotide triphosphates (dNTPs)-implicates enzymatic dNTP depletion in innate antiviral immunity. Here we show that the allosteric binding sites of the enzyme are plastic and can accommodate oligonucleotides in place of the allosteric activators, GTP and dNTP. SAMHD1 displays a preference for oligonucleotides containing phosphorothioate bonds in the Rp configuration located 3' to G nucleotides (GpsN), the modification pattern that occurs in a mechanism of antiviral defense in prokaryotes. In the presence of GTP and dNTPs, binding of GpsN-containing oligonucleotides promotes formation of a distinct tetramer with mixed occupancy of the allosteric sites. Mutations that impair formation of the mixed-occupancy complex abolish the antiretroviral activity of SAMHD1, but not its ability to deplete dNTPs. The findings link nucleic acid binding to the antiretroviral activity of SAMHD1, shed light on the immunomodulatory effects of synthetic phosphorothioated oligonucleotides and raise questions about the role of nucleic acid phosphorothioation in human innate immunity.
Asunto(s)
Nucleótidos/metabolismo , Proteína 1 que Contiene Dominios SAM y HD/metabolismo , Humanos , Inmunidad Innata/genética , Inmunidad Innata/fisiología , Mutación/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Proteína 1 que Contiene Dominios SAM y HD/genéticaRESUMEN
ETS family transcription factors control development of different cell types in humans, whereas deregulation of these proteins leads to severe developmental syndromes and cancers. One of a few members of the ETS family that are known to act solely as repressors, ERF, is required for normal osteogenesis and hematopoiesis. Another important function of ERF is acting as a tumor suppressor by antagonizing oncogenic fusions involving other ETS family factors. The structure of ERF and the DNA binding properties specific to this protein have not been elucidated. In this study, we determined two crystal structures of the complexes of the DNA binding domain of ERF with DNA. In one, ERF is in a distinct dimeric form, with Cys72 in a reduced state. In the other, two dimers of ERF are assembled into a tetramer that is additionally locked by two Cys72-Cys72 disulfide bonds across the dimers. In the tetramer, the ERF molecules are bound to a pseudocontinuous DNA on the same DNA face at two GGAA binding sites on opposite strands. Sedimentation velocity analysis showed that this tetrameric assembly forms on continuous DNA containing such tandem sites spaced by 7 bp. Our bioinformatic analysis of three previously reported sets of ERF binding loci across entire genomes showed that these loci were enriched in such 7 bp spaced tandem sites. Taken together, these results strongly suggest that the observed tetrameric assembly is a functional state of ERF in the human cell.
RESUMEN
MtmOIV and MtmW catalyze the final two reactions in the mithramycin (MTM) biosynthetic pathway, the Baeyer-Villiger opening of the fourth ring of premithramycinâ B (PMB), creating the C3 pentyl side chain, strictly followed by reduction of the distal keto group on the new side chain. Unexpectedly this results in a C2 stereoisomer of mithramycin, iso-mithramycin (iso-MTM). Iso-MTM undergoes a non-enzymatic isomerization to MTM catalyzed by Mg2+ ions. Crystal structures of MtmW and its complexes with co-substrate NADPH and PEG, suggest a catalytic mechanism of MtmW. The structures also show that a tetrameric assembly of this enzyme strikingly resembles the ring-shaped ß subunit of a vertebrate ion channel. We show that MtmW and MtmOIV form a complex in the presence of PMB and NADPH, presumably to hand over the unstable MtmOIV product to MtmW, yielding iso-MTM, as a potential self-resistance mechanism against MTM toxicity.
