Biochemical Society transactions最新文献

Several biomolecular condensates assemble in mammalian cells in response to viral infection. The most studied of these are stress granules (SGs), which have been proposed to promote antiviral innate immune signaling pathways, including the RLR-MAVS, the protein kinase R (PKR), and the OAS-RNase L pathways. However, recent studies have demonstrated that SGs either negatively regulate or do not impact antiviral signaling. Instead, the SG-nucleating protein, G3BP1, may function to perturb viral RNA biology by condensing viral RNA into viral-aggregated RNA condensates, thus explaining why viruses often antagonize G3BP1 or hijack its RNA condensing function. However, a recently identified condensate, termed double-stranded RNA-induced foci, promotes the activation of the PKR and OAS-RNase L antiviral pathways. In addition, SG-like condensates known as an RNase L-induced bodies (RLBs) have been observed during many viral infections, including SARS-CoV-2 and several flaviviruses. RLBs may function in promoting decay of cellular and viral RNA, as well as promoting ribosome-associated signaling pathways. Herein, we review these recent advances in the field of antiviral biomolecular condensates, and we provide perspective on the role of canonical SGs and G3BP1 during the antiviral response.

Pulmonary arterial hypertension (PAH) is a rare and life-threatening vascular disorder, characterised by abnormal remodelling of the pulmonary vessels and elevated pulmonary artery pressure, leading to right ventricular hypertrophy and right-sided heart failure. The importance of bone morphogenetic protein (BMP) signalling in the pathogenesis of PAH is demonstrated by human genetic studies. Many PAH risk genes are involved in the BMP signalling pathway and are highly expressed or preferentially act on vascular endothelial cells. Endothelial dysfunction is recognised as an initial trigger for PAH, and endothelial BMP signalling plays a crucial role in the maintenance of endothelial integrity. BMPR2 is the most prevalent PAH gene, found in over 80% of heritable cases. As BMPRII protein is the major type II receptor for a large family of BMP ligands and expressed ubiquitously in many tissues, dysregulated BMP signalling in other cells may also contribute to PAH pathobiology. Sotatercept, which contains the extracellular domain of another transforming growth factor-β family type II receptor ActRIIA fused to immunoglobin Fc domain, was recently approved by the FDA as a treatment for PAH. Neither its target cells nor its mechanism of action is fully understood. This review will revisit BMPRII function and its extracellular regulation, summarise how dysregulated BMP signalling in endothelial cells and smooth muscle cells may contribute to PAH pathogenesis, and discuss how novel therapeutics targeting the extracellular regulation of BMP signalling, such as BMP9 and Sotatercept, can be related to restoring BMPRII function.

The long non-coding RNA (lncRNA) Xist is crucially involved in a process called X chromosome inactivation (XCI), the transcriptional silencing of one of the two X chromosomes in female mammals to achieve X dosage compensation between the sexes. Because Xist RNA silences the X chromosome from which it is transcribed, the activation of Xist transcription marks the initiation of the XCI process and thus, mechanisms and players that activate this gene are of central importance to the XCI process. During female mouse embryogenesis, XCI occurs in two steps. At the 2-4 cell stages imprinted XCI (iXCI) silences exclusively the paternally inherited X chromosome (Xp). While extraembryonic cells including trophoblasts keep the Xp silenced, epiblast cells that give rise to the embryo proper reactivate the Xp and undergo random XCI (rXCI) around implantation. Both iXCI and rXCI are dependent on Xist. Rlim, also known as Rnf12, is an X-linked E3 ubiquitin ligase that is involved in the transcriptional activation of Xist. However, while data on the crucial involvement of Rlim during iXCI appear clear, its role in rXCI has been controversial. This review discusses data leading to this disagreement and recent evidence for a regulatory switch of Xist transcription in epiblasts of implanting embryos, partially reconciling the roles of Rlim during Xist activation.

Imprinted genes provide an attractive paradigm to unravel links between transcription and genome architecture. The parental allele-specific expression of these essential genes - which are clustered in chromosomal domains - is mediated by parental methylation imprints at key regulatory DNA sequences. Recent chromatin conformation capture (3C)-based studies show differential organization of topologically associating domains between the parental chromosomes at imprinted domains, in embryonic stem and differentiated cells. At several imprinted domains, differentially methylated regions show allelic binding of the insulator protein CTCF, and linked focal retention of cohesin, at the non-methylated allele only. This generates differential patterns of chromatin looping between the parental chromosomes, already in the early embryo, and thereby facilitates the allelic gene expression. Recent research evokes also the opposite scenario, in which allelic transcription contributes to the differential genome organization, similarly as reported for imprinted X chromosome inactivation. This may occur through epigenetic effects on CTCF binding, through structural effects of RNA Polymerase II, or through imprinted long non-coding RNAs that have chromatin repressive functions. The emerging picture is that epigenetically-controlled differential genome architecture precedes and facilitates imprinted gene expression during development, and that at some domains, conversely, the mono-allelic gene expression also influences genome architecture.

