High-Sensitivity Cardiac Troponin I Enhances Preeclampsia Prediction Beyond Maternal Factors and the sFlt-1/PlGF Ratio.

BACKGROUND: Preeclampsia shares numerous risk factors with cardiovascular diseases. Here, we aimed to assess the potential utility of high-sensitivity cardiac troponin I (hs-cTnI) values during pregnancy in predicting preeclampsia occurrence. METHODS: This study measured hs-cTnI levels in 3721 blood samples of 2245 pregnant women from 4 international, prospective cohorts. Three analytical approaches were used: (1) a cross-sectional analysis of all women using a single blood sample, (2) a longitudinal analysis of hs-cTnI trajectories in women with multiple samples, and (3) analyses of prediction models incorporating hs-cTnI, maternal factors, and the sFlt-1 (soluble fms-like tyrosine kinase 1)/PlGF (placental growth factor) ratio. RESULTS: Women with hs-cTnI levels in the upper quarter had higher odds ratios for preeclampsia occurrence compared with women with levels in the lower quarter. Associations were driven by preterm preeclampsia (odds ratio, 5.78 [95% CI, 2.73-12.26]) and remained significant when using hs-cTnI as a continuous variable adjusted for confounders. Between-trimester hs-cTnI trajectories were independent of subsequent preeclampsia occurrence. A prediction model incorporating a practical hs-cTnI level of detection cutoff (≥1.9 pg/mL) alongside maternal factors provided comparable performance with the sFlt-1/PlGF ratio. A comprehensive model including sFlt-1/PlGF, maternal factors, and hs-cTnI provided added value (cross-validated area under the receiver operator characteristic, 0.78 [95% CI, 0.73-0.82]) above the sFlt-1/PlGF ratio alone (cross-validated area under the receiver operator characteristic, 0.70 [95% CI, 0.65-0.76]; P=0.027). As assessed by likelihood ratio tests, the addition of hs-cTnI to each prediction model significantly improved the respective prediction model not incorporating hs-cTnI, particularly for preterm preeclampsia. Net reclassification improvement analyses indicated that incorporating hs-cTnI improved risk prediction predominantly by correctly reclassifying women with subsequent preeclampsia occurrence. CONCLUSIONS: These exploratory findings uncover a potential role for hs-cTnI as a complementary biomarker in the prediction of preeclampsia. After validation in prospective studies, hs-cTnI, alongside maternal factors, may either be considered as a substitute for angiogenic biomarkers in health care systems where they are sparce or unavailable, or as an enhancement to established prediction models using angiogenic markers.

A pH Switch Controls Zinc Binding in Tomato Copper-Zinc Superoxide Dismutase.

Copper-zinc superoxide dismutase (SOD1) is a major antioxidant metalloenzyme that protects cells from oxidative damage by superoxide anions (O2-). Structural, biophysical, and other characteristics have in the past been compiled for mammalian SOD1s and for the highly homologous fungal and bovine SOD1s. Here, we characterize the biophysical properties of a plant SOD1 from tomato chloroplasts and present several of its crystal structures. The most unusual of these structures is a structure at low pH in which tSOD1 harbors zinc in the copper-binding site but contains no metal in the zinc-binding site. The side chain of D83, normally a zinc ligand, adopts an alternate rotameric conformation to form an unusual bidentate hydrogen bond with the side chain of D124, precluding metal binding in the zinc-binding site. This alternate conformation of D83 appears to be responsible for the previously observed pH-dependent loss of zinc from the zinc-binding site of SOD1. Titrations of cobalt into apo tSOD1 at a similar pH support the lack of an intact zinc-binding site. Further characterization of tSOD1 reveals that it is a weaker dimer relative to human SOD1 and that it can be activated in vivo through a copper chaperone for the SOD1-independent mechanism.

Contribution of surface salt bridges to protein stability: guidelines for protein engineering.

The small globular protein, ubiquitin, contains a pair of oppositely charged residues, K11 and E34, that according to the three-dimensional structure are located on the surface of this protein with a spatial orientation characteristic of a salt bridge. We investigated the strength of this salt bridge and its contribution to the global stability of the ubiquitin molecule. Using the "double mutant cycle" analysis, the strength of the pairwise interactions between K11 and E34 was estimated to be favorable by 3.6kJ/mol. Further, the salt bridge of the reverse orientation, i.e. E11/K34, can be formed and is found to have a strength (3.8kJ/mol) similar to that of the K11/E34 pair. However, the global stability of the K11/E34 variant of ubiquitin is 2.2kJ/mol higher than that of the E11/K34 variant. The difference in the contribution of the opposing salt bridge orientations to the overall stability of the ubiquitin molecule is attributed to the difference in the charge-charge interactions between residues forming the salt bridge and the rest of the ionizable groups in this protein. On the basis of these results, we concluded that surface salt bridges are stabilizing, but their contribution to the overall protein stability is strongly context-dependent, with charge-charge interactions being the largest determinant. Analysis of 16 salt bridges from six different proteins, for which detailed experimental data on energetics have been reported, support the conclusions made from the analysis of the salt bridge in ubiquitin. Implications of these findings for engineering proteins with enhanced thermostability are discussed.

Hydrophobic interactions at the Ccap position of the C-capping motif of alpha-helices.

We investigated the possible role of residues at the Ccap position in an alpha-helix on protein stability. A set of 431 protein alpha-helices containing a C'-Gly from the Protein Data Bank (PDB) was analyzed, and the normalized frequencies for finding particular residues at the Ccap position, the average fraction of buried surface area, and the hydrogen bonding patterns of the Ccap residue side-chain were calculated. We found that on average the Ccap position is 70% buried and noted a significant correlation (R=0.8) between the relative burial of this residue and its hydrophobicity as defined by the Gibbs energy of transfer from octanol or cyclohexane to water. Ccap residues with polar side-chains are commonly involved in hydrogen bonding. The hydrogen bonding pattern is such that, the longer side-chains of Glu, Gln, Arg, Lys, His form hydrogen bonds with residues distal (>+/-4) in sequence, while the shorter side-chains of Asp, Asn, Ser, Thr exhibit hydrogen bonds with residues close in sequence (<+/-4), mainly involving backbone atoms. Experimentally we determined the thermodynamic propensities of residues at the Ccap position using the protein ubiquitin as a model system. We observed a large variation in the stability of the ubiquitin variants depending on the nature of the Ccap residue. Furthermore, the measured changes in stability of the ubiquitin variants correlate with the hydrophobicity of the Ccap residue. The experimental results, together with the statistical analysis of protein structures from the PDB, indicate that the key hydrophobic capping interactions between a helical residue (C3 or C4) and a residue outside the helix (C", C3' or C4') are frequently enhanced by the hydrophobic interactions with Ccap residues.