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1.
iScience ; 27(3): 109302, 2024 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-38450154

RESUMO

Protein phosphatase 2A (PP2A) is an essential Ser/Thr phosphatase. The PP2A holoenzyme complex comprises a scaffolding (A), regulatory (B), and catalytic (C) subunit, with PPP2CA being the principal catalytic subunit. The full scope of PP2A substrates in cells remains to be defined. To address this, we employed dTAG proteolysis-targeting chimeras to efficiently and selectively degrade dTAG-PPP2CA in homozygous knock-in HEK293 cells. Unbiased global phospho-proteomics identified 2,204 proteins with significantly increased phosphorylation upon dTAG-PPP2CA degradation, implicating them as potential PPP2CA substrates. A vast majority of these are novel. Bioinformatic analyses revealed involvement of the potential PPP2CA substrates in spliceosome function, cell cycle, RNA transport, and ubiquitin-mediated proteolysis. We identify a pSP/pTP motif as a predominant target for PPP2CA and confirm some of our phospho-proteomic data with immunoblotting. We provide an in-depth atlas of potential PPP2CA substrates and establish targeted degradation as a robust tool to unveil phosphatase substrates in cells.

2.
Trends Pharmacol Sci ; 44(11): 786-801, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37778939

RESUMO

Targeted protein degradation (TPD) is an emerging modality for research and therapeutics. Most TPD approaches harness cellular ubiquitin-dependent proteolytic pathways. Proteolysis-targeting chimeras (PROTACs) and molecular glue (MG) degraders (MGDs) represent the most advanced TPD approaches, with some already used in clinical settings. Despite these advances, TPD still faces many challenges, pertaining to both the development of effective, selective, and tissue-penetrant degraders and understanding their mode of action. In this review, we focus on progress made in addressing these challenges. In particular, we discuss the utility and application of recent proteomic approaches as indispensable tools to enable insights into degrader development, including target engagement, degradation selectivity, efficacy, safety, and mode of action.


Assuntos
Quimera de Direcionamento de Proteólise , Proteômica , Humanos , Proteólise , Ubiquitina-Proteína Ligases
3.
4.
Cell Chem Biol ; 30(10): 1261-1276.e7, 2023 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-37591251

RESUMO

Targeted protein degradation (TPD), induced by enforcing target proximity to an E3 ubiquitin ligase using small molecules has become an important drug discovery approach for targeting previously undruggable disease-causing proteins. However, out of over 600 E3 ligases encoded by the human genome, just over 10 E3 ligases are currently utilized for TPD. Here, using the affinity-directed protein missile (AdPROM) system, in which an anti-GFP nanobody was linked to an E3 ligase, we screened over 30 E3 ligases for their ability to degrade 4 target proteins, K-RAS, STK33, ß-catenin, and FoxP3, which were endogenously GFP-tagged. Several new E3 ligases, including CUL2 diGly receptor KLHDC2, emerged as effective degraders, suggesting that these E3 ligases can be taken forward for the development of small-molecule degraders, such as proteolysis targeting chimeras (PROTACs). As a proof of concept, we demonstrate that a KLHDC2-recruiting peptide-based PROTAC connected to chloroalkane is capable of degrading HALO-GFP protein in cells.


Assuntos
Fatores de Transcrição , beta Catenina , Humanos , beta Catenina/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteólise , Descoberta de Drogas , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo
5.
Methods Enzymol ; 681: 61-79, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36764764

RESUMO

Targeted protein degradation (TPD) is a useful approach in dissecting protein function and therapeutics. Technologies such as RNA interference or gene knockout that are routinely used rely on protein turnover. However, RNA interference takes a long time to deplete target proteins and is not suitable for long-lived proteins, while a genetic knockout is irreversible, takes a long time to achieve and is not suitable for essential genes. TPD has the potential to overcome the limitations of RNA interference and gene editing approaches. We have established the Affinity directed PROtein Missile (AdPROM) system, which harnesses nanobodies or binders of target proteins to redirect E3 ubiquitin ligase activity to the target protein to induce TPD through the ubiquitin proteasome system. Here we provide a step-by-step protocol for using the AdPROM system for targeted proteolysis of endogenously GFP-tagged K-RAS through an anti-GFP nanobody. This protocol can be amended to target a wide range of different proteins of interest (POIs) either by replacing the anti-GFP nanobody with a nanobody recognising the POI or by endogenously tagging the POI with GFP through CRISPR/Cas9 genome editing.


