The Purification Of Inflammatory Caspases And Its Effect On TPD54

Cells can die in different ways.Each of these death modes has its own characteristics and corresponding physiological effects.Compared with apoptosis,which was observed earlier and fully studied,pyroptosis is a phenomenon that has been gradually recognized as a relatively passive form of programmed cell death,it is a defensive mechanism triggered by pathogen infection.Pyroptosis is caused by activation of a pathogen by a pattern recognition receptor in the cell,which activates a special type of caspase protein,that is,the inflammatory caspase protein,they cleaves GSDMD and causes a hole in the cell membrane,causing the cell to swell and rupture and release cell contents.The inflammatory cysteine protease caspase is a key protein factor that initiates the process of cell pyroptosis.They also have similar cleavage activity and substrate selectivity as other caspase enzymes,along with the study of cell pyroptosis.In addition to GSDMD,the interaction of inflammatory caspase which activated in pyroptosis with other proteins,as well as the survival of cells and the impact on the whole body are worth studying.In order to explore the proteolytic factors that may interact with inflammatory caspase in the cell's pyroptosis,we designed and purified the inflammatory caspase protein caspase-1 and caspase-5,and used immunoprecipitation to detect some possible substrate proteins.Through our research,we have found that a protein factor TPD54,which is involved in cell adhesion and may contribute to apoptosis,has a site in its amino acid sequence that may be cleaved by caspase-1.We first transferred the two overexpression vectors carrying caspase-1 and TPD54 genes together into 293T cells,then TPD54 gene was single-transformed,a certain amount of cells were collected,purified TPD54 protein was obtained by co-immunoprecipitation,and purified caspase-1 was used in vitro.Cutting experiments were performed to detect the interaction between the two proteins.The results showed that no obvious cutting marks were found between caspase-1 and TPD54 that were co-expressed in vivo.However,when the purity of TPD54 was increased after the in vitro cutting experiment,cut bands of the expected size appeared.The discovery of this part of the experiment allows us to know a new caspase-1 protein cleavage substrate TPD54,opening up new ideas for further experiments to explore the function of inflammatory caspase.The function of TPD54 in cancer cells leads us to speculate that it may be stimulated by caspase-1 to promote the secretion of interleukins,and may also affect the choice of cells between the pyroptic and apoptotic pathways.These conjectures need follow-up experiments to provide further proof.The improvement of these theories will deepen our understanding of the physiological phenomenon of programmed cell death and provide new ideas for the treatment and prevention of various diseases..