42 research outputs found
Anthracenyl isoxazole amides (AIMs) stabilize quadruplex DNA structures in telomeric and c-MYC promotor sequences
Approximately 23,000 people are affected by malignant brain and CNS tumors in the United States each year and those afflicted have a median survival rate of only 12β15 months due to the limited treatment options available. The anthracenyl isoxazole amides (AIMs) are a novel class of compounds that have been shown to possess significant anti tumor activity in the NCI 60 cell line panel and to inhibit growth of SNB-19 glioblastoma cells at low micromolar and nanomolar concentrations. The goal of our current research is to characterize the mechanism underlying the anti tumor activity of the AIMs. We hypothesize the mechanism of growth inhibition to involve binding and stabilization of a DNA tertiary structure known as a guanine quadruplex. Various regulatory regions of DNA, such the c-MYC oncogene promoter sequence and repeating sequences formed at the end of telomeres, adopt the quadruplex conformation. Stabilization of quadruplex structures by small-molecule binding ligands has been reported to modulate the expression of genes and inhibit telomerase activity. Down-regulation of certain oncogenes or the inhibition of telomerase can cause tumor cells to undergo apoptosis or become unable to efficiently replicate. To establish whether interactions between the AIMs and quadruplex-forming sequences act to stabilize the quadruplex tertiary structure, circular dichroism spectroscopy (CD) was employed. CD is a method that utilizes the differential absorbance of left and right circularly polarized light to examine the chiral structure of molecules. CD thermal melting studies were conducted to determine whether the AIMs would increase the melting temperature of quadruplex forming sequences as an indication of increased stability. Our results demonstrated the AIMs, at two equivalents, increase the melting temperature (Tm) of both the c-MYC promoter and telomeric sequences by approximately 2β3 Β°C with strong statistical significance and reproducibility. Utilizing CD allows the use of low micromolar concentrations of DNA and this method will be used in the future to rapidly develop additional structure-activity relationships between novel AIMs and quadruplex forming sequences. Our laboratory has also shown chemical shifts in the imino region upon treatment with the AIMs for both the c-MYC promotor and telomeric sequence by NMR, providing additional evidence of the AIMs interaction with quadruplex structures. Interestingly, fluorescence microscopy of SNB-19 cells treated with AIMs show their localization is primarily in the mitochondria, and mitochondrial DNA contains several other important quadruplex forming sequences. Mitochondrial-dependent apoptosis has been suggested for other quadruplex binding ligands and therefore our future work will examine the potential stabilization of mitochondrial quadruplexes by these and other novel AIMs
Auriculocondylar syndrome 2 results from the dominant-negative action of PLCB4 variants.
Auriculocondylar syndrome 2 (ARCND2) is a rare autosomal dominant craniofacial malformation syndrome linked to multiple genetic variants in the coding sequence of phospholipase C Ξ²4 (PLCB4). PLCB4 is a direct signaling effector of the endothelin receptor type A (EDNRA)-Gq/11 pathway, which establishes the identity of neural crest cells (NCCs) that form lower jaw and middle ear structures. However, the functional consequences of PLCB4 variants on EDNRA signaling is not known. Here, we show, using multiple signaling reporter assays, that known PLCB4 variants resulting from missense mutations exert a dominant-negative interference over EDNRA signaling. In addition, using CRISPR/Cas9, we find that F0 mouse embryos modeling one PLCB4 variant have facial defects recapitulating those observed in hypomorphic Ednra mouse models, including a bone that we identify as an atavistic change in the posterior palate/oral cavity. Remarkably, we have identified a similar osseous phenotype in a child with ARCND2. Our results identify the disease mechanism of ARCND2, demonstrate that the PLCB4 variants cause craniofacial differences and illustrate how minor changes in signaling within NCCs may have driven evolutionary changes in jaw structure and function. This article has an associated First Person interview with the first author of the paper
