RESUMO
Human ALC1 is an oncogene-encoded chromatin-remodeling enzyme required for DNA repair that possesses a poly(ADP-ribose) (PAR)-binding macro domain. Its engagement with PARylated PARP1 activates ALC1 at sites of DNA damage, but the underlying mechanism remains unclear. Here, we establish a dual role for the macro domain in autoinhibition of ALC1 ATPase activity and coupling to nucleosome mobilization. In the absence of DNA damage, an inactive conformation of the ATPase is maintained by juxtaposition of the macro domain against predominantly the C-terminal ATPase lobe through conserved electrostatic interactions. Mutations within this interface displace the macro domain, constitutively activate the ALC1 ATPase independent of PARylated PARP1, and alter the dynamics of ALC1 recruitment at DNA damage sites. Upon DNA damage, binding of PARylated PARP1 by the macro domain induces a conformational change that relieves autoinhibitory interactions with the ATPase motor, which selectively activates ALC1 remodeling upon recruitment to sites of DNA damage.
Assuntos
Montagem e Desmontagem da Cromatina , Dano ao DNA , DNA Helicases/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Nucleossomos/enzimologia , Domínio Catalítico , Linhagem Celular Tumoral , DNA Helicases/química , DNA Helicases/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Ativação Enzimática , Humanos , Microscopia Eletrônica , Simulação de Dinâmica Molecular , Mutação , Nucleossomos/química , Poli(ADP-Ribose) Polimerase-1/química , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli ADP Ribosilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Espalhamento a Baixo Ângulo , Eletricidade Estática , Relação Estrutura-Atividade , Fatores de Tempo , Difração de Raios XRESUMO
Zinc finger E-box binding homeobox 1 (ZEB1) is a transcriptional regulator involved in embryonic development and cancer progression. ZEB1 induces epithelial-mesenchymal transition (EMT). Triple-negative human breast cancers express high ZEB1 mRNA levels and exhibit features of EMT. In the human triple-negative breast cancer cell model Hs578T, ZEB1 associates with almost 2,000 genes, representing many cellular functions, including cell polarity regulation (DLG2 and FAT3). By introducing a CRISPR-Cas9-mediated 30 bp deletion into the ZEB1 second exon, we observed reduced migratory and anchorage-independent growth capacity of these tumor cells. Transcriptomic analysis of control and ZEB1 knockout cells, revealed 1,372 differentially expressed genes. The TIMP metallopeptidase inhibitor 3 and the teneurin transmembrane protein 2 genes showed increased expression upon loss of ZEB1, possibly mediating pro-tumorigenic actions of ZEB1. This work provides a resource for regulators of cancer progression that function under the transcriptional control of ZEB1. The data confirm that removing a single EMT transcription factor, such as ZEB1, is not sufficient for reverting the triple-negative mesenchymal breast cancer cells into more differentiated, epithelial-like clones, but can reduce tumorigenic potential, suggesting that not all pro-tumorigenic actions of ZEB1 are linked to the EMT.
Assuntos
Regulação Neoplásica da Expressão Gênica/genética , Neoplasias de Mama Triplo Negativas/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Transição Epitelial-Mesenquimal/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
We previously established a mechanism of negative regulation of transforming growth factor ß signaling mediated by the nuclear ADP-ribosylating enzyme poly-(ADP-ribose) polymerase 1 (PARP1) and the deribosylating enzyme poly-(ADP-ribose) glycohydrolase (PARG), which dynamically regulate ADP-ribosylation of Smad3 and Smad4, two central signaling proteins of the pathway. Here we demonstrate that the bone morphogenetic protein (BMP) pathway can also be regulated by the opposing actions of PARP1 and PARG. PARG positively contributes to BMP signaling and forms physical complexes with Smad5 and Smad4. The positive role PARG plays during BMP signaling can be neutralized by PARP1, as demonstrated by experiments where PARG and PARP1 are simultaneously silenced. In contrast to PARG, ectopic expression of PARP1 suppresses BMP signaling, whereas silencing of endogenous PARP1 enhances signaling and BMP-induced differentiation. The two major Smad proteins of the BMP pathway, Smad1 and Smad5, interact with PARP1 and can be ADP-ribosylated in vitro, whereas PARG causes deribosylation. The overall outcome of this mode of regulation of BMP signal transduction provides a fine-tuning mechanism based on the two major enzymes that control cellular ADP-ribosylation.
