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1.
Cell Mol Life Sci ; 80(4): 112, 2023 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-37004621

RESUMO

Recently, a number of reports on the importance of USP35 in cancer have been published. However, very little is known about the exact mechanism by which USP35 activity is regulated. Here, we show the possible regulation of USP35 activity and the structural specificity affecting its function by analyzing various fragments of USP35. Interestingly, the catalytic domain of USP35 alone does not exhibit deubiquitinating activity; in contrast, the C-terminal domain and insertion region in the catalytic domain is required for full USP35 activity. Additionally, through its C-terminal domain, USP35 forms a homodimer that prevents USP35 degradation. CHIP bound to HSP90 interacts with and ubiquitinates USP35. However, when fully functional USP35 undergoes auto-deubiquitination, which attenuates CHIP-mediated ubiquitination. Finally, USP35 dimer is required for deubiquitination of the substrate Aurora B and regulation of faithful mitotic progression. The properties of USP35 identified in this study are a unique homodimer structure, regulation of deubiquitinating activity through this, and utilization of a novel E3 ligase involved in USP35 auto-deubiquitination, which adds another complexity to the regulation of deubiquitinating enzymes.


Assuntos
Neoplasias , Ubiquitina-Proteína Ligases , Humanos , Ubiquitina-Proteína Ligases/metabolismo , Endopeptidases/genética , Endopeptidases/metabolismo , Ubiquitinação
2.
Int J Mol Sci ; 22(9)2021 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-33946990

RESUMO

Ubiquitination and deubiquitination are protein post-translational modification processes that have been recognized as crucial mediators of many complex cellular networks, including maintaining ubiquitin homeostasis, controlling protein stability, and regulating several signaling pathways. Therefore, some of the enzymes involved in ubiquitination and deubiquitination, particularly E3 ligases and deubiquitinases, have attracted attention for drug discovery. Here, we review recent findings on USP15, one of the deubiquitinases, which regulates diverse signaling pathways by deubiquitinating vital target proteins. Even though several basic previous studies have uncovered the versatile roles of USP15 in different signaling networks, those have not yet been systematically and specifically reviewed, which can provide important information about possible disease markers and clinical applications. This review will provide a comprehensive overview of our current understanding of the regulatory mechanisms of USP15 on different signaling pathways for which dynamic reverse ubiquitination is a key regulator.


Assuntos
Processamento de Proteína Pós-Traducional/fisiologia , Transdução de Sinais/fisiologia , Proteases Específicas de Ubiquitina/fisiologia , Animais , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Proteínas Morfogenéticas Ósseas/fisiologia , Complexo do Signalossomo COP9/fisiologia , Humanos , Imunidade Inata , Masculino , Camundongos , NF-kappa B/metabolismo , Neoplasias da Próstata/metabolismo , Domínios Proteicos , Isoformas de Proteínas , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Precursores de RNA/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Proteases Específicas de Ubiquitina/química , Proteases Específicas de Ubiquitina/genética , Ubiquitinação , Via de Sinalização Wnt/fisiologia , Proteínas de Xenopus/metabolismo
3.
Biochem Biophys Res Commun ; 523(2): 542-547, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-31928715

RESUMO

The MDM2-p53 pathway is crucial for maintenance of p53 homeostasis. Some ribosomal proteins (RPs) play critical roles in regulating p53 by interacting with MDM2. However, the role and functional mechanism of each RP in MDM2-p53 pathway still remain unknown. In this study, we found that Ribosomal Protein S2 (RPS2) is a new regulator of MDM2-P53 signaling pathway to regulate p53 protein level. Here, we characterized that RPS2 interacts with MDM2 through the RING finger domain of MDM2. RPS2 is ubiquitinated by MDM2 and the ubiquitinated status of RPS2 regulates the stability of p53, which is activated in response to cellular stresses such as DNA damage, oxidative stress, and especially ribosomal stress. In addition, p53 is not induced in RPS2 knockdown even in the ribosomal stressed condition, indicating that RPS2 is essential for the stabilization of p53. Collectively, our data suggest that RPS2 plays a critical role in the regulation of p53 signaling including the ribosomal stress response.


