ABSTRACT
TRIM proteins constitute a large, diverse and ancient protein family which play a key role in processes including cellular differentiation, autophagy, apoptosis, DNA repair, and tumour suppression. Mostly known and studied through the lens of their ubiquitination activity as E3 ligases, it has recently emerged that many of these proteins are involved in direct RNA binding through their NHL or PRY/SPRY domains. We summarise the current knowledge concerning the mechanism of RNA binding by TRIM proteins and its biological role. We discuss how RNA-binding relates to their previously described functions such as E3 ubiquitin ligase activity, and we will consider the potential role of enrichment in membrane-less organelles.
Subject(s)
RNA/metabolism , Ubiquitin-Protein Ligases/classification , Ubiquitin-Protein Ligases/metabolism , Binding Sites , Humans , RNA/chemistry , Ubiquitin-Protein Ligases/chemistryABSTRACT
Bromodomain and extra-terminal (BET) family of proteins are one of the major readers of epigenetic marks and an important target class in oncology and other disease areas. The importance of the BET family of proteins is manifested by the explosion in the number of inhibitors against these targets that have successfully entered clinical trials. One important BET family member is bromodomain containing protein 4 (BRD4). Structural and biophysical studies of BRD4 are complicated by its tertiary-structure consisting of two bromodomains connected by a flexible inter-domain linker of approximately 180 amino acids. A detailed understanding of the interplay of these bromodomains will be key to rational drug design in BRD4, yet there are no reported three-dimensional structures of the multi-domain BRD4 and NMR studies of the tandem domain are hampered by the size of the protein. Here, we present a method for rapid Sortase A-mediated segmental labelling of the individual bromodomains of BRD4 that provides a powerful strategy that will enable NMR studies of ligand-bromodomain interactions with atomic detail. In our labelling strategy, we have used U-[2H,15N]-isotope labelling on the C-terminal bromodomain with selective introduction of 13CH3 methyl groups on Ile (δ1), Val and Leu, whereas the N-terminal bromodomain remained unlabelled. This labelling scheme resulted in significantly simplified NMR spectra and will allow for high-resolution interaction, structure and dynamics studies in the presence of ligands.
Subject(s)
Aminoacyltransferases , Bacterial Proteins , Cysteine Endopeptidases , Isotope Labeling/methods , Nuclear Proteins/chemistry , Transcription Factors/chemistry , Amino Acid Sequence , Biophysical Phenomena , Cell Cycle Proteins , Humans , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Nuclear Proteins/genetics , Protein Domains , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Transcription Factors/geneticsABSTRACT
Plasmodium falciparum is responsible for causing cerebral malaria in humans. IMP1 is an immunogenic protein, present in the parasite, which has been shown to induce an immune response against apicomplexan parasites in a species-specific manner. Here, we report the complete NMR assignments of PfIMP1.
