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1.
EMBO Rep ; 23(12): e55044, 2022 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-36278408

RESUMO

FBXW7, which encodes a substrate-specific receptor of an SCF E3 ligase complex, is a frequently mutated human tumor suppressor gene known to regulate the post-translational stability of various proteins involved in cellular proliferation. Here, using genome-wide CRISPR screens, we report a novel synthetic lethal genetic interaction between FBXW7 and CCNL1 and describe CCNL1 as a new substrate of the SCF-FBXW7 E3 ligase. Further analysis showed that the CCNL1-CDK11 complex is critical at the G2-M phase of the cell cycle since defective CCNL1 accumulation, resulting from FBXW7 mutation, leads to shorter mitotic time. Cells harboring FBXW7 loss-of-function mutations are hypersensitive to treatment with a CDK11 inhibitor, highlighting a genetic vulnerability that could be leveraged for cancer treatment.


Assuntos
Ciclinas , Proteína 7 com Repetições F-Box-WD , Ubiquitina-Proteína Ligases , Humanos , Proteína 7 com Repetições F-Box-WD/genética , Mutação , Ubiquitina-Proteína Ligases/genética , Ciclinas/metabolismo , Ubiquitinação
2.
Nat Commun ; 13(1): 2057, 2022 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-35440108

RESUMO

The AKT kinases have emerged as promising therapeutic targets in oncology and both allosteric and ATP-competitive AKT inhibitors have entered clinical investigation. However, long-term efficacy of such inhibitors will likely be challenged by the development of resistance. We have established prostate cancer models of acquired resistance to the allosteric inhibitor MK-2206 or the ATP-competitive inhibitor ipatasertib following prolonged exposure. While alterations in AKT are associated with acquired resistance to MK-2206, ipatasertib resistance is driven by rewired compensatory activity of parallel signaling pathways. Importantly, MK-2206 resistance can be overcome by treatment with ipatasertib, while ipatasertib resistance can be reversed by co-treatment with inhibitors of pathways including PIM signaling. These findings demonstrate that distinct resistance mechanisms arise to the two classes of AKT inhibitors and that combination approaches may reverse resistance to ATP-competitive inhibition.


Assuntos
Antineoplásicos , Proteínas Proto-Oncogênicas c-akt , Trifosfato de Adenosina/farmacologia , Inibidores da Angiogênese/farmacologia , Antineoplásicos/farmacologia , Humanos , Masculino , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais
3.
Commun Biol ; 4(1): 475, 2021 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-33846513

RESUMO

COVID-19 is a respiratory illness caused by a novel coronavirus called SARS-CoV-2. The viral spike (S) protein engages the human angiotensin-converting enzyme 2 (ACE2) receptor to invade host cells with ~10-15-fold higher affinity compared to SARS-CoV S-protein, making it highly infectious. Here, we assessed if ACE2 polymorphisms can alter host susceptibility to SARS-CoV-2 by affecting this interaction. We analyzed over 290,000 samples representing >400 population groups from public genomic datasets and identified multiple ACE2 protein-altering variants. Using reported structural data, we identified natural ACE2 variants that could potentially affect virus-host interaction and thereby alter host susceptibility. These include variants S19P, I21V, E23K, K26R, T27A, N64K, T92I, Q102P and H378R that were predicted to increase susceptibility, while variants K31R, N33I, H34R, E35K, E37K, D38V, Y50F, N51S, M62V, K68E, F72V, Y83H, G326E, G352V, D355N, Q388L and D509Y were predicted to be protective variants that show decreased binding to S-protein. Using biochemical assays, we confirmed that K31R and E37K had decreased affinity, and K26R and T92I variants showed increased affinity for S-protein when compared to wildtype ACE2. Consistent with this, soluble ACE2 K26R and T92I were more effective in blocking entry of S-protein pseudotyped virus suggesting that ACE2 variants can modulate susceptibility to SARS-CoV-2.


