Faculty Bibliography

Prokaryotes and eukaryotes alike endogenously generate the gaseous molecule hydrogen sulfide (H₂S). Bacterial H₂S acts as a cytoprotectant against antibiotics-induced stress and promotes redox homeostasis. In E. coli, endogenous H₂S production is primarily dependent on 3-mercaptopyruvate sulfurtransferase (3MST), encoded by mstA. Here, we show that cells lacking 3MST acquire a phenotypic suppressor mutation resulting in compensatory H₂S production and tolerance to antibiotics and oxidative stress. Using whole genome sequencing, we identified a non-synonymous mutation within an uncharacterized LacI-type transcription factor, ycjW. We then mapped regulatory targets of YcjW and discovered it controls the expression of carbohydrate metabolic genes and thiosulfate sulfurtransferase PspE. Induction of pspE expression in the suppressor strain provides an alternative mechanism for H₂S biosynthesis. Our results reveal a complex interaction between carbohydrate metabolism and H₂S production in bacteria and the role, a hitherto uncharacterized transcription factor, YcjW, plays in linking the two.

The essential histone H3 lysine 79 methyltransferase Dot1L regulates transcription and genomic stability and is deregulated in leukemia. The activity of Dot1L is stimulated by mono-ubiquitination of histone H2B on lysine 120 (H2BK120Ub); however, the detailed mechanism is not understood. We report cryo-EM structures of human Dot1L bound to (1) H2BK120Ub and (2) unmodified nucleosome substrates at 3.5 Å and 4.9 Å, respectively. Comparison of both structures, complemented with biochemical experiments, provides critical insights into the mechanism of Dot1L stimulation by H2BK120Ub. Both structures show Dot1L binding to the same extended surface of the histone octamer. In yeast, this surface is used by silencing proteins involved in heterochromatin formation, explaining the mechanism of their competition with Dot1. These results provide a strong foundation for understanding conserved crosstalk between histone modifications found at actively transcribed genes and offer a general model of how ubiquitin might regulate the activity of chromatin enzymes.

Introduction: Dimethyl sulfoxide (DMSO), propylene glycol (PG) and miglyol (MG) are common organic solvents often used to dissolve neuro-pharmacological agents for in vivo assays in animal models of various pediatric brain disorders. Nonetheless, these compounds were reported to exhibit pharmacological and pathological effects on the central nervous system of their own. Here we report chronic effects of these solvents on behavior, motoric functions and brain morphology in young rats (P35-37), following neonatal exposure to one of the compounds (P6).
Method(s): Compounds were administered intraperitoneally (DMSO, 2 or 4 ml/kg; MG, 2 ml/kg) or per os (PG, 2.5 ml/kg) at concentrations considered safe and non-interfering with neuroscience research. Age/sex matched controls received phosphate-buffered saline (PBS). Animals were sacrificed following behavioral and locomotor assays (P40) and their brains were subjected to pathological analyses.
Result(s): Rats exposed to DMSO (n = 10; 4 ml/kg only), spent significantly more time in the open field center and traveled shorter distance (p < 0.05) vs. controls (n = 10). Rats exposed to DMSO and MG exhibited shorter social interactions vs. controls (p < 0.05). CatWalk gait analysis showed various disturbances (p < 0.05) in rats exposed to any of the three compounds (DMSO, 4 ml/kg only). Brain pathological analyses revealed increased expression of microglia (Iba-1+) and reactive astrocytes (GFAP+) in rats exposed to DMSO (p < 0.05).
Conclusion(s): Observed chronic behavioral, motoric and morphologic sequelae of neonatal exposure to DMSO, PG or MG at concentrations that are generally considered safe raise concerns about under-appreciated neuro-toxicity of these common organic solvents, which warrants further exploration in larger translational studies

The biological mediator hydrogen sulfide (H₂S) is produced by bacteria and has been shown to be cytoprotective against oxidative stress and to increase the sensitivity of various bacteria to a range of antibiotic drugs. Here we evaluated whether bacterial H₂S provides resistance against the immune response, using two bacterial species that are common sources of nosocomial infections, Escherichia coli and Staphylococcus aureus Elevations in H₂S increased the resistance of both species to immune-mediated killing. Clearance of infections with wild type and genetically H₂S-deficient E. coli and S. aureus was compared in vitro and in mouse models of abdominal sepsis and burn wound infection. Also, inhibitors of H₂S-producing enzymes were used to assess bacterial killing by leukocytes. We found that inhibition of bacterial H₂S production can increase susceptibility of both bacterial species to rapid killing by immune cells and can improve bacterial clearance after severe burn, an injury that increases susceptibility to opportunistic infections. These findings support the role of H₂S as a bacterial defense mechanism against the host response and implicate bacterial H₂S inhibition as a potential therapeutic intervention in the prevention or treatment of infections.

