RESUMEN
BACKGROUND: Custom genes have become a common resource in recombinant biology over the last 20 years due to the plummeting cost of DNA synthesis. These genes are often "optimized" to non-native sequences for overexpression in a non-native host by substituting synonymous codons within the coding DNA sequence (CDS). A handful of studies have compared native and optimized CDSs, reporting different levels of soluble product due to the accumulation of misfolded aggregates, variable activity of enzymes, and (at least one report of) a change in substrate specificity. No study, to the best of our knowledge, has performed a practical comparison of CDSs generated from different codon optimization algorithms or reported the corresponding protein yields. RESULTS: In our efforts to understand what factors constitute an optimized CDS, we identified that there is little consensus among codon-optimization algorithms, a roughly equivalent chance that an algorithm-optimized CDS will increase or diminish recombinant yields as compared to the native DNA, a near ubiquitous use of a codon database that was last updated in 2007, and a high variability of output CDSs by some algorithms. We present a case study, using KRas4B, to demonstrate that a median codon frequency may be a better predictor of soluble yields than the more commonly utilized CAI metric. CONCLUSIONS: We present a method for visualizing, analyzing, and comparing algorithm-optimized DNA sequences for recombinant protein expression. We encourage researchers to consider if DNA optimization is right for their experiments, and work towards improving the reproducibility of published recombinant work by publishing non-native CDSs.
Asunto(s)
Codón/análisis , Expresión Génica , Análisis de Secuencia de ADN/métodos , Algoritmos , HumanosRESUMEN
Cytotoxic molecules can kill cancer cells by disrupting critical cellular processes or by inducing novel activities. 6-(4-(Diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one (DNMDP) is a small molecule that kills cancer cells by generation of novel activity. DNMDP induces complex formation between phosphodiesterase 3A (PDE3A) and schlafen family member 12 (SLFN12) and specifically kills cancer cells expressing elevated levels of these two proteins. Here, we examined the characteristics and covariates of the cancer cell response to DNMDP. On average, the sensitivity of human cancer cell lines to DNMDP is correlated with PDE3A expression levels. However, DNMDP could also bind the related protein, PDE3B, and PDE3B supported DNMDP sensitivity in the absence of PDE3A expression. Although inhibition of PDE3A catalytic activity did not account for DNMDP sensitivity, we found that expression of the catalytic domain of PDE3A in cancer cells lacking PDE3A is sufficient to confer sensitivity to DNMDP, and substitutions in the PDE3A active site abolish compound binding. Moreover, a genome-wide CRISPR screen identified the aryl hydrocarbon receptor-interacting protein (AIP), a co-chaperone protein, as required for response to DNMDP. We determined that AIP is also required for PDE3A-SLFN12 complex formation. Our results provide mechanistic insights into how DNMDP induces PDE3A-SLFN12 complex formation, thereby killing cancer cells with high levels of PDE3A and SLFN12 expression.
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neoplasias/patología , Secuencia de Bases , Biomarcadores de Tumor/metabolismo , Sistemas CRISPR-Cas/genética , Dominio Catalítico , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/química , Mutación del Sistema de Lectura/genética , Genoma , Heterocigoto , Humanos , Unión Proteica/efectos de los fármacos , Piridazinas/farmacologíaRESUMEN
Beclin-1 (BECN1) is an essential component of macroautophagy. This process is a highly conserved survival mechanism that recycles damaged cellular components or pathogens by encasing them in a bilayer vesicle that fuses with a lysosome to allow degradation of the vesicular contents. Mutations or altered expression profiles of BECN1 have been linked to various cancers and neurodegenerative diseases. Viruses, including HIV and herpes simplex virus 1 (HSV-1), are also known to specifically target BECN1 as a means of evading host defense mechanisms. Autophagy is regulated by the interaction between BECN1 and Bcl-2, a pro-survival protein in the apoptotic pathway that stabilizes the BECN1 homodimer. Disruption of the homodimer by phosphorylation or competitive binding promotes autophagy through an unknown mechanism. We report here the first recombinant synthesis (3-5 mg/L in an Escherichia coli culture) and characterization of full-length, human BECN1. Our analysis reveals that full-length BECN1 exists as a soluble homodimer (KD â¼ 0.45 µM) that interacts with Bcl-2 (KD = 4.3 ± 1.2 µM) and binds to lipid membranes. Dimerization is proposed to be mediated by a coiled-coil region of BECN1. A construct lacking the C-terminal BARA domain but including the coiled-coil region exhibits a homodimer KD 3.5-fold weaker than that of full-length BECN1, indicating that both the BARA domain and the coiled-coil region of BECN1 contribute to dimer formation. Using site-directed mutagenesis, we show that residues at the C-terminus of the coiled-coil region previously shown to interact with the BARA domain play a key role in dimerization and mutations weaken the interface by â¼5-fold.
