RESUMO
Harmaline is a naturally occurring ß-carboline alkaloid that is isolated from Peganum harmala. It has shown efficacy in treating Parkinson's disease and has been reported to exhibit antimicrobial and anticancer properties. However, the molecular mechanism of harmaline in the context of esophageal squamous cell carcinoma (ESCC) has not been characterized. Here, we report that harmaline attenuates ESCC growth by directly targeting the mammalian target of rapamycin (mTOR). Harmaline strongly reduced cell proliferation and anchorage-independent cell growth. Additionally, harmaline treatment induced G2/M phase cell-cycle arrest through upregulation of p27. The results of in vitro and cell-based assays showed that harmaline directly inhibited the activity of mTOR kinase and the phosphorylation of its downstream pathway components. Depletion of mTOR using an shRNA-mediated strategy in ESCC cell lines indicated that reduced mTOR protein expression levels are correlated with decreased cell proliferation. Additionally, we observed that the inhibitory effect of harmaline was dependent upon mTOR expression. Notably, oral administration of harmaline suppressed ESCC patient-derived tumor growth in vivo. Taken together, harmaline is a potential mTOR inhibitor that might be used for therapeutically treating ESCC.
Assuntos
Neoplasias Esofágicas , Carcinoma de Células Escamosas do Esôfago , Neoplasias de Cabeça e Pescoço , Peganum , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Esofágicas/tratamento farmacológico , Carcinoma de Células Escamosas do Esôfago/tratamento farmacológico , Harmalina/farmacologia , Humanos , Sirolimo , Serina-Treonina Quinases TORRESUMO
ß-Catenin is a multifunctional protein and participates in numerous processes required for embryonic development, cell proliferation, and homeostasis through various molecular interactions and signaling pathways. To date, however, there is no direct evidence that ß-catenin contributes to cytokinesis. Here, we identify a novel p-S60 epitope on ß-catenin generated by Plk1 kinase activity, which can be found at the actomyosin contractile ring of early telophase cells and at the midbody of late telophase cells. Depletion of ß-catenin leads to cytokinesis-defective phenotypes, which eventually result in apoptotic cell death. In addition, phosphorylation of ß-catenin Ser60 by Plk1 is essential for the recruitment of Ect2 to the midbody, activation of RhoA, and interaction between ß-catenin, Plk1, and Ect2. Time-lapse image analysis confirmed the importance of ß-catenin phospho-Ser60 in furrow ingression and the completion of cytokinesis. Taken together, we propose that phosphorylation of ß-catenin Ser60 by Plk1 in cooperation with Ect2 is essential for the completion of cytokinesis. These findings may provide fundamental knowledge for the research of cytokinesis failure-derived human diseases.
Assuntos
Actomiosina , Citocinese , Actomiosina/metabolismo , Proteínas de Ciclo Celular/metabolismo , Células HeLa , Humanos , Fosforilação , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/metabolismo , Fuso Acromático/metabolismo , beta Catenina/metabolismo , Quinase 1 Polo-LikeRESUMO
Cancer-associated fibroblasts (CAFs) are important in tumor progression. The autophagy adaptor protein, p62/SQSTM1/Sequestosome-1, is up-regulated in tumors, but down-regulated in CAFs in the early stages of lung adenocarcinoma. We investigated whether p62-induced autophagy might control CAF activation. Under CAF-inducing conditions, like hypoxia or cancer cell co-cultures, p62 ablation or autophagy inhibition with hydroxychloroquine (HCQ) impaired CAF activation and reduced transforming growth factor beta (TGFß) production, which impeded tumor growth. During CAF activation, p62-induced autophagy up-regulated the expression of the anti-oxidant signaling protein, nuclear factor erythroid 2-related factor 2 (Nrf2), and the ER-stress response regulator, activating transcription factor 6 (ATF6). Genetically or pharmacologically inhibiting the Nrf2-ATF6 pathway totally blocked CAF activation and tumor progression. These results demonstrate that p62 is a key modulator of primary lung adenocarcinoma progression. Thus, targeting the p62-Nrf2 autophagy signaling pathway might be a novel, stroma-focused, cancer prevention and/or treatment strategy.
