The Role Of DDRGK1 In Regulating Apoptosis And Proliferation Of Embryonic Fibroblasts And Inflammation Of Mammary Epithelial Cells Via Autophagy Pathway

DDRGK1（DDRGK domain-containing protein 1）is an important member of the newly discovered Ufm1（Ubiquitin-Fold Modifier 1）-conjugating system（also called UFMylation system）.It is reported that DDRGK1 deficiency would cause a variety of physiological and pathological diseases,including impairment of hematopoietic stem cell differentiation and development,intestinal homeostasis dysfunction,skeletal dysplasia and neurodevelopment disorders.In addition,DDRGK1 knockout mice results in embryonic lethality,suggesting a critical role of DDRGK1 in maintaining normal physiological processes.However,the underlying mechanism is poorly understood.Autophagy is a crucial process for maintaining cellular homeostasis,which has been reported to be involved in almost every physiological process in the body.As proteomic analysis showed an abundant number of differentially expressed proteins enriched in“Lysosome”and“Regulation of autophagy”pathways during DDRGK1 deletion,we speculate that the physiological dysfunction mediated by DDRGK1 loss may also be relevant to autophagy.Thus,it is necessary to explore the relationship between DDRGK1 and autophagy.In addition,with the deepening of research on UFMylation system,its role in livestock production became increasingly prominent.Bovine mastitis is a severe problem restricting the development of dairy industry in animal husbandry.Although the cases of mastitis has been reduced with the use of antibiotics,the problem of drug resistance remains inevitable.Therefore,it has become the research focus in prevention and therapy of mastitis about how to improve the natural anti-inflammatory ability of dairy cows.However,whether DDRGK1 is involved in the mastitis regulation and the specific regulatory mechanism remains to be verified.Therefore,this study took DDRGK1 as the entry point and took mouse embryonic fibroblast cells（MEFs）and bovine mammary epithelial cells（b MEC）as the research objects respectively to explore the role of DDRGK1 in cell fate regulation of MEFs and the inflammatory response of bovine mammary epithelial cells by using the techniques including i TRAQ-based proteomic,western blotting,q RT-PCR,immunofluorescence,co-immunoprecipitation,living cell staining,flow cytometry analysis,nuclear and cytoplasmic extraction,etc.,aiming to enrich the theoretical research on the biological function of DDRGK1 and UFMylation modification system and provide a theoretical basis for the development of prevention and therapy of cow mastitis and breeding schemes for mastitis resistance in the dairy industry.This study is divided into three parts,and the main conclusions are as follows:1.DDRGK1 loss aggravates apoptosis of MEFs by compromised autophagic degradation.In this study,we utilized an immortalized mouse embryonic fibroblasts（MEFs）harvested from the DDRGK1^F/F;ROSA26-Cre ERT2 mice,in which DDRGK1 depletion can be induced by 4-hydroxytamoxifen（4-OHT）treatment.Firstly,proteomic analysis suggested that DDRGK1 may participate in autophagy process as we identified an abundant number of differentially expressed proteins enriched in“lysosome”and“Regulation of autophagy”pathways during DDRGK1 deletion.Further biochemical tests showed that DDRGK1 deficiency in MEFs has a dual effect on autophagy.On one hand,DDRGK1deficiency promotes autophagy induction by regulating m TOR and AMPK signalings,on the other hand,it blocks autophagic degradation by inhibiting autophagosome-lysosome fusion.This dual effect of DDRGK1 depletion on autophagy ultimately impairs autophagic degradation,leading to a significant accumulation of autophagosomes and autophagic substrate SQSTM1.In addition,we found that autophagic degradation dysfunction caused by DDRGK1 loss aggravates apoptosis in MEFs,which could be effectively alleviate with si Atg7 transfection.Further studies reveal that DDRGK1 loss is correlated with suppressed lysosomal function,including impaired Cathepsin D（CTSD）expression and aberrant lysosomal p H.Importantly,we found DDRGK1 interacts with Atp6v0d1 and maintains the stable