RESUMO
Long noncoding RNAs (lncRNAs) are involved in diverse cellular processes through multiple mechanisms. Here, we describe a previously uncharacterized human lncRNA, CONCR (cohesion regulator noncoding RNA), that is transcriptionally activated by MYC and is upregulated in multiple cancer types. The expression of CONCR is cell cycle regulated, and it is required for cell-cycle progression and DNA replication. Moreover, cells depleted of CONCR show severe defects in sister chromatid cohesion, suggesting an essential role for CONCR in cohesion establishment during cell division. CONCR interacts with and regulates the activity of DDX11, a DNA-dependent ATPase and helicase involved in DNA replication and sister chromatid cohesion. These findings unveil a direct role for an lncRNA in the establishment of sister chromatid cohesion by modulating DDX11 enzymatic activity.
Assuntos
Cromátides/metabolismo , Replicação do DNA , DNA de Neoplasias/biossíntese , Neoplasias/metabolismo , RNA Longo não Codificante/metabolismo , Células A549 , Animais , Apoptose , Proliferação de Células , Cromátides/genética , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , DNA de Neoplasias/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Células HeLa , Humanos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Neoplasias/genética , Neoplasias/patologia , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Interferência de RNA , RNA Longo não Codificante/genética , Fatores de Tempo , Transcrição Gênica , Ativação Transcricional , Transfecção , Carga Tumoral , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
As the powerhouses of the eukaryotic cell, mitochondria must maintain their genomes which encode proteins essential for energy production. Mitochondria are characterized by guanine-rich DNA sequences that spontaneously form unusual three-dimensional structures known as G-quadruplexes (G4). G4 structures can be problematic for the essential processes of DNA replication and transcription because they deter normal progression of the enzymatic-driven processes. In this study, we addressed the hypothesis that mitochondrial G4 is a source of mutagenesis leading to base-pair substitutions. Our computational analysis of 2757 individual genomes from two Italian population cohorts (SardiNIA and InCHIANTI) revealed a statistically significant enrichment of mitochondrial mutations within sequences corresponding to stable G4 DNA structures. Guided by the computational analysis results, we designed biochemical reconstitution experiments and demonstrated that DNA synthesis by two known mitochondrial DNA polymerases (Pol γ, PrimPol) in vitro was strongly blocked by representative stable G4 mitochondrial DNA structures, which could be overcome in a specific manner by the ATP-dependent G4-resolving helicase Pif1. However, error-prone DNA synthesis by PrimPol using the G4 template sequence persisted even in the presence of Pif1. Altogether, our results suggest that genetic variation is enriched in G-quadruplex regions that impede mitochondrial DNA replication.
Assuntos
DNA Helicases/genética , DNA Polimerase gama/genética , DNA Primase/genética , Replicação do DNA/genética , DNA Polimerase Dirigida por DNA/genética , Quadruplex G , Enzimas Multifuncionais/genética , DNA Mitocondrial/genética , Genoma Mitocondrial/genética , Guanina/metabolismo , Humanos , Itália , Mitocôndrias/genética , Mutagênese/genética , Mutação/genética , Conformação de Ácido Nucleico , Sequenciamento Completo do GenomaRESUMO
Fanconi Anemia (FA) is characterized by bone marrow failure, congenital abnormalities, and cancer. Of over 20 FA-linked genes, FANCJ uniquely encodes a DNA helicase and mutations are also associated with breast and ovarian cancer. fancj-/- cells are sensitive to DNA interstrand cross-linking (ICL) and replication fork stalling drugs. We delineated the molecular defects of two FA patient-derived FANCJ helicase domain mutations. FANCJ-R707C was compromised in dimerization and helicase processivity, whereas DNA unwinding by FANCJ-H396D was barely detectable. DNA binding and ATP hydrolysis was defective for both FANCJ-R707C and FANCJ-H396D, the latter showing greater reduction. Expression of FANCJ-R707C or FANCJ-H396D in fancj-/- cells failed to rescue cisplatin or mitomycin sensitivity. Live-cell imaging demonstrated a significantly compromised recruitment of FANCJ-R707C to laser-induced DNA damage. However, FANCJ-R707C expressed in fancj-/- cells conferred resistance to the DNA polymerase inhibitor aphidicolin, G-quadruplex ligand telomestatin, or DNA strand-breaker bleomycin, whereas FANCJ-H396D failed. Thus, a minimal threshold of FANCJ catalytic activity is required to overcome replication stress induced by aphidicolin or telomestatin, or to repair bleomycin-induced DNA breakage. These findings have implications for therapeutic strategies relying on DNA cross-link sensitivity or heightened replication stress characteristic of cancer cells.
