RESUMO
Brain region-specific degeneration and somatic expansions of the mutant Huntingtin (mHTT) CAG tract are key features of Huntington's disease (HD). However, the relationships among CAG expansions, death of specific cell types and molecular events associated with these processes are not established. Here, we used fluorescence-activated nuclear sorting (FANS) and deep molecular profiling to gain insight into the properties of cell types of the human striatum and cerebellum in HD and control donors. CAG expansions arise at mHTT in striatal medium spiny neurons (MSNs), cholinergic interneurons and cerebellar Purkinje neurons, and at mutant ATXN3 in MSNs from SCA3 donors. CAG expansions in MSNs are associated with higher levels of MSH2 and MSH3 (forming MutSß), which can inhibit nucleolytic excision of CAG slip-outs by FAN1. Our data support a model in which CAG expansions are necessary but may not be sufficient for cell death and identify transcriptional changes associated with somatic CAG expansions and striatal toxicity.
Assuntos
Corpo Estriado , Doença de Huntington , Humanos , Animais , Cerebelo/metabolismo , Doença de Huntington/genética , Modelos Animais de DoençasRESUMO
Brain region-specific degeneration and somatic expansions of the mutant Huntingtin (mHTT) CAG tract are key features of Huntington's disease (HD). However, the relationships between CAG expansions, death of specific cell types, and molecular events associated with these processes are not established. Here we employed fluorescence-activated nuclear sorting (FANS) and deep molecular profiling to gain insight into the properties of cell types of the human striatum and cerebellum in HD and control donors. CAG expansions arise in striatal medium spiny neurons (MSNs) and cholinergic interneurons, in cerebellar Purkinje neurons, and at mATXN3 in MSNs from SCA3 donors. CAG expansions in MSNs are associated with higher levels of MSH2 and MSH3 (forming MutSß), which can inhibit nucleolytic excision of CAG slip-outs by FAN1 in a concentration-dependent manner. Our data indicate that ongoing CAG expansions are not sufficient for cell death, and identify transcriptional changes associated with somatic CAG expansions and striatal toxicity.
RESUMO
Ongoing inchworm-like CAG and CGG repeat expansions in brains, arising by aberrant processing of slipped DNAs, may drive Huntington's disease, fragile X syndrome, and autism. FAN1 nuclease modifies hyper-expansion rates by unknown means. We show that FAN1, through iterative cycles, binds, dimerizes, and cleaves slipped DNAs, yielding striking exo-nuclease pauses along slip-outs: 5'-C↓A↓GC↓A↓G-3' and 5'-C↓T↓G↓C↓T↓G-3'. CAG excision is slower than CTG and requires intra-strand A·A and T·T mismatches. Fully paired hairpins arrested excision, whereas disease-delaying CAA interruptions further slowed excision. Endo-nucleolytic cleavage is insensitive to slip-outs. Rare FAN1 variants are found in individuals with autism with CGG/CCG expansions, and CGG/CCG slip-outs show exo-nuclease pauses. The slip-out-specific ligand, naphthyridine-azaquinolone, which induces contractions of expanded repeats in vivo, requires FAN1 for its effect, and protects slip-outs from FAN1 exo-, but not endo-, nucleolytic digestion. FAN1's inchworm pausing of slip-out excision rates is well suited to modify inchworm expansion rates, which modify disease onset and progression.
Assuntos
Transtorno do Espectro Autista/genética , Reparo de Erro de Pareamento de DNA , Endodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/metabolismo , Instabilidade Genômica , Doença de Huntington/genética , Enzimas Multifuncionais/metabolismo , Ataxias Espinocerebelares/genética , Expansão das Repetições de Trinucleotídeos , Animais , Transtorno do Espectro Autista/enzimologia , Linhagem Celular Tumoral , Progressão da Doença , Endodesoxirribonucleases/genética , Exodesoxirribonucleases/genética , Predisposição Genética para Doença , Humanos , Doença de Huntington/enzimologia , Enzimas Multifuncionais/genética , Mutação , Conformação de Ácido Nucleico , Fenótipo , Ligação Proteica , Células Sf9 , Ataxias Espinocerebelares/enzimologiaRESUMO
Existing cancer therapies are often associated with drug resistance and toxicity, which results in poor prognosis and recurrence of cancer. This necessitates the identification and development of novel therapeutics against existing as well as novel cellular targets. In this study, a novel class of Benzocoumarin-Stilbene hybrid molecules were synthesized and evaluated for their antiproliferative activity against various cancer cell lines followed by in vivo antitumor activity in a mouse model of cancer. The most promising molecule among the series, i.e. compound (E)-4-(3,5-dimethoxystyryl)-2H-benzo[h]chromen-2-one (19) showed maximum antiproliferative activity in breast cancer cell lines (MDA-MB-231 and 4T1) and decreased the tumor size in the in-vivo 4T1 cell-induced orthotopic syngeneic mouse breast cancer model. The mechanistic studies of compound 19 by various biochemical, cell biology and biophysical approaches suggest that the compound binds to and inhibits the human DNA ligase I enzyme activity that might be the cause for significant reduction in tumor growth and may constitute a promising next-generation therapy against breast cancers.
