RESUMEN
The transition from transcription initiation to elongation at promoters of primary response genes (PRGs) in metazoan cells is controlled by inducible transcription factors, which utilize P-TEFb to phosphorylate RNA polymerase II (Pol II) in response to stimuli. Prior to stimulation, a fraction of P-TEFb is recruited to promoter-proximal regions in a catalytically inactive state bound to the 7SK small nuclear ribonucleoprotein (snRNP) complex. However, it remains unclear how and why the 7SK snRNP is assembled at these sites. Here we report that the transcriptional regulator KAP1 continuously tethers the 7SK snRNP to PRG promoters to facilitate P-TEFb recruitment and productive elongation in response to stimulation. Remarkably, besides PRGs, genome-wide studies revealed that KAP1 and 7SK snRNP co-occupy most promoter-proximal regions containing paused Pol II. Collectively, we provide evidence of an unprecedented mechanism controlling 7SK snRNP delivery to promoter-proximal regions to facilitate "on-site" P-TEFb activation and Pol II elongation.
Asunto(s)
Regulación Viral de la Expresión Génica , VIH/metabolismo , Regiones Promotoras Genéticas , ARN Polimerasa II/metabolismo , Proteínas Represoras/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Elongación de la Transcripción Genética , Sitios de Unión , Activación Enzimática , Células HCT116 , Células HEK293 , VIH/genética , Humanos , Células Jurkat , Complejos Multiproteicos , Factor B de Elongación Transcripcional Positiva/metabolismo , Interferencia de ARN , ARN Polimerasa II/genética , Proteínas Represoras/genética , Ribonucleoproteínas Nucleares Pequeñas/genética , Factores de Tiempo , Transfección , Proteína 28 que Contiene Motivos Tripartito , Activación ViralRESUMEN
Spontaneous deamination of DNA cytosine and adenine into uracil and hypoxanthine, respectively, causes C to T and A to G transition mutations if left unrepaired. Endonuclease Q (EndoQ) initiates the repair of these premutagenic DNA lesions in prokaryotes by cleaving the phosphodiester backbone 5' of either uracil or hypoxanthine bases or an apurinic/apyrimidinic (AP) lesion generated by the excision of these damaged bases. To understand how EndoQ achieves selectivity toward these structurally diverse substrates without cleaving undamaged DNA, we determined the crystal structures of Pyrococcus furiosus EndoQ bound to DNA substrates containing uracil, hypoxanthine, or an AP lesion. The structures show that substrate engagement by EndoQ depends both on a highly distorted conformation of the DNA backbone, in which the target nucleotide is extruded out of the helix, and direct hydrogen bonds with the deaminated bases. A concerted swing motion of the zinc-binding and C-terminal helical domains of EndoQ toward its catalytic domain allows the enzyme to clamp down on a sharply bent DNA substrate, shaping a deep active-site pocket that accommodates the extruded deaminated base. Within this pocket, uracil and hypoxanthine bases interact with distinct sets of amino acid residues, with positioning mediated by an essential magnesium ion. The EndoQ-DNA complex structures reveal a unique mode of damaged DNA recognition and provide mechanistic insights into the initial step of DNA damage repair by the alternative excision repair pathway. Furthermore, we demonstrate that the unique activity of EndoQ is useful for studying DNA deamination and repair in mammalian systems.
