RESUMEN
ATP-dependent chromatin remodellers regulate access to genetic information by controlling nucleosome positions in vivo. However, the mechanism by which remodellers discriminate between different nucleosome substrates is poorly understood. Many chromatin remodelling proteins possess conserved protein domains that interact with nucleosomal features. Here we used a quantitative high-throughput approach, based on the use of a DNA-barcoded mononucleosome library, to profile the biochemical activity of human ISWI family remodellers in response to a diverse set of nucleosome modifications. We show that accessory (non-ATPase) subunits of ISWI remodellers can distinguish between differentially modified nucleosomes, directing remodelling activity towards specific nucleosome substrates according to their modification state. Unexpectedly, we show that the nucleosome acidic patch is necessary for maximum activity of all ISWI remodellers evaluated. This dependence also extends to CHD and SWI/SNF family remodellers, suggesting that the acidic patch may be generally required for chromatin remodelling. Critically, remodelling activity can be regulated by modifications neighbouring the acidic patch, signifying that it may act as a tunable interaction hotspot for ATP-dependent chromatin remodellers and, by extension, many other chromatin effectors that engage this region of the nucleosome surface.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Ensamble y Desensamble de Cromatina , Nucleosomas/química , Nucleosomas/metabolismo , Especificidad por Sustrato , Factores de Transcripción/metabolismo , Código de Barras del ADN Taxonómico , Histonas/metabolismo , Humanos , Modelos Moleculares , Nucleosomas/genética , Subunidades de Proteína/metabolismoRESUMEN
Protein lysine crotonylation has emerged as an important post-translational modification (PTM) in the regulation of gene transcription through epigenetic mechanisms. Here we introduce a chemical probe, based on a water-soluble phosphine warhead, which reacts with the crotonyl modification. We show that this reagent is complementary to antibody-based tools allowing detection of endogenous cellular proteins such as histones carrying the crotonylation PTM. The tool is also used to show that the histone acylation activity of the transcriptional coactivator, p300, can be activated by pre-existing lysine crotonylation through a positive feedback mechanism. This reagent provides a versatile and sensitive probe for the analysis of this PTM.
Asunto(s)
Proteína p300 Asociada a E1A/análisis , Sondas Moleculares/química , Fosfinas/química , Proteína p300 Asociada a E1A/genética , Proteína p300 Asociada a E1A/metabolismo , Histonas/metabolismo , Humanos , Lisina/metabolismo , Procesamiento Proteico-PostraduccionalRESUMEN
Supramolecular anchoring of transition metal complexes to a protein scaffold is an attractive approach to the construction of artificial metalloenzymes since this is conveniently achieved by self-assembly. Here, we report a novel design for supramolecular artificial metalloenzymes that exploits the promiscuity of the central hydrophobic cavity of the transcription factor Lactococcal multidrug resistance Regulator (LmrR) as a generic binding site for planar coordination complexes that do not provide specific protein binding interactions. The success of this approach is manifested in the excellent enantioselectivities that are achieved in the Cu(II) catalyzed enantioselective Friedel-Crafts alkylation of indoles.
Asunto(s)
Proteínas Bacterianas/química , Materiales Biomiméticos/química , Metaloproteínas/química , Multimerización de Proteína , Lactococcus lactis , Estructura Cuaternaria de ProteínaRESUMEN
Membrane chromatography is routinely used to remove host cell proteins, viral particles, and aggregates during antibody downstream processing. The application of membrane chromatography to the field of antibody-drug conjugates (ADCs) has been applied in a limited capacity and in only specialized scenarios. Here, we utilized the characteristics of the membrane adsorbers, Sartobind® S and Phenyl, for aggregate and payload clearance while polishing the ADC in a single chromatographic run. The Sartobind® S membrane was used in the removal of excess payload, while the Sartobind® Phenyl was used to polish the ADC by clearance of unwanted drug-to-antibody ratio (DAR) species and aggregates. The Sartobind® S membrane reproducibly achieved log-fold clearance of free payload with a 10 membrane-volume wash. Application of the Sartobind® Phenyl decreased aggregates and higher DAR species while increasing DAR homogeneity. The Sartobind® S and Phenyl membranes were placed in tandem to simplify the process in a single chromatographic run. With the optimized binding, washing, and elution conditions, the tandem membrane approach was performed in a shorter timescale with minimum solvent consumption and high yield. The application of the tandem membrane chromatography system presents a novel and efficient purification scheme that can be realized during ADC manufacturing.
RESUMEN
Site creation: Enantioselective artificial metalloenzymes have been created by grafting a new active site onto bovine pancreatic polypeptide through the introduction of an amino acid capable of coordinating a copper(II) ion. This hybrid catalyst gave good enantioselectivities in the Diels-Alder and Michael addition reactions in water (see scheme) and displayed a very high substrate selectivity.
Asunto(s)
Metaloproteínas/química , Polipéptido Pancreático/química , Secuencia de Aminoácidos , Animales , Catálisis , Bovinos , Cobre/química , Datos de Secuencia Molecular , EstereoisomerismoAsunto(s)
Proteínas Bacterianas/química , Cobre/metabolismo , Lactococcus lactis/química , Metaloproteínas/química , Factores de Transcripción/química , 2,2'-Dipiridil/química , 2,2'-Dipiridil/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Ligandos , Metaloproteínas/metabolismo , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Fenantrolinas/química , Fenantrolinas/metabolismo , Multimerización de Proteína , Estereoisomerismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoAsunto(s)
Oligonucleótidos/metabolismo , Tetrahidrofolato Deshidrogenasa/metabolismo , Animales , Secuencia de Bases , Biocatálisis , Ratones , NADP/metabolismo , Hibridación de Ácido Nucleico , Oligonucleótidos/química , Oligonucleótidos/genética , Ingeniería de Proteínas , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Tetrahidrofolato Deshidrogenasa/química , Tetrahidrofolato Deshidrogenasa/genéticaRESUMEN
Artificial metalloenzymes have emerged over the last decades as an attractive approach towards combining homogeneous catalysis and biocatalysis. A wide variety of catalytic transformations have been established by artificial metalloenzymes, thus establishing proof of concept. The field is now slowly transforming to take on new challenges. These include novel designs, novel catalytic reactions, some of which have no equivalent in both homogenous catalysis and biocatalysis and the incorporation of artificial metalloenzymes in chemoenzymatic cascades. Some of these developments represent promising steps towards integrating artificial metalloenzymes in biological systems. This review will focus on advances in this field and perspectives discussed.