RESUMO
HIV-1 integration into the human genome is dependent on 3'-processing of the viral DNA. Recently, we reported that the cellular Three Prime Repair Exonuclease 1 (TREX1) enhances HIV-1 integration by degrading the unprocessed viral DNA, while the integration-competent 3'-processed DNA remained resistant. Here, we describe the mechanism by which the 3'-processed HIV-1 DNA resists TREX1-mediated degradation. Our kinetic studies revealed that the rate of cleavage (kcat) of the 3'-processed DNA was significantly lower (approximately 2-2.5-fold) than the unprocessed HIV-1 DNA by TREX1. The kcat values of human TREX1 for the processed U5 and U3 DNA substrates were 3.8 s-1 and 4.5 s-1, respectively. In contrast, the unprocessed U5 and U3 substrates were cleaved at 10.2 s-1 and 9.8 s-1, respectively. The efficiency of degradation (kcat/Km) of the 3'-processed DNA (U5-70.2 and U3-28.05 pM-1s-1) was also significantly lower than the unprocessed DNA (U5-103.1 and U3-65.3 pM-1s-1). Furthermore, the binding affinity (Kd) of TREX1 was markedly lower (â¼2-fold) for the 3'-processed DNA than the unprocessed DNA. Molecular docking and dynamics studies revealed distinct conformational binding modes of TREX1 with the 3'-processed and unprocessed HIV-1 DNA. Particularly, the unprocessed DNA was favorably positioned in the active site with polar interactions with the catalytic residues of TREX1. Additionally, a stable complex was formed between TREX1 and the unprocessed DNA compared the 3'-processed DNA. These results pinpoint the mechanism by which TREX1 preferentially degrades the integration-incompetent HIV-1 DNA and reveal the unique structural and conformational properties of the integration-competent 3'-processed HIV-1 DNA.
Assuntos
DNA Viral , Exodesoxirribonucleases , HIV-1 , Fosfoproteínas , Exodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/química , Exodesoxirribonucleases/genética , HIV-1/metabolismo , Humanos , Fosfoproteínas/metabolismo , Fosfoproteínas/química , Fosfoproteínas/genética , DNA Viral/metabolismo , DNA Viral/genética , DNA Viral/química , Cinética , Integração Viral , TermodinâmicaRESUMO
SAMHD1 dNTP hydrolase activity places it at the crossroad of several important biological pathways, such as viral restriction, cell cycle regulation, and innate immunity. Recently, a dNTPase independent function for SAMHD1 in homologous recombination (HR) of DNA double-strand breaks has been identified. SAMHD1 function and activity is regulated by several post-translational modifications, including protein oxidation. Here, we showed that oxidation of SAMHD1 increases ssDNA binding affinity and occurs in a cell cycle-dependent manner during S phase consistent with a role in HR. We determined the structure of oxidized SAMHD1 in complex with ssDNA. The enzyme binds ssDNA at the regulatory sites at the dimer interface. We propose a mechanism that oxidation of SAMHD1 acts as a functional switch to toggle between dNTPase activity and DNA binding.
Assuntos
Modelos Moleculares , Proteína 1 com Domínio SAM e Domínio HD , Oxirredução , Proteína 1 com Domínio SAM e Domínio HD/química , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Ligação Proteica , DNA de Cadeia Simples/metabolismo , Estrutura Terciária de Proteína , Células PC-3 , HumanosRESUMO
Three prime repair exonuclease 1 (TREX1) is the most abundant 3'â5' exonuclease in mammalian cells. It has been suggested that TREX1 degrades HIV-1 DNA to enable the virus to evade the innate immune system. However, the exact role of TREX1 during early steps of HIV-1 infection is not clearly understood. In this study, we report that HIV-1 infection is associated with upregulation, perinuclear accumulation, and nuclear localization of TREX1. However, TREX1 overexpression did not affect reverse transcription or nuclear entry of the virus. Surprisingly, HIV-1 DNA integration was increased in TREX1-overexpressing cells, suggesting a role of the exonuclease in the post-nuclear entry step of infection. Accordingly, preintegration complexes (PICs) extracted from TREX1-overexpressing cells retained higher levels of DNA integration activity. TREX1 depletion resulted in reduced levels of proviral integration, and PICs formed in TREX1-depleted cells retained lower DNA integration activity. Addition of purified TREX1 to PICs also enhanced DNA integration activity, suggesting that TREX1 promotes HIV-1 integration by stimulating PIC activity. To understand the mechanism, we measured TREX1 exonuclease activity on substrates containing viral DNA ends. These studies revealed that TREX1 preferentially degrades the unprocessed viral DNA, but the integration-competent 3'-processed viral DNA remains resistant to degradation. Finally, we observed that TREX1 addition stimulates the activity of HIV-1 intasomes assembled with the unprocessed viral DNA but not that of intasomes containing the 3'-processed viral DNA. These biochemical analyses provide a mechanism by which TREX1 directly promotes HIV-1 integration. Collectively, our study demonstrates that HIV-1 infection upregulates TREX1 to facilitate viral DNA integration. IMPORTANCE Productive HIV-1 infection is dependent on a number of cellular factors. Therefore, a clear understanding of how the virus exploits the cellular machinery will identify new targets for inhibiting HIV-1 infection. The three prime repair exonuclease 1 (TREX1) is the most active cellular exonuclease in mammalian cells. It has been reported that TREX1 prevents accumulation of HIV-1 DNA and enables the virus to evade the host innate immune response. Here, we show that HIV-1 infection results in the upregulation, perinuclear accumulation, and nuclear localization of TREX1. We also provide evidence that TREX1 promotes HIV-1 integration by preferentially degrading viral DNAs that are incompatible with chromosomal insertion. These observations identify a novel role of TREX1 in a post-nuclear entry step of HIV-1 infection.