Assuntos
Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteínas Ribossômicas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Células HeLa , Humanos , Domínios Proteicos , Estabilidade Proteica , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Ribossômicas/genética , Transdução de Sinais , Estresse Fisiológico , Proteína Supressora de Tumor p53/genética , Ubiquitinação
4.
Int J Mol Sci ; 21(16)2020 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-32781716

RESUMO

Deubiquitinating enzymes regulate various cellular processes, particularly protein degradation, localization, and protein-protein interactions. The dysregulation of deubiquitinating enzyme (DUB) activity has been linked to several diseases; however, the function of many DUBs has not been identified. Therefore, the development of methods to assess DUB activity is important to identify novel DUBs, characterize DUB selectivity, and profile dynamic DUB substrates. Here, we review various methods of evaluating DUB activity using cell lysates or purified DUBs, as well as the types of probes used in these methods. In addition, we introduce some techniques that can deliver DUB probes into the cells and cell-permeable activity-based probes to directly visualize and quantify DUB activity in live cells. This review could contribute to the development of DUB inhibitors by providing important information on the characteristics and applications of various probes used to evaluate and detect DUB activity in vitro and in vivo.


Assuntos
Enzimas Desubiquitinantes/metabolismo , Ensaios Enzimáticos/métodos , Animais , Polarização de Fluorescência , Humanos , Sondas Moleculares/química , Ubiquitina/metabolismo , Ubiquitinação
5.
Int J Mol Sci ; 21(11)2020 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-32512887

RESUMO

Ubiquitination and deubiquitination play a critical role in all aspects of cellular processes, and the enzymes involved are tightly regulated by multiple factors including posttranslational modifications like most other proteins. Dysfunction or misregulation of these enzymes could have dramatic physiological consequences, sometimes leading to diseases. Therefore, it is important to have a clear understanding of these regulatory processes. Here, we have reviewed the posttranslational modifications of deubiquitinating enzymes and their consequences on the catalytic activity, stability, abundance, localization, and interaction with the partner proteins.


Assuntos
Enzimas Desubiquitinantes/metabolismo , Processamento de Proteína Pós-Traducional , Catálise , Humanos , Fosforilação , Transporte Proteico , Ubiquitina/metabolismo , Ubiquitinação
6.
Int J Mol Sci ; 21(24)2020 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-33317068

RESUMO

Inhibition of the molecular chaperone heat shock protein 90 (Hsp90) represents a promising approach for cancer treatment. BIIB021 is a highly potent Hsp90 inhibitor with remarkable anticancer activity; however, its clinical application is limited by lack of potency and response. In this study, we aimed to investigate the impact of replacing the hydrophobic moiety of BIIB021, 4-methoxy-3,5-dimethylpyridine, with various five-membered ring structures on the binding to Hsp90. A focused array of N7/N9-substituted purines, featuring aromatic and non-aromatic rings, was designed, considering the size of hydrophobic pocket B in Hsp90 to obtain insights into their binding modes within the ATP binding site of Hsp90 in terms of π-π stacking interactions in pocket B as well as outer α-helix 4 configurations. The target molecules were synthesized and evaluated for their Hsp90α inhibitory activity in cell-free assays. Among the tested compounds, the isoxazole derivatives 6b and 6c, and the sole six-membered derivative 14 showed favorable Hsp90α inhibitory activity, with IC50 values of 1.76 µM, 0.203 µM, and 1.00 µM, respectively. Furthermore, compound 14 elicited promising anticancer activity against MCF-7, SK-BR-3, and HCT116 cell lines. The X-ray structures of compounds 4b, 6b, 6c, 8, and 14 bound to the N-terminal domain of Hsp90 were determined in order to understand the obtained results and to acquire additional structural insights, which might enable further optimization of BIIB021.


Assuntos
Antineoplásicos/síntese química , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Adenina/análogos & derivados , Adenina/química , Adenina/farmacologia , Animais , Antineoplásicos/farmacologia , Sítios de Ligação , Células HCT116 , Proteínas de Choque Térmico HSP90/química , Humanos , Isoxazóis/química , Células MCF-7 , Camundongos , Ligação Proteica , Piridinas/química , Piridinas/farmacologia , Relação Estrutura-Atividade
7.
J Biol Chem ; 293(3): 847-862, 2018 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-29191835

