Xueshu Xie

The post-translational modification, lysine succinylation is implicated in the regulation of various metabolic pathways. However, its biological relevance remains uncertain due to methodological difficulties in determining high-impact succinylation sites. In the present study, using stable isotope labeling and data-independent acquisition mass spectrometry, we quantified lysine succinylation stoichiometries in mouse livers. Despite the low overall stoichiometry of lysine succinylation, several high stoichiometry sites were identified, especially upon deletion of the desuccinylase SIRT5. In particular, multiple high stoichiometry lysine sites identified in argininosuccinate synthase (ASS1), a key enzyme in urea cycle, are regulated by SIRT5. Mutation of the high stoichiometry lysine in ASS1 to succinyl-mimetic glutamic acid significantly decreased its enzymatic activity. Metabolomics profiling confirms that SIRT5 deficiency decreases urea cycle activity in liver. Importantly, SIRT5 deficiency compromises ammonia tolerance and reduces locomotor and exploratory activity in male mice upon high-ammonium diet feeding. Therefore, lysine succinylation is functionally important in ammonia metabolism.

Post-translational modifications (PTM) decorate proteins to provide functional heterogeneity to an existing proteome. The large number of known PTMs highlights the many ways that cells can modify their proteins to respond to diverse stimuli. Recently, PTMs have begun to receive increased interest because new sensitive proteomics workflows and structural methodologies now allow researchers to obtain large-scale, in-depth and unbiased information concerning PTM type and site localization. However, few PTMs have been extensively assessed for functional consequences, leaving a large knowledge gap concerning the inner workings of the cell. Here, we review understanding of N-𝜀-lysine acetylation in bacteria, a PTM that was largely ignored in bacteria until a decade ago. Acetylation is a modification that can dramatically change the function of a protein through alteration of its properties, including hydrophobicity, solubility, and surface properties, all of which may influence protein conformation and interactions with substrates, cofactors and other macromolecules. Most bacteria carry genes predicted to encode the lysine acetyltransferases and lysine deacetylases that add and remove acetylations, respectively. Many bacteria also exhibit acetylation activities that do not depend on an enzyme, but instead on direct transfer of acetyl groups from the central metabolites acetyl coenzyme A or acetyl phosphate. Regardless of mechanism, most central metabolic enzymes possess lysines that are acetylated in a regulated fashion and many of these regulated sites are conserved across the spectrum of bacterial phylogeny. The interconnectedness of acetylation and central metabolism suggests that acetylation may be a response to nutrient availability or the energy status of the cell. However, this and other hypotheses related to acetylation remain untested.

Posttranslational modification of a protein, either alone or in combination with other modifications, can control properties of that protein, such as enzymatic activity, localization, stability, or interactions with other molecules. N -ε-Lysine acetylation is one such modification that has gained attention in recent years, with a prevalence and significance that rival those of phosphorylation.

Isotopic compositions of reactants affect the rates of chemical and biochemical reactions. Usually it is assumed that heavy stable isotope enrichment leads to progressively slower reactions. Yet the effect of stable isotopes may be nonlinear, as exemplified by the “isotopic resonance” phenomenon. Since the isotopic compositions of other planets of Solar system, including Mars and Venus, are markedly different from terrestrial (e.g., deuterium content is ≈5 and ≈100 times higher, respectively), it is far from certain that terrestrial life will thrive in these isotopic conditions. Here we found that Martian deuterium content negatively affected survival of shrimp in semi-closed biosphere on a year-long time scale. Moreover, the bacterium Escherichia coli grows slower at Martian isotopic compositions and even slower at Venus’s compositions. Thus, the biological impact of varying stable isotope compositions needs to be taken into account when planning interplanetary missions.

Sixty years after the seminal Miller-Urey experiment that abiotically produced a mixture of racemized amino acids, we provide a definite proof that this primordial soup, when properly cooked, was edible for primitive organisms. Direct admixture of even small amounts of Miller-Urey mixture strongly inhibitsE. colibacteria growth due to the toxicity of abundant components, such as cyanides. However, these toxic compounds are both volatile and extremely reactive, while bacteria are highly capable of adaptation. Consequently, after bacterial adaptation to a mixture of the two most abundant abiotic amino acids, glycine and racemized alanine, dried and reconstituted MU soup was found to support bacterial growth and even accelerate it compared to a simple mixture of the two amino acids. Therefore, primordial Miller-Urey soup was perfectly suitable as a growth media for early life forms.

Isotopic composition of reactants affects the rates of chemical and biochemical reactions. As a rule, enrichment of heavy stable isotopes leads to progressively slower reactions. But the recent isotopic resonance hypothesis suggests that the dependence of the reaction rate upon the enrichment degree is not monotonous. Instead, at some “resonance” isotopic compositions, the kinetics increases, while at “off-resonance” compositions the same reactions progress slower. To test the predictions of this hypothesis for the elements C, H, N and O, we designed a precise (standard error ±0.05%) experiment that measures the parameters of bacterial growth in minimal media with varying isotopic composition. A number of predicted resonance conditions were tested, with significant enhancements in kinetics discovered at these conditions. The combined statistics extremely strongly supports the validity of the isotopic resonance phenomenon (p ≪ 10⁻¹⁵). This phenomenon has numerous implications for the origin of life studies and astrobiology and possible applications in agriculture, biotechnology, medicine, chemistry and other areas.

Using very precise (±0.05%) measurements of the growth parameters for bacteria E. coli grown on minimal media, we aimed to determine the lowest deuterium concentration at which the adverse effects that are prominent at higher enrichments start to become noticeable. Such a threshold was found at 0.5% D, a surprisingly high value, while the ultralow deuterium concentrations (≤0.25% D) showed signs of the opposite trend. Bacterial adaptation for 400 generations in isotopically different environment confirmed preference for ultralow (≤0.25% D) enrichment. This effect appears to be similar to those described in sporadic but multiple earlier reports. Possible explanations include hormesis and isotopic resonance phenomena, with the latter explanation being favored.

Electrospray ionization (ESI) is increasingly used to measure binding strengths, but it is not always clear whether the ESI process introduces artifacts. Here we propose a model monomer–dimer equilibrium system based on DNA oligonucleotides to systematically explore biomolecular self-association with the ESI-mass spectrometry (MS) titration method. The oligonucleotides are designed to be self-complementary and have the same chemical composition and mass, allowing for equal ionization probability, ion transmission, and detection efficiency in ESI-MS. The only difference is the binding strength, which is determined by the nucleotide sequence and can be tuned to cover a range of dissociation constant values. This experimental design allows one to focus on the impact of ESI on the chemical equilibrium and to avoid the other typical sources of variation in ESI-MS signal responses, which yields a direct comparison of samples with different binding strengths. For a set of seven model DNA oligonucleotides, the monomer–dimer binding equilibrium was probed with the ESI-MS titration method in both positive and negative ion modes. A mathematical model describing the dependence of the monomer-to-dimer peak intensity ratio on the DNA concentration was proposed and used to extract apparent K_d values and the fraction of DNA duplex that irreversibly dissociates in the gas phase. The K_d values determined via ESI-MS titration were compared to those determined in solution with isothermal titration calorimetry and equilibrium thermal denaturation methods and were found to be significantly lower. The observed discrepancy was attributed to a greater electrospray response of dimers relative to that of monomers.