Assuntos
DNA Viral/metabolismo , Exodesoxirribonucleases/metabolismo , Infecções por HIV/virologia , HIV-1/fisiologia , Imunidade Inata/imunologia , Fosfoproteínas/metabolismo , Integração Viral , Replicação Viral , Núcleo Celular , DNA Viral/genética , Exodesoxirribonucleases/genética , Células HEK293 , Infecções por HIV/genética , Células HeLa , Humanos , Fosfoproteínas/genéticaRESUMO
Many of the pathogenic species of the genus Bordetella have an absolute requirement for nicotinic acid (NA) for laboratory growth. These Gram-negative bacteria also harbor a gene cluster homologous to the nic cluster of Pseudomonas putida which is involved in the aerobic degradation of NA and its transcriptional control. We report here that BpsR, a negative regulator of biofilm formation and Bps polysaccharide production, controls the growth of Bordetella bronchiseptica by repressing the expression of nic genes. The severe growth defect of the ΔbpsR strain in Stainer-Scholte medium was restored by supplementation with NA, which also functioned as an inducer of nic genes at low micromolar concentrations that are usually present in animals and humans. Purified BpsR protein bound to the nic promoter region, and its DNA binding activity was inhibited by 6-hydroxynicotinic acid (6-HNA), the first metabolite of the NA degradative pathway. Reporter assays with the isogenic mutant derivative of the wild-type (WT) strain harboring deletion in nicA, which encodes a putative nicotinic acid hydroxylase responsible for conversion of NA to 6-HNA, showed that 6-HNA is the actual inducer of the nic genes in the bacterial cell. Gene expression profiling further showed that BpsR dually activated and repressed the expression of genes associated with pathogenesis, transcriptional regulation, metabolism, and other cellular processes. We discuss the implications of these findings with respect to the selection of pyridines such as NA and quinolinic acid for optimum bacterial growth depending on the ecological niche.IMPORTANCE BpsR, the previously described regulator of biofilm formation and Bps polysaccharide production, controls Bordetella bronchiseptica growth by regulating the expression of genes involved in the degradation of nicotinic acid (NA). 6-Hydroxynicotinic acid (6-HNA), the first metabolite of the NA degradation pathway prevented BpsR from binding to DNA and was the actual in vivo inducer. We hypothesize that BpsR enables Bordetella bacteria to efficiently and selectively utilize NA for their survival depending on the environment in which they reside. The results reported herein lay the foundation for future investigations of how BpsR and the alteration of its activity by NA orchestrate the control of Bordetella growth, metabolism, biofilm formation, and pathogenesis.
Assuntos
Proteínas de Bactérias/metabolismo , Bordetella bronchiseptica/crescimento & desenvolvimento , Bordetella bronchiseptica/metabolismo , Regulação Bacteriana da Expressão Gênica , Niacina/metabolismo , Proteínas de Bactérias/genética , Sequência de Bases , Bordetella bronchiseptica/genética , Deleção de Genes , Genes Reguladores , Transcrição GênicaRESUMO
The dNTP triphosphohydrolase SAMHD1 is a regulator of cellular dNTP pools. Given its central role in nucleotide metabolism, SAMHD1 performs important functions in cellular homeostasis, cell cycle regulation, and innate immunity. It therefore represents a high-profile target for small molecule drug design. SAMHD1 has a complex mechanism of catalytic activation that makes the design of an activating compound challenging. However, an inhibitor of SAMHD1 could serve multiple therapeutic roles, including the potentiation of antiviral and anticancer drug regimens. The lack of high-throughput screens that directly measure SAMHD1 catalytic activity has impeded efforts to identify inhibitors of SAMHD1. Here we describe a novel high-throughput screen that directly measures SAMHD1 catalytic activity. This assay results in a colorimetric end point that can be read spectrophotometrically and utilizes bis(4-nitrophenyl) phosphate as the substrate and Mn2+ as the activating cation that facilitates catalysis. When used to screen a library of Food and Drug Administration-approved drugs, this HTS identified multiple novel compounds that inhibited SAMHD1 dNTPase activity at micromolar concentrations.