Asunto(s)
Autofagia , Beclina-1/química , Multimerización de Proteína , Secuencia de Aminoácidos , Beclina-1/biosíntesis , Beclina-1/genética , Escherichia coli , Humanos , Mutagénesis Sitio-Dirigida , Dominios Proteicos , Estructura Secundaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMEN
RNase L is part of the innate immune response to viral infection. It is activated by a small oligonucleotide (2-5A) whose synthesis is initiated as part of the interferon response. Binding of 2-5A to the N-terminal regulatory region, the ANK domain, of RNase L activates its ribonuclease activity and results in cleavage of RNA in the cell, which ultimately leads to apoptosis of the infected cell. The mechanism by which 2-5A activates the ribonuclease activity of RNase L is currently unclear but 2-5A has been shown to induce dimerization of RNase L. To investigate the importance of dimerization of RNase L, we developed a 15kDa dimerization-inducing protein domain that was fused to the N-terminus of RNase L. From these studies we provide direct evidence that dimerization of RNase L occurs at physiologically relevant protein concentrations and correlates with activation of ribonuclease activity. We also show that the binding of 2-5A to RNase L promotes dimerization of the ANK domain and suggest how this could transmit a signal to the rest of the protein to activate ribonuclease activity. Finally, we show that the dimerization-inducing domain can be used as a general fusion partner to aid in protein expression and purification.
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Endorribonucleasas/química , Endorribonucleasas/metabolismo , ARN/metabolismo , Adenosina Trifosfato/metabolismo , Repetición de Anquirina , Cromatografía en Gel , Dicroismo Circular , Endorribonucleasas/aislamiento & purificación , Unión Proteica , Multimerización de Proteína , Estructura Terciaria de ProteínaRESUMEN
Protein phosphatase, Mg2+/Mn2+ dependent 1D (PPM1D), is a serine/threonine phosphatase that is recurrently activated in cancer, regulates the DNA damage response (DDR), and suppresses the activation of p53. Consistent with its oncogenic properties, genetic loss or pharmacologic inhibition of PPM1D impairs tumor growth and sensitizes cancer cells to cytotoxic therapies in a wide range of preclinical models. Given the therapeutic potential of targeting PPM1D specifically and the DDR and p53 pathway more generally, we sought to deepen our biological understanding of PPM1D as a drug target and determine how PPM1D inhibition differs from other therapeutic approaches to activate the DDR. We performed a high throughput screen to identify new allosteric inhibitors of PPM1D, then generated and optimized a suite of enzymatic, cell-based, and in vivo pharmacokinetic and pharmacodynamic assays to drive medicinal chemistry efforts and to further interrogate the biology of PPM1D. Importantly, this drug discovery platform can be readily adapted to broadly study the DDR and p53. We identified compounds distinct from previously reported allosteric inhibitors and showed in vivo on-target activity. Our data suggest that the biological effects of inhibiting PPM1D are distinct from inhibitors of the MDM2-p53 interaction and standard cytotoxic chemotherapies. These differences also highlight the potential therapeutic contexts in which targeting PPM1D would be most valuable. Therefore, our studies have identified a series of new PPM1D inhibitors, generated a suite of in vitro and in vivo assays that can be broadly used to interrogate the DDR, and provided important new insights into PPM1D as a drug target.