Asunto(s)
Productos Biológicos/metabolismo , Plicamicina/biosíntesis , CatálisisRESUMEN
RNA binding proteins have emerged as critical oncogenic factors and potential targets in cancer therapy. In this study, we evaluated Musashi1 (Msi1) targeting as a strategy to treat glioblastoma (GBM); the most aggressive brain tumor type. Msi1 expression levels are often high in GBMs and other tumor types and correlate with poor clinical outcome. Moreover, Msi1 has been implicated in chemo- and radio-resistance. Msi1 modulates a range of cancer relevant processes and pathways and regulates the expression of stem cell markers and oncogenic factors via mRNA translation/stability. To identify Msi1 inhibitors capable of blocking its RNA binding function, we performed a ~ 25,000 compound fluorescence polarization screen. NMR and LSPR were used to confirm direct interaction between Msi1 and luteolin, the leading compound. Luteolin displayed strong interaction with Msi1 RNA binding domain 1 (RBD1). As a likely consequence of this interaction, we observed via western and luciferase assays that luteolin treatment diminished Msi1 positive impact on the expression of pro-oncogenic target genes. We tested the effect of luteolin treatment on GBM cells and showed that it reduced proliferation, cell viability, colony formation, migration and invasion of U251 and U343 GBM cells. Luteolin also decreased the proliferation of patient-derived glioma initiating cells (GICs) and tumor-organoids but did not affect normal astrocytes. Finally, we demonstrated the value of combined treatments with luteolin and olaparib (PARP inhibitor) or ionizing radiation (IR). Our results show that luteolin functions as an inhibitor of Msi1 and demonstrates its potential use in GBM therapy.
Asunto(s)
Glioblastoma/tratamiento farmacológico , Luteolina/farmacología , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas de Unión al ARN/química , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Terapia Combinada , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glioblastoma/genética , Glioblastoma/patología , Glioblastoma/radioterapia , Humanos , Luteolina/química , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Fenotipo , Ftalazinas/administración & dosificación , Piperazinas/administración & dosificación , ARN/química , ARN/genética , Proteínas de Unión al ARN/antagonistas & inhibidores , Proteínas de Unión al ARN/genética , Radiación Ionizante , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
SAMHD1 is a dNTP triphosphohydrolase (dNTPase) that impairs retroviral replication in a subset of non-cycling immune cells. Here we show that SAMHD1 is a redox-sensitive enzyme and identify three redox-active cysteines within the protein: C341, C350, and C522. The three cysteines reside near one another and the allosteric nucleotide binding site. Mutations C341S and C522S abolish the ability of SAMHD1 to restrict HIV replication, whereas the C350S mutant remains restriction competent. The C522S mutation makes the protein resistant to inhibition by hydrogen peroxide but has no effect on the tetramerization-dependent dNTPase activity of SAMHD1 in vitro or on the ability of SAMHD1 to deplete cellular dNTPs. Our results reveal that enzymatic activation of SAMHD1 via nucleotide-dependent tetramerization is not sufficient for the establishment of the antiviral state and that retroviral restriction depends on the ability of the protein to undergo redox transformations.
Asunto(s)
Cisteína/metabolismo , Retroviridae/fisiología , Proteína 1 que Contiene Dominios SAM y HD/metabolismo , Replicación Viral/fisiología , Cisteína/genética , Células HEK293 , Humanos , Mutación , Oxidación-Reducción , Proteína 1 que Contiene Dominios SAM y HD/genética , Células U937RESUMEN
Restriction factors are intrinsic cellular defense proteins that have evolved to block microbial infections. Retroviruses such as HIV-1 are restricted by TRIM5 proteins, which recognize the viral capsid shell that surrounds, organizes, and protects the viral genome. TRIM5α uses a SPRY domain to bind capsids with low intrinsic affinity (KD of >1 mM) and therefore requires higher-order assembly into a hexagonal lattice to generate sufficient avidity for productive capsid recognition. TRIMCyp, on the other hand, binds HIV-1 capsids through a cyclophilin A domain, which has a well-defined binding site and higher affinity (KD of â¼10 µM) for isolated capsid subunits. Therefore, it has been argued that TRIMCyp proteins have dispensed with the need for higher-order assembly to function as antiviral factors. Here, we show that, consistent with its high degree of sequence similarity with TRIM5α, the TRIMCyp B-box 2 domain shares the same ability to self-associate and facilitate assembly of a TRIMCyp hexagonal lattice that can wrap about the HIV-1 capsid. We also show that under stringent experimental conditions, TRIMCyp-mediated restriction of HIV-1 is indeed dependent on higher-order assembly. Both forms of TRIM5 therefore use the same mechanism of avidity-driven capsid pattern recognition.IMPORTANCE Rhesus macaques and owl monkeys are highly resistant to HIV-1 infection due to the activity of TRIM5 restriction factors. The rhesus macaque TRIM5α protein blocks HIV-1 through a mechanism that requires self-assembly of a hexagonal TRIM5α lattice around the invading viral core. Lattice assembly amplifies very weak interactions between the TRIM5α SPRY domain and the HIV-1 capsid. Assembly also promotes dimerization of the TRIM5α RING E3 ligase domain, resulting in synthesis of polyubiquitin chains that mediate downstream steps of restriction. In contrast to rhesus TRIM5α, the owl monkey TRIM5 homolog, TRIMCyp, binds isolated HIV-1 CA subunits much more tightly through its cyclophilin A domain and therefore was thought to act independently of higher-order assembly. Here, we show that TRIMCyp shares the assembly properties of TRIM5α and that both forms of TRIM5 use the same mechanism of hexagonal lattice formation to promote viral recognition and restriction.