expression of Atp6v0d1 and Atp6v1a by regulating their proteasomal degradation,while DDRGK1 loss leads to the abnormal accumulation of both,which might be an important trigger for lysosomal dysfunction.In conclusion,our findings put forward a novel role of DDRGK1 in autophagy regulation via modulating lysosomal function,providing a novel insight into the molecular and cellular mechanism of DDRGK1 function.2.DDRGK1 deficiency-induced G2/M arrest is correlated with impaired autophagic degradationDuring the process of DDRGK1 deletion,we found MEFs with DDRGK1 deficiency showed a significant growth arrest,suggesting that DDRGK1 deficiency may affect cell proliferation.Therefore,in this study,we also took the above-mentioned MEFs to explore the role of DDRGK1 in cell proliferation and cell cycle progression.DDRGK1 deficiency was found to induce G2/M arrest in MEFs.Quantification of proportion of M phase cells and immunofluorescence staining with Phospho-H3-Ser10 protein further confirm that DDRGK1 deletion impairs mitotic entry.Further investigations show that DDRGK1 loss would cause DNA damage and inhibit CDK1 activity through ATM/ATR-Chk2-CDC25signaling cascade.Meanwhile,DDRGK1 deficiency also mediated the phosphorylation of Wee1 and Myt1 kinases and caused CDK1 inactivation,leading to G2/M arrest and subsequent inhibition of cell proliferation ultimately.Notably,DDRGK1 deletion-induced suppression of autophagic degradation was found to be involved in the regulation of cell cycle progression.Pharmacological or genetic rescue of impaired autophagic degradation alleviated DDRGK1 loss-mediated G2/M arrest,while blockage of autophagic degradation by Baf-A1 treatment had no further effect on G2/M arrest.In conclusion,our study reveals a potential relationship between DDRGK1 deletion-induced G2/M blockade and impaired autophagic degradation,which may provide a potential therapeutic target and strategy for diseases related to DDRGK1 deficiency.3.DDRGK1 plays an anti-inflammatory role in LTA-mediated inflammation of bovine mammary epithelial cells by regulating NF-κB pathwayIn this study,the regulation of DDRGK1 on mammary inflammation was explored by constructing in vitro mastitis models.Results showed that the expression of DDRGK1decreased for a short period of time,and then recovered after a 12-hour incubation during the LTA-induced b MEC inflammation.Furthermore,whether treated with LTA or not,DDRGK1 deficiency led to a significant increase in the expression of pro-inflammatory cytokines,suggesting that DDRGK1 plays an anti-inflammatory role in the regulation of mammary inflammation.Moreover,both nuclear and cytoplasmic extraction analysis and immunofluorescence staining of NF-κB showed that DDRGK1 loss promoted nuclear translocation of NF-κB.Meanwhile,western blotting analysis showed that DDRGK1knockdown promoted the phosphorylation of NF-κB,suggesting that DDRGK1 deletion contributes to transcriptional activation of NF-κB.In addition,autophagy is also found to play a role in the regulation of mammary inflammation related to DDRGK1.Taken together,this study took MEFs and bovine mammary epithelial cells as research objects to explore the key role of DDRGK1 in maintaining cell physiological functions.Firstly,we determined the regulatory relationship between DDRGK1 and autophagy in MEFs,and proved that DDRGK1 loss leads to impaired autophagic degradation by mediating lysosomal dysfunction,which ultimately results in apoptosis of MEF cells.Moreover,this impaired autophagic degradation mediated by DDRGK1 loss exacerbates DNA damage and induces G2/M arrest of MEFs by initiating the DNA damage checkpoint mechanism,resulting in inhibition of cell proliferation.In addition,DDRGK1 deficiency exacerbates LTA-induced inflammatory responses in bovine mammary epithelial cells via activating NF-κB pathway,suggesting that DDRGK1 may exert an anti-inflammatory role in bovine mastitis.All in all,this study provides a new insight into the function and regulatory mechanism of DDRGK1 and a theoretical basis for the development of non-antibiotic dependent prevention and therapy of cow mastitis and breeding schemes for mastitis resistance in the dairy industry.