RESUMEN
The discovery and implementation of thiopurine methyltransferase (TPMT) pharmacogenetics has been a success story and has reduced the suffering from serious adverse reactions during thiopurine treatment of childhood leukaemia and inflammatory bowel disease. This MiniReview summarizes four studies included in Dr Zimdahl Kahlin's doctoral thesis as well as the current knowledge on this field of research. The genotype-phenotype concordance of TPMT in a cohort of 12 663 individuals with clinically analysed TPMT status is described. Notwithstanding the high concordance, the benefits of combined genotyping and phenotyping for TPMT status determination are discussed. The results from the large cohort also demonstrate that the factors of gender and age affect TPMT enzyme activity. In addition, characterization of four previously undescribed TPMT alleles (TPMT*41, TPMT*42, TPMT*43 and TPMT*44) shows that a defective TPMT enzyme could be caused by several different mechanisms. Moreover, the folate analogue methotrexate (MTX), used in combination with thiopurines during maintenance therapy of childhood leukaemia, affects the metabolism of thiopurines and interacts with TPMT, not only by binding and inhibiting the enzyme activity but also by regulation of its gene expression.
Asunto(s)
Metotrexato/metabolismo , Metiltransferasas/genética , Variantes Farmacogenómicas , Purinas/metabolismo , Sitios de Unión , Biotransformación , Interacciones Farmacológicas , Regulación Enzimológica de la Expresión Génica , Genotipo , Humanos , Metotrexato/farmacocinética , Metiltransferasas/antagonistas & inhibidores , Metiltransferasas/metabolismo , Farmacogenética , Fenotipo , Unión Proteica , Purinas/farmacocinéticaRESUMEN
Transcription factor c-MYC is a potent oncoprotein; however, the mechanism of transcriptional regulation via MYC-protein interactions remains poorly understood. The TATA-binding protein (TBP) is an essential component of the transcription initiation complex TFIID and is required for gene expression. We identify two discrete regions mediating MYC-TBP interactions using structural, biochemical and cellular approaches. A 2.4 -Å resolution crystal structure reveals that human MYC amino acids 98-111 interact with TBP in the presence of the amino-terminal domain 1 of TBP-associated factor 1 (TAF1TAND1). Using biochemical approaches, we have shown that MYC amino acids 115-124 also interact with TBP independently of TAF1TAND1. Modeling reveals that this region of MYC resembles a TBP anchor motif found in factors that regulate TBP promoter loading. Site-specific MYC mutants that abrogate MYC-TBP interaction compromise MYC activity. We propose that MYC-TBP interactions propagate transcription by modulating the energetic landscape of transcription initiation complex assembly.
Asunto(s)
Mapas de Interacción de Proteínas , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteína de Unión a TATA-Box/metabolismo , Línea Celular Tumoral , Cristalografía por Rayos X , Histona Acetiltransferasas/química , Histona Acetiltransferasas/metabolismo , Humanos , Modelos Moleculares , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Proto-Oncogénicas c-myc/química , Factores Asociados con la Proteína de Unión a TATA/química , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Proteína de Unión a TATA-Box/química , Factor de Transcripción TFIID/química , Factor de Transcripción TFIID/metabolismoRESUMEN
Thiopurines are widely used in the treatment of leukemia and inflammatory bowel diseases. Thiopurine metabolism varies among individuals because of differences in the polymorphic enzyme thiopurine methyltransferase (TPMT, EC 2.1.1.67), and to avoid severe adverse reactions caused by incorrect dosing it is recommended that the patient's TPMT status be determined before the start of thiopurine treatment. This study describes the concordance between genotyping for common TPMT alleles and phenotyping in a Swedish cohort of 12,663 patients sampled before or during thiopurine treatment. The concordance between TPMT genotype and enzyme activity was 94.5%. Compared to the genotype, the first measurement of TPMT enzyme activity was lower than expected for 4.6% of the patients. Sequencing of all coding regions of the TPMT gene in genotype/phenotype discrepant individuals led to the identification of rare and novel TPMT alleles. Fifteen individuals (0.1%) with rare or novel genotypes were identified, and three TPMT alleles (TPMT*42, *43, and *44) are characterized here for the first time. These 15 patients would not have been detected as carrying a deviating TPMT genotype if only genotyping of the most common TPMT variants had been performed. This study highlights the benefit of combining TPMT genotype and phenotype determination in routine testing. More accurate dose recommendations can be made, which might decrease the number of adverse reactions and treatment failures during thiopurine treatment.
