RESUMEN
Foldback priming at DNA double-stranded breaks is one mechanism proposed to initiate palindromic gene amplification, a common feature of cancer cells. Here, we show that small (5-9 bp) inverted repeats drive the formation of large palindromic duplications, the major class of chromosomal rearrangements recovered from yeast cells lacking Sae2 or the Mre11 nuclease. RPA dysfunction increased the frequency of palindromic duplications in Sae2 or Mre11 nuclease-deficient cells by â¼ 1,000-fold, consistent with intra-strand annealing to create a hairpin-capped chromosome that is subsequently replicated to form a dicentric isochromosome. The palindromic duplications were frequently associated with duplication of a second chromosome region bounded by a repeated sequence and a telomere, suggesting the dicentric chromosome breaks and repairs by recombination between dispersed repeats to acquire a telomere. We propose secondary structures within single-stranded DNA are potent instigators of genome instability, and RPA and Mre11-Sae2 play important roles in preventing their formation and propagation, respectively.
Asunto(s)
Endodesoxirribonucleasas/metabolismo , Endonucleasas/metabolismo , Exodesoxirribonucleasas/metabolismo , Amplificación de Genes , Secuencias Invertidas Repetidas , Proteína de Replicación A/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Cromosomas Fúngicos/genética , Cromosomas Fúngicos/metabolismo , Roturas del ADN , ADN de Hongos/genética , ADN de Hongos/metabolismo , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Endodesoxirribonucleasas/genética , Endonucleasas/genética , Exodesoxirribonucleasas/genética , Inestabilidad Genómica/genética , Isocromosomas/genética , Proteína de Replicación A/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Tissue engineering scaffolds are intended to provide mechanical and biological support for cells to migrate, engraft and ultimately regenerate the tissue. Development of scaffolds with sustained delivery of growth factors and chemokines would enhance the therapeutic benefits, especially in wound healing. In this study, we incorporated our previously designed therapeutic particles, composed of fusion of elastin-like peptides (ELPs) as the drug delivery platform to keratinocyte growth factor (KGF), into a tissue scaffold, alloderm. The results demonstrated that sustained KGF-ELP release was achieved and the bioactivity of the released therapeutic particles was shown via cell proliferation assay, as well as a mouse pouch model in vivo, where higher cellular infiltration and vascularization were observed in scaffolds functionalized with KGF-ELPs.
Asunto(s)
Biopolímeros/química , Colágeno/química , Elastina/química , Andamios del Tejido/química , Animales , Biopolímeros/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Colágeno/farmacología , Factor 7 de Crecimiento de Fibroblastos/metabolismo , Humanos , Ratones , Neovascularización Fisiológica/efectos de los fármacos , Ingeniería de TejidosRESUMEN
Lipid bilayer-coated mesoporous silica nanoparticles are unique core-shell nanomaterials currently being developed as drug delivery vehicles. To improve cargo loading and biocirculation, the pore structure and surface chemistry of the particle have been modified and well characterized. However, an understanding of cargo release mechanisms from cellular uptake pathways remains largely unexplored. Here, we present a study of the release mechanism of lipid bilayer-coated silica particles induced by endosomal-like pH change from 7.4 to 5.0. We found that this relatively small pH change produces rapid deformation of the supported lipid bilayer that ultimately results in holes in the membrane. Using a combination of dye release studies, wide-field and confocal fluorescence microscopies, and surface area modeling analysis, we determined that small blister-like structures are formed, which lead to lateral membrane displacement and hole formation. Possible mechanisms for the blister formation, which include curvature effects and interfacial interactions, are discussed.