RESUMO
Protein arginylation is a critical regulator of a variety of biological processes. The ability to uncover the global arginylation pattern and its associated signaling pathways would enable us to identify novel disease targets. Here, we report the development of a tool able to capture the N-terminal arginylome. This tool, termed R-catcher, is based on the ZZ domain of p62, which was previously shown to bind N-terminally arginylated proteins. Mutating the ZZ domain enhanced its binding specificity and affinity for Nt-Arg. R-catcher pulldown coupled to LC-MS/MS led to the identification of 59 known and putative arginylated proteins. Among these were a subgroup of novel ATE1-dependent arginylated ER proteins that are linked to diverse biological pathways, including cellular senescence and vesicle-mediated transport as well as diseases, such as Amyotrophic Lateral Sclerosis and Alzheimer's disease. This study presents the first molecular tool that allows the unbiased identification of arginylated proteins, thereby unlocking the arginylome and provide a new path to disease biomarker discovery.
Assuntos
Aminoaciltransferases/metabolismo , Arginina/metabolismo , Retículo Endoplasmático/metabolismo , Vetores Genéticos/genética , Proteínas de Membrana/metabolismo , Processamento de Proteína Pós-Traducional , Aminoaciltransferases/química , Aminoaciltransferases/genética , Arginina/química , Arginina/genética , Células HeLa , Humanos , Proteínas de Membrana/genética , Especificidade por SubstratoRESUMO
3,4,5-Trihydroxycinnamic acid (THCA) has been reported to possess anti-inflammatory activity. However, the effect of THCA for treating allergic asthma was unknown. Therefore, in the present study, the anti-asthmatic effects of THCA were studied in both in vitro and in vivo studies. In phorbol 12-myristate 13-acetate (PMA)-stimulated A549 airway epithelial cells, THCA pretreatment decreased the mRNA expression and secretion of interleukin (IL)-8, monocyte chemoattractant protein-1 (MCP-1), and intercellular adhesion molecules 1 (ICAM-1), and reduced the mRNA expression of matrix metalloproteinase 9 (MMP-9). THCA also inhibited PMA-induced protein kinase B (AKT), mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) activation in A549 cells. In lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages, THCA pretreatment suppressed the mRNA expression of ICAM-1 and MMP-9. In addition, THCA suppressed the adhesion of EOL and A549 cells. In ovalbumin (OVA)-administered asthmatic mice, THCA exerted inhibitory activity on IL-5, IL-13, and MCP-1 in bronchoalveolar lavage fluid (BALF) and on OVA-specific immunoglobulin E (IgE) in serum. THCA attenuated the numbers of inflammatory cells in BALF and the influx of inflammatory cell in lung tissues. Furthermore, THCA downregulated the levels of inducible nitric oxide (iNOS), cyclooxygenase-2 (COX-2), and leukotriene B4 (LTB4) expression, mucus production and CREB phosphorylation as well as Penh value. These effects were accompanied by suppression of AKT, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and NF-κB activation. Therefore, the results of the current study suggest that THCA may be a valuable adjuvant or therapeutic in the prevention or treatment of allergic asthma.
Assuntos
Asma/induzido quimicamente , Asma/tratamento farmacológico , Cinamatos/farmacologia , Macrófagos/efeitos dos fármacos , Animais , Adesão Celular/efeitos dos fármacos , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Tumoral , Quimiocinas/genética , Quimiocinas/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/toxicidade , Células RAW 264.7 , Distribuição AleatóriaRESUMO
In the past, several microtubule targeting agents (MTAs) have been developed into successful anticancer drugs. However, the usage of these drugs has been limited by the acquisition of drug resistance in many cancers. Therefore, there is a constant demand for the development of new therapeutic drugs. Here we report the discovery of 5-5 (3-cchlorophenyl)-N-(3-pyridinyl)-2-furamide (CPPF), a novel microtubule targeting anticancer agent. Using both 2D and 3D culture systems, we showed that CPPF was able to suppress the proliferation of diverse cancer cell lines. In addition, CPPF was able to inhibit the growth of multidrug-resistant cell lines that are resistant to other MTAs, such as paclitaxel and colchicine. Our results showed that CPPF inhibited growth by depolymerizing microtubules leading to mitotic arrest and apoptosis. We also confirmed CPPF anticancer effects in vivo using both a mouse xenograft and a two-step skin cancer mouse model. Using established zebrafish models, we showed that CPPF has low toxicity in vivo. Overall, our study proves that CPPF has the potential to become a successful anticancer chemotherapeutic drug.