Assuntos
Inibidores Enzimáticos/farmacologia , Nitrofenóis/farmacologia , Proteína 1 com Domínio SAM e Domínio HD/antagonistas & inibidores , Descoberta de Drogas , Inibidores Enzimáticos/química , Ensaios de Triagem em Larga Escala , Humanos , Manganês/farmacologia , Multimerização ProteicaRESUMO
The TREX1 gene encodes a potent DNA exonuclease, and mutations in TREX1 cause a spectrum of lupus-like autoimmune diseases. Most lupus patients develop autoantibodies to double-stranded DNA (dsDNA), but the source of DNA antigen is unknown. The TREX1 D18N mutation causes a monogenic, cutaneous form of lupus called familial chilblain lupus, and the TREX1 D18N enzyme exhibits dysfunctional dsDNA-degrading activity, providing a link between dsDNA degradation and nucleic acid-mediated autoimmune disease. We determined the structure of the TREX1 D18N protein in complex with dsDNA, revealing how this exonuclease uses a novel DNA-unwinding mechanism to separate the polynucleotide strands for single-stranded DNA (ssDNA) loading into the active site. The TREX1 D18N dsDNA interactions coupled with catalytic deficiency explain how this mutant nuclease prevents dsDNA degradation. We tested the effects of TREX1 D18N in vivo by replacing the TREX1 WT gene in mice with the TREX1 D18N allele. The TREX1 D18N mice exhibit systemic inflammation, lymphoid hyperplasia, vasculitis, and kidney disease. The observed lupus-like inflammatory disease is associated with immune activation, production of autoantibodies to dsDNA, and deposition of immune complexes in the kidney. Thus, dysfunctional dsDNA degradation by TREX1 D18N induces disease in mice that recapitulates many characteristics of human lupus. Failure to clear DNA has long been linked to lupus in humans, and these data point to dsDNA as a key substrate for TREX1 and a major antigen source in mice with dysfunctional TREX1 enzyme.
Assuntos
Pérnio/enzimologia , Pérnio/genética , Dano ao DNA , DNA/metabolismo , Exodesoxirribonucleases/genética , Inflamação/patologia , Lúpus Eritematoso Cutâneo/enzimologia , Lúpus Eritematoso Cutâneo/genética , Fosfoproteínas/genética , Alelos , Animais , Anticorpos/imunologia , Autoimunidade/imunologia , Sequência de Bases , Pérnio/patologia , DNA/química , DNA/genética , Exodesoxirribonucleases/química , Humanos , Lúpus Eritematoso Cutâneo/patologia , Camundongos , Dados de Sequência Molecular , Mutação/genética , Conformação de Ácido Nucleico , Fenótipo , Fosfoproteínas/química , Biossíntese de ProteínasRESUMO
Many regulated epigenetic elements and base lesions found in genomic DNA can both directly impact gene expression and play a role in disease processes. However, due to their noncanonical nature, they are challenging to assess with conventional technologies. Here, we present a new approach for the targeted detection of diverse modified bases in DNA. We first use enzymatic components of the DNA base excision repair pathway to install an individual affinity label at each location of a selected modified base with high yield. We then probe the resulting material with a solid-state nanopore assay capable of discriminating labeled DNA from unlabeled DNA. The technique features exceptional modularity via selection of targeting enzymes, which we establish through the detection of four DNA base elements: uracil, 8-oxoguanine, T:G mismatch, and the methyladenine analog 1,N6-ethenoadenine. Our results demonstrate the potential for a quantitative nanopore assessment of a broad range of base modifications.
