Translocation of HSP47 and generation of mitochondrial reactive oxygen species in human neuroblastoma SK-N-SH cells following electron and X-ray irradiation.

Generation of mitochondrial reactive oxygen species (ROS), lipid peroxidation, 4-hydroxy-2-nonenal, and heat-shock protein (HSP) 47 after electron and X-ray irradiations were detected in the human neuroblastoma cell line SK-N-SH. After 10 Gy electron irradiation and 15 Gy X-ray irradiation, mitochondrial ROS production and lipid peroxidation were significantly increased. Additionally, we observed a significant increase in the levels of HSP47 after 3 and 10 Gy electron irradiation as well as 15 Gy X-ray irradiation. Furthermore, myristoylation and farnesylation were increased after 10 Gy electron and 15 Gy X-ray irradiations. We found that the level of HSP47 increased in the mitochondria after 10 Gy electron and 15 Gy X-ray irradiations. HSP47 coexisted with myristoylation and farnesylation. Furthermore, HSP47 overexpression increased mitochondrial ROS production. These results suggest that HSP47 plays an important role in mitochondria and induces mitochondrial ROS production in SK-N-SH cells.

LITESEC-T3SS - Light-controlled protein delivery into eukaryotic cells with high spatial and temporal resolution.

Many bacteria employ a type III secretion system (T3SS) injectisome to translocate proteins into eukaryotic host cells. Although the T3SS can efficiently export heterologous cargo proteins, a lack of target cell specificity currently limits its application in biotechnology and healthcare. In this study, we exploit the dynamic nature of the T3SS to govern its activity. Using optogenetic interaction switches to control the availability of the dynamic cytosolic T3SS component SctQ, T3SS-dependent effector secretion can be regulated by light. The resulting system, LITESEC-T3SS (Light-induced translocation of effectors through sequestration of endogenous components of the T3SS), allows rapid, specific, and reversible activation or deactivation of the T3SS upon illumination. We demonstrate the light-regulated translocation of heterologous reporter proteins, and induction of apoptosis in cultured eukaryotic cells. LITESEC-T3SS constitutes a new method to control protein secretion and translocation into eukaryotic host cells with unparalleled spatial and temporal resolution.

NLRC4 Mutation in flagellin-derived peptide CBLB502 ligand-binding domain reduces the inflammatory response but not radioprotective activity.

Bacterial flagellin is a pathogen-associated molecular pattern recognized by surface-localized Toll-like receptor 5 (TLR5) and cytosolic NOD-like receptor protein 4 (NLRC4). CBLB502, derived from Salmonella flagellin, exhibits high radioprotective efficacy in mice and primates by regulating TLR5 and the nuclear factor kappa B (NF-κB) signaling pathway. In this study, we examined the effects of CBLB502 and mutations in its NLRC4- and TLR5-binding domains on radioprotective efficacy and the immune inflammatory response. The results showed that CBLB502 mutation with I213A in the TLR5-binding domain significantly reduced NF-κB activity and radioprotective activity, whereas CBLB502 mutation with L292A in NLRC4-binding domain did not. Additionally, CBLB502 with both mutations greatly reduced NF-κB activity and eliminated radioprotection in mice. In contrast, NLRC4-binding domain mutation reduced the secretion of inflammatory interleukin-1ß and interleukin-18. CBLB502 exerts its radioprotective effects through both the TLR5 and NLRC4 pathways. Additionally, deletion in the NLRC4-binding domain did not reduce radioprotective activity but reduced the inflammatory response.

Identification of intracellular cavin target proteins reveals cavin-PP1alpha interactions regulate apoptosis.

Caveolae are specialized domains of the plasma membrane. Formation of these invaginations is dependent on the expression of Caveolin-1 or -3 and proteins of the cavin family. In response to stress, caveolae disassemble and cavins are released from caveolae, allowing cavins to potentially interact with intracellular targets. Here, we describe the intracellular (non-plasma membrane) cavin interactome using biotin affinity proteomics and mass spectrometry. We validate 47 potential cavin-interactor proteins using a cell-free expression system and protein-protein binding assays. These data, together with pathway analyses, reveal unknown roles for cavin proteins in metabolism and stress signaling. We validated the interaction between one candidate interactor protein, protein phosphatase 1 alpha (PP1α), and Cavin-1 and -3 and show that UV treatment causes release of Cavin3 from caveolae allowing interaction with, and inhibition of, PP1α. This interaction increases H2AX phosphorylation to stimulate apoptosis, identifying a pro-apoptotic signaling pathway from surface caveolae to the nucleus.

