RESUMO
Protein ubiquitylation controls diverse processes within eukaryotic cells, including protein degradation, and is often dysregulated in disease. Moreover, small-molecule degraders that redirect ubiquitylation activities toward disease targets are an emerging and promising therapeutic class. Over 600 E3 ubiquitin ligases are expressed in humans, but their substrates remain largely elusive, necessitating the development of new methods for their discovery. Here we report the development of E3-substrate tagging by ubiquitin biotinylation (E-STUB), a ubiquitin-specific proximity labeling method that biotinylates ubiquitylated substrates in proximity to an E3 ligase of interest. E-STUB accurately identifies the direct ubiquitylated targets of protein degraders, including collateral targets and ubiquitylation events that do not lead to substrate degradation. It also detects known substrates of E3 ligase CRBN and VHL with high specificity. With the ability to elucidate proximal ubiquitylation events, E-STUB may facilitate the development of proximity-inducing therapeutics and act as a generalizable method for E3-substrate mapping.
Assuntos
Biotinilação , Ubiquitina-Proteína Ligases , Ubiquitina , Ubiquitinação , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/química , Humanos , Ubiquitina/metabolismo , Ubiquitina/química , Especificidade por Substrato , Células HEK293 , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , ProteóliseRESUMO
The protein kinase maternal and embryonic leucine zipper kinase (MELK) is critical for mitotic progression of cancer cells; however, its mechanisms of action remain largely unknown. By combined approaches of immunoprecipitation/mass spectrometry and peptide library profiling, we identified the eukaryotic translation initiation factor 4B (eIF4B) as a MELK-interacting protein during mitosis and a bona fide substrate of MELK. MELK phosphorylates eIF4B at Ser406, a modification found to be most robust in the mitotic phase of the cell cycle. We further show that the MELK-eIF4B signaling axis regulates protein synthesis during mitosis. Specifically, synthesis of myeloid cell leukemia 1 (MCL1), an antiapoptotic protein known to play a role in cancer cell survival during cell division, depends on the function of MELK-elF4B. Inactivation of MELK or eIF4B results in reduced protein synthesis of MCL1, which, in turn, induces apoptotic cell death of cancer cells. Our study thus defines a MELK-eIF4B signaling axis that regulates protein synthesis during mitosis, and consequently influences cancer cell survival.
Assuntos
Fatores de Iniciação em Eucariotos/genética , Mitose , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Biossíntese de Proteínas , Proteínas Serina-Treonina Quinases/genética , Sequência de Aminoácidos , Animais , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Sequência Conservada , Fatores de Iniciação em Eucariotos/antagonistas & inibidores , Fatores de Iniciação em Eucariotos/metabolismo , Células HEK293 , Humanos , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Biblioteca de Peptídeos , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Alinhamento de Sequência , Transdução de SinaisRESUMO
The related NUAK1 and NUAK2 are members of the AMPK (AMP-activated protein kinase) family of protein kinases that are activated by the LKB1 (liver kinase B1) tumour suppressor kinase. Recent work suggests they play important roles in regulating key biological processes including Myc-driven tumorigenesis, senescence, cell adhesion and neuronal polarity. In the present paper we describe the first highly specific protein kinase inhibitors of NUAK kinases namely WZ4003 and HTH-01-015. WZ4003 inhibits both NUAK isoforms (IC50 for NUAK1 is 20 nM and for NUAK2 is 100 nM), whereas HTH-01-015 inhibits only NUAK1 (IC50 is 100 nM). These compounds display extreme selectivity and do not significantly inhibit the activity of 139 other kinases that were tested including ten AMPK family members. In all cell lines tested, WZ4003 and HTH-01-015 inhibit the phosphorylation of the only well-characterized substrate, MYPT1 (myosin phosphate-targeting subunit 1) that is phosphorylated by NUAK1 at Ser(445). We also identify a mutation (A195T) that does not affect basal NUAK1 activity, but renders it ~50-fold resistant to both WZ4003 and HTH-01-015. Consistent with NUAK1 mediating the phosphorylation of MYPT1 we find that in cells overexpressing drug-resistant NUAK1[A195T], but not wild-type NUAK1, phosphorylation of MYPT1 at Ser(445) is no longer suppressed by WZ4003 or HTH-01-015. We also demonstrate that administration of WZ4003 and HTH-01-015 to MEFs (mouse embryonic fibroblasts) significantly inhibits migration in a wound-healing assay to a similar extent as NUAK1-knockout. WZ4003 and HTH-01-015 also inhibit proliferation of MEFs to the same extent as NUAK1 knockout and U2OS cells to the same extent as NUAK1 shRNA knockdown. We find that WZ4003 and HTH-01-015 impaired the invasive potential of U2OS cells in a 3D cell invasion assay to the same extent as NUAK1 knockdown. The results of the present study indicate that WZ4003 and HTH-01-015 will serve as useful chemical probes to delineate the biological roles of the NUAK kinases.