Asunto(s)
Estudios de Asociación Genética/métodos , Genotipo , Metiltransferasas/genética , Metiltransferasas/metabolismo , Farmacogenética/métodos , Fenotipo , Polimorfismo de Nucleótido Simple/genética , Adulto , Hipersensibilidad a las Drogas/diagnóstico , Hipersensibilidad a las Drogas/epidemiología , Hipersensibilidad a las Drogas/genética , Femenino , Humanos , Masculino , Persona de Mediana Edad , Errores Innatos del Metabolismo de la Purina-Pirimidina/diagnóstico , Errores Innatos del Metabolismo de la Purina-Pirimidina/epidemiología , Errores Innatos del Metabolismo de la Purina-Pirimidina/genética , Suecia/epidemiologíaRESUMEN
Thiopurine induced toxicity is associated with defects in the thiopurine methyltransferase (TPMT) gene. TPMT is a polymorphic enzyme, with most of the single nucleotide polymorphisms (SNPs) causing an amino acid change, altering the enzymatic activity of the TPMT protein. In this study, we characterize a novel patient allele c.719A > C, named TPMT*41, together with the more common variant *3C c.719A > G, resulting in an amino acid shift at tyrosine 240 to serine, p.Y240S and cysteine, p.Y240C respectively. We show that the patient heterozygote for c.719A > C has intermediate enzymatic activity in red blood cells. Furthermore, in vitro studies, using recombinant protein, show that TPMT p.Y240S is less stable than both TPMTwt and TPMT p.Y240C. The addition of SAM increases the stability and, in agreement with Isothermal Titration Calorimetry (ITC) data, higher molar excess of SAM is needed in order to stabilize TPMT p.Y240C and TPMT p.Y240S compared to TPMTwt. Molecular dynamics simulations show that the loss of interactions is most severe for Y240S, which agrees with the thermal stability of the mutations. In conclusion, our study shows that SAM increases the stability of TPMT and that changing only one amino acid can have a dramatic effect on TPMT stability and activity.
Asunto(s)
Alelos , Metiltransferasas/genética , Mutación , Polimorfismo de Nucleótido Simple , Genotipo , HumanosRESUMEN
Hierarchic phosphorylation and concomitant Pin1-mediated proline isomerization of the oncoprotein c-Myc controls its cellular stability and activity. However, the molecular basis for Pin1 recognition and catalysis of c-Myc and other multisite, disordered substrates in cell regulation and disease is unclear. By nuclear magnetic resonance, surface plasmon resonance, and molecular modeling, we show that Pin1 subdomains jointly pre-anchor unphosphorylated c-Myc1-88 in the Pin1 interdomain cleft in a disordered, or "fuzzy", complex at the herein named Myc Box 0 (MB0) conserved region N-terminal to the highly conserved Myc Box I (MBI). Ser62 phosphorylation in MBI intensifies previously transient MBI-Pin1 interactions in c-Myc1-88 binding, and increasingly engages Pin1PPIase and its catalytic region with maintained MB0 interactions. In cellular assays, MB0 mutated c-Myc shows decreased Pin1 interaction, increased protein half-life, but lowered rates of Myc-driven transcription and cell proliferation. We propose that dynamic Pin1 recognition of MB0 contributes to the regulation of c-Myc activity in cells.
Asunto(s)
Isomerasa de Peptidilprolil/química , Proteínas Proto-Oncogénicas c-myc/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Humanos , Datos de Secuencia Molecular , Mutación , Peptidilprolil Isomerasa de Interacción con NIMA , Isomerasa de Peptidilprolil/genética , Isomerasa de Peptidilprolil/metabolismo , Fosforilación , Unión Proteica , Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas c-myc/químicaRESUMEN
We present the software CDpal that is used to analyze thermal and chemical denaturation data to obtain information on protein stability. The software uses standard assumptions and equations applied to two-state and various types of three-state denaturation models in order to determine thermodynamic parameters. It can analyze denaturation monitored by both circular dichroism and fluorescence spectroscopy and is extremely flexible in terms of input format. Furthermore, it is intuitive and easy to use because of the graphical user interface and extensive documentation. As illustrated by the examples herein, CDpal should be a valuable tool for analysis of protein stability.
Asunto(s)
Biología Computacional/métodos , Proteínas/química , Dicroismo Circular , Modelos Moleculares , Desnaturalización Proteica , Estabilidad Proteica , Programas Informáticos , Espectrometría de Fluorescencia , TemperaturaRESUMEN
The Myc oncoprotein is a key contributor to the development of many human cancers. As such, understanding its molecular activities and biological functions has been a field of active research since its discovery more than three decades ago. Genome-wide studies have revealed Myc to be a global regulator of gene expression. The identification of its DNA-binding partner protein, Max, launched an area of extensive research into both the protein-protein interactions and protein structure of Myc. In this review, we highlight key insights with respect to Myc interactors and protein structure that contribute to the understanding of Myc's roles in transcriptional regulation and cancer. Structural analyses of Myc show many critical regions with transient structures that mediate protein interactions and biological functions. Interactors, such as Max, TRRAP, and PTEF-b, provide mechanistic insight into Myc's transcriptional activities, while others, such as ubiquitin ligases, regulate the Myc protein itself. It is appreciated that Myc possesses a large interactome, yet the functional relevance of many interactors remains unknown. Here, we discuss future research trends that embrace advances in genome-wide and proteome-wide approaches to systematically elucidate mechanisms of Myc action. This article is part of a Special Issue entitled: Myc proteins in cell biology and pathology.