Assuntos
Anticorpos de Domínio Único , Proteólise , Anticorpos de Domínio Único/genética , Proteínas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitinação , Ubiquitina-Proteína Ligases/metabolismo
6.
Cell Chem Biol ; 30(2): 188-202.e6, 2023 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-36720221

RESUMO

Reversible protein phosphorylation, catalyzed by protein kinases and phosphatases, is a fundamental process that controls protein function and intracellular signaling. Failure of phospho-control accounts for many human diseases. While a kinase phosphorylates multiple substrates, a substrate is often phosphorylated by multiple kinases. This renders phospho-control at the substrate level challenging, as it requires inhibition of multiple kinases, which would thus affect other kinase substrates. Here, we describe the development and application of the affinity-directed phosphatase (AdPhosphatase) system for targeted dephosphorylation of specific phospho-substrates. By deploying the Protein Phosphatase 1 or 2A catalytic subunits conjugated to an antigen-stabilized anti-GFP nanobody, we can promote the dephosphorylation of two independent phospho-proteins, FAM83D or ULK1, knocked in with GFP-tags using CRISPR-Cas9, with exquisite specificity. By redirecting protein phosphatases to neo-substrates through nanobody-mediated proximity, AdPhosphatase can alter the phospho-status and function of target proteins and thus, offers a new modality for potential drug discovery approaches.


Assuntos
Proteínas Quinases , Proteína Fosfatase 2 , Humanos , Proteínas de Ciclo Celular/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Fosforilação , Proteínas Quinases/metabolismo , Proteína Fosfatase 2/metabolismo , Especificidade por Substrato , Monoéster Fosfórico Hidrolases/metabolismo
7.
Cell Chem Biol ; 29(10): 1482-1504.e7, 2022 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-36075213

RESUMO

Proteolysis-targeting chimeras (PROTACs) bring a protein of interest (POI) into spatial proximity of an E3 ubiquitin ligase, promoting POI ubiquitylation and proteasomal degradation. PROTACs rely on endogenous cellular machinery to mediate POI degradation, therefore the subcellular location of the POI and access to the E3 ligase being recruited potentially impacts PROTAC efficacy. To interrogate whether the subcellular context of the POI influences PROTAC-mediated degradation, we expressed either Halo or FKBP12F36V (dTAG) constructs consisting of varying localization signals and tested the efficacy of their degradation by von Hippel-Lindau (VHL)- or cereblon (CRBN)-recruiting PROTACs targeting either Halo or dTAG. POIs were localized to the nucleus, cytoplasm, outer mitochondrial membrane, endoplasmic reticulum, Golgi, peroxisome or lysosome. Differentially localized Halo or FKBP12F36V proteins displayed varying levels of degradation using the same respective PROTACs, suggesting therefore that the subcellular context of the POI can influence the efficacy of PROTAC-mediated POI degradation.


Assuntos
Proteína 1A de Ligação a Tacrolimo , Ubiquitina-Proteína Ligases , Proteólise , Proteína 1A de Ligação a Tacrolimo/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
8.
SLAS Discov ; 26(7): 885-895, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34041938

RESUMO

Targeted protein degradation is an emerging new strategy for the modulation of intracellular protein levels with applications in chemical biology and drug discovery. One approach to enable this strategy is to redirect the ubiquitin-proteasome system to mark and degrade target proteins of interest (POIs) through the use of proteolysis targeting chimeras (PROTACs). Although great progress has been made in enabling PROTACs as a platform, there are still a limited number of E3 ligases that have been employed for PROTAC design. Herein we report a novel phenotypic screening approach for the identification of E3 ligase binders. The key concept underlying this approach is the high-throughput modification of screening compounds with a chloroalkane moiety to generate HaloPROTACs in situ, which were then evaluated for their ability to degrade a GFP-HaloTag fusion protein in a cellular context. As proof of concept, we demonstrated that we could generate and detect functional HaloPROTACs in situ, using a validated Von Hippel-Lindau (VHL) binder that successfully degraded the GFP-HaloTag fusion protein in living cells. We then used this method to prepare and screen a library of approximately 2000 prospective E3 ligase-recruiting molecules.