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Using Synthetic ApoC-II Peptides and nAngptl4 Fragments to Measure Lipoprotein Lipase Activity in Radiometric and Fluorescent Assays
Lipoprotein lipase (LPL) plays a crucial role in preventing dyslipidemia by hydrolyzing triglycerides (TGs) in packaged lipoproteins. Since hypertriglyceridemia (HTG) is a major risk factor for cardiovascular disease (CVD), the leading cause of death worldwide, methods that accurately quantify the hydrolytic activity of LPL in clinical and pre-clinical samples are much needed. To date, the methods used to determine LPL activity vary considerably in their approach, in the LPL substrates used, and in the source of LPL activators and inhibitors used to quantify LPL-specific activity, rather than other lipases, e.g., hepatic lipase (HL) or endothelial lipase (EL) activity. Here, we describe methods recently optimized in our laboratory, using a synthetic ApoC-II peptide to activate LPL, and an n-terminal Angiopoietin-Like 4 fragment (nAngptl4) to inhibit LPL, presenting a cost-effective and reproducible method to measure LPL activity in human post-heparin plasma (PHP) and in LPL-enriched heparin released (HR) fractions from LPL secreting cells. We also describe a modified version of the triolein-based assay using human serum as a source of endogenous activators and inhibitors and to determine the relative abundance of circulating factors that regulate LPL activity. Finally, we describe how an ApoC-II peptide and nAngptl4 can be applied to high-throughput measurements of LPL activity using the EnzChek™ fluorescent TG analog substrate with PHP, bovine LPL, and HR LPL enriched fractions. In summary, this manuscript assesses the current methods of measuring LPL activity and makes new recommendations for measuring LPL-mediated hydrolysis in pre-clinical and clinical samples.
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Neoamphimedine Circumvents Metnase-Enhanced DNA Topoisomerase IIΞ± Activity Through ATP-Competitive Inhibition
Type IIΞ± DNA topoisomerase (TopoIIΞ±) is among the most important clinical drug targets for the treatment of cancer. Recently, the DNA repair protein Metnase was shown to enhance TopoIIΞ± activity and increase resistance to TopoIIΞ± poisons. Using in vitro DNA decatenation assays we show that neoamphimedine potently inhibits TopoIIΞ±-dependent DNA decatenation in the presence of Metnase. Cell proliferation assays demonstrate that neoamphimedine can inhibit Metnase-enhanced cell growth with an IC50 of 0.5 ΞΌM. Additionally, we find that the apparent Km of TopoIIΞ± for ATP increases linearly with higher concentrations of neoamphimedine, indicating ATP-competitive inhibition, which is substantiated by molecular modeling. These findings support the continued development of neoamphimedine as an anticancer agent, particularly in solid tumors that over-express Metnase
Characterization of 4-HNE Modified L-FABP Reveals Alterations in Structural and Functional Dynamics
4-Hydroxynonenal (4-HNE) is a reactive Ξ±,Ξ²-unsaturated aldehyde produced during oxidative stress and subsequent lipid peroxidation of polyunsaturated fatty acids. The reactivity of 4-HNE towards DNA and nucleophilic amino acids has been well established. In this report, using proteomic approaches, liver fatty acid-binding protein (L-FABP) is identified as a target for modification by 4-HNE. This lipid binding protein mediates the uptake and trafficking of hydrophobic ligands throughout cellular compartments. Ethanol caused a significant decrease in L-FABP protein (P<0.001) and mRNA (P<0.05), as well as increased poly-ubiquitinated L-FABP (P<0.001). Sites of 4-HNE adduction on mouse recombinant L-FABP were mapped using MALDI-TOF/TOF mass spectrometry on apo (Lys57 and Cys69) and holo (Lys6, Lys31, His43, Lys46, Lys57 and Cys69) L-FABP. The impact of 4-HNE adduction was found to occur in a concentration-dependent manner; affinity for the fluorescent ligand, anilinonaphthalene-8-sulfonic acid, was reduced from 0.347 Β΅M to Kd1β=β0.395 Β΅M and Kd2β=β34.20 Β΅M. Saturation analyses revealed that capacity for ligand is reduced by approximately 50% when adducted by 