Asunto(s)
Proteínas Arqueales/química , ADN de Archaea/química , Endonucleasas/química , Pyrococcus furiosus/enzimología , Proteínas Arqueales/genética , Dominio Catalítico , ADN de Archaea/genética , Desaminación , Endonucleasas/genética , Pyrococcus furiosus/genéticaRESUMEN
Inducible transcriptional programs mediate the regulation of key biological processes and organismal functions. Despite their complexity, cells have evolved mechanisms to precisely control gene programs in response to environmental cues to regulate cell fate and maintain normal homeostasis. Upon stimulation with proinflammatory cytokines such as tumor necrosis factor-α (TNF), the master transcriptional regulator nuclear factor (NF)-κB utilizes the PPM1G/PP2Cγ phosphatase as a coactivator to normally induce inflammatory and cell survival programs. However, how PPM1G activity is precisely regulated to control NF-κB transcription magnitude and kinetics remains unknown. Here, we describe a mechanism by which the ARF tumor suppressor binds PPM1G to negatively regulate its coactivator function in the NF-κB circuit thereby promoting insult resolution. ARF becomes stabilized upon binding to PPM1G and forms a ternary protein complex with PPM1G and NF-κB at target gene promoters in a stimuli-dependent manner to provide tunable control of the NF-κB transcriptional program. Consistently, loss of ARF in colon epithelial cells leads to up-regulation of NF-κB antiapoptotic genes upon TNF stimulation and renders cells partially resistant to TNF-induced apoptosis in the presence of agents blocking the antiapoptotic program. Notably, patient tumor data analysis validates these findings by revealing that loss of ARF strongly correlates with sustained expression of inflammatory and cell survival programs. Collectively, we propose that PPM1G emerges as a therapeutic target in a variety of cancers arising from ARF epigenetic silencing, to loss of ARF function, as well as tumors bearing oncogenic NF-κB activation.
Asunto(s)
Inflamación/metabolismo , FN-kappa B/genética , Neoplasias/metabolismo , Proteína Fosfatasa 2C/metabolismo , Proteína p14ARF Supresora de Tumor/metabolismo , Apoptosis/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Epiteliales/patología , Humanos , Inflamación/genética , Complejos Multiproteicos , FN-kappa B/metabolismo , Neoplasias/genética , Neoplasias/patología , Regiones Promotoras Genéticas , Dominios Proteicos , Mapas de Interacción de Proteínas , Proteína Fosfatasa 2C/química , Proteína Fosfatasa 2C/genética , Transcripción Genética , Factor de Necrosis Tumoral alfa/farmacología , Proteína p14ARF Supresora de Tumor/genéticaRESUMEN
Human immunodeficiency virus type 1 (HIV-1) Vif recruits a cellular ubiquitin ligase complex to degrade antiviral APOBEC3 enzymes (APOBEC3C-H) and PP2A phosphatase regulators (PPP2R5A to PPP2R5E). While APOBEC3 antagonism is the canonical function of HIV-1 Vif, this viral accessory protein is also known to trigger G2/M cell cycle arrest. Vif initiates G2/M arrest by degrading multiple PPP2R5 family members, an activity prevalent among diverse HIV-1 and simian immunodeficiency virus (SIV) isolates. Here, computational protein-protein docking was used to delineate a Vif/CBF-ß/PPP2R5 complex in which Vif is predicted to bind the same PPP2R5 surface as physiologic phosphatase targets. This model was tested using targeted mutagenesis of amino acid residues within or adjacent to the putative interface to show loss or retention, respectively, of Vif-induced PPP2R5 degradation activity. Additionally, expression of a peptide that mimics cellular targets of PPP2R5s robustly inhibited Vif-mediated degradation of PPP2R5A but not APOBEC3G. Moreover, live-cell imaging studies examining Vif-mediated degradation of PPP2R5A and APOBEC3G within the same cell revealed that PPP2R5A degradation kinetics are comparable to those of APOBEC3G with a half-life of roughly 6 h postinfection, demonstrating that Vif can concurrently mediate the degradation of distinct cellular substrates. Finally, experiments with a panel of patient-derived Vif isolates indicated that PPP2R5A degradation activity is common in patient-derived isolates. Taken together, these results support a model in which PPP2R5 degradation and global changes in the cellular phosphoproteome are likely to be advantageous for viral pathogenesis.IMPORTANCE A critical function of HIV-1 Vif is to counteract the family of APOBEC3 innate immune proteins. It is also widely accepted that Vif induces G2/M cell cycle arrest in several different cell types. Recently, it has been shown that Vif degrades multiple PPP2R5 phosphoregulators to induce the G2/M arrest phenotype. Here, computational approaches are used to test a structural model of the Vif/PPP2R5 complex. In addition, imaging studies are used to show that Vif degrades these PPP2R5 substrates in roughly the same time frame as APOBEC3 degradation and that this activity is prevalent in patient-derived Vif isolates. These studies are important by further defining PPP2R5 proteins as a bona fide substrate of HIV-1 Vif.
