Phosphorylation-dependent BRD4 dimerization and implications for therapeutic inhibition of BET family proteins.

Funder: AstraZenecaFunder: AstraZeneca postdoc fundBromodomain-containing protein 4 (BRD4) is an epigenetic reader and oncology drug target that regulates gene transcription through binding to acetylated chromatin via bromodomains. Phosphorylation by casein kinase II (CK2) regulates BRD4 function, is necessary for active transcription and is involved in resistance to BRD4 drug inhibition in triple-negative breast cancer. Here, we provide the first biophysical analysis of BRD4 phospho-regulation. Using integrative structural biology, we show that phosphorylation by CK2 modulates the dimerization of human BRD4. We identify two conserved regions, a coiled-coil motif and the Basic-residue enriched Interaction Domain (BID), essential for the BRD4 structural rearrangement, which we term the phosphorylation-dependent dimerization domain (PDD). Finally, we demonstrate that bivalent inhibitors induce a conformational change within BRD4 dimers in vitro and in cancer cells. Our results enable the proposal of a model for BRD4 activation critical for the characterization of its protein-protein interaction network and for the development of more specific therapeutics

A distinct Golgi-targeting mechanism of dGM130 in Drosophila neurons

GM130 is a matrix protein that is conserved in metazoans and involved in the architecture of the Golgi apparatus. In neurons, Golgi apparatus and dendritic Golgi outposts (GOs) have different compartmental organizations, and GM130 localization is present in both, indicating that GM130 has a unique Golgi-targeting mechanism. Here, we investigated the Golgi-targeting mechanism of the GM130 homologue, dGM130, using in vivo imaging of Drosophila dendritic arborization (da) neurons. The results showed that two independent Golgi-targeting domains (GTDs) with different Golgi localization characteristics in dGM130, together determined the precise localization of dGM130 in both the soma and dendrites. GTD1, covering the first coiled-coil region, preferentially targeted to somal Golgi rather than GOs; whereas GTD2, containing the second coiled-coil region and C-terminus, dynamically targeted to Golgi in both soma and dendrites. These findings suggest that there are two distinct mechanisms by which dGM130 targets to the Golgi apparatus and GOs, underlying the structural differences between them, and further provides new insights into the formation of neuronal polarity

RNF41 interacts with the VPS52 subunit of the GARP and EARP complexes

RNF41 (Ring Finger Protein 41) is an E3 ubiquitin ligase involved in the intracellular sorting and function of a diverse set of substrates. Next to BRUCE and Parkin, RNF41 can directly ubiquitinate ErbB3, IL-3, EPO and RARa receptors or downstream signaling molecules such as Myd88, TBK1 and USP8. In this way it can regulate receptor signaling and routing. To further elucidate the molecular mechanism behind the role of RNF41 in intracellular transport we performed an Array MAPPIT (Mammalian Protein-Protein Interaction Trap) screen using an extensive set of proteins derived from the human ORFeome collection. This paper describes the identification of VPS52, a subunit of the GARP (Golgi-Associated Retrograde Protein) and the EARP (Endosome-Associated Recycling Protein) complexes, as a novel interaction partner of RNF41. Through interaction via their coiled coil domains, RNF41 ubiquitinates and relocates VPS52 away from VPS53, a common subunit of the GARP and EARP complexes, towards RNF41 bodies

Tunable thermal bioswitches for in vivo control of microbial therapeutics

Temperature is a unique input signal that could be used by engineered microbial therapeutics to sense and respond to host conditions or spatially targeted external triggers such as focused ultrasound. To enable these possibilities, we present two families of tunable, orthogonal, temperature-dependent transcriptional repressors providing switch-like control of bacterial gene expression at thresholds spanning the biomedically relevant range of 32–46°C. We integrate these molecular bioswitches into thermal logic circuits and demonstrate their utility in three in vivo microbial therapy scenarios, including spatially precise activation using focused ultrasound, modulation of activity in response to a host fever, and self-destruction after fecal elimination to prevent environmental escape. This technology provides a critical capability for coupling endogenous or applied thermal signals to cellular function in basic research, biomedical and industrial applications

Biochemical properties of bacterial reverse transcriptase-related (rvt) gene products : multimerization, protein priming, and nucleotide preference

Author Posting. © The Author(s), 2018. This is the author's version of the work. It is posted here by permission of Springer for personal use, not for redistribution. The definitive version was published in Current Genetics 64 (2018): 1287-1301, doi:10.1007/s00294-018-0844-6.Cellular reverse transcriptase-related (rvt) genes represent a novel class of reverse transcriptases (RTs), which are only distantly related to RTs of retrotransposons and retroviruses, but, similarly to telomerase RTs, are immobilized in the genome as single-copy genes. They have been preserved by natural selection throughout the evolutionary history of large taxonomic groups, including most fungi, a few plants and invertebrates, and even certain bacteria, being the only RTs present across different domains of life. Bacterial rvt genes are exceptionally rare but phylogenetically related, consistent with common origin of bacterial rvt genes rather than eukaryote-to-bacteria transfer. To investigate biochemical properties of bacterial RVTs, we conducted in vitro studies of recombinant HaRVT protein from the filamentous gliding bacterium Herpetosiphon aurantiacus (Chloroflexi). Although HaRVT does not utilize externally added standard primer-template combinations, in the presence of divalent manganese it can polymerize very short products, using dNTPs rather than NTPs, with a strong preference for dCTP incorporation. Further, we investigated the highly conserved N- and C-terminal domains, which distinguish RVT proteins from other RTs. We show that the N-terminal coiled-coil motif, which is present in nearly all RVTs, is responsible for the ability of HaRVT to multimerize in solution, forming up to octamers. The C-terminal domain may be capable of protein priming, which is abolished by site-directed mutagenesis of the catalytic aspartate and greatly reduced in the absence of the conserved tyrosine residues near the C-terminus. The unusual biochemical properties displayed by RVT in vitro will provide the basis for understanding its biological function in vivo.This work was supported by the U.S. National Science Foundation grant MCB-1121334 to I.A.2019-05-1