Assuntos
Enzima de Conversão de Angiotensina 2/genética , COVID-19/genética , Predisposição Genética para Doença/genética , Mutação de Sentido Incorreto/genética , Polimorfismo Genético , Receptores Virais/genética , Sequência de Aminoácidos , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19/metabolismo , COVID-19/virologia , Interações Hospedeiro-Patógeno , Humanos , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Receptores Virais/química , Receptores Virais/metabolismo , SARS-CoV-2/metabolismo , SARS-CoV-2/fisiologia , Homologia de Sequência de Aminoácidos , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Internalização do Vírus
4.
J Cell Biol ; 219(2)2020 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-31881079

RESUMO

Activation of Wnt signaling entails ßcatenin protein stabilization and translocation to the nucleus to regulate context-specific transcriptional programs. The majority of colorectal cancers (CRCs) initiate following APC mutations, resulting in Wnt ligand-independent stabilization and nuclear accumulation of ßcatenin. The mechanisms underlying ßcatenin nucleocytoplasmic shuttling remain incompletely defined. Using a novel, positive selection, functional genomic strategy, DEADPOOL, we performed a genome-wide CRISPR screen and identified IPO11 as a required factor for ßcatenin-mediated transcription in APC mutant CRC cells. IPO11 (Importin-11) is a nuclear import protein that shuttles cargo from the cytoplasm to the nucleus. IPO11-/- cells exhibit reduced nuclear ßcatenin protein levels and decreased ßcatenin target gene activation, suggesting IPO11 facilitates ßcatenin nuclear import. IPO11 knockout decreased colony formation of CRC cell lines and decreased proliferation of patient-derived CRC organoids. Our findings uncover a novel nuclear import mechanism for ßcatenin in cells with high Wnt activity.


Assuntos
Proteína da Polipose Adenomatosa do Colo/genética , Neoplasias Colorretais/genética , beta Catenina/genética , beta Carioferinas/genética , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Neoplasias Colorretais/patologia , Regulação Neoplásica da Expressão Gênica/genética , Células HEK293 , Humanos , Mutação , Via de Sinalização Wnt/genética
5.
Elife ; 82019 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-31452509

RESUMO

Secreted Wnt proteins regulate development and adult tissue homeostasis by binding and activating cell-surface Frizzled receptors and co-receptors including LRP5/6. The hydrophobicity of Wnt proteins has complicated their purification and limited their use in basic research and as therapeutics. We describe modular tetravalent antibodies that can recruit Frizzled and LRP5/6 in a manner that phenocopies the activities of Wnts both in vitro and in vivo. The modular nature of these synthetic Frizzled and LRP5/6 Agonists, called FLAgs, enables tailored engineering of specificity for one, two or multiple members of the Frizzled family. We show that FLAgs underlie differentiation of pluripotent stem cells, sustain organoid growth, and activate stem cells in vivo. Activation of Wnt signaling circuits with tailored FLAgs will enable precise delineation of functional outcomes directed by distinct receptor combinations and could provide a new class of therapeutics to unlock the promise of regenerative medicine.


Assuntos
Anticorpos/metabolismo , Receptores Frizzled/agonistas , Via de Sinalização Wnt/efeitos dos fármacos , Animais , Linhagem Celular , Humanos , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/agonistas , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/agonistas , Camundongos , Organoides/efeitos dos fármacos , Organoides/crescimento & desenvolvimento , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/fisiologia , Ligação Proteica
6.
Cell ; 163(6): 1515-26, 2015 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-26627737

RESUMO

The ability to perturb genes in human cells is crucial for elucidating gene function and holds great potential for finding therapeutic targets for diseases such as cancer. To extend the catalog of human core and context-dependent fitness genes, we have developed a high-complexity second-generation genome-scale CRISPR-Cas9 gRNA library and applied it to fitness screens in five human cell lines. Using an improved Bayesian analytical approach, we consistently discover 5-fold more fitness genes than were previously observed. We present a list of 1,580 human core fitness genes and describe their general properties. Moreover, we demonstrate that context-dependent fitness genes accurately recapitulate pathway-specific genetic vulnerabilities induced by known oncogenes and reveal cell-type-specific dependencies for specific receptor tyrosine kinases, even in oncogenic KRAS backgrounds. Thus, rigorous identification of human cell line fitness genes using a high-complexity CRISPR-Cas9 library affords a high-resolution view of the genetic vulnerabilities of a cell.