RESUMEN
The eukaryotic initiation factor 4E (eIF4E) is the key component of the translational initiation complex that recruits mRNA by binding to a unique "cap" structure located at the 5' end of the mRNA. Overexpression of eIF4E has been implicated in the development of cancer, potentially as a result of increasing the cellular levels of proteins involved in processes that include proliferation and regulation of apoptosis. As a result, the cap-binding site of eIF4E has become a target for the development of anti-cancer therapeutics. The structure of eIF4E bound to the cap mimic 7-methyl-GDP revealed that two tryptophans from different loops in eIF4E sandwiched the 7-methylguanine group between them. This interaction gives rise to a strong exciton coupling signal between the two tryptophans that can be visualized by CD spectroscopy. eIF4E is a challenging protein to work with because of a propensity to aggregate under conditions used in biophysical techniques. CD spectroscopy provides a gentle, solution-based approach to study binding to the cap-binding site of eIF4E. Evidence is provided that the exciton coupling signal can be used to both qualitatively and quantitatively analyze the binding of cap analogs to eIF4E.
Asunto(s)
Dicroismo Circular , Factor 4E Eucariótico de Iniciación/metabolismo , Caperuzas de ARN/metabolismo , Sitios de Unión , Factor 4E Eucariótico de Iniciación/química , Factor 4E Eucariótico de Iniciación/genética , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/química , Guanosina Difosfato/metabolismo , Nucleótidos/química , Nucleótidos/metabolismo , Unión Proteica , Replegamiento Proteico , Estructura Terciaria de Proteína , Análogos de Caperuza de ARN/química , Análogos de Caperuza de ARN/metabolismo , Caperuzas de ARN/química , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Ribavirina/química , Ribavirina/metabolismo , Soluciones/químicaRESUMEN
Corin is a transmembrane tethered enzyme best known for processing the hormone atrial natriuretic peptide (ANP) in cardiomyocytes to control electrolyte balance and blood pressure. Loss of function mutations in Corin prevent ANP processing and lead to hypertension. Curiously, Corin loss of function variants also result in lighter coat color pigmentation in multiple species. Corin pigmentation effects are dependent on a functional Agouti locus encoding the agouti-signaling protein (ASIP) based on a genetic interaction. However, the nature of this conserved role of Corin has not been defined. Here we report that ASIP is a direct proteolytic substrate of the Corin enzyme.
RESUMEN
PPM1D encodes a serine/threonine phosphatase that regulates numerous pathways including the DNA damage response and p53. Activating mutations and amplification of PPM1D are found across numerous cancer types. GSK2830371 is a potent and selective allosteric inhibitor of PPM1D, but its mechanism of binding and inhibition of catalytic activity are unknown. Here we use computational, biochemical and functional genetic studies to elucidate the molecular basis of GSK2830371 activity. These data confirm that GSK2830371 binds an allosteric site of PPM1D with high affinity. By further incorporating data from hydrogen deuterium exchange mass spectrometry and sedimentation velocity analytical ultracentrifugation, we demonstrate that PPM1D exists in an equilibrium between two conformations that are defined by the movement of the flap domain, which is required for substrate recognition. A hinge region was identified that is critical for switching between the two conformations and was directly implicated in the high-affinity binding of GSK2830371 to PPM1D. We propose that the two conformations represent active and inactive forms of the protein reflected by the position of the flap, and that binding of GSK2830371 shifts the equilibrium to the inactive form. Finally, we found that C-terminal truncating mutations proximal to residue 400 result in destabilization of the protein via loss of a stabilizing N- and C-terminal interaction, consistent with the observation from human genetic data that nearly all PPM1D mutations in cancer are truncating and occur distal to residue 400. Taken together, our findings elucidate the mechanism by which binding of a small molecule to an allosteric site of PPM1D inhibits its activity and provides insights into the biology of PPM1D.
