John W. R. Schwabe’s research while affiliated with Cambridge University Hospitals NHS Foundation Trust and other places

Reversible modification of the histone H3 N-terminal tail is critical in regulating the chromatin structure, gene expression, and cell states, while its dysregulation contributes to disease pathogenesis. Understanding the crosstalk between H3 tail modifications in nucleosomes constitutes a central challenge in epigenetics. Here, we describe an engineered sortase transpeptidase, cW11, that displays highly favorable properties for introducing scarless H3 tails onto nucleosomes. This approach significantly accelerates the production of both symmetrically and asymmetrically modified nucleosomes. We demonstrate the utility of asymmetrically modified nucleosomes produced in this way in dissecting the impact of multiple modifications on eraser enzyme processing and molecular recognition by a reader protein. Moreover, we show that cW11 sortase is very effective at cutting and tagging histone H3 tails from endogenous histones, facilitating multiplex “cut-and-paste” middle-down proteomics with tandem mass tags. This cut-and-paste proteomics approach permits the quantitative analysis of histone H3 modification crosstalk after treatment with different histone deacetylase inhibitors. We propose that these chemoenzymatic tail isolation and modification strategies made possible with cW11 sortase will broadly power epigenetic discovery and therapeutic development.

Cereblon-recruiting PROTACs can degrade HDAC1 with selectivity over HDAC3.

Fetal hemoglobin (HbF) induction is a well-known strategy for the treatment of hemoglobinopathies such as Sickle Cell Disease (SCD) and Thalassemia, worldwide life-threatening conditions. Increased levels of HbF are able to prevent HbS polymerization and mitigate disease manifestations, resulting in lower morbidity and mortality in SCD. Nowadays there are only four drugs approved by the FDA for SCD treatment: Hydroxyurea, voxelotor, L-glutamine and crizanlizumab, while the search for new effective and safe treatments are scarce. Controlling the expression of the gamma-globin gene by inhibiting or degrading epigenetic targets has been shown to be promising for HbF induction, and histone deacetylases (HDACs 1 and 2) stand out as potential targets. Targeted protein degradation (TPD) was explored in this work with the synthesis of new cereblon-based molecules able to anchor to cereblon and HDACs 1 and 2 simultaneously, leading to their dose dependent decrease via polyubiquitination and proteasome degradation. Six new cereblon derivatives containing a HDAC inhibitor subunit were designed, synthesized and evaluated in HCT-116 cells for HDAC 1, 2 and 3 degradation via quantitative western blot at concentrations ranging from 0.01 to 10 µM. The promising results showed that three of these compounds (ARP-26, ARP-37 and ARP-49) were able to degrade HDAC-1 more selectively, when compared to HDAC-2 and HDAC-3, achieving DC50 values of 2.5 µM. A derivative with no ability to bind to cereblon was also synthesized resulting in no degradation of HDACs 1-3, suggesting that these compounds act via cereblon binding. ARP-49 was then selected for further studies in HUDEP-2 cell culture at concentrations of 500 and 750nM, and incubation time of 72 and 96h. After the treatment, cells were stained with HbF-antibody and the percentage of HbF positive cells was assessed by flow cytometry. The quantification of HBG1/2 mRNA was obtained by qPCR. HUDEP-2 cells exhibit a basal level of HbF positive cells lower than 1%, then the results obtained from our compounds were normalized based on the control. At 72h, the concentrations of 500nM and 750nM were able to significantly increase the HBG1/2 mRNA levels by 26.9-fold and 35.5-fold, respectively (CTRL = 0.027 ± 0.006 arbitrary units (a.u.) vs 500nM = 0.73 ±0.25 a.u. and 750nM = 0.96 ± 0.11 a.u., p<0.0001, n=4). At 96h, both concentrations were also able to significantly induce the expression of the HBG1/2 genes by 34-fold and 44.5-fold, respectively (CTRL = 0.034 ± 0.012 a.u. vs 500nM = 1.12 ± 0.35 a.u. and 750nM = 1.52 ± 0.37 a.u., p<0.0001, n=4). HbF-positive cells at 72h presented a significantly increase of 7.5-fold and 9-fold at 500 and 750nM, respectively (CTRL = 0.58 ± 0.15 % vs 500nM = 4.43 ± 0.88 % and 750nM = 5.29 ± 1.52 %, p<0.0001, n=4), while at 96h the increase was 8.37-fold and 10.6-fold, respectively (CTRL = 0.65 ± 0.15 % vs 500nM = 5.44 ± 0.79 % and 750nM = 6.89 ± 1.05 %, p<0.0001, n=4). HUDEP-2 cells were also treated with HU at 100 µM, reaching its highest level of HBG1/2 expression at 72h (10-fold), when compared to control, and an increase in the HbF-positive cells population by 8.5-fold and 8.9-fold at 72h and 96h, respectively (CTRL 72h = 0.66 ± 0.23% vs HU 72h = 5.65 ± 0.59%, CTRL 96h = 0.64 ± 0.15% vs HU 96h = 5.73 ± 0.52%; p<0.0001, n=2). Compounds obtained in this study present a degradation selectivity towards HDAC-1 with no previous description in the literature. Furthermore, the ability to significantly induce HBG1/2 expression in HUDEP-2 cells was superior to the one seen for the standard SCD treatment, HU, with levels of HBG1/2 expression 2.6-fold higher (500nM, 72h), at a concentration 200-fold lower. These findings support that ARP-49 shows promising potential for further pre-clinical studies for increasing the production of HbF.

