Idendification And Functional Study Of Human UFM1-Specific Protease UFSP1

Posttranslational modification refers to the chemical modification of proteins after translation,which can rapidly respond to the internal and external changes in cells,regulate a variety of signaling pathways,and play important functions in vivo.Common posttranslational modifications include methylation,acetylation,phosphorylation,ubiquitination,and ubiquitin-like modification.The modification system of UFM1（UFMylation）is a recently discovered class of post-translational ubiquitin-like modification.Deficiency in this modification leads to embryonic lethality in mice and diseases in humans,suggesting that Ufmylation has essential biological functions.UFM1 is sequentially catalyzed by UFM1-activating enzyme 5（UBA5,E1）,UFM1-binding enzyme 1（UFC1,E2）,and UFM1-specific ligase 1（UFL1,E3）to covalently conjugate to the target protein.In addition,both UFM1 maturation and de-UFMylation are required UFM1 specific proteases.In human genome there are two UFSP genes（UFSP1 and UFSP2）.However,hUFSP1（human UFSP1）has long been thought to be inactive or non-functional.In NCBI database,hUFSP1 has been annotated as“inactive”,because it is believed that the UFSP1 gene is translated from ⁴⁴⁵AUG as the first translation codon and therefore it lacks a specific protease domain.Therefore,UFSP2 is considered to be the only active protease that mediates the UFM1 precursor（pro-UFM1）maturation and de-UFMylation in human cells.Interestingly,some studies have shown that knockout of UFSP2 leads to a significant increase in UFMylation in human cells,suggesting that there are other active UFM1-specific proteases mediating pro-UFM1 maturation in human cells.This project aims to identify the UFM1 specific protease responsible for UFM1 maturation and de-Ufmylation,besides the UFSP2.Given that only UFSP1 and UFSP2 are present in the human genome,we are naturally concerned about the true identity of UFSP1 in the process of UFMylation/de-UFMylation.AlthoughUFSP1 is currently considered to be enzymatically inactive in most species,including primates,the possibility of non-AUG translation initiation of hUFSP1 protein is predicted by comparing the DNA and amino acid sequences of UFSP1 gene in rats,mice,humans and other species.It has been known since 1980’s that translation can be initiated at codons other than AUG.Usually start with a near-cognate start codon,and in most cases use a near-cognate codon that is only one nucleotide different from AUG（for example CUG,GUG,UUG）.In this study,by using a series of eukaryotic expression constructs,protein expression and purification,mass spectrometry analysis,in vitro enzyme activity detection,and Ufmylation detection assay,we found that hUFSP1 protein does not translate from the codon of ⁴⁴⁵AUG as the translation start site,but ²¹⁷CUG（non-AUG）codon upstream is used as the translation start site.Furthermore,we showed that hUFSP1 protein translated from upstream ²¹⁷CUG contains the cysteine protease active domain with both UFM1 maturation and de-Ufmylation activity.These results solved a long-standing puzzle in the human Ufmylation modification and provide new insights not only in the mechanistic action of Ufmylation but also in the translation of genes employing nontraditional start codons.