Light-Enabled Identification of the Neuronal Substrates of Alkylphenol Anesthetics

General anesthetics are a critical class of drugs in modern medicine; however, the precise mechanisms by which they cause unconsciousness and unwanted side effects are largely undefined. In order to understand pharmacologic mechanisms of anesthetic action, drug interactions with macromolecular substrates and the subsequent functional consequences must be characterized. Analogs of general anesthetics that function as photolabels have been developed to assist in the identification of molecular targets. One such photolabel, meta-azi-propofol (AziPm), is an analog of the clinically used alkylphenol anesthetic propofol. In this work, AziPm is employed in a variety of experiments that aim to identify molecular substrates of propofol. Two proteins identified as propofol targets are more thoroughly examined: (1) the sirtuin deacetylase SIRT2 and (2) the mitochondrial voltage-dependent anion channel (VDAC). The binding sites of propofol on these proteins, and the in vitro functional consequences of propofol binding, are determined. Also described are the molecular interactions of VDAC with a separate ligand, cholesterol, which shares a binding site with propofol. In addition to molecular studies, a novel in vivo photolabeling technique, called optoanesthesia, that utilizes AziPm is introduced, and the behavioral phenotype induced by optoanesthesia in Xenopus laevis tadpoles is characterized. Finally, optoanesthesia is demonstrated with other ligands, including a photoactive analog of an anthracene anesthetic, and mechanistic insight into the pharmacology of this anthracene is revealed

Replication Protein A (RPA) Targeting of Uracil DNA Glycosylase (UNG2)

Replication Protein A (RPA) is a single stranded DNA binding protein which stabilizes ssDNA for replication and repair. One function of RPA is to bind the DNA repair enzyme uracil DNA glycosylase (UNG2) and direct its activity towards ssDNA dsDNA junctions. UNG2 removes uracil bases from DNA which can appear through dUMP misincorporation or through cytosine deamination. If uracil is present instead of a cytosine, then the original GC pair becomes a GU pair. The uracil will then base pair to adenine in the replicated daughter strand. This results in a GC → AT mutation that could contribute to cancer formation. RPA is known to target UNG2 towards individual uracil bases. We hypothesize that RPA will target UNG2 to uracil bases in DNA regardless of the number of uracils in the DNA strand

Interaction of Fluorescent Probes with Sirtuin Proteins

Sirtuins are a class of proteins belonging to the Sir2 (Silencing information regulator 2) family of NAD+ dependent protein lysine deacylases. Different Isoforms (SIRT1-SIRT7) differ in their specific deacylase activity and cellular location. They have roles in DNA repair, glucose metabolism, and cellular proliferation which make them highly desirable targets for carcinoma therapeutics. We previously used 1-aminoanthracene’s (AMA) fluorescent properties when bound with SIRT2 (Kd of 37 μM) to develop a high-throughput screen to identify novel ligands that inhibit SIRT2’s enzymatic activities. We hope to reveal other potential probes for future high-throughput screening with all the sirtuin isotopes. 1-AMA’s fluorescence along with fluorescent labeled peptides “Cy3-PEG4-H4K16(myr)” and “FAM-PEG4-H4K16(myr)” were used in binding assays to determine their affinities with SIRT2, SIRT3, and SIRT6. Further, we determined 1-AMA’s ability to bind sirtuin isoforms when they were equilibrated with 100 μM of various acyl-peptides. 1-AMA displays weak binding to SIRT3 and SIRT6 when compared to SIRT2. FAM-PEG4-H4K16(myr) binds SIRT2 with a Kd of 7nM which is much higher than its interaction with SIRT3 and SIRT6 (Kd of 6 μM and 2 μM, respectively). Cy3-PEG4-H4K16(myr) binds as expected SIRT2,3,6 although its affinity for SIRT6 changes minimally with the addition of ADP-ribose, which suggests Cy3 may facilitate binding in the absence of SIRT6’s co-factor. Future work will test additional probes with the other sirtuin proteins and establish their competency to be utilized for high-throughput screening

Effect of Uracil DNA Glycosylase Activity on the Efficacy of Thymidylate Synthase Inhibitor/HDAC Inhibitor Combination Therapies in Colon Cancer