Legumes house nitrogen-fixing endosymbiotic rhizobia in specialised polyploid cells within root nodules. This results in a mutualistic relationship whereby the plant host receives fixed nitrogen from the bacteria in exchange for dicarboxylic acids. This plant-microbe interaction requires the regulation of multiple metabolic and physiological processes in both the host and symbiont in order to achieve highly efficient symbiosis. Recent studies have showed that the success of symbiosis is influenced by the circadian clock of the plant host. Medicago and soybean plants with altered clock mechanisms showed compromised nodulation and reduced plant growth. Furthermore, transcriptomic analyses revealed that multiple genes with key roles in recruitment of rhizobia to plant roots, infection and nodule development were under circadian control, suggesting that appropriate timing of expression of these genes may be important for nodulation. There is also evidence for rhythmic gene expression of key nitrogen fixation genes in the rhizobium symbiont, and temporal coordination between nitrogen fixation in the bacterial symbiont and nitrogen assimilation in the plant host may be important for successful symbiosis. Understanding of how circadian regulation impacts on nodule establishment and function will identify key plant-rhizobial connections and regulators that could be targeted to increase the efficiency of this relationship.

Chemokine receptors are integral to the immune system and prime targets in drug discovery that have undergone extensive structural elucidation in recent years. We outline a timeline of these structural achievements, discuss the intracellular negative allosteric modulation of chemokine receptors, analyze the mechanisms of orthosteric receptor activation, and report on the emerging concept of biased signaling. Additionally, we highlight differences of G-protein binding among chemokine receptors. Intracellular allosteric modulators in chemokine receptors interact with a conserved motif within transmembrane helix 7 and helix 8 and exhibit a two-fold inactivation mechanism that can be harnessed for drug-discovery efforts. Chemokine recognition is a multi-step process traditionally explained by a two-site model within chemokine recognition site 1 (CRS1) and CRS2. Recent structural studies have extended our understanding of this complex mechanism with the identification of CRS1.5 and CRS3. CRS3 is implicated in determining ligand specificity and surrounds the chemokine by almost 180°. Within CRS3 we identified the extracellular loop 2 residue 45.51 as a key interaction mediator for chemokine binding. Y2917.43 on the other hand was shown in CCR1 to be a key determinant of signaling bias which, along with specific chemokine-dependent phosphorylation ensembles at the G-protein coupled receptors (GPCR's) C-terminus, seems to play a pivotal role in determining the direction of signal bias in GPCRs.

Sample preparation can present a significant hurdle within single particle cryo-electron microscopy (cryoEM), resulting in issues with reproducibility, data quality or an inability to visualise the sample. There are several factors which can influence this, including sample or buffer composition, grid type, route of sample preparation and interactions with the air-water interface (AWI). Here, we review some of the current routes for sample preparation and the associated challenges. We discuss a range of approaches for overcoming these challenges, such as minimising the grid preparation time, surfactants, grid type and biochemical approaches such as nanomagnetic beads. Finally, we discuss how a set of commercially available protein samples may serve as a benchmark suite for future technologies. This provides a route to compare techniques' abilities not just to generate high-resolution structures but also to overcome the challenges traditionally associated with cryoEM. As the field continues to produce new approaches to sample preparation and we start to better understand the underlying principles behind the behaviour of proteins within a thin film and in response to different environments, especially grid composition, it is hoped that more universal solutions can be provided that make the intractable systems tractable, improve resolution and, importantly, speed up data collection and reduce the currently required dataset sizes.

Reproducible tissue morphology is a fundamental feature of embryonic development. To ensure such robustness during tissue morphogenesis, inherent noise in biological processes must be buffered. While redundant genes, parallel signaling pathways and intricate network topologies are known to reduce noise, over the last few years, mechanical properties of tissues have been shown to play a vital role. Here, taking the example of somite shape changes, I will discuss how tissues are highly plastic in their ability to change shapes leading to increased precision and reproducibility.

Conformational changes of catalytically-important structural elements are a key feature of the regulation mechanisms of protein kinases and are important for dictating inhibitor binding modes and affinities. The lack of widely applicable methods for tracking kinase conformational changes in solution has hindered our understanding of kinase regulation and our ability to design conformationally selective inhibitors. Here we provide an overview of two recently developed methods that detect conformational changes of the regulatory activation loop and αC-helix of kinases and that yield complementary information about allosteric mechanisms. An intramolecular Förster resonance energy transfer-based approach provides a scalable platform for detecting and classifying structural changes in high-throughput, as well as quantifying ligand binding cooperativity, shedding light on the energetics governing allostery. The pulsed electron paramagnetic resonance technique double electron-electron resonance provides lower throughput but higher resolution information on structural changes that allows for unambiguous assignment of conformational states and quantification of population shifts. Together, these methods are shedding new light on kinase regulation and drug interactions and providing new routes for the identification of novel kinase inhibitors and allosteric modulators.

Mitochondrial DNA replication is initiated by the transcription of mitochondrial RNA polymerase (mtRNAP), as mitochondria lack a dedicated primase. However, the mechanism determining the switch between continuous transcription and premature termination to generate RNA primers for mitochondrial DNA (mtDNA) replication remains unclear. The pentatricopeptide repeat domain of mtRNAP exhibits exoribonuclease activity, which is required for the initiation of mtDNA replication in Drosophila. In this review, we explain how this exonuclease activity contributes to primer synthesis in strand-coupled mtDNA replication, and discuss how its regulation might co-ordinate mtDNA replication and transcription in both Drosophila and mammals.