Peptide Mimics of the Cysteine-Rich Regions of HapX and SreA Bind a [2Fe-2S] Cluster In Vitro.

HapX and SreA are transcription factors that regulate the response of the fungus Aspergillus fumigatus to the availability of iron. During iron starvation, HapX represses genes involved in iron consuming pathways and upon a shift to iron excess, HapX activates these same genes. SreA blocks the expression of genes needed for iron uptake during periods of iron availability. Both proteins possess cysteine-rich regions (CRR) that are hypothesized to be necessary for the sensing of iron levels. However, the contribution of each of these domains to the function of the protein has remained unclear. Here, the ability of peptide analogs of each CRR is determined to bind an iron-sulfur cluster in vitro. UV-vis and resonance Raman (RR) spectroscopies reveal that each CRR is capable of coordinating a [2Fe-2S] cluster with comparable affinities. The iron-sulfur cluster coordinated to the CRR-B domain of HapX displays particularly high stability. The data are consistent with HapX and SreA mediating responses to cellular iron levels through the direct coordination of [2Fe-2S] clusters. The high stability of the CRR-B peptide may also find use as a starting point for the development of new green catalysts.

Integrating extracellular vesicle and circulating cell-free DNA analysis using a single plasma aliquot improves the detection of HER2 positivity in breast cancer patients.

Multi-analyte liquid biopsies represent an emerging opportunity for non-invasive cancer assessment. We developed ONCE (ONe Aliquot for Circulating Elements), an approach for the isolation of extracellular vesicles (EV) and cell-free DNA (cfDNA) from a single aliquot of blood. We assessed ONCE performance to classify HER2-positive early-stage breast cancer (BrCa) patients by combining EV-associated RNA (EV-RNA) and cfDNA signals on n=64 healthy donors (HD) and non-metastatic BrCa patients. Specifically, we isolated EV-enriched samples by a charge-based (CB) method and investigated EV-RNA and cfDNA by next-generation sequencing (NGS) and by digital droplet PCR (ddPCR). Sequencing of cfDNA and EV-RNA from HER2- and HER2+ patients demonstrated concordance with in situ molecular analyses of matched tissues. Combined analysis of the two circulating analytes by ddPCR showed increased sensitivity in ERBB2/HER2 detection compared to single nucleic acid components. Multi-analyte liquid biopsy prediction performance was comparable to tissue-based sequencing results from TCGA. Also, imaging flow cytometry analysis revealed HER2 protein on the surface of EV isolated from the HER2+ BrCa plasma, thus corroborating the potential relevance of studying EV as companion analyte to cfDNA. This data confirms the relevance of combining cfDNA and EV-RNA for HER2 cancer assessment and supports the ONCE as a valuable tool for multi-analytes liquid biopsies' clinical implementation.

Histidine Ligated Iron-Sulfur Peptides.

Iron-sulfur clusters are thought to be ancient cofactors that could have played a role in early protometabolic systems. Thus far, redox active, prebiotically plausible iron-sulfur clusters have always contained cysteine ligands to the cluster. However, extant iron-sulfur proteins can be found to exploit other modes of binding, including ligation by histidine residues, as seen with [2Fe-2S] Rieske and MitoNEET proteins. Here, we investigated the ability of cysteine- and histidine-containing peptides to coordinate a mononuclear Fe2+ center and a [2Fe-2S] cluster and compare their properties with purified iron-sulfur proteins. The iron-sulfur peptides were characterized by UV-vis, circular dichroism, and paramagnetic NMR spectroscopies and cyclic voltammetry. Small (≤6 amino acids) peptides can coordinate [2Fe-2S] clusters through a combination of cysteine and histidine residues with similar reduction potentials as their corresponding proteins. Such complexes may have been important for early cell-like systems.

Prebiotic Environments with Mg2+ and Thiophilic Metal Ions Increase the Thermal Stability of Cysteine and Non-cysteine Peptides.

Wet-dry cycles driven by heating to high temperatures are frequently invoked for the prebiotic synthesis of peptides. Similarly, iron-sulfur clusters are often cited as an example of an ancient catalyst that helped prune early chemical systems into metabolic-like pathways. Because extant iron-sulfur clusters are metallocofactors of protein enzymes and nearly ubiquitous across biology, a reasonable hypothesis is that prebiotic iron-sulfur peptides formed on the early Earth. However, iron-sulfur clusters are coordinated by multiple cysteine residues, and the stability of cysteines to the heat steps of wet-dry cycles has not been determined. It, therefore, has remained unclear if the peptides needed to stabilize the formation of iron-sulfur clusters could have formed. If not, then iron-sulfur-dependent activity may have emerged later, when milder, more biological-like peptide synthesis machinery took hold. Here, we report the thermal stability of cysteine-containing peptides. We show that temperatures of 150 °C lead to the rapid degradation of cysteinyl peptides. However, the presence of Mg2+ at environmentally reasonable concentrations leads to significant protection. Thiophilic metal ions also protect against degradation at 150 °C but require concentrations not frequently observed in the environment. Nevertheless, cysteine-containing peptides are stable at lower, prebiotically plausible temperatures in seawater, carbonate lake, and ferrous lake conditions. The data are consistent with the persistence of cysteine-containing peptides on the early Earth in environments rich in metal ions. High concentrations of Mg2+ are common intra- and extra-cellularly, suggesting that the protection afforded by Mg2+ may reflect conditions that were present on the prebiotic Earth.

Duplications of an iron-sulphur tripeptide leads to the formation of a protoferredoxin.

Based on UV-Vis, NMR, and EPR spectroscopies and DFT and molecular dynamics calculations, a model prebiotic [2Fe-2S] tripeptide was shown to accept and donate electrons. Duplications of the tripeptide sequence led to a protoferredoxin with increased stability. Duplications of primitive peptides may have contributed to the formation of contemporary ferredoxins.

Cysteine containing dipeptides show a metal specificity that matches the composition of seawater.

Model prebiotic dipeptide sequences were identified by bioinformatics and DFT and molecular dynamics calculations. The peptides were then synthesized and evaluated for metal affinity and specificity. Cysteine containing dipeptides were not associated with metal affinities that followed the Irving-Williams series but did follow the concentration trends found in seawater.