Asunto(s)
Desaminasa APOBEC-3G/química , VIH-1/genética , Proteína Fosfatasa 2/química , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/química , Desaminasa APOBEC-3G/genética , Desaminasa APOBEC-3G/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Sitios de Unión , Expresión Génica , Regulación de la Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Células HEK293 , Infecciones por VIH/virología , VIH-1/aislamiento & purificación , VIH-1/metabolismo , Células HeLa , Interacciones Huésped-Patógeno/genética , Humanos , Cinética , Modelos Moleculares , Mutación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteína Fosfatasa 2/genética , Proteína Fosfatasa 2/metabolismo , Estructura Secundaria de Proteína , Proteolisis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal , Especificidad por Sustrato , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
Apolipoprotein B mRNA editing enzyme catalytic subunit-like protein 3B (APOBEC3B or A3B), as other APOBEC3 members, is a single-stranded (ss)DNA cytosine deaminase with antiviral activity. A3B is also overexpressed in multiple tumor types, such as carcinomas of the bladder, cervix, lung, head/neck, and breast. A3B generates both dispersed and clustered C-to-T and C-to-G mutations in intrinsically preferred trinucleotide motifs (TCA/TCG/TCT). A3B-catalyzed mutations are likely to promote tumor evolution and cancer progression and, as such, are associated with poor clinical outcomes. However, little is known about cellular processes that regulate A3B. Here, we used a proteomics approach involving affinity purification coupled to MS with human 293T cells to identify cellular proteins that interact with A3B. This approach revealed a specific interaction with cyclin-dependent kinase 4 (CDK4). We validated and mapped this interaction by co-immunoprecipitation experiments. Functional studies and immunofluorescence microscopy experiments in multiple cell lines revealed that A3B is not a substrate for CDK4-Cyclin D1 phosphorylation nor is its deaminase activity modulated. Instead, we found that A3B is capable of disrupting the CDK4-dependent nuclear import of Cyclin D1. We propose that this interaction may favor a more potent antiviral response and simultaneously facilitate cancer mutagenesis.
Asunto(s)
Ciclina D1/metabolismo , Quinasa 4 Dependiente de la Ciclina/metabolismo , Citidina Desaminasa/metabolismo , Antígenos de Histocompatibilidad Menor/metabolismo , Secuencia de Aminoácidos , Ciclina D1/genética , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 4 Dependiente de la Ciclina/genética , Citidina Desaminasa/antagonistas & inhibidores , Citidina Desaminasa/genética , Células HEK293 , Humanos , Inmunoprecipitación , Espectrometría de Masas , Microscopía Fluorescente , Antígenos de Histocompatibilidad Menor/genética , Péptidos/análisis , Péptidos/química , Fosforilación , Unión Proteica , Dominios Proteicos , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Alineación de SecuenciaRESUMEN
The single-stranded DNA cytosine-to-uracil deaminase APOBEC3B is an antiviral protein implicated in cancer. However, its substrates in cells are not fully delineated. Here APOBEC3B proteomics reveal interactions with a surprising number of R-loop factors. Biochemical experiments show APOBEC3B binding to R-loops in cells and in vitro. Genetic experiments demonstrate R-loop increases in cells lacking APOBEC3B and decreases in cells overexpressing APOBEC3B. Genome-wide analyses show major changes in the overall landscape of physiological and stimulus-induced R-loops with thousands of differentially altered regions, as well as binding of APOBEC3B to many of these sites. APOBEC3 mutagenesis impacts genes overexpressed in tumors and splice factor mutant tumors preferentially, and APOBEC3-attributed kataegis are enriched in RTCW motifs consistent with APOBEC3B deamination. Taken together with the fact that APOBEC3B binds single-stranded DNA and RNA and preferentially deaminates DNA, these results support a mechanism in which APOBEC3B regulates R-loops and contributes to R-loop mutagenesis in cancer.