Human uracil DNA glycosylase (UNG2) is responsible for removing uracil bases from DNA and initiates base excision repair pathways. Accumulation of uracil or its fluorinated analogs in DNA is one of the killing mechanisms of thymidylate synthase (TS) inhibitors in cancer cells, and depletion of UNG2 often enhances the toxicity of these anticancer drugs. We tested the effect of UNG2 KO on the efficacy of multiple TS inhibitors (5-fluorouracil, fluorodeoxyuridine, and pemetrexed) and we determined that, except for 5-fluorouracil, all other TS inhibitors were significantly more potent in UNG2 KO cells compared to wild-type HT29 cells. Interestingly, UNG2 protein levels can also be depleted by the HDAC inhibitors SAHA and MS275, providing a pharmacologic strategy to reduce UNG2 activity in cells. Unexpectedly, the HDAC inhibitors synergized with 5-fluorouracil but not fluorodeoxyuridine in both wild-type and UNG2-knockout cells. Similarly, HDAC inhibitors synergized with pemetrexed in wild-type HT29 but not UNG2-knockout cells. This suggested that HDAC inhibitors sensitized cells to 5-fluorouracil through an UNG2-independent mechanism. Interestingly, SAHA depleted the UNG2 level, whereas TS inhibitors alone and their combination with SAHA upregulated the level of UNG2 at 24 hours. This suggests HDAC inhibitors deplete UNG2 but, when combined with TS inhibitors, it did not affect UNG2, at least at a concentration of 100nM. Our future aim is to study these pharmacological drug combinations targeting UNG2 activity in cells and elucidate exact mechanisms of cell death

UNG2 and RPA Activity on ssDNA-dsDNA Junctions

Uracil DNA glycosylase, or UNG2, is an enzyme that is involved in DNA repair. Its primary job is to eliminate harmful uracil bases from DNA strands. To do this, the enzyme is assisted by replication protein A (RPA). RPA helps UNG2 in the identification of uracil bases by targeting UNG2 activity near ssDNA-dsDNA junctions (1-3). The results from assays presented here agree with published findings that showed UNG2 is heavily targeted by RPA to uracil bases that are close to ssDNA-dsDNA junctions (for example, uracil located 9 bps from the junction as opposed to 33 bps) (1,2). However, these previous experiments were performed in the absence of a macromolecular crowding agent. Inert compounds such as PEG8K can be used experimentally to better represent the physiologic environment inside a cell, which is very crowded with proteins and other small molecules and differs from dilute conditions often used in enzyme assays. In the presence of a crowding agent (PEG8K), we found that RPA balances UNG2’s selectivity for uracil sites near ssDNA-dsDNA junctions that contain a 5’ ssDNA section. In other words, UNG2 becomes less targeted to uracils that are very close to the junction (i.e., U9). Interestingly, this effect was not seen when we examined RPA effects on UNG2 activity using ssDNA-dsDNA junctions substrates that contain a 3’ ssDNA section

Resin infusion of layered metal/composite hybrid and resulting metal/composite hybrid laminate

A method of fabricating a metal/composite hybrid laminate is provided. One or more layered arrangements are stacked on a solid base to form a layered structure. Each layered arrangement is defined by a fibrous material and a perforated metal sheet. A resin in its liquid state is introduced along a portion of the layered structure while a differential pressure is applied across the laminate structure until the resin permeates the fibrous material of each layered arrangement and fills perforations in each perforated metal sheet. The resin is cured thereby yielding a metal/composite hybrid laminate

Substrate-Dependent Modulation of SIRT2 by a Fluorescent Probe, 1-Aminoanthracene