Assuntos
Adenosina Difosfato Ribose/metabolismo , Proteínas Morfogenéticas Ósseas/farmacologia , Proteínas de Ligação a DNA/metabolismo , Glicosídeo Hidrolases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Linhagem Celular , Células Cultivadas , Proteínas de Ligação a DNA/genética , Expressão Gênica/efeitos dos fármacos , Glicosídeo Hidrolases/genética , Células HEK293 , Humanos , Immunoblotting , Camundongos Knockout , Ligação Proteica , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Proteína Smad4/genética , Proteína Smad4/metabolismo , Proteína Smad5/genética , Proteína Smad5/metabolismoRESUMO
The transcription factor SNAI1 mediates epithelial-mesenchymal transition, fibroblast activation and controls inter-tissue migration. High SNAI1 expression characterizes metastatic triple-negative breast carcinomas, and its knockout by CRISPR/Cas9 uncovered an epithelio-mesenchymal phenotype accompanied by reduced signaling by the cytokine TGFß. The SNAI1 knockout cells exhibited plasticity in differentiation, drifting towards the luminal phenotype, gained stemness potential and could differentiate into acinar mammospheres in 3D culture. Loss of SNAI1 de-repressed the transcription factor FOXA1, a pioneering factor of mammary luminal progenitors. FOXA1 induced a specific gene program, including the androgen receptor (AR). Inhibiting AR via a specific antagonist regenerated the basal phenotype and blocked acinar differentiation. Thus, loss of SNAI1 in the context of triple-negative breast carcinoma cells promotes an intermediary luminal progenitor phenotype that gains differentiation plasticity based on the dual transcriptional action of FOXA1 and AR. This function of SNAI1 provides means to separate cell invasiveness from progenitor cell de-differentiation as independent cellular programs.
Assuntos
Neoplasias da Mama , Neoplasias de Mama Triplo Negativas , Linhagem Celular Tumoral , Plasticidade Celular/genética , Transição Epitelial-Mesenquimal/genética , Feminino , Humanos , Receptores Androgênicos/metabolismo , Fatores de Transcrição da Família Snail/genética , Fator de Crescimento Transformador beta , Neoplasias de Mama Triplo Negativas/genéticaRESUMO
Spheroid cultures of primary human hepatocytes (PHH) are used in studies of hepatic drug metabolism and toxicity. The cultures are maintained under different conditions, with possible confounding results. We performed an in-depth analysis of the influence of various culture conditions to find the optimal conditions for the maintenance of an in vivo like phenotype. The formation, protein expression, and function of PHH spheroids were followed for three weeks in a high-throughput 384-well format. Medium composition affected spheroid histology, global proteome profile, drug metabolism and drug-induced toxicity. No epithelial-mesenchymal transition was observed. Media with fasting glucose and insulin levels gave spheroids with phenotypes closest to normal PHH. The most expensive medium resulted in PHH features most divergent from that of native PHH. Our results provide a protocol for culture of healthy PHH with maintained function - a prerequisite for studies of hepatocyte homeostasis and more reproducible hepatocyte research.
RESUMO
The number of transporter proteins that are not fully characterized is immense. Here, we used Drosophila melanogaster and human cell lines to perform a first in-depth characterization of CG4928, an ortholog to the human UNC93A, of which little is known. Solute carriers regulate and maintain biochemical pathways important for the body, and malfunctioning transport is associated with multiple diseases. Based on phylogenetic analysis, CG4928 is closely related to human UNC93A and has a secondary and a tertiary protein structure and folding similar to major facilitator superfamily transporters. Ubiquitous knockdown of CG4928 causes flies to have a reduced secretion rate from the Malpighian tubules; altering potassium content in the body and in the Malpighian tubules, homologous to the renal system; and results in the development of edema. The edema could be rescued by using amiloride, a common diuretic, and by maintaining the flies on ion-free diets. CG4928-overexpressing cells did not facilitate the transport of sugars and amino acids; however, proximity ligation assay revealed that CG4928 co-localized with TASK1 channels. Overexpression of CG4928 resulted in induced apoptosis and cytotoxicity, which could be restored when cells were kept in high-sodium media. Furthermore, the basal membrane potential was observed to be disrupted. Taken together, the results indicate that CG4928 is of importance for generating the cellular membrane potential by an unknown manner. However, we speculate that it most likely acts as a regulator or transporter of potassium flows over the membrane.