Liver X receptor-α (LXRα) regulates cellular cholesterol abundance and potently activates hepatic lipogenesis. Here we show that at least 1 in 450 people in the UK Biobank carry functionally impaired mutations in LXRα, which is associated with biochemical evidence of hepatic dysfunction. On a western diet, male and female mice homozygous for a dominant negative mutation in LXRα have elevated liver cholesterol, diffuse cholesterol crystal accumulation and develop severe hepatitis and fibrosis, despite reduced liver triglyceride and no steatosis. This phenotype does not occur on low-cholesterol diets and can be prevented by hepatocyte-specific overexpression of LXRα. LXRα knockout mice exhibit a milder phenotype with regional variation in cholesterol crystal deposition and inflammation inversely correlating with steatosis. In summary, LXRα is necessary for the maintenance of hepatocyte health, likely due to regulation of cellular cholesterol content. The inverse association between steatosis and both inflammation and cholesterol crystallization may represent a protective action of hepatic lipogenesis in the context of excess hepatic cholesterol.

Reversible modification of the histone H3 N-terminal tail is critical in regulating chromatin structure, gene expression, and cell states, while its dysregulation contributes to disease pathogenesis. Understanding the crosstalk between H3 tail modifications in nucleosomes constitutes a central challenge in epigenetics. Here we describe an engineered sortase transpeptidase, cW11, that displays highly favorable properties for introducing scarless H3 tails onto nucleosomes. This approach significantly accelerates the production of both symmetrically and asymmetrically modified nucleosomes. We demonstrate the utility of asymmetrically modified nucleosomes produced in this way in dissecting the impact of multiple modifications on eraser enzyme processing and molecular recognition by a reader protein. Moreover, we show that cW11 sortase is very effective at cutting and tagging histone H3 tails from endogenous histones, facilitating multiplex “cut-and-paste” middle down proteomics with tandem mass tags. This cut-and-paste proteomics approach permits the quantitative analysis of histone H3 modification crosstalk after treatment with different histone deacetylase inhibitors. We propose that these chemoenzymatic tail isolation and modification strategies made possible with cW11 sortase will broadly power epigenetics discovery and therapeutic development.