Assuntos
Quebras de DNA de Cadeia Dupla , DNA Helicases/genética , DNA Helicases/metabolismo , Reparo do DNA , Replicação do DNA , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , RNA Helicases/genética , RNA Helicases/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Animais , Afidicolina/toxicidade , Linhagem Celular , Quinase 1 do Ponto de Checagem/metabolismo , Galinhas , Cisplatino/toxicidade , DNA de Cadeia Simples , Anemia de Fanconi/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/química , Quadruplex G , Mutação de Sentido Incorreto , Oxazóis/toxicidade , RNA Helicases/química , Rad51 Recombinase/análise , Recombinases/genética , Recombinases/metabolismo , Proteína de Replicação A/metabolismo , Estresse FisiológicoRESUMO
Cockayne syndrome is a neurodegenerative accelerated aging disorder caused by mutations in the CSA or CSB genes. Although the pathogenesis of Cockayne syndrome has remained elusive, recent work implicates mitochondrial dysfunction in the disease progression. Here, we present evidence that loss of CSA or CSB in a neuroblastoma cell line converges on mitochondrial dysfunction caused by defects in ribosomal DNA transcription and activation of the DNA damage sensor poly-ADP ribose polymerase 1 (PARP1). Indeed, inhibition of ribosomal DNA transcription leads to mitochondrial dysfunction in a number of cell lines. Furthermore, machine-learning algorithms predict that diseases with defects in ribosomal DNA (rDNA) transcription have mitochondrial dysfunction, and, accordingly, this is found when factors involved in rDNA transcription are knocked down. Mechanistically, loss of CSA or CSB leads to polymerase stalling at non-B DNA in a neuroblastoma cell line, in particular at G-quadruplex structures, and recombinant CSB can melt G-quadruplex structures. Indeed, stabilization of G-quadruplex structures activates PARP1 and leads to accelerated aging in Caenorhabditis elegans In conclusion, this work supports a role for impaired ribosomal DNA transcription in Cockayne syndrome and suggests that transcription-coupled resolution of secondary structures may be a mechanism to repress spurious activation of a DNA damage response.
Assuntos
DNA Helicases/genética , Enzimas Reparadoras do DNA/genética , DNA de Neoplasias/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , Fatores de Transcrição/genética , Transcrição Gênica , Linhagem Celular Tumoral , Síndrome de Cockayne/genética , Síndrome de Cockayne/metabolismo , Dano ao DNA , DNA Helicases/metabolismo , Reparo do DNA , Enzimas Reparadoras do DNA/metabolismo , DNA de Neoplasias/química , DNA de Neoplasias/metabolismo , DNA Ribossômico/genética , Quadruplex G , Técnicas de Silenciamento de Genes , Humanos , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Warsaw breakage syndrome (WBS) is a recently recognized DDX11-related rare cohesinopathy, characterized by severe prenatal and postnatal growth restriction, microcephaly, developmental delay, cochlear anomalies, and sensorineural hearing loss. Only seven cases have been reported in the English literature, and thus the information on the phenotype and genotype of this interesting condition is limited. We provide clinical and molecular information on five additional unrelated patients carrying novel bi-allelic variants in the DDX11 gene, identified via whole exome sequencing. One of the variants was found to be a novel Saudi founder variant. All identified variants were classified as pathogenic or likely pathogenic except for one that was initially classified as a variant of unknown significance (VOUS) (p.Arg378Pro). Functional characterization of this VOUS using heterologous expression of wild type and mutant DDX11 revealed a marked effect on protein stability, thus confirming pathogenicity of this variant. The phenotypic data of the seven WBS reported patients were compared to our patients for further phenotypic delineation. Although all the reported patients had cochlear hypoplasia, one patient also had posterior labyrinthine anomaly. We conclude that while the cardinal clinical features in WBS (microcephaly, growth retardation, and cochlear anomalies) are almost universally present, the breakage phenotype is highly variable and can be absent in some cases. This report further expands the knowledge of the phenotypic and molecular features of WBS.
