Interdependence of Bad and Puma during Ionizing-Radiation-Induced Apoptosis

Ionizing radiation (IR)-induced DNA double-strand breaks trigger an extensive cellular signaling response that involves the coordination of hundreds of proteins to regulate DNA repair, cell cycle arrest and apoptotic pathways. The cellular outcome often depends on the level of DNA damage as well as the particular cell type. Proliferating zebrafish embryonic neurons are highly sensitive to IR-induced apoptosis, and both p53 and its transcriptional target puma are essential mediators of the response. The BH3-only protein Puma has previously been reported to activate mitochondrial apoptosis through direct interaction with the pro-apoptotic Bcl-2 family proteins Bax and Bak, thus constituting the role of an “activator” BH3-only protein. This distinguishes it from BH3-only proteins like Bad that are thought to indirectly promote apoptosis through binding to anti-apoptotic Bcl-2 family members, thereby preventing the sequestration of activator BH3-only proteins and allowing them to directly interact with and activate Bax and Bak. We have shown previously that overexpression of the BH3-only protein Bad in zebrafish embryos supports normal embryonic development but greatly sensitizes developing neurons to IR-induced apoptosis. While Bad has previously been shown to play only a minor role in promoting IR-induced apoptosis of T cells in mice, we demonstrate that Bad is essential for robust IR-induced apoptosis in zebrafish embryonic neural tissue. Moreover, we found that both p53 and Puma are required for Bad-mediated radiosensitization in vivo. Our findings show the existence of a hierarchical interdependence between Bad and Puma whereby Bad functions as an essential sensitizer and Puma as an essential activator of IR-induced mitochondrial apoptosis specifically in embryonic neural tissue

p63 Mediates an Apoptotic Response to Pharmacological and Disease-Related ER Stress in the Developing Epidermis

SummaryEndoplasmic reticulum (ER) stress triggers tissue-specific responses that culminate in either cellular adaptation or apoptosis, but the genetic networks distinguishing these responses are not well understood. Here we demonstrate that ER stress induced in the developing zebrafish causes rapid apoptosis in the brain, spinal cord, tail epidermis, lens, and epiphysis. Focusing on the tail epidermis, we uncover an apoptotic response that depends on Puma, but not on p53 or Chop. puma is transcriptionally activated during this ER stress response in a p53-independent manner, and is an essential mediator of epidermal apoptosis. We demonstrate that the p63 transcription factor is upregulated to initiate this apoptotic pathway and directly activates puma transcription in response to ER stress. We also show that a mutation of human Connexin 31, which causes erythrokeratoderma variabilis, induces ER stress and p63-dependent epidermal apoptosis in the zebrafish embryo, thus implicating this pathway in the pathogenesis of inherited disease

Ccdc94 Protects Cells from Ionizing Radiation by Inhibiting the Expression of p53

DNA double-strand breaks (DSBs) represent one of the most deleterious forms of DNA damage to a cell. In cancer therapy, induction of cell death by DNA DSBs by ionizing radiation (IR) and certain chemotherapies is thought to mediate the successful elimination of cancer cells. However, cancer cells often evolve to evade the cytotoxicity induced by DNA DSBs, thereby forming the basis for treatment resistance. As such, a better understanding of the DSB DNA damage response (DSB–DDR) pathway will facilitate the design of more effective strategies to overcome chemo- and radioresistance. To identify novel mechanisms that protect cells from the cytotoxic effects of DNA DSBs, we performed a forward genetic screen in zebrafish for recessive mutations that enhance the IR–induced apoptotic response. Here, we describe radiosensitizing mutation 7 (rs7), which causes a severe sensitivity of zebrafish embryonic neurons to IR–induced apoptosis and is required for the proper development of the central nervous system. The rs7 mutation disrupts the coding sequence of ccdc94, a highly conserved gene that has no previous links to the DSB–DDR pathway. We demonstrate that Ccdc94 is a functional member of the Prp19 complex and that genetic knockdown of core members of this complex causes increased sensitivity to IR–induced apoptosis. We further show that Ccdc94 and the Prp19 complex protect cells from IR–induced apoptosis by repressing the expression of p53 mRNA. In summary, we have identified a new gene regulating a dosage-sensitive response to DNA DSBs during embryonic development. Future studies in human cancer cells will determine whether pharmacological inactivation of CCDC94 reduces the threshold of the cancer cell apoptotic response

A novel luminescence-based high-throughput approach for cellular resolution of protein ubiquitination using tandem ubiquitin binding entities (TUBEs).