The nuclear receptor Liver X Receptor-α (LXR α) activates lipogenic gene expression in hepatocytes. Its inhibition has therefore been proposed as a strategy to treat metabolic dysfunction-associated steatotic liver disease (MASLD). In order to understand the impact of reducing LXRα activity on human health we first examined the association between the carriage of rare loss of function mutations in NR1H3 (encoding LXRα) and metabolic and hepatic phenotypes. We identified 63 rare predicted damaging variants in the ligand binding domain of LXRα in 454,787 participants in UK Biobank. On functional characterisation, 42 of these were found to be severely impaired. Consistent with loss of the lipogenic actions of LXRα, carriers of damaging mutations in LXRα had reduced serum triglycerides (Beta=-0.13 s.d. [plusmn]0.03, P=2.7x10 ⁻⁵ , N(carriers)=971). Surprisingly, these carriers also have elevated concentrations of serum liver enzymes (e.g. ALT: Beta=0.17s.d.±0.03, P=1.1x10 ⁻⁸ , N(carriers)=972) with a 35% increased risk of clinically significant elevations in ALT (OR=1.32, 95%CI:1.15-1.53, P=1.2x10 ⁻⁴ , N(carriers)=972), suggestive of hepatotoxicity. We generated a knock-in mouse carrying one of the most severely damaging mutations (Nr1h3 p.W441R) which we demonstrated to have dominant negative properties. Homozygous knock-in mice rapidly developed severe hepatitis and fibrotic liver injury following exposure to western diet despite markedly reduced steatosis, liver triglycerides and lipogenic gene expression. This phenotype was completely rescued by viral over-expression of wildtype LXRα specifically in hepatocytes, indicating a cell-autonomous effect of the mutant on hepatocyte health. While homozygous LXRα knockout mice showed some evidence of hepatocyte injury under similar dietary conditions, the phenotype of the LXRα W441R/W441R mouse was much more severe, suggesting that dominant negative mutations that actively co-repress target genes can result in pathological impacts significantly more severe than those seen with simple absence of the receptor. In summary, our results show that loss of function mutations in LXRα occur in at least 1/450 people and are associated with evidence of liver dysfunction. These findings implicate LXRα in the maintenance of human liver health, identify a new murine model of rapidly progressive fibrotic liver disease and caution against LXR antagonism as a therapeutic strategy for MASLD.

Intestinal absorption is an important contributor to systemic cholesterol homeostasis. Niemann-Pick C1 Like 1 (NPC1L1) assists in the initial step of dietary cholesterol uptake, but how cholesterol moves downstream of NPC1L1 is unknown. We show that Aster-B and Aster-C are critical for nonvesicular cholesterol movement in enterocytes. Loss of NPC1L1 diminishes accessible plasma membrane (PM) cholesterol and abolishes Aster recruitment to the intestinal brush border. Enterocytes lacking Asters accumulate PM cholesterol and show endoplasmic reticulum cholesterol depletion. Aster-deficient mice have impaired cholesterol absorption and are protected against diet-induced hypercholesterolemia. Finally, the Aster pathway can be targeted with a small-molecule inhibitor to manipulate cholesterol uptake. These findings identify the Aster pathway as a physiologically important and pharmacologically tractable node in dietary lipid absorption.

Histone deacetylases 1 and 2 (HDAC1/2) serve as the catalytic subunit of six distinct families of nuclear complexes. These complexes repress gene transcription through removing acetyl groups from lysine residues in histone tails. In addition to the deacetylase subunit, these complexes typically contain transcription factor and/or chromatin binding activities. The MIER:HDAC complex has hitherto been poorly characterized. Here, we show that MIER1 unexpectedly co-purifies with an H2A:H2B histone dimer. We show that MIER1 is also able to bind a complete histone octamer. Intriguingly, we found that a larger MIER1:HDAC1:BAHD1:C1QBP complex additionally co-purifies with an intact nucleosome on which H3K27 is either di- or tri-methylated. Together this suggests that the MIER1 complex acts downstream of PRC2 to expand regions of repressed chromatin and could potentially deposit histone octamer onto nucleosome-depleted regions of DNA.

About a quarter of total human cancers carry mutations in Ras isoforms. Accumulating evidence suggests that small GTPases, RalA and RalB, and their activators, Ral guanine nucleotide exchange factors (RalGEFs), play an essential role in oncogenic Ras-induced signalling. We studied the interaction between human K-Ras4B and the Ras association (RA) domain of Rgl2 (Rgl2 RA ), one of the RA-containing RalGEFs. We show that the G12V oncogenic K-Ras4B mutation increases the affinity with Rgl2 RA . The crystal structure of the K-Ras4B G12V : Rgl2 RA complex shows a 2:2 heterotetramer where the Switch I and Switch II regions of each K-Ras G12V interact with both Rgl2 RA molecules. This structural arrangement is highly similar to the H-Ras E31K :RALGDS RA crystal structure and is distinct from the well-characterised Ras:Raf complex. Importantly, the G12V mutation was found at the dimer interface of K-Ras4B G12V with its partner. Our study reveals a potentially distinct mode of Ras:effector complex formation by RalGEFs, and offers a possible mechanistic explanation for how the oncogenic K-Ras4B G12V hyperactivates the RalA/B pathway. Summary Blurb The work reports that the K-RAS4B.G12V oncogenic mutation increases the affinity with Rgl2, an activator of RalA/B and provides a structural explanation by analysing the K-Ras4B(G12V):Rgl2 complex crystal structure where the G12V mutation resides at the interface of the complex.