Structure-activity relationship study of small-molecule inhibitor of Atg12-Atg3 protein-protein interaction.

Autophagy is a catabolic process that was described to play a critical role in advanced stages of cancer, wherein it maintains tumor cell homeostasis and growth by supplying nutrients. Autophagy is also described to support alternative cellular trafficking pathways, providing a non-canonical autophagy-dependent inflammatory cytokine secretion mechanism. Therefore, autophagy inhibitors have high potential in the treatment of cancer and acute inflammation.

Loss of EIF4G2 mediates aggressiveness in distinct human endometrial cancer subpopulations with poor survival outcome in patients.

The non-canonical translation initiation factor EIF4G2 plays essential roles in cellular stress responses via translation of selective mRNA cohorts. Currently there is limited and conflicting information regarding its involvement in cancer development and progression. Here we assessed its role in endometrial cancer (EC), in a cohort of 280 EC patients across different types, grades, and stages, and found that low EIF4G2 expression highly correlated with poor overall- and recurrence-free survival in Grade 2 EC patients, monitored over a period of up to 12 years.

Loss-of-function cancer-linked mutations in the EIF4G2 non-canonical translation initiation factor.

Tumor cells often exploit the protein translation machinery, resulting in enhanced protein expression essential for tumor growth. Since canonical translation initiation is often suppressed because of cell stress in the tumor microenvironment, non-canonical translation initiation mechanisms become particularly important for shaping the tumor proteome. EIF4G2 is a non-canonical translation initiation factor that mediates internal ribosome entry site (IRES)- and uORF-dependent initiation mechanisms, which can be used to modulate protein expression in cancer.

Identifying a selective inhibitor of autophagy that targets ATG12-ATG3 protein-protein interaction.

Macroautophagy/autophagy is a catabolic process by which cytosolic content is engulfed, degraded and recycled. It has been implicated as a critical pathway in advanced stages of cancer, as it maintains tumor cell homeostasis and continuous growth by nourishing hypoxic or nutrient-starved tumors. Autophagy also supports alternative cellular trafficking pathways, providing a mechanism of non-canonical secretion of inflammatory cytokines.

Electron Tomography and Machine Learning for Understanding the Highly Ordered Structure of Leafhopper Brochosomes.

Insects known as leafhoppers (Hemiptera: Cicadellidae) produce hierarchically structured nanoparticles known as brochosomes that are exuded and applied to the insect cuticle, thereby providing camouflage and anti-wetting properties to aid insect survival. Although the physical properties of brochosomes are thought to depend on the leafhopper species, the structure-function relationships governing brochosome behavior are not fully understood. Brochosomes have complex hierarchical structures and morphological heterogeneity across species, due to which a multimodal characterization approach is required to effectively elucidate their nanoscale structure and properties.

DAP5 drives translation of specific mRNA targets with upstream ORFs in human embryonic stem cells.

Death associated protein 5 (DAP5/eIF4G2/NAT1) is a member of the eIF4G translation initiation factors that has been shown to mediate noncanonical and/or cap-independent translation. It is essential for embryonic development and for differentiation of embryonic stem cells (ESCs), specifically its ability to drive translation of specific target mRNAs. In order to expand the repertoire of DAP5 target mRNAs, we compared ribosome profiles in control and DAP5 knockdown (KD) human ESCs (hESCs) to identify mRNAs with decreased ribosomal occupancy upon DAP5 silencing.

A new function for the serine protease HtrA2 in controlling radiation-induced senescence in cancer cells.

Radiation therapy can induce cellular senescence in cancer cells, leading to short-term tumor growth arrest but increased long-term recurrence. To better understand the molecular mechanisms involved, we developed a model of radiation-induced senescence in cultured cancer cells. The irradiated cells exhibited a typical senescent phenotype, including upregulation of p53 and its main target, p21, followed by a sustained reduction in cellular proliferation, changes in cell size and cytoskeleton organization, and senescence-associated beta-galactosidase activity.

Epiblast fragmentation by shedding-a novel mechanism to eliminate cells in post-implantation mouse embryos.

The role of programmed cell death during embryonic development has been described previously, but its specific contribution to peri- and post-implantation stages is still debatable. Here, we used transmission electron microscopy and immunostaining of E5.5-7.

Engineering insects from the endosymbiont out.

