A structural basis for the activation of peroxisome proliferator-activated receptor gamma (PPARγ) by perfluorooctanoic acid (PFOA).

Perfluorooctanoic acid (PFOA) is a widespread environmental pollutant of the perfluoroalkyl substance (PFAS) class that is extremely resistant to environmental and metabolic degradation, leading to bioaccumulation. PFOA exposure has been linked to many health effects including endocrine disruption and metabolic dysregulation, but our understanding of the molecular mechanisms resulting in these outcomes remains incomplete. One target affected by PFOA is the ligand regulated nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) which plays a critical role in controlling metabolic homeostasis through regulating processes such as adipogenesis, glucose homeostasis, inflammation and osteogenesis. It has been previously established that PFOA activates PPARγ through binding to the PPARγ ligand binding domain (PPARγ LBD) leading to increased expression of PPARγ controlled target genes. However, the mechanism by which PFOA achieves this has remained elusive. Here, we employed a combination of X-ray crystallography and fluorescence polarization assays to provide a structural basis for PFOA mediated activation of PPARγ via binding to the PPARγ LBD. Using X-ray crystallography, the cocrystal structure of the PPARγ LBD:PFOA complex was solved. This revealed that PFOA occupies three distinct sites, two within the PPARγ LBD and one within the activation function 2 (AF2) on the protein surface. Structural comparison of PFOA binding with previously reported PPARγ:ligand complexes supports that PFOA activates PPARγ by a partial agonist mechanism at micromolar concentrations. Fluorescence polarization assays also revealed that PFOA binding to the AF2 is unlikely to occur in a cellular context and confirmed that PFOA behaves as a partial agonist in vitro, weakly recruiting a coactivator peptide to the AF2 of the PPARγ LBD. This discovery provides an advancement in understanding PFOA mediated regulation of PPARγ, giving new insight regarding regulation of PPARγ by PFAS and PFAS substitutes in general and can be applied to the design and assessment of safer PFAS.

Production of recombinant human proliferating cellular nuclear antigen (PCNA) for structural and biophysical characterization.

Human proliferating cell nuclear antigen (hPCNA) is a DNA replication processivity factor, which acts as a docking platform, allowing proteins to have access to the replication fork and increasing the affinity of DNA interacting proteins, making it critical for cell survival. The trimer forms a ring-shaped oligomer allowing DNA to pass through the middle and interacting proteins to dock on the outside of the ring. Without this structural formation, there is a loss of DNA replication and repair in the cell. Due to the location of subunit-subunit termini, the addition of a purification tag can hamper crystallography and biophysical experiments, as the trimer complex folding can be impeded. To avoid these complications, a tag-less, step-wise purification was implemented, which resulted in 17.6 mg from 2 L culture of pure hPCNA with a 260 nm/280 nm value of 0.43. The produced crystal structure reveals a correctly formed oligomer. The clear depletion of the tracer binding and probe protein interaction in a fluorescence polarisation competition-based assay demonstrates the purification method produces a protein structure with a functional binding site. This purification method presents a reliable and simple method for producing hPCNA for biophysical characterisation.

PPARG Post-translational Modifications Regulate Bone Formation and Bone Resorption.

The peroxisome proliferator-activated receptor gamma (PPARγ) regulates osteoblast and osteoclast differentiation, and is the molecular target of thiazolidinediones (TZDs), insulin sensitizers that enhance glucose utilization and adipocyte differentiation. However, clinical use of TZDs has been limited by side effects including a higher risk of fractures and bone loss. Here we demonstrate that the same post-translational modifications at S112 and S273, which influence PPARγ pro-adipocytic and insulin sensitizing activities, also determine PPARγ osteoblastic (pS112) and osteoclastic (pS273) activities. Treatment of either hyperglycemic or normoglycemic animals with SR10171, an inverse agonist that blocks pS273 but not pS112, increased trabecular and cortical bone while normalizing metabolic parameters. Additionally, SR10171 treatment modulated osteocyte, osteoblast, and osteoclast activities, and decreased marrow adiposity. These data demonstrate that regulation of bone mass and energy metabolism shares similar mechanisms suggesting that one pharmacologic agent could be developed to treat both diabetes and metabolic bone disease.

A mechanistic study on the inhibition of α-chymotrypsin by a macrocyclic peptidomimetic aldehyde.

Here we describe an NMR and X-ray crystallography-based characterisation of the mechanism by which a new class of macrocyclic peptidomimetic aldehyde inhibits α-chymotrypsin. In particular, a (13)C-labelled analogue of the inhibitor was prepared and used in NMR experiments to confirm formation of a hemiacetal intermediate on binding with α-chymotrypsin. Analysis of an X-ray crystallographic structure in complex with α-chymotrypsin reveals that the backbone adopts a stable ß-strand conformation as per its design. Binding is further stabilised by interaction with the oxyanion hole near the S1 subsite and multiple hydrogen bonds.