RESUMEN
The Mre11-Rad50-Xrs2/NBS1 (MRX/N) nuclease/ATPase complex plays structural and catalytic roles in the repair of DNA double-strand breaks (DSBs) and is the DNA damage sensor for Tel1/ATM kinase activation. Saccharomyces cerevisiae Sae2 can function with MRX to initiate 5'-3' end resection and also plays an important role in attenuation of DNA damage signaling. Here we describe a class of mre11 alleles that suppresses the DNA damage sensitivity of sae2Δ cells by accelerating turnover of Mre11 at DNA ends, shutting off the DNA damage checkpoint and allowing cell cycle progression. The mre11 alleles do not suppress the end resection or hairpin-opening defects of the sae2Δ mutant, indicating that these functions of Sae2 are not responsible for DNA damage resistance. The purified M(P110L)RX complex shows reduced binding to single- and double-stranded DNA in vitro relative to wild-type MRX, consistent with the increased turnover of Mre11 from damaged sites in vivo. Furthermore, overproduction of Mre11 causes DNA damage sensitivity only in the absence of Sae2. Together, these data suggest that it is the failure to remove Mre11 from DNA ends and attenuate Rad53 kinase signaling that causes hypersensitivity of sae2Δ cells to clastogens.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Quinasa de Punto de Control 2/metabolismo , Daño del ADN , Proteínas de Unión al ADN/metabolismo , Endodesoxirribonucleasas/metabolismo , Endonucleasas/metabolismo , Exodesoxirribonucleasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Quinasa de Punto de Control 2/genética , Roturas del ADN de Doble Cadena , Reparación del ADN , ADN de Hongos/genética , ADN de Hongos/metabolismo , Proteínas de Unión al ADN/genética , Endodesoxirribonucleasas/genética , Endonucleasas/genética , Exodesoxirribonucleasas/genética , Microscopía Fluorescente , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Mutación , Unión Proteica , Proteínas de Saccharomyces cerevisiae/genética , Transducción de Señal/genéticaRESUMEN
Replication protein A (RPA) is the main eukaryotic single-stranded DNA (ssDNA) binding protein, having essential roles in all DNA metabolic reactions involving ssDNA. RPA binds ssDNA with high affinity, thereby preventing the formation of secondary structures and protecting ssDNA from the action of nucleases, and directly interacts with other DNA processing proteins. Here, we discuss recent results supporting the idea that one function of RPA is to prevent annealing between short repeats that can lead to chromosome rearrangements by microhomology-mediated end joining or the formation of hairpin structures that are substrates for structure-selective nucleases. We suggest that replication fork catastrophe caused by depletion of RPA could result from cleavage of secondary structures by nucleases, and that failure to cleave hairpin structures formed at DNA ends could lead to gene amplification. These studies highlight the important role RPA plays in maintaining genome integrity.
Asunto(s)
Proteína de Replicación A/fisiología , Animales , Emparejamiento Base , Reparación del ADN , Replicación del ADN , Inestabilidad Genómica , Recombinación Homóloga , Humanos , Conformación de Ácido Nucleico , Homología de Secuencia de Ácido NucleicoRESUMEN
Elastin-like peptides (ELPs) are a versatile platform for tissue engineering and drug delivery. Here, micelle forming ELP chains are genetically fused to three therapeutic molecules, keratinocyte growth factor (KGF), stromal cell-derived growth factor 1 (SDF1), and cathelicidin (LL37), to be used in wound healing. Chronic wounds represent a growing problem worldwide. A combinatorial therapy approach targeting different aspects of wound healing would be beneficial, providing a controlled and sustained release of active molecules, while simultaneously protecting these therapeutics from the surrounding harsh wound environment. The results of this study demonstrate that the conjugation of the growth factors KGF and SDF1 and the antimicrobial peptide LL37 to ELPs does not affect the micelle structure and that all three therapeutic moieties retain their bioactivity in vitro. Importantly, when the combination of these micelle ELP nanoparticles are applied to wounds in diabetic mice, over 90 % wound closure is observed, which is significantly higher than when the therapeutics are applied in their naked forms. The application of the nanoparticles designed here is the first report of targeting different aspect of wound healing synergistically.
