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.

A Magnesium Binding Site And The Anomeric Effect Regulate The Abiotic Redox Chemistry Of Nicotinamide Nucleotides.

Nicotinamide adenine dinucleotide (NAD+) is a redox active molecule that is universally found in biology. Despite the importance and simplicity of this molecule, few reports exist that investigate which molecular features are important for the activity of this ribodinucleotide. By exploiting the nonenzymatic reduction and oxidation of NAD+ by pyruvate and methylene blue, respectively, we were able to identify key molecular features necessary for the intrinsic activity of NAD+ through kinetic analysis. Such features may explain how NAD+ could have been selected early during the emergence of life. Simpler molecules, such as nicotinamide, that lack an anomeric carbon are incapable of accepting electrons from pyruvate. The phosphate moiety inhibits activity in the absence of metal ions but facilitates activity at physiological pH and model prebiotic conditions by recruiting catalytic Mg2+. Reduction proceeds through consecutive single electron transfer events. Of the derivatives tested, including nicotinamide mononucleotide, nicotinamide riboside, 3-(aminocarbonyl)-1-(2,3-dihydroxypropyl)pyridinium, 1-methylnicotinamide, and nicotinamide, only NAD+ and nicotinamide mononucleotide would be capable of efficiently accepting and donating electrons within a nonenzymatic electron transport chain. The data are consistent with early metabolic chemistry exploiting NAD+ or nicotinamide mononucleotide and not simpler molecules.

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.

Cyclophospholipids Enable a Protocellular Life Cycle.

There is currently no plausible path for the emergence of a self-replicating protocell, because prevalent formulations of model protocells are built with fatty acid vesicles that cannot withstand the concentrations of Mg2+ needed for the function and replication of nucleic acids. Although prebiotic chelates increase the survivability of fatty acid vesicles, the resulting model protocells are incapable of growth and division. Here, we show that protocells made of mixtures of cyclophospholipids and fatty acids can grow and divide in the presence of Mg2+-citrate. Importantly, these protocells retain encapsulated nucleic acids during growth and division, can acquire nucleotides from their surroundings, and are compatible with the nonenzymatic extension of an RNA oligonucleotide, chemistry needed for the replication of a primitive genome. Our work shows that prebiotically plausible mixtures of lipids form protocells that are active under the conditions necessary for the emergence of Darwinian evolution.

Autoregulatory control of mitochondrial glutathione homeostasis.

Mitochondria must maintain adequate amounts of metabolites for protective and biosynthetic functions. However, how mitochondria sense the abundance of metabolites and regulate metabolic homeostasis is not well understood. In this work, we focused on glutathione (GSH), a critical redox metabolite in mitochondria, and identified a feedback mechanism that controls its abundance through the mitochondrial GSH transporter, SLC25A39. Under physiological conditions, SLC25A39 is rapidly degraded by mitochondrial protease AFG3L2. Depletion of GSH dissociates AFG3L2 from SLC25A39, causing a compensatory increase in mitochondrial GSH uptake. Genetic and proteomic analyses identified a putative iron-sulfur cluster in the matrix-facing loop of SLC25A39 as essential for this regulation, coupling mitochondrial iron homeostasis to GSH import. Altogether, our work revealed a paradigm for the autoregulatory control of metabolic homeostasis in organelles.

Model Atmospheric Aerosols Convert to Vesicles upon Entry into Aqueous Solution.

Aerosols are abundant on the Earth and likely played a role in prebiotic chemistry. Aerosol particles coagulate, divide, and sample a wide variety of conditions conducive to synthesis. While much work has centered on the generation of aerosols and their chemistry, little effort has been expended on their fate after settling. Here, using a laboratory model, we show that aqueous aerosols transform into cell-sized protocellular structures upon entry into aqueous solution containing lipid. Such processes provide for a heretofore unexplored pathway for the assembly of the building blocks of life from disparate geochemical regions within cell-like vesicles with a lipid bilayer in a manner that does not lead to dilution. The efficiency of aerosol to vesicle transformation is high with prebiotically plausible lipids, such as decanoic acid and decanol, that were previously shown to be capable of forming growing and dividing vesicles. The high transformation efficiency with 10-carbon lipids in landing solutions is consistent with the surface properties and dynamics of short-chain lipids. Similar processes may be operative today as fatty acids are common constituents of both contemporary aerosols and the sea. Our work highlights a new pathway that may have facilitated the emergence of the Earth's first cells.

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.

Protometabolism as out-of-equilibrium chemistry.

It is common to compare life with machines. Both consume fuel and release waste to run. In biology, the engine that drives the living system is referred to as metabolism. However, attempts at deciphering the origins of metabolism do not focus on this energetic relationship that sustains life but rather concentrate on nonenzymatic reactions that produce all the intermediates of an extant metabolic pathway. Such an approach is akin to studying the molecules produced from the burning of coal instead of deciphering how the released energy drives the movement of pistons and ultimately the train when investigating the mechanisms behind locomotion. Theories that do explicitly invoke geological chemical gradients to drive metabolism most frequently feature hydrothermal vent conditions, but hydrothermal vents are not the only regions of the early Earth that could have provided the fuel necessary to sustain the Earth's first (proto)cells. Here, we give examples of prior reports on protometabolism and highlight how more recent investigations of out-of-equilibrium systems may point to alternative scenarios more consistent with the majority of prebiotic chemistry data accumulated thus far. This article is part of the theme issue 'Emergent phenomena in complex physical and socio-technical systems: from cells to societies'.

