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1.
J Biol Chem ; 290(18): 11601-10, 2015 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-25771539

RESUMO

The protein N-terminal methyltransferase 1 (NTMT1) catalyzes the transfer of the methyl group from the S-adenosyl-l-methionine to the protein α-amine, resulting in formation of S-adenosyl-l-homocysteine and α-N-methylated proteins. NTMT1 is an interesting potential anticancer target because it is overexpressed in gastrointestinal cancers and plays an important role in cell mitosis. To gain insight into the biochemical mechanism of NTMT1, we have characterized the kinetic mechanism of recombinant NTMT1 using a fluorescence assay and mass spectrometry. The results of initial velocity, product, and dead-end inhibition studies indicate that methylation by NTMT1 proceeds via a random sequential Bi Bi mechanism. In addition, our processivity studies demonstrate that NTMT1 proceeds via a distributive mechanism for multiple methylations. Together, our studies provide new knowledge about the kinetic mechanism of NTMT1 and lay the foundation for the development of mechanism-based inhibitors.


Assuntos
Proteínas Metiltransferases/metabolismo , Sequência de Aminoácidos , Biocatálise , Inibidores Enzimáticos/farmacologia , Humanos , Cinética , Metilação , Proteínas Metiltransferases/antagonistas & inibidores , Proteínas Metiltransferases/química
2.
Anal Biochem ; 478: 59-64, 2015 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-25778392

RESUMO

Protein methylation and acetylation play important roles in biological processes, and misregulation of these modifications is involved in various diseases. Therefore, it is critical to understand the activities of the enzymes responsible for these modifications. Herein we describe a sensitive method for ratiometric quantification of methylated and acetylated peptides via MALDI-MS by direct spotting of enzymatic methylation and acetylation reaction mixtures without tedious purification procedures. The quantifiable detection limit for peptides with our method is approximately 10 fmol. This is achieved by increasing the signal-to-noise ratio through the addition of NH4H2PO4 to the matrix solution and reduction of the matrix α-cyanohydroxycinnamic acid concentration to 2 mg/ml. We have demonstrated the application of this method in enzyme kinetic analysis and inhibition studies. The unique feature of this method is the simultaneous quantification of multiple peptide species for investigation of processivity mechanisms. Its wide buffer compatibility makes it possible to be adapted to investigate the activity of any protein methyltransferase or acetyltransferase.


Assuntos
Acetiltransferases/metabolismo , Peptídeos/metabolismo , Proteínas Metiltransferases/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Acetilação , Sequência de Aminoácidos , Humanos , Cinética , Metilação , Peptídeos/análise
3.
J Am Chem Soc ; 136(17): 6355-61, 2014 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-24702247

RESUMO

A working hypothesis for the pathogenesis of myotonic dystrophy type 1 (DM1) involves the aberrant sequestration of an alternative splicing regulator, MBNL1, by expanded CUG repeats, r(CUG)(exp). It has been suggested that a reversal of the myotonia and potentially other symptoms of the DM1 disease can be achieved by inhibiting the toxic MBNL1-r(CUG)(exp) interaction. Using rational design, we discovered an RNA-groove binding inhibitor (ligand 3) that contains two triaminotriazine units connected by a bisamidinium linker. Ligand 3 binds r(CUG)12 with a low micromolar affinity (K(d) = 8 ± 2 µM) and disrupts the MBNL1-r(CUG)12 interaction in vitro (K(i) = 8 ± 2 µM). In addition, ligand 3 is cell and nucleus permeable, exhibits negligible toxicity to mammalian cells, dissolves MBNL1-r(CUG)(exp) ribonuclear foci, and restores misregulated splicing of IR and cTNT in a DM1 cell culture model. Importantly, suppression of r(CUG)(exp) RNA-induced toxicity in a DM1 Drosophila model was observed after treatment with ligand 3. These results suggest ligand 3 as a lead for the treatment of DM1.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Imidazóis/química , Imidazóis/farmacologia , Distrofia Miotônica/genética , Proteínas de Ligação a RNA/metabolismo , RNA/genética , Expansão das Repetições de Trinucleotídeos/efeitos dos fármacos , Processamento Alternativo/efeitos dos fármacos , Animais , Sequência de Bases , Proteínas de Ligação a DNA/antagonistas & inibidores , Drosophila , Descoberta de Drogas , Células HeLa , Humanos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Terapia de Alvo Molecular , Distrofia Miotônica/tratamento farmacológico , Distrofia Miotônica/metabolismo , Conformação de Ácido Nucleico/efeitos dos fármacos , RNA/antagonistas & inibidores , RNA/química , RNA/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores
4.
ACS Comb Sci ; 22(6): 306-310, 2020 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-32418423