Asunto(s)
Neoplasias , Estructuras R-Loop , Humanos , ADN de Cadena Simple/genética , Estudio de Asociación del Genoma Completo , Mutagénesis , Neoplasias/genética , Neoplasias/patología , Citidina Desaminasa/genética , Antígenos de Histocompatibilidad Menor/genética , Antígenos de Histocompatibilidad Menor/metabolismoRESUMEN
Although CRISPR/Cas9 technology has created a renaissance in genome engineering, particularly for gene knockout generation, methods to introduce precise single base changes are also highly desirable. The covalent fusion of a DNA-editing enzyme such as APOBEC to a Cas9 nickase complex has heightened hopes for such precision genome engineering. However, current cytosine base editors are prone to undesirable off-target mutations, including, most frequently, target-adjacent mutations. Here, we report a method to "attract" the DNA deaminase, APOBEC3B, to a target cytosine base for specific editing with minimal damage to adjacent cytosine bases. The key to this system is fusing an APOBEC-interacting protein (not APOBEC itself) to Cas9n, which attracts nuclear APOBEC3B transiently to the target site for editing. Several APOBEC3B interactors were tested and one, hnRNPUL1, demonstrated proof-of-concept with successful C-to-T editing of episomal and chromosomal substrates and lower frequencies of target-adjacent events.
Asunto(s)
Citidina Desaminasa/genética , Edición Génica/métodos , Ingeniería Genética/métodos , Ribonucleoproteínas Nucleares Heterogéneas/genética , Antígenos de Histocompatibilidad Menor/genética , Proteínas Nucleares/genética , Factores de Transcripción/genética , Desaminasas APOBEC/genética , Proteína 9 Asociada a CRISPR/genética , Sistemas CRISPR-Cas/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Citosina/química , ADN/genética , Desoxirribonucleasa I/genética , Genoma/genética , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Proteínas Nucleares/metabolismo , Prueba de Estudio Conceptual , Factores de Transcripción/metabolismoRESUMEN
APOBEC3B (A3B)-catalyzed DNA cytosine deamination contributes to the overall mutational landscape in breast cancer. Molecular mechanisms responsible for A3B upregulation in cancer are poorly understood. Here we show that a single E2F cis-element mediates repression in normal cells and that expression is activated by its mutational disruption in a reporter construct or the endogenous A3B gene. The same E2F site is required for A3B induction by polyomavirus T antigen indicating a shared molecular mechanism. Proteomic and biochemical experiments demonstrate the binding of wildtype but not mutant E2F promoters by repressive PRC1.6/E2F6 and DREAM/E2F4 complexes. Knockdown and overexpression studies confirm the involvement of these repressive complexes in regulating A3B expression. Altogether, these studies demonstrate that A3B expression is suppressed in normal cells by repressive E2F complexes and that viral or mutational disruption of this regulatory network triggers overexpression in breast cancer and provides fuel for tumor evolution.
Asunto(s)
Citidina Desaminasa/genética , Factores de Transcripción E2F/genética , Antígenos de Histocompatibilidad Menor/genética , Transducción de Señal , Citidina Desaminasa/metabolismo , Factores de Transcripción E2F/metabolismo , Células HEK293 , Humanos , Células MCF-7 , Antígenos de Histocompatibilidad Menor/metabolismo , Unión ProteicaRESUMEN
HIV-1 Vif hijacks a cellular ubiquitin ligase complex to degrade antiviral APOBEC3 enzymes and PP2A phosphatase regulators (PPP2R5A-E). APOBEC3 counteraction is essential for viral pathogenesis. However, Vif also functions through an unknown mechanism to induce G2 cell cycle arrest. Here, deep mutagenesis is used to define the Vif surface required for PPP2R5 degradation and isolate a panel of separation-of-function mutants (PPP2R5 degradation-deficient and APOBEC3G degradation-proficient). Functional studies with Vif and PPP2R5 mutants were combined to demonstrate that PPP2R5 is, in fact, the target Vif degrades to induce G2 arrest. Pharmacologic and genetic approaches show that direct modulation of PP2A function or depletion of specific PPP2R5 proteins causes an indistinguishable arrest phenotype. Vif function in the cell cycle checkpoint is present in common HIV-1 subtypes worldwide and likely advantageous for viral pathogenesis.