4-HNE. Thermal stability curves of apo L-FABP was also found to be significantly affected by 4-HNE adduction (ΞTmβ=β5.44Β°C, P<0.01). Computational-based molecular modeling simulations of adducted protein revealed minor conformational changes in global protein structure of apo and holo L-FABP while more apparent differences were observed within the internal binding pocket, revealing reduced area and structural integrity. New solvent accessible portals on the periphery of the protein were observed following 4-HNE modification in both the apo and holo state, suggesting an adaptive response to carbonylation. The results from this study detail the dynamic process associated with L-FABP modification by 4-HNE and provide insight as to how alterations in structural integrity and ligand binding may a contributing factor in the pathogenesis of ALD
Genetic Variants of Lipoprotein Lipase and Regulatory Factors Associated with Alzheimerβs Disease Risk
Lipoprotein lipase (LPL) is a key enzyme in lipid and lipoprotein metabolism. The canonical role of LPL involves the hydrolysis of triglyceride-rich lipoproteins for the provision of FFAs to metabolic tissues. However, LPL may also contribute to lipoprotein uptake by acting as a molecular bridge between lipoproteins and cell surface receptors. Recent studies have shown that LPL is abundantly expressed in the brain and predominantly expressed in the macrophages and microglia of the human and murine brain. Moreover, recent findings suggest that LPL plays a direct role in microglial function, metabolism, and phagocytosis of extracellular factors such as amyloid- beta (Aβ). Although the precise function of LPL in the brain remains to be determined, several studies have implicated LPL variants in Alzheimer’s disease (AD) risk. For example, while mutations shown to have a deleterious effect on LPL function and expression (e.g., N291S, HindIII, and PvuII) have been associated with increased AD risk, a mutation associated with increased bridging function (S447X) may be protective against AD. Recent studies have also shown that genetic variants in endogenous LPL activators (ApoC-II) and inhibitors (ApoC-III) can increase and decrease AD risk, respectively, consistent with the notion that LPL may play a protective role in AD pathogenesis. Here, we review recent advances in our understanding of LPL structure and function, which largely point to a protective role of functional LPL in AD neuropathogenesis
A Mechanistic and Structural Analysis of the Inhibition of the 90-kDa Heat Shock Protein by the Benzoquinone and Hydroquinone AnsamycinsSβ
The benzoquinone ansamycins inhibit the ATPase activity of the 90-kDa heat shock protein (Hsp90), disrupting the function of numerous client proteins involved in oncogenesis. In this study, we examine the role of NAD(P)H:quinone oxidoreductase 1 (NQO1) in the metabolism of trans- and cis-amide isomers of the benzoquinone ansamycins and their mechanism of Hsp90 inhibition. Inhibition of purified human Hsp90 by a series of benzoquinone ansamycins was examined in the presence and absence of NQO1, and their relative rate of NQO1-mediated reduction was determined. Computational-based molecular docking simulations indicated that the trans- but not the cis-amide isomers of the benzoquinone ansamycins could be accommodated by the NQO1 active site, and the ranking order of binding energies correlated with the relative reduction rate using purified human NQO1. The trans-cis isomerization of the benzoquinone ansamycins in Hsp90 inhibition has been disputed in recent reports. Previous computational studies have used the closed or cocrystallized Hsp90 structures in an attempt to explore this isomerization step; however, we have successfully docked both the trans- and cis-amide isomers of the benzoquinone ansamycins into the open Hsp90 structure. The results of these studies indicate that both trans- and cis-amide isomers of the hydroquinone ansamycins exhibited increased binding affinity for Hsp90 relative to their parent quinones. Our data support a mechanism in which trans- rather than cis-amide forms of benzoquinone ansamycins are metabolized by NQO1 to hydroquinone ansamycins and that Hsp90-mediated trans-cis isomerization via tautomerization plays an important role in subsequent Hsp90 inhibition