RESUMO

Peptide macrocyclization is typically associated with the development of higher affinity and more protease stable protein ligands, and, as such, is an important tool in peptide drug discovery. Yet, within the context of a diverse library, does cyclization give inherent advantages over linear peptides? Here, we used mRNA display to create a peptide library of diverse ring sizes and topologies (monocyclic, bicyclic, and linear). Several rounds of in vitro selection against streptavidin were performed and the winning peptide sequences were analyzed for their binding affinities and overall topologies. The effect of adding a protease challenge on the enrichment of various peptides was also investigated. Taken together, the selection output yields insights about the relative abundance of binders of various topologies within a structurally diverse library.


Assuntos
Biblioteca de Peptídeos , Peptídeos/química , RNA Mensageiro , Sequência de Aminoácidos , Descoberta de Drogas , Peptídeos/farmacologia
5.
Chem Commun (Camb) ; 55(61): 8959-8962, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31290487

RESUMO

Hydrocarbon stapled peptides are promising therapeutics for inhibition of intracellular protein-protein interactions. Here we develop a new high-throughput strategy for hydrocarbon stapled peptide discovery based on mRNA display of peptides containing α-methyl cysteine and cyclized with m-dibromoxylene. We focus on development of a peptide binder to the HPV16 E2 protein.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Evolução Molecular Direcionada/métodos , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Peptídeos/metabolismo , Engenharia de Proteínas/métodos , Fatores de Transcrição/metabolismo , Alquilação , Sequência de Aminoácidos , Proteínas de Ciclo Celular , Ciclização , Cisteína/química , Hidrocarbonetos Bromados/química , Biblioteca de Peptídeos , Peptídeos/química , Ligação Proteica/efeitos dos fármacos , RNA Mensageiro/química
6.
Curr Opin Chem Biol ; 46: 172-179, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30077877

RESUMO

The ability to introduce non-canonical amino acids into peptides and proteins is facilitated by working within in vitro translation systems. Non-canonical amino acids can be introduced into these systems using sense codon reprogramming, stop codon suppression, and by breaking codon degeneracy. Here, we review how these techniques have been used to create proteins with novel properties and how they facilitate sophisticated studies of protein function. We also discuss how researchers are using in vitro translation experiments with non-canonical amino acids to explore the tolerance of the translation apparatus to artificial building blocks. Finally, we give several examples of how non-canonical amino acids can be combined with mRNA-displayed peptide libraries for the creation of protease-stable, macrocyclic peptide libraries for ligand discovery.


Assuntos
Código Genético , Biblioteca de Peptídeos , Peptídeos/genética , Animais , Códon/genética , Descoberta de Drogas/métodos , Humanos , Ligantes , Compostos Macrocíclicos/química , Peptídeos/química , Peptídeos Cíclicos/química , Peptídeos Cíclicos/genética , Biossíntese de Proteínas , Engenharia de Proteínas/métodos , Proteínas/genética
8.
Virology ; 508: 180-187, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28570919

RESUMO

Human papillomaviruses are causative agents in several human diseases ranging from genital warts to ano-genital and oropharyngeal cancers. Currently only symptoms of HPV induced disease are treated; there are no antivirals available that directly target the viral life cycle. Previously, we determined that the cellular protein TopBP1 interacts with the HPV16 replication/transcription factor E2. This E2-TopBP1 interaction is essential for optimal E1-E2 DNA replication and for the viral life cycle. The drug calcein disrupts the interaction of TopBP1 with itself and other host proteins to promote cell death. Here we demonstrate that calcein blocks HPV16 E1-E2 DNA replication via blocking the viral replication complex forming at the origin of replication. This occurs at non-toxic levels of calcein and demonstrates specificity as it does not block the ability of E2 to regulate transcription. We propose that calcein or derivatives could be developed as an anti-HPV therapeutic.


Assuntos
Antivirais/farmacologia , Replicação do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Fluoresceínas/farmacologia , Papillomavirus Humano 16/efeitos dos fármacos , Proteínas Oncogênicas Virais/metabolismo , Infecções por Papillomavirus/virologia , Origem de Replicação/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/metabolismo , Humanos , Proteínas Oncogênicas Virais/genética , Ligação Proteica
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