The study of death receptor (DR) signaling has led to the discovery of new signaling paradigms, including the first example of direct receptor-mediated activation of a protease (caspase-8) that ...functions as a second messenger to initiate a 'death cascade' of downstream protease activation. More recently, this receptor system has underscored the importance of ubiquitin modification in NF-kappaB activation. Both degradative lysine 48-linked polyubiquitin and scaffolding lysine 63-linked polyubiquitin have an essential role in signal propagation. Remarkably, a negative feedback process, termed ubiquitin editing, regulates signaling that emanates from certain DRs. Ubiquitin editing is mediated by a complex interplay between the ubiquitination and deubiquitination machinery, resulting in the replacement of signal enhancing lysine 63-linked polyubiquitin with signal extinguishing lysine 48-linked polyubiquitin. The ubiquitination machinery and its regulation in the context of DR signaling are discussed herein.
The anti-apoptotic protein MCL-1 is a key regulator of cancer cell survival and a known resistance factor for small-molecule BCL-2 family inhibitors such as ABT-263 (navitoclax), making it an ...attractive therapeutic target. However, directly inhibiting this target requires the disruption of high-affinity protein-protein interactions, and therefore designing small molecules potent enough to inhibit MCL-1 in cells has proven extremely challenging. Here, we describe a series of indole-2-carboxylic acids, exemplified by the compound A-1210477, that bind to MCL-1 selectively and with sufficient affinity to disrupt MCL-1-BIM complexes in living cells. A-1210477 induces the hallmarks of intrinsic apoptosis and demonstrates single agent killing of multiple myeloma and non-small cell lung cancer cell lines demonstrated to be MCL-1 dependent by BH3 profiling or siRNA rescue experiments. As predicted, A-1210477 synergizes with the BCL-2/BCL-XL inhibitor navitoclax to kill a variety of cancer cell lines. This work represents the first description of small-molecule MCL-1 inhibitors with sufficient potency to induce clear on-target cellular activity. It also demonstrates the utility of these molecules as chemical tools for dissecting the basic biology of MCL-1 and the promise of small-molecule MCL-1 inhibitors as potential therapeutics for the treatment of cancer.
Arabidopsis thaliana De-etiolated-1 (AtDET1) is a highly conserved protein, with orthologs in vertebrate and invertebrate organisms. AtDET1 negatively regulates photomorphogenesis, but its ...biochemical mechanism and function in other species are unknown. We report that human DET1 (hDET1) promotes ubiquitination and degradation of the proto-oncogenic transcription factor c-Jun by assembling a multisubunit ubiquitin ligase containing DNA Damage Binding Protein-1 (DDB1), cullin 4A (CUL4A), Regulator of Cullins-1 (ROC1), and constitutively photomorphogenic-1. Ablation of any subunit by RNA interference stabilized c-Jun and increased c-Jun-activated transcription. These findings characterize a c-Jun ubiquitin ligase and define a specific function for hDET1 in mammalian cells.
The experimental study and manipulation of programmed cell death has been greatly assisted by the identification of genetic and pharmacological tools that can either induce or block cell lethality. ...This review discusses new insights into the molecular sensing of perturbations induced by such tools, as well as the possible consequences of this detection in determining cell survival.
The educational potential of games has captured the ongoing interest of scholars and educators who have sought to understand when, how, and under what conditions games support the teaching and ...learning process. General knowledge of how games support literacy, scientific thinking, or social learning has been theorized and researched, but some applications of game-based learning remain unexplored. One area where much remains to be learned is within online doctoral education and particularly in the poorly understood area of research methods education. In this study, three doctoral students and an instructor collaboratively field-tested a set of instructional activities within World of Warcraft that were designed to promote understanding of qualitative research methods. A duoethnographic approach was used to promote dual-perspective dialogue about the merits and challenges of using online gaming environments as field sites where research methods can be practiced and developed. Results illuminate merits, challenges, and areas of development as researchers that surfaced while completing the research methods activities. Directions for further research are suggested.
