Project description:BCL-2-associated X protein (BAX) is a promising therapeutic target for activating or restraining apoptosis in diseases of pathologic cell survival or cell death, respectively. In response to cellular stress, BAX transforms from a quiescent cytosolic monomer into a toxic oligomer that permeabilizes the mitochondria, releasing key apoptogenic factors. The mitochondrial lipid trans-2-hexadecenal (t-2-hex) sensitizes BAX activation by covalent derivatization of cysteine 126 (C126). Here, we performed a disulfide tethering screen to discover C126-reactive molecules that modulate BAX activity. We identified covalent BAX inhibitor 1 (CBI1) as a compound that selectively derivatizes BAX at C126 and inhibits BAX activation by the physiologic ligand tBID and point mutagenesis. Biochemical and structural analyses revealed that CBI1 can inhibit BAX by a dual mechanism of action: conformational constraint and competitive blockade of lipidation. Hydrogen deuterium exchange mass spectrometry (HDX MS) showed that CBI1 derivatization of BAX C126 reversed the conformational changes caused by the auto-activating mutant F116A These data inform a pharmacologic strategy for blocking apoptosis in diseases of unwanted cell death by covalent targeting of BAX C126.
Project description:BAX is a pro-apoptotic member of the BCL-2 family, which regulates the balance between cellular life and death. During homeostasis, BAX predominantly resides in the cytosol as a latent monomer but, in response to stress, transforms into an oligomeric protein that permeabilizes the mitochondria, leading to cell death. Because renegade BAX activation poses a grave risk to the cell, the architecture of BAX must ensure monomeric stability yet simultaneously enable conformational change upon stress signaling. The specific structural features that afford both stability and dynamic flexibility remain ill-defined and represent a critical control point of BAX regulation. Here, we identified a nexus of interactions involving four discrete residues of the BAX core α5 helix that are individually essential to maintaining the structure and latency of monomeric BAX and are collectively required for dimeric assembly. We compared the HDX MS profile of full-length recombinant wild-type BAX in solution with that of the BAX L113A, F114A, Y115A, and F116A single point mutants. In each case, we observed striking, regiospecific consequences of replacing the bulky hydrophobic residue with alanine. We also compared HDX in a construct of α2-α5 for the wt protein as well as mutants. The dual yet distinct roles of these residues reveals the intricacy of BAX conformational regulation and opportunities for therapeutic modulation.
Project description:Acidic activation domains are intrinsically disordered regions of transcription factors that bind coactivators. The intrinsic disorder and low evolutionary conservation of activation domains have made it difficult to identify the sequence features controlling AD activity. To address this problem, we designed thousands of variants in seven acidic activation domains and measured their activities with a new high-throughput assay in human cell culture. We found that strong activation domain activity required a balance between the number of acidic residues and aromatic and leucine residues. These findings motivated a predictor of activation domains that scans the human proteome for clusters of aromatic and leucine residues embedded in regions of high acidity. This predictor identifies known activation domains and accurately predicts new ones. Our results support a flexible model of activation domains in which acidic residues solubilize hydrophobic motifs so that they can interact with coactivators.
Project description:KEAP1, a cytoplasmic repressor of the oxidative stress responsive transcription factor NRF2, senses the presence of electrophilic agents by modification of its sensor cysteine residues. In addition to xenobiotics, several reactive metabolites have been shown to covalently modify key cysteines on KEAP1, although the full repertoire of these molecules and their respective modifications remains undefined. Here, we report the discovery of sAKZ692, a small molecule identified by high throughput screening that stimulates NRF2 transcriptional activity in cells by inhibiting the glycolytic enzyme pyruvate kinase. sAKZ692 treatment promotes the buildup of glyceraldehyde 3-phosphate, a metabolite which leads to S-lactate modification of cysteine sensor residues of KEAP1, resulting in NRF2 dependent transcription. This work identifies a novel posttranslational modification of cysteine derived from a reactive central carbon metabolite and helps further define the complex relationship between metabolism and the oxidative stress sensing machinery of the cell.
Project description:Cordyceps participates in various pharmacological activities including anti-tumor, and is involved in the regulation of NF-κB signaling pathway. However, the detailed role of cordycepin in suppression of NF-κB signaling pathway is less clear. In this study, we first analyzed the effect of cordyceps on NF-κB activity in TK-10 cells, and found that cordyceps resulted in a dose-dependent reduction in TNF-α-induced NF-κB activation. Here, we show that cordyceps mediated NF-kB inhibition induces apoptosis in TK-10 cells involved the serial activation of caspases. Moreover, we demonstrate that in addition to activating caspases, the cordyceps negatively modulates TNF-α-mediated NF-κB signaling to promote JNK activation, which results in apoptosis, and that NF-kB regulates antiapoptotic factor GADD45b and the JNK kinase MKK7. When the TNFα cytokine binds to the TNF receptor, IκB dissociates from NF-κB. As a result, the active NF-κB translocates to the nucleus. Cordyceps clearly prevented NF-κB from mobilizing to the nucleus, resulting in downregulation of GADD45b, whereas upregulation of MKK7 and phosphorylation of JNK (p-JNK). This increased Bax activation, leading to marked cordyceps-induced apoptosis. Bax subfamily proteins induced apoptosis through caspase-3. Furthermore, siRNA mediated inhibition of MKK7 downregulated p-JNK and The JNK inhibitor SP600125 strongly inhibited Bax. Thus, these results indicate that cordyceps inhibits NF-κB/GADD45b signaling activation to upregulate MKK7-JNK signaling pathway to induce apoptosis in TK-10 cells and support the potential of cordyceps as a therapeutic agent for renal cancer.
Project description:The objective of this pharmacokinetic study is to exclude a possible influence of CETUX on the plasma disposition and metabolic activation of Capecitabine (CCB) and when this regimen is given combined with Oxaliplatin (OxPt).
Project description:The type III receptor tyrosine kinase, FLT3 is frequently mutated in acute myeloid leukemia. Phosphorylation of FLT3 on tyrosine residues is a critical event for downstream signaling. Here we studied the role of Y842 residue of FLT3, which is located in the activation loop.