Sirtuin isoform 2 (SIRT2) is an enzyme that catalyzes the removal of acyl groups from lysine residues. SIRT2’s catalytic domain has a hydrophobic tunnel where its substrate acyl groups bind. Here, we report that the fluorescent probe 1-aminoanthracene (AMA) binds within SIRT2’s hydrophobic tunnel in a substrate-dependent manner. AMA’s interaction with SIRT2 was characterized by its enhanced fluorescence upon protein binding (\u3e10-fold). AMA interacted weakly with SIRT2 alone in solution (Kd = 37 μM). However, when SIRT2 was equilibrated with a decanoylated peptide substrate, AMA’s affinity for SIRT2 was enhanced ∼10-fold (Kd = 4μM). The peptide’s decanoyl chain and AMA co-occupied SIRT2’s hydrophobic tunnel when bound to the protein. In contrast, binding of AMA to SIRT2 was competitive with a myristoylated substrate whose longer acyl chain occluded the entire tunnel. AMA competitively inhibited SIRT2 demyristoylase activity with an IC50 of 21 μM, which was significantly more potent than its inhibition of other deacylase activities. Finally, binding and structural analysis suggests that the AMA binding site in SIRT2’s hydrophobic tunnel was structurally stabilized when SIRT2 interacted with a decanoylated or 4-oxononanoylated substrate, but AMA’s binding site was less stable when SIRT2 was bound to an acetylated substrate. Our use of AMA to explore changes in SIRT2’s hydrophobic tunnel that are induced by interactions with specific has implications for developing ligands that modulate SIRT2’s substrate specificity

Modeling Biphasic, Non-Sigmoidal Dose-Response Relationships: Comparison of Brain-Cousens and Cedergreen Models for a Biochemical Dataset

Biphasic, non-sigmoidal dose-response relationships are frequently observed in biochemistry and pharmacology, but they are not always analyzed with appropriate statistical methods. Here, we examine curve fitting methods for "hormetic" dose-response relationships where low and high doses of an effector produce opposite responses. We provide the full dataset used for modeling, and we provide the code for analyzing the dataset in SAS using two established mathematical models of hormesis, the Brain-Cousens model and the Cedergreen model. We show how to obtain and interpret curve parameters such as the ED50 that arise from modeling, and we discuss how curve parameters might change in a predictable manner when the conditions of the dose-response assay are altered. In addition to modeling the raw dataset that we provide, we also model the dataset after applying common normalization techniques, and we indicate how this affects the parameters that are associated with the fit curves. The Brain-Cousens and Cedergreen models that we used for curve fitting were similarly effective at capturing quantitative information about the biphasic dose-response relationships

Modeling Biphasic, Non-Sigmoidal Dose-Response Relationships: Comparison of Brain- Cousens and Cedergreen Models for a Biochemical Dataset

Biphasic, non-sigmoidal dose-response relationships are frequently observed in biochemistry and pharmacology, but they are not always analyzed with appropriate statistical methods. Here, we examine curve fitting methods for “hormetic” dose-response relationships where low and high doses of an effector produce opposite responses. We provide the full dataset used for modeling, and we provide the code for analyzing the dataset in SAS using two established mathematical models of hormesis, the Brain-Cousens model and the Cedergreen model. We show how to obtain and interpret curve parameters such as the ED50 that arise from modeling, and we discuss how curve parameters might change in a predictable manner when the conditions of the dose-response assay are altered. In addition to modeling the raw dataset that we provide, we also model the dataset after applying common normalization techniques, and we indicate how this affects the parameters that are associated with the fit curves. The Brain-Cousens and Cedergreen models that we used for curve fitting were similarly effective at capturing quantitative information about the biphasic dose-response relationships

A Conserved Mechanism for Hormesis in Molecular Systems

Hormesis refers to dose-response phenomena where low dose treatments elicit a response that is opposite the response observed at higher doses. Hormetic dose-response relationships have been observed throughout all of biology, but the underlying determinants of many reported hormetic dose-responses have not been identified. In this report, we describe a conserved mechanism for hormesis on the molecular level where low dose treatments enhance a response that becomes reduced at higher doses. The hormetic mechanism relies on the ability of protein homo-multimers to simultaneously interact with a substrate and a competitor on different subunits at low doses of competitor. In this case, hormesis can be observed if simultaneous binding of substrate and competitor enhances a response of the homo-multimer. We characterized this mechanism of hormesis in binding experiments that analyzed the interaction of homotrimeric proliferating cell nuclear antigen (PCNA) with uracil DNA glycosylase (UNG2) and a fluorescein-labeled peptide. Additionally, the basic features of this molecular mechanism appear to be conserved with at least two enzymes that are stimulated by low doses of inhibitor: dimeric BRAF and octameric glutamine synthetase 2 (GS2). Identifying such molecular mechanisms of hormesis may help explain specific hormetic responses of cells and organisms treated with exogenous compounds