RESUMO
Epithelial cells contribute to the development of various vital organs by generating tubular and/or glandular architectures. The fully developed forms of ductal organs depend on processes of branching morphogenesis, whereby frequency, total number, and complexity of the branching tissue define the final architecture in the organ. Some ductal tissues, like the mammary gland during pregnancy and lactation, disintegrate and regenerate through periodic cycles. Differentiation of branched epithelia is driven by antagonistic actions of parallel growth factor systems that mediate epithelial-mesenchymal communication. Transforming growth factor-ß (TGF-ß) family members and their extracellular antagonists are prominently involved in both normal and disease-associated (e.g., malignant or fibrotic) ductal tissue patterning. Here, we discuss collective knowledge that permeates the roles of TGF-ß family members in the control of the ductal tissues in the vertebrate body.
Assuntos
Morfogênese , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta/fisiologia , Animais , Transição Epitelial-Mesenquimal , Feminino , Humanos , Pulmão/embriologia , Masculino , Glândulas Mamárias Animais/embriologia , Glândulas Mamárias Humanas/embriologia , Organogênese , Pâncreas/embriologia , Próstata/embriologia , Glândulas Salivares/embriologiaRESUMO
Transcriptional regulation mediated by the zinc finger protein Snail1 controls early embryogenesis. By binding to the epithelial tumor suppressor CDH1 gene, Snail1 initiates the epithelial-mesenchymal transition (EMT). The EMT generates stem-like cells and promotes invasiveness during cancer progression. Accordingly, Snail1 mRNA and protein is abundantly expressed in triple-negative breast cancers with enhanced metastatic potential and phenotypic signs of the EMT. Such high endogenous Snail1 protein levels permit quantitative chromatin immunoprecipitation-sequencing (ChIP-seq) analysis. Snail1 associated with 185 genes at cis regulatory regions in the Hs578T triple-negative breast cancer cell model. These genes include morphogenetic regulators and signaling components that control polarized differentiation. Using the CRISPR/Cas9 system in Hs578T cells, a double deletion of 10 bp each was engineered into the first exon and into the second exon-intron junction of Snail1, suppressing Snail1 expression and causing misregulation of several hundred genes. Specific attention to regulators of chromatin organization provides a possible link to new phenotypes uncovered by the Snail1 loss-of-function mutation. On the other hand, genetic inactivation of Snail1 was not sufficient to establish a full epithelial transition to these tumor cells. Thus, Snail1 contributes to the malignant phenotype of breast cancer cells via diverse new mechanisms.
Assuntos
Genoma Humano , Fatores de Transcrição da Família Snail/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Sequência de Bases , Proteína Morfogenética Óssea 6/metabolismo , Osso e Ossos/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Inativação de Genes , Células HEK293 , Homeostase , Humanos , Mesoderma/metabolismo , Fenótipo , Ligação Proteica , Transcriptoma/genéticaRESUMO
A large fraction of the adult population is on lifelong medication for cardiovascular disorders, but the metabolic consequences are largely unknown. This study determines the effects of common anti-hypertensive and lipid lowering drugs on circulating plasma protein biomarkers. We studied 425 proteins in plasma together with anthropometric and lifestyle variables, and the genetic profile in a cross-sectional cohort. We found 8406 covariate-protein associations, and a two-stage GWAS identified 17253 SNPs to be associated with 109 proteins. By computationally removing variation due to lifestyle and genetic factors, we could determine that medication, per se, affected the abundance levels of 35.7% of the plasma proteins. Medication either affected a single, a few, or a large number of protein, and were found to have a negative or positive influence on known disease pathways and biomarkers. Anti-hypertensive or lipid lowering drugs affected 33.1% of the proteins. Angiotensin-converting enzyme inhibitors showed the strongest lowering effect by decreasing plasma levels of myostatin. Cell-culture experiments showed that angiotensin-converting enzyme inhibitors reducted myostatin RNA levels. Thus, understanding the effects of lifelong medication on the plasma proteome is important both for sharpening the diagnostic precision of protein biomarkers and in disease management.