Asunto(s)
Neoplasias , Proteína Fosfatasa 2C , Sitio Alostérico , Aminopiridinas/farmacología , Dipéptidos/farmacología , Humanos , Mutación , Neoplasias/tratamiento farmacológico , Neoplasias/enzimología , Neoplasias/genética , Conformación Proteica , Proteína Fosfatasa 2C/antagonistas & inhibidores , Proteína Fosfatasa 2C/química , Proteína Fosfatasa 2C/genética , Proteína Fosfatasa 2C/metabolismo , Serina/genética , Serina/metabolismo , Relación Estructura-ActividadRESUMEN
BACKGROUND: Corin is a protease expressed in cardiomyocytes that plays a key role in salt handling and intravascular volume homeostasis via activation of natriuretic peptides. It is unknown if Corin loss-of-function (LOF) is causally associated with risk of coronary artery disease (CAD). METHODS: We analyzed all coding CORIN variants in an Italian case-control study of CAD. We functionally tested all 64 rare missense mutations in Western Blot and Mass Spectroscopy assays for proatrial natriuretic peptide cleavage. An expanded rare variant association analysis for Corin LOF mutations was conducted in whole exome sequencing data from 37 799 CAD cases and 212 184 controls. RESULTS: We observed LOF variants in CORIN in 8 of 1803 (0.4%) CAD cases versus 0 of 1725 controls (P, 0.007). Of 64 rare missense variants profiled, 21 (33%) demonstrated <30% of wild-type activity and were deemed damaging in the 2 functional assays for Corin activity. In a rare variant association study that aggregated rare LOF and functionally validated damaging missense variants from the Italian study, we observed no association with CAD-21 of 1803 CAD cases versus 12 of 1725 controls with adjusted odds ratio of 1.61 ([95% CI, 0.79-3.29]; P=0.17). In the expanded sequencing dataset, there was no relationship between rare LOF variants with CAD was also observed (odds ratio, 1.15 [95% CI, 0.89-1.49]; P=0.30). Consistent with the genetic analysis, we observed no relationship between circulating Corin concentrations with incident CAD events among 4744 participants of a prospective cohort study-sex-stratified hazard ratio per SD increment of 0.96 ([95% CI, 0.87-1.07], P=0.48). CONCLUSIONS: Functional testing of missense mutations improved the accuracy of rare variant association analysis. Despite compelling pathophysiology and a preliminary observation suggesting association, we observed no relationship between rare damaging variants in CORIN or circulating Corin concentrations with risk of CAD.
Asunto(s)
Enfermedad de la Arteria Coronaria/genética , Genómica , Mutación Missense , Análisis de Secuencia de ADN , Serina Endopeptidasas/genética , Adulto , Enfermedad de la Arteria Coronaria/epidemiología , Femenino , Humanos , Italia/epidemiología , Masculino , Factores de RiesgoRESUMEN
DNMDP and related compounds, or velcrins, induce complex formation between the phosphodiesterase PDE3A and the SLFN12 protein, leading to a cytotoxic response in cancer cells that express elevated levels of both proteins. The mechanisms by which velcrins induce complex formation, and how the PDE3A-SLFN12 complex causes cancer cell death, are not fully understood. Here, we show that PDE3A and SLFN12 form a heterotetramer stabilized by binding of DNMDP. Interactions between the C-terminal alpha helix of SLFN12 and residues near the active site of PDE3A are required for complex formation, and are further stabilized by interactions between SLFN12 and DNMDP. Moreover, we demonstrate that SLFN12 is an RNase, that PDE3A binding increases SLFN12 RNase activity, and that SLFN12 RNase activity is required for DNMDP response. This new mechanistic understanding will facilitate development of velcrin compounds into new cancer therapies.