Assuntos
Técnicas Biossensoriais/métodos , Dano ao DNA , DNA/análise , Nanoporos , Neoplasias/genética , Adenina/análogos & derivados , Pareamento Incorreto de Bases , DNA/genética , Reparo do DNA , Epigênese Genética , Guanina/análogos & derivados , Guanina/análise , Humanos , Modelos Moleculares , Nanoporos/ultraestrutura , Nanotecnologia/métodos , Uracila/análiseRESUMO
Acquisition of adaptive mutations is essential for microbial persistence during chronic infections. This is particularly evident during chronic Pseudomonas aeruginosa lung infections in cystic fibrosis (CF) patients. Thus far, mutagenesis has been attributed to the generation of reactive species by polymorphonucleocytes (PMN) and antibiotic treatment. However, our current studies of mutagenesis leading to P. aeruginosa mucoid conversion have revealed a potential new mutagen. Our findings confirmed the current view that reactive oxygen species can promote mucoidy in vitro, but revealed PMNs are proficient at inducing mucoid conversion in the absence of an oxidative burst. This led to the discovery that cationic antimicrobial peptides can be mutagenic and promote mucoidy. Of specific interest was the human cathelicidin LL-37, canonically known to disrupt bacterial membranes leading to cell death. An alternative role was revealed at sub-inhibitory concentrations, where LL-37 was found to induce mutations within the mucA gene encoding a negative regulator of mucoidy and to promote rifampin resistance in both P. aeruginosa and Escherichia coli. The mechanism of mutagenesis was found to be dependent upon sub-inhibitory concentrations of LL-37 entering the bacterial cytosol and binding to DNA. LL-37/DNA interactions then promote translesion DNA synthesis by the polymerase DinB, whose error-prone replication potentiates the mutations. A model of LL-37 bound to DNA was generated, which reveals amino termini α-helices of dimerized LL-37 bind the major groove of DNA, with numerous DNA contacts made by LL-37 basic residues. This demonstrates a mutagenic role for antimicrobials previously thought to be insusceptible to resistance by mutation, highlighting a need to further investigate their role in evolution and pathoadaptation in chronic infections.
Assuntos
Adaptação Fisiológica , Peptídeos Catiônicos Antimicrobianos/metabolismo , DNA Bacteriano/metabolismo , Mutagênese , Infecções por Pseudomonas/metabolismo , Pseudomonas aeruginosa/metabolismo , Adolescente , Adulto , Peptídeos Catiônicos Antimicrobianos/química , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Criança , Doença Crônica , Fibrose Cística/genética , Fibrose Cística/metabolismo , Fibrose Cística/patologia , DNA Bacteriano/química , DNA Bacteriano/genética , Evolução Molecular , Feminino , Humanos , Masculino , Modelos Moleculares , Infecções por Pseudomonas/genética , Infecções por Pseudomonas/patologia , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/genética , CatelicidinasRESUMO
TREX1 acts in concert with the SET complex in granzyme A-mediated apoptosis, and mutations in TREX1 cause Aicardi-Goutières syndrome and familial chilblain lupus. Here, we report monoallelic frameshift or missense mutations and one 3' UTR variant of TREX1 present in 9/417 individuals with systemic lupus erythematosus but absent in 1,712 controls (P = 4.1 x 10(-7)). We demonstrate that two mutant TREX1 alleles alter subcellular targeting. Our findings implicate TREX1 in the pathogenesis of SLE.
Assuntos
Exodesoxirribonucleases/genética , Lúpus Eritematoso Cutâneo/genética , Mutação , Fosfoproteínas/genética , Regiões 3' não Traduzidas/genética , Endossomos/metabolismo , Exodesoxirribonucleases/química , Exodesoxirribonucleases/metabolismo , Mutação da Fase de Leitura , Frequência do Gene , Predisposição Genética para Doença , Genótipo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Lúpus Eritematoso Cutâneo/enzimologia , Mutação de Sentido Incorreto , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
TREX1 is a 3'-deoxyribonuclease that degrades single- and double-stranded DNA (ssDNA and dsDNA) to prevent inappropriate nucleic acid-mediated immune activation. More than 40 different disease-causing TREX1 mutations have been identified exhibiting dominant and recessive genetic phenotypes in a spectrum of autoimmune disorders. Mutations in TREX1 at positions Asp-18 and Asp-200 to His and Asn exhibit dominant autoimmune phenotypes associated with the clinical disorders familial chilblain lupus and Aicardi-Goutières syndrome. Our previous biochemical studies showed that the TREX1 dominant autoimmune disease phenotype depends upon an intact DNA-binding process coupled with dysfunctional active site chemistry. Studies here show that the TREX1 Arg-62 residues extend across the dimer interface into the active site of the opposing protomer to coordinate substrate DNA and to affect catalysis in the opposing protomer. The TREX1(R62A/R62A) homodimer exhibits â¼50-fold reduced ssDNA and dsDNA degradation activities relative to TREX1(WT). The TREX1 D18H, D18N, D200H, and D200N dominant mutant enzymes were prepared as compound heterodimers with the TREX1 R62A substitution in the opposing protomer. The TREX1(D18H/R62A), TREX1(D18N/R62A), TREX1(D200H/R62A), and TREX1(D200N/R62A) compound heterodimers exhibit higher levels of ss- and dsDNA degradation activities than the homodimers demonstrating the requirement for TREX1 Arg-62 residues to provide necessary structural elements for full catalytic activity in the opposing TREX1 protomer. This concept is further supported by the loss of dominant negative effects in the TREX1 D18H, D18N, D200H, and D200N compound heterodimers. These data provide compelling evidence for the required TREX1 dimeric structure for full catalytic function.