Isocitrate dehydrogenase (IDH) mutation have been reported to impose in gliomas a shortage of NADPH required to maintain a redox state and may rely on cysteine (Cys) availability for biosynthesis of glutathione (GSH) to ensure antioxidant levels. Cys may be replenished via extracellular intake or by de novo intracellular synthesis via transsulfuration (TS) pathway. The aim of this study was to investigate alterations of Cys metabolism in genetic variants of high-grade gliomas (HGG). Seventeen tumor samples from 15 adult patients (11 M / 4 F; average age 57 years, range 25 - 81 years), who underwent surgical resection for newly diagnosed or recurrent HGG were analyzed by HPLC. Levels of Cys, homocysteine and GSH were correlated with the genetic signature of HGG (wild-types vs. IDH1 mutation, PTEN deletion, EGFR amplification and MGMT methylation). Cys levels were significantly higher (2.1 fold increase; p=0.0038) in IDH1-mut (n=4) vs. IDH1-wt HGG (n=13), with comparable homocysteine and GSH levels. PTEN deletion and EGFR amplification did not significantly alter Cys metabolites with comparable levels of Cys, homocysteine and GSH detected in PTEN-del (n=7) and PTEN-intact (n=6) HGG, as well as in EGFR-amp (n=7) and EGFR-non amp (n=9) HGG. Significantly higher Cys levels (3.2 fold increase; p=0.0186) were also found in MGMT methylated (n=4) vs. non-methylated (n=3) HGG, with comparable levels of homocysteine and GSH. Increased Cys levels detected in IDH1-mut and MGMT methylated HGG support the hypothesis that these tumors may preferentially use the TS pathway for GSH synthesis. These findings are consistent with our report of increased TS pathway enzyme cystathionine B-synthase (CBS) in HGG, but concurrent increased intake of Cys cannot be excluded. Our results suggest utilizing Cys metabolites as potential markers and/or therapeutic targets in some genetic variants of HGG, a hypothesis that should be further explored in larger translational trials

New chemical inhibitors of protein-protein interactions are needed to propel advances in molecular pharmacology. Peptoids are peptidomimetic oligomers with the capability to inhibit protein-protein interactions by mimicking protein secondary structure motifs. Here we report the in silico design of a macrocycle primarily composed of peptoid subunits that targets the β-catenin:TCF interaction. The β-catenin:TCF interaction plays a critical role in the Wnt signaling pathway which is over-activated in multiple cancers, including prostate cancer. Using the Rosetta suite of protein design algorithms, we evaluate how different macrocycle structures can bind a pocket on β-catenin that associates with TCF. The in silico designed macrocycles are screened in vitro using luciferase reporters to identify promising compounds. The most active macrocycle inhibits both Wnt and AR-signaling in prostate cancer cell lines, and markedly diminishes their proliferation. In vivo potential is demonstrated through a zebrafish model, in which Wnt signaling is potently inhibited.

Glycogen is synthesized and stored to maintain postprandial blood glucose homeostasis and to ensure an uninterrupted energy supply between meals. Although the regulation of glycogen turnover has been well studied, the effects of glycogen on aging and disease development have been largely unexplored. In Caenorhabditis elegans fed a high sugar diet, glycogen potentiates resistance to oxidants, but paradoxically, shortens lifespan. Depletion of glycogen by oxidants or inhibition of glycogen synthesis extends the lifespan of worms by an AMPK-dependent mechanism. Thus, glycogen is not merely an inert storage molecule, but also an active regulator of energy balance and aging. Its depletion by oxidants may be beneficial in the treatment of hyperglycemia and glycogen-related diseases.

Fast photochemical oxidation of proteins (FPOP) may be used to characterize changes in protein structure by measuring differences in the apparent rate of peptide oxidation by hydroxyl radicals. The variability between replicates is high for some peptides and limits the statistical power of the technique, even using modern methods controlling variability in radical dose and quenching. Currently, the root cause of this variability has not been systematically explored, and it is unknown if the major source(s) of variability are structural heterogeneity in samples, remaining irreproducibility in FPOP oxidation, or errors in LC-MS quantification of oxidation. In this work, we demonstrate that coefficient of variation of FPOP measurements varies widely at low peptide signal intensity, but stabilizes to ≈ 0.13 at higher peptide signal intensity. We dramatically reduced FPOP variability by increasing the total sample loaded onto the LC column, indicating that the major source of variability in FPOP measurements is the difficulties in quantifying oxidation at low peptide signal intensities. This simple method greatly increases the sensitivity of FPOP structural comparisons, an important step in applying the technique to study subtle conformational changes and protein-ligand interactions. Graphical Abstract ᅟ.