Asunto(s)
Diabetes Mellitus Experimental , Nanopartículas , Animales , Elastina/química , Elastina/metabolismo , Ratones , Micelas , Nanopartículas/química , Ingeniería de Tejidos , Cicatrización de HeridasRESUMEN
Eukaryotic nitric oxide (NO) signaling involves modulation of cyclic GMP (cGMP) levels through activation of the soluble isoform of guanylate cyclase (sGC). sGC is a heterodimeric hemoprotein that contains a Heme-Nitric oxide and OXygen binding (H-NOX) domain, a Per/ARNT/Sim (PAS) domain, a coiled-coil (CC) domain, and a catalytic domain. To evaluate the role of these domains in regulating the ligand binding properties of the heme cofactor of NO-sensitive sGC, we constructed chimeras by swapping the rat ß1 H-NOX domain with the homologous region of H-NOX domain-containing proteins from Thermoanaerobacter tengcongensis, Vibrio cholerae, and Caenorhabditis elegans (TtTar4H, VCA0720, and Gcy-33, respectively). Characterization of ligand binding by electronic absorption and resonance Raman spectroscopy indicates that the other rat sGC domains influence the bacterial and worm H-NOX domains. Analysis of cGMP production in these proteins reveals that the chimeras containing bacterial H-NOX domains exhibit guanylate cyclase activity, but this activity is not influenced by gaseous ligand binding to the heme cofactor. The rat-worm chimera containing the atypical sGC Gcy-33 H-NOX domain was weakly activated by NO, CO, and O(2), suggesting that atypical guanylate cyclases and NO-sensitive guanylate cyclases have a common molecular mechanism for enzyme activation. To probe the influence of the other sGC domains on the mammalian sGC heme environment, we generated heme pocket mutants (Pro118Ala and Ile145Tyr) in the ß1 H-NOX construct (residues 1-194), the ß1 H-NOX-PAS-CC construct (residues 1-385), and the full-length α1ß1 sGC heterodimer (ß1 residues 1-619). Spectroscopic characterization of these proteins shows that interdomain communication modulates the coordination state of the heme-NO complex and the heme oxidation rate. Taken together, these findings have important implications for the allosteric mechanism of regulation within H-NOX domain-containing proteins.
Asunto(s)
Dominio Catalítico , Guanilato Ciclasa/química , Guanilato Ciclasa/metabolismo , Receptores Citoplasmáticos y Nucleares/química , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Caenorhabditis elegans/enzimología , Guanilato Ciclasa/genética , Hemo/química , Hemo/metabolismo , Cinética , Ligandos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación , Óxido Nítrico/metabolismo , Oxígeno/metabolismo , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Ratas , Receptores Citoplasmáticos y Nucleares/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Guanilil Ciclasa Soluble , Thermoanaerobacter/enzimología , Vibrio cholerae/enzimologíaRESUMEN
Nitric oxide (NO) is the physiologically relevant activator of the mammalian hemoprotein soluble guanylate cyclase (sGC). The heme cofactor of alpha1beta1 sGC has a high affinity for NO but has never been observed to form a complex with oxygen. Introduction of a key tyrosine residue in the sGC heme binding domain beta1(1-385) is sufficient to produce an oxygen-binding protein, but this mutation in the full-length enzyme did not alter oxygen affinity. To evaluate ligand binding specificity in full-length sGC we mutated several conserved distal heme pocket residues (beta1 Val-5, Phe-74, Ile-145, and Ile-149) to introduce a hydrogen bond donor in proximity to the heme ligand. We found that the NO coordination state, NO dissociation, and enzyme activation were significantly affected by the presence of a tyrosine in the distal heme pocket; however, the stability of the reduced porphyrin and the proteins affinity for oxygen were unaltered. Recently, an atypical sGC from Drosophila, Gyc-88E, was shown to form a stable complex with oxygen. Sequence analysis of this protein identified two residues in the predicted heme pocket (tyrosine and glutamine) that may function to stabilize oxygen binding in the atypical cyclase. The introduction of these residues into the rat beta1 distal heme pocket (Ile-145 --> Tyr and Ile-149 --> Gln) resulted in an sGC construct that oxidized via an intermediate with an absorbance maximum at 417 nm. This absorbance maximum is consistent with globin Fe(II)-O(2) complexes and is likely the first observation of a Fe(II)-O(2) complex in the full-length alpha1beta1 protein. Additionally, these data suggest that atypical sGCs stabilize O(2) binding by a hydrogen bonding network involving tyrosine and glutamine.