Assuntos
Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Microtúbulos/metabolismo , Neoplasias/tratamento farmacológico , Células A549 , Animais , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Colchicina/farmacologia , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Células HeLa , Células Hep G2 , Humanos , Células Jurkat , Células K562 , Células MCF-7 , Masculino , Camundongos , Mitose/efeitos dos fármacos , Neoplasias/metabolismo , Células PC-3 , Paclitaxel/farmacologia , Células U937 , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Peixe-ZebraRESUMO
Primary cilium is an antenna-like microtubule-based cellular sensing structure. Abnormal regulation of the dynamic assembly and disassembly cycle of primary cilia is closely related to ciliopathy and cancer. The Wnt signaling pathway plays a major role in embryonic development and tissue homeostasis, and defects in Wnt signaling are associated with a variety of human diseases, including cancer. In this study, we provide direct evidence of Wnt3a-induced primary ciliogenesis, which includes a continuous pathway showing that the stimulation of Wnt3a, a canonical Wnt ligand, promotes the generation of ß-catenin p-S47 epitope by CK1δ, and these events lead to the reorganization of centriolar satellites resulting in primary ciliogenesis. We have also confirmed the application of our findings in MCF-7/ADR cells, a multidrug-resistant tumor cell model. Thus, our data provide a Wnt3a-induced primary ciliogenesis pathway and may provide a clue on how to overcome multidrug resistance in cancer treatment.
Assuntos
Centríolos/metabolismo , Cílios/metabolismo , Organogênese , Proteína Wnt3A/metabolismo , beta Catenina/metabolismo , Sequência de Aminoácidos , Animais , Caseína Quinases/metabolismo , Centrossomo/metabolismo , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Epitopos/metabolismo , Células HEK293 , Células HeLa , Humanos , Ligantes , Células MCF-7 , Camundongos , Fosforilação , Fosfosserina/metabolismo , Proteína Wnt3A/químicaRESUMO
Bone homeostasis is regulated by a balance of bone formation and bone resorption; dysregulation of bone homeostasis may cause bone-related diseases (e.g., osteoporosis, osteopetrosis, bone fracture). Members of the nuclear factor of activated T cells (NFAT) family of transcription factors play crucial roles in the regulation of immune system, inflammatory responses, cardiac formation, skeletal muscle development, and bone homeostasis. Of these, NFATc1 is a key transcription factor mediating osteoclast differentiation, which is regulated by phosphorylation by distinct NFAT kinases including casein kinase 1 (CK1), glycogen synthase kinase 3 (GSK3), and dual-specificity tyrosine-phosphorylation-regulated kinases (DYRKs). In this study, we report that cell division control protein 2 homolog (cdc2) is a novel NFAT protein kinase that inhibits NFATc1 activation by direct phosphorylation of the NFATc1 S263 residue. Cdc2 inhibitors such as Roscovitine and BMI-1026 induce reduction of phosphorylation of NFATc1, and this process leads to the inhibition of NFATc1 translocation from the nucleus to the cytoplasm, consequently increasing the nuclear pool of NFATc1. Additionally, the inhibition of cdc2-mediated NFATc1 phosphorylation causes an elevation of osteoclast differentiation or TRAP-positive staining in zebrafish scales. Our results suggest that cdc2 is a novel NFAT protein kinase that negatively regulates osteoclast differentiation.
