181 research outputs found
Inke NĂ€thke:The ABCs of APC
NĂ€thke investigates the many functions of adenomatous polyposis coli protein and its contribution to human disease
Measuring the Interaction of Transcription Factor Nrf2 with Its Negative Regulator Keap1 in Single Live Cells by an Improved FRET/FLIM Analysis
Transcription
factor NF-E2 p45-related factor 2 (Nrf2) and its
principal negative regulator, Kelch-like ECH-associated protein 1
(Keap1), comprise a molecular effector and sensor system that robustly
responds to perturbations of the cellular redox homeostasis by orchestrating
a comprehensive cytoprotective program. Under homeostatic conditions,
Nrf2 is a short-lived protein, which is targeted for ubiquitination
and proteasomal degradation. Upon encounter of electrophiles, oxidants,
or pro-inflammatory stimuli, the cysteine sensors in Keap1 are chemically
modified, rendering Keap1 unable to target Nrf2 for degradation, and
consequently leading to accumulation of the transcription factor and
enhanced transcription of cytoprotective genes. A detailed understanding
of the proteinâprotein interactions between Nrf2 and Keap1
has been achieved by use of various in vitro systems, but few assays
are available to assess these interactions in the context of the living
cell. We previously developed an imaging-based FLIM/FRET methodology
to visualize and measure the interaction between Nrf2 and Keap1 in
single cells. Here, our goal was to improve this methodology in order
to increase throughput and precision, and decrease cell-to-cell variability.
To eliminate the possibility of orientation bias, we incorporated
a flexible linker between Keap1 and the FRET acceptor fluorescent
protein tag. To ensure the correct image capture of Nrf2 fused to
the FRET donor fluorescent protein tag, we matched the maturation
time of the fluorescent tag to the half-life of the endogenous Nrf2,
by using sfGFP as the FRET donor. Using a global binning approach
increased the assay throughput, whereas including the measured instrument
response function in the analysis improved precision. The application
of this methodology revealed a strong covariation of the results with
the expression level of the acceptor. Taking the acceptor level into
account circumvented cell-to-cell variability and enhanced sensitivity
of the measurements of the Keap1âNrf2 interaction in live cells
Loss of APC induces polyploidy as a result of a combination of defects in mitosis and apoptosis
Mutations in the adenomatous polyposis coli (APC) tumor suppressor gene initiate a majority of colorectal cancers. Acquisition of chromosomal instability is an early event in these tumors. We provide evidence that the loss of APC leads to a partial loss of interkinetochore tension at metaphase and alters mitotic progression. Furthermore, we show that inhibition of APC in U2OS cells compromises the mitotic spindle checkpoint. This is accompanied by a decrease in the association of the checkpoint proteins Bub1 and BubR1 with kinetochores. Additionally, APC depletion reduced apoptosis. As expected from this combination of defects, tetraploidy and polyploidy are consequences of APC inhibition in vitro and in vivo. The removal of APC produced the same defects in HCT116 cells that have constitutively active ÎČ-catenin. These data show that the loss of APC immediately induces chromosomal instability as a result of a combination of mitotic and apoptotic defects. We suggest that these defects amplify each other to increase the incidence of tetra- and polyploidy in early stages of tumorigenesis
Tissue environment, not ontogeny, defines murine intestinal intraepithelial T lymphocytes
Tissue-resident intestinal intraepithelial T lymphocytes (T-IEL) patrol the gut and have important roles in regulating intestinal homeostasis. T-IEL include both induced T-IEL, derived from systemic antigen-experienced lymphocytes, and natural T-IEL, which are developmentally targeted to the intestine. While the processes driving T-IEL development have been elucidated, the precise roles of the different subsets and the processes driving activation and regulation of these cells remain unclear. To gain functional insights into these enigmatic cells, we used high-resolution, quantitative mass spectrometry to compare the proteomes of induced T-IEL and natural T-IEL subsets, with naive CD8(+) T cells from lymph nodes. This data exposes the dominant effect of the gut environment over ontogeny on T-IEL phenotypes. Analyses of protein copy numbers of >7000 proteins in T-IEL reveal skewing of the cell surface repertoire towards epithelial interactions and checkpoint receptors; strong suppression of the metabolic machinery indicating a high energy barrier to functional activation; upregulated cholesterol and lipid metabolic pathways, leading to high cholesterol levels in T-IEL; suppression of T cell antigen receptor signalling and expression of the transcription factor TOX, reminiscent of chronically activated T cells. These novel findings illustrate how T-IEL integrate multiple tissue-specific signals to maintain their homeostasis and potentially function
