PEGylation is a promising approach to address the central challenge of applying biologics, i.e., lack of protein stability in the demanding environment of the human body. Wider application is hindered by lack of atomic level understanding of protein-PEG interactions, preventing design of conjugates with predicted properties. We deployed an integrative structural and biophysical approach to address this critical challenge with the PEGylated carbohydrate recognition domain of human galectin-3 (Gal3C), a lectin essential for cell adhesion and potential biologic. PEGylation dramatically increased Gal3C thermal stability, forming a stable intermediate and redirecting its unfolding pathway. Structural details revealed by NMR pointed to a potential role of PEG localization facilitated by charged residues. Replacing these residues subtly altered the protein-PEG interface and thermal unfolding behavior, providing insight into rationally designing conjugates while preserving PEGylation benefits.

Skin tumorigenesis results from DNA damage, increased inflammation, and evasion of apoptosis. The peroxisome proliferator-activated receptors (PPARs) can modulate these mechanisms in non-melanoma skin cancer. However, limited data exists regarding the role of PPARs in melanoma. This study examined the effect of proliferator-activated receptor-β/δ (PPARβ/δ) and PPARγ on cell proliferation, anchorage-dependent clonogenicity, and ectopic xenografts in the UACC903 human melanoma cell line. Stable overexpression of either PPARβ/δ or PPARγ enhanced ligand-induced expression of a PPARβ/δ/PPARγ target gene in UACC903 cell lines as compared with controls. The induction of target gene expression by ligand activation of PPARγ was not altered by overexpression of PPARβ/δ, or vice versa. Stable overexpression of either PPARβ/δ or PPARγ reduced the percentage of cells in the G1 and S phase of the cell cycle, and increased the percentage of cells in the G2/M phase of the cell cycle in UACC903 cell lines as compared with controls. Ligand activation of PPARβ/δ did not further alter the distribution of cells within each phase of the cell cycle. By contrast, ligand activation of PPARγ enhanced these changes in stable UACC903 cells overexpressing PPARγ compared with controls. Stable overexpression of either PPARβ/δ or PPARγ and/or ligand activation of either PPARβ/δ or PPARγ inhibited cell proliferation, and anchorage-dependent clonogenicity of UACC903 cell lines as compared with controls. Further, overexpression of either PPARβ/δ or PPARγ and/or ligand activation of either PPARβ/δ or PPARγ inhibited ectopic xenograft tumorigenicity derived from UACC903 melanoma cells as compared with controls, and this was likely due in part to induction of apoptosis. Results from these studies demonstrate the antitumorigenic effects of both PPARβ/δ and PPARγ and suggest that targeting these receptors may be useful for primary or secondary melanoma chemoprevention.

The relative hydrophilicity at the interface of a nanoparticle was measured utilizing electron paramagnetic resonance (EPR) spectroscopy. The supramolecular structure was assembled from spin-labeled peptide amphiphiles (PA) derived from N-carboxy anhydrides (NCA). Cyanuric chloride, or 2,4,6-trichloro-1,3,5-triazine (TCT), was used as a modular platform to synthesize the spin-labeled, lipidmimetic macroinitiator used for the ring-opening polymerization of γ-benzyl-L-glutamic acid NCA to produce polyglutamate-b-dodecanethiol 2 . Through static and dynamic light scattering, as well as transmission electron microscopy, PAs with DP of 50 and 17 were shown to assemble into stable nanoparticles with an average hydrodynamic radius of 117 and 84 nm, respectively. Continuous wave EPR spectroscopy revealed that the mobility parameter (h −1 /h 0 ) and 2A iso of the nitroxide radical increased with increasing pH, in concert with the deprotonation of the PE side chains and associated helix−coil transition. These results are consistent with an increase in the relative hydration and polarity at the nanoparticle interface, which would be dependent on the secondary structure of the polypeptide. This research suggests that a pH stimulus could be used to facilitate water diffusion through the membrane.

Protein−polymer conjugates are widely used in many clinical and industrial applications, but lack of experimental data relating protein−polymer interactions to improved protein stability prevents their rational design. Advances in synthetic chemistry have expanded the palette of polymer designs, including development of nonlinear architectures, novel monomer chemical scaffolds, and control of hydrophobicity, but more experimental data are needed to transform advances in chemistry into next generation conjugates. Using an integrative biophysical approach, we investigated the molecular basis for polymer-based thermal stabilization of a human galectin protein, Gal3C, conjugated with polymers of linear and nonlinear architectures, different degrees of polymerization, and varying hydrophobicities. Independently varying the degree of polymerization and polymer architecture enabled delineation of specific polymer properties contributing to improved protein stability. Insights from NMR spectroscopy of the polymer-conjugated Gal3C backbone revealed patterns of protein−polymer interactions shared between linear and nonlinear polymer architectures for thermally stabilized conjugates. Despite large differences in polymer chemical scaffolds, protein−polymer interactions resulting in thermal stabilization appear conserved. We observed a clear relation between polymer length and protein− polymer thermal stability shared among chemically different polymers. Our data indicate a wide range of polymers may be useful for engineering conjugate properties and provide conjugate design criteria.