Characterization of temperature-dependent hemin uptake receptors HupA and HvtA in Vibrio vulnificus.

The Gram-negative pathogen Vibrio vulnificus produces several iron-sequestration systems including a hemin uptake system in response to iron limitation as a means to acquire this essential element. Strains of this organism are capable of causing serious septicemia in humans and eels, where hemin is abundant and an advantageous source of iron. Vibrio vulnificus hemin uptake systems consist of HupA, a well studied outer membrane protein, and a recently identified HvtA protein receptor. In this study, we confirmed that the expression of the hvtA gene is iron-regulated in a fur-dependent manner. When analyzed for virulence in a hemin-overloaded murine model system, the hupA gene was more important for establishing infection than the hvtA gene. Transcriptional profiling of these genes using strains of two different biotypes, biotype 1 (human pathogen) and biotype 2 (eel pathogen), showed that the expression of the two receptors was also regulated in response to temperature. The expression of hupA was highly induced in elevated temperatures in the human pathogenic strain when tested in iron-depleted conditions. Conversely, hvtA expression was induced significantly in the eel pathogenic strain at a lower temperature, a condition where the hupA locus was relatively repressed. Our results indicate that although both hupA and hvtA are involved for optimal hemin uptake in V. vulnificus, their expression is dually regulated by the environmental cues of iron concentration and temperature. Together, these data suggest that the virulence genes hupA and hvtA are tightly regulated and strictly induced during iron limitation combined with the physiological temperature of the host organism.

Stable Acinar Progenitor Cell Model Identifies Treacle-Dependent Radioresistance.

Radiotherapy for head and neck cancers can result in extensive damage to the salivary glands, significantly affecting patient quality of life. However, the salivary gland can recover in patients receiving lower doses of radiation. In addition, there is considerable interest in delineating the mechanisms by which stem cells survive radiation exposure and promote tissue regeneration. In this study, we isolated stable radioresistant acinar progenitor cells from the submaxillary gland of the Sprague Dawley rat. Progenitor cells are characterized as c-Kithigh/alpha-amylase+ and are resistant to X rays (≤5 Gy).We further isolated a radiosensitive acinar counterpart, characterized as c-Kitlow/alpha-amylase+, which is effectively killed by exposure to 2 Gy X ray of radiation. Phosphopeptides with homology to the treacle protein (TCOF1) were disproportionately increased in progenitor cells, compared to their radiosensitive counterparts. Silencing of TCOF1 expression (shRNA) radiosensitized progenitor cells, a response conserved in human cells with TCOF1 knockdown. Collectively, these observations indicate that radiation resistance is an intrinsic property of c-Kithigh salivary gland progenitor cells. Since human salivary gland stem cells with c-Kit expression are believed to have enhanced regenerative potencies, our model system provides a stable platform to investigate molecular features associated with c-Kit expression that may contribute to protection or stabilization of the stem cell niche.

Radiation-promoted CDC6 protein stability contributes to radioresistance by regulating senescence and epithelial to mesenchymal transition.