RESUMO

Elevated expression of human enhancer filamentation 1 (HEF1; also known as NEDD9 or Cas-L) is an essential stimulus for the metastatic process of various solid tumors. This process requires HEF1 localization to focal adhesions (FAs). Although the association of HEF1 with FAs is considered to play a role in cancer cell migration, the mechanism targeting HEF1 to FAs remains unclear. Moreover, up-regulation of Polo-like kinase 1 (Plk1) positively correlates with human cancer metastasis, yet how Plk1 deregulation promotes metastasis remains elusive. Here, we report that casein kinase 1δ (CK1δ) phosphorylates HEF1 at Ser-780 and Thr-804 and that these phosphorylation events promote a physical interaction between Plk1 and HEF1. We found that this interaction is critical for HEF1 translocation to FAs and for inducing migration of HeLa cells. Plk1-docking phosphoepitopes were mapped/confirmed in HEF1 by various methods, including X-ray crystallography, and mutated for functional analysis in HeLa cells. In summary, our results reveal the role of a phosphorylation-dependent HEF1-Plk1 complex in HEF1 translocation to FAs to induce cell migration. Our findings provide critical mechanistic insights into the HEF1-Plk1 complex-dependent localization of HEF1 to FAs underlying the metastatic process and may therefore contribute to the development of new cancer therapies.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Adesões Focais/metabolismo , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular , Proliferação de Células/genética , Proliferação de Células/fisiologia , Adesões Focais/genética , Células HeLa , Humanos , Immunoblotting , Imunoprecipitação , Fosfoproteínas/genética , Fosforilação/genética , Fosforilação/fisiologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Quinase 1 Polo-Like
8.
Nucleic Acids Res ; 45(8): 4866-4880, 2017 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-28088760

RESUMO

Post-translational modifications contribute to the spliceosome dynamics by facilitating the physical rearrangements of the spliceosome. Here, we report USP15, a deubiquitinating enzyme, as a regulator of protein-protein interactions for the spliceosome dynamics. We show that PRP31, a component of U4 snRNP, is modified with K63-linked ubiquitin chains by the PRP19 complex and deubiquitinated by USP15 and its substrate targeting factor SART3. USP15SART3 makes a complex with USP4 and this ternary complex serves as a platform to deubiquitinate PRP31 and PRP3. The ubiquitination and deubiquitination status of PRP31 regulates its interaction with the U5 snRNP component PRP8, which is required for the efficient splicing of chromosome segregation related genes, probably by stabilizing the U4/U6.U5 tri-snRNP complex. Collectively, our data suggest that USP15 plays a key role in the regulation of dynamic protein-protein interactions of the spliceosome.


Assuntos
Antígenos de Neoplasias/genética , Mapas de Interação de Proteínas/genética , Proteínas de Ligação a RNA/genética , Proteases Específicas de Ubiquitina/genética , Enzimas Reparadoras do DNA/genética , Células HeLa , Humanos , Complexos Multiproteicos/genética , Proteínas Nucleares/genética , Processamento de Proteína Pós-Traducional , Fatores de Processamento de RNA/genética , Ribonucleoproteína Nuclear Pequena U4-U6/genética , Spliceossomos/genética , Ubiquitinação/genética
9.
Int J Mol Sci ; 20(23)2019 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-31795161

RESUMO

Mitosis is a complex and dynamic process that is tightly regulated by a large number of mitotic proteins. Dysregulation of these proteins can generate daughter cells that exhibit genomic instability and aneuploidy, and such cells can transform into tumorigenic cells. Thus, it is important for faithful mitotic progression to regulate mitotic proteins at specific locations in the cells at a given time in each phase of mitosis. Ubiquitin-dependent modifications play critical roles in this process by regulating the degradation, translocation, or signal transduction of mitotic proteins. Here, we review how ubiquitination and deubiquitination regulate the progression of mitosis. In addition, we summarize the substrates and roles of some deubiquitinating enzymes (DUBs) crucial for mitosis and describe how they contribute error correction during mitosis and control the transition between the mitotic phases.