Asunto(s)
Glutamina/química , Guanilato Ciclasa/química , Hemo/química , Óxido Nítrico/química , Oxígeno/química , Receptores Citoplasmáticos y Nucleares/química , Tirosina/química , Secuencia de Aminoácidos , Animales , Guanosina Trifosfato/química , Cinética , Datos de Secuencia Molecular , Porfirinas/química , Unión Proteica , Ratas , Homología de Secuencia de Aminoácido , Guanilil Ciclasa Soluble , Espectrofotometría/métodosRESUMEN
Projections of the stage of the Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) pandemic and local, regional and national public health policies to limit coronavirus spread as well as "reopen" cities and states, are best informed by serum neutralizing antibody titers measured by reproducible, high throughput, and statically credible antibody (Ab) assays. To date, a myriad of Ab tests, both available and FDA authorized for emergency, has led to confusion rather than insight per se. The present study reports the results of a rapid, point-in-time 1,000-person cohort study using serial blood donors in the New York City metropolitan area (NYC) using multiple serological tests, including enzyme-linked immunosorbent assays (ELISAs) and high throughput serological assays (HTSAs). These were then tested and associated with assays for neutralizing Ab (NAb). Of the 1,000 NYC blood donor samples in late June and early July 2020, 12.1% and 10.9% were seropositive using the Ortho Total Ig and the Abbott IgG HTSA assays, respectively. These serological assays correlated with neutralization activity specific to SARS-CoV-2. The data reported herein suggest that seroconversion in this population occurred in approximately 1 in 8 blood donors from the beginning of the pandemic in NYC (considered March 1, 2020). These findings deviate with an earlier seroprevalence study in NYC showing 13.7% positivity. Collectively however, these data demonstrate that a low number of individuals have serologic evidence of infection during this "first wave" and suggest that the notion of "herd immunity" at rates of ~60% or higher are not near. Furthermore, the data presented herein show that the nature of the Ab-based immunity is not invariably associated with the development of NAb. While the blood donor population may not mimic precisely the NYC population as a whole, rapid assessment of seroprevalence in this cohort and serial reassessment could aid public health decision making.
Asunto(s)
COVID-19/epidemiología , SARS-CoV-2/inmunología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/inmunología , Donantes de Sangre , COVID-19/inmunología , Estudios de Cohortes , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Humanos , Inmunoglobulina G/sangre , Masculino , Persona de Mediana Edad , Ciudad de Nueva York/epidemiología , SARS-CoV-2/patogenicidad , Sensibilidad y Especificidad , Seroconversión/fisiología , Estudios Seroepidemiológicos , Pruebas Serológicas/métodos , Glicoproteína de la Espiga del Coronavirus/inmunologíaRESUMEN
Soluble guanylate cyclase (sGC) serves as a receptor for the signaling agent nitric oxide (NO). sGC synthesis of cGMP is regulated by NO, GTP, ATP, and allosteric activators such as YC-1. The guanylate cyclase activity and adenylate cyclase activity of full-length sGC and the sGC catalytic domain constructs (alpha1(cat)beta1(cat)) are reported here. ATP is a mixed-type inhibitor of cGMP production for both sGC and alpha1(cat)beta1(cat), indicating that the C-terminus of sGC contains an allosteric nucleotide binding site. YC-1 did not activate alpha1(cat)beta1(cat) or compete with ATP inhibition of cGMP synthesis, which suggests that YC-1 and ATP bind to distinct sites. alpha1(cat)beta1(cat) and NO-stimulated sGC also synthesize cAMP, but this activity is inhibited by ATP via noncompetitive substrate inhibition and by GTP via mixed-type inhibition. Additionally, the adenylate cyclase activity of purified sGC was inhibited by PC12 lysate, suggesting that an intracellular small molecule or protein regulates this activity in vivo.