Asunto(s)
Neoplasias/genética , Mapas de Interacción de Proteínas/genética , Proteoma , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Regulación de la Expresión Génica , Genoma Humano , Humanos , Neoplasias/patología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Conformación Proteica , Procesamiento Proteico-Postraduccional/genética , Proteínas Proto-Oncogénicas c-myc/biosíntesis , Proteínas Proto-Oncogénicas c-myc/metabolismoRESUMEN
In this paper, we describe the structure of a N-terminal domain motif in nuclear-localized FKBP251-73, a member of the FKBP family, together with the structure of a sequence-related subdomain of the E3 ubiquitin ligase HectD1 that we show belongs to the same fold. This motif adopts a compact 5-helix bundle which we name the Basic Tilted Helix Bundle (BTHB) domain. A positively charged surface patch, structurally centered around the tilted helix H4, is present in both FKBP25 and HectD1 and is conserved in both proteins, suggesting a conserved functional role. We provide detailed comparative analysis of the structures of the two proteins and their sequence similarities, and analysis of the interaction of the proposed FKBP25 binding protein YY1. We suggest that the basic motif in BTHB is involved in the observed DNA binding of FKBP25, and that the function of this domain can be affected by regulatory YY1 binding and/or interactions with adjacent domains.
Asunto(s)
Estructura Terciaria de Proteína , Proteínas de Unión a Tacrolimus/química , Ubiquitina-Proteína Ligasas/química , Secuencia de Aminoácidos , Humanos , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Estructura Secundaria de Proteína , Homología de Secuencia de Aminoácido , Proteínas de Unión a Tacrolimus/metabolismo , Factor de Transcripción YY1/metabolismoRESUMEN
The general transcription factor TFIID provides a regulatory platform for transcription initiation. Here we present the crystal structure (1.97 Å) and NMR analysis of yeast TAF1 N-terminal domains TAND1 and TAND2 bound to yeast TBP, together with mutational data. We find that yeast TAF1-TAND1, which in itself acts as a transcriptional activator, binds TBP's concave DNA-binding surface by presenting similar anchor residues to TBP as does Mot1 but from a distinct structural scaffold. Furthermore, we show how TAF1-TAND2 uses an aromatic and acidic anchoring pattern to bind a conserved TBP surface groove traversing the basic helix region, and we find highly similar TBP-binding motifs also presented by the structurally distinct TFIIA, Mot1 and Brf1 proteins. Our identification of these anchoring patterns, which can be easily disrupted or enhanced, provides insight into the competitive multiprotein TBP interplay critical to transcriptional regulation.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Modelos Moleculares , Conformación Proteica , Proteínas de Saccharomyces cerevisiae/química , Factores Asociados con la Proteína de Unión a TATA/química , Proteína de Unión a TATA-Box/química , Factor de Transcripción TFIID/química , Transcripción Genética/fisiología , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Cristalización , Unión Proteica , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Proteína de Unión a TATA-Box/metabolismo , Factor de Transcripción TFIID/metabolismo , Factor de Transcripción TFIIIB/química , Factor de Transcripción TFIIIB/metabolismoRESUMEN
The crucial role of Myc as an oncoprotein and as a key regulator of cell growth makes it essential to understand the molecular basis of Myc function. The N-terminal region of c-Myc coordinates a wealth of protein interactions involved in transformation, differentiation and apoptosis. We have characterized in detail the intrinsically disordered properties of Myc-1-88, where hierarchical phosphorylation of S62 and T58 regulates activation and destruction of the Myc protein. By nuclear magnetic resonance (NMR) chemical shift analysis, relaxation measurements and NOE analysis, we show that although Myc occupies a very heterogeneous conformational space, we find transiently structured regions in residues 22-33 and in the Myc homology box I (MBI; residues 45-65); both these regions are conserved in other members of the Myc family. Binding of Bin1 to Myc-1-88 as assayed by NMR and surface plasmon resonance (SPR) revealed primary binding to the S62 region in a dynamically disordered and multivalent complex, accompanied by population shifts leading to altered intramolecular conformational dynamics. These findings expand the increasingly recognized concept of intrinsically disordered regions mediating transient interactions to Myc, a key transcriptional regulator of major medical importance, and have important implications for further understanding its multifaceted role in gene regulation.