Assuntos
Anormalidades Múltiplas/genética , Quebra Cromossômica , Sequência de Aminoácidos , Criança , Pré-Escolar , RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/genética , DNA Helicases/química , DNA Helicases/genética , Orelha Interna/diagnóstico por imagem , Fácies , Feminino , Regulação da Expressão Gênica , Humanos , Lactente , Recém-Nascido , Imageamento por Ressonância Magnética , Masculino , Modelos Moleculares , Fenótipo , Inibidores de Proteassoma/farmacologia , Estabilidade Proteica , Síndrome , Tomografia Computadorizada por Raios XRESUMO
We present evidence that Tim establishes a physical and functional interaction with DDX11, a super-family 2 iron-sulfur cluster DNA helicase genetically linked to the chromosomal instability disorder Warsaw breakage syndrome. Tim stimulates DDX11 unwinding activity on forked DNA substrates up to 10-fold and on bimolecular anti-parallel G-quadruplex DNA structures and three-stranded D-loop approximately 4-5-fold. Electrophoretic mobility shift assays revealed that Tim enhances DDX11 binding to DNA, suggesting that the observed stimulation derives from an improved ability of DDX11 to interact with the nucleic acid substrate. Surface plasmon resonance measurements indicate that DDX11 directly interacts with Tim. DNA fiber track assays with HeLa cells exposed to hydroxyurea demonstrated that Tim or DDX11 depletion significantly reduced replication fork progression compared to control cells; whereas no additive effect was observed by co-depletion of both proteins. Moreover, Tim and DDX11 are epistatic in promoting efficient resumption of stalled DNA replication forks in hydroxyurea-treated cells. This is consistent with the finding that association of the two endogenous proteins in the cell extract chromatin fraction is considerably increased following hydroxyurea exposure. Overall, our studies provide evidence that Tim and DDX11 physically and functionally interact and act in concert to preserve replication fork progression in perturbed conditions.
Assuntos
Proteínas de Ciclo Celular/metabolismo , RNA Helicases DEAD-box/metabolismo , DNA Helicases/metabolismo , Replicação do DNA , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Sequência de Bases , Proteínas de Ciclo Celular/genética , RNA Helicases DEAD-box/genética , DNA/química , DNA/metabolismo , DNA Helicases/genética , Replicação do DNA/genética , Quadruplex G , Células HeLa/efeitos dos fármacos , Humanos , Hidroxiureia/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Dados de Sequência Molecular , Conformação de Ácido NucleicoRESUMO
Mutations in the c10orf2 gene encoding the human mitochondrial DNA replicative helicase Twinkle are linked to several rare genetic diseases characterized by mitochondrial defects. In this study, we have examined the catalytic activity of Twinkle helicase on model replication fork and DNA repair structures. Although Twinkle behaves as a traditional 5' to 3' helicase on conventional forked duplex substrates, the enzyme efficiently dissociates D-loop DNA substrates irrespective of whether it possesses a 5' or 3' single-stranded tailed invading strand. In contrast, we report for the first time that Twinkle branch-migrates an open-ended mobile three-stranded DNA structure with a strong 5' to 3' directionality preference. To determine how well Twinkle handles potential roadblocks to mtDNA replication, we tested the ability of the helicase to unwind substrates with site-specific oxidative DNA lesions or bound by the mitochondrial transcription factor A. Twinkle helicase is inhibited by DNA damage in a unique manner that is dependent on the type of oxidative lesion and the strand in which it resides. Novel single molecule FRET binding and unwinding assays show an interaction of the excluded strand with Twinkle as well as events corresponding to stepwise unwinding and annealing. TFAM inhibits Twinkle unwinding, suggesting other replisome proteins may be required for efficient removal. These studies shed new insight on the catalytic functions of Twinkle on the key DNA structures it would encounter during replication or possibly repair of the mitochondrial genome and how well it tolerates potential roadblocks to DNA unwinding.