Asunto(s)
Proteínas de la Cápside/metabolismo , Cápside/metabolismo , Proteínas Portadoras/metabolismo , VIH-1/genética , Proteínas/metabolismo , Secuencia de Aminoácidos , Animales , Factores de Restricción Antivirales , Aotidae , Proteínas de la Cápside/ultraestructura , Proteínas Portadoras/genética , VIH-1/metabolismo , Células HeLa , Humanos , Macaca mulatta , Dominios Proteicos , Multimerización de Proteína , Proteínas/genética , Proteínas de Motivos Tripartitos , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
The small-molecule 6-(tert-butyl)-4-phenyl-4-(trifluoromethyl)-1H,3H-1,3,5-triazin-2-one (3G11) inhibits HIV-1 replication in the human T cell line MT-2. Here, we showed that 3G11 specifically and potently blocks HIV-1 infection. By contrast, 3G11 did not block other retroviruses such as HIV-2, simian immunodeficiency virus (SIVmac ), bovine immunodeficiency virus, feline immunodeficiency virus, equine infectious anemia virus, N-tropic murine leukemia virus, B-tropic murine leukemia virus, and Moloney murine leukemia virus. Analysis of DNA metabolism by real-time PCR revealed that 3G11 blocks the formation of HIV-1 late reverse transcripts during infection prior to the first-strand transfer step. In agreement, an in vitro assay revealed that 3G11 blocks the enzymatic activity of HIV-1 reverse transcriptase as strong as nevirapine. Docking of 3G11 to the HIV-1 reverse transcriptase enzyme suggested a direct interaction between residue L100 and 3G11. In agreement, an HIV-1 virus bearing the reverse transcriptase change L100I renders HIV-1 resistant to 3G11, which suggested that the reverse transcriptase enzyme is the viral determinant for HIV-1 sensitivity to 3G11. Although NMR experiments revealed that 3G11 binds to the HIV-1 capsid, functional experiments suggested that capsid is not the viral determinant for sensitivity to 3G11. Overall, we described a novel non-nucleoside reverse transcription inhibitor that blocks HIV-1 infection.
Asunto(s)
Transcriptasa Inversa del VIH/antagonistas & inhibidores , Inhibidores de la Transcriptasa Inversa/farmacología , Triazinas/farmacología , Animales , Línea Celular , Perros , VIH-1/efectos de los fármacos , Humanos , Espectroscopía de Resonancia Magnética , Simulación del Acoplamiento Molecular , Inhibidores de la Transcriptasa Inversa/química , Triazinas/químicaRESUMEN
IgG autoantibodies mediate pathology in systemic lupus patients and lupus-prone mice. In this study, we showed that the class-switched IgG autoantibody response in MRL/Faslpr/lpr and C57/Sle1Sle2Sle2 mice was blocked by the CID 1067700 compound, which specifically targeted Ras-related in brain 7 (Rab7), an endosome-localized small GTPase that was upregulated in activated human and mouse lupus B cells, leading to prevention of disease development and extension of lifespan. These were associated with decreased IgG-expressing B cells and plasma cells, but unchanged numbers and functions of myeloid cells and T cells. The Rab7 inhibitor suppressed T cell-dependent and T cell-independent Ab responses, but it did not affect T cell-mediated clearance of Chlamydia infection, consistent with a B cell-specific role of Rab7. Indeed, B cells and plasma cells were inherently sensitive to Rab7 gene knockout or Rab7 activity inhibition in class switching and survival, respectively, whereas proliferation/survival of B cells and generation of plasma cells were not affected. Impairment of NF-κB activation upon Rab7 inhibition, together with the rescue of B cell class switching and plasma cell survival by enforced NF-κB activation, indicated that Rab7 mediates these processes by promoting NF-κB activation, likely through signal transduction on intracellular membrane structures. Thus, a single Rab7-inhibiting small molecule can target two stages of B cell differentiation to dampen the pathogenic autoantibody response in lupus.