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/química , Péptidos y Proteínas de Señalización Intracelular/química , Piridazinas/química , Adenosina Monofosfato/química , Rastreo Diferencial de Calorimetría , Dominio Catalítico , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Microscopía por Crioelectrón , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/genética , Endorribonucleasas/química , Células HEK293 , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Cinética , Espectrometría de Masas , Complejos Multienzimáticos/ultraestructura , Mutación , Unión Proteica , Conformación Proteica en Hélice alfa , Multimerización de Proteína , Piridazinas/farmacología , Proteínas Recombinantes , Tetrahidroisoquinolinas/químicaRESUMEN
Anti-cancer uses of non-oncology drugs have occasionally been found, but such discoveries have been serendipitous. We sought to create a public resource containing the growth inhibitory activity of 4,518 drugs tested across 578 human cancer cell lines. We used PRISM, a molecular barcoding method, to screen drugs against cell lines in pools. An unexpectedly large number of non-oncology drugs selectively inhibited subsets of cancer cell lines in a manner predictable from the cell lines' molecular features. Our findings include compounds that killed by inducing PDE3A-SLFN12 complex formation; vanadium-containing compounds whose killing depended on the sulfate transporter SLC26A2; the alcohol dependence drug disulfiram, which killed cells with low expression of metallothioneins; and the anti-inflammatory drug tepoxalin, which killed via the multi-drug resistance protein ABCB1. The PRISM drug repurposing resource (https://depmap.org/repurposing) is a starting point to develop new oncology therapeutics, and more rarely, for potential direct clinical translation.
Asunto(s)
Neoplasias , Línea Celular , Disulfiram , Reposicionamiento de Medicamentos , Humanos , Neoplasias/tratamiento farmacológicoRESUMEN
The RFX complex is key component of a multi-protein complex that regulates the expression of the Major Histocompatibility Class II (MHCII) genes, whose products are essential for the initiation and development of the adaptive immune response. The RFX complex is comprised of three proteins--RFX5, RFXAP, and RFXB--all of which are required for expression of MHCII genes. We have used electrophoretic mobility shift assays to characterize the DNA binding of RFX5 and the complexes it forms with RFXB and RFXAP, to the proximal regulatory region of the MHCII promoter. DNA binding of RFX5 is inhibited by domains flanking its DNA binding domain, and both RFXAP and RFXB are required to overcome the inhibition of both domains. We provide evidence that a single RFX complex binds to the proximal regulatory region of the MHCII promoter and identify regions of the DNA that are important for high affinity binding of the RFX complex. Together, our results provide the most detailed view to date of the assembly of the RFX complex on the MHCII promoter and how its DNA binding is regulated.
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Proteínas de Unión al ADN/metabolismo , Genes MHC Clase II , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismo , Secuencia de Bases , Relación Dosis-Respuesta a Droga , Humanos , Sistema Inmunológico , Cinética , Modelos Genéticos , Datos de Secuencia Molecular , Oligonucleótidos/química , Unión Proteica , Factores de Transcripción del Factor Regulador X , Análisis de Secuencia de ADNRESUMEN
Major histocompatibility complex class II (MHCII) molecules have a central role in the mammalian adaptive immune response against infection. The level of the immune response is directly related to the concentration of MHCII molecules in the cell, which have a central role in initiating the immune response. MHCII molecules are therefore a potential target for the development of immunosuppressant drugs for the treatment of organ transplant rejection and autoimmune disease. The expression of MHCII molecules is regulated by a cell specific multiprotein complex. The RFX complex is the key DNA binding component of this complex. The RFX complex is composed of three proteins-RFX5, RFXAP, and RFXB-all of which are required for activation of expression of the MHCII genes. Little is currently known about the precise regions of the RFX proteins that are required for complex formation, or their structure. We have therefore identified the key regions of RFX5, RFXAP, and RFXB, which are required to form the RFX complex and have characterized the individual domains and the complexes they form using NMR and circular dichroism spectroscopy and isothermal titration calorimetry. Our results support a model for the assembly of the RFX complex in which the interaction between RFX5 and RFXAP promote folding of a poorly structured region ofRFXAP, which is required for high affinity binding of RFXB to the RFX5.RFXAP complex.
