Rapid reconstitution of ubiquitinated nucleosome using a non-denatured histone octamer ubiquitylation approach.

Background: Histone ubiquitination modification is emerging as a critical epigenetic mechanism involved in a range of biological processes. In vitro reconstitution of ubiquitinated nucleosomes is pivotal for elucidating the influence of histone ubiquitination on chromatin dynamics.

Results: In this study, we introduce a Non-Denatured Histone Octamer Ubiquitylation (NDHOU) approach for generating ubiquitin or ubiquitin-like modified histone octamers.

Development of high affinity broadly reactive aptamers for spike protein of multiple SARS-CoV-2 variants.

We have developed broadly reactive aptamers against multiple variants by alternating the target between spike proteins from different SARS-CoV-2 variants during the selection process. In this process we have developed aptamers which can recognise all variants, from the original wild-type 'Wuhan' strain to Omicron, with high affinity ( values in the pM range).

Reversible structural changes in the influenza hemagglutinin precursor at membrane fusion pH.

The subunits of the influenza hemagglutinin (HA) trimer are synthesized as single-chain precursors (HA0s) that are proteolytically cleaved into the disulfide-linked polypeptides HA1 and HA2. Cleavage is required for activation of membrane fusion at low pH, which occurs at the beginning of infection following transfer of cell-surface-bound viruses into endosomes. Activation results in extensive changes in the conformation of cleaved HA.

Evolution of the SARS-CoV-2 spike protein in the human host.

Recently emerged variants of SARS-CoV-2 contain in their surface spike glycoproteins multiple substitutions associated with increased transmission and resistance to neutralising antibodies. We have examined the structure and receptor binding properties of spike proteins from the B.1.

The effect of the D614G substitution on the structure of the spike glycoprotein of SARS-CoV-2.

The majority of currently circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viruses have mutant spike glycoproteins that contain the D614G substitution. Several studies have suggested that spikes with this substitution are associated with higher virus infectivity. We use cryo-electron microscopy to compare G614 and D614 spikes and show that the G614 mutant spike adopts a range of more open conformations that may facilitate binding to the SARS-CoV-2 receptor, ACE2, and the subsequent structural rearrangements required for viral membrane fusion.

Structure and binding properties of Pangolin-CoV spike glycoprotein inform the evolution of SARS-CoV-2.

Coronaviruses of bats and pangolins have been implicated in the origin and evolution of the pandemic SARS-CoV-2. We show that spikes from Guangdong Pangolin-CoVs, closely related to SARS-CoV-2, bind strongly to human and pangolin ACE2 receptors. We also report the cryo-EM structure of a Pangolin-CoV spike protein and show it adopts a fully-closed conformation and that, aside from the Receptor-Binding Domain, it resembles the spike of a bat coronavirus RaTG13 more than that of SARS-CoV-2.

Antibody-mediated disruption of the SARS-CoV-2 spike glycoprotein.

The CR3022 antibody, selected from a group of SARS-CoV monoclonal antibodies for its ability to cross-react with SARS-CoV-2, has been examined for its ability to bind to the ectodomain of the SARS-CoV-2 spike glycoprotein. Using cryo-electron microscopy we show that antibody binding requires rearrangements in the S1 domain that result in dissociation of the spike.

Receptor binding and priming of the spike protein of SARS-CoV-2 for membrane fusion.

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is initiated by virus binding to the ACE2 cell-surface receptors, followed by fusion of the virus and cell membranes to release the virus genome into the cell. Both receptor binding and membrane fusion activities are mediated by the virus spike glycoprotein. As with other class-I membrane-fusion proteins, the spike protein is post-translationally cleaved, in this case by furin, into the S1 and S2 components that remain associated after cleavage.

Author Correction: SARS-CoV-2 and bat RaTG13 spike glycoprotein structures inform on virus evolution and furin-cleavage effects.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Hemagglutinin Structure and Activities.

Hemagglutinins (HAs) are the receptor-binding and membrane fusion glycoproteins of influenza viruses. They recognize sialic acid-containing, cell-surface glycoconjugates as receptors but have limited affinity for them, and, as a consequence, virus attachment to cells requires their interaction with several virus HAs. Receptor-bound virus is transferred into endosomes where membrane fusion by HAs is activated at pH between 5 and 6.

Structural transitions in influenza haemagglutinin at membrane fusion pH.

Infection by enveloped viruses involves fusion of their lipid envelopes with cellular membranes to release the viral genome into cells. For HIV, Ebola, influenza and numerous other viruses, envelope glycoproteins bind the infecting virion to cell-surface receptors and mediate membrane fusion. In the case of influenza, the receptor-binding glycoprotein is the haemagglutinin (HA), and following receptor-mediated uptake of the bound virus by endocytosis, it is the HA that mediates fusion of the virus envelope with the membrane of the endosome.