Assuntos
DNA de Cadeia Simples/química , Exodesoxirribonucleases/química , Fosfoproteínas/química , Multimerização Proteica/fisiologia , Subunidades Proteicas/química , Substituição de Aminoácidos , Arginina/química , Arginina/genética , Arginina/metabolismo , Doenças Autoimunes do Sistema Nervoso/enzimologia , Doenças Autoimunes do Sistema Nervoso/genética , Catálise , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Humanos , Mutação de Sentido Incorreto , Malformações do Sistema Nervoso/enzimologia , Malformações do Sistema Nervoso/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Estrutura Quaternária de Proteína , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismoRESUMO
TREX1 is an autonomous 3'-exonuclease that degrades DNA to prevent inappropriate immune activation. The TREX1 protein is composed of 314 amino acids; the N-terminal 242 amino acids contain the catalytic domain, and the C-terminal region (CTR) localizes TREX1 to the cytosolic compartment. In this study, we show that TREX1 modification by ubiquitination is controlled by a highly conserved sequence in the CTR to affect cellular localization. Transfection of TREX1 deletion constructs into human cells demonstrated that this sequence is required for ubiquitination at multiple lysine residues through a "non-canonical" ubiquitin linkage. A proteomic approach identified ubiquilin 1 as a TREX1 CTR-interacting protein, and this interaction was verified in vitro and in vivo. Cotransfection studies indicated that ubiquilin 1 localizes TREX1 to cytosolic punctate structures dependent upon the TREX1 CTR and lysines within the TREX1 catalytic core. Several TREX1 mutants linked to the autoimmune diseases Aicardi-Goutières syndrome and systemic lupus erythematosus that exhibit full catalytic function were tested for altered ubiquitin modification and cellular localization. Our data show that these catalytically competent disease-causing TREX1 mutants exhibit differential levels of ubiquitination relative to WT TREX1, suggesting a novel mechanism of dysfunction. Furthermore, these differentially ubiquitinated disease-causing mutants also exhibit altered ubiquilin 1 co-localization. Thus, TREX1 post-translational modification indicates an additional mechanism by which mutations disrupt TREX1 biology, leading to human autoimmune disease.
Assuntos
Exodesoxirribonucleases/química , Exodesoxirribonucleases/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Ubiquitinação , Proteínas Adaptadoras de Transdução de Sinal , Doenças Autoimunes do Sistema Nervoso/metabolismo , Proteínas Relacionadas à Autofagia , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Células HEK293 , Humanos , Lúpus Eritematoso Sistêmico/metabolismo , Lisina/metabolismo , Proteínas Mutantes/metabolismo , Malformações do Sistema Nervoso/metabolismo , Ligação Proteica , Processamento de Proteína Pós-Traducional , Transporte Proteico , Relação Estrutura-AtividadeRESUMO
AmrZ, a member of the Ribbon-Helix-Helix family of DNA binding proteins, functions as both a transcriptional activator and repressor of multiple genes encoding Pseudomonas aeruginosa virulence factors. The expression of these virulence factors leads to chronic and sustained infections associated with worsening prognosis. In this study, we present the X-ray crystal structure of AmrZ in complex with DNA containing the repressor site, amrZ1. Binding of AmrZ to this site leads to auto-repression. AmrZ binds this DNA sequence as a dimer-of-dimers, and makes specific base contacts to two half sites, separated by a five base pair linker region. Analysis of the linker region shows a narrowing of the minor groove, causing significant distortions. AmrZ binding assays utilizing sequences containing variations in this linker region reveals that secondary structure of the DNA, conferred by the sequence of this region, is an important determinant in binding affinity. The results from these experiments allow for the creation of a model where both intrinsic structure of the DNA and specific nucleotide recognition are absolutely necessary for binding of the protein. We also examined AmrZ binding to the algD promoter, which results in activation of the alginate exopolysaccharide biosynthetic operon, and found the protein utilizes different interactions with this site. Finally, we tested the in vivo effects of this differential binding by switching the AmrZ binding site at algD, where it acts as an activator, for a repressor binding sequence and show that differences in binding alone do not affect transcriptional regulation.