Assuntos
Anilidas/farmacologia , Benzodiazepinonas/farmacologia , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirimidinas/farmacologia , Proteínas Repressoras/antagonistas & inibidores , Quinases Proteína-Quinases Ativadas por AMP , Animais , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos , Ativação Enzimática/efeitos dos fármacos , Células HEK293 , Humanos , Camundongos , Neoplasias/patologia , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Repressoras/agonistas , Proteínas Repressoras/genética , Especificidade por Substrato , Proteínas Supressoras de Tumor/fisiologiaRESUMO
Tauopathies are neurodegenerative diseases characterized by aberrant forms of tau protein accumulation leading to neuronal death in focal brain areas. Positron emission tomography (PET) tracers that bind to pathological tau are used in diagnosis, but there are no current therapies to eliminate these tau species. We employed targeted protein degradation technology to convert a tau PET-probe into a functional degrader of pathogenic tau. The hetero-bifunctional molecule QC-01-175 was designed to engage both tau and Cereblon (CRBN), a substrate-receptor for the E3-ubiquitin ligase CRL4CRBN, to trigger tau ubiquitination and proteasomal degradation. QC-01-175 effected clearance of tau in frontotemporal dementia (FTD) patient-derived neuronal cell models, with minimal effect on tau from neurons of healthy controls, indicating specificity for disease-relevant forms. QC-01-175 also rescued stress vulnerability in FTD neurons, phenocopying CRISPR-mediated MAPT-knockout. This work demonstrates that aberrant tau in FTD patient-derived neurons is amenable to targeted degradation, representing an important advance for therapeutics.
Assuntos
Demência Frontotemporal/tratamento farmacológico , Neurônios/metabolismo , Fármacos Neuroprotetores/metabolismo , Proteólise , Tauopatias/tratamento farmacológico , Proteínas tau/metabolismo , Linhagem Celular , Humanos , Modelos Teóricos , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/síntese químicaRESUMO
The Hippo-YAP signaling pathway is a critical regulator of proliferation, apoptosis, and cell fate. The main downstream effector of this pathway, YAP, has been shown to be misregulated in human cancer and has emerged as an attractive target for therapeutics. A significant insufficiency in our understanding of the pathway is the identity of transcriptional targets of YAP that drive its potent growth phenotypes. Here, using liver cancer as a model, we identify NUAK2 as an essential mediator of YAP-driven hepatomegaly and tumorigenesis in vivo. By evaluating several human cancer cell lines we determine that NUAK2 is selectively required for YAP-driven growth. Mechanistically, we found that NUAK2 participates in a feedback loop to maximize YAP activity via promotion of actin polymerization and myosin activity. Additionally, pharmacological inactivation of NUAK2 suppresses YAP-dependent cancer cell proliferation and liver overgrowth. Importantly, our work here identifies a specific, potent, and actionable target for YAP-driven malignancies.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Carcinogênese/genética , Carcinoma Hepatocelular/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/genética , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinases/genética , Actinas/genética , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Antineoplásicos/farmacologia , Benzodiazepinonas/farmacologia , Carcinogênese/efeitos dos fármacos , Carcinogênese/metabolismo , Carcinogênese/patologia , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Retroalimentação Fisiológica , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Nus , Miosinas/genética , Miosinas/metabolismo , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas de Sinalização YAPRESUMO
Heterobifunctional molecules that recruit E3 ubiquitin ligases, such as cereblon, for targeted protein degradation represent an emerging pharmacological strategy. A major unanswered question is how generally applicable this strategy is to all protein targets. In this study, we designed a multi-kinase degrader by conjugating a highly promiscuous kinase inhibitor with a cereblon-binding ligand, and used quantitative proteomics to discover 28 kinases, including BTK, PTK2, PTK2B, FLT3, AURKA, AURKB, TEC, ULK1, ITK, and nine members of the CDK family, as degradable. This set of kinases is only a fraction of the intracellular targets bound by the degrader, demonstrating that successful degradation requires more than target engagement. The results guided us to develop selective degraders for FLT3 and BTK, with potentials to improve disease treatment. Together, this study demonstrates an efficient approach to triage a gene family of interest to identify readily degradable targets for further studies and pre-clinical developments.