Ionizing radiation (IR) is a conventional cancer therapeutic, to which cancer cells develop radioresistance with exposure. The residual cancer cells after radiation treatment also have increased metastatic potential. The mechanisms by which cancer cells develop radioresistance and gain metastatic potential are still unknown. In this study acute IR exposure induced cancer cell senescence and apoptosis, but after long-term IR exposure, cancer cells exhibited radioresistance. The proliferation of radioresistant cells was retarded, and most cells were arrested in G0/G1 phase. The radioresistant cells simultaneously showed resistance to further IR-induced apoptosis, premature senescence, and epithelial to mesenchymal transformation (EMT). Acute IR exposure steadily elevated CDC6 protein levels due to the attenuation of ubiquitination, while CDC6 overexpression was observed in the radioresistant cells because the insufficiency of CDC6 phosphorylation blocked protein translocation from nucleus to cytoplasm, resulting in subcellular protein accumulation when the cells were arrested in G0/G1 phase. CDC6 ectopic overexpression in CNE2 cells resulted in apoptosis resistance, G0/G1 cell cycle arrest, premature senescence, and EMT, similar to the characteristics of radioresistant CNE2-R cells. Targeting CDC6 with siRNA promoted IR-induced senescence, sensitized cancer cells to IR-induced apoptosis, and reversed EMT. Furthermore, CDC6 depletion synergistically repressed the growth of CNE2-R xenografts when combined with IR. The study describes for the first time cell models for IR-induced senescence, apoptosis resistance, and EMT, three major mechanisms by which radioresistance develops. CDC6 is a novel radioresistance switch regulating senescence, apoptosis, and EMT. These studies suggest that CDC6highKI67low represents a new diagnostic marker of radiosensitivity, and CDC6 represents a new therapeutic target for cancer radiosensitization.

Epigallocatechin-3-gallate Enhances Radiation Sensitivity in Colorectal Cancer Cells Through Nrf2 Activation and Autophagy.

BACKGROUND/AIM: Epigallocatechin-3-gallate (EGCG) is a major polyphenolic component of green tea. EGCG plays a potential role in radio-sensitizing cancer cells. The combined effect of EGCG and radiation was investigated in a colorectal cancer cell line, focusing on nuclear factor (erythroid-derived 2)-like 2 (Nrf2) autophagy signalling. MATERIALS AND METHODS: HCT-116 cells were treated with 12.5 µM EGCG for different periods of time, 2 Gy radiation, or both. Cell viability was determined with the WST-8 assay. The number of colonies was determined with the colony formation assay. mRNA expression of LC3 and caspase-9 was analyzed with quantitative real-time polymerase chain reaction. RESULTS: Combination treatment with EGCG and radiation significantly decreased the growth of HCT-116 cells. The number of colonies was reduced to 34.2% compared to the control group. Immunofluorescence microscopy images showed that nuclear translocation of Nrf2 was significantly increased when cells were treated with the combination of EGCG and radiation compared to the control and single-treatment groups. Combined treatment with EGCG and radiation significantly induced LC3 and caspase-9 mRNA expression. CONCLUSION: EGCG increased the sensitivity of colorectal cancer cells to radiation by inhibiting cell proliferation and inducing Nrf2 nuclear translocation and autophagy.

Receptor tyrosine kinase signaling in cancer radiotherapy and its targeting for tumor radiosensitization.

PURPOSE: One of the most important implications of 'Radiation Biology' research is to improve cancer radiotherapy with minimum side effects. In this regard, combination of chemotherapy with radiation has significantly improved tumor control as well as overall survival in a variety of cancers. However, this has been achieved at the cost of significant normal tissue toxicity, due to the lack of specificity of chemotherapy. Membrane-localized receptor tyrosine kinases (RTKs) have been found to play a driving role in various hallmarks of cancer. Moreover, an early successful clinical trial using RTK-antagonist (cetuximab) to improve tumor radiosensitivity has led to an advancement in this field of research. However, a comprehensive review integrating these findings of various oncogenic RTKs, from basic radiobiology-to-radiotherapy clinical trials, is lacking in literature. Therefore, the present review analyses relevant in-vitro, in-vivo, preclinical/clinical studies and postulates the concept of 'Radiation Biology of RTKs in Cancer'. CONCLUSIONS: The present review elucidates the effect of IR on various oncogenic RTKs and their mechanisms, downstream signaling, intracellular translocations, their role in the repair of radiation-induced DNA damage and post-irradiation survival. Based on the knowledge derived from RTK biology and the analysis of relevant clinical trials, this review attempts to identify radiobiological considerations, which could be implemented in future trials, combining radiotherapy with RTK-antagonist. Additionally, we identify the radiosensitizing potential of recently developed RTK-targeted nanoformulations. This review would probably change the Radiation Oncologist's view for translation of tumor-specific radiosensitization in clinic.

Physcomitrella patens MAX2 characterization suggests an ancient role for this F-box protein in photomorphogenesis rather than strigolactone signalling.