Assuntos
Descoberta de Drogas/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Proteólise/efeitos dos fármacos , Humanos , Ligação Proteica , Bibliotecas de Moléculas Pequenas , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
9.
Life Sci Alliance ; 4(2)2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33361109

RESUMO

The function of the FAM83F protein, like the functions of many members of the FAM83 family, is poorly understood. Here, we show that injection of Fam83f mRNA into Xenopus embryos causes axis duplication, a phenotype indicative of enhanced Wnt signalling. Consistent with this, overexpression of FAM83F activates Wnt signalling, whereas ablation of FAM83F from human colorectal cancer (CRC) cells attenuates it. We demonstrate that FAM83F is farnesylated and interacts and co-localises with CK1α at the plasma membrane. This interaction with CK1α is essential for FAM83F to activate Wnt signalling, and FAM83F mutants that do not interact with CK1α fail to induce axis duplication in Xenopus embryos and to activate Wnt signalling in cells. FAM83F acts upstream of GSK-3ß because the attenuation of Wnt signalling caused by loss of FAM83F can be rescued by GSK-3 inhibition. Introduction of a farnesyl-deficient mutant of FAM83F in cells through CRISPR/Cas9 genome editing redirects the FAM83F-CK1α complex away from the plasma membrane and significantly attenuates Wnt signalling, indicating that FAM83F exerts its effects on Wnt signalling at the plasma membrane.


Assuntos
Caseína Quinase Ialfa/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Neoplasias/metabolismo , Via de Sinalização Wnt , Animais , Linhagem Celular , Membrana Celular/metabolismo , Desenvolvimento Embrionário/genética , Imunofluorescência , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Genes Reporter , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Prenilação , Ligação Proteica , Transporte Proteico , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis
10.
Life Sci Alliance ; 4(2)2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33361334

RESUMO

Immunomodulatory imide drugs (IMiDs) bind CRBN, a substrate receptor of the Cul4A E3 ligase complex, enabling the recruitment of neo-substrates, such as CK1α, and their degradation via the ubiquitinproteasome system. Here, we report FAM83F as such a neo-substrate. The eight FAM83 proteins (A-H) interact with and regulate the subcellular distribution of CK1α. We demonstrate that IMiD-induced FAM83F degradation requires its association with CK1α. However, no other FAM83 protein is degraded by IMiDs. We have recently identified FAM83F as a mediator of the canonical Wnt signalling pathway. The IMiD-induced degradation of FAM83F attenuated Wnt signalling in colorectal cancer cells and removed CK1α from the plasma membrane, mirroring the phenotypes observed with genetic ablation of FAM83F. Intriguingly, the expression of FAM83G, which also binds to CK1α, appears to attenuate the IMiD-induced degradation of CK1α, suggesting a protective role for FAM83G on CK1α. Our findings reveal that the efficiency and extent of target protein degradation by IMiDs depends on the nature of inherent multiprotein complex in which the target protein is part of.


Assuntos
Caseína Quinase Ialfa/metabolismo , Imidas/farmacologia , Fatores Imunológicos/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Neoplasias/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Bases , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Técnicas de Introdução de Genes , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Estabilidade Proteica , Proteólise/efeitos dos fármacos , Ubiquitina-Proteína Ligases/metabolismo
11.
Biochem J ; 477(23): 4603-4621, 2020 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-33306089

RESUMO

Regarded as constitutively active enzymes, known to participate in many, diverse biological processes, the intracellular regulation bestowed on the CK1 family of serine/threonine protein kinases is critically important, yet poorly understood. Here, we provide an overview of the known CK1-dependent cellular functions and review the emerging roles of CK1-regulating proteins in these processes. We go on to discuss the advances, limitations and pitfalls that CK1 researchers encounter when attempting to define relationships between CK1 isoforms and their substrates, and the challenges associated with ascertaining the correct physiological CK1 isoform for the substrate of interest. With increasing interest in CK1 isoforms as therapeutic targets, methods of selectively inhibiting CK1 isoform-specific processes is warranted, yet challenging to achieve given their participation in such a vast plethora of signalling pathways. Here, we discuss how one might shut down CK1-specific processes, without impacting other aspects of CK1 biology.