Asunto(s)
Puntos de Control del Ciclo Celular , Proteína Fosfatasa 2/metabolismo , Proteolisis , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/metabolismo , Línea Celular , Puntos de Control de la Fase G2 del Ciclo Celular , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Fosforilación , Unión Proteica , Reproducibilidad de los Resultados , Electricidad Estática , Especificidad por SustratoRESUMEN
APOBEC3B is a single-stranded DNA cytosine deaminase with beneficial innate antiviral functions. However, misregulated APOBEC3B can also be detrimental by inflicting APOBEC signature C-to-T and C-to-G mutations in genomic DNA of multiple cancer types. Polyomavirus and papillomavirus oncoproteins induce APOBEC3B overexpression, perhaps to their own benefit, but little is known about the cellular mechanisms hijacked by these viruses to do so. Here we investigate the molecular mechanism of APOBEC3B upregulation by the polyomavirus large T antigen. First, we demonstrate that the upregulated APOBEC3B enzyme is strongly nuclear and partially localized to virus replication centers. Second, truncated T antigen (truncT) is sufficient for APOBEC3B upregulation, and the RB-interacting motif (LXCXE), but not the p53-binding domain, is required. Third, genetic knockdown of RB1 alone or in combination with RBL1 and/or RBL2 is insufficient to suppress truncT-mediated induction of APOBEC3B Fourth, CDK4/6 inhibition by palbociclib is also insufficient to suppress truncT-mediated induction of APOBEC3B Last, global gene expression analyses in a wide range of human cancers show significant associations between expression of APOBEC3B and other genes known to be regulated by the RB/E2F axis. These experiments combine to implicate the RB/E2F axis in promoting APOBEC3B transcription, yet they also suggest that the polyomavirus RB-binding motif has at least one additional function in addition to RB inactivation for triggering APOBEC3B upregulation in virus-infected cells.IMPORTANCE The APOBEC3B DNA cytosine deaminase is overexpressed in many different cancers and correlates with elevated frequencies of C-to-T and C-to-G mutations in 5'-TC motifs, oncogene activation, acquired drug resistance, and poor clinical outcomes. The mechanisms responsible for APOBEC3B overexpression are not fully understood. Here, we show that the polyomavirus truncated T antigen (truncT) triggers APOBEC3B overexpression through its RB-interacting motif, LXCXE, which in turn likely modulates the binding of E2F family transcription factors to promote APOBEC3B expression. This work strengthens the mechanistic linkage between active cell cycling, APOBEC3B overexpression, and cancer mutagenesis. Although this mutational mechanism damages cellular genomes, viruses may leverage it to promote evolution, immune escape, and pathogenesis. The cellular portion of the mechanism may also be relevant to nonviral cancers, where genetic mechanisms often activate the RB/E2F axis and APOBEC3B mutagenesis contributes to tumor evolution.
Asunto(s)
Antígenos Virales de Tumores/metabolismo , Citidina Desaminasa/biosíntesis , Interacciones Huésped-Patógeno , Antígenos de Histocompatibilidad Menor/biosíntesis , Poliomavirus/crecimiento & desarrollo , Proteína p53 Supresora de Tumor/metabolismo , Regulación hacia Arriba , Antígenos Virales de Tumores/genética , Sitios de Unión , Células Cultivadas , Factores de Transcripción E2F/metabolismo , Perfilación de la Expresión Génica , Humanos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Neoplasias/patología , Proteínas de Unión a Retinoblastoma/metabolismoRESUMEN
The apolipoprotein B messenger RNA editing enzyme, catalytic polypeptide-like (APOBEC) family of single-stranded DNA (ssDNA) cytosine deaminases provides innate immunity against virus and transposon replication1-4. A well-studied mechanism is APOBEC3G restriction of human immunodeficiency virus type 1, which is counteracted by a virus-encoded degradation mechanism1-4. Accordingly, most work has focused on retroviruses with obligate ssDNA replication intermediates and it is unclear whether large double-stranded DNA (dsDNA) viruses may be similarly susceptible to restriction. Here, we show that the large dsDNA herpesvirus Epstein-Barr virus (EBV), which is the causative agent of infectious mononucleosis and multiple cancers5, utilizes a two-pronged approach to counteract restriction by APOBEC3B. Proteomics studies and immunoprecipitation experiments showed that the ribonucleotide reductase large subunit of EBV, BORF26,7, binds APOBEC3B. Mutagenesis mapped the interaction to the APOBEC3B catalytic domain, and biochemical studies demonstrated that BORF2 stoichiometrically inhibits APOBEC3B DNA cytosine deaminase activity. BORF2 also caused a dramatic relocalization of nuclear APOBEC3B to perinuclear bodies. On lytic reactivation, BORF2-null viruses were susceptible to APOBEC3B-mediated deamination as evidenced by lower viral titres, lower infectivity and hypermutation. The Kaposi's sarcoma-associated herpesvirus homologue, ORF61, also bound APOBEC3B and mediated relocalization. These data support a model where the genomic integrity of human γ-herpesviruses is maintained by active neutralization of the antiviral enzyme APOBEC3B.