Assuntos
Anticolesterolemiantes/uso terapêutico , Anti-Hipertensivos/uso terapêutico , Biomarcadores/sangue , Proteínas Sanguíneas/análise , Doenças Cardiovasculares/sangue , Hipolipemiantes/uso terapêutico , Doenças Cardiovasculares/tratamento farmacológico , Doenças Cardiovasculares/patologia , Estudos de Coortes , Estudos Transversais , Humanos , Prognóstico , Fatores de RiscoRESUMO
The shelterin proteins are required for telomere integrity. Shelterin dysfunction can lead to initiation of unwarranted DNA damage and repair pathways at chromosomal termini. Interestingly, many shelterin accessory proteins are involved in DNA damage signaling and repair. We demonstrate here that in normal human fibroblasts, telomeric ends are protected by phosphorylation of CGG triplet repeat-binding protein 1 (CGGBP1) at serine 164 (S164). We show that serine 164 is a major phosphorylation site on CGGBP1 with important functions. We provide evidence that one of the kinases that can phosphorylate S164 CGGBP1 is ATR. Overexpression of S164A phospho-deficient CGGBP1 exerted a dominant-negative effect, causing telomeric dysfunction, accelerated telomere shortening, enhanced fusion of telomeres, and crisis. However, overexpression of wild-type or phospho-mimicking S164E CGGBP1 did not cause these effects. This telomere damage was associated with reduced binding of the shelterin protein POT1 to telomeric DNA. Our results suggest that CGGBP1 phosphorylation at S164 is a novel telomere protection signal, which can affect telomere-protective function of the shelterin complex.
Assuntos
Dano ao DNA/genética , Proteínas de Ligação a DNA/genética , Serina/genética , Telômero/genética , Proteínas de Ciclo Celular/genética , Regulação da Expressão Gênica , Humanos , Fosforilação , Complexo Shelterina , Transdução de Sinais/genética , Proteínas de Ligação a Telômeros/genéticaRESUMO
BACKGROUND: Initiation, amplitude, duration and termination of transforming growth factor ß (TGFß) signaling via Smad proteins is regulated by post-translational modifications, including phosphorylation, ubiquitination and acetylation. We previously reported that ADP-ribosylation of Smads by poly(ADP-ribose) polymerase 1 (PARP-1) negatively influences Smad-mediated transcription. PARP-1 is known to functionally interact with PARP-2 in the nucleus and the enzyme poly(ADP-ribose) glycohydrolase (PARG) can remove poly(ADP-ribose) chains from target proteins. Here we aimed at analyzing possible cooperation between PARP-1, PARP-2 and PARG in regulation of TGFß signaling. METHODS: A robust cell model of TGFß signaling, i.e. human HaCaT keratinocytes, was used. Endogenous Smad3 ADP-ribosylation and protein complexes between Smads and PARPs were studied using proximity ligation assays and co-immunoprecipitation assays, which were complemented by in vitro ADP-ribosylation assays using recombinant proteins. Real-time RT-PCR analysis of mRNA levels and promoter-reporter assays provided quantitative analysis of gene expression in response to TGFß stimulation and after genetic perturbations of PARP-1/-2 and PARG based on RNA interference. RESULTS: TGFß signaling rapidly induces nuclear ADP-ribosylation of Smad3 that coincides with a relative enhancement of nuclear complexes of Smads with PARP-1 and PARP-2. Inversely, PARG interacts with Smads and can de-ADP-ribosylate Smad3 in vitro. PARP-1 and PARP-2 also form complexes with each other, and Smads interact and activate auto-ADP-ribosylation of both PARP-1 and PARP-2. PARP-2, similar to PARP-1, negatively regulates specific TGFß target genes (fibronectin, Smad7) and Smad transcriptional responses, and PARG positively regulates these genes. Accordingly, inhibition of TGFß-mediated transcription caused by silencing endogenous PARG expression could be relieved after simultaneous depletion of PARP-1. CONCLUSION: Nuclear Smad function is negatively regulated by PARP-1 that is assisted by PARP-2 and positively regulated by PARG during the course of TGFß signaling.
Assuntos
Glicosídeo Hidrolases/fisiologia , Poli(ADP-Ribose) Polimerases/fisiologia , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/fisiologia , Fibronectinas/genética , Fibronectinas/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Humanos , Poli(ADP-Ribose) Polimerase-1 , Processamento de Proteína Pós-Traducional , Transdução de Sinais , Proteína Smad2/metabolismo , Proteína Smad7/genética , Proteína Smad7/metabolismo , Transcrição GênicaRESUMO
Human POT1, a widely studied telomere protector protein is perceived to be expressed as a single 70kDa form. A survey of the literature as well as different commercially available antibodies against POT1 suggests occurrence of multiple forms of POT1. Knowledge about possible various forms of an important protein like POT1 is necessary for our understanding about its function. We have discovered that POT1 exists in at least three consistently occurring forms; 90, 70 and 45kDa. The unexpected molecular weights of POT1 seem to be associated with SUMO1 and ubiquitin conjugation; the latter occurring at a double lysine residue at 289-KK-290. We also present evidence that the relative abundance of the different POT1 forms can be altered by experimental modulation of POT1 nuclear localization. We thus present strong evidence that there are post-translational modifications of POT1 that can affect its molecular weight as well as intracellular localization and function.