Mitotic stress is an integral part of the oncogene-induced senescence program that promotes multinucleation and cell cycle arrest
Oncogene-induced senescence (OIS) is a tumor suppression mechanism that blocks cell proliferation in response to oncogenic signaling. OIS is frequently accompanied by multinucleation; however, the origin of this is unknown. Here, we show that multinucleate OIS cells originate mostly from failed mitosis. Prior to senescence, mutant H-RasV12 activation in primary human fibroblasts compromised mitosis, concordant with abnormal expression of mitotic genes functionally linked to the observed mitotic spindle and chromatin defects. Simultaneously, H-RasV12 activation enhanced survival of cells with damaged mitoses, culminating in extended mitotic arrest and aberrant exit from mitosis via mitotic slippage. ERK-dependent transcriptional upregulation of Mcl1 was, at least in part, responsible for enhanced survival and slippage of cells with mitotic defects. Importantly, mitotic slippage and oncogene signaling cooperatively induced senescence and key senescence effectors p21 and p16. In summary, activated Ras coordinately triggers mitotic disruption and enhanced cell survival to promote formation of multinucleate senescent cells
The isoquinoline PRL-295 increases the thermostability of Keap1 and disrupts its interaction with Nrf2
Transcription factor Nrf2 and its negative regulator Keap1 orchestrate a cytoprotective response against oxidative, metabolic, and inflammatory stress. Keap1 is a drug target, with several small molecules in drug development. Here, we show that the isoquinoline PRL-295 increased Keap1 thermostability in lysates from cells expressing fluorescently tagged Keap1. The thermostability of endogenous Keap1 also increased in intact cells and murine liver following PRL-295 treatment. Fluorescence Lifetime ImagingâFörster Resonance Energy Transfer (FLIM-FRET) experiments in cells co-expressing sfGFP-Nrf2 and Keap1-mCherry further showed that PRL-295 prolonged the donor fluorescence lifetime, indicating disruption of the Keap1-Nrf2 protein complex. Orally administered PRL-295 to mice activated the Nrf2transcriptional target NAD(P)H:quinone oxidoreductase 1 (NQO1) in liver and decreased the levels of plasma alanine aminotransferase and aspartate aminotransferase upon acetaminophen-induced hepatic injury. Thus, PRL-295 engages the Keap1 protein target in cells and in vivo, disrupting its interaction with Nrf2, leading to activation of Nrf2-dependent transcription and hepatocellular protection
Phenyl Bis-Sulfonamide Keap1-Nrf2 ProteinâProtein Interaction Inhibitors with an Alternative Binding Mode
Inhibitors of Kelch-like ECH-associated protein 1 (Keap1) increase the activity of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) by stalling its ubiquitination and degradation. This enhances the expression of genes encoding proteins involved in drug detoxification, redox homeostasis, and mitochondrial function. Nrf2 activation offers a potential therapeutic approach for conditions including Alzheimerâs and Parkinsonâs diseases, vascular inflammation, and chronic obstructive airway disease. Non-electrophilic Keap1-Nrf2 proteinâprotein interaction (PPI) inhibitors may have improved toxicity profiles and different pharmacological properties to cysteine-reactive electrophilic inhibitors. Here, we describe and characterize a series of phenyl bis-sulfonamide PPI inhibitors that bind to Keap1 at submicromolar concentrations. Structural studies reveal that the compounds bind to Keap1 in a distinct âpeptidomimeticâ conformation that resembles the Keap1-Nrf2 ETGE peptide complex. This is different to other small molecule Keap1-Nrf2 PPI inhibitors, including bicyclic aryl bis-sulfonamides, offering a starting point for new design approaches to Keap1 inhibitors
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