Assuntos
Genes Essenciais , Teorema de Bayes , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Técnicas de Inativação de Genes , Biblioteca Gênica , Humanos , Mutação
7.
Mol Cell Proteomics ; 14(7): 1781-95, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25900982

RESUMO

The identification of ubiquitin E3 ligase substrates has been challenging, due in part to low-affinity, transient interactions, the rapid degradation of targets and the inability to identify proteins from poorly soluble cellular compartments. SCF(ß-TrCP1) and SCF(ß-TrCP2) are well-studied ubiquitin E3 ligases that target substrates for proteasomal degradation, and play important roles in Wnt, Hippo, and NFκB signaling. Combining 26S proteasome inhibitor (MG132) treatment with proximity-dependent biotin labeling (BioID) and semiquantitative mass spectrometry, here we identify SCF(ß-TrCP1/2) interacting partners. Based on their enrichment in the presence of MG132, our data identify over 50 new putative SCF(ß-TrCP1/2) substrates. We validate 12 of these new substrates and reveal previously unsuspected roles for ß-TrCP in the maintenance of nuclear membrane integrity, processing (P)-body turnover and translational control. Together, our data suggest that ß-TrCP is an important hub in the cellular stress response. The technique presented here represents a complementary approach to more standard IP-MS methods and should be broadly applicable for the identification of substrates for many ubiquitin E3 ligases.


Assuntos
Biotina/metabolismo , Proteína Fosfatase 1/metabolismo , Proteínas Ligases SKP Culina F-Box/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Contendo Repetições de beta-Transducina/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Complexos Multiproteicos/metabolismo , Membrana Nuclear/metabolismo , Fosforilação , Estabilidade Proteica , Reprodutibilidade dos Testes , Especificidade por Substrato , Ubiquitina/metabolismo
8.
Biochem Biophys Res Commun ; 447(4): 696-701, 2014 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-24755078

RESUMO

Mdm2 and MdmX are important negative regulators of the tumor suppressor p53. Structurally homologous Mdm2 and MdmX inhibit p53 by directly blocking p53 transcriptional activation. Mdm2 also modifies and targets p53 for 26S proteasome dependent protein degradation through E3 ligase activity mediated by its C-terminal RING domain. However, MdmX lacks intrinsic E3 ligase activity and fails to catalyze ubiquitination of p53 despite containing a conserved RING domain. Thus, a comparative structural analysis between the Mdm2 and MdmX RING domains offers a unique way to elucidate the distinct functions of the two proteins in ubiquitination. We performed site-directed mutagenesis of the MdmX RING domain and found that the substitution of the residue N448 for cysteine and the substitution of the residue K478 for arginine granted MdmX RING domain ubiquitination activity. The structural analysis of the Mdm2 and MdmX RING domains revealed that the residue C449 of Mdm2 (structurally homologous to MdmX RING N448) located at the Mdm2 RING dimer interface is critical for the stability of the RING dimer structure, while the residue R479 (structurally homologous to MdmX RING K478) plays a role in recruiting and activating the ubiquitin E2 conjugating enzyme. This study provides new insight into the molecular mechanism of Mdm2 RING domain mediated ubiquitination.


Assuntos
Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas c-mdm2/química , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação/genética , Proteínas de Ciclo Celular , Genes p53 , Humanos , Ligação de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Nucleares/metabolismo , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Ativação Transcricional , Proteína Supressora de Tumor p53/metabolismo , Ubiquitinação
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