Assuntos
Reabsorção Óssea , Osteoclastos , Animais , Diferenciação Celular , Quinase 3 da Glicogênio Sintase , Fatores de Transcrição NFATC/metabolismo , Osteoclastos/metabolismo , Fosforilação , Ligante RANK , Peixe-Zebra/metabolismoRESUMO
The initiation of centrosome duplication is regulated by the Plk4/STIL/hsSAS-6 axis; however, the involvement of other centrosomal proteins in this process remains unclear. In this study, we demonstrate that Cep131 physically interacts with Plk4 following phosphorylation of residues S21 and T205. Localizing at the centriole, phosphorylated Cep131 has an increased capability to interact with STIL, leading to further activation and stabilization of Plk4 for initiating centrosome duplication. Moreover, we found that Cep131 overexpression resulted in centrosome amplification by excessive recruitment of STIL to the centriole and subsequent stabilization of Plk4, contributing to centrosome amplification. The xenograft mouse model also showed that both centrosome amplification and colon cancer growth were significantly increased by Cep131 overexpression. These findings demonstrate that Cep131 is a novel substrate of Plk4, and that phosphorylation or dysregulated Cep131 overexpression promotes Plk4 stabilization and therefore centrosome amplification, establishing a perspective in understanding a relationship between centrosome amplification and cancer development.
Assuntos
Proteínas de Ciclo Celular/genética , Centrossomo/metabolismo , Neoplasias do Colo/genética , Proteínas do Citoesqueleto/genética , Proteínas Serina-Treonina Quinases/genética , Animais , Linhagem Celular Tumoral , Neoplasias do Colo/patologia , Progressão da Doença , Regulação Neoplásica da Expressão Gênica/genética , Células HCT116 , Células HEK293 , Xenoenxertos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Fosforilação/genéticaRESUMO
Methylation is a primary epigenetic mechanism regulating gene expression. 5-aza-2'-deoxycytidine is an FDA-approved drug prescribed for treatment of cancer by inhibiting DNA-Methyl-Transferase 1 (DNMT1). Results of this study suggest that prolonged treatment with 5-aza-2'-deoxycytidine could induce centrosome abnormalities in cancer cells and that CEP131, a centrosome protein, is regulated by DNMT1. Interestingly, cancer cell growth was attenuated in vitro and in vivo by inhibiting the expression of Cep131. Finally, Cep131-deficient cells were more sensitive to treatment with DNMT1 inhibitors. These findings suggest that Cep131 is a potential novel anti-cancer target. Agents that can inhibit this protein may be useful alone or in combination with DNMT1 inhibitors to treat cancer. [BMB Reports 2019; 52(5): 342-347].
Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferase 1/antagonistas & inibidores , Decitabina/farmacologia , Proteínas dos Microtúbulos/antagonistas & inibidores , Neoplasias do Colo do Útero/tratamento farmacológico , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proteínas do Citoesqueleto , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Metilação de DNA/efeitos dos fármacos , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Epigênese Genética , Feminino , Células HEK293 , Células HeLa , Humanos , Proteínas dos Microtúbulos/genética , Proteínas dos Microtúbulos/metabolismo , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismoRESUMO
Hypoxia-inducible factor-1α (HIF-1α) mediates tumor cell adaptation to hypoxic conditions and is a potentially important anticancer therapeutic target. We previously developed a method for synthesizing a benzofuran-based natural product, (R)-(-)-moracin-O, and obtained a novel potent analog, MO-460 that suppresses the accumulation of HIF-1α in Hep3B cells. However, the molecular target and underlying mechanism of action of MO-460 remained unclear. In the current study, we identified heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) as a molecular target of MO-460. MO-460 inhibits the initiation of HIF-1α translation by binding to the C-terminal glycine-rich domain of hnRNPA2B1 and inhibiting its subsequent binding to the 3'-untranslated region of HIF-1α mRNA. Moreover, MO-460 suppresses HIF-1α protein synthesis under hypoxic conditions and induces the accumulation of stress granules. The data provided here suggest that hnRNPA2B1 serves as a crucial molecular target in hypoxia-induced tumor survival and thus offer an avenue for the development of novel anticancer therapies.