Assuntos
Caseína Quinase I/metabolismo , Transdução de Sinais , Animais , Caseína Quinase I/genética , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Especificidade por Substrato
12.
Cell Chem Biol ; 27(9): 1151-1163.e6, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32668202

RESUMO

K-RAS is known as the most frequently mutated oncogene. However, the development of conventional K-RAS inhibitors has been extremely challenging, with a mutation-specific inhibitor reaching clinical trials only recently. Targeted proteolysis has emerged as a new modality in drug discovery to tackle undruggable targets. Our laboratory has developed a system for targeted proteolysis using peptidic high-affinity binders, called "AdPROM." Here, we used CRISPR/Cas9 technology to knock in a GFP tag on the native K-RAS gene in A549 adenocarcinoma (A549GFPKRAS) cells and constructed AdPROMs containing high-affinity GFP or H/K-RAS binders. Expression of GFP-targeting AdPROM in A549GFPKRAS led to robust proteasomal degradation of endogenous GFP-K-RAS, while expression of anti-HRAS-targeting AdPROM in different cell lines resulted in the degradation of both GFP-tagged and untagged K-RAS, and untagged H-RAS. Our findings imply that endogenous RAS proteins can be targeted for proteolysis, supporting the idea of an alternative therapeutic approach to these undruggable targets.


Assuntos
Proteólise , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Células A549 , Marcadores de Afinidade , Sistemas CRISPR-Cas/genética , Linhagem Celular , Proliferação de Células , Técnicas de Introdução de Genes , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Microscopia de Fluorescência , Peptídeos/química , Peptídeos/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
13.
Cell Chem Biol ; 27(9): 1164-1180.e5, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32668203

RESUMO

The affinity-directed protein missile (AdPROM) system utilizes specific polypeptide binders of intracellular proteins of interest (POIs) conjugated to an E3 ubiquitin ligase moiety to enable targeted proteolysis of the POI. However, a chemically tuneable AdPROM system is more desirable. Here, we use Halo-tag/VHL-recruiting proteolysis-targeting chimera (HaloPROTAC) technology to develop a ligand-inducible AdPROM (L-AdPROM) system. When we express an L-AdPROM construct consisting of an anti-GFP nanobody conjugated to the Halo-tag, we achieve robust degradation of GFP-tagged POIs only upon treatment of cells with the HaloPROTAC. For GFP-tagged POIs, ULK1, FAM83D, and SGK3 were knocked in with a GFP-tag using CRISPR/Cas9. By substituting the anti-GFP nanobody for a monobody that binds H- and K-RAS, we achieve robust degradation of unmodified endogenous RAS proteins only in the presence of the HaloPROTAC. Through substitution of the polypeptide binder, the highly versatile L-AdPROM system is useful for the inducible degradation of potentially any intracellular POI.


Assuntos
Proteólise , Anticorpos de Cadeia Única/metabolismo , Marcadores de Afinidade , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Sistemas CRISPR-Cas/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Técnicas de Introdução de Genes , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/imunologia , Proteínas de Fluorescência Verde/metabolismo , Humanos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/genética , Ubiquitinação , Proteínas ras/metabolismo
14.
Cell Signal ; 72: 109632, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32289446

RESUMO

The majority of mutations identified in patients with amelogenesis imperfecta have been mapped to FAM83H. As FAM83H expression is not limited to the enamel, how FAM83H contributes to amelogenesis is still largely unknown. We previously reported that members of the FAM83 family of proteins interact with and regulate the subcellular distribution of the promiscuous serine-threonine protein kinase CK1 family, through their shared N-terminal DUF1669 domains. FAM83H co-localises with CK1 isoforms to speckle-like structures in both the cytoplasm and nucleus. In this report, we show FAM83H, unlike other FAM83 proteins, interacts and colocalises with NCK1/2 tyrosine kinase adaptor proteins. This interaction is mediated by proline-rich motifs within the C-terminus of FAM83H, specifically interacting with the second and third SH3 domains of NCK1/2. Moreover, FAM83H pathogenic AI mutant proteins, which trigger C-terminal truncations of FAM83H, retain their interactions with CK1 isoforms but lose interaction with NCK1/2. These AI mutant FAM83H proteins acquire a nuclear localisation, and recruit CK1 isoforms to the nucleus where CK1 retains its kinase activity. As understanding the constituents of the FAM83H-localised speckles may hold the key to unravelling potential substrates of FAM83H-associated CK1 substrates, we employed a TurboID-based proximity labelling approach and uncovered several proteins including Iporin and BAG3 as potential constituents of the speckles.