Asunto(s)
Regulación de la Expresión Génica , Factores de Transcripción/química , Calorimetría , Dicroismo Circular , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Vectores Genéticos , Humanos , Espectroscopía de Resonancia Magnética , Mutagénesis Sitio-Dirigida , Reacción en Cadena de la Polimerasa , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Factores de Transcripción del Factor Regulador X , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Corin (atrial natriuretic peptide-converting enzyme, EC 3.4.21) is a transmembrane serine protease expressed in cardiomyocytes. Corin exerts its cardioprotective effects via the proteolytic cleavage and activation of pro-atrial natriuretic peptide (pro-ANP) to ANP. We recently described an ANP reporter cell line stably expressing the ANP receptor, a cGMP-dependent cation channel used as a real-time cGMP biosensor, and the Ca2+-sensitive photoprotein aequorin. Here, we describe the generation of a novel reporter cell line expressing the calcium biosensor GCaMP6 instead of aequorin. In contrast to the luminescence-based assay, ANP stimulation of our novel GCaMP6 reporter cell resulted in stable, long-lasting fluorescence signals. Using this novel reporter system, we were able to detect pro-ANP to ANP conversion by purified, soluble wildtype corin (solCorin), but not the active site mutant solCorin(S985A), resulting in left-shifted concentration-response curves. Furthermore, cellular pro-ANPase activity could be detected on HEK 293 cells after transient expression of wildtype corin. In contrast, corin activity was not detected after transfection with the inactive corin(S985A) variant. In supernatants from cardiomyocyte-derived HL-1 cells pro-ANP to ANP conversion could also be detected, while in HL-1 corin knockout cells no conversion was observed. These findings underline the role of corin as the pro-ANP convertase. Our novel fluorescence-based ANP reporter cell line is well-suited for the sensitive detection of corin activity, and may be used for the identification and characterization of novel corin modulators.
Asunto(s)
Factor Natriurético Atrial/metabolismo , Canales Catiónicos Regulados por Nucleótidos Cíclicos/metabolismo , Receptores del Factor Natriurético Atrial/metabolismo , Serina Endopeptidasas/metabolismo , Animales , Factor Natriurético Atrial/genética , Factor Natriurético Atrial/farmacología , Calcio/metabolismo , Línea Celular , GMP Cíclico/metabolismo , Canales Catiónicos Regulados por Nucleótidos Cíclicos/genética , Células HEK293 , Humanos , Ratones , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Receptores del Factor Natriurético Atrial/genética , Serina Endopeptidasas/genéticaRESUMEN
Pax-5, a member of the paired domain family of transcription factors, is a key regulator of B lymphocyte-specific transcription and differentiation. A major target of Pax-5-mediated activation is the mb-1 gene, which encodes the essential transmembrane signaling protein Ig-alpha. Pax-5 recruits three members of the Ets family of transcription factors: Ets-1, Fli-1 and GABPalpha (with GABPbeta1), to assemble ternary complexes on the mb-1 promoter in vitro. Using the Pax-5:Ets-1:DNA crystal structure as a guide, we defined amino acid requirements for transcriptional activation of endogenous mb-1 genes using a novel cell-based assay. Mutations in the beta-hairpin/beta-turn of the DNA-binding domain of Pax-5 demonstrated its importance for DNA sequence recognition and activation of mb-1 transcription. Mutations of amino acids contacting Ets-1 in the crystal structure reduced or blocked mb-1 promoter activation. One of these mutations, Q22A, resulted in greatly reduced mb-1 gene transcript levels, concurrent with the loss of its ability to recruit Fli-1 to bind the promoter in vitro. In contrast, the mutation had no effect on recruitment of the related Ets protein GABPalpha (with GABPbeta1). These data further define requirements for Pax-5 function in vivo and reveal the complexity of interactions required for cooperative partnerships between transcription factors.
