Identification And Functional Analysis Of F-box Protein Fbp1 Substrate Ade16 Of Cryptococcus Neoformans

F-box proteins are responsible for specific recognition and degradation of substrates in SCF（F-box）E3 ubiquitin ligase complexes.Previous studies have shown that F-box proteins play an important role in the pathogenicity of fungi.Fbp1 is the first F-box protein identified in Cryptococcus neoformans,and when the FBP1 gene was knocked out,the sexual reproduction of C.neoformans would be blocked.As a novel virulence factor,Fbp1 also affects the immunogenicity of C.neoformans.However,the specific function of Fbp1 and the regulation mechanism of its substrate in C.neoformans are still unclear.Therefore,it is crucial to explore the substrates of Fbp1and studying the function of these substrates;it is very important for us to understand how the F-box proteins affect the sexual reproduction or pathogenicity of fungi.Meanwhile,it is also helpful for us to better explain the mechanism of the ubiquitin-proteasome degradation pathway in the pathogenicity of C.neoformans.To identify the substrates of Fbp1,we performed a quantitative proteomic analysis of two strains of H99 and fbp1Δmutants by i TRAQ technology to screen the proteins that may interact with Fbp1.Among the high-abundance proteins in the background of fbp1Δmutant,a protein CNAG＿00700,named Ade16,that may interact with Fbp1 was identified.In this study,we mainly analyzed the interaction between F-box protein Fbp1and Ade16 of C.neoformans,the mechanism and function of Ade16 in C.neoformans.The main findings are as follows:First,we analyzed the gene sequence of ADE16,and found that it is an ATIC bifunctionalenzymes（5-aminoimidazole-4-carboxamideribonucleotide formyltransferase/IMP cyclohydrolase,AICARFT/IMPCHase,ATIC）.The gene is2396 bp in length and contains five exons and four introns,encoding a protein with 605amino acids.The domain analysis showed that Ade16 contains an MGS domain and an ATIC bifunctional enzymes domain,respectively.It is similar to the domains of isozymes Ade16 and Ade17 domains encoding ATIC bifunctional enzymes in Saccharomyces cerevisiae,and the sequence identity is as high as about 80.42%.Secondly,we proved that Ade16 interacts with Fbp1 through the Yeast two-hybrid interaction and immunoprecipitation assays.The protein stability and ubiquitination of Ade16 protein in the wild-type and fbp1Δmutants backgrounds were also detected.The results show that Ade16 is a substrate of Fbp1 and its ubiquitination is regulated by Fbp1.Then the RT-q PCR analysis showed that in the early stage of C.neoformans mating,the expression level of ADE16 was increased first and then decreased,and it was expressed at all stages of C.neoformans mating.The fluorescence observation of GFP-Ade16 fusion-expressing strains showed that the Ade16 was located in the cytoplasm at all stages of C.neoformans during mating,and the cytoplasmic localization is not affected by stress conditions.Finally,to study the function of Ade16 in C.neoformans,we have repeatedly tried to knock out the ADE16 gene by biolistic transformation but failed.After replacing the native promoter of ADE16 with an inducible promoter,we found that ADE16 is an essential gene in C.neoformans;and then we constructed the iADE16 interference strains and ADE16^OE overexpression strains.Virulence factors assays showed that,compared with wild-type strains,the capsule of the iADE16 interfering strain was significantly smaller,and the capsule of overexpression strain significantly enlarged.However,the melanin production and growth ability at 37°C of the Cryptococcus strains were not affected.Growth assays of the Cryptococcus strains under stress conditions showed that iADE16 interfence strains were sensitive to high concentrations of KCl and Na Cl,indicating that the Ade16 may play a role in the response of C.neoformans to some high-salt and hyperosmotic stress.iADE16 interference strains are insensitive to SDS and Congo red,but ADE16^OEoverexpression strains are sensitive to SDS and insensitive to Congo red.This is consistent with the stress phenotype of fbp1Δmutant strains,indicating that the intracellular protein expression levels of ADE16 overexpression were similar to that of the FBP1 knock out strains,thus simulating the phenotype of fbp1Δmutant strains.The pathogenicity of the iADE16 interference strains and ADE16^OE overexpression strains to mice was no different from that of the wild-type strains.The iADE16interfering strains can mate and produce spores normally.However,ADE16^OE strains can produce mating hyphae but failed to produce spores after mating.The phenotype is consistent with the fbp1Δmutant strains,which indicates that Fbp1 is likely to affect the sexual reproduction of C.neoformans by regulating Ade16.To sum up,our study suggests that Ade16 is a substrate of Fbp1,and the stability and ubiquitination of Ade16 are regulated by Fbp1.Ade16 plays a specific role in capsule production and stress resistance of C.neoformans.High levels of Ade16proteins may affect the integrity of the cell membrane of C.neoformans.The ADE16^OEoverexpression strains do not produce spores after mating,indicating that Fbp1 may regulate Ade16 through the ubiquitin-proteasome system,thereby affecting the sexual reproduction process of C.neoformans.