Author Correction: G-tract RNA removes Polycomb repressive complex 2 from genes.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

G-tract RNA removes Polycomb repressive complex 2 from genes.

Polycomb repressive complex 2 (PRC2) maintains repression of cell-type-specific genes but also associates with genes ectopically in cancer. While it is currently unknown how PRC2 is removed from genes, such knowledge would be useful for the targeted reversal of deleterious PRC2 recruitment events. Here, we show that G-tract RNA specifically removes PRC2 from genes in human and mouse cells.

Influenza hemagglutinin membrane anchor.

Viruses with membranes fuse them with cellular membranes, to transfer their genomes into cells at the beginning of infection. For Influenza virus, the membrane glycoprotein involved in fusion is the hemagglutinin (HA), the 3D structure of which is known from X-ray crystallographic studies. The soluble ectodomain fragments used in these studies lacked the "membrane anchor" portion of the molecule.

The structure of the RbBP5 β-propeller domain reveals a surface with potential nucleic acid binding sites.

The multi-protein complex WRAD, formed by WDR5, RbBP5, Ash2L and Dpy30, binds to the MLL SET domain to stabilize the catalytically active conformation required for histone H3K4 methylation. In addition, the WRAD complex contributes to the targeting of the activated complex to specific sites on chromatin. RbBP5 is central to MLL catalytic activation, by making critical contacts with the other members of the complex.

Phosphorylation of AMPK by upstream kinases is required for activity in mammalian cells.

AMP-activated protein kinase (AMPK) plays a major role in regulating metabolism and has attracted significant attention as a therapeutic target for treating metabolic disorders. AMPK activity is stimulated more than 100-fold by phosphorylation of threonine 172 (Thr). Binding of AMP to the γ subunit allosterically activates the kinase.

Comment on "Structural basis of histone H3K27 trimethylation by an active polycomb repressive complex 2".

Jiao and Liu (Research Articles, 16 October 2015, aac4383) reported the crystal structure of the protein complex polycomb repressive complex 2 from Chaetomium thermophilum This landmark structure has brought invaluable insights into the activation mechanism of this essential methyltransferase. However, the analysis of the x-ray data discussed below suggests that the description of oncogenic H3K27M peptide binding to the active site is incorrect.

Identification of (R)-N-((4-Methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-methyl-1-(1-(1-(2,2,2-trifluoroethyl)piperidin-4-yl)ethyl)-1H-indole-3-carboxamide (CPI-1205), a Potent and Selective Inhibitor of Histone Methyltransferase EZH2, Suitable for Phase I Clinical Trials for B-Cell Lymphomas.

Polycomb repressive complex 2 (PRC2) has been shown to play a major role in transcriptional silencing in part by installing methylation marks on lysine 27 of histone 3. Dysregulation of PRC2 function correlates with certain malignancies and poor prognosis. EZH2 is the catalytic engine of the PRC2 complex and thus represents a key candidate oncology target for pharmacological intervention.

Structural basis of oncogenic histone H3K27M inhibition of human polycomb repressive complex 2.

Polycomb repressive complex 2 (PRC2) silences gene expression through trimethylation of K27 of histone H3 (H3K27me3) via its catalytic SET domain. A missense mutation in the substrate of PRC2, histone H3K27M, is associated with certain pediatric brain cancers and is linked to a global decrease of H3K27me3 in the affected cells thought to be mediated by inhibition of PRC2 activity. We present here the crystal structure of human PRC2 in complex with the inhibitory H3K27M peptide bound to the active site of the SET domain, with the methionine residue located in the pocket that normally accommodates the target lysine residue.

Evolving Catalytic Properties of the MLL Family SET Domain.

Methylation of histone H3 lysine-4 is a hallmark of chromatin associated with active gene expression. The activity of H3K4-specific modification enzymes, in higher eukaryotes the MLL (or KMT2) family, is tightly regulated. The MLL family has six members, each with a specialized function.

Structures of complexes formed by H5 influenza hemagglutinin with a potent broadly neutralizing human monoclonal antibody.

H5N1 avian influenza viruses remain a threat to public health mainly because they can cause severe infections in humans. These viruses are widespread in birds, and they vary in antigenicity forming three major clades and numerous antigenic variants. The most important features of the human monoclonal antibody FLD194 studied here are its broad specificity for all major clades of H5 influenza HAs, its high affinity, and its ability to block virus infection, in vitro and in vivo.