Protein turnover is highly regulated by the post-translational process of ubiquitination. Deregulation of the ubiquitin proteasome system (UPS) has been implicated in cancer and neurodegenerative diseases, and modulating this system has proven to be a viable approach for therapeutic intervention. Development of novel technologies that enable high-throughput studies of substrate protein ubiquitination is essential for drug discovery in the UPS. Conventional approaches for studying ubiquitination either require high amounts of starting protein or rely on exogenous or modified ubiquitin moieties and thus limiting their utility. In order to circumvent these issues, we developed a high-throughput live-cell assay which combines the NanoBiT luminescence-based technology with tandem ubiquitin-binding entities (TUBE) to resolve substrate ubiquitination. To demonstrate the effectiveness and utility of this assay, we studied the compound-induced ubiquitination of G To S Phase Transition 1 (GSPT1) protein. Using this new assay, we characterized compounds with varying levels of GSPT1 ubiquitination activity. This method provides a novel cell based approach for assaying substrate ubiquitination in living cells that can be adapted to study the kinetics of ubiquitin transfer onto a substrate protein of interest. In addition, our results show that this approach is portable for studying ubiquitination of target proteins with varying functions

p53 is required for Bad-mediated sensitivity to IR but not wortmannin.

<p>(A) Shown are lateral views of representative tails from 27-hpf wild-type or <i>p53</i> mutant embryos injected with 50 pg of <i>mcherry</i> (cntl) or <i>hBAD</i> mRNA. Embryos were exposed (or not) to 8 Gy IR at 24 hpf and analyzed three hours later by the Casp3 assay. Apoptosis was observed in the spinal cord after <i>hBAD</i> mRNA was injected into wild-type (arrowheads), but not mutant, <i>p53</i> embryos. (B) Fluorescence intensity was measured in the spinal cords of at least 10 embryos from each group in (A). Data represent one experiment, but the experiment was independently performed three times with similar results. (C) One-cell stage wild-type or <i>p53</i> mutant embryos were injected with 50 pg of mRNA encoding either <i>mcherry</i> control (cntl) or the constitutively active mutant <i>zbad 2SA</i>. At 8 hpf, embryos were analyzed for survival (defined by a beating heart) as performed previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088151#pone.0088151-Sorrells1" target="_blank">[34]</a>. Data represent one experiment, but the experiment was independently performed three times with similar results. (D) One-cell stage wild-type embryos were injected with 50 pg of mRNA encoding either zebrafish <i>bad</i> or the apoptotically-inactive <i>zbad bh3 mut</i>. At 8 hpf, embryos were treated with increasing concentrations of wortmannin, or DMSO vehicle alone. At 48 hpf, embryos were examined for survival. At least 10 embryos were analyzed per group in three independent experiments. E) Shown are lateral views of tails from <i>p53</i> wild-type (left) or mutant (right) embryos after injection with 25 pg of mRNA encoding either <i>mcherry</i> (cntl), <i>zbad</i>, or <i>zbad bh3 mut</i>. Embryos were split into two groups and treated with either 0.3 µM wortmannin or DMSO vehicle beginning at 8 hpf and analyzed at 24 hpf by the Casp3 assay. Wild-type Bad synergizes with wortmannin to induce apoptosis in multiple tissues in a <i>p53</i>-independent manner (arrowheads).</p

Puma is required for Bad-mediated radiosensitization.

