John Offer

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.

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 viruss surface. Intriguingly, however, recent findings suggest that these carbohydrates, which have evolved to protect HIV and promote its transmission, are also attractive therapeutic targets.

A new scaffold for amide ligation

Highly chemoselective amide forming ligation reactions have facilitated the synthetic access to proteins and other amide-linked bioconjugates. In order to generalize this approach, a N(alpha)-2-phenyl ethanethiol scaffold has been developed to promote S to N acyl transfer in a manner analogous to native chemical ligation with N-terminal cysteine residues. Analysis of scaffold-mediated ligation reactions in aqueous solution indicate that the ligation rate at Xaa-Gly junctions is sufficient for the synthesis of large polypeptides. In addition, it was found that the ligation rate is independent of the stereocenter in the scaffold and S- to N-acyl transfer is rate limiting. These studies indicate that the N(alpha)-2-phenyl ethanethiol scaffold is a good candidate for the development of a ligation chemistry for the formation of Xaa-Gly peptides and other unhindered amides.

In situ thioester formation for protein ligation using α-methylcysteine

The progress of total chemical protein synthesis has been hampered by difficulties in preparing peptide thioesters by standard Fmoc peptide synthesis. The amino acid, α-methylcysteine, sited at the C-terminus of a peptide can substitute for a thioester in peptide ligation reactions. C-terminal α-methylcysteine is fully compatible with Fmoc peptide synthesis and its use in ligation is very simple and robust. Its potential is demonstrated with the synthesis of model proteins.

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.

Cholesterol seco‐Sterol‐Induced Aggregation of Methylated Amyloid‐β Peptides—Insights into Aldehyde‐Initiated Fibrillization of Amyloid‐β

The nucleation and aggregation of amyloid-b (Ab) peptides, Ab(1–40) and Ab(1–42), into neurotoxic oligomers is considered a primary event in Alzheimer!s disease (AD) pathogenesis. The vast majority of this disease is sporadic in origin (> 85%), involving the oligomerization of native Ab peptide oligomers, therefore, research is ongoing to classify the in vivo environmental triggers of AD onset that facilitate intracellular and extracellular nucleation, aggregation and deposition of native Ab peptide. As part of our ongoing research in this area, we discovered cholesterol seco-sterol aldehyde 1 (termed atheronal-B) in vivo. Aldehyde 1 is quantifiable in all human plasma and central nervous system (CNS), but is significantly elevated in the plasma and inflamed arteries of patients with advanced atherosclerosis, and in the CNS of patients with inflammatory neurological disease. We have further shown that adduction of 1 to apoB-100, a protein component of lowdensity lipoprotein (LDL), causes this protein to misfold in vitro, a misfolding process that renders LDL particles susceptible for uptake into macrophages. In addition, we have shown that 1 accelerates the aggregation of Ab(1–40) and Ab(1–42) in vitro hinting that 1 could be a plausible chemical factor linking the known AD association with atherosclerosis. An important and largely unanswered question regarding lipid aldehyde-induced protein misfolding is the nature of the interaction between the aldehyde and the protein and how this facilitates protein aggregation. Herein we show, by kinetic analyses of the atheronal-B (1)-induced oligomerization and fibrillization of a panel of synthetic mono-, bisand tris-N,N-dimethylamine-containing Ab(1–40) protein sequences 2b–f (Figure 1b), that the aggregation of Ab(1–40) peptide 2a is accelerated by 1 only when the aldehyde adducts to the e-amino group of Lys16; no initiation in oligomerization of 2a is observed when aldehyde 1 adducts to either the e-amino group of Lys28 or the a-amino group amine of Asp1. In addition, the atheronal-B-induced aggregation of peptide Ab(1–40) 2a is inhibited by cholesterol. Both data combine to suggest that the atheronal-B-induced aggregation of Ab(1–40) involves a high degree of structural recognition between the lipid and the peptide that involves, in part, binding of 1 into the putative cholesterol-binding domain of 2a. Atheronal-B (1) was synthesized as outlined previously. Peptides 2a–f were synthesized by Boc/benzyl solid phase peptide synthesis (SPPS) using in situ neutralization. N,NDimethyl Lys (K*) substitutions were incorporated using Boc-Lys(Me)2-CO2H. The N ,N-dimethyl Asp group required for peptides 2d and 2 f was incorporated using Me2N-Asp(OcHx)-CO2H, prepared by reductive amination Figure 1. Atheronal-B(1)-induced aggregation of Ab(1–40) peptide (2a). a) Schiff-base equilibrium between Lys16 of Ab(1–40) (2a) and aldehyde 1. Similar equilibria exist for Schiff-base formation at the eamino group of Lys28 and the a-amino group of Asp1. b) Amino acid sequences of Ab(1–40) (2a) and N,N-dimethylamino-containing peptide sequences (2b–f) synthesized for these studies. The central hydrophobic cluster (CHC) of Ab is underlined. K* = N,N-dimethyl Lys.

Automated synthesis of backbone protected peptides

The automated introduction of removable substitution along a peptide backbone prevents chain-association and synthesis failure.

Cholesterol Secosterol Adduction Inhibits the Misfolding of a Mutant Prion Protein Fragment that Induces Neurodegeneration

Cholesterol secosterol aldehydes inhibit the misfolding of a prion protein fragment that induces GSS in mice. Atheronal-B completely inhibits the α to β-form transformation of MoPrP(89-143, P101L) via a mechanism that involves adduction to the protein. This result offers a paradigm shift in lipid aldehyde induced protein misfolding and offers a new molecular scaffold on which to develop new potential prion disease therapeutics

A chemical approach to immunoprotein engineering: chemoselective functionalization of thioester proteins in their native state

Less than 6 feet under: Serum proteins C3, C4, and α2M each contain a thioester domain buried within a hydrophobic pocket, which is thought to shield the labile thioester from hydrolysis. Herein, we make use of the inherent reactivity of the hydrazide for thioester moieties to chemoselectively label these crucial serum regulators in their native conformation; this demonstrates that access to the thioester site is much greater than previously supposed.

Orthogonal ligation: a three piece assembly of a PNA–peptide–PNA conjugate

A PNA-peptide-PNA conjugate was assembled from three fragments using a combination of native chemical ligation and an orthogonal, auxiliary-mediated ligation.