Assuntos
Proteínas de Bactérias/biossíntese , Genes Bacterianos/fisiologia , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidade , Elementos de Resposta/fisiologia , Fatores de Transcrição/metabolismo , Fatores de Virulência/biossíntese , Proteínas de Bactérias/genética , Óperon/fisiologia , Ligação Proteica , Multimerização Proteica/fisiologia , Pseudomonas aeruginosa/genética , Fatores de Transcrição/genética , Fatores de Virulência/genéticaRESUMO
Solid-state (SS-) nanopore sensing has gained tremendous attention in recent years, but it has been constrained by its intrinsic lack of selectivity. To address this, we previously established a novel SS-nanopore assay that produces translocation signals only when a target biotinylated nucleic acid fragment binds to monovalent streptavidin (MS), a protein variant with a single high-affinity biotin-binding domain. While this approach has enabled selective quantification of diverse nucleic acid biomarkers, sensitivity enhancements are needed to improve the detection of low-abundance translational targets. Because the translocation dynamics that determine assay efficacy are largely governed by constituent charge characteristics, we here incorporate a polyhistidine-tagged MS (hMS) to alter the component detectability. We investigate the effects of buffer pH, salt concentration, and SS-nanopore diameter on the performance with the alternate reagent, achieve significant improvements in measurement sensitivity and selectivity, and expand the range of device dimensions viable for the assay. We used this improvement to detect as little as 1 nM miRNA spiked into human plasma. Overall, our findings improve the potential for broader applications of SS-nanopores in the quantitative analyses of molecular biomarkers.
Assuntos
Histidina , Nanoporos , Ácidos Nucleicos , Humanos , Estreptavidina/química , BiomarcadoresRESUMO
HIV-1 integration into the human genome is dependent on 3'-processing of the reverse transcribed viral DNA. Recently, we reported that the cellular Three Prime Repair Exonuclease 1 (TREX1) enhances HIV-1 integration by degrading the unprocessed viral DNA, while the integration-competent 3'-processed DNA remained resistant. Here, we describe the mechanism by which the 3'-processed HIV-1 DNA resists TREX1-mediated degradation. Our kinetic studies revealed that the rate of cleavage (kcat) of the 3'-processed DNA was significantly lower than the unprocessed HIV-1 DNA by TREX1. The efficiency of degradation (kcat/KM) of the 3'-processed DNA was also significantly lower than the unprocessed DNA. Furthermore, the binding affinity (Kd) of TREX1 was markedly lower to the 3'-processed DNA compared to the unprocessed DNA. Molecular docking and dynamics studies revealed distinct conformational binding modes of TREX1 with the 3'-processed and unprocessed HIV-1 DNA. Particularly, the unprocessed DNA was favorably positioned in the active site with polar interactions with the catalytic residues of TREX1. Additionally, a stable complex was formed between TREX1 and the unprocessed DNA compared the 3'-processed DNA. These results pinpoint the biochemical mechanism by which TREX1 preferentially degrades the integration-incompetent HIV-1 DNA and reveal the unique structural and conformational properties of the integration-competent 3'-processed HIV-1 DNA.
RESUMO
Mutations in the three genes encoding the heterotrimeric RNase H2 complex cause Aicardi-Goutières Syndrome (AGS). Our mouse RNase H2 structure revealed that the catalytic RNase H2A subunit interfaces mostly with the RNase H2C subunit that is intricately interwoven with the RNase H2B subunit. We mapped the positions of AGS-causing RNase H2A mutations using the mouse RNase H2 structure and proposed that these mutations cause varied effects on catalytic potential. To determine the functional consequences of these mutations, heterotrimeric human RNase H2 complexes containing the RNase H2A subunit mutations were prepared, and catalytic efficiencies and nucleic acid binding properties were compared with the wild-type (WT) complex. These analyses reveal a dramatic range of effects with mutations at conserved positions G37S, R186W, and R235Q, reducing enzymatic activities and substrate binding affinities by as much as a 1000-fold, whereas mutations at non-conserved positions R108W, N212I, F230L, T240M, and R291H reduced activities and binding modestly or not at all. All mutants purify as three-subunit complexes, further supporting the required heterotrimeric structure in eukaryotic RNase H2. These kinetic properties reveal varied functional consequences of AGS-causing mutations in the catalytic RNase H2A subunit and reflect the complex mechanisms of nuclease dysfunction that include catalytic deficiencies and altered protein-nucleic acid interactions relevant in AGS.
