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Revealing disulfide bond formation in GSDME upon UVC irradiation under non-reducing conditions

ABSTRACT: It is widely acknowledged that gasdermin family proteins, which are known as the executors of pyroptosis, undergo protease-mediated cleavage prior to inducing pyroptosis. Here, we unexpectedly discovered a non-canonical form of pyroptosis mediated by full-length GSDME (FL-GSDME) without any proteolytic cleavage. Upon intense ultraviolet (UV) irradiation-triggered DNA damage, hyperactivation of nuclear PARP1 led to extensive formation of poly(ADP-ribose) (PAR) polymers and then release to the cytoplasm.These PAR polymers activate PARP5 to catalyze GSDME PARylation, resulted in a conformational change in GSDME that relieved autoinhibition imposed by its C terminus on the N terminus. On the other hand, intense UV irradiation boosted mitochondrial fission-dependent generation of mitochondrial reactive oxygen species (mito-ROS), further promoting cytochrome c-catalyzed peroxidation of cardiolipin. This lipid-ROS signal was then sensed by PARylated-GSDME and then induced oxidative oligomerization of GSDME, which facilitated FL-GSDME plasma membrane targeting for perforation, eventually inducing pyroptosis. Reagents that concurrently stimulate PARPs activity and lipid-ROS also induced sequential modifications i.e., PARylation and oxidation of FL-GSDME and synergistically promoted pyroptotic cell death. Overall, our findings elucidate a novel mechanism underlying cleavage-independent function of GSDME in executing cell demise, further enriching the paradigms and cognition of FL-GSDME-mediated non-canonical pyroptosis.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hela Cell

SUBMITTER: Bo Zhou

LAB HEAD: Hang-zi Chen

PROVIDER: PXD051840 | Pride | 2024-06-17

REPOSITORIES: Pride

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Project description:Today, nanoparticles are used in various commercial products. One of the most common nanoparticles is titanium dioxide (TiO2). It has a catalytic activity and UV absorption (λ<400 nm), and it generates reactive oxygen species (ROS). The catalytic activity of TiO2 nanoparticle is capable of killing a wide range of microorganisms. In the environment, nanoparticles form structures consisting of primary particles, and their aggregates and agglomerates. These compounds are defined NOAA (nano-objects, and their aggregates and agglomerates greater than 100 nm). The unique properties of TiO2 nanoparticles can be maintained in the environment, thus, the growing use of TiO2 nanoparticles is raising concerns about the environmental risks. The assessment of biological and ecological effects of TiO2-NOAA is necessary. In our previous study, we assessed the effect of TiO2-NOAA on microbes by using Saccharomyces cerevisiae and Escherichia coli. It was shown that TiO2-NOAA decomposed methylene blue under UV irradiation. It suggested that TiO2-NOAA generated ROS under UV irradiation. However, TiO2-NOAA did not show growth inhibition in minimal agar medium under UV irradiation. By adding TiO2-NOAA in medium, colony formation was observed with UV intensity that inactivates microbes. Moreover, TiO2-NOAA adsorbed microbes. These results suggested that the amount of ROS generated by TiO2-NOAA was not enough to inactivate microbes, and TiO2-NOAA might protect microbes from UV. In this study, we assessed the effect of TiO2-NOAA in more detail by using S. cerevisiae. We used DNA microarray analysis for qualitative assessment. Further, we carried out quantitative assessment by using Real Time RT-PCR method for characteristic genes in DNA microarray analysis. To compare yeast cells in various conditions, six kinds of treatment conditions were prepared (Condition 1. adsorbed fraction to TiO2-NOAA under UV, 2. non-adsorbed fraction to TiO2-NOAA under UV, 3. adsorbed fraction to TiO2-NOAA without UV, 4. non-adsorbed fraction to TiO2-NOAA without UV, 5. irradiated UV and 6. negative control). From the result of DNA microarray analysis, the most number of genes was altered in Condition 1, followed by Condition 3 and 5. The genes related to oxidative stress, and the genes related to synthesis of trehalose and glycogen were significantly up-regulated of yeast cells in Condition 1 and 5, and Condition 1 and 3, respectively. These results suggest that yeast cells suffer oxidative stress by TiO2-NOAA under UV, and they also suffer membrane damage by TiO2-NOAA itself, as a result, they reserve energy sources. From the result of Real Time RT-PCR, genes related to oxidative stress (GRE2, SOD2) were up-regulated in Condition 1 and 3, however, these expression levels in each condition were not significant. And genes related to synthesis of trehalose and glycogen (GSY1, TPS2) were up-regulated in Condition 1 and 3. These results suggest that oxidative stress is caused not by TiO2-NOAA but by UV. It is also suggested that yeast cells were damaged at their membranes by TiO2-NOAA, as a result, genes related to synthesis of trehalose and glycogen were up-regulated. Thus, we suggest that the effect of TiO2-NOAA on yeast cells under UV irradiation is greater due to TiO2-NOAA itself than due to ROS generated by TiO2-NOAA.

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Revealing disulfide bond formation in GSDME upon UVC irradiation under non-reducing conditions

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