Treatment of Wound Infections in a Mouse Model Using Zn2+-Releasing Phage Bound to Gold Nanorods.

Infections caused by drug-resistant bacteria, particularly Gram-negative organisms, are increasingly difficult to treat using antibiotics. A potential alternative is "phage therapy", in which phages infect and lyse the bacterial host. However, phage therapy poses serious drawbacks and safety concerns, such as the risk of genetic transduction of antibiotic resistance genes, inconsistent pharmacokinetics, and unknown evolutionary potential. In contrast, metallic nanoparticles possess precise, tunable properties, including efficient conversion of electronic excitation into heat. In this work, we demonstrate that engineered phage-nanomaterial conjugates that target the Gram-negative pathogen Pseudomonas aeruginosa are highly effective as a treatment of infected wounds in mice. Photothermal heating, performed as a single treatment (15 min) or as two treatments on consecutive days, rapidly reduced the bacterial load and released Zn2+ to promote wound healing. The phage-nanomaterial treatment was significantly more effective than systemic standard-of-care antibiotics, with a >10× greater reduction in bacterial load and ∼3× faster healing as measured by wound size reduction when compared to fluoroquinolone treatment. Notably, the phage-nanomaterial was also effective against a P. aeruginosa strain resistant to polymyxins, a last-line antibiotic therapy. Unlike these antibiotics, the phage-nanomaterial showed no detectable toxicity or systemic effects in mice, consistent with the short duration and localized nature of phage-nanomaterial treatment. Our results demonstrate that phage therapy controlled by inorganic nanomaterials can be a safe and effective antimicrobial strategy in vivo.

Metals Are Integral to Life as We Know It.

Investigations of biology and the origins of life regularly focus on the components of the central dogma and thus the elements that compose nucleic acids and peptides. Less attention is given to the inorganic components of a biological cell, which are required for biological polymers to function. The Earth was and continues to be rich in metals, and so investigations of the emergence and evolution of life must account for the role that metal ions play. Evolution is shaped by what is present, and not all elements of the periodic table are equally accessible. The presence of metals, the solubility of their ions, and their intrinsic reactivity all impacted the composition of the cells that emerged. Geological and bioinformatic analyses clearly show that the suite of accessible metal ions changed over the history of the Earth; however, such analyses tend to be interpreted in comparison to average oceanic conditions, which do not represent well the many niche environments present on the Earth. While there is still debate concerning the sequence of events that led to extant biology, what is clear is that life as we know it requires metals, and that past and current metal-dependent events remain, at least partially, imprinted in the chemistry of the cell.

Origins of life: Encapsulating Darwinian evolution.

Encapsulation of RNA within model protocells promotes folding, promotes the binding of substrates, promotes catalysis, and protects against denaturation. A new study argues for an active role of lipid vesicles in the origins of life.

Racing toward Fast and Effective 17O Isotopic Labeling and Nuclear Magnetic Resonance Spectroscopy of N-Formyl-MLF-OH and Associated Building Blocks.

Solid-state 1H, 13C, and 15N nuclear magnetic resonance (NMR) spectroscopy has been an essential analytical method in studying complex molecules and biomolecules for decades. While oxygen-17 (17O) NMR is an ideal and robust candidate to study hydrogen bonding within secondary and tertiary protein structures for example, it continues to elude many. We discuss an improved multiple-turnover labeling procedure to develop a fast and cost-effective method to 17O label fluoroenylmethyloxycarbonyl (Fmoc)-protected amino acid building blocks. This approach allows for inexpensive ($0.25 USD/mg) insertion of 17O labels, an important barrier to overcome for future biomolecular studies. The 17O NMR results of these building blocks and a site-specific strategy for labeled N-acetyl-MLF-OH and N-formyl-MLF-OH tripeptides are presented. We showcase growth in NMR development for maximizing sensitivity gains using emerging sensitivity enhancement techniques including population transfer, high-field dynamic nuclear polarization, and cross-polarization magic-angle spinning cryoprobes.

Protometabolic Reduction of NAD+ with α-Keto Acids.

Deciphering the origins of the chemistry that supports life has frequently centered on determining prebiotically plausible paths that produce the molecules found in biology. What has been less investigated is how the energy released from the breakdown of foodstuff is coupled to the persistence of the protocell. To gain better insight into how such coupled chemistry could have emerged prebiotically, we probed the reactivity of the ribodinucleotide NAD+ with small organic molecules that were previously identified as potential constituents of protometabolism. We find that NAD+ is readily reduced nonenzymatically by α-keto acids, such as pyruvate and oxaloacetate, during oxidative decarboxylation. In the presence of FAD and a terminal electron acceptor, the consumption of α-keto acids by NAD+ initiates a plausible prebiotic electron transport chain. The observed reactivity suggests that components of the RNA world were capable of initiating the chemistry needed to capture the energy released from catabolism to drive anabolism.

