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1.
Proc Natl Acad Sci U S A ; 120(35): e2305037120, 2023 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-37603740

RESUMO

Polo-like kinase 1 (Plk1) is considered an attractive target for anticancer therapy. Over the years, studies on the noncatalytic polo-box domain (PBD) of Plk1 have raised the expectation of generating highly specific protein-protein interaction inhibitors. However, the molecular nature of the canonical PBD-dependent interaction, which requires extensive water network-mediated interactions with its phospholigands, has hampered efforts to identify small molecules suitable for Plk1 PBD drug discovery. Here, we report the identification of the first allosteric inhibitor of Plk1 PBD, called Allopole, a prodrug that can disrupt intracellular interactions between PBD and its cognate phospholigands, delocalize Plk1 from centrosomes and kinetochores, and induce mitotic block and cancer cell killing. At the structural level, its unmasked active form, Allopole-A, bound to a deep Trp-Phe-lined pocket occluded by a latch-like loop, whose adjoining region was required for securely retaining a ligand anchored to the phospho-binding cleft. Allopole-A binding completely dislodged the L2 loop, an event that appeared sufficient to trigger the dissociation of a phospholigand and inhibit PBD-dependent Plk1 function during mitosis. Given Allopole's high specificity and antiproliferative potency, this study is expected to open an unexplored avenue for developing Plk1 PBD-specific anticancer therapeutic agents.


Assuntos
Proteínas de Ciclo Celular , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas , Divisão do Núcleo Celular , Quinase 1 Polo-Like
2.
Commun Biol ; 6(1): 712, 2023 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-37433832

RESUMO

Proper organization of intracellular assemblies is fundamental for efficient promotion of biochemical processes and optimal assembly functionality. Although advances in imaging technologies have shed light on how the centrosome is organized, how its constituent proteins are coherently architected to elicit downstream events remains poorly understood. Using multidisciplinary approaches, we showed that two long coiled-coil proteins, Cep63 and Cep152, form a heterotetrameric building block that undergoes a stepwise formation into higher molecular weight complexes, ultimately generating a cylindrical architecture around a centriole. Mutants defective in Cep63•Cep152 heterotetramer formation displayed crippled pericentriolar Cep152 organization, polo-like kinase 4 (Plk4) relocalization to the procentriole assembly site, and Plk4-mediated centriole duplication. Given that the organization of pericentriolar materials (PCM) is evolutionarily conserved, this work could serve as a model for investigating the structure and function of PCM in other species, while offering a new direction in probing the organizational defects of PCM-related human diseases.


Assuntos
Centríolos , Centrossomo , Proteínas Serina-Treonina Quinases , Humanos , Ciclo Celular , Peso Molecular , Domínios Proteicos , Proteínas Serina-Treonina Quinases/metabolismo
4.
ACS Pharmacol Transl Sci ; 6(3): 422-446, 2023 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-36926457

RESUMO

Polo-like kinase 1 (Plk1), a mitotic kinase whose activity is widely upregulated in various human cancers, is considered an attractive target for anticancer drug discovery. Aside from the kinase domain, the C-terminal noncatalytic polo-box domain (PBD), which mediates the interaction with the enzyme's binding targets or substrates, has emerged as an alternative target for developing a new class of inhibitors. Various reported small molecule PBD inhibitors exhibit poor cellular efficacy and/or selectivity. Here, we report structure-activity relationship (SAR) studies on triazoloquinazolinone-derived inhibitors, such as 43 (a 1-thioxo-2,4-dihydrothieno[2,3-e][1,2,4]triazolo[4,3-a]pyrimidin-5(1H)-one) that effectively block Plk1, but not Plk2 and Plk3 PBDs, with improved affinity and drug-like properties. The range of prodrug moieties needed for thiol group masking of the active drugs has been expanded to increase cell permeability and mechanism-based cancer cell (L363 and HeLa) death. For example, a 5-thio-1-methyl-4-nitroimidazolyl prodrug 80, derived from 43, showed an improved cellular potency (GI50 4.1 µM). As expected, 80 effectively blocked Plk1 from localizing to centrosomes and kinetochores and consequently induced potent mitotic block and apoptotic cell death. Another prodrug 78 containing 9-fluorophenyl in place of the thiophene-containing heterocycle in 80 also induced a comparable degree of anti-Plk1 PBD effect. However, orally administered 78 was rapidly converted in the bloodstream to parent drug 15, which was shown be relatively stable toward in vivo oxidation due to its 9-fluorophenyl group in comparison to unsubstituted phenyl. Further derivatization of these inhibitors, particularly to improve the systemic prodrug stability, could lead to a new class of therapeutics against Plk1-addicted cancers.