Assuntos
Enzimas Desubiquitinantes/metabolismo , Mitose , Animais , Humanos , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
10.
Nucleic Acids Res ; 44(11): 5424-37, 2016 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-27060135

RESUMO

Squamous cell carcinoma antigen recognized by T-cells 3 (SART3) is a U4/U6 recycling factor as well as a targeting factor of USP4 and USP15. However, the details of how SART3 recognizes these deubiquitinases and how they get subsequently translocated into the nucleus are not known. Here, we present the crystal structures of the SART3 half-a-tetratricopeptide (HAT) repeat domain alone and in complex with the domain present in ubiquitin-specific protease (DUSP)-ubiquitin-like (UBL) domains of ubiquitin specific protease 4 (USP4). The 12 HAT repeats of SART3 are in two sub-domains (HAT-N and HAT-C) forming a dimer through HAT-C. USP4 binds SART3 at the opposite surface of the HAT-C dimer interface utilizing the ß-structured linker between the DUSP and the UBL domains. The binding affinities of USP4 and USP15 to SART3 are 0.9 µM and 0.2 µM, respectively. The complex structure of SART3 nuclear localization signal (NLS) and importin-α reveals bipartite binding, and removal of SART3 NLS prevents the entry of USP4 (and USP15) into the nucleus and abrogates the subsequent deubiquitinase activity of USP4.


Assuntos
Antígenos de Neoplasias/química , Antígenos de Neoplasias/metabolismo , Modelos Moleculares , Conformação Proteica , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Spliceossomos/metabolismo , Ubiquitina Tiolesterase/química , Ubiquitina Tiolesterase/metabolismo , Sequência de Aminoácidos , Antígenos de Neoplasias/genética , Cristalografia por Raios X , Humanos , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Sinais de Localização Nuclear/química , Sinais de Localização Nuclear/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas de Ligação a RNA/genética , Relação Estrutura-Atividade , Sequências de Repetição em Tandem , Ubiquitina Tiolesterase/genética , Proteases Específicas de Ubiquitina/metabolismo , alfa Carioferinas/química , alfa Carioferinas/metabolismo
11.
Anal Chem ; 89(4): 2390-2397, 2017 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-28192940

RESUMO

We employed modified glass nanocapillaries to investigate interactions between the RNA-binding protein, known as cell carcinoma antigen recognized by T cells-3 (SART3), and the noncoding spliceosome component, U6 small nuclear RNA (snRNA), at the single-molecule level. We functionalized the nanocapillaries with U6 snRNA fragments, which were hybridized to DNA molecules and then covalently attached to the nanocapillary surface. When transported through the modified nanocapillaries, two different SART3-derived constructs, HAT-RRM1-RRM2 and RRM1-RRM2, exhibited resistive ionic current pulses with different dwell times, which represented their different binding affinities to tethered U6 snRNAs. The dissociation constants (KD), estimated from the bias voltage dependence of translocation events, were approximately 1.9 µM and 201 µM for HAT-RRM1-RRM2 and RRM1-RRM2, respectively. These values were comparable to corresponding values obtained with isothermal titration calorimetry, demonstrating that the modified glass nanocapillaries are applicable to analyses of protein-ligand interactions at the single-molecule level.


Assuntos
Antígenos de Neoplasias/metabolismo , Calorimetria , Nanotubos/química , RNA Nuclear Pequeno/metabolismo , Proteínas de Ligação a RNA/metabolismo , Antígenos de Neoplasias/química , Eletricidade , Humanos , Concentração de Íons de Hidrogênio , Cinética , Peptídeos/química , Peptídeos/metabolismo , Proteínas de Ligação a RNA/química
12.
Biochem Biophys Res Commun ; 482(4): 968-974, 2017 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-27908729

RESUMO

TM0800 from Thermotoga maritima is one of the hypothetical proteins with unknown function. The crystal structure determined at 2.3 Å resolution reveals a two domain structure: the N-terminal domain forming a barrel and the C-terminal forming a lid. One FMN is bound between the two domains with the phosphate making intricate hydrogen bonds with protein and three tightly bound water molecules, and the isoalloxazine ring packed against the side chains of Met22 and Met276. The structure is almost identical to that of FabK (enoyl-acyl carrier protein (ACP) reductase, ENR II), a key enzyme in bacterial type II fatty-acid biosynthesis that catalyzes the final step in each elongation cycle; and the enzymatic activity confirms that TM0800 is an ENR. Enzymatic activity was almost completely abolished when the helices connecting the barrel and the lid were deleted. Also, the Met276Ala and Ser280Ala mutants showed a significant reduction in enzymatic activity. The crystal structure of Met276Ala mutant at 1.9 Å resolution showed an absence of FMN suggesting that FMN plays a role in catalysis, and Met276 is important in positioning FMN. TmFabK exists as a dimer in both solution and crystal. Together this study provides molecular basis for the catalytic activity of FabK.