Assuntos
DNA Helicases/metabolismo , DNA/metabolismo , Proteínas Mitocondriais/metabolismo , DNA/química , Dano ao DNA , Transferência Ressonante de Energia de Fluorescência , Humanos , Oxirredução , Especificidade por SubstratoRESUMO
Now a day's meat and meat products are not only generating convenience trends; they have been recognized as core of meat industry. Meat spread is a convenience cooked spreadable product prepared with meat and non-meat additives. Response surface methodology was used to investigate the effects of three different levels of honey (10, 15, 20 g), vinegar (2.0, 6.0, 10.0 ml) and tomato powder (0.5, 1.0, 1.5 g) on response variables viz. color/appearance, flavor, spreadability, texture, after taste, adhesiveability, overall acceptability, while standardizing the process of development of sweet and sour chicken meat spread box-behnken experimental design was used in which 17 different runs with 5 trials of three similar centre point. A second order polynomial was fitted to all the response variables and surface plots as well as equations were conducted. All the processing variables significantly affected the response variables either linearly or quadratically whereas the "Lack of Fit" was non-significant relative to the pure error. For optimization, target values were set in the form of ranges of all the processing and response variables. While applying multiple regression analysis, a total of 43 workable solutions was found, out of which the product with 14.28% honey, 5.38% vinegar and 1.39% tomato powder was selected. The responses for color/appearance, flavor, spreadability, texture, after taste, adhesiveability, overall acceptability, were predicted at 7.11, 6.72, 7.00, 6.99, 6.61, 6.94 and 6.79 respectively, with a desirability value of 1.
RESUMO
Mitochondrial DNA deletions are prominent in human genetic disorders, cancer, and aging. It is thought that stalling of the mitochondrial replication machinery during DNA synthesis is a prominent source of mitochondrial genome instability; however, the precise molecular determinants of defective mitochondrial replication are not well understood. In this work, we performed a computational analysis of the human mitochondrial genome using the "Pattern Finder" G-quadruplex (G4) predictor algorithm to assess whether G4-forming sequences reside in close proximity (within 20 base pairs) to known mitochondrial DNA deletion breakpoints. We then used this information to map G4P sequences with deletions characteristic of representative mitochondrial genetic disorders and also those identified in various cancers and aging. Circular dichroism and UV spectral analysis demonstrated that mitochondrial G-rich sequences near deletion breakpoints prevalent in human disease form G-quadruplex DNA structures. A biochemical analysis of purified recombinant human Twinkle protein (gene product of c10orf2) showed that the mitochondrial replicative helicase inefficiently unwinds well characterized intermolecular and intramolecular G-quadruplex DNA substrates, as well as a unimolecular G4 substrate derived from a mitochondrial sequence that nests a deletion breakpoint described in human renal cell carcinoma. Although G4 has been implicated in the initiation of mitochondrial DNA replication, our current findings suggest that mitochondrial G-quadruplexes are also likely to be a source of instability for the mitochondrial genome by perturbing the normal progression of the mitochondrial replication machinery, including DNA unwinding by Twinkle helicase.
Assuntos
DNA Helicases/metabolismo , Replicação do DNA , DNA Mitocondrial/genética , Doença/genética , Quadruplex G , Mitocôndrias/enzimologia , Proteínas Mitocondriais/metabolismo , Deleção de Sequência/genética , Envelhecimento/genética , Animais , Sequência de Bases , Dicroísmo Circular , Biologia Computacional , Sequência Conservada/genética , Dano ao DNA , Evolução Molecular , Genoma Mitocondrial/genética , Humanos , Dados de Sequência Molecular , Neoplasias/genética , Desnaturação de Ácido Nucleico , Motivos de Nucleotídeos/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Telômero/metabolismo , Raios UltravioletaRESUMO
In 2010, a new recessive cohesinopathy disorder, designated Warsaw breakage syndrome (WABS), was described. The individual with WABS displayed microcephaly, pre- and postnatal growth retardation, and abnormal skin pigmentation. Cytogenetic analysis revealed mitomycin C (MMC)-induced chromosomal breakage; however, an additional sister chromatid cohesion defect was also observed. WABS is genetically linked to bi-allelic mutations in the ChlR1/DDX11 gene which encodes a protein of the conserved family of Iron-Sulfur (Fe-S) cluster DNA helicases. Mutations in the budding yeast ortholog of ChlR1, known as Chl1, were known to cause sister chromatid cohesion defects, indicating a conserved function of the gene. In 2012, three affected siblings were identified with similar symptoms to the original WABS case, and found to have a homozygous mutation in the conserved Fe-S domain of ChlR1, confirming the genetic linkage. Significantly, the clinically relevant mutations perturbed ChlR1 DNA unwinding activity. In addition to its genetic importance in human disease, ChlR1 is implicated in papillomavirus genome maintenance and cancer. Although its precise functions in genome homeostasis are still not well understood, ongoing molecular studies of ChlR1 suggest the helicase plays a critically important role in cellular replication and/or DNA repair.