5.
Nat Commun ; 13(1): 5439, 2022 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-36114179

RESUMO

Serine/threonine phosphorylation of insulin receptor substrate (IRS) proteins is well known to modulate insulin signaling. However, the molecular details of this process have mostly been elusive. While exploring the role of phosphoserines, we have detected a direct link between Tyr-flanking Ser/Thr phosphorylation sites and regulation of specific phosphotyrosine phosphatases. Here we present a concise structural study on how the activity of SHP2 phosphatase is controlled by an asymmetric, dual phosphorylation of its substrates. The structure of SHP2 has been determined with three different substrate peptides, unveiling the versatile and highly dynamic nature of substrate recruitment. What is more, the relatively stable pre-catalytic state of SHP2 could potentially be useful for inhibitor design. Our findings not only show an unusual dependence of SHP2 catalytic activity on Ser/Thr phosphorylation sites in IRS1 and CD28, but also suggest a negative regulatory mechanism that may also apply to other tyrosine kinase pathways as well.


Assuntos
Insulina , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Antígenos CD28/metabolismo , Insulina/metabolismo , Proteínas Substratos do Receptor de Insulina/metabolismo , Fosfotirosina , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptor de Insulina/metabolismo , Serina/química , Treonina
6.
Nat Commun ; 11(1): 5769, 2020 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-33188182

RESUMO

Transcription factor phosphorylation at specific sites often activates gene expression, but how environmental cues quantitatively control transcription is not well-understood. Activating protein 1 transcription factors are phosphorylated by mitogen-activated protein kinases (MAPK) in their transactivation domains (TAD) at so-called phosphoswitches, which are a hallmark in response to growth factors, cytokines or stress. We show that the ATF2 TAD is controlled by functionally distinct signaling pathways (JNK and p38) through structurally different MAPK binding sites. Moreover, JNK mediated phosphorylation at an evolutionarily more recent site diminishes p38 binding and made the phosphoswitch differently sensitive to JNK and p38 in vertebrates. Structures of MAPK-TAD complexes and mechanistic modeling of ATF2 TAD phosphorylation in cells suggest that kinase binding motifs and phosphorylation sites line up to maximize MAPK based co-regulation. This study shows how the activity of an ancient transcription controlling phosphoswitch became dependent on the relative flux of upstream signals.


Assuntos
Fator 2 Ativador da Transcrição/metabolismo , Regulação da Expressão Gênica , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Transcrição Gênica , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Fator 2 Ativador da Transcrição/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Células HEK293 , Humanos , Luciferases/metabolismo , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Fosforilação , Ligação Proteica , Dedos de Zinco
7.
Structure ; 28(10): 1101-1113.e5, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-32649858

RESUMO

Mitogen-activated protein kinases (MAPKs) control essential eukaryotic signaling pathways. While much has been learned about MAPK activation, much less is known about substrate recruitment and specificity. MAPK substrates may be other kinases that are crucial to promote a further diversification of the signaling outcomes. Here, we used a variety of molecular and cellular tools to investigate the recruitment of two substrate kinases, RSK1 and MK2, to three MAPKs (ERK2, p38α, and ERK5). Unexpectedly, we identified that kinase heterodimers form structurally and functionally distinct complexes depending on the activation state of the MAPK. These may be incompatible with downstream signaling, but naturally they may also form structures that are compatible with the phosphorylation of the downstream kinase at the activation loop, or alternatively at other allosteric sites. Furthermore, we show that small-molecule inhibitors may affect the quaternary arrangement of kinase heterodimers and thus influence downstream signaling in a specific manner.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/química , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Ativação Enzimática , Células HEK293 , Humanos , Espectroscopia de Ressonância Magnética , Proteína Quinase 1 Ativada por Mitógeno/química , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 14 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 14 Ativada por Mitógeno/química , Proteína Quinase 14 Ativada por Mitógeno/genética , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Proteína Quinase 7 Ativada por Mitógeno/química , Proteína Quinase 7 Ativada por Mitógeno/genética , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Fosforilação , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Multimerização Proteica , Estrutura Quaternária de Proteína , Espalhamento a Baixo Ângulo , Difração de Raios X
8.
Adv Exp Med Biol ; 896: 315-26, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27165334