Assuntos
Mutação , Ribonuclease H/química , Animais , Doenças Autoimunes do Sistema Nervoso/enzimologia , Catálise , Domínio Catalítico , Relação Dose-Resposta a Droga , Humanos , Cinética , Camundongos , Mutagênese Sítio-Dirigida , Malformações do Sistema Nervoso/enzimologia , Ligação Proteica , Estrutura Terciária de Proteína , Especificidade por SubstratoRESUMO
The SAMHD1 protein is an HIV-1 restriction factor that is targeted by the HIV-2 accessory protein Vpx in myeloid lineage cells. Mutations in the SAMHD1 gene cause Aicardi-Goutières syndrome, a genetic disease that mimics congenital viral infection. To determine the physiological function of the SAMHD1 protein, the SAMHD1 gene was cloned, recombinant protein was produced, and the catalytic activity of the purified enzyme was identified. We show that SAMHD1 contains a dGTP-regulated deoxynucleotide triphosphohydrolase. We propose that Vpx targets SAMHD1 for degradation in a viral strategy to control cellular deoxynucleotide levels for efficient replication.
Assuntos
Doenças Autoimunes do Sistema Nervoso/genética , HIV-1/genética , Proteínas Monoméricas de Ligação ao GTP/genética , Células Mieloides/citologia , Malformações do Sistema Nervoso/genética , Proteínas Virais Reguladoras e Acessórias/metabolismo , Animais , Doenças Autoimunes/genética , Doenças Autoimunes/metabolismo , Catálise , Bovinos , Nucleotídeos de Desoxiguanina/química , Relação Dose-Resposta a Droga , HIV-1/metabolismo , Humanos , Camundongos , Proteínas Monoméricas de Ligação ao GTP/química , Nucleosídeos/química , Monoéster Fosfórico Hidrolases/química , Proteína 1 com Domínio SAM e Domínio HD , Replicação ViralRESUMO
TREX1 is a potent 3' â 5' exonuclease that degrades single- and double-stranded DNA (ssDNA and dsDNA). TREX1 mutations at amino acid positions Asp-18 and Asp-200 in familial chilblain lupus and Aicardi-Goutières syndrome elicit dominant immune dysfunction phenotypes. Failure to appropriately disassemble genomic DNA during normal cell death processes could lead to persistent DNA signals that trigger the innate immune response and autoimmunity. We tested this concept using dsDNA plasmid and chromatin and show that the TREX1 exonuclease locates 3' termini generated by endonucleases and degrades the nicked DNA polynucleotide. A competition assay was designed using TREX1 dominant mutants and variants to demonstrate that an intact DNA binding process, coupled with dysfunctional chemistry in the active sites, explains the dominant phenotypes in TREX1 D18N, D200N, and D200H alleles. The TREX1 residues Arg-174 and Lys-175 positioned adjacent to the active sites act with the Arg-128 residues positioned in the catalytic cores to facilitate melting of dsDNA and generate ssDNA for entry into the active sites. Metal-dependent ssDNA binding in the active sites of the catalytically inactive dominant TREX1 mutants contributes to DNA retention and precludes access to DNA 3' termini by active TREX1 enzyme. Thus, the dominant disease genetics exhibited by the TREX1 D18N, D200N, and D200H alleles parallel precisely the biochemical properties of these TREX1 dimers during dsDNA degradation of plasmid and chromatin DNA in vitro. These results support the concept that failure to degrade genomic dsDNA is a principal pathway of immune activation in TREX1-mediated autoimmune disease.
Assuntos
Doenças Autoimunes do Sistema Nervoso/enzimologia , Pérnio/enzimologia , DNA de Cadeia Simples/metabolismo , Exodesoxirribonucleases/metabolismo , Genes Dominantes , Lúpus Eritematoso Cutâneo/enzimologia , Mutação de Sentido Incorreto , Malformações do Sistema Nervoso/enzimologia , Fosfoproteínas/metabolismo , Alelos , Substituição de Aminoácidos , Doenças Autoimunes do Sistema Nervoso/genética , Doenças Autoimunes do Sistema Nervoso/imunologia , Morte Celular/genética , Morte Celular/imunologia , Pérnio/genética , Pérnio/imunologia , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/imunologia , Exodesoxirribonucleases/química , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/imunologia , Humanos , Imunidade Inata/genética , Lúpus Eritematoso Cutâneo/genética , Lúpus Eritematoso Cutâneo/imunologia , Malformações do Sistema Nervoso/genética , Malformações do Sistema Nervoso/imunologia , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/imunologiaRESUMO
Mutations in the TREX1 gene cause Aicardi-Goutières syndrome (AGS) and are linked to the autoimmune disease systemic lupus erythematosus. The TREX1 protein is a dimeric 3' DNA exonuclease that degrades DNA to prevent inappropriate immune activation. One of the most common TREX1 mutations, R114H, causes AGS as a homozygous and compound heterozygous mutation and is found as a heterozygous mutation in systemic lupus erythematosus. The TREX1 proteins containing R114H and the insertion mutations aspartate at position 201 (D201ins) and alanine at position 124 (A124ins), found in compound heterozygous AGS with R114H, were prepared and the DNA degradation activities were tested. The homodimer TREX1(R114H/R114H) exhibits a 23-fold reduced single-stranded DNA (ssDNA) exonuclease activity relative to TREX1(WT). The TREX1(D201ins/D201ins) and TREX1(A124ins/A124ins) exhibit more than 10,000-fold reduced ssDNA degradation activities. However, the TREX1(R114H/D201ins) and TREX1(R114H/A124ins) compound heterodimers exhibit activities 10-fold greater than the TREX1(R114H/R114H) homodimer during ssDNA and double-stranded DNA (dsDNA) degradation. These higher levels of activities measured in the TREX1(R114H/D201ins) and TREX1(R114H/A124ins) compound heterodimers are attributed to Arg-114 residues of TREX1(D201ins) and TREX1(A124ins) positioned at the dimer interface contributing to the active sites of the opposing TREX1(R114H) protomer. This interpretation is further supported by exonuclease activities measured for TREX1 enzymes containing R114A and R114K mutations. These biochemical data provide direct evidence for TREX1 residues in one protomer contributing to DNA degradation catalyzed in the opposing protomer and help to explain the dimeric TREX1 structure required for full catalytic competency.
