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Purpose: The use of clinical radiation for cancer treatment is limited by damage to underlying normal tissue including to the vascular endothelium. We investigated the mechanisms of X-ray-induced cell damage to endothelial cells.
Direct cleavage and activation of gasdermin B by asthma trigger allergensTo the Editor:Recent fine-mapping studies have pointed to gasdermn B (GSDMB ) as a potential asthma susceptibility gene in 17q21 locus, the strongest and most highly replicated signal in genome-wide association studies1. The GSDMB protein is a member of the gasdermin family that, when cleaved, triggers an inflammatory cell death known as pyroptosis2. Caspase-1 and granzyme A have been shown to cut GSDMB at specific sites to release the N-terminal fragment of the protein (GSDMB-NT) that has the ability to induce pyroptosis in cells, including airway epithelial cells3,4. These findings suggest that the role of GSDMB in asthma lies in its ability to be activated through cleavage to induce pyroptosis; however, it remains unclear whether GSDMB cleavage and activation occur in the context of asthma.Common asthma trigger allergens often possess protease activities that cause airway epithelial injury and inflammation5,6. We thus tested whether the allergens directly cleave GSDMB. Incubation of extracts from house dust mite (HDM), a common asthma trigger, with lysates from human bronchial epithelial cells, which express endogenous GSDMB3, resulted in GSDMB cleavage as evidenced by the appearance of a smaller protein around 17kD (Figure 1A). Since the GSDMB antibody used in the Western blotting targets the C-terminus of the protein, the 17kD protein band likely represents the C-terminal GSDMB fragment. Such GSDMB cleavage was also observed when lysates from cells expressing C-terminal-FLAG-tagged GSDMB were mixed with HDM extract (Figure 1B). Furthermore, mold or cockroach extract also cleaved tagged GSDMB (Figure 1C). The cleavage of GSDMB protein by all allergen extracts resulted in a single product of similar size (about 17 kD), suggesting a specific cutting site.To identify the cleavage site, we incubated recombinant full-length GSDMB with HDM extract and resolved the cleaved protein products on SDS-PAGE (Figure 1D). We excised the putative 17 kD C-terminal fragment (GSDMB-CT, Figure 1D) and determined the N-terminal amino acid sequence of the fragment via Edman sequencing (Supplemental Figure S1, Figure 1E). Despite some ambiguities, the first ten amino acid residues of the 17 kD GSDMB-CT largely map to position 245 to 254 (SLGSEDSRNM) of the full length GSDMB protein (Figure 1E). This result indicates that GSDMB was cleaved immediately after the lysine residue at position 244 (K244). Interestingly, granzyme A also cuts GSDMB at the same K244 site4. To confirm K244 as the site of cleavage, we mutated lysine 244 to alanine (K244A) in GSDMB and tested whether the mutant protein can be cleaved by HDM. As shown by Western blotting, HDM was able to cleave wild type (WT) GSDMB but failed to cleave K244A GSDMB as evidenced by the absence of the 17 kD fragment (Figure 1F).The cleavage of GSDMB by HDM is expected to release an N-terminal fragment of 244 amino acids (GSDMB-NT-K244) (Figure 2A). We next tested whether GSDMB-NT-K244 triggers pyroptosis. Transfection of GSDMB-NT-K244 induced cell morphological changes characteristic of pyroptosis, including rounding up and detachment (Figure 2B). LDH release assay confirmed increased toxicity in these cells (~3.4 fold) as compared to cells transfected with the full-length GSDMB (Figure 2C). Consistent with our previous finding on GSDMB-NT shortened by a functional asthma-associated splice variant3, transfection of a truncated GSDMB-NT from the variant (NT-K231var) did not induce pyroptosis (Figure 2B,C).While future studies are needed to identify the specific proteases within the allergen extracts that cleave GSDMB, our current study demonstrates that asthma triggers such as HDM can directly cleave and activate GSDMB, thus providing biochemical evidence linking GSDMB-mediated pyroptosis to asthma.
Significance Dysregulated endoplasmic reticulum (ER) stress response contributes to the pathogenesis of myriad diseases. The molecular pathways leading to ER stress-induced cell death are well characterized; however, much less is known about how cells suppress excessive ER stress response to avoid apoptosis and restore homeostasis. Using a CRISPR-based loss-of-function genetic screen, our study uncovered multiple suppressors of ER stress response. These suppressors include a polycomb protein complex that directly inhibits the expression of the transcriptional factor central to ER stress-induced cell death and a microRNA that targets IRE1, a canonical ER stress pathway component. Our study reveals regulatory mechanisms that ameliorate potentially damaging stress response and provides potential therapeutic targets for pathologies whose etiology is linked to overactive ER stress response.
Radiation damage to biological systems is determined by the type of radiation, the total dosage of exposure, the dose rate, and the region of the body exposed. Three modes of cell death-necrosis, apoptosis, and autophagy-as well as accelerated senescence have been demonstrated to occur in vitro and in vivo in response to radiation in cancer cells as well as in normal cells. The basis for cellular selection for each mode depends on various factors including the specific cell type involved, the dose of radiation absorbed by the cell, and whether it is proliferating and/or transformed. Here we review the signaling mechanisms activated by radiation for the induction of toxicity in transformed and normal cells. Understanding the molecular mechanisms of radiation toxicity is critical for the development of radiation countermeasures as well as for the improvement of clinical radiation in cancer treatment.
Abstract : Exposure to high doses of ionizing radiation (IR) causes serious biological damage that can lead to death. During the course of radiotherapy, the use of IR for the treatment of thoracic cancers is limited by IR-induced cell death to the underlying normal lung tissue potentially leading to pneumonitis and/or fibrotic remodeling of the lung. Studies on the mechanisms of IR-induced cell death have been exponentially increasing, however, most of these studies are conducted on immortalized cancer cell lines that are not lung-derived and therefore do not represent the biological status of normal lung cells. We investigated the mechanisms of IR-induced cell death in low passage cultures of primary pulmonary artery endothelial cells (PAEC) exposed to varying doses of X-rays. We observed that irradiated PAEC undergo accelerated senescence as the primary mode of cell death at doses examined.