Assuntos
Proteínas de Bactérias/química , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/química , Thermotoga maritima/química , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/metabolismo , Modelos Moleculares , Conformação Proteica , Multimerização Proteica , Thermotoga maritima/metabolismo
13.
Biochem Biophys Res Commun ; 494(3-4): 452-459, 2017 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-29061303

RESUMO

Evogliptin ((R)-4-((R)-3-amino-4-(2,4,5-trifluorophenyl)butanoyl)-3-(tert-butoxymethyl) piperazine-2-one)) is a highly potent selective inhibitor of dipeptidyl peptidase IV (DPP4) that was approved for the treatment of type 2 diabetes in South Korea. In this study, we report the crystal structures of Evogliptin, DA-12166, and DA-12228 (S,R diastereomer of Evogliptin) complexed to human DPP4. Analysis of both the structures and inhibitory activities suggests that the binding of the trifluorophenyl moiety in the S1 pocket and the piperazine-2-one moiety have hydrophobic interactions with Phe357 in the S2 extensive subsite, and that the multiple hydrogen bonds made by the (R)-ß-amine group in the S2 pocket and the contacts made by the (R)-tert-butyl group with Arg125 contribute to the high potency observed for Evogliptin.


Assuntos
Dipeptidil Peptidase 4/química , Inibidores da Dipeptidil Peptidase IV/química , Modelos Químicos , Modelos Moleculares , Piperazinas/química , Sítios de Ligação , Ativação Enzimática , Humanos , Ligação Proteica
14.
Mol Carcinog ; 56(8): 1992-1998, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28370334

RESUMO

The transforming growth factor-ß (TGF-ß) signaling pathway is associated with carcinogenesis and various biological processes. SMAD2 and SMAD4, which are putative tumor suppressors, have an important role in TGF-ß signaling. The aberrant expression of these genes is implicated in some cancers. However, the mechanisms of SMAD2 and SMAD4 dysregulation are poorly understood. In this study, we observed that miR-27a was upregulated in lung cancer cell lines and patients. In addition, SMAD2 and SMAD4 genes were identified as targets of miR-27a by several target prediction databases and experimental validation. Functional studies revealed that miR-27a overexpression decreased SMAD2 and SMAD4 mRNA and protein levels. Furthermore, miR-27a contributed to cell proliferation and invasion by inhibiting TGF-ß-induced cell cycle arrest. These results suggest that miR-27a may function as an oncogene by regulating SMAD2 and SMAD4 in lung cancer. Thus, miR-27a may be a potential target for cancer therapy.


Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , MicroRNAs/genética , Proteína Smad2/genética , Proteína Smad4/genética , Fator de Crescimento Transformador beta/metabolismo , Ciclo Celular , Linhagem Celular Tumoral , Humanos , Pulmão/metabolismo , Pulmão/patologia , Neoplasias Pulmonares/metabolismo , MicroRNAs/metabolismo , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Transdução de Sinais
15.
Bioorg Med Chem Lett ; 27(2): 237-241, 2017 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-27914802

RESUMO

A novel series of heat shock protein 90 (Hsp90) inhibitors was identified by X-ray crystal analysis of complex structures at solvent-exposed exit pocket C. The 2-amino-pyrrolo[2,3-d]pyrimidine derivatives, 7-deazapurines substituted with a benzyl moiety at C5, showed potent Hsp90 inhibition and broad-spectrum antiproliferative activity against NCI-60 cancer cell lines. The most potent compound, 6a, inhibited Hsp90 with an IC50 of 36nM and showed a submicromolar mean GI50 value against NCI-60 cell lines. The interaction of 6a at the ATP-binding pocket of Hsp90 was confirmed by X-ray crystallography and Western blot analysis.


