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1.
Anal Biochem ; 555: 67-72, 2018 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-29733811

RESUMO

Neddylation is a posttranslational modification that regulates protein stability, activity, and subcellular localization. Here, we describe a new tool for exploring the neddylation cycle of cullin1 (Cul1) directly in a cellular context. This assay utilizes the NanoLuc® Binary Technology (NanoBiT) to monitor the covalent neddylation status of Cul1. A stable clonal cell line derived from HEK293 was developed that expressed a C-terminus LgBiT tagged-Cul1 and N-terminus SmBiT tagged-Nedd8. Using this cell line, we screened inhibitors that are known to disrupt Nedd8 biology and demonstrated that both inhibitors of Nedd8-activating enzyme (NAE) and Constitutive photomorphogenesis 9 signalosome (CSN) complex produce concentration and time dependent signal decreases and increases, respectively. The kinetics of both responses could be monitored in real time and demonstrated that modulation of the Nedd8 pathway occurs rapidly. Further characterization of the cellular components of this cell line was performed in order to quantify the various levels of Cul1, Nedd8 and NAE and determined to be near endogenous levels. There was no difference between control and stably transfected cell lines in viability studies of NAE and CSN inhibitors. Taken together, these results suggest that the NanoBiT assay can be used to monitor Cul1 neddylation specifically and in real time.


Assuntos
Bioensaio/métodos , Proteínas Culina/metabolismo , Proteína NEDD8/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Culina/genética , Células HCT116 , Células HEK293 , Humanos , Proteína NEDD8/genética
2.
J Biol Chem ; 289(33): 22648-22658, 2014 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-24966333

RESUMO

E1 enzymes activate ubiquitin or ubiquitin-like proteins (Ubl) via an adenylate intermediate and initiate the enzymatic cascade of Ubl conjugation to target proteins or lipids. Ubiquitin-fold modifier 1 (Ufm1) is activated by the E1 enzyme Uba5, and this pathway is proposed to play an important role in the endoplasmic reticulum (ER) stress response. However, the mechanisms of Ufm1 activation by Uba5 and subsequent transfer to the conjugating enzyme (E2), Ufc1, have not been studied in detail. In this work, we found that Uba5 activated Ufm1 via a two-step mechanism and formed a binary covalent complex of Uba5∼Ufm1 thioester. This feature contrasts with the three-step mechanism and ternary complex formation in ubiquitin-activating enzyme Uba1. Uba5 displayed random ordered binding with Ufm1 and ATP, and its ATP-pyrophosphate (PPi) exchange activity was inhibited by both AMP and PPi. Ufm1 activation and Uba5∼Ufm1 thioester formation were stimulated in the presence of Ufc1. Furthermore, binding of ATP to Uba5∼Ufm1 thioester was required for efficient transfer of Ufm1 from Uba5 to Ufc1 via transthiolation. Consistent with the two-step activation mechanism, the mechanism-based pan-E1 inhibitor, adenosine 5'-sulfamate (ADS), reacted with the Uba5∼Ufm1 thioester and formed a covalent, tight-binding Ufm1-ADS adduct in the active site of Uba5, which prevented further substrate binding or catalysis. ADS was also shown to inhibit the Uba5 conjugation pathway in the HCT116 cells through formation of the Ufm1-ADS adduct. This suggests that further development of more selective Uba5 inhibitors could be useful in interrogating the roles of the Uba5 pathway in cells.