Assuntos
Benzofuranos/farmacologia , Produtos Biológicos/farmacologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Regiões 3' não Traduzidas , Benzofuranos/química , Produtos Biológicos/química , Linhagem Celular Tumoral , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Estrutura Molecular , Ligação Proteica , Biossíntese de Proteínas/efeitos dos fármacos , Domínios e Motivos de Interação entre Proteínas , Estresse Fisiológico/efeitos dos fármacos , Transcrição GênicaRESUMO
The N-end rule pathway is a proteolytic system in which single N-terminal residues of proteins act as N-degrons. These degrons are recognized by N-recognins, facilitating substrate degradation via the ubiquitin (Ub) proteasome system (UPS) or autophagy. We have previously identified a set of N-recognins [UBR1, UBR2, UBR4 (also known as p600) and UBR5 (also known as EDD)] that bind N-degrons through their UBR boxes to promote proteolysis by the proteasome. Here, we show that the 570â kDa N-recognin UBR4 is associated with maturing endosomes through an interaction with Ca2+-bound calmodulin. The endosomal recruitment of UBR4 is essential for the biogenesis of early endosomes (EEs) and endosome-related processes, such as the trafficking of endocytosed protein cargos and degradation of extracellular cargos by endosomal hydrolases. In mouse embryos, UBR4 marks and plays a role in the endosome-lysosome pathway that mediates the heterophagic proteolysis of endocytosed maternal proteins into amino acids. By screening 9591 drugs through the DrugBank database, we identify picolinic acid as a putative ligand for UBR4 that inhibits the biogenesis of EEs. Our results suggest that UBR4 is an essential modulator in the endosome-lysosome system.This article has an associated First Person interview with the first author of the paper.
Assuntos
Proteínas de Ligação a Calmodulina/metabolismo , Proteínas do Citoesqueleto/metabolismo , Endossomos/metabolismo , Cálcio/metabolismo , Calmodulina/genética , Calmodulina/metabolismo , Proteínas de Ligação a Calmodulina/genética , Proteínas do Citoesqueleto/genética , Endossomos/genética , Humanos , Lisossomos/genética , Lisossomos/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Biogênese de Organelas , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína LigasesRESUMO
The N-end rule pathway is a proteolytic system in which single N-terminal amino acids of proteins act as a class of degrons (N-degrons) that determine the half-lives of proteins. We have previously identified a family of mammals N-recognins (termed UBR1, UBR2, UBR4/p600, and UBR5/EDD) whose conserved UBR boxes bind N-degrons to facilitate substrate ubiquitination and proteasomal degradation via the ubiquitin-proteasome system (UPS). Amongst these N-recognins, UBR1 and UBR2 mediate ubiquitination and proteolysis of short-lived regulators and misfolded proteins. Here, we characterized the null phenotypes of UBR4-deficient mice in which the UBR box of UBR4 was deleted. We show that the mutant mice die around embryonic days 9.5-10.5 (E9.5-E10.5) associated with abnormalities in various developmental processes such as neurogenesis and cardiovascular development. These developmental defects are significantly attributed to the inability to maintain cell integrity and adhesion, which significantly correlates to the severity of null phenotypes. UBR4-loss induces the depletion of many, but not all, proteins from the plasma membrane, suggesting that UBR4 is involved in proteome-wide turnover of cell surface proteins. Indeed, UBR4 is associated with and required to generate the multivesicular body (MVB) which transiently store endocytosed cell surface proteins before their targeting to autophagosomes and subsequently lysosomes. Our results suggest that the N-recognin UBR4 plays a role in the homeostasis of cell surface proteins and, thus, cell adhesion and integrity.