Assuntos
Antineoplásicos/farmacologia , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Pirimidinas/farmacologia , Pirróis/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Modelos Moleculares , Estrutura Molecular , Pirimidinas/síntese química , Pirimidinas/química , Pirróis/síntese química , Pirróis/química , Relação Estrutura-Atividade
16.
Biochem Biophys Res Commun ; 477(4): 717-722, 2016 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-27353378

RESUMO

Peroxiredoxins (Prxs) are a family of antioxidant enzymes found ubiquitously. Prxs function not only as H2O2 scavengers but also as highly sensitive H2O2 sensors and signal transducers. Since reactive oxygen species are involved in many cellular metabolic and signaling processes, Prxs play important roles in various diseases. Prxs can be hyperoxidized to the sulfinic acid (SO2H) or sulfonic acid (SO3H) forms in the presence of high concentrations of H2O2. It is known that oligomerization of Prx is changed accompanying oxidation states, and linked to the function. Among the six Prxs in mammals, Prx6 is the only 1-Cys Prx. It is found in all organs in humans, unlike some 2-Cys Prxs, and is present in all species from bacteria to humans. In addition, Prx6 has Ca(2+)-independent phospholipase A2 (PLA2) activity. Thus far only the crystal structure of Prx in the oxidized state has been reported. In this study, we present the crystal structures of human Prx6 in the reduced (SH) and the sulfinic acid (SO2H) forms.


Assuntos
Cisteína/química , Oxigênio/química , Peroxirredoxina VI/química , Peroxirredoxina VI/ultraestrutura , Sítios de Ligação , Dimerização , Ativação Enzimática , Humanos , Oxirredução , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade
17.
Mol Pharm ; 13(4): 1197-205, 2016 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-26886576

RESUMO

LB30870, a new direct thrombin inhibitor, showed 80% reduction in oral bioavailability in fed state. The present study aims to propose trypsin binding as a mechanism for such negative food effect and demonstrate a prodrug approach to mitigate food effect. Effect of food composition on fed state oral bioavailability of LB30870 was studied in dogs. Various prodrugs were synthesized, and their solubility, permeability, and trypsin binding affinity were measured. LB30870 and prodrugs were subject to cocrystallization with trypsin, and the X-ray structures of cocrystals were determined. Food effect was studied in dogs for selected prodrugs. Protein or lipid meal appeared to affect oral bioavailability of LB30870 in dogs more than carbohydrate meal. Blocking both carboxyl and amidine groups of LB30870 resulted in trypsin Ki values orders of magnitude higher than that of LB30870. Prodrugs belonged to either Biopharmaceutical Classification System I, II, or III. X-ray crystallography revealed that prodrugs did not bind to trypsin, but instead their hydrolysis product at the amidine blocking group formed cocrystal with trypsin. A prodrug with significantly less food effect than LB30870 was identified. Binding of prodrugs to food components such as dietary fiber appeared to counteract the positive effect brought with the prodrug approach. Further formulation research is warranted to enhance the oral bioavailability of prodrugs. In conclusion, this study is the first to demonstrate that the negative food effect of LB30870 can be attributed to trypsin binding. Trypsin binding study is proposed as a screening tool during lead optimization to minimize food effect.


Assuntos
Antitrombinas/química , Pró-Fármacos/química , Trombina/antagonistas & inibidores , Amidinas/química , Animais , Anticoagulantes/química , Células CACO-2 , Cristalografia por Raios X , Dipeptídeos/química , Cães , Estabilidade de Medicamentos , Fluoracetatos , Humanos , Solubilidade
18.
Bioorg Med Chem Lett ; 26(14): 3307-3312, 2016 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-27241691

RESUMO

A series of new 2-anilinoquinolines possessing 3-(morpholino or 4-methylpiperazin-1-yl)propoxy moiety at C5 of quinoline has been designed and synthesized as potential anticancer agents. Their antiproliferative activities were evaluated against a panel of 60 cancer cell lines at NCI and compared with gefitinib as a reference compound. Most of the tested compounds displayed potent and broad spectrum antiproliferative activities. Compounds 7d, 7f and 7g showed strong inhibitory and lethal effects at 10µM concentration. Moreover, they manifested superior potencies and efficacies than gefitinib across the most tested cell lines. Compound 7d, with 4-chloro-3-trifluoromethylphenyl group, proved to be the most potent and efficacious derivative in this series, with mean GI50 and TGI values of 1.62µM and 3.47µM, respectively. Kinase screening of 7d against a panel of 47 oncogenic kinases revealed its selective inhibitory effect (96% inhibition) towards TrkA kinase. Furthermore, the most potent compounds showed low cytotoxic effects against HFF-1 normal cell line.