Assuntos
Complexos Multiproteicos , Proteínas , Enzimas Ativadoras de Ubiquitina , Trifosfato de Adenosina/química , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Domínio Catalítico , Linhagem Celular , Ativação Enzimática , Humanos , Modelos Químicos , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Ligação Proteica , Estrutura Quaternária de Proteína , Proteínas/química , Proteínas/genética , Proteínas/metabolismo , Enzimas Ativadoras de Ubiquitina/química , Enzimas Ativadoras de Ubiquitina/genética , Enzimas Ativadoras de Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo
3.
Cell Biochem Biophys ; 67(1): 139-47, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23754621

RESUMO

Ubiquitin (Ub) and ubiquitin-like (Ubl) proteins regulate a variety of important cellular processes by forming covalent conjugates with target proteins or lipids. Ubl conjugation is catalyzed by a cascade of proteins including activating enzymes (E1), conjugating enzymes (E2), and in many cases ligation enzymes (E3). The discovery of MLN4924 (Brownell et al., Mol Cell 37: 102-111, 1), an investigational small molecule that is a mechanism-based inhibitor of NEDD8-activating enzyme (NAE), reveals a promising strategy of targeting E1/Ubl pathway for therapeutic purposes. In order to better understand, the biochemical dynamics of Ubl conjugation in cells and tissues, we have developed a mass spectrometry-based method to quantify E1 and Ubls using isotope-labeled proteins as internal standards. Furthermore, we have used the described method to quantify levels of the covalent Nedd8-inhibitor adduct formed in MLN4924 treated cells and tissues. The Nedd8-MLN4924 adduct is a tight-binding inhibitor of NAE, and its cellular concentration represents an indirect pharmacodynamic readout of NAE/Nedd8 pathway inhibition.


Assuntos
Ciclopentanos/química , Pirimidinas/química , Enzimas Ativadoras de Ubiquitina/química , Ubiquitinas/química , Animais , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Feminino , Células HCT116 , Células HeLa , Humanos , Marcação por Isótopo , Proteína NEDD8 , Nanotecnologia , Peptídeos/análise , Ratos , Ratos Nus , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Espectrometria de Massas em Tandem , Ubiquitina/química , Ubiquitina/metabolismo , Enzimas Ativadoras de Ubiquitina/genética , Enzimas Ativadoras de Ubiquitina/metabolismo , Ubiquitinas/metabolismo
4.
J Biol Chem ; 287(19): 15512-22, 2012 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-22427669

RESUMO

Uba6 is a homolog of the ubiquitin-activating enzyme, Uba1, and activates two ubiquitin-like proteins (UBLs), ubiquitin and FAT10. In this study, biochemical and biophysical experiments were performed to understand the mechanisms of how Uba6 recognizes two distinct UBLs and catalyzes their activation and transfer. Uba6 is shown to undergo a three-step activation process and form a ternary complex with both UBLs, similar to what has been observed for Uba1. The catalytic mechanism of Uba6 is further supported by inhibition studies using a mechanism-based E1 inhibitor, Compound 1, which forms covalent adducts with both ubiquitin and FAT10. In addition, pre-steady state kinetic analysis revealed that the rates of UBL-adenylate (step 1) and thioester (step 2) formation are similar between ubiquitin and FAT10. However, distinct kinetic behaviors were also observed for ubiquitin and FAT10. FAT10 binds Uba6 with much higher affinity than ubiquitin while demonstrating lower catalytic activity in both ATP-PP(i) exchange and E1-E2 transthiolation assays. Also, Compound 1 is less potent with FAT10 as the UBL compared with ubiquitin in ATP-PP(i) exchange assays, and both a slow rate of covalent adduct formation and weak adduct binding to Uba6 contribute to the diminished potency observed for FAT10. Together with expression level analysis in IM-9 cells, this study sheds light on the potential role of cytokine-induced FAT10 expression in regulating Uba6 pathways.


Assuntos
Trifosfato de Adenosina/metabolismo , Difosfatos/metabolismo , Enzimas Ativadoras de Ubiquitina/metabolismo , Ubiquitinas/metabolismo , Monofosfato de Adenosina/química , Monofosfato de Adenosina/metabolismo , Monofosfato de Adenosina/farmacologia , Sequência de Aminoácidos , Animais , Western Blotting , Linhagem Celular , Ativação Enzimática/efeitos dos fármacos , Humanos , Interferon gama/farmacologia , Cinética , Espectrometria de Massas , Dados de Sequência Molecular , Estrutura Molecular , Ligação Proteica/efeitos dos fármacos , Spodoptera , Especificidade por Substrato , Compostos de Sulfidrila/química , Compostos de Sulfidrila/metabolismo , Ressonância de Plasmônio de Superfície , Fator de Necrose Tumoral alfa/farmacologia , Enzimas Ativadoras de Ubiquitina/química , Enzimas Ativadoras de Ubiquitina/genética , Ubiquitinas/química , Ubiquitinas/genética
5.
J Biol Chem ; 286(47): 40867-77, 2011 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-21969368

