Jarid2 Methylation via the PRC2 Complex Regulates H3K27me3 Deposition during Cell Differentiation.

Polycomb Group (PcG) proteins maintain transcriptional repression throughout development, mostly by regulating chromatin structure. Polycomb Repressive Complex 2 (PRC2), a component of the Polycomb machinery, is responsible for the methylation of histone H3 lysine 27 (H3K27me2/3). Jarid2 was previously identified as a cofactor of PRC2, regulating PRC2 targeting to chromatin and its enzymatic activity. Deletion of Jarid2 leads to impaired orchestration of gene expression during cell lineage commitment. Here, we reveal an unexpected crosstalk between Jarid2 and PRC2, with Jarid2 being methylated by PRC2. This modification is recognized by the Eed core component of PRC2 and triggers an allosteric activation of PRC2's enzymatic activity. We show that Jarid2 methylation is important to promote PRC2 activity at a locus devoid of H3K27me3 and for the correct deposition of this mark during cell differentiation. Our results uncover a regulation loop where Jarid2 methylation fine-tunes PRC2 activity depending on the chromatin context.

Hypoxic regulation of hand1 controls the fetal-neonatal switch in cardiac metabolism.

Cardiomyocytes are vulnerable to hypoxia in the adult, but adapted to hypoxia in utero. Current understanding of endogenous cardiac oxygen sensing pathways is limited. Myocardial oxygen consumption is determined by regulation of energy metabolism, which shifts from glycolysis to lipid oxidation soon after birth, and is reversed in failing adult hearts, accompanying re-expression of several "fetal" genes whose role in disease phenotypes remains unknown. Here we show that hypoxia-controlled expression of the transcription factor Hand1 determines oxygen consumption by inhibition of lipid metabolism in the fetal and adult cardiomyocyte, leading to downregulation of mitochondrial energy generation. Hand1 is under direct transcriptional control by HIF1α. Transgenic mice prolonging cardiac Hand1 expression die immediately following birth, failing to activate the neonatal lipid metabolising gene expression programme. Deletion of Hand1 in embryonic cardiomyocytes results in premature expression of these genes. Using metabolic flux analysis, we show that Hand1 expression controls cardiomyocyte oxygen consumption by direct transcriptional repression of lipid metabolising genes. This leads, in turn, to increased production of lactate from glucose, decreased lipid oxidation, reduced inner mitochondrial membrane potential, and mitochondrial ATP generation. We found that this pathway is active in adult cardiomyocytes. Up-regulation of Hand1 is protective in a mouse model of myocardial ischaemia. We propose that Hand1 is part of a novel regulatory pathway linking cardiac oxygen levels with oxygen consumption. Understanding hypoxia adaptation in the fetal heart may allow development of strategies to protect cardiomyocytes vulnerable to ischaemia, for example during cardiac ischaemia or surgery.

Advances in Fmoc solid-phase peptide synthesis.

Today, Fmoc SPPS is the method of choice for peptide synthesis. Very-high-quality Fmoc building blocks are available at low cost because of the economies of scale arising from current multiton production of therapeutic peptides by Fmoc SPPS. Many modified derivatives are commercially available as Fmoc building blocks, making synthetic access to a broad range of peptide derivatives straightforward. The number of synthetic peptides entering clinical trials has grown continuously over the last decade, and recent advances in the Fmoc SPPS technology are a response to the growing demand from medicinal chemistry and pharmacology. Improvements are being continually reported for peptide quality, synthesis time and novel synthetic targets. Topical peptide research has contributed to a continuous improvement and expansion of Fmoc SPPS applications.

A backbone amide protecting group for overcoming difficult sequences and suppressing aspartimide formation.

A backbone amide bond protecting group, 2-hydroxy-4-methoxy-5-nitrobenzyl (Hmnb), improved the synthesis of aggregation and aspartimide-prone peptides. Introduction of Hmnb is automated and carried out during peptide assembly by addition of 4-methoxy-5-nitrosalicylaldehyde to the peptidyl-resin and on-resin reduction to the secondary amine. Acylation of the hindered secondary amine is aided by the formation of an internal nitrophenol ester that undergoes a favourable O,N intramolecular acyl transfer. This activated ester participates in the coupling and generally gives complete reaction with standard coupling conditions. Hmnb is easily available in a single preparative step from commercially available material. Different methods for removing the amide protecting group were explored. The protecting group is labile to acidolysis, following reduction of the nitro group to the aniline. The two main uses of backbone protection of preventing aspartimide formation and of overcoming difficult sequences are demonstrated, first with the synthesis of a challenging aspartimide-prone test sequence and then with the classic difficult sequence ACP (65-74) and a 23-mer homopolymer of polyalanine.

HF-Free Boc Synthesis of Peptide Thioesters for Ligation and Cyclization.

We have developed a convenient method for the direct synthesis of peptide thioesters, versatile intermediates for peptide ligation and cyclic peptide synthesis. The technology uses a modified Boc SPPS strategy that avoids the use of anhydrous HF. Boc in situ neutralization protocols are used in combination with Merrifield hydroxymethyl resin and TFA/TMSBr cleavage. Avoiding HF extends the scope of Boc SPPS to post-translational modifications that are compatible with the milder cleavage conditions, demonstrated here with the synthesis of the phosphorylated protein CHK2. Peptide thioesters give easy, direct, access to cyclic peptides, illustrated by the synthesis of cyclorasin, a KRAS inhibitor.

Exploiting the defensive sugars of HIV-1 for drug and vaccine design.

