Chibby drives β catenin cytoplasmic accumulation leading to activation of the unfolded protein response in BCR-ABL1+ cells

Chronic myeloid leukemia (CML) is a myeloproliferative disease caused by the constitutive tyrosine kinase (TK) activity of the BCR-ABL fusion protein. However, the phenotype of leukemic stem cells (LSC) is sustained by β catenin rather than by the BCR-ABL TK. β catenin activity in CML is contingent upon its stabilization proceeding from the BCR-ABL-induced phosphorylation at critical residues for interaction with the Adenomatous polyposis coli (APC)/Axin/glycogen synthase kinase 3 (GSK3) destruction complex or GSK3 inactivating mutations. Here we studied the impact of β catenin antagonist Chibby (CBY) on β catenin signaling in BCR-ABL1+ cells. CBY is a small conserved protein which interacts with β catenin and impairs β catenin-mediated transcriptional activation through two distinct molecular mechanisms: 1) competition with T cell factor (TCF) or lymphoid enhancer factor (LEF) for β catenin binding; and 2) nuclear export of β catenin via interaction with 14-3-3. We found that its enforced expression in K562 cell line promoted β catenin cytoplasmic translocation resulting in inhibition of target gene transcription. Moreover, cytoplasmic accumulation of β catenin activated the endoplasmic reticulum (ER) stress-associated pathway known as unfolded protein response (UPR). CBY-driven cytoplasmic accumulation of β catenin is also a component of BCR-ABL1+ cell response to the TK inhibitor Imatinib (IM). It evoked the UPR activation leading to the induction of BCL2-interacting mediator of cell death (BIM) by UPR sensors. BIM, in turn, contributed to the execution phase of apoptosis in the activation of ER resident caspase 12 and mobilization of Ca2+ stores

Transcriptomic, proteomic and metabolomic analysis of UV-B signaling in maize

<p>Abstract</p> <p>Background</p> <p>Under normal solar fluence, UV-B damages macromolecules, but it also elicits physiological acclimation and developmental changes in plants. Excess UV-B decreases crop yield. Using a treatment twice solar fluence, we focus on discovering signals produced in UV-B-irradiated maize leaves that translate to systemic changes in shielded leaves and immature ears.</p> <p>Results</p> <p>Using transcriptome and proteomic profiling, we tracked the kinetics of transcript and protein alterations in exposed and shielded organs over 6 h. In parallel, metabolic profiling identified candidate signaling molecules based on rapid increase in irradiated leaves and increased levels in shielded organs; pathways associated with the synthesis, sequestration, or degradation of some of these potential signal molecules were UV-B-responsive. Exposure of just the top leaf substantially alters the transcriptomes of both irradiated and shielded organs, with greater changes as additional leaves are irradiated. Some phenylpropanoid pathway genes are expressed only in irradiated leaves, reflected in accumulation of pathway sunscreen molecules. Most protein changes detected occur quickly: approximately 92% of the proteins in leaves and 73% in immature ears changed after 4 h UV-B were altered by a 1 h UV-B treatment.</p> <p>Conclusions</p> <p>There were significant transcriptome, proteomic, and metabolomic changes under all conditions studied in both shielded and irradiated organs. A dramatic decrease in transcript diversity in irradiated and shielded leaves occurs between 0 h and 1 h, demonstrating the susceptibility of plants to short term UV-B spikes as during ozone depletion. Immature maize ears are highly responsive to canopy leaf exposure to UV-B.</p

A calpain-cleaved fragment of \u3b2-catenin promotes BCRABL1+ cell survival evoked by autophagy induction in response to imatinib

Autophagy protects chronic myeloid leukemia stem cells from tyrosine kinase inhibitors hence supporting the disease persistence under therapy. However, the signals involved in autophagy regulation relative to BCR-ABL1 are still elusive. The autophagic flux proceeding from the inhibition of BCR-ABL1 tyrosine kinase represents a regulatory mechanism of \u3b2-catenin stability through events encompassing the activation of calpain, which targets \u3b2-catenin for proteasome-independent degradation. Accordingly, its inactivation may contribute to induce autophagy and autophagy induction may, in turn, promote \u3b2-catenin autolysosomal degradation to originate a regulatory loop where \u3b2-catenin plays a central role in cell decision between life and death. Here we proved that the cytoplasmic accumulation of \u3b2-catenin driven by up-regulation of its antagonist Chibby1 is a component of autophagy induction in response to imatinib in BCR-ABL1+ cells opposing the apoptotic death. It is contingent upon ER stress and elevation of free Ca(2+) cytosolic concentration and results in the calpain cleavage into a 28kDa fragment implicated in \u3b2-catenin proteasome-independent degradation. More important for BCR-ABL1+ cell survival and proliferation following IM treatment, might be the calpain-mediated cleavage of \u3b2-catenin accumulated within the cytoplasmic compartment into a 75kDa fragment, still owning TCF-dependent transcriptional activity. Such a \u3b2-catenin fragment might be crucial for BCR-ABL1+ cell survival following the fusion protein TK inhibition

Histone Acetylation and Chromatin Remodeling Are Required for UV-B–Dependent Transcriptional Activation of Regulated Genes in Maize[W]