Insects are an incredibly diverse group of animals with species that benefit and harm natural ecosystems, agriculture, and human health. Many insects have consequential associations with microbes: bacterial symbionts may be embedded in different insect tissues and cell types, inherited across insect generations, and required for insect survival and reproduction. Genetically engineering insect symbionts is key to understanding and harnessing these associations.

Ser289 phosphorylation activates both DAPK1 and DAPK2 but in response to different intracellular signaling pathways.

DAPK1 and DAPK2 are calmodulin (CaM)-regulated protein kinases that share a high degree of homology in their catalytic and CaM regulatory domains. Both kinases function as tumor suppressors, and both have been implicated in autophagy regulation. Over the years, common regulatory mechanisms for the two kinases as well as kinase-specific ones have been identified.

A cancer associated somatic mutation in LC3B attenuates its binding to E1-like ATG7 protein and subsequent lipidation.

Macroautophagy/autophagy is a conserved catabolic process that maintains cellular homeostasis under basal growth and stress conditions. In cancer, autophagy can either prevent or promote tumor growth, at early or advanced stages, respectively. We screened public databases to identify autophagy-related somatic mutations in cancer, using a computational approach to identify cancer mutational target sites, employing exact statistics.

Autophagy-dependent cell death - where, how and why a cell eats itself to death.

Autophagy as a means of cell killing was first advanced by Clark's phenotypic description of 'Type II autophagic cell death' in 1990. However, this phenomenon later came into question, because the presence of autophagosomes in dying cells does not necessarily signify that autophagy is the cause of demise, but rather may reflect the efforts of the cell to prevent it. Resolution of this issue comes from a more careful definition of autophagy-dependent cell death (ADCD) as a regulated cell death that is shown experimentally to require different components of the autophagy machinery without involvement of alternative cell death pathways.

Non-canonical activation of DAPK2 by AMPK constitutes a new pathway linking metabolic stress to autophagy.

Autophagy is an intracellular degradation process essential for adaptation to metabolic stress. DAPK2 is a calmodulin-regulated protein kinase, which has been implicated in autophagy regulation, though the mechanism is unclear. Here, we show that the central metabolic sensor, AMPK, phosphorylates DAPK2 at a critical site in the protein structure, between the catalytic and the calmodulin-binding domains.

Death by over-eating: The Gaucher disease associated gene GBA1, identified in a screen for mediators of autophagic cell death, is necessary for developmental cell death in Drosophila midgut.

Autophagy is critical for homeostasis and cell survival during stress, but can also lead to cell death, a little understood process that has been shown to contribute to developmental cell death in lower model organisms, and to human cancer cell death. We recently reported on our thorough molecular and morphologic characterization of an autophagic cell death system involving resveratrol treatment of lung carcinoma cells. To gain mechanistic insight into this death program, we performed a signalome-wide RNAi screen for genes whose functions are necessary for resveratrol-induced death.

Signalome-wide RNAi screen identifies GBA1 as a positive mediator of autophagic cell death.

Activating alternative cell death pathways, including autophagic cell death, is a promising direction to overcome the apoptosis resistance observed in various cancers. Yet, whether autophagy acts as a death mechanism by over consumption of intracellular components is still controversial and remains undefined at the ultrastructural and the mechanistic levels. Here we identified conditions under which resveratrol-treated A549 lung cancer cells die by a mechanism that fulfills the previous definition of autophagic cell death.

Changes in cIAP2, survivin and BimEL expression characterize the switch from autophagy to apoptosis in prolonged starvation.

Background: Autophagy is a catabolic process involving the engulfment of cytoplasmic content within autophagosomes followed by their delivery to lysosomes. This process is a survival mechanism, enabling cells to cope with nutrient deprivation by degradation and recycling of macromolecules. Yet during continued stress such as prolonged starvation, a switch from autophagy to apoptosis is often detected.

Cap-independent translation by DAP5 controls cell fate decisions in human embryonic stem cells.

Multiple transcriptional and epigenetic changes drive differentiation of embryonic stem cells (ESCs). This study unveils an additional level of gene expression regulation involving noncanonical, cap-independent translation of a select group of mRNAs. This is driven by death-associated protein 5 (DAP5/eIF4G2/NAT1), a translation initiation factor mediating IRES-dependent translation.