Asunto(s)
Adenosina Trifosfato/química , Guanosina Trifosfato/química , Guanilato Ciclasa/metabolismo , Subunidades de Proteína/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Adenosina Trifosfato/antagonistas & inhibidores , Adenosina Trifosfato/fisiología , Adenilil Ciclasas/metabolismo , Regulación Alostérica/fisiología , Sitio Alostérico/fisiología , Animales , Dominio Catalítico , Guanosina Trifosfato/metabolismo , Guanosina Trifosfato/fisiología , Guanilato Ciclasa/antagonistas & inhibidores , Guanilato Ciclasa/química , Células PC12 , Conformación Proteica , Subunidades de Proteína/antagonistas & inhibidores , Subunidades de Proteína/química , Ratas , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Receptores Citoplasmáticos y Nucleares/química , Guanilil Ciclasa Soluble , Especificidad por SustratoRESUMEN
CYP3A4, a cytochrome P450 enzyme regulated by the nuclear receptor PXR, is involved in most of the drug metabolizing pathways. Studying the regulation/induction of CYP3A4 and PXR is critical in toxicology and drug-drug interaction (DDI) studies. Primary human hepatocytes constitute the preferred in vitro platform for drug development efforts. However, they are expensive, scarce and heterogeneous. Hepatic cell lines, such as Huh7, could provide a cost-effective alternative, however, they express negligible amounts of CYP450s and PXR. In this study, we show that dinaciclib, a potent cyclin dependent kinase inhibitor, significantly increases the basal CYP3A4 and PXR levels in 24 hours. We also demonstrated that matured Huh7s can be used for drug induction studies, where CYP3A4, CYP1A2, CYP2C9, and CYP2C19 inductions were achieved following rifampicin treatment. More importantly, through a direct demonstration using amiodarone and rifampicin as model drugs, we showed that matured Huh7s present a suitable platform for DDI studies.
Asunto(s)
Amiodarona/farmacología , Sistema Enzimático del Citocromo P-450/biosíntesis , Hígado/metabolismo , Receptor X de Pregnano/metabolismo , Rifampin/farmacología , Línea Celular Tumoral , Inducción Enzimática/efectos de los fármacos , Humanos , Hígado/citologíaRESUMEN
Mammalian polymerase theta (Polθ) is a multifunctional enzyme that promotes error-prone DNA repair by alternative nonhomologous end joining (alt-NHEJ). Here we present structure-function analyses that reveal that, in addition to the polymerase domain, Polθ-helicase activity plays a central role during double-strand break (DSB) repair. Our results show that the helicase domain promotes chromosomal translocations by alt-NHEJ in mouse embryonic stem cells and also suppresses CRISPR-Cas9- mediated gene targeting by homologous recombination (HR). In vitro assays demonstrate that Polθ-helicase activity facilitates the removal of RPA from resected DSBs to allow their annealing and subsequent joining by alt-NHEJ. Consistent with an antagonistic role for RPA during alt-NHEJ, inhibition of RPA1 enhances end joining and suppresses recombination. Taken together, our results reveal that the balance between HR and alt-NHEJ is controlled by opposing activities of Polθ and RPA, providing further insight into the regulation of repair-pathway choice in mammalian cells.
Asunto(s)
Dominio Catalítico/genética , Reparación del ADN por Unión de Extremidades/genética , ADN Polimerasa Dirigida por ADN/genética , Células Madre Embrionarias/citología , Proteína de Replicación A/antagonistas & inhibidores , Animales , Sistemas CRISPR-Cas/genética , Línea Celular , Roturas del ADN de Doble Cadena , Recombinación Homóloga/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína de Replicación A/genética , Relación Estructura-Actividad , Translocación Genética/genética , ADN Polimerasa thetaRESUMEN
Microhomology-mediated end joining (MMEJ) is a Ku- and ligase IV-independent mechanism for the repair of DNA double-strand breaks that contributes to chromosome rearrangements. Here we used a chromosomal end-joining assay to determine the genetic requirements for MMEJ in Saccharomyces cerevisiae. We found that end resection influences the ability to expose microhomologies; however, it is not rate limiting for MMEJ in wild-type cells. The frequency of MMEJ increased by up to 350-fold in rfa1 hypomorphic mutants, suggesting that replication protein A (RPA) bound to the single-stranded DNA (ssDNA) overhangs formed by resection prevents spontaneous annealing between microhomologies. In vitro, the mutant RPA complexes were unable to fully extend ssDNA and were compromised in their ability to prevent spontaneous annealing. We propose that the helix-destabilizing activity of RPA channels ssDNA intermediates from mutagenic MMEJ to error-free homologous recombination, thus preserving genome integrity.