The nuclear proteomes of maize (Zea mays) lines that differ in UV-B tolerance were compared by two-dimensional gel electrophoresis after UV light treatment. Differential accumulation of chromatin proteins, particularly histones, constituted the largest class identified by mass spectrometry. UV-B–tolerant landraces and the B73 inbred line show twice as many protein changes as the UV-B–sensitive b, pl W23 inbred line and transgenic maize expressing RNA interference constructs directed against chromatin factors. Mass spectrometic analysis of posttranslational modifications on histone proteins demonstrates that UV-B–tolerant lines exhibit greater acetylation on N-terminal tails of histones H3 and H4 after irradiation. These acetylated histones are enriched in the promoter and transcribed regions of the two UV-B–upregulated genes examined; radiation-sensitive lines lack this enrichment. DNase I and micrococcal nuclease hypersensitivity assays indicate that chromatin adopts looser structures around the selected genes in the UV-B–tolerant samples. Chromatin immunoprecipitation experiments identified additional chromatin factor changes associated with the nfc102 test gene after UV-B treatment in radiation-tolerant lines. Chromatin remodeling is thus shown to be a key process in acclimation to UV-B, and lines deficient in this process are more sensitive to UV-B

Transcriptomic, proteomic and metabolomic analysis of maize responses to UV-B: Comparison of greenhouse and field growth conditions

UV-B radiation from normal solar fluence elicits physiological and developmental changes in plants under fluctuating environmental conditions. Most UV photobiology studies in plants utilize controlled greenhouse and growth chamber environments in which few conditions vary except the brief presence of UV-B radiation. Our purpose was to compare responses to UV-B in irradiated and shielded maize organs in field (natural solar plus 2x solar supplementation for defined periods) and greenhouse (2x solar supplementation only) conditions during a 4 h exposure. Three parameters were assessed—transcripts, proteins and metabolites—to determine the degree of overlap in maize responses in field and greenhouse conditions. We assessed irradiated leaves, and both shielded leaves and immature ears. After comparing transcriptome, proteome and metabolome profiles, we find there are more differences than similarities between field and greenhouse responses

Tensile Microstrain Fluctuations in the BaPbO Units in Superconducting BaPb_1−xBi_xO₃ by Scanning Dispersive Micro-XANES

BaPb1−xBixO3 (BPBO) bismuthate, showing high TC superconductivity for 0.05 3] centered nanoscale units (BBO) coexist with BaPbO3 centered (BPO) units in the BPBO perovskite; therefore, we expect a tensile microstrain in BPO units due the misfit strain between the two different lattices. Here, we report the measurement of the spatial micro-fluctuations of the local tensile microstrain ε in the BaPO units in superconducting Ba(Pb1−xBix)O3 crystals with x1 = 0.19 an x2 = 0.28. We show here the feasibility of applying the scanning dispersive micro-X-ray absorption near edge structure (SdμXANES) technique, using focused synchrotron radiation, to probe the microscale spatial fluctuations of the microstrain in BPO units. This unconventional real-space SdμXANES microscopy at the Pb L3 edge has been collected in the dispersive mode. Our experimental method allows us to measure either the local Bi chemical concentration x and the local lattice microstrain of local BBO and BPO units. The 5 × 5 micron-size spots from the focused X-ray beam allowed us to obtain maps of 1600 points covering an area of 200 × 200 microns. The mapping shows a substantial difference between the spatial fluctuations of the microstrain ε and the chemical inhomogeneity x. Moreover, we show the different relations ε(x) in samples with lower (x1 = 0.19) and higher (x2 = 0.28) doping respect to the optimum doping (x = 0.25)

14-3-3 Binding and Sumoylation Concur to the Down-Modulation of <i>β-catenin</i> Antagonist <i>chibby 1</i> in Chronic Myeloid Leukemia

<div><p>The down-modulation of the β-catenin antagonist Chibby 1 (CBY1) associated with the <i>BCR-ABL1</i> fusion gene of chronic myeloid leukemia (CML) contributes to the aberrant activation of β-catenin, particularly in leukemic stem cells (LSC) resistant to tyrosine kinase (TK) inhibitors. It is, at least partly, driven by transcriptional events and gene promoter hyper-methylation. Here we demonstrate that it also arises from reduced protein stability upon binding to 14-3-3σ adapter protein. CBY1/14-3-3σ interaction in BCR-ABL1+ cells is mediated by the fusion protein TK and AKT phosphorylation of CBY1 at critical serine 20, and encompasses the 14-3-3σ binding modes I and II involved in the binding with client proteins. Moreover, it is impaired by c-Jun N-terminal kinase (JNK) phosphorylation of 14-3-3σ at serine 186, which promotes dissociation of client proteins. The ubiquitin proteasome system UPS participates in reducing stability of CBY1 bound with 14-3-3σ through enhanced SUMOylation. Our results open new routes towards the research on molecular pathways promoting the proliferative advantage of leukemic hematopoiesis over the normal counterpart.</p></div

IM promotes changes in CBY1 and β-catenin expression associated with the dissolution of CBY1 and β-catenin binding with 14-3-3σ.

<p> A- Cytoplasmic and nuclear protein analysis was performed after 4 and 24 hours of exposure to IM. In first instance, <i>BCR-ABL1</i> de-phosphorylation at the critical residue for constitutive activation of the fusion protein enzymatic activity (tytosine-Y-245) was assessed, hence proving IM inhibitory effect on its target at the time other protein expression and interactions were investigated; B- WB and IP/IB have been performed according to published procedures and confirmed in three independent experiments. Densitometric analysis of signal intensities shows a statistically significance difference (p<0.05; Student’s t test) in treated vs untreated cells. Actin and histone H1 were used as control for loading of cytoplasmic and nuclear proteins, respectively. Lack of IM off target effects is shown in the Supplementary section, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131074#pone.0131074.s002" target="_blank">S2 Fig</a></p