Assuntos
Anormalidades Múltiplas/genética , RNA Helicases DEAD-box/genética , DNA Helicases/genética , RNA Helicases DEAD-box/fisiologia , Quebras de DNA , DNA Helicases/fisiologia , Quadruplex G , Instabilidade Genômica , Homeostase , Humanos , Mitomicina , Mutação , Neoplasias/genética , Papillomaviridae/genética , Fenótipo , Especificidade por Substrato , SíndromeRESUMO
G-quadruplex (G4) DNA, an alternate structure formed by Hoogsteen hydrogen bonds between guanines in G-rich sequences, threatens genomic stability by perturbing normal DNA transactions including replication, repair, and transcription. A variety of G4 topologies (intra- and intermolecular) can form in vitro, but the molecular architecture and cellular factors influencing G4 landscape in vivo are not clear. Helicases that unwind structured DNA molecules are emerging as an important class of G4-resolving enzymes. The BRCA1-associated FANCJ helicase is among those helicases able to unwind G4 DNA in vitro, and FANCJ mutations are associated with breast cancer and linked to Fanconi anemia. FANCJ belongs to a conserved iron-sulfur (Fe S) cluster family of helicases important for genomic stability including XPD (nucleotide excision repair), DDX11 (sister chromatid cohesion), and RTEL (telomere metabolism), genetically linked to xeroderma pigmentosum/Cockayne syndrome, Warsaw breakage syndrome, and dyskeratosis congenita, respectively. To elucidate the role of FANCJ in genomic stability, its molecular functions in G4 metabolism were examined. FANCJ efficiently unwound in a kinetic and ATPase-dependent manner entropically favored unimolecular G4 DNA, whereas other Fe-S helicases tested did not. The G4-specific ligands Phen-DC3 or Phen-DC6 inhibited FANCJ helicase on unimolecular G4 â¼1000-fold better than bi- or tetramolecular G4 DNA. The G4 ligand telomestatin induced DNA damage in human cells deficient in FANCJ but not DDX11 or XPD. These findings suggest FANCJ is a specialized Fe-S cluster helicase that preserves chromosomal stability by unwinding unimolecular G4 DNA likely to form in transiently unwound single-stranded genomic regions.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/química , Proteínas de Grupos de Complementação da Anemia de Fanconi/química , Quadruplex G , Regulação da Expressão Gênica , Instabilidade Genômica , Proteínas Ferro-Enxofre/química , Fatores de Transcrição de Zíper de Leucina Básica/genética , DNA/química , DNA Helicases/genética , Reparo do DNA , Replicação do DNA , Escherichia coli/metabolismo , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Guanina/química , Humanos , Concentração Inibidora 50 , Ligantes , Interferência de RNA , Proteínas Recombinantes/química , Thermoplasma/metabolismoRESUMO
About 60% to 70% of people with dementia have Alzheimer's Disease (AD), a neuro-degenerative illness. One reason for this disorder is the misfolding of naturally occurring proteins in the human brain, specifically ß-amyloid (Aß) and tau. Certain diagnostic imaging techniques, such as amyloid PET imaging, tau PET imaging, Magnetic Resonance Imaging (MRI), Comput-erized Tomography (CT), and others, can detect biomarkers in blood, plasma, and cerebral spinal fluids, like an increased level of ß-amyloid, plaques, and tangles. In order to create new pharma-cotherapeutics for Alzheimer's disease, researchers must have a thorough and detailed knowledge of amyloid beta misfolding and other related aspects. Dolopezil, rivastigmine, galantamine, and other acetylcholinesterase inhibitors are among the medications now used to treat Alzheimer's disease. Another medication that can temporarily alleviate dementia symptoms is memantine, which blocks the N-methyl-D-aspartate (NMDA) receptor. However, it is not able to halt or re-verse the progression of the disease. Medication now on the market can only halt its advance-ment, not reverse it. Interventions to alleviate behavioral and psychological symptoms, exhibit an-ti-neuroinflammation and anti-tau effects, induce neurotransmitter alteration and cognitive en-hancement, and provide other targets have recently been developed. For some Alzheimer's pa-tients, the FDA-approved monoclonal antibody, aducanumab, is an option; for others, phase 3 clinical studies are underway for drugs, like lecanemab and donanemab, which have demonstrat-ed potential in eliminating amyloid protein. However, additional study is required to identify and address these limitations in order to reduce the likelihood of side effects and maximize the thera-peutic efficacy.