Asunto(s)
Citidina Desaminasa/antagonistas & inhibidores , Herpesvirus Humano 4/metabolismo , Herpesvirus Humano 8/metabolismo , Ribonucleótido Reductasas/metabolismo , Proteínas Virales/metabolismo , Sistemas CRISPR-Cas , Dominio Catalítico/genética , Línea Celular , Genoma Viral/genética , Células HEK293 , Herpesvirus Humano 4/crecimiento & desarrollo , Humanos , Antígenos de Histocompatibilidad Menor , Interferencia de ARN , ARN Interferente Pequeño/genética , Ribonucleótido Reductasas/genética , Proteínas Virales/genéticaRESUMEN
The APOBEC3 family of cytosine deaminases catalyzes the conversion of cytosines-to-uracils in single-stranded DNA. Traditionally, these enzymes are associated with antiviral immunity and restriction of DNA-based pathogens. However, a role for these enzymes in tumor evolution and metastatic disease has also become evident. The primary APOBEC3 candidate in cancer mutagenesis is APOBEC3B (A3B) for three reasons: (1) A3B mRNA is upregulated in several different cancers, (2) A3B expression and mutational loads correlate with poor clinical outcomes, and (3) A3B is the only family member known to be constitutively nuclear. Previous studies have mapped non-canonical A3B nuclear localization determinants to a single surface-exposed patch within the N-terminal domain (NTD). Here, we show that A3B has an additional, distinct, surface-exposed NTD region that contributes to nuclear localization. Disruption of residues within the first 30 amino acids of A3B (import surface 1) or loop 5/α-helix 3 (import surface 2) completely abolish nuclear localization. These import determinants also graft into NTDs of related family members and mediate re-localization from cell-wide-to-nucleus or cytoplasm-to-nucleus. These findings demonstrate that both sets of residues are required for non-canonical A3B nuclear localization and describe unique surfaces that may serve as novel therapeutic targets.
Asunto(s)
Núcleo Celular/metabolismo , Citidina Desaminasa/química , Citidina Desaminasa/metabolismo , Antígenos de Histocompatibilidad Menor/química , Antígenos de Histocompatibilidad Menor/metabolismo , Señales de Localización Nuclear/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Núcleo Celular/genética , Citidina Desaminasa/genética , Células HEK293 , Células HeLa , Humanos , Antígenos de Histocompatibilidad Menor/genética , Mutación , Conformación Proteica , Dominios Proteicos , Homología de SecuenciaRESUMEN
APOBEC enzymes are DNA cytosine deaminases that normally serve as virus restriction factors, but several members, including APOBEC3H, also contribute to cancer mutagenesis. Despite their importance in multiple fields, little is known about cellular processes that regulate these DNA mutating enzymes. We show that APOBEC3H exists in two distinct subcellular compartments, cytoplasm and nucleolus, and that the structural determinants for each mechanism are genetically separable. First, native and fluorescently tagged APOBEC3Hs localize to these two compartments in multiple cell types. Second, a series of genetic, pharmacologic, and cell biological studies demonstrate active cytoplasmic and nucleolar retention mechanisms, whereas nuclear import and export occur through passive diffusion. Third, APOBEC3H cytoplasmic retention determinants relocalize APOBEC3A from a passive cell-wide state to the cytosol and, additionally, endow potent HIV-1 restriction activity. These results indicate that APOBEC3H has a structural zipcode for subcellular localization and selecting viral substrates for restriction.
