PEGylation Increases the Strength of a Nearby NH-π Hydrogen Bond in the WW Domain.

Here we show that an NH-π interaction between a highly conserved Asn and a nearby Trp stabilizes the WW domain of the human protein Pin1. The strength of this NH-π interaction depends on the structure of the arene, with NH-π interactions involving Trp or naphthylalanine being substantially more stabilizing than those involving Tyr or Phe. Calculations suggest arene size and polarizability are key structural determinants of NH-π interaction strength. Methylation or PEGylation of the Asn side-chain amide nitrogen each strengthens the associated NH-π interaction, though likely for different reasons. We hypothesize that methylation introduces steric clashes that destabilize conformations in which the NH-π interaction is not possible, whereas PEGylation strengthens the NH-π interaction via localized desolvation of the protein surface.

PEGylation near a Patch of Nonpolar Surface Residues Increases the Conformational Stability of the WW Domain.

Many proteins have one or more surface-exposed patches of nonpolar residues; our observations here suggest that PEGylation near such locations might be a useful strategy for increasing protein conformational stability. Specifically, we show that conjugating a PEG-azide to a propargyloxyphenylalanine via the copper(I)-catalyzed azide-alkyne cycloaddition can increase the conformational stability of the WW domain due to a favorable synergistic effect that depends on the hydrophobicity of a nearby patch of nonpolar surface residues.

Influence of PEGylation on the Strength of Protein Surface Salt Bridges.

Conjugation of polyethylene glycol (PEGylation) is a well-known strategy for extending the serum half-life of protein drugs and for increasing their resistance to proteolysis and aggregation. We previously showed that PEGylation can increase protein conformational stability; the extent of PEG-based stabilization depends on the PEGylation site, the structure of the PEG-protein linker, and the ability of PEG to release water molecules from the surrounding protein surface to the bulk solvent. The strength of a noncovalent interaction within a protein depends strongly on its microenvironment, with salt-bridge and hydrogen-bond strength increasing in nonpolar versus aqueous environments. Accordingly, we wondered whether partial desolvation by PEG of the surrounding protein surface might result in measurable increases in the strength of a salt bridge near a PEGylation site. Here we explore this possibility using triple-mutant box analysis to assess the impact of PEGylation on the strength of nearby salt bridges at specific locations within three peptide model systems. The results indicate that PEG can increase the nearby salt-bridge strength, though this effect is not universal, and its precise structural prerequisites are not a simple function of secondary structural context, of the orientation and distance between the PEGylation site and salt bridge, or of salt-bridge residue identity. We obtained high-resolution X-ray diffraction data for a PEGylated peptide in which PEG enhances the strength of a nearby salt bridge. Comparing the electron density map of this PEGylated peptide versus that of its non-PEGylated counterpart provides evidence of localized protein surface desolvation as a mechanism for PEG-based salt-bridge stabilization.

Simvastatin Inhibits Epithelial-to-Mesenchymal Transition Through Induction of HO-1 in Cultured Renal Proximal Tubule Cells.

BACKGROUND/AIM: Studies have shown that simvastatin (SIM) inhibits epithelial-mesenchymal transition (EMT), a key step in fibrosis, and activates the anti-fibrotic heme oxygenase-1 (HO-1) gene in renal proximal tubule cells independent of its lipid-lowering. We tested the hypothesis that SIM inhibits EMT via HO-1-dependent suppression of reactive oxygen species (ROS) release. MATERIALS AND METHODS: Renal proximal tubule cells were treated with either 10 µM SIM or 10 ng/ml transforming growth factor-ß1 (TGFß1) or with their combination and promoter activity of the alpha-smooth muscle actin (α-SMA) gene, stress fiber formation (markers of EMT), as well as ROS production were determined. HO-1 was manipulated via genetic and pharmacologic means. RESULTS: SIM prevented TGFß1-dependent EMT and ROS production. Inhibition/knockdown of HO-1 reversed, while induction/overexpression of HO-1 emulated beneficial effects of SIM. CONCLUSION: SIM, via HO-1, suppresses TGFß1-dependent ROS production and, hence, EMT. Further evaluation of the anti-fibrotic nature of SIM in the kidney would be useful in the treatment of chronic kidney disease.

Intrauterine growth restriction programs an accelerated age-related increase in cardiovascular risk in male offspring.

Placental insufficiency programs an increase in blood pressure associated with a twofold increase in serum testosterone in male growth-restricted offspring at 4 mo of age. Population studies indicate that the inverse relationship between birth weight and blood pressure is amplified with age. Thus, we tested the hypothesis that intrauterine growth restriction programs an age-related increase in blood pressure in male offspring. Growth-restricted offspring retained a significantly higher blood pressure at 12 but not at 18 mo of age compared with age-matched controls. Blood pressure was significantly increased in control offspring at 18 mo of age relative to control counterparts at 12 mo; however, blood pressure was not increased in growth-restricted at 18 mo relative to growth-restricted counterparts at 12 mo. Serum testosterone levels were not elevated in growth-restricted offspring relative to control at 12 mo of age. Thus, male growth-restricted offspring no longer exhibited a positive association between blood pressure and testosterone at 12 mo of age. Unlike hypertension in male growth-restricted offspring at 4 mo of age, inhibition of the renin-angiotensin system with enalapril (250 mg/l for 2 wk) did not abolish the difference in blood pressure in growth-restricted offspring relative to control counterparts at 12 mo of age. Therefore, these data suggest that intrauterine growth restriction programs an accelerated age-related increase in blood pressure in growth-restricted offspring. Furthermore, this study suggests that the etiology of increased blood pressure in male growth-restricted offspring at 12 mo of age differs from that at 4 mo of age.