Assuntos
Doenças Autoimunes do Sistema Nervoso/enzimologia , DNA de Cadeia Simples/metabolismo , Exodesoxirribonucleases/metabolismo , Lúpus Eritematoso Sistêmico/enzimologia , Mutação de Sentido Incorreto , Malformações do Sistema Nervoso/enzimologia , Fosfoproteínas/metabolismo , Multimerização Proteica , Substituição de Aminoácidos , Doenças Autoimunes do Sistema Nervoso/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Exodesoxirribonucleases/química , Exodesoxirribonucleases/genética , Humanos , Lúpus Eritematoso Sistêmico/genética , Malformações do Sistema Nervoso/genética , Fosfoproteínas/química , Fosfoproteínas/genética , Regiões Promotoras Genéticas/genética , Estrutura Quaternária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato/genéticaRESUMO
The AmrZ protein from the pathogenic bacterium Pseudomonas aeruginosa is a transcription factor that activates and represses the genes for several potent virulence factors, which gives the bacteria a selective advantage in infection. AmrZ was crystallized in complex with DNA containing the amrZ1 repressor binding site. Obtaining crystals of the complex required the integration of a number of well known techniques along with the development of new methods. Here, these processes are organized and combined into a comprehensive method which yielded diffraction-quality crystals. Part of this method included thorough data mining of the crystallization conditions of protein-DNA complexes to create a new directed crystallization screen. An optimized technique for the verification of protein-DNA complexes in crystals is also presented. Taken together, the methods described in this article attempt to streamline the difficult process of obtaining diffraction-quality crystals of protein-DNA complexes through the organization of older methods combined with the introduction of new techniques.
Assuntos
Proteínas de Bactérias/química , Cristalização/métodos , DNA/química , Pseudomonas aeruginosa/química , Fatores de Transcrição/química , Sequência de Aminoácidos , Proteínas de Bactérias/análise , Cristalografia por Raios X , DNA/análise , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Fatores de Transcrição/análiseRESUMO
The goal of this study is to identify pharmacological inhibitors that target a recently identified novel mediator of breast cancer brain metastasis (BCBM), truncated glioma-associated oncogene homolog 1 (tGLI1). Inhibitors of tGLI1 are not yet available. To identify compounds that selectively kill tGLI1-expressing breast cancer, we screened 1527 compounds using two sets of isogenic breast cancer and brain-tropic breast cancer cell lines engineered to stably express the control, GLI1, or tGLI1 vector, and identified the FDA-approved antifungal ketoconazole (KCZ) to selectively target tGLI1-positive breast cancer cells and breast cancer stem cells, but not tGLI1-negative breast cancer and normal cells. KCZ's effects are dependent on tGLI1. Two experimental mouse metastasis studies have demonstrated that systemic KCZ administration prevented the preferential brain metastasis of tGLI1-positive breast cancer and suppressed the progression of established tGLI1-positive BCBM without liver toxicities. We further developed six KCZ derivatives, two of which (KCZ-5 and KCZ-7) retained tGLI1-selectivity in vitro. KCZ-7 exhibited higher blood-brain barrier penetration than KCZ/KCZ-5 and more effectively reduced the BCBM frequency. In contrast, itraconazole, another FDA-approved antifungal, failed to suppress BCBM. The mechanistic studies suggest that KCZ and KCZ-7 inhibit tGLI1's ability to bind to DNA, activate its target stemness genes Nanog and OCT4, and promote tumor proliferation and angiogenesis. Our study establishes the rationale for using KCZ and KCZ-7 for treating and preventing BCBM and identifies their mechanism of action.