RESUMO

Signaling complexes within the cell convert extracellular cues into physiological outcomes. Their assembly involves signaling enzymes, allosteric regulators and scaffold proteins that often contain long stretches of disordered protein regions, display multi-domain architectures, and binding affinity between individual components is low. These features are indispensable for their central roles as dynamic information processing hubs, on the other hand they also make reconstruction of structurally homogeneous complex samples highly challenging. In this present chapter we discuss protein machinery which influences extracellular signal reception, intracellular pathway activity, and cytoskeletal or transcriptional activity.


Assuntos
Mapeamento de Interação de Proteínas , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Animais , Sítios de Ligação , Cristalografia por Raios X , Humanos , Modelos Moleculares , Complexos Multiproteicos , Ligação Proteica , Engenharia de Proteínas/métodos , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estrutura Quaternária de Proteína , Subunidades Proteicas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Relação Estrutura-Atividade
9.
Mol Syst Biol ; 11(11): 837, 2015 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-26538579

RESUMO

Mitogen-activated protein kinases (MAPK) are broadly used regulators of cellular signaling. However, how these enzymes can be involved in such a broad spectrum of physiological functions is not understood. Systematic discovery of MAPK networks both experimentally and in silico has been hindered because MAPKs bind to other proteins with low affinity and mostly in less-characterized disordered regions. We used a structurally consistent model on kinase-docking motif interactions to facilitate the discovery of short functional sites in the structurally flexible and functionally under-explored part of the human proteome and applied experimental tools specifically tailored to detect low-affinity protein-protein interactions for their validation in vitro and in cell-based assays. The combined computational and experimental approach enabled the identification of many novel MAPK-docking motifs that were elusive for other large-scale protein-protein interaction screens. The analysis produced an extensive list of independently evolved linear binding motifs from a functionally diverse set of proteins. These all target, with characteristic binding specificity, an ancient protein interaction surface on evolutionarily related but physiologically clearly distinct three MAPKs (JNK, ERK, and p38). This inventory of human protein kinase binding sites was compared with that of other organisms to examine how kinase-mediated partnerships evolved over time. The analysis suggests that most human MAPK-binding motifs are surprisingly new evolutionarily inventions and newly found links highlight (previously hidden) roles of MAPKs. We propose that short MAPK-binding stretches are created in disordered protein segments through a variety of ways and they represent a major resource for ancient signaling enzymes to acquire new regulatory roles.


Assuntos
Proteínas Quinases Ativadas por Mitógeno/química , Proteínas Quinases Ativadas por Mitógeno/ultraestrutura , Estrutura Terciária de Proteína , Sequência de Aminoácidos , Animais , Biologia Computacional , Humanos , Simulação de Acoplamento Molecular , Alinhamento de Sequência , Transdução de Sinais , Propriedades de Superfície
10.
Plant Sci ; 180(1): 61-8, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21421348

RESUMO

The effect of cold acclimation and abscisic acid (ABA) treatment on the free amino acid composition was compared in Chinese Spring chromosome 5A substitution lines with different levels of freezing tolerance. The total amino acid content gradually increased during the 3-week cold acclimation period, while the effect of ABA became visible only after 7 d. The ratio of members of the glutamate family increased during cold acclimation and the ratio of amino acids belonging to the aspartate family decreased. Opposite changes were observed after treatment with ABA. Consistently with these results, ABA only induced a major increase in the Asn content, while the Asp, Glu, Gln and Pro levels were greatly induced by cold. A corresponding alteration at the gene expression level was only found for Pro and Glu. With the exception of Pro, cold- or ABA-induced changes in the amino acid levelsor Pro, did not correlate with the freezing tolerance of the three genotypes examined and were not affected by chromosome 5A. Since cold acclimation induced the accumulation of most of the amino acids, while ABA had a significant effect only on Asn, the cold-induced changes in free amino acid levels were probably not mediated by ABA.


Assuntos
Ácido Abscísico/farmacologia , Aminoácidos/metabolismo , Temperatura Baixa , Triticum/efeitos dos fármacos , Triticum/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa
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