DAPK2 is a novel regulator of mTORC1 activity and autophagy.

Autophagy is a tightly regulated catabolic process, which is upregulated in cells in response to many different stress signals. Inhibition of mammalian target of rapmaycin complex 1 (mTORC1) is a crucial step in induction of autophagy, yet the mechanisms regulating the fine tuning of its activity are not fully understood. Here we show that death-associated protein kinase 2 (DAPK2), a Ca(2+)-regulated serine/threonine kinase, directly interacts with and phosphorylates mTORC1, and has a part in suppressing mTOR activity to promote autophagy induction.

Discovering protein-protein interactions within the programmed cell death network using a protein-fragment complementation screen.

Apoptosis and autophagy are distinct biological processes, each driven by a different set of protein-protein interactions, with significant crosstalk via direct interactions among apoptotic and autophagic proteins. To measure the global profile of these interactions, we adapted the Gaussia luciferase protein-fragment complementation assay (GLuc PCA), which monitors binding between proteins fused to complementary fragments of a luciferase reporter. A library encompassing 63 apoptotic and autophagic proteins was constructed for the analysis of ∼3,600 protein-pair combinations.

Infestation of urban populations of the Northern white-breasted hedgehog, Erinaceus roumanicus, by Ixodes spp. ticks in Poland.

Infestation by the nest-dwelling Ixodes hexagonus Leach and the exophilic Ixodes ricinus (Linnaeus) (Ixodida: Ixodidae) on the Northern white-breasted hedgehog, Erinaceus roumanicus (Erinaceomorpha: Erinaceidae), was investigated during a 4-year study in residential areas of the city of Poznań, west-central Poland. Of 341 hedgehogs, 303 (88.9%) hosted 10 061 Ixodes spp.

DAP-kinase and autophagy.

DAP-kinase (DAPK) is a Ca(2+)-calmodulin regulated kinase with various, diverse cellular activities, including regulation of apoptosis and caspase-independent death programs, cytoskeletal dynamics, and immune functions. Recently, DAPK has also been shown to be a critical regulator of autophagy, a catabolic process whereby the cell consumes cytoplasmic contents and organelles within specialized vesicles, called autophagosomes. Here we present the latest findings demonstrating how DAPK modulates autophagy.

The DAP-kinase interactome.

DAP-kinase (DAPK) is a Ca(2+)/calmodulin regulated Ser/Thr kinase that activates a diverse range of cellular activities. It is subject to multiple layers of regulation involving both intramolecular signaling, and interactions with additional proteins, including other kinases and phosphatases. Its protein stability is modulated by at least three distinct ubiquitin-dependent systems.

The DAPK family: a structure-function analysis.

DAP-kinase (DAPK) is the founding member of a family of highly related, death associated Ser/Thr kinases that belongs to the calmodulin (CaM)-regulated kinase superfamily. The family includes DRP-1 and ZIP-kinase (ZIPK), both of which share significant homology within the common N-terminal kinase domain, but differ in their extra-catalytic domains. Both DAPK and DRP-1 possess a conserved CaM autoregulatory domain, and are regulated by calcium-activated CaM and by an inhibitory auto-phosphorylation within the domain.

The translation initiation factor DAP5 promotes IRES-driven translation of p53 mRNA.

Translational regulation of the p53 mRNA can determine the ratio between p53 and its N-terminal truncated isoforms and therefore has a significant role in determining p53-regulated signaling pathways. Although its importance in cell fate decisions has been demonstrated repeatedly, little is known about the regulatory mechanisms that determine this ratio. Two internal ribosome entry sites (IRESs) residing within the 5'UTR and the coding sequence of p53 mRNA drive the translation of full-length p53 and Δ40p53 isoform, respectively.

Biochemical and functional characterization of the ROC domain of DAPK establishes a new paradigm of GTP regulation in ROCO proteins.

DAPK (death-associated protein kinase) is a newly recognized member of the mammalian family of ROCO proteins, characterized by common ROC (Ras of complex proteins) and COR (C-terminal of ROC) domains. In the present paper, we review our recent work showing that DAPK is functionally a ROCO protein; its ROC domain binds and hydrolyses GTP. Furthermore, GTP binding regulates DAPK catalytic activity in a novel manner by enhancing autophosphorylation on inhibitory Ser308, thereby promoting the kinase 'off' state.