Assuntos
Amelogênese Imperfeita/genética , Mutação/genética , Proteínas/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Células HEK293 , Humanos , Proteínas Oncogênicas/metabolismo , Ligação Proteica , Domínios Proteicos , Proteínas/química , Proteínas/metabolismo
15.
Cell Cycle ; 19(5): 505-524, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32048898

RESUMO

The coordinated activities of many protein kinases, acting on multiple protein substrates, ensures the error-free progression through mitosis of eukaryotic cells. Enormous research effort has thus been devoted to studying the roles and regulation of these mitotic kinases, and to the identification of their physiological substrates. Central for the timely deployment of specific protein kinases to their appropriate substrates during the cell division cycle are the many anchoring proteins, which serve critical regulatory roles. Through direct association, anchoring proteins are capable of modulating the catalytic activity and/or sub-cellular distribution of the mitotic kinases they associate with. The key roles of some anchoring proteins in cell division are well-established, whilst others are still being unearthed. Here, we review the current knowledge on anchoring proteins for some mitotic kinases, and highlight how targeting anchoring proteins for inhibition, instead of the mitotic kinases themselves, could be advantageous for disrupting the cell division cycle.


Assuntos
Mitose , Proteínas Quinases/metabolismo , Animais , Aurora Quinases/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Humanos , Pontos de Checagem da Fase M do Ciclo Celular , Proteínas Associadas aos Microtúbulos/metabolismo , Mitose/efeitos dos fármacos
16.
Cell Death Dis ; 11(1): 49, 2020 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-31969556

RESUMO

The signalling pathways initiated by members of the transforming growth factor-ß (TGFß) family of cytokines control many metazoan cellular processes, including proliferation and differentiation, epithelial-mesenchymal transition (EMT) and apoptosis. TGFß signalling is therefore strictly regulated to ensure appropriate context-dependent physiological responses. In an attempt to identify novel regulatory components of the TGFß signalling pathway, we performed a pharmacological screen by using a cell line engineered to report the endogenous transcription of the TGFß-responsive target gene PAI-1. The screen revealed that small molecule inhibitors of salt-inducible kinases (SIKs) attenuate TGFß-mediated transcription of PAI-1 without affecting receptor-mediated SMAD phosphorylation, SMAD complex formation or nuclear translocation. We provide evidence that genetic inactivation of SIK isoforms also attenuates TGFß-dependent transcriptional responses. Pharmacological inhibition of SIKs by using multiple small-molecule inhibitors potentiated apoptotic cell death induced by TGFß stimulation. Our data therefore provide evidence for a novel function of SIKs in modulating TGFß-mediated transcriptional and cellular responses.


Assuntos
Apoptose/genética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Serpina E2/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/genética , Núcleo Celular/metabolismo , Citoplasma/efeitos dos fármacos , Citoplasma/genética , Citoplasma/metabolismo , Técnicas de Inativação de Genes , Inativação Gênica , Humanos , Indanos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Pirimidinas/farmacologia , Serpina E2/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Proteínas Smad/metabolismo
17.
Wellcome Open Res ; 4: 133, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31656861

RESUMO

Background: Two recessive mutations in the FAM83G gene, causing A34E and R52P amino acid substitutions in the DUF1669 domain of the PAWS1 protein, are associated with palmoplantar keratoderma (PPK) in humans and dogs respectively. We have previously reported that PAWS1 associates with the Ser/Thr protein kinase CK1α through the DUF1669 domain to mediate canonical Wnt signalling. Methods: Co-immunoprecipitation was used to investigate possible changes to PAWS1 interactors caused by the mutations. We also compared the stability of wild-type and mutant PAWS1 in cycloheximide-treated cells. Effects on Wnt signalling were determined using the TOPflash luciferase reporter assay in U2OS cells expressing PAWS1 mutant proteins. The ability of PAWS1 to induce axis duplication in Xenopus embryos was also tested. Finally, we knocked-in the A34E mutation at the native gene locus and measured Wnt-induced AXIN2 gene expression by RT-qPCR. Results: We show that these PAWS1 A34E and PAWS1 R52P mutants fail to interact with CK1α but, like the wild-type protein, do interact with CD2AP and SMAD1. Like cells carrying a PAWS1 F296A mutation, which also abolishes CK1α binding, cells carrying the A34E and R52P mutants respond poorly to Wnt signalling to an extent resembling that observed in FAM83G gene knockout cells. Consistent with this observation, these mutants, in contrast to the wild-type protein, fail to induce axis duplication in Xenopus embryos. We also found that the A34E and R52P mutant proteins are less abundant than the native protein and appear to be less stable, both when overexpressed in FAM83G-knockout cells and when knocked-in at the native FAM83G locus. Ala 34 of PAWS1 is conserved in all FAM83 proteins and mutating the equivalent residue in FAM83H (A31E) also abolishes interaction with CK1 isoforms. Conclusions: We propose that mutations in PAWS1 cause PPK pathogenesis through disruption of the CK1α interaction and attenuation of Wnt signalling.