Asunto(s)
Aminohidrolasas/metabolismo , VIH-1/fisiología , Transporte Activo de Núcleo Celular/fisiología , Secuencia de Aminoácidos , Carcinogénesis/metabolismo , Línea Celular , Línea Celular Tumoral , Núcleo Celular/metabolismo , Citidina Desaminasa/metabolismo , Citoplasma/metabolismo , Células HEK293 , Células HeLa , HumanosRESUMEN
Human APOBEC3H (A3H) is a single-stranded DNA cytosine deaminase that inhibits HIV-1. Seven haplotypes (I-VII) and four splice variants (SV154/182/183/200) with differing antiviral activities and geographic distributions have been described, but the genetic and mechanistic basis for variant expression and function remains unclear. Using a combined bioinformatic/experimental analysis, we find that SV200 expression is specific to haplotype II, which is primarily found in sub-Saharan Africa. The underlying genetic mechanism for differential mRNA splicing is an ancient intronic deletion [del(ctc)] within A3H haplotype II sequence. We show that SV200 is at least fourfold more HIV-1 restrictive than other A3H splice variants. To counteract this elevated antiviral activity, HIV-1 protease cleaves SV200 into a shorter, less restrictive isoform. Our analyses indicate that, in addition to Vif-mediated degradation, HIV-1 may use protease as a counter-defense mechanism against A3H in >80% of sub-Saharan African populations.
Asunto(s)
Empalme Alternativo/inmunología , Aminohidrolasas/inmunología , Proteasa del VIH/inmunología , VIH-1/inmunología , Haplotipos/inmunología , Empalme Alternativo/genética , Secuencia de Aminoácidos , Aminohidrolasas/genética , Aminohidrolasas/metabolismo , Secuencia de Bases , Células HEK293 , Proteasa del VIH/metabolismo , VIH-1/metabolismo , Haplotipos/genética , Humanos , Isoenzimas/genética , Isoenzimas/inmunología , Isoenzimas/metabolismo , Polimorfismo de Nucleótido Simple/genética , Polimorfismo de Nucleótido Simple/inmunología , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Replicación Viral/inmunología , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/inmunología , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
APOBEC-catalyzed cytosine-to-uracil deamination of single-stranded DNA (ssDNA) has beneficial functions in immunity and detrimental effects in cancer. APOBEC enzymes have intrinsic dinucleotide specificities that impart hallmark mutation signatures. Although numerous structures have been solved, mechanisms for global ssDNA recognition and local target-sequence selection remain unclear. Here we report crystal structures of human APOBEC3A and a chimera of human APOBEC3B and APOBEC3A bound to ssDNA at 3.1-Å and 1.7-Å resolution, respectively. These structures reveal a U-shaped DNA conformation, with the specificity-conferring -1 thymine flipped out and the target cytosine inserted deep into the zinc-coordinating active site pocket. The -1 thymine base fits into a groove between flexible loops and makes direct hydrogen bonds with the protein, accounting for the strong 5'-TC preference. These findings explain both conserved and unique properties among APOBEC family members, and they provide a basis for the rational design of inhibitors to impede the evolvability of viruses and tumors.
Asunto(s)
Citidina Desaminasa/química , Antígenos de Histocompatibilidad Menor/química , Proteínas/química , Aminación , Secuencia de Bases , Sitios de Unión , Dominio Catalítico , Secuencia de Consenso , Cristalografía por Rayos X , Citidina Desaminasa/fisiología , Citosina , ADN de Cadena Simple/química , Humanos , Enlace de Hidrógeno , Cinética , Antígenos de Histocompatibilidad Menor/fisiología , Modelos Moleculares , Mutagénesis , Unión Proteica , Conformación Proteica en Hélice alfa , Proteínas/fisiología , Especificidad por SustratoRESUMEN
Cytosine mutations within TCA/T motifs are common in cancer. A likely cause is the DNA cytosine deaminase APOBEC3B (A3B). However, A3B-null breast tumours still have this mutational bias. Here we show that APOBEC3H haplotype I (A3H-I) provides a likely solution to this paradox. A3B-null tumours with this mutational bias have at least one copy of A3H-I despite little genetic linkage between these genes. Although deemed inactive previously, A3H-I has robust activity in biochemical and cellular assays, similar to A3H-II after compensation for lower protein expression levels. Gly105 in A3H-I (versus Arg105 in A3H-II) results in lower protein expression levels and increased nuclear localization, providing a mechanism for accessing genomic DNA. A3H-I also associates with clonal TCA/T-biased mutations in lung adenocarcinoma suggesting this enzyme makes broader contributions to cancer mutagenesis. These studies combine to suggest that A3B and A3H-I, together, explain the bulk of 'APOBEC signature' mutations in cancer.