<p>(A) Shown are lateral views of representative tails from 27-hpf embryos exposed to 8 Gy IR after injection at the one-cell stage with 50 pg of mRNA encoding <i>mcherry</i> (cntl) or <i>hBAD</i> in addition to either 100 nmol of <i>puma</i> MO or mismatch (mm) control MO. Analysis of Caspase 3 activity shows that <i>hBAD</i>-mediated radiosensitivity (arrowheads) is dependent on <i>puma</i> expression. (B) Fluorescence intensity was measured in the spinal cords of at least 10 embryos from each group in (A). Data represent one experiment, but the experiment was independently performed three times with similar results.</p

Bad does not augment p53 transcriptional activity.

<p>p53 transcriptional activity was analyzed in embryos injected with 50<i>mcherry</i> (cntl) or <i>hBAD</i>. Embryos were exposed to 8 Gy (or not) at 24 hpf. RNA was harvested from each group at 27 hpf and analyzed for gene expression changes by qPCR. Expression of the <i>gapdh</i> gene was measured to normalize <i>puma</i> and <i>p21</i> mRNA levels. All data was compared to unirradiated wild-type control-mRNA-injected data, which was adjusted to a value of 1. Control-injected <i>p53</i> mutant embryos irradiated at 24 hpf and harvested at 27 hpf were included as a negative control for p53-mediated transcriptional induction. Data represent one experiment, but the experiment was independently performed three times with similar results.</p

Bad is required for IR-induced apoptosis in zebrafish embryonic neural tissue.

<p>(A) Shown are lateral views of 27-hpf embryos (head is top left in each panel) either uninjected or injected with 200 nmol of <i>bad ATG</i>, <i>bad e2i2</i> or mismatch (mm) MO. Half of each group of embryos were exposed to 15 Gy IR, and all were analyzed by the Casp3 assay. In control embryos (no inj and mm), IR-induced apoptosis occurs predominantly in the brain and all along the spinal cord (white arrowheads), whereas in <i>bad</i>-deficient embryos (ATG and e2i2), residual apoptosis is only observed in the head (arrowheads). (B) Fluorescence intensity, reflecting level of Caspase 3 activity, was measured in the spinal cords of at least 10 embryos from each group in (A) as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088151#pone.0088151-Sorrells1" target="_blank">[34]</a>. The fluorescence intensity in irradiated mismatch-MO-injected embryos was normalized to 1. (C) One-cell stage zebrafish embryos were injected with 100 nmol of <i>bad ATG</i>, <i>bad e2i2</i> or mm MO as indicated (“++” indicates that 200 nmol was injected to keep total concentration of MO constant between experimental groups) and irradiated and analyzed as in (A-B). Data represent one experiment, and the experiment was independently performed three times with similar results.</p

Advancements in assay technologies and strategies to enable drug discovery

ABSTRACT: Assays drive drug discovery from the exploratory phases to the clinical testing of drug candidates. As such, numerous assay technologies and methodologies have arisen to support drug discovery efforts. Robust identification and characterization of tractable chemical matter requires biochemical, biophysical, and cellular approaches and often bene-fits from high-throughput methods. To increase throughput, efforts have been made to provide assays in miniaturized volumes which can be arrayed in microtiter plates to support the testing of as many as 100,000 samples/day. Alongside these efforts has been the growth of microtiter plate-free formats with encoded libraries that can support the screening billions of compounds, a hunt for new drug modalities, as well as the emphasis on more disease relevant formats using complex cell models of disease states. This review will focus on recent developments in high-throughput assay technolo-gies applied to identify starting points for drug discovery. We also provide recommendations on strategies for implement-ing various assay types to select high quality leads for drug development

An IMiD-inducible degron provides reversible regulation for chimeric antigen receptor expression and activity

The recent development of successful CAR (chimeric antigen receptor) T cell therapies has been accompanied by a need to better control potentially fatal toxicities that can arise from adverse immune reactions. Here we present a ligand-controlled CAR system, based on the IKZF3 ZF2 β-hairpin IMiD-inducible degron, which allows for the reversible control of expression levels of type I membrane proteins, including CARs. Testing this system in an established mouse xenotransplantation model for acute lymphoblastic leukemia, we validate the ability of the CAR19-degron to target and kill CD19-positive cells displaying complete control/clearance of the tumor. We also demonstrate that the activity of CAR19-degron can be regulated in vivo when dosing a US Food and Drug Administration-approved drug, lenalidomide