RESUMO
INTRODUCTION: PIM Kinases (PIM-1, PIM-2, and PIM-3) have been reported to play crucial role in signaling cascades that govern cell survival, proliferation, and differentiation. Over-expression of these kinases leads to hematological malignancies such as diffuse large B cell lymphomas (DLBCL), multiple myeloma, leukemia, lymphoma and prostate cancer etc. PIM kinases as biomarkers and potential therapeutic targets have shown promise toward precision cancer therapy. The selective PIM-1, PIM-2, and/or PIM-3 isoform inhibitors have shown significant results in patients with advanced stages of cancer including relapsed/refractory cancer. AREAS COVERED: A comprehensive literature review of PIM Kinases (PIM-1, PIM-2, and PIM-3) in oncogenesis, the patented PIM kinase inhibitors (2016-Present), and their pharmacological and structural insights have been highlighted. EXPERT OPINION: Recently, PIM kinases viz. PIM-1, PIM-2, and PIM-3 (members of the serine/threonine protein kinase family) as therapeutic targets have attracted considerable interest in oncology especially in hematological malignancies. The patented PIM kinase inhibitors comprised of heterocyclic (fused)ring structure(s) like indole, pyridine, pyrazine, pyrazole, pyridazine, piperazine, thiazole, oxadiazole, quinoline, triazolo-pyridine, pyrazolo-pyridine, imidazo-pyridazine, oxadiazole-thione, pyrazolo-pyrimidine, triazolo-pyridazine, imidazo-pyridazine, pyrazolo-quinazoline and pyrazolo-pyridine etc. showed promising results in cancer chemotherapy.
Assuntos
Antineoplásicos , Neoplasias , Patentes como Assunto , Inibidores de Proteínas Quinases , Proteínas Proto-Oncogênicas c-pim-1 , Humanos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Antineoplásicos/farmacologia , Animais , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Neoplasias/enzimologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , Neoplasias Hematológicas/tratamento farmacológico , Neoplasias Hematológicas/patologia , Terapia de Alvo Molecular , Desenvolvimento de Medicamentos , Desenho de Fármacos , Proteínas Serina-Treonina QuinasesRESUMO
Mutations in the gene encoding the iron-sulfur-containing DNA helicase DDX11 (ChlR1) were recently identified as a cause of a new recessive cohesinopathy, Warsaw breakage syndrome (WABS), in a single patient with severe microcephaly, pre- and postnatal growth retardation, and abnormal skin pigmentation. Here, using homozygosity mapping in a Lebanese consanguineous family followed by exome sequencing, we identified a novel homozygous mutation (c.788G>A [p.R263Q]) in DDX11 in three affected siblings with severe intellectual disability and many of the congenital abnormalities reported in the WABS original case. Cultured lymphocytes from the patients showed increased mitomycin C-induced chromosomal breakage, as found in WABS. Biochemical studies of purified recombinant DDX11 indicated that the p.R263Q mutation impaired DDX11 helicase activity by perturbing its DNA binding and DNA-dependent ATP hydrolysis. Our findings thus confirm the involvement of DDX11 in WABS, describe its phenotypical spectrum, and provide novel insight into the structural requirement for DDX11 activity.
Assuntos
Anormalidades Múltiplas/genética , RNA Helicases DEAD-box/genética , DNA Helicases/genética , Predisposição Genética para Doença/genética , Mutação de Sentido Incorreto , Anormalidades Múltiplas/patologia , Sequência de Aminoácidos , Sequência de Bases , Quebra Cromossômica , Consanguinidade , RNA Helicases DEAD-box/metabolismo , DNA Helicases/metabolismo , Exoma/genética , Saúde da Família , Feminino , Humanos , Deficiência Intelectual , Masculino , Linhagem , Análise de Sequência de DNA , SíndromeRESUMO
Uracil DNA glycosylase (Ung) initiates the uracil excision repair pathway. We have earlier characterized the Y66W and Y66H mutants of Ung and shown that they are compromised by approximately 7- and approximately 170-fold, respectively in their uracil excision activities. In this study, fluorescence anisotropy measurements show that compared with the wild-type, the Y66W protein is moderately compromised and attenuated in binding to AP-DNA. Allelic exchange of ung in Escherichia coli with ung::kan, ungY66H:amp or ungY66W:amp alleles showed approximately 5-, approximately 3.0- and approximately 2.0-fold, respectively increase in mutation frequencies. Analysis of mutations in the rifampicin resistance determining region of rpoB revealed that the Y66W allele resulted in an increase in A to G (or T to C) mutations. However, the increase in A to G mutations was mitigated upon expression of wild-type Ung from a plasmid borne gene. Biochemical and computational analyses showed that the Y66W mutant maintains strict specificity for uracil excision from DNA. Interestingly, a strain deficient in AP-endonucleases also showed an increase in A to G mutations. We discuss these findings in the context of a proposal that the residency of DNA glycosylase(s) onto the AP-sites they generate shields them until recruitment of AP-endonucleases for further repair.