Assuntos
Proteínas de Ligação a Calmodulina/metabolismo , Membrana Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Homeostase/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Neurogênese/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Animais , Autofagossomos/metabolismo , Proteínas de Ligação a Calmodulina/genética , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Proteínas do Citoesqueleto/genética , Endocitose/fisiologia , Fibroblastos/metabolismo , Células HEK293 , Células HeLa , Humanos , Lisossomos/metabolismo , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/genética , Corpos Multivesiculares/metabolismo , Proteoma , Ubiquitina-Proteína Ligases/genéticaRESUMO
Atopic dermatitis (AD) is a chronic inflammatory skin disease whose prevalence is increasing worldwide. Filaggrin (FLG) is essential for the development of the skin barrier, and its genetic mutations are major predisposing factors for AD. In this study, we developed a convenient and practical method to detect FLG mutations in AD patients using peptide nucleic acid (PNA) probes labelled with fluorescent markers for rapid analysis. Fluorescence melting curve analysis (FMCA) precisely identified FLG mutations based on the distinct difference in the melting temperatures of the wild-type and mutant allele. Moreover, PNA probe-based FMCA easily and accurately verified patient samples with both heterozygote and homozygote FLG mutations, providing a high-throughput method to reliable screen AD patients. Our method provides a convenient, rapid and accurate diagnostic tool to identify potential AD patients allowing for early preventive treatment, leading to lower incidence rates of AD, and reducing total healthcare expenses.
Assuntos
Análise Mutacional de DNA/métodos , Sondas de DNA , Dermatite Atópica/diagnóstico , Dermatite Atópica/genética , Proteínas de Filamentos Intermediários/genética , Alelos , Estudos de Casos e Controles , Proteínas Filagrinas , Fluorescência , Heterozigoto , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Homozigoto , Humanos , Mutação , Ácidos Nucleicos Peptídicos/genética , Temperatura de TransiçãoRESUMO
The novel synthetic compound designated STK899704 (PubChem CID: 5455708) suppresses the proliferation of a broad range of cancer cell types. However, the details of its effect on lung cancer cells are unclear. We investigated the precise anticancer effect of STK899704 on senescence and growth arrest of A549 human non-small cell lung cancer (NSCLC) cells. STK899704 affected NSCLC cell cycle progression and decreased cell viability in a dose-dependent manner. Immunofluorescence staining revealed that STK899704 destabilized microtubules. Cell cycle analysis showed an increase in the population of NSCLC cells in the sub-G1 and G2/M phases, indicating that STK899704 might cause DNA damage via tubulin aggregation. Furthermore, we observed increased mitotic catastrophe in STK899704-treated cells. As STK899704 led to elevated levels of the p53 pathway-associated proteins, it would likely affect the core DNA damage response pathway. Moreover, STK899704 promoted senescence of NSCLC cells by inducing the p53-associated DNA damage response pathways. These findings suggest that the novel anti-proliferative small molecule STK899704 promotes cell death by inducing DNA damage response pathways and senescence after cell cycle arrest, being a potential drug for treating human lung cancers.
RESUMO
The conjugation of amino acids to the protein N termini is universally observed in eukaryotes and prokaryotes, yet its functions remain poorly understood. In eukaryotes, the amino acid l-arginine (l-Arg) is conjugated to N-terminal Asp (Nt-Asp), Glu, Gln, Asn, and Cys, directly or associated with posttranslational modifications. Following Nt-arginylation, the Nt-Arg is recognized by UBR boxes of N-recognins such as UBR1, UBR2, UBR4/p600, and UBR5/EDD, leading to substrate ubiquitination and proteasomal degradation via the N-end rule pathway. It has been a mystery, however, why studies for the past five decades identified only a handful of Nt-arginylated substrates in mammals, although five of 20 principal amino acids are eligible for arginylation. Here, we show that the Nt-Arg functions as a bimodal degron that directs substrates to either the ubiquitin (Ub)-proteasome system (UPS) or macroautophagy depending on physiological states. In normal conditions, the arginylated forms of proteolytic cleavage products, D101-CDC6 and D1156-BRCA1, are targeted to UBR box-containing N-recognins and degraded by the proteasome. However, when proteostasis by the UPS is perturbed, their Nt-Arg redirects these otherwise cellular wastes to macroautophagy through its binding to the ZZ domain of the autophagic adaptor p62/STQSM/Sequestosome-1. Upon binding to the Nt-Arg, p62 acts as an autophagic N-recognin that undergoes self-polymerization, facilitating cargo collection and lysosomal degradation of p62-cargo complexes. A chemical mimic of Nt-Arg redirects Ub-conjugated substrates from the UPS to macroautophagy and promotes their lysosomal degradation. Our results suggest that the Nt-Arg proteome of arginylated proteins contributes to reprogramming global proteolytic flux under stresses.