RESUMO

Ubiquitin-activating enzyme (UAE or E1) activates ubiquitin via an adenylate intermediate and catalyzes its transfer to a ubiquitin-conjugating enzyme (E2). MLN4924 is an adenosine sulfamate analogue that was identified as a selective, mechanism-based inhibitor of NEDD8-activating enzyme (NAE), another E1 enzyme, by forming a NEDD8-MLN4924 adduct that tightly binds at the active site of NAE, a novel mechanism termed substrate-assisted inhibition (Brownell, J. E., Sintchak, M. D., Gavin, J. M., Liao, H., Bruzzese, F. J., Bump, N. J., Soucy, T. A., Milhollen, M. A., Yang, X., Burkhardt, A. L., Ma, J., Loke, H. K., Lingaraj, T., Wu, D., Hamman, K. B., Spelman, J. J., Cullis, C. A., Langston, S. P., Vyskocil, S., Sells, T. B., Mallender, W. D., Visiers, I., Li, P., Claiborne, C. F., Rolfe, M., Bolen, J. B., and Dick, L. R. (2010) Mol. Cell 37, 102-111). In the present study, substrate-assisted inhibition of human UAE (Ube1) by another adenosine sulfamate analogue, 5'-O-sulfamoyl-N(6)-[(1S)-2,3-dihydro-1H-inden-1-yl]-adenosine (Compound I), a nonselective E1 inhibitor, was characterized. Compound I inhibited UAE-dependent ATP-PP(i) exchange activity, caused loss of UAE thioester, and inhibited E1-E2 transthiolation in a dose-dependent manner. Mechanistic studies on Compound I and its purified ubiquitin adduct demonstrate that the proposed substrate-assisted inhibition via covalent adduct formation is entirely consistent with the three-step ubiquitin activation process and that the adduct is formed via nucleophilic attack of UAE thioester by the sulfamate group of Compound I after completion of step 2. Kinetic and affinity analysis of Compound I, MLN4924, and their purified ubiquitin adducts suggest that both the rate of adduct formation and the affinity between the adduct and E1 contribute to the overall potency. Because all E1s are thought to use a similar mechanism to activate their cognate ubiquitin-like proteins, the substrate-assisted inhibition by adenosine sulfamate analogues represents a promising strategy to develop potent and selective E1 inhibitors that can modulate diverse biological pathways.


Assuntos
Inibidores Enzimáticos/farmacologia , Ácidos Sulfônicos/farmacologia , Enzimas Ativadoras de Ubiquitina/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Animais , Bovinos , Difosfatos/metabolismo , Humanos , Hidrólise/efeitos dos fármacos , Cinética , Compostos de Sulfidrila/metabolismo , Enzimas Ativadoras de Ubiquitina/metabolismo
6.
Mol Cell ; 37(1): 102-11, 2010 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-20129059

RESUMO

The NEDD8-activating enzyme (NAE) initiates a protein homeostatic pathway essential for cancer cell growth and survival. MLN4924 is a selective inhibitor of NAE currently in clinical trials for the treatment of cancer. Here, we show that MLN4924 is a mechanism-based inhibitor of NAE and creates a covalent NEDD8-MLN4924 adduct catalyzed by the enzyme. The NEDD8-MLN4924 adduct resembles NEDD8 adenylate, the first intermediate in the NAE reaction cycle, but cannot be further utilized in subsequent intraenzyme reactions. The stability of the NEDD8-MLN4924 adduct within the NAE active site blocks enzyme activity, thereby accounting for the potent inhibition of the NEDD8 pathway by MLN4924. Importantly, we have determined that compounds resembling MLN4924 demonstrate the ability to form analogous adducts with other ubiquitin-like proteins (UBLs) catalyzed by their cognate-activating enzymes. These findings reveal insights into the mechanism of E1s and suggest a general strategy for selective inhibition of UBL conjugation pathways.