Assuntos
DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Uracila-DNA Glicosidase/metabolismo , Alelos , Substituição de Aminoácidos , Dano ao DNA , Reparo do DNA , RNA Polimerases Dirigidas por DNA , Escherichia coli/enzimologia , Proteínas de Escherichia coli/genética , Genoma Bacteriano , Mutação , Uracila-DNA Glicosidase/genéticaRESUMO
Phosphatidyl-inositol-3-kinase (PI3K) has emerged as a potential therapeutic target for the development of novel anticancer drugs. The dysregulation of PI3K has been associated with many human malignancies such as breast, colon, endometrial, brain, and prostate cancers. The PI3K kinases in their different isoforms namely α, ß, δ, and γ, encode PIK3CA, PIK3CB, PIK3CD, and PIK3CG genes. Specific gene mutation or overexpression of the protein is responsible for therapeutic failure of current therapeutics. Recently, various PI3K signaling pathway inhibitors have been identified which showed promising therapeutic results by acting on specific isoforms of the kinase too. Several inhibitors containing medicinally privileged scaffolds like oxadiazole, pyrrolotriazine, quinazoline, quinazolinone, quinazoline-chalcone hybrids, quinazoline-sulfonamide, pyrazolochalcone, quinolone hydroxamic acid, benzofuropyridinone, imidazopyridine, benzoxazines, dibenzoxanthene, indoloderivatives, benzimidazole, and benzothiazine derivatives have been developed to target PI3K pathway and/or a specific isoform. The PI3K inhibitors which are under clinical trial studies include GDC-0032, INK1117 for PI3K-α, and AZD8186 for PI3K-ß. This review primarily focuses on the structural insights and structure anticancer activity relationship studies of recent PI3K inhibitors including their clinical stages of development and therapeutic values.
RESUMO
Phosphatidyl-inositol-3-kinase (PI3K) has emerged as a potential therapeutic target for the development of novel anticancer drugs. The dysregulation of PI3K has been associated with many human malignancies such as breast, colon, endometrial, brain, and prostate cancers. The PI3K kinases in their different isoforms, namely α, ß, δ, and γ, encode PIK3CA, PIK3CB, PIK3CD, and PIK3CG genes. Specific gene mutation or overexpression of the protein is responsible for the therapeutic failure of current therapeutics. Recently, various PI3K signaling pathway inhibitors have been identified, which showed promising therapeutic results by acting on specific isoforms of the kinase too. Several inhibitors containing medicinally privileged scaffolds like oxadiazole, pyrrolotriazine, quinazoline, quinazolinone, quinazoline-chalcone hybrids, quinazoline-sulfonamide, pyrazolochalcone, quinolone hydroxamic acid, benzofuropyridinone, imidazopyridine, benzoxazines, dibenzoxanthene, indoloderivatives, benzimidazole, and benzothiazine derivatives have been developed to target the PI3K pathway and/or a specific isoform. The PI3K inhibitors under clinical trial studies include GDC-0032, INK1117 for PI3K-α, and AZD8186 for PI3K-ß. This review primarily focuses on the structural insights, anticancer activities, and structure-activity relationship (SARs) studies of recent PI3K inhibitors, including their clinical stages of development and therapeutic values.