Asunto(s)
Autoanticuerpos/inmunología , Linfocitos B/inmunología , Compuestos Heterocíclicos con 2 Anillos/farmacología , Cambio de Clase de Inmunoglobulina/efectos de los fármacos , Lupus Eritematoso Sistémico/inmunología , Células Plasmáticas/fisiología , Tiourea/análogos & derivados , Proteínas de Unión al GTP rab/antagonistas & inhibidores , Animales , Linfocitos B/efectos de los fármacos , Linfocitos B/fisiología , Proliferación Celular/efectos de los fármacos , Supervivencia Celular , Infecciones por Chlamydia/inmunología , Femenino , Regulación de la Expresión Génica , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Lupus Eritematoso Sistémico/fisiopatología , Activación de Linfocitos , Ratones , Ratones Endogámicos MRL lpr , FN-kappa B/efectos de los fármacos , FN-kappa B/metabolismo , Células Plasmáticas/efectos de los fármacos , Células Plasmáticas/inmunología , Linfocitos T/inmunología , Tiourea/farmacología , Regulación hacia Arriba , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/inmunología , Proteínas de Unión a GTP rab7RESUMEN
Transcription factors have been considered undruggable, but this paradigm has been recently challenged. DNA binding natural product mithramycin (MTM) is a potent antagonist of oncogenic transcription factor EWS-FLI1. Structural details of MTM recognition of DNA, including the FLI1 binding sequence GGA(A/T), are needed to understand how MTM interferes with EWS-FLI1. We report a crystal structure of an MTM analogue MTM SA-Trp bound to a DNA oligomer containing a site GGCC, and two structures of a novel analogue MTM SA-Phe in complex with DNA. MTM SA-Phe is bound to sites AGGG and GGGT on one DNA, and to AGGG and GGGA(T) (a FLI1 binding site) on the other, revealing how MTM recognizes different DNA sequences. Unexpectedly, at sub-micromolar concentrations MTMs stabilize FLI1-DNA complex on GGAA repeats, which are critical for the oncogenic function of EWS-FLI1. We also directly demonstrate by nuclear magnetic resonance formation of a ternary FLI1-DNA-MTM complex on a single GGAA FLI1/MTM binding site. These biochemical and structural data and a new FLI1-DNA structure suggest that MTM binds the minor groove and perturbs FLI1 bound nearby in the major groove. This ternary complex model may lead to development of novel MTM analogues that selectively target EWS-FLI1 or other oncogenic transcription factors, as anti-cancer therapeutics.
Asunto(s)
ADN/química , Plicamicina/química , Proteína Proto-Oncogénica c-fli-1/química , Secuencia de Bases , ADN/metabolismo , Modelos Moleculares , Conformación Molecular , Estructura Molecular , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/metabolismo , Plicamicina/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteína Proto-Oncogénica c-fli-1/metabolismo , Relación Estructura-ActividadRESUMEN
SAMHD1, a dNTP triphosphohydrolase, contributes to interferon signaling and restriction of retroviral replication. SAMHD1-mediated retroviral restriction is thought to result from the depletion of cellular dNTP pools, but it remains controversial whether the dNTPase activity of SAMHD1 is sufficient for restriction. The restriction ability of SAMHD1 is regulated in cells by phosphorylation on T592. Phosphomimetic mutations of T592 are not restriction competent, but appear intact in their ability to deplete cellular dNTPs. Here we use analytical ultracentrifugation, fluorescence polarization and NMR-based enzymatic assays to investigate the impact of phosphomimetic mutations on SAMHD1 tetramerization and dNTPase activity in vitro. We find that phosphomimetic mutations affect kinetics of tetramer assembly and disassembly, but their effects on tetramerization equilibrium and dNTPase activity are insignificant. In contrast, the Y146S/Y154S dimerization-defective mutant displays a severe dNTPase defect in vitro, but is indistinguishable from WT in its ability to deplete cellular dNTP pools and to restrict HIV replication. Our data suggest that the effect of T592 phosphorylation on SAMHD1 tetramerization is not likely to explain the retroviral restriction defect, and we hypothesize that enzymatic activity of SAMHD1 is subject to additional cellular regulatory mechanisms that have not yet been recapitulated in vitro.