Assuntos
Antracenos , Antineoplásicos/química , Antineoplásicos/farmacologia , DNA Ligase Dependente de ATP/antagonistas & inibidores , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Estilbenos , Animais , Antracenos/química , Apoptose/efeitos dos fármacos , Neoplasias da Mama , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dano ao DNA , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Estrutura Molecular , Transdução de Sinais/efeitos dos fármacos , Estilbenos/química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Human Flap endonuclease1 (FEN1) is an enzyme that is indispensable for DNA replication and repair processes and inhibition of its Flap cleavage activity results in increased cellular sensitivity to DNA damaging agents (cisplatin, temozolomide, MMS, etc.), with the potential to improve cancer prognosis. Reports of the high expression levels of FEN1 in several cancer cells support the idea that FEN1 inhibitors may target cancer cells with minimum side effects to normal cells. In this study, we used large publicly available, high-throughput screening data of small molecule compounds targeted against FEN1. Two machine learning algorithms, Support Vector Machine (SVM) and Random Forest (RF), were utilized to generate four classification models from huge PubChem bioassay data containing probable FEN1 inhibitors and non-inhibitors. We also investigated the influence of randomly selected Zinc-database compounds as negative data on the outcome of classification modelling. The results show that the SVM model with inactive compounds was superior to RF with Matthews's correlation coefficient (MCC) of 0.67 for the test set. A Maybridge database containing approximately 53 000 compounds was screened and top ranking 5 compounds were selected for enzyme and cell-based in vitro screening. The compound JFD00950 was identified as a novel FEN1 inhibitor with in vitro inhibition of flap cleavage activity as well as cytotoxic activity against a colon cancer cell line, DLD-1.
Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Células Epiteliais/efeitos dos fármacos , Endonucleases Flap/antagonistas & inibidores , Aprendizado de Máquina , Antineoplásicos/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Colo/efeitos dos fármacos , Colo/enzimologia , Colo/patologia , Bases de Dados de Compostos Químicos , Descoberta de Drogas , Inibidores Enzimáticos/química , Células Epiteliais/enzimologia , Células Epiteliais/patologia , Endonucleases Flap/genética , Endonucleases Flap/metabolismo , Expressão Gênica , Células HEK293 , Humanos , Concentração Inibidora 50 , Naftoquinonas/química , Naftoquinonas/farmacologia , Especificidade de ÓrgãosRESUMO
DNA replication is a complex phenomenon that requires the concerted action of several enzymes, together with their protein and non-protein cofactors. In the nucleus, the two DNA strands are duplicated by two completely independent methods due to their anti-parallel orientation and the restrictive nature of DNA polymerases that allow DNA synthesis in the 5'-3' direction only. In this review, we focus on the proteins that are involved in the more complex and discontinuous process of lagging strand DNA synthesis by the formation of small DNA fragments called Okazaki fragments which are later sealed to form a continuous strand of DNA. We try and connect all the protein-protein interactions important for lagging strand synthesis in the S-phase of the cell cycle, describe the dynamics of these interactions and go on to discuss the post-translational modifications that affect them. We also look at how mutations in any of the players of the lagging strand synthesis can cause genomic instability leading to cancer and discuss if any of the players may be targeted for cancer therapy.
Assuntos
Replicação do DNA , Instabilidade Genômica , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Sequência de Aminoácidos , Animais , Humanos , Proteínas de Neoplasias/química , Neoplasias/metabolismo , Processamento de Proteína Pós-TraducionalRESUMO
Living organisms belonging to all three domains of life, viz., eubacteria, archaeabacteria, and eukaryotes encode one or more DNA ligases. DNA ligases are indispensable in various DNA repair and replication processes and a deficiency or an inhibition of their activity can lead to accumulation of DNA damage and strand breaks. DNA damage, specially strand breaks at unsustainable levels can lead to replication block and/or cell death. DNA ligases as potential anticancer targets have been realized only recently. There is enough rationale to suggest that ligases have a tremendous potential for novel therapeutics including anticancer and antibacterial therapy, specially when the world is facing acute problems of drug resistance and chemotherapy failure, with an immediate need for new therapeutic targets. Here, we review the current state of the art in the development of human ligase inhibitors, their structures, molecular mechanisms, physiological effects, and their potential in future cancer therapy. Citing examples, we focus on strategies for improving the activity and specificity of existing and novel inhibitors by using structure-based rational approaches. In the end, we describe potential new sites on the ligase I protein that can be targeted for the development of novel inhibitors. This is the first comprehensive review to compile all known human ligase inhibitors and to provide a rationale for the further development of ligase inhibitors for cancer therapy.