Strigolactones (SLs) are key hormonal regulators of flowering plant development and are widely distributed amongst streptophytes. In Arabidopsis, SLs signal via the F-box protein MORE AXILLARY GROWTH2 (MAX2), affecting multiple aspects of development including shoot branching, root architecture and drought tolerance. Previous characterization of a Physcomitrella patens moss mutant with defective SL synthesis supports an ancient role for SLs in land plants, but the origin and evolution of signalling pathway components are unknown. Here we investigate the function of a moss homologue of MAX2, PpMAX2, and characterize its role in SL signalling pathway evolution by genetic analysis. We report that the moss Ppmax2 mutant shows very distinct phenotypes from the moss SL-deficient mutant. In addition, the Ppmax2 mutant remains sensitive to SLs, showing a clear transcriptional SL response in dark conditions, and the response to red light is also altered. These data suggest divergent evolutionary trajectories for SL signalling pathway evolution in mosses and vascular plants. In P. patens, the primary roles for MAX2 are in photomorphogenesis and moss early development rather than in SL response, which may require other, as yet unidentified, factors.

Optochemical Control of Protein Localization and Activity within Cell-like Compartments.

We report inducible dimerization strategies for controlling protein positioning, enzymatic activity, and organelle assembly inside synthetic cell-like compartments upon photostimulation. Using a photocaged TMP-Haloligand compound, we demonstrate small molecule and light-induced dimerization of DHFR and Haloenzyme to localize proteins to a compartment boundary and reconstitute tripartite sfGFP assembly. Using photocaged rapamycin and fragments of split TEV protease fused to FRB and FKBP, we establish optical triggering of protease activity inside cell-size compartments. We apply light-inducible protease activation to initiate assembly of membraneless organelles, demonstrating the applicability of these tools for characterizing cell biological processes in vitro. This modular toolkit, which affords spatial and temporal control of protein function in a minimal cell-like system, represents a critical step toward the reconstitution of a tunable synthetic cell, built from the bottom up.

Near-infrared light-controlled systems for gene transcription regulation, protein targeting and spectral multiplexing.

Near-infrared (NIR, 740-780 nm) optogenetic systems are well-suited to spectral multiplexing with blue-light-controlled tools. Here, we present two protocols, one for regulation of gene transcription and another for control of protein localization, that use a NIR-responsive bacterial phytochrome BphP1-QPAS1 optogenetic pair. In the first protocol, cells are transfected with the optogenetic constructs for independently controlling gene transcription by NIR (BphP1-QPAS1) and blue (LightOn) light. The NIR and blue-light-controlled gene transcription systems show minimal spectral crosstalk and induce a 35- to 40-fold increase in reporter gene expression. In the second protocol, the BphP1-QPAS1 pair is combined with a light-oxygen-voltage-sensing (LOV) domain-based construct into a single optogenetic tool, termed iRIS. This dual-light-controllable protein localization tool allows tridirectional protein translocation among the cytoplasm, nucleus and plasma membrane. Both procedures can be performed within 3-5 d. Use of NIR light-controlled optogenetic systems should advance basic and biomedical research.

Arabidopsis Blue Light Receptor Phototropin 1 Undergoes Blue Light-Induced Activation in Membrane Microdomains.

Phototropin (phot)-mediated signaling initiated by blue light (BL) plays a critical role in optimizing photosynthetic light capture at the plasma membrane (PM) in plants. However, the mechanisms underlying the regulation of phot activity at the PM in response to BL remain largely unclear. In this study, by single-particle tracking and stepwise photobleaching analysis of phot1-GFP proteins we demonstrated that in the dark phot1 proteins remain in an inactive state and mostly exist as monomers. Dimerization and the diffusion rate of phot1-GFP increased in a dose-dependent manner in response to BL. In contrast, BL did not affect the lateral diffusion of kinase-inactive phot1D806N-GFP but did enhance its dimerization, suggesting that phot1 dimerization is independent of phosphorylation. Förster resonance energy transfer-fluorescence lifetime imaging microscopy analysis revealed that the interaction between phot1-GFP and a marker of sterol-rich lipid environments, AtRem1.3-mCherry, was enhanced with increased time of BL treatment. However, this BL-dependent interaction was not obvious in plants co-expressing phot1D806N-GFP and AtRem1.3-mCherry, indicating that BL facilitates the translocation of functional phot1-GFP into AtRem1.3-labeled microdomains to activate phot-mediated signaling. Conversely, sterol depletion attenuated phot1-GFP dynamics, dimerization, and phosphorylation. Taken together, these results indicate that membrane microdomains act as organizing platforms essential for the proper function of activated phot1 at the PM.