The genome of the Pacific oyster Crassostrea gigas brings new insights on the massive expansion of the C1q gene family in Bivalvia

C1q domain-containing (C1qDC) proteins are regarded as important players in the innate immunity of bivalve mollusks and other invertebrates and their highly adaptive binding properties indicate them as efficient pathogen recognition molecules. Although experimental studies support this view, the molecular data available at the present time are not sufficient to fully explain the great molecular diversification of this family, present in bivalves with hundreds of C1q coding genes. Taking advantage of the fully sequenced genome of the Pacific oyster Crassostrea gigas and more than 100 transcriptomic datasets, we: (i) re-annotated the oyster C1qDC loci, thus identifying the correct genomic organization of 337 C1qDC genes, (ii) explored the expression pattern of oyster C1qDC genes in diverse developmental stages and adult tissues of unchallenged and experimentally treated animals; (iii) investigated the expansion of the C1qDC gene family in all major bivalve subclasses.Overall, we provide a broad description of the functionally relevant features of oyster C1qDC genes, their comparative expression levels and new evidence confirming that a gene family expansion event has occurred during the course of Bivalve evolution, leading to the diversification of hundreds of different C1qDC genes in both the Pteriomorphia and Heterodonta subclasses

CCBuilder:An interactive web-based tool for building, designing and assessing coiled-coil protein assemblies

Motivation: The ability to accurately model protein structures at the atomistic level underpins efforts to understand protein folding, to engineer natural proteins predictably and to design proteins de novo . Homology-based methods are well established and produce impressive results. However, these are limited to structures presented by and resolved for natural proteins. Addressing this problem more widely and deriving truly ab initio models requires mathematical descriptions for protein folds; the means to decorate these with natural, engineered or de novo sequences; and methods to score the resulting models. Results: We present CCBuilder, a web-based application that tackles the problem for a defined but large class of protein structure, the α-helical coiled coils. CCBuilder generates coiled-coil backbones, builds side chains onto these frameworks and provides a range of metrics to measure the quality of the models. Its straightforward graphical user interface provides broad functionality that allows users to build and assess models, in which helix geometry, coiled-coil architecture and topology and protein sequence can be varied rapidly. We demonstrate the utility of CCBuilder by assembling models for 653 coiled-coil structures from the PDB, which cover >96% of the known coiled-coil types, and by generating models for rarer and de novo coiled-coil structures. Availability and implementation: CCBuilder is freely available, without registration, at http://coiledcoils.chm.bris.ac.uk/app/cc_builder

Electron Microscopy Structural Insights into CPAP Oligomeric Behavior:A Plausible Assembly Process of a Supramolecular Scaffold of the Centrosome

Centrosomal P4.1-associated protein (CPAP) is a cell cycle regulated protein fundamental for centrosome assembly and centriole elongation. In humans, the region between residues 897–1338 of CPAP mediates interactions with other proteins and includes a homodimerization domain. CPAP mutations cause primary autosomal recessive microcephaly and Seckel syndrome. Despite of the biological/clinical relevance of CPAP, its mechanistic behavior remains unclear and its C-terminus (the G-box/TCP domain) is the only part whose structure has been solved. This situation is perhaps due in part to the challenges that represent obtaining the protein in a soluble, homogeneous state for structural studies. Our work constitutes a systematic structural analysis on multiple oligomers of HsCPAP897−1338, using single-particle electron microscopy (EM) of negatively stained (NS) samples. Based on image classification into clearly different regular 3D maps (putatively corresponding to dimers and tetramers) and direct observation of individual images representing other complexes of HsCPAP897−1338 (i.e., putative flexible monomers and higher-order multimers), we report a dynamic oligomeric behavior of this protein, where different homo-oligomers coexist in variable proportions. We propose that dimerization of the putative homodimer forms a putative tetramer which could be the structural unit for the scaffold that either tethers the pericentriolar material to centrioles or promotes procentriole elongation. A coarse fitting of atomic models into the NS 3D maps at resolutions around 20 Å is performed only to complement our experimental data, allowing us to hypothesize on the oligomeric composition of the different complexes. In this way, the current EM work represents an initial step toward the structural characterization of different oligomers of CPAP, suggesting further insights to understand how this protein works, contributing to the elucidation of control mechanisms for centriole biogenesis.This work was supported by the Comunidad de Madrid through grant CAM (S2010/BMD- 2305) and the Spanish Ministry of Economy and Competitiveness through Grants AIC-A-2011-0638 and BIO2013-44647-R.Peer reviewedPeer Reviewe

Tunable thermal bioswitches for in vivo control of microbial therapeutics

Temperature is a unique input signal that could be used by engineered microbial therapeutics to sense and respond to host conditions or spatially targeted external triggers such as focused ultrasound. To enable these possibilities, we present two families of tunable, orthogonal, temperature-dependent transcriptional repressors providing switch-like control of bacterial gene expression at thresholds spanning the biomedically relevant range of 32–46°C. We integrate these molecular bioswitches into thermal logic circuits and demonstrate their utility in three in vivo microbial therapy scenarios, including spatially precise activation using focused ultrasound, modulation of activity in response to a host fever, and self-destruction after fecal elimination to prevent environmental escape. This technology provides a critical capability for coupling endogenous or applied thermal signals to cellular function in basic research, biomedical and industrial applications