Assuntos
Monofosfato de Adenosina/metabolismo , Ciclopentanos/metabolismo , Inibidores Enzimáticos/metabolismo , Pirimidinas/metabolismo , Ubiquitinas/metabolismo , Monofosfato de Adenosina/química , Sítios de Ligação , Ligação Competitiva , Linhagem Celular Tumoral , Cristalografia por Raios X , Ciclopentanos/química , Ciclopentanos/farmacologia , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Proteína NEDD8 , Estrutura Terciária de Proteína , Pirimidinas/química , Pirimidinas/farmacologia , Ubiquitinas/química
7.
Assay Drug Dev Technol ; 4(6): 661-9, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17199504

RESUMO

Adenine phosphoribosyltransferase plays a role in purine salvage by catalyzing the direct conversion of adenine to adenosine monophosphate. The involvement of the purine salvage pathway in tumor proliferation and angiogenesis makes adenine phosphoribosyltransferase a potential target for oncology drug discovery. We have expressed and characterized recombinant, N-terminally His-tagged human adenine phosphoribosyltransferase. Two assay formats were assessed for use in a high throughput screen: a spectrophotometric-based enzyme-coupled assay system and a radiometric ionic capture scintillation proximity bead assay format. Ultimately, the scintillation proximity assay format was chosen because of automated screening compatibility limitations of the coupled assay. We describe here the biochemical characterization of adenine phosphoribosyltransferase and the development of a robust, homogeneous, 384-well assay suitable for high throughput screening.


Assuntos
Adenina Fosforribosiltransferase/metabolismo , Contagem de Cintilação/métodos , Adenina/metabolismo , Adenina Fosforribosiltransferase/antagonistas & inibidores , Adenilato Quinase/metabolismo , Humanos , L-Lactato Desidrogenase/metabolismo , NAD/metabolismo , Piruvato Quinase/metabolismo , Proteínas Recombinantes/metabolismo , Trítio
8.
Assay Drug Dev Technol ; 3(5): 533-41, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16305310

RESUMO

NAD synthetase is responsible for the conversion of nicotinic acid adenine dinucleotide to nicotinamide adenine dinucleotide. This reaction provides a biosynthetic route of the coenzyme and, thus, a source of cellular reducing equivalents. Alterations in the oxidative reductive potential of the cell have been implicated as a contributing factor in many disease states. Thus, this enzyme represents a new class of potential drug targets, and, hence, our efforts were focused upon developing a robust assay for utilization in a high throughput screen. Toward that end, we describe a coupled enzyme assay format for the measurement of recombinant human NAD synthetase by employing lactate dehydrogenase in a cycling/amplification reaction linked ultimately to the fluorescence generation of resorufin from resazurin via diaphorase. We present kinetics of the reaction of NAD synthetase in the coupled assay format, optimization conditions, and inhibition of the reaction by gossypol [1,1',6,6',7,7'-hexahydroxy-3,3'-dimethyl-5,5'-bis(1-methylethyl)-[2,2'- binaphthalene]-8,8'-dicarboxaldehyde] and illustrate the robustness of the assay by demonstrating 384-well microtiter plate uniformity statistics. Collectively, our results show that the assay method is both robust and well suited for this class of enzymes involved in the NAD+ biosynthetic pathway.


Assuntos
Amida Sintases/análise , Amida Sintases/química , Bioensaio/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Proteínas Recombinantes/análise , Proteínas Recombinantes/química , Espectrometria de Fluorescência/métodos , Amida Sintases/genética , Ativação Enzimática , Corantes Fluorescentes , Humanos , Engenharia de Proteínas/métodos , Coloração e Rotulagem/métodos
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