The use of the vaccinia virus complement control protein (VCP) in the rat retina.

The complement system is highly implicated in both the prevalence and progression of Age-Related Macular Degeneration (AMD). Complement system inhibitors therefore have potential therapeutic value in managing excessive activation of the complement pathways in retinal degenerations. The vaccinia virus complement control protein (VCP) has been shown to be effective as a complement inhibitor in neuroinflammatory models including traumatic brain injury and spinal cord injury. We aimed to investigate the potential of VCP as a therapeutic molecule for retinal degenerations. In this study, we investigated the effect, localisation and delivery of VCP to the rodent retina. Complement inhibition activity of VCP was tested using a hemolytic assay. Photoreceptor cell death, inflammation and retinal stress were assayed to determine if any retinal toxicity was induced by an intravitreal injection of VCP. The effect of VCP was investigated in a model of photo-oxidative retinal degeneration. Localisation of VCP after injection was determined using a fluorescein-tagged form of VCP, as well as immunohistochemistry. Finally, a copolymer resin (Elvax) was trialled for the slow-release delivery of VCP to the retina. We found that a dose equivalent to 20µg VCP when intravitreally injected into the rat eye did not cause any photoreceptor cell death or immune cell recruitment, but led to an increase in GFAP. In photo-oxidative damaged retinas, there were no differences in photoreceptor loss, retinal stress (Gfap) and inflammation (Ccl2 and C3) between VCP and saline-injected groups; however, Jun expression was reduced in VCP-treated retinas. After VCP was injected into the eye, it was taken up in all layers of the retina but was cleared within 1-3 hours of delivery. This study indicates that a method to sustain the delivery of VCP to the retina is necessary to further investigate the effect of VCP as a complement inhibitor for retinal degenerations.

Nuclear GSK3ß induces DNA double-strand break repair by phosphorylating 53BP1 in glioblastoma.

Glioblastoma is the most malignant and lethal subtype brain tumors with high risk of recurrence and therapeutic resistance. Emerging evidence has indicated that glycogen synthesis kinase 3 (GSK3)ß plays oncogenic roles in multiple tumor types; however, the underlying mechanisms remain largely unknown. It has also been demonstrated that p53 binding protein 1 (53BP1) plays a central role in DNA double-strand break (DSB) repair. This study aimed to reveal the significance of GSK3ß translocation from the cytoplasm to the nucleus, and to determine whether GSK3ß induces DNA DSB repair in the nuclei of glioblastoma cells via phospho-53BP1. By performing in vitro experiments, we found that GSK3ß translocated from the cytoplasm to the nucleus, and it then bound to 53BP1 following exposure to IR (IR). In addition, 53BP1-mediated DNA DSB repair was observed to be abrogated by the inhibition of GSK3ß. Further experiments on the phosphorylation site of 53BP1 by GSK3ß revealed that the S/T-Q motif may play a critical role. Importantly, our in vivo and in vitro data clearly indicated that GSK3ß induced the phosphorylation of 53BP1 at the Ser166 site. Moreover, brain tumor xenograft models revealed that following exposure to IR plus SB216763, a specific GSK3ß inhibitor, tumor growth was markedly inhibited and the survival of mice markedly increased. Based on these results, we concluded that the phosphorylation of 53BP1 by GSK3ß was indispensable for DNA DSB repair. Our study also suggested that the inhibition of GSK3ß by SB216763 significantly inhibited the proliferation and induced the apoptosis of glioblastoma cells. Taken together, our data indicate that GSK3ß, a key phosphorylation protein for 53BP1, may be a potential target for enhancing the sensitivity of glioblastoma cells to radiation.

Ultraviolet-Ray-Induced Sea Cucumber (Stichopus japonicus) Melting Is Mediated by the Caspase-Dependent Mitochondrial Apoptotic Pathway.