Asunto(s)
Antígenos CD/genética , Proteínas de Unión al ADN/metabolismo , Receptores de Antígenos de Linfocitos B/genética , Factores de Transcripción/metabolismo , Activación Transcripcional , Animales , Antígenos CD/biosíntesis , Antígenos CD79 , Línea Celular , ADN/química , ADN/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Factor de Transcripción de la Proteína de Unión a GA , Sustancias Macromoleculares , Modelos Moleculares , Mutación , Factor de Transcripción PAX5 , Estructura Secundaria de Proteína , Proteína Proto-Oncogénica c-ets-1 , Proteína Proto-Oncogénica c-fli-1 , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-ets , Receptores de Antígenos de Linfocitos B/biosíntesis , Transactivadores/metabolismo , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
Circulating low-density lipoprotein cholesterol (LDLc) is regulated by membrane-bound LDL receptor (LDLr). Upon LDLc and LDLr interaction the complex is internalized by the cell, leading to LDLc degradation and LDLr recycling back to the cell surface. The proprotein convertase subtilisin/kexin type 9 (PCSK9) protein regulates this cycling. PCSK9 is secreted from the cell and binds LDLr. When the complex is internalized, PCSK9 prevents LDLr from shuttling back to the surface and instead targets it for degradation. PCSK9 is a serine protease expressed as a zymogen that undergoes autoproteolysis, though the two resulting protein domains remain stably associated as a heterodimer. This PCSK9 autoprocessing is required for the protein to be secreted from the cell. To date, direct analysis of PCSK9 autoprocessing has proven challenging, as no catalytically active zymogen has been isolated. A PCSK9 loss-of-function point mutation (Q152H) that reduces LDLc levels two-fold was identified in a patient population. LDLc reduction was attributed to a lack of PCSK9(Q152H) autoprocessing preventing secretion of the protein. We have isolated a zymogen form of PCSK9, PCSK9(Q152H), and a related mutation (Q152N), that can undergo slow autoproteolysis. We show that the point mutation prevents the formation of the mature form of PCSK9 by hindering folding, reducing the rate of autoproteolysis, and destabilizing the heterodimeric form of the protein. In addition, we show that the zymogen form of PCSK9 adopts a structure that is distinct from the processed form and is unable to bind a mimetic peptide based on the EGF-A domain of the LDLr.
Asunto(s)
Péptidos/química , Mutación Puntual , Proproteína Convertasa 9/química , Multimerización de Proteína , Receptores de LDL/química , Sustitución de Aminoácidos , Humanos , Péptidos/genética , Péptidos/metabolismo , Proproteína Convertasa 9/genética , Proproteína Convertasa 9/metabolismo , Unión Proteica , Dominios Proteicos , Receptores de LDL/genética , Receptores de LDL/metabolismoRESUMEN
We present the results of in vitro DNA-binding assays for a mutant protein (Q44K) of the E. coli methionine repressor, MetJ, as well as the crystal structure at 2.2 A resolution of the apo-mutant bound to a 10-mer oligonucleotide encompassing an 8 bp met-box sequence. The wild-type protein binds natural operators co-operatively with respect to protein concentration forming at least a dimer of repressor dimers along operator DNAs. The minimum operator length is thus 16 bp, each MetJ dimer interacting with a single met-box site. In contrast, the Q44K mutant protein can also bind stably as a single dimer to 8 bp target sites, in part due to additional contacts made to the phosphodiester backbone outside the 8 bp target via the K44 side-chains. Protein-protein co-operativity in the mutant is reduced relative to the wild-type allowing the properties of an intermediate on the pathway to operator site saturation to be examined for the first time. The crystal structure of the decamer complex shows a unique conformation for the protein bound to the single met-box site, possibly explaining the reduced protein-protein co-operativity. In both the extended and minimal DNA complexes formed, the mutant protein makes slightly different contacts to the edges of DNA base-pairs than the wild-type, even though the site of amino acid substitution is distal from the DNA-binding motif. Quantitative binding assays suggest that this is not due to artefacts caused by the crystallisation conditions but is most likely due to the relatively small contribution of such direct contacts to the overall binding energy of DNA-protein complex formation, which is dominated by sequence-dependent distortions of the DNA duplex and by the protein-protein contact between dimers.