Assuntos
Arginina/metabolismo , Autofagia/fisiologia , Proteínas de Ciclo Celular/metabolismo , Proteínas Nucleares/metabolismo , Proteólise , Proteínas de Ligação a RNA/metabolismo , Aminoaciltransferases/genética , Aminoaciltransferases/metabolismo , Animais , Autofagia/efeitos dos fármacos , Proteína BRCA1/metabolismo , Feminino , Células HEK293 , Células HeLa , Humanos , Hidroxicloroquina/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Complexo de Endopeptidases do Proteassoma/metabolismo , Domínios Proteicos , Ubiquitina/metabolismoRESUMO
Skin cancer is the most common type of cancer. The incidence rate of skin cancer has continuously increased over the past decades. In an effort to discover novel anticancer agents, we identified a novel tubulin inhibitor STK899704, which is structurally distinct from other microtubule-binding agents such as colchicine, vinca alkaloids and taxanes. STK899704 inhibited microtubule polymerization leading to mitotic arrest and suppressed the proliferation of various cancer cell lines as well as multidrug resistance cancer cell lines. In this study, our investigation is further extended into animal model to evaluate the effect of STK899704 on skin carcinogenesis in vivo. Surprisingly, almost 80% of the tumors treated with STK899704 were regressed with a one-fifth reduction in tumor volume. Furthermore, the efficacy of STK899704 was nearly 2 times higher than that of 5-fluorouracil, a widely used skin cancer therapeutic. Overall, our results suggest that STK899704 is a promising anticancer chemotherapeutic that may replace existing therapies, particularly for skin cancer.
Assuntos
Benzofuranos/uso terapêutico , Neoplasias Cutâneas/tratamento farmacológico , Moduladores de Tubulina/uso terapêutico , 9,10-Dimetil-1,2-benzantraceno , Animais , Antimetabólitos Antineoplásicos/uso terapêutico , Carcinogênese , Colchicina/uso terapêutico , Modelos Animais de Doenças , Fluoruracila/uso terapêutico , Masculino , Camundongos , Neoplasias Cutâneas/induzido quimicamente , Neoplasias Cutâneas/patologia , Acetato de Tetradecanoilforbol , Tubulina (Proteína)/metabolismoRESUMO
BiP and other endoplasmic reticulum (ER)-resident proteins are thought to be metabolically stable and to function primarily in the ER lumen. We sought to assess how the abundance of these proteins dynamically fluctuates in response to various stresses and how their subpopulations are relocated to non-ER compartments such as the cytosol. We showed that the molecular chaperone BiP (also known as GRP78) was short-lived under basal conditions and ER stress. The turnover of BiP was in part driven by its amino-terminal arginylation (Nt-arginylation) by the arginyltransferase ATE1, which generated an autophagic N-degron of the N-end rule pathway. ER stress elicited the formation of R-BiP, an effect that was increased when the proteasome was also inhibited. Nt-arginylation correlated with the cytosolic relocalization of BiP under the types of stress tested. The cytosolic relocalization of BiP did not require the functionality of the unfolded protein response or the Sec61- or Derlin1-containing translocon. A key inhibitor of the turnover and Nt-arginylation of BiP was HERP (homocysteine-responsive ER protein), a 43-kDa ER membrane-integrated protein that is an essential component of ER-associated protein degradation. Pharmacological inhibition of the ER-Golgi secretory pathway also suppressed R-BiP formation. Finally, we showed that cytosolic R-BiP induced by ER stress and proteasomal inhibition was routed to autophagic vacuoles and possibly additional metabolic fates. These results suggest that Nt-arginylation is a posttranslational modification that modulates the function, localization, and metabolic fate of ER-resident proteins.