bioRxiv Science⌕ Search

Biology subjects

Romartinez-Alonso, B.

Publications and source records attributed to Romartinez-Alonso, B..

3 recordsLinked to original sources

Damaging mutations in LXRα uncouple lipogenesis from hepatotoxicity and implicate hepatic cholesterol sensing in human liver health

The nuclear receptor Liver X Receptor- (LXR) 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 LXR activity on human health we first examined the association between the carriage of rare loss of function mutations in NR1H3 (encoding LXR) and metabolic and hepatic phenotypes. We identified 63 rare predicted damaging variants in the ligand binding domain of LXR 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 LXR, carriers of damaging mutations in LXR had reduced serum triglycerides ({beta}=-0.13 s.d. {+/-}0.03, P=2.7x10-5, N(carriers)=971). Surprisingly, these carriers also had 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 LXR specifically in hepatocytes, indicating a cell-autonomous effect of the mutant on hepatocyte health. While homozygous LXR knockout mice showed some evidence of hepatocyte injury under similar dietary conditions, the phenotype of the LXRW441R/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 LXR occur in at least 1/450 people and are associated with evidence of liver dysfunction. These findings implicate LXR 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.

genetics↗

Characterising the Protein-Protein Interaction Between MDM2 and 14-3-3σ; Proof of Concept for Small Molecule Stabilisation

Mouse Double Minute 2 (MDM2) is a key negative regulator of the tumour suppressor protein p53. MDM2 overexpression occurs in many types of cancer and results in the suppression of wild type p53. The 14-3-3 family of adaptor proteins are known to bind to MDM2 and the 14-3-3{sigma} isoform controls MDM2 cellular localisation and stability to inhibit its activity. Therefore, small molecule stabilisation of the 14-3-3{sigma}/MDM2 protein-protein interaction (PPI) is a potential therapeutic strategy for the treatment of cancer. In this work we provide a detailed biophysical and structural characterisation of the phosphorylation-dependent interaction between 14-3-3{sigma} and peptides that mimic the 14-3-3 binding motifs within MDM2. The data show that di-phosphorylation of MDM2 at S166 and S186 is essential for high affinity 14-3-3 binding and that the binary complex formed involves one MDM2 di-phosphorylated peptide bound to a dimer of 14-3-3{sigma}. Each of the two phosphorylated stretches of MDM2 occupies one of the two binding grooves of a 14-3-3{sigma} dimer, a novel model for binding of di-phosphorylated peptides to 14-3-3 proteins. In addition, we show that the 14-3-3{sigma}/MDM2 interaction is amenable to small molecule stabilisation. The natural product fusicoccin A forms a ternary complex with a 14-3-3{sigma} dimer and an MDM2 di-phosphorylated peptide resulting in stablisation of the 14-3-3{sigma}/MDM2 PPI. This work serves as a proof-of-concept of the drugability of the 14-3-3/MDM2 PPI and paves the way toward the development of more selective and efficacious small molecule stabilisers.

biochemistry↗

Structural and biochemical insights into heterotetramer formation between human oncogenic K-Ras4BG12V and Rgl2, a RalA/B activator

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 KRas4B and the Ras association (RA) domain of Rgl2 (Rgl2RA), one of the RA-containing RalGEFs. We show that the G12V oncogenic KRas4B mutation changes the interaction kinetics with Rgl2RA. The crystal structure of the KRas4BG12V: Rgl2RA complex shows a 2:2 heterotetramer where the Switch I and Switch II regions of each KRasG12V interact with both Rgl2RA molecules. This structural arrangement is highly similar to the HRasE31K:RALGDSRA crystal structure and is distinct from the well-characterised Ras:Raf complex. Interestingly, the G12V mutation was found at the dimer interface of KRas4BG12V 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 KRas4BG12V hyperactivates the RalA/B pathway.

biochemistry↗