The Function Study Of Uba6 And Uba7 In Zebrafish

Ubiquitination is a multistep enzymatic cascade that is comprised of ubiquitin-activating enzymes(Els),ubiquitin-conjugating enzymes(E2s)and ubiquitin ligases(E3s).Firstly,the ubiquitin-activating enzyme UBE1(El)uses ATP to adenylate and then bind an ubiquitin molecule.Subsequently,a second ubiquitin molecule is then adenylated and bound to a different site of the same El enzyme.The E1 enzyme then transfers an ubiquitin molecule to the ubiquitin-conjugating enzyme E2.In the final step,the E2 enzyme transfers the ubiquitin to the target protein with the help of the ubiquitin ligase E3,resulting in ubiquitination of the target proteins.Protein with multiple Lysine-48(K48)of ubiquitin will be degraded by proteasome,while through Lysine63(K63)or monoubiquitination of proteins will not promote protein degradation but rather regulates processes such as receptor internalization,endocytosis,transcription,and DNA repair.In vertebrates,in addition to the canonical E1,UBE1,UBA6 was identified as another E1,which can activate both ubiquitin and FAT10.The El for ISG15 is Ubell(also called Uba7),and the E1 SAE-UBA2、NAE1-UBA3 for SUMO and NEDD8.UBE1 and UBA6 has 40%homology,but UBE1 is more close to UBA7.In cell lines and in vitro and,UBA6 can activate ubiquitin,and transfer ubiquitin to an UBA6-specific E2,USE1.UBA6 and USE1 in human and zebrafish,and sea urchins are all present,but does not exist in the worm,flies and yeast.It has been reported that Uba6 knock out mice was lethal,which implied that Uba6 was essential for mice embryonic development.Deletion of Uba6 in mice caused embryonic lethality is likely due to some defects in certain ubiquitination pathways that cannot be compensated by E1 and is not related to FAT 10.ISG15 is a 17 KD ubiquitin-like modifier that is rapidly induced by type I interferon signaling.Like ubiquitin and ubiquitin-like modifiers,ISG15 can exist as a free protein or as a covalent conjugate to target proteins.ISG15 conjugation and deconjugation processes are controlled by a canonical set of enzymes,including the ISG15-activating enzyme UBA7,ISG15-conjugating enzyme UbcH8 and the E3 ligases and ISG15-deconjugating enzyme UBP43.Unlike the ubiquitin system,the enzymes involved in ISG15 conjugation is IFN inducible.Protein ISG15 modification(ISGylation)is strongly induced upon IFN treatment or viral and bacterial infections.Whether protein ISGylation is involved in the diverse downstream responses of IFN signaling triggered by microbes or other stress remains to be determined it indicates that ISG15 pathway may play a role in innate immunity.It has been reported that,UBA 7 knock out mice has defected in ISG15 conjugation,homozygous of Uba7 knock out mice is fertile and healthy.UBA7 deficient cells did not show any abnormal responses to IFN treatment,and UBA7 +/+ and UBA7-/-cells exhibited similar susceptibility to vesicular stomatitis virus(VSV)and lymphocytic choriomeningitis virus(LCMV)infection,indicating that UBA7 and protein ISGylation are not essential for IFN signaling.UBA7 mRNA expression is often reduced in lung cancer cells,and in HBE cells UBA7can reduced cyclinD1 expression.This finding implicated UBA7 as a growth or tumor suppressor.UBA7 promotes the complex between ISG15 and cyclinDl,and inhibits the expression of cyclinDl.UBP43,the deconjugation enzyme antagonized this effect.UBA7 knockdown increased cyclin D1 expression.UBA7 confers growth suppression by preferentially targeting cyclin D1.UBA7-ISG15 as a growth suppressive pathway exerting anti-neoplastic effects by targeting cyclin D1 for repression.Protein modification by ubiquitin or ubiquitin like modifier is a cascade reaction of E1 and E2,E3 enzymes.El plays a crucial role in ubiquitination.UBA6 and UBA7 are E1 like proteins.Previous literature reports that UBA6 participate in ubiquitination.UBA7 is the sole E1 for ISGylation and the main E2,E3 are UbcH8 and Herc5 respectively.In this paper,we explored the function of gene uba6 and uba7 in zebrafish.By insitu hybridization of zebrafish embryos,we detect the development of digestive organ in early embryos which the gene uba6 and uba7 were knocked out by TALEN.The probe we used include ifabp,Ifabp,trypsin,insulin.And we find that the liver,intestine,pancreas are all developing as normal.The result shows that gene uba6 and uba7 are not essential for early embryonic development.We also research the role of zebrafish Uba7-ISG15 pathway in innate immune.But the ISGylation in zebrafish did not increase obviously after injection with bacteria,LPS,poly(I:C)as well as overexpression of type ⅠIFN,such as IL6 and ILlb.It still need more research.