Sea cucumber body-wall melting occurs under certain circumstances. We have shown that apoptosis but not autolysis plays a critical role in the initial stage. However, it is still unclear how apoptosis is triggered in this process. In this study, we examined the levels of reactive oxygen species (ROS), the levels of B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X (Bax) proteins, the depolarization of mitochondrial transmembrane potentials, and cytochrome c (Cyt c) release during sea cucumber melting induced by ultraviolet (UV) exposure. We also investigated the contribution of caspase in this process by injecting a pan-caspase inhibitor. Our data showed that UV exposure stimulates ROS production, dysfunction of mitochondria, and the release of Cyt c in sea cucumber coelomic fluid cells and body walls. We found a decrease of Bcl-2 and increase of Bax in the mitochondria after UV exposure. We also demonstrated that these changes are associated with elevated caspase-9 and -3 activity. Finally, our data showed that the inhibition of caspases-9 and -3 using an inhibitor suppresses UV-induced sea cucumber melting. These results suggest that apoptosis during sea cucumber melting is mediated by mitochondrial dysfunction and follows the activation of the caspase-signaling pathway. This study presents a novel insight into the mechanism of sea cucumber melting.

TIE2 Associates with Caveolae and Regulates Caveolin-1 To Promote Their Nuclear Translocation.

DNA repair pathways are aberrant in cancer, enabling tumor cells to survive standard therapies-chemotherapy and radiotherapy. Our group previously reported that, upon irradiation, the membrane-bound tyrosine kinase receptor TIE2 translocates into the nucleus and phosphorylates histone H4 at Tyr51, recruiting ABL1 to the DNA repair complexes that participate in the nonhomologous end-joining pathway. However, no specific molecular mechanisms of TIE2 endocytosis have been reported. Here, we show that irradiation or ligand-induced TIE2 trafficking is dependent on caveolin-1, the main component of caveolae. Subcellular fractionation and confocal microscopy demonstrated TIE2/caveolin-1 complexes in the nucleus, and using inhibitor or small interfering RNAs (siRNAs) against caveolin-1 or Tie2 inhibited their trafficking. TIE2 was found in caveolae and directly phosphorylated caveolin-1 at Tyr14 in vitro and in vivo This modification regulated the generation of TIE2/caveolin-1 complexes and was essential for TIE2/caveolin-1 nuclear translocation. Our data further demonstrate that the combination of TIE2 and caveolin-1 inhibitors resulted in significant radiosensitization of malignant glioma cells, which will guide the development of combinatorial treatment with radiotherapy for patients with glioblastoma.

A Proinflammatory Function of Toll-Like Receptor 2 in the Retinal Pigment Epithelium as a Novel Target for Reducing Choroidal Neovascularization in Age-Related Macular Degeneration.

Current treatments for choroidal neovascularization, a major cause of blindness for patients with age-related macular degeneration, treat symptoms but not the underlying causes of the disease. Inflammation has been strongly implicated in the pathogenesis of choroidal neovascularization. We examined the inflammatory role of Toll-like receptor 2 (TLR2) in age-related macular degeneration. TLR2 was robustly expressed by the retinal pigment epithelium in mouse and human eyes, both normal and with macular degeneration/choroidal neovascularization. Nuclear localization of NF-κB, a major downstream target of TLR2 signaling, was detected in the retinal pigment epithelium of human eyes, particularly in eyes with advanced stages of age-related macular degeneration. TLR2 antagonism effectively suppressed initiation and growth of spontaneous choroidal neovascularization in a mouse model, and the combination of anti-TLR2 and antivascular endothelial growth factor receptor 2 yielded an additive therapeutic effect on both area and number of spontaneous choroidal neovascularization lesions. Finally, in primary human fetal retinal pigment epithelium cells, ligand binding to TLR2 induced robust expression of proinflammatory cytokines, and end products of lipid oxidation had a synergistic effect on TLR2 activation. Our data illustrate a functional role for TLR2 in the pathogenesis of choroidal neovascularization, likely by promoting inflammation of the retinal pigment epithelium, and validate TLR2 as a novel therapeutic target for reducing choroidal neovascularization.