Assuntos
Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteômica , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Tirosina Quinase da Agamaglobulinemia/genética , Tirosina Quinase da Agamaglobulinemia/metabolismo , Humanos , Inibidores de Proteínas Quinases/química , Proteólise , Tirosina Quinase 3 Semelhante a fms/genética , Tirosina Quinase 3 Semelhante a fms/metabolismoRESUMO
Thorough preclinical target validation is essential for the success of drug discovery efforts. In this study, we combined chemical and genetic perturbants, including the development of a novel selective maternal embryonic leucine zipper kinase (MELK) inhibitor HTH-01-091, CRISPR/Cas9-mediated MELK knockout, a novel chemical-induced protein degradation strategy, RNA interference and CRISPR interference to validate MELK as a therapeutic target in basal-like breast cancers (BBC). In common culture conditions, we found that small molecule inhibition, genetic deletion, or acute depletion of MELK did not significantly affect cellular growth. This discrepancy to previous findings illuminated selectivity issues of the widely used MELK inhibitor OTSSP167, and potential off-target effects of MELK-targeting short hairpins. The different genetic and chemical tools developed here allow for the identification and validation of any causal roles MELK may play in cancer biology, which will be required to guide future MELK drug discovery efforts. Furthermore, our study provides a general framework for preclinical target validation.
Assuntos
Neoplasias da Mama/patologia , Proliferação de Células , Proteínas Serina-Treonina Quinases/análise , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Humanos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genéticaRESUMO
Activating mutations involving the PI3K pathway occur frequently in human cancers. However, PI3K inhibitors primarily induce cell cycle arrest, leaving a significant reservoir of tumor cells that may acquire or exhibit resistance. We searched for genes that are required for the survival of PI3K mutant cancer cells in the presence of PI3K inhibition by conducting a genome scale shRNA-based apoptosis screen in a PIK3CA mutant human breast cancer cell. We identified 5 genes (PIM2, ZAK, TACC1, ZFR, ZNF565) whose suppression induced cell death upon PI3K inhibition. We showed that small molecule inhibitors of the PIM2 and ZAK kinases synergize with PI3K inhibition. In addition, using a microscale implementable device to deliver either siRNAs or small molecule inhibitors in vivo, we showed that suppressing these 5 genes with PI3K inhibition induced tumor regression. These observations identify targets whose inhibition synergizes with PI3K inhibitors and nominate potential combination therapies involving PI3K inhibition.
Assuntos
Apoptose , Sinergismo Farmacológico , Inibidores Enzimáticos/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , MAP Quinase Quinase Quinases , Camundongos SCID , Neoplasias Experimentais/terapia , Transplante Heterólogo , Resultado do TratamentoRESUMO
A general and efficient approach to important fluorinated azaheterocycles has been developed by incorporating nucleophilic fluorination into alkene difunctionalization. This intramolecular aminofluorination transformation of alkenes has been achieved via the aminocyclization of reactive unsaturated N-iodoamines, which can be generated in situ from either unsaturated N-chloramines or their amine precursors in a one-pot protocol.
Assuntos
Alcenos/química , Aminas/química , Compostos Aza/síntese química , Hidrocarbonetos Fluorados/síntese química , Compostos Aza/química , Catálise , Ciclização , Hidrocarbonetos Fluorados/química , Estrutura Molecular , EstereoisomerismoRESUMO
Described here is a stable isotope labeling protocol that can be used with a chemical modification- and mass spectrometry-based protein-ligand binding assay for detecting and quantifying both the direct and indirect binding events that result from protein-ligand binding interactions. The protocol utilizes an H(2) (16)O(2) and H(2) (18)O(2) labeling strategy to evaluate the chemical denaturant dependence of methionine oxidation in proteins both in the presence and absence of a target ligand. The differential denaturant dependence to the oxidation reactions performed in the presence and absence of ligand provides a measure of the protein stability changes that occur as a result of direct interactions of proteins with the target ligand and/or as a result of indirect interactions involving other protein-ligand interactions that are either induced or disrupted by the ligand. The described protocol utilizes the (18)O/(16)O ratio in the oxidized protein samples to quantify the ligand-induced protein stability changes. The ratio is determined using the isotopic distributions observed for the methionine-containing peptides used for protein identification in the LC-MS-based proteomics readout. The strategy is applied to a multi-component protein mixture in this proof-of-principle experiment, which was designed to evaluate the technique's ability to detect and quantify the direct binding interaction between cyclosporin A and cyclophilin A and to detect the indirect binding interaction between cyclosporin A and calcineurin (i.e., the protein-protein interaction between cyclophilin A and calcineurin that is induced by cyclosporin A binding to cyclophilin A).