Population-Level Membrane Diversity Triggers Growth and Division of Protocells.

To date, multiple mechanisms have been described for the growth and division of model protocells, all of which exploit the lipid dynamics of fatty acids. In some examples, the more heterogeneous aggregate consisting of fatty acid and diacyl phospholipid or fatty acid and peptide grows at the expense of the more homogeneous aggregate containing a restricted set of lipids with similar dynamics. Imbalances between surface area and volume during growth can generate filamentous vesicles, which are typically divided by shear forces. Here, we describe another pathway for growth and division that depends simply on differences in the compositions of fatty acid membranes without additional components. Growth is driven by the thermodynamically favorable mixing of lipids between two populations, i.e., the system as a whole proceeds toward equilibrium. Division is the result of growth-induced curvature. Importantly, growth and division do not require a specific composition of lipids. For example, vesicles made from one type of lipid, e.g., short-chain fatty acids, grow and divide when fed with vesicles consisting of another type of lipid, e.g., long-chain fatty acids, and vice versa. After equilibration, additional rounds of growth and division could potentially proceed by the introduction of compositionally distinct aggregates. Since prebiotic synthesis likely gave rise to mixtures of lipids, the data are consistent with the presence of growing and dividing protocells on the prebiotic Earth.

Spectral decomposition of iron-sulfur clusters.

The near universal availability of UV-Visible spectrophotometers makes this instrument a highly exploited tool for the inexpensive, rapid examination of iron-sulfur clusters. Yet, the analysis of iron-sulfur cluster reconstitution experiments by UV-Vis spectroscopy is notoriously difficult due to the presence of broad, ill-defined peaks. Other types of spectroscopies, such as electron paramagnetic resonance spectroscopy and Mössbauer spectroscopy, are superior in characterizing the type of cluster present and their associated electronic transitions but require expensive, less readily available equipment. Here, we describe a tool that utilizes the accessible and convenient platform of Microsoft Excel to allow for the semi-quantitative analysis of iron-sulfur clusters by UV-Vis spectroscopy. This tool, which we call Fit-FeS, could potentially be used to additionally decompose spectra of solutions containing chromophores other than iron-sulfur clusters.

Cell-Free Synthesis of Dopamine and Serotonin in Two Steps with Purified Enzymes.

The synthesis of serotonin and dopamine with purified enzymes is described. Both pathways start from an amino acid substrate and synthesize the monoamine neurotransmitter in two enzymatic steps. The enzymes human tryptophan hydroxylase isoform 2, Rattus norvegicus tyrosine hydroxylase, Chlamydia pneumoniae Cpn1046, and aromatic amino acid decarboxylase from Drosophila melanogaster are recombinantly expressed, purified, and shown to be functional in vitro. The hydroxylases efficiently convert L-DOPA (L-dihydroxy-phenylalanine) and 5-HTP (5-hydroxytryptophan) from L-tyrosine and L-tryptophan, respectively. A single aromatic amino acid decarboxylase is capable of converting both hydroxylated intermediates into the final neurotransmitter. The platform described here may facilitate future efforts to generate medically useful artificial cells and nanofactories.

Artificial cells drive neural differentiation.

We report the construction of artificial cells that chemically communicate with mammalian cells under physiological conditions. The artificial cells respond to the presence of a small molecule in the environment by synthesizing and releasing a potent protein signal, brain-derived neurotrophic factor. Genetically controlled artificial cells communicate with engineered human embryonic kidney cells and murine neural stem cells. The data suggest that artificial cells are a versatile chassis for the in situ synthesis and on-demand release of chemical signals that elicit desired phenotypic changes of eukaryotic cells, including neuronal differentiation. In the future, artificial cells could be engineered to go beyond the capabilities of typical smart drug delivery vehicles by synthesizing and delivering specific therapeutic molecules tailored to distinct physiological conditions.

Cyclophospholipids Increase Protocellular Stability to Metal Ions.

Model protocells have long been constructed with fatty acids, because these lipids are prebiotically plausible and can, at least theoretically, support a protocell life cycle. However, fatty acid protocells are stable only within a narrow range of pH and metal ion concentration. This instability is particularly problematic as the early Earth would have had a range of conditions, and extant life is completely reliant on metal ions for catalysis and the folding and activity of biological polymers. Here, prebiotically plausible monoacyl cyclophospholipids are shown to form robust vesicles that survive a broad range of pH and high concentrations of Mg2+ , Ca2+ , and Na+ . Importantly, stability to Mg2+ and Ca2+ is improved by the presence of environmental concentrations of Na+ . These results suggest that cyclophospholipids, or lipids with similar characteristics, may have played a central role during the emergence of Darwinian evolution.