Asunto(s)
VIH/fisiología , Nucleótidos/metabolismo , Proteína 1 que Contiene Dominios SAM y HD/genética , Proteína 1 que Contiene Dominios SAM y HD/metabolismo , Tirosina/genética , Regulación Alostérica , Sustitución de Aminoácidos , Línea Celular , Humanos , Modelos Moleculares , Mutación , Fosforilación , Multimerización de Proteína , Proteína 1 que Contiene Dominios SAM y HD/química , Relación Estructura-ActividadRESUMEN
SAMHD1 is a dNTP hydrolase, whose activity is required for maintaining low dNTP concentrations in non-cycling T cells, dendritic cells, and macrophages. SAMHD1-dependent dNTP depletion is thought to impair retroviral replication in these cells, but the relationship between the dNTPase activity and retroviral restriction is not fully understood. In this study, we investigate allosteric activation of SAMHD1 by deoxynucleotide-dependent tetramerization and measure how the lifetime of the enzymatically active tetramer is affected by different dNTP ligands bound in the allosteric site. The EC50dNTP values for SAMHD1 activation by dNTPs are in the 2-20 µm range, and the half-life of the assembled tetramer after deoxynucleotide depletion varies from minutes to hours depending on what dNTP is bound in the A2 allosteric site. Comparison of the wild-type SAMHD1 and the T592D mutant reveals that the phosphomimetic mutation affects the rates of tetramer dissociation, but has no effect on the equilibrium of allosteric activation by deoxynucleotides. Collectively, our data suggest that deoxynucleotide-dependent tetramerization contributes to regulation of deoxynucleotide levels in cycling cells, whereas in non-cycling cells restrictive to retroviral replication, SAMHD1 activation is likely to be achieved through a distinct mechanism.
Asunto(s)
Desoxirribonucleótidos/química , Proteínas de Unión al GTP Monoméricas/química , Mutación Missense , Multimerización de Proteína , Regulación Alostérica , Sustitución de Aminoácidos , Desoxirribonucleótidos/metabolismo , Activación Enzimática , Humanos , Proteínas de Unión al GTP Monoméricas/genética , Proteínas de Unión al GTP Monoméricas/metabolismo , Proteína 1 que Contiene Dominios SAM y HD , Linfocitos T/enzimologíaRESUMEN
The human antigen R (HuR) stabilizes many mRNAs of proto-oncogene, transcription factors, cytokines and growth factors by recognizing AU-rich elements (AREs) presented in their 3' or 5' untranslated region (UTR). Multiple lines of experimental evidence suggest that this process plays a key role in cancer development. Thus, destabilizing HuR/RNA interaction by small molecules presents an opportunity for cancer treatment/prevention. Here we present an integrated approach to identify inhibitors of HuR/RNA interaction using a combination of fluorescence-based and NMR-based high throughput screening (HTS). The HTS assay with fluorescence polarization readout and Z'-score of 0.8 was used to perform a screen of the NCI diversity set V library in a 384 well plate format. An NMR-based assay with saturation transfer difference (STD) detection was used for hits validation. Protein NMR spectroscopy was used to demonstrate that some hit compounds disrupt formation of HuR oligomer, whereas others block RNA binding. Thus, our integrated high throughput approach provides a new avenue for identification of small molecules targeting HuR/RNA interaction.
Asunto(s)
Proteína 1 Similar a ELAV/metabolismo , Polarización de Fluorescencia/métodos , Espectroscopía de Resonancia Magnética/métodos , ARN/metabolismo , Bibliotecas de Moléculas Pequeñas/aislamiento & purificación , Proteína 1 Similar a ELAV/química , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Modelos Moleculares , Conformación de Ácido Nucleico , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Proto-Oncogenes Mas , ARN/química , Reproducibilidad de los Resultados , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
Members of the tripartite motif (TRIM) protein family of RING E3 ubiquitin (Ub) ligases promote innate immune responses by catalyzing synthesis of polyubiquitin chains linked through lysine 63 (K63). Here, we investigate the mechanism by which the TRIM5α retroviral restriction factor activates Ubc13, the K63-linkage-specific E2. Structural, biochemical, and functional characterization of the TRIM5α:Ubc13-Ub interactions reveals that activation of the Ubc13-Ub conjugate requires dimerization of the TRIM5α RING domain. Our data explain how higher-order oligomerization of TRIM5α, which is promoted by the interaction with the retroviral capsid, enhances the E3 Ub ligase activity of TRIM5α and contributes to its antiretroviral function. This E3 mechanism, in which RING dimerization is transient and depends on the interaction of the TRIM protein with the ligand, is likely to be conserved in many members of the TRIM family and may have evolved to facilitate recognition of repetitive epitope patterns associated with infection.