Assuntos
Antineoplásicos , Inibidores de Fosfoinositídeo-3 Quinase , Antineoplásicos/farmacologia , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Isoformas de Proteínas/metabolismo , Quinazolinas , Relação Estrutura-AtividadeRESUMO
The present study was aimed to develop Manihot esculenta and Carrageenan bio-based composite active film functionalized with anise, caraway, and nutmeg essential oils (EOs) and to assess the shelf life of chicken nuggets wrapped with the developed film at refrigeration storage. Overall, the Minimum Inhibitory Concentration (MIC) values of the three EOs ranged from 0.4 to 0.8% v/v of which nutmeg EO was found most effective. Incorporation of EOs in the film resulted in significant (P Ë 0.05) decrease in tensile strength and water activity while elongation at break was significantly (P Ë 0.05) increased. No significant (P Ë 0.05) changes in thickness and Water Vapor Transmission Rate (WVTR) were observed. Based on physicomechanical and sensory evaluation, films incorporated with 0.5, 1, and 1% concentration of anise, nutmeg, and caraway EO were selected. Chicken nuggets overwrapped with aforementioned films were stored aerobically at refrigeration temperature (4 ± 1 °C) to evaluate antimicrobial, antioxidant, and sensory characteristics. The result indicated that pH, peroxide, free fatty acid (FFA), and thiobarbituric acide (TBA) value of treatments were significantly (P Ë 0.05) lower than controls however significantly (P < 0.05) higher DPPH activity was observed in all treatments. The total plate count, psychrophilic count and, yeast and mold count were also significantly (P Ë 0.01) lower in treatment groups and were within the permissible limits. The treated samples were well acceptable during whole storage period of 15 days. The application of composite, active edible bio-based film was found proficient in confining product quality attributes throughout storage. PRACTICAL APPLICATION: The majority of films used for packaging of meat and meat products are derived from synthetic "plastic" materials. The demerits associated with plastics have eventually led to explore natural alternatives such as edible films. The composite-active bio-based films have a huge potential to be molded for specific film properties based on requirements of product-specific packaging conditions.
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Anti-Infecciosos/farmacologia , Antioxidantes/análise , Filmes Comestíveis , Embalagem de Alimentos/instrumentação , Produtos da Carne/análise , Óleos Voláteis/análise , Polímeros/química , Animais , Antioxidantes/farmacologia , Bactérias/efeitos dos fármacos , Bactérias/crescimento & desenvolvimento , Carragenina/química , Galinhas , Embalagem de Alimentos/métodos , Armazenamento de Alimentos , Fungos/efeitos dos fármacos , Fungos/crescimento & desenvolvimento , Humanos , Manihot/química , Produtos da Carne/microbiologia , Testes de Sensibilidade Microbiana , Óleos Voláteis/farmacologia , Polímeros/síntese química , Refrigeração , PaladarRESUMO
The PIM kinase, also known as serine/threonine kinase plays an important role in cancer biology and is found in three different isoforms namely PIM-1, PIM-2, and PIM-3. They are extensively distributed and are implicated in a variety of biological processes, including cell proliferation, cell differentiation, and apoptosis. They act as weak oncogene and whenever expressed in exacerbating forms are responsible for different types of human cancer. Recently, different isoforms of PIM kinase have been identified as a clinical biomarker and potential therapeutic target for personalized treatment of advanced cancer. The inhibition of PIM kinase has become a scientific interest and some inhibitors have been developed and/or are under different phases of clinical trials. Several medicinally privileged heterocyclic ring scaffolds such as pyrrole, pyrimidine, thiazolidine, benzofuran, indole, triazole, oxadiazole, and quinoline derivatives have been synthesized and evaluated for their PIM inhibitory activity. This review comprehensively focuses on pharmacological implications of PIM kinases in oncogenesis, structural insights of PIM inhibitors and their structure-activity relationships (SARs).
Assuntos
Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Relação Estrutura-AtividadeRESUMO
INTRODUCTION: Chalcone or benzylideneacetophenone or 1,3-diphenyl-2-propene-1-one is a natural product comprising of two aromatic rings connected together by a three-carbon α, ß unsaturated carbonyl bridge. It is one of the most privileged scaffolds in medicinal chemistry that can be synthesized in the laboratory and can be converted into several therapeutically active heterocyclic scaffolds. It exhibits multifarious pharmacological activities and also plays a key role in several non-pharmacological scientific applications. AREAS COVERED: The present article comprehensively focuses on the approved patents during the time duration 2014-2018 on various chalcone molecules with diverse pharmacological activities. EXPERT OPINION: The study puts forward the latest updated therapeutic applications of chalcone-based compounds as antiproliferative, antidiabetic, anti-infective, anti-inflammatory, antioxidant, antiaging, neuroprotective, and cardioprotective agents. The type, position, and the number of substituents present on the chalcone scaffold have been perceived to play an imperative function in interacting with molecular targets (receptor, enzyme, and/or channel) to express the biological responses. In the majority of the studies, the overall activity of the ligand administered as pharmaceutically acceptable salt is found to be better than that of standard marketed drug preparation. The article will certainly attract (medicinal)-chemists actively involved in the development of therapeutically active scaffolds.