Asunto(s)
Adenocarcinoma/metabolismo , Aminohidrolasas/genética , Aminohidrolasas/metabolismo , Neoplasias de la Mama/genética , Predisposición Genética a la Enfermedad , Neoplasias Pulmonares/genética , Adenocarcinoma/genética , ADN/genética , Femenino , Haplotipos , Humanos , MutaciónRESUMEN
Overexpression of the antiviral DNA cytosine deaminase APOBEC3B has been linked to somatic mutagenesis in many cancers. Human papillomavirus infection accounts for APOBEC3B upregulation in cervical and head/neck cancers, but the mechanisms underlying nonviral malignancies are unclear. In this study, we investigated the signal transduction pathways responsible for APOBEC3B upregulation. Activation of protein kinase C (PKC) by the diacylglycerol mimic phorbol-myristic acid resulted in specific and dose-responsive increases in APOBEC3B expression and activity, which could then be strongly suppressed by PKC or NF-κB inhibition. PKC activation caused the recruitment of RELB, but not RELA, to the APOBEC3B promoter, implicating noncanonical NF-κB signaling. Notably, PKC was required for APOBEC3B upregulation in cancer cell lines derived from multiple tumor types. By revealing how APOBEC3B is upregulated in many cancers, our findings suggest that PKC and NF-κB inhibitors may be repositioned to suppress cancer mutagenesis, dampen tumor evolution, and decrease the probability of adverse outcomes, such as drug resistance and metastasis.
Asunto(s)
Citidina Desaminasa/biosíntesis , Neoplasias/metabolismo , Proteína Quinasa C/metabolismo , Factor de Transcripción ReIA/metabolismo , Factor de Transcripción ReIB/metabolismo , Línea Celular Tumoral , Citidina Desaminasa/genética , Humanos , Antígenos de Histocompatibilidad Menor , Subunidad p50 de NF-kappa B/biosíntesis , Subunidad p52 de NF-kappa B/biosíntesis , Neoplasias/genética , Infecciones por Papillomavirus/patología , Regiones Promotoras Genéticas/genética , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/genética , Transducción de Señal , Acetato de Tetradecanoilforbol/análogos & derivados , Acetato de Tetradecanoilforbol/farmacología , Factor de Transcripción ReIA/antagonistas & inhibidores , Factor de Transcripción ReIB/antagonistas & inhibidores , Activación TranscripcionalRESUMEN
The transition from transcription initiation into elongation is controlled by transcription factors, which recruit positive transcription elongation factor b (P-TEFb) to promoters to phosphorylate RNA polymerase II. A fraction of P-TEFb is recruited as part of the inhibitory 7SK small nuclear ribonucleoprotein particle (snRNP), which inactivates the kinase and prevents elongation. However, it is unclear how P-TEFb is captured from the promoter-bound 7SK snRNP to activate elongation. Here, we describe a mechanism by which transcription factors mediate the enzymatic release of P-TEFb from the 7SK snRNP at promoters to trigger activation in a gene-specific manner. We demonstrate that Tat recruits PPM1G/PP2Cγ to locally disassemble P-TEFb from the 7SK snRNP at the HIV promoter via dephosphorylation of the kinase T loop. Similar to Tat, nuclear factor (NF)-κB recruits PPM1G in a stimulus-dependent manner to activate elongation at inflammatory-responsive genes. Recruitment of PPM1G to promoter-assembled 7SK snRNP provides a paradigm for rapid gene activation through transcriptional pause release.