NAD(P)H Quinone Oxidoreductase Protects TAp63γ from Proteasomal Degradation and Regulates TAp63γ-Dependent Growth Arrest

BACKGROUND: p63 is a member of the p53 transcription factor family. p63 is expressed from two promoters resulting in proteins with opposite functions: the transcriptionally active TAp63 and the dominant-negative DeltaNp63. Similar to p53, the TAp63 isoforms induce cell cycle arrest and apoptosis. The DeltaNp63 isoforms are dominant-negative variants opposing the activities of p53, TAp63 and TAp73. To avoid unnecessary cell death accompanied by proper response to stress, the expression of the p53 family members must be tightly regulated. NAD(P)H quinone oxidoreductase (NQO1) has recently been shown to interact with and inhibit the degradation of p53. Due to the structural similarities between p53 and p63, we were interested in studying the ability of wild-type and polymorphic, inactive NQO1 to interact with and stabilize p63. We focused on TAp63gamma, as it is the most potent transcription activator and it is expected to have a role in tumor suppression. PRINCIPAL FINDINGS: We show that TAp63gamma can be degraded by the 20S proteasomes. Wild-type but not polymorphic, inactive NQO1 physically interacts with TAp63gamma, stabilizes it and protects it from this degradation. NQO1-mediated TAp63gamma stabilization was especially prominent under stress. Accordingly, we found that downregulation of NQO1 inhibits TAp63gamma-dependant p21 upregulation and TAp63gamma-induced growth arrest stimulated by doxorubicin. CONCLUSIONS/SIGNIFICANCE: Our report is the first to identify this new mechanism demonstrating a physical and functional relationship between NQO1 and the most potent p63 isoform, TAp63gamma. These findings appoint a direct role for NQO1 in the regulation of TAp63gamma expression, especially following stress and may therefore have clinical implications for tumor development and therapy

Characterization of 4-HNE Modified L-FABP Reveals Alterations in Structural and Functional Dynamics

4-Hydroxynonenal (4-HNE) is a reactive α,β-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 µM to Kd1 = 0.395 µM and Kd2 = 34.20 µM. 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 (ΔTm = 5.44°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

AIMing towards improved antitumor efficacy Dedicated to the memory of Professor Albert I. Meyers

Using the structure-activity relationship emerging from previous Letter, and guided by pharmacokinetic properties, new AIMs have been prepared with both improved efficacy against human glioblastoma cells and cell permeability as determined by fluorescent confocal microscopy. We present our first unambiguous evidence for telomeric G4-forming oligonucleotide anisotropy by NMR resulting from direct interaction with AIMs, which is consistent with both our G4 melting studies by CD, and our working hypothesis. Finally, we show that AIMs induce apoptosis in SNB-19 cells