The sustained effort towards developing an antibody vaccine against HIV/AIDS has provided much of our understanding of viral immunology. It is generally accepted that one of the main barriers to antibody neutralization of HIV is the array of protective structural carbohydrates that covers the antigens on the virus's surface. Intriguingly, however, recent findings suggest that these carbohydrates, which have evolved to protect HIV and promote its transmission, are also attractive therapeutic targets.

Crystal structures of histone demethylase JMJD2A reveal basis for substrate specificity.

Post-translational histone modification has a fundamental role in chromatin biology and is proposed to constitute a 'histone code' in epigenetic regulation. Differential methylation of histone H3 and H4 lysyl residues regulates processes including heterochromatin formation, X-chromosome inactivation, genome imprinting, DNA repair and transcriptional regulation. The discovery of lysyl demethylases using flavin (amine oxidases) or Fe(II) and 2-oxoglutarate as cofactors (2OG oxygenases) has changed the view of methylation as a stable epigenetic marker. However, little is known about how the demethylases are selective for particular lysyl-containing sequences in specific methylation states, a key to understanding their functions. Here we reveal how human JMJD2A (jumonji domain containing 2A), which is selective towards tri- and dimethylated histone H3 lysyl residues 9 and 36 (H3K9me3/me2 and H3K36me3/me2), discriminates between methylation states and achieves sequence selectivity for H3K9. We report structures of JMJD2A-Ni(II)-Zn(II) inhibitor complexes bound to tri-, di- and monomethyl forms of H3K9 and the trimethyl form of H3K36. The structures reveal a lysyl-binding pocket in which substrates are bound in distinct bent conformations involving the Zn-binding site. We propose a mechanism for achieving methylation state selectivity involving the orientation of the substrate methyl groups towards a ferryl intermediate. The results suggest distinct recognition mechanisms in different demethylase subfamilies and provide a starting point to develop chemical tools for drug discovery and to study and dissect the complexity of reversible histone methylation and its role in chromatin biology.

On welfare pluralism, social policy and the contribution of sociology: Revisiting Robert Pinker.

On occasion it makes sound sense to undertake a retrospective review of a late colleague's contribution to his or her subject area. This applies to Robert Pinker, Professor of Social Administration at the London School of Economics, who died at the age of 89 in February 2021. Over a long life he made a major impact on working for press freedom and to social work studies, but this article concerns his work on social policy, and particularly on the idea of welfare pluralism, a many-faceted idea the exploration of which powered two pathbreaking books Social Theory and Social Policy (1971) and The Idea of Welfare (1979). In the twentieth century many states including the United Kingdom had greatly expanded their welfare provisions for their citizens, and, in some, an academic subject area, often called social administration or social policy had grown in response. Pinker started writing in the 1960s, dissatisfied with the conventional approach of Richard Titmuss and others, almost exclusively concerned with the state and welfare. He made the case for a radical rebalance toward including everyday experiences of obligations and how familial informal welfare practices are strengthened, weakened or modified by formal social services. However, ahead of his time, Pinker was arguing for an enhanced sociological imagination in the study of social policy and on the very idea of "welfare". This article has sections reflecting the facets of Pinker's thinking about welfare pluralism, including "social policy's past", "exchange and stigma", "taking informal welfare seriously", "divergent views of altruism", "comparative studies", "on a mixture of means to welfare" and "aspects of Pinker's legacy". The idea of welfare pluralism is now familiar. But Pinker's crucial pioneering role, depth of understanding of the issues and grasp of their intertwining is seldom recalled. This article should help to meet the need for his contribution to be reinserted into the mainstream of sociological thought on welfare, so enriching new research.

The influence of different lipid environments on the structure and function of the hepatitis C virus p7 ion channel protein.

Abstract The hepatitis C virus (HCV) encodes the p7 protein that oligomerizes to form an ion channel. The 63 amino acid long p7 monomer is an integral membrane protein predominantly found in the endoplasmic reticulum (ER). Although it is currently unknown whether p7 is incorporated into secreted virions, its presence is crucial for the release of infectious virus. The molecular and biophysical mechanism employed by the p7 ion channel is largely unknown, but in vivo it is likely to be embedded in membranes undergoing changes in lipid composition. In this study we analyze the influence of the lipid environment on p7 ion channel structure and function using electrophysiology and synchrotron radiation circular dichroism (SRCD) spectroscopy. We incorporated chemically synthesized p7 polypeptides into artificial planar membranes of various lipid compositions. A lipid bilayer composition comprising phosphatidylcholine (PC) and phosphatidylethanolamine (PE) (4:1 PC:PE) led to burst-like patterns in the channel recordings with channel openings lasting up to 0.5 s. The reverse ratio of PC:PE (1:4) gave rise to individual channels continuously opening for up to 8 s. SRCD spectroscopy of p7 embedded into liposomes of corresponding lipid compositions suggests there is a structural effect of the lipid composition on the p7 protein.

A Shortcut to the Synthesis of Peptide Thioesters.

Peptide thioesters serve as fundamental building blocks for the synthesis of proteins and cyclic peptides. Classically, methods to synthesize thioesters have been based on acid-labile amino-protecting groups for which final side-chain deprotection required the use of hazardous hydrogen fluoride (HF). Alternative protection schemes based on base-labile amino-protecting groups have become preferred methods but are not suitable due to the lability of thioester bonds toward bases. In this method, we employ a trifluoracetic acid/trimethylsilyl bromide (TFA/TMSBr) protocol using a hydroxymethyl resin obviating the need for HF. TFA/TMSBr is volatile enough to be easily removed yet less hazardous than HF, making it more practical for general peptide chemists. We describe optimized cleavage procedures and appropriate protecting group schemes and discuss in situ neutralization protocols. The method is relatively simple, straightforward, and easily scalable, allowing the facile preparation of alkyl and aryl thioesters.