Asunto(s)
Proteínas Portadoras/metabolismo , Poliubiquitina/biosíntesis , Multimerización de Proteína/fisiología , Enzimas Ubiquitina-Conjugadoras/metabolismo , Animales , Factores de Restricción Antivirales , Proteínas Portadoras/genética , Células Cultivadas , Perros , Poliubiquitina/genética , Retroviridae/genética , Retroviridae/metabolismo , Proteínas de Motivos Tripartitos , Enzimas Ubiquitina-Conjugadoras/genética , Ubiquitina-Proteína Ligasas , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Upon infection of susceptible cells by HIV-1, the conical capsid formed by â¼250 hexamers and 12 pentamers of the CA protein is delivered to the cytoplasm. The capsid shields the RNA genome and proteins required for reverse transcription. In addition, the surface of the capsid mediates numerous host-virus interactions, which either promote infection or enable viral restriction by innate immune responses. In the intact capsid, there is an intermolecular interface between the N-terminal domain (NTD) of one subunit and the C-terminal domain (CTD) of the adjacent subunit within the same hexameric ring. The NTD-CTD interface is critical for capsid assembly, both as an architectural element of the CA hexamer and pentamer and as a mechanistic element for generating lattice curvature. Here we report biochemical experiments showing that PF-3450074 (PF74), a drug that inhibits HIV-1 infection, as well as host proteins cleavage and polyadenylation specific factor 6 (CPSF6) and nucleoporin 153 kDa (NUP153), bind to the CA hexamer with at least 10-fold higher affinities compared with nonassembled CA or isolated CA domains. The crystal structure of PF74 in complex with the CA hexamer reveals that PF74 binds in a preformed pocket encompassing the NTD-CTD interface, suggesting that the principal inhibitory target of PF74 is the assembled capsid. Likewise, CPSF6 binds in the same pocket. Given that the NTD-CTD interface is a specific molecular signature of assembled hexamers in the capsid, binding of NUP153 at this site suggests that key features of capsid architecture remain intact upon delivery of the preintegration complex to the nucleus.
Asunto(s)
Cápside/química , VIH-1/química , Indoles/química , Fenilalanina/análogos & derivados , Factores de Escisión y Poliadenilación de ARNm/química , Cápside/metabolismo , Cristalografía por Rayos X , Infecciones por VIH , VIH-1/metabolismo , Indoles/farmacología , Proteínas de Complejo Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/metabolismo , Fenilalanina/química , Fenilalanina/farmacología , Unión Proteica , Factores de Escisión y Poliadenilación de ARNm/metabolismoRESUMEN
The binding of the TRIM5α restriction factor to the HIV capsid is mediated by the C-terminal SPRY domain of TRIM5α. Atomic-level details of this host-pathogen interaction, which involves mobile variable loops of the SPRY domain, remain unclear. Some of the key determinants of restriction are encompassed by the long and disordered v1 loop of the SPRY domain. We applied molecular modeling to elucidate the conformational repertoire of the v1 loop and its role in the interaction with the capsid. All-atom replica exchange molecular dynamics revealed multiple transient, interconverting states of the v1 loop consistent with the intrinsic disorder observed experimentally. The docking of the SPRY conformations representing 10 most populated states onto the high-resolution model of the assembled HIV-1 capsid revealed that a subset of v1 conformations produced plausible binding poses, in which the SPRY domain binds close to the pseudo-2-fold symmetry axis and the v1 loop spans the interhexamer gap. Such binding mode is well supported by the NMR binding data and known escape mutants. We speculate that the binding mode that involves interaction of the capsid with a subset of preexisting SPRY conformations arising from the intrinsic disorder of the v1 loop may explain the remarkable ability of TRIM5α to resist viral evasion by mutagenesis and to restrict divergent retroviruses.