Apoptosis inhibition rather than enhanced cellular proliferation occurs in prostate cancer (CaP), the most commonly diagnosed malignancy in American men. Therefore, it is important to characterize ...residual apoptotic pathways in CaP cells. When intracellular Ca2+ stores are released and plasma membrane “store-operated” Ca2+ entry channels subsequently open, cytosolic Ca2+ increases and is thought to induce apoptosis. However, cells incapable of releasing Ca2+ stores are resistant to apoptotic stimuli, indicating that Ca2+ store release is also important. We investigated whether release of intracellular Ca2+ stores is sufficient to induce apoptosis of the CaP cell line LNCaP. We developed a method to release stored Ca2+ without elevating cytosolic Ca2+; this stimulus induced LNCaP cell apoptosis. We compared the apoptotic pathways activated by intracellular Ca2+ store release with the dual insults of store release and cytosolic Ca2+ elevation. Earlier processing of caspases-3 and -7 occurred when intracellular store release was the sole Ca2+ perturbation. Apoptosis was attenuated in both conditions in stable transfected cells expressing antiapoptotic proteins BclxL and catalytically inactive caspase-9, and in both scenarios inactive caspase-9 became complexed with caspase-7. Thus, intracellular Ca2+ store release initiates an apoptotic pathway similar to that elicited by the dual stimuli of cytosolic Ca2+ elevation and intracellular store release.
Posttranslational modification of proteins with polyubiquitin occurs in diverse signaling pathways and is tightly regulated to ensure cellular homeostasis. Studies employing ubiquitin mutants suggest ...that the fate of polyubiquitinated proteins is determined by which lysine within ubiquitin is linked to the C terminus of an adjacent ubiquitin. We have developed linkage-specific antibodies that recognize polyubiquitin chains joined through lysine 63 (K63) or 48 (K48). A cocrystal structure of an anti-K63 linkage Fab bound to K63-linked diubiquitin provides insight into the molecular basis for specificity. We use these antibodies to demonstrate that RIP1, which is essential for tumor necrosis factor-induced NF-κB activation, and IRAK1, which participates in signaling by interleukin-1β and Toll-like receptors, both undergo polyubiquitin editing in stimulated cells. Both kinase adaptors initially acquire K63-linked polyubiquitin, while at later times K48-linked polyubiquitin targets them for proteasomal degradation. Polyubiquitin editing may therefore be a general mechanism for attenuating innate immune signaling.
The experimental study and manipulation of programmed cell death has been greatly assisted by the identification of genetic and pharmacological tools that can either induce or block cell lethality. ...This review discusses new insights into the molecular sensing of perturbations induced by such tools, as well as the possible consequences of this detection in determining cell survival.
COP1 (constitutively photomorphogenic 1) is a RING-finger-containing protein that functions to repress plant photomorphogenesis, the light-mediated programme of plant development. Mutants of COP1 are ...constitutively photomorphogenic, and this has been attributed to their inability to negatively regulate the proteins LAF1 (ref. 1) and HY5 (ref. 2). The role of COP1 in mammalian cells is less well characterized. Here we identify the tumour-suppressor protein p53 as a COP1-interacting protein. COP1 increases p53 turnover by targeting it for degradation by the proteasome in a ubiquitin-dependent fashion, independently of MDM2 or Pirh2, which are known to interact with and negatively regulate p53. Moreover, COP1 serves as an E3 ubiquitin ligase for p53 in vitro and in vivo, and inhibits p53-dependent transcription and apoptosis. Depletion of COP1 by short interfering RNA (siRNA) stabilizes p53 and arrests cells in the G1 phase of the cell cycle. Furthermore, we identify COP1 as a p53-inducible gene, and show that the depletion of COP1 and MDM2 by siRNA cooperatively sensitizes U2-OS cells to ionizing-radiation-induced cell death. Overall, these results indicate that COP1 is a critical negative regulator of p53 and represents a new pathway for maintaining p53 at low levels in unstressed cells.
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