18.
EMBO Rep ; 20(9): e47495, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31338967

RESUMO

The concerted action of many protein kinases helps orchestrate the error-free progression through mitosis of mammalian cells. The roles and regulation of some prominent mitotic kinases, such as cyclin-dependent kinases, are well established. However, these and other known mitotic kinases alone cannot account for the extent of protein phosphorylation that has been reported during mammalian mitosis. Here we demonstrate that CK1α, of the casein kinase 1 family of protein kinases, localises to the spindle and is required for proper spindle positioning and timely cell division. CK1α is recruited to the spindle by FAM83D, and cells devoid of FAM83D, or those harbouring CK1α-binding-deficient FAM83DF283A/F283A knockin mutations, display pronounced spindle positioning defects, and a prolonged mitosis. Restoring FAM83D at the endogenous locus in FAM83D-/- cells, or artificially delivering CK1α to the spindle in FAM83DF283A/F283A cells, rescues these defects. These findings implicate CK1α as new mitotic kinase that orchestrates the kinetics and orientation of cell division.


Assuntos
Caseína Quinase I/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Fuso Acromático/metabolismo , Animais , Caseína Quinase I/genética , Ciclo Celular/genética , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Citometria de Fluxo , Células HeLa , Humanos , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/genética , Mitose/genética , Mitose/fisiologia
19.
Cell Mol Life Sci ; 76(14): 2761-2777, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31030225

RESUMO

Protein silencing is often employed as a means to aid investigations in protein function and is increasingly desired as a therapeutic approach. Several types of protein silencing methodologies have been developed, including targeting the encoding genes, transcripts, the process of translation or the protein directly. Despite these advances, most silencing systems suffer from limitations. Silencing protein expression through genetic ablation, for example by CRISPR/Cas9 genome editing, is irreversible, time consuming and not always feasible. Similarly, RNA interference approaches warrant prolonged treatments, can lead to incomplete protein depletion and are often associated with off-target effects. Targeted proteolysis has the potential to overcome some of these limitations. The field of targeted proteolysis has witnessed the emergence of many methodologies aimed at targeting specific proteins for degradation in a spatio-temporal manner. In this review, we provide an appraisal of the different targeted proteolytic systems and discuss their applications in understanding protein function, as well as their potential in therapeutics.


Assuntos
Edição de Genes , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas/metabolismo , Proteólise , Humanos , Complexo de Endopeptidases do Proteassoma/genética , Proteínas/genética , Ubiquitinação
20.
Methods Mol Biol ; 1891: 29-35, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30414124

RESUMO

Transcriptional reporter systems allow researchers to investigate the function and regulation of transcription factors. Conventional systems employ artificial cDNA overexpression vectors containing either a promoter fragment or specific nucleotide sequence repeats upstream of firefly luciferase or fluorescent reporters, such as green fluorescence protein (GFP) cDNA. These systems suffer mainly from the lack of chromatin context. Here, we describe the rapid generation of endogenous transcriptional reporter cells for the bone morphogenetic protein (BMP) pathway using CRISPR/Cas9 genome editing. In principle, our methodology can be applied to any cell line. The endogenous reporters will provide a robust system for the investigation of BMP transcriptional activity in the context of native chromatin landscape and facilitate chemical and genetic screens.


Assuntos
Proteínas Morfogenéticas Ósseas/genética , Sistemas CRISPR-Cas , Edição de Genes , Genes Reporter , Transcrição Gênica , Proteínas Morfogenéticas Ósseas/metabolismo , Clonagem Molecular , Humanos , Regiões Promotoras Genéticas , RNA Guia de Cinetoplastídeos , Proteínas Recombinantes , Transdução de Sinais
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