Asunto(s)
Proteínas Bacterianas/química , Escherichia coli/metabolismo , Regiones Operadoras Genéticas , Proteínas Represoras/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Emparejamiento Base , Cristalografía por Rayos X , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Oligonucleótidos/química , Oligonucleótidos/metabolismo , Unión Proteica , Estructura Cuaternaria de Proteína , Proteínas Represoras/genética , Proteínas Represoras/metabolismoRESUMEN
Members of the ETS transcription factor family have been implicated in several cancers, where they are often dysregulated by genomic derangement. ETS variant 1 (ETV1) is an ETS factor gene that undergoes chromosomal translocation in prostate cancers and Ewing sarcomas, amplification in melanomas, and lineage dysregulation in gastrointestinal stromal tumors. Pharmacologic perturbation of ETV1 would be appealing in these cancers; however, oncogenic transcription factors are often deemed "undruggable" by conventional methods. Here, we used small-molecule microarray screens to identify and characterize drug-like compounds that modulate the biologic function of ETV1. We identified the 1,3,5-triazine small molecule BRD32048 as a top candidate ETV1 perturbagen. BRD32048 binds ETV1 directly, modulating both ETV1-mediated transcriptional activity and invasion of ETV1-driven cancer cells. Moreover, BRD32048 inhibits p300-dependent acetylation of ETV1, thereby promoting its degradation. These results point to a new avenue for pharmacologic ETV1 inhibition and may inform a general means to discover small molecule perturbagens of transcription factor oncoproteins.
Asunto(s)
Compuestos de Anilina/administración & dosificación , Proteínas de Unión al ADN/metabolismo , Terapia Molecular Dirigida , Neoplasias/tratamiento farmacológico , Neoplasias de la Próstata/tratamiento farmacológico , Factores de Transcripción/metabolismo , Triazinas/administración & dosificación , Línea Celular Tumoral , Proteínas de Unión al ADN/antagonistas & inhibidores , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Neoplasias/metabolismo , Regiones Promotoras Genéticas , Neoplasias de la Próstata/patología , Bibliotecas de Moléculas Pequeñas , Resonancia por Plasmón de Superficie , Factores de Transcripción/antagonistas & inhibidoresRESUMEN
The mammalian immune response is mediated by a heterotetrameric transcriptional control complex, called regulatory factor X (RFX), that regulates the expression of major histocompatibility complex class II genes. RFX comprises three proteins: RFX5 (two copies), RFXAP, and RFXB, and mutations and deletions that prevent the assembly of the RFX complex have been linked to a severe immunodeficiency disorder. Two RFX5 molecules and one RFXAP molecule assemble in the cytoplasm prior to nuclear localization, a process mediated by an N-terminal "dimerization domain" of RFX5 (RFX5(N)) and a C-terminal domain of RFXAP (RFXAP(C)). We previously presented evidence that RFXAP(C) is unstructured in the absence of RFX5(N) but adopts a regular structure in the RFX5(N)(2)-RFXAP(C) complex and that the RFX5(N)(2)-RFXAP(C) complex binds RFXB with high affinity. We now report the structure of the RFX5(N)(2)-RFXAP(C) complex, determined in solution by (15)N- and (13)C-edited NMR spectroscopy. RFX5(N) consists of a long central helix flanked by two shorter helices. The central helices of the two RFX5(N) molecules form an antiparallel coiled coil, and the flanking helices pack at the ends of the long helices in a perpendicular arrangement such that the RFX5(N) dimer is shaped like a staple. RFXAP(C) consists of two α-helices that form a V-shaped structure that packs within the RFX5(N)(2) staple. Leucine residues in the leucine-rich region of RFX5(N) (62-LYLYLQL-68) that are critical for major histocompatibility complex class II gene expression in vivo contribute to both the dimer (Leu64 and Leu68) and the RFX5(N)-RFXAP(C) interfaces (Leu62 and Leu66). The clustering of hydrophobic residues from different regions of RFXAP(C) suggests a potential binding site for RFXB.