Assuntos
Antineoplásicos/farmacologia , Morfolinos/farmacologia , Naftalenos/farmacologia , Fosfotransferases/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Quinolinas/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura Molecular , Morfolinos/química , Naftalenos/química , Fosfotransferases/metabolismo , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Quinolinas/química , Relação Estrutura-Atividade
19.
Proc Natl Acad Sci U S A ; 110(48): 19360-5, 2013 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-24218616

RESUMO

Protein kinase CK2 is a ubiquitous kinase that can phosphorylate hundreds of cellular proteins and plays important roles in cell growth and development. Deregulation of CK2 is related to a variety of human cancers, and CK2 is regarded as a suppressor of apoptosis; therefore, it is a target of anticancer therapy. Nucleolar phosphoprotein 140 (Nopp140), which is an intrinsically disordered protein, interacts with CK2 and inhibits the latter's catalytic activity in vitro. Interestingly, the catalytic activity of CK2 is recovered in the presence of d-myo-inositol 1,2,3,4,5,6-hexakisphosphate (IP6). IP6 is widely distributed in animal cells, but the molecular mechanisms that govern its cellular functions in animal cells have not been completely elucidated. In this study, the crystal structure of CK2 in complex with IP6 showed that the lysine-rich cluster of CK2 plays an important role in binding to IP6. The biochemical experiments revealed that a Nopp140 fragment (residues 568-596) and IP6 competitively bind to the catalytic subunit of CK2 (CK2α), and phospho-Ser574 of Nopp140 significantly enhances its interaction with CK2α. Substitutions of K74E, K76E, and K77E in CK2α significantly reduced the interactions of CK2α with both IP6 and the Nopp140-derived peptide. Our study gives an insight into the regulation of CK2. In particular, our work suggests that CK2 activity is inhibited by Nopp140 and reactivated by IP6 by competitive binding at the substrate recognition site of CK2.


Assuntos
Caseína Quinase II/química , Caseína Quinase II/metabolismo , Regulação da Expressão Gênica/fisiologia , Substâncias Macromoleculares/química , Modelos Moleculares , Proteínas Nucleares/química , Fosfoproteínas/química , Ácido Fítico/química , Substituição de Aminoácidos , Cristalização , Humanos , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Ácido Fítico/metabolismo , Conformação Proteica , Difração de Raios X
20.
J Biol Chem ; 289(18): 12264-74, 2014 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-24610782

RESUMO

Valosin-containing protein (VCP), also known as p97, is an AAA(+) ATPase that plays an essential role in a broad array of cellular processes including the endoplasmic reticulum-associated degradation (ERAD) pathway. Recently, ERAD-specific deubiquitinating enzymes have been reported to be physically associated with VCP, although the exact mechanism is not yet clear. Among these enzymes is ovarian tumor domain-containing protein 1 (OTU1). Here, we report the structural basis for interaction between VCP and OTU1. The crystal structure of the ubiquitin regulatory X-like (UBXL) domain of OTU1 (UBXLOTU1) complexed to the N-terminal domain of VCP (NVCP) at 1.8-Å resolution reveals that UBXLOTU1 adopts a ubiquitin-like fold and binds at the interface of two subdomains of NVCP using the (39)GYPP(42) loop of UBXLOTU1 with the two prolines in cis- and trans-configurations, respectively. A mutagenesis study shows that this loop is not only critical for the interaction with VCP but also for its role in the ERAD pathway. Negative staining EM shows that one molecule of OTU1 binds to one VCP hexamer, and isothermal titration calorimetry suggests that the two proteins bind with a KD of 0.71 µM. Analytical size exclusion chromatography and isothermal titration calorimetry demonstrates that OTU1 can bind VCP in both the presence and absence of a heterodimer formed by ubiquitin fusion degradation protein 1 and nuclear localization protein 4.


Assuntos
Adenosina Trifosfatases/química , Proteínas de Ciclo Celular/química , Estrutura Terciária de Proteína , Proteínas de Saccharomyces cerevisiae/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Sítios de Ligação/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Degradação Associada com o Retículo Endoplasmático/genética , Células HEK293 , Humanos , Immunoblotting , Microscopia Eletrônica , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Mutação , Ligação Proteica , Mapeamento de Interação de Proteínas , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais/genética , Proteína com Valosina
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