Establishment And Phenotype Analysis Of Skp2^G72S/G72S Knock-in Mouse Model

The degradation of intracellular proteins are mainly composed of two systems:lysosome system and ubiquitin proteasome system.The ubiquitin proteasome system includes ubiquitin activating enzyme?E1?,ubiquitin-conjugating enzyme?E2?and ubiquitin ligase?E3?.The substrate specificity for ubiquitination is predominantly manipulated by E3 ligase.Until now,there are more than 600 E3 ligases reported,which can be divided into three families according to their protein sequence characteristics.the HECT family,the RING finger family and the RBR family.Among the RING type E3 ligase,the SCF complex has been extensively studied.The SCF complex is consist of the scaffold protein Cullin1,the RING finger protein Rbx1/2,the adaptor protein Skp1 and F-box protein.F-box proteins is consist of three major subfamily:FBXW,FBXL and FBXO subfamilies.These F-box proteins target a wide range of protein substrates and are involved in the regulation of cellular processes such as cell cycle,cell proliferation,apoptosis,angiogenesis,and metastasis.Skp2 belongs to the FBXL family and is one of the most widely studied F-box protein.Akt and Cdk2phosphorylate Skp2 at residues Ser72 and Ser64,and the phosphorylation of Skp2 on these residues inhibit the binding of Cdh1 to Skp2,thereby weakening Skp2ubiquitination and degradation,leading to increased stability of Skp2,triggering SCF E3 complex formation and activity.Importantly,it mediates the relocalization of Skp2to the cytoplasm,promoting cell migration and cell proliferation.I n addition,overexpression of Skp2 in mice leads to tumor progression.Moreover,Skp2 plays an important role in regulating cell senescence.Since the phosphorylation modification of Skp2 protein,especially the phosphorylation of Ser72 residue of Skp2 protein,plays an important role in regulating cell proliferation and aging.However,much literatures of this field are mainly focused on cell-level studies but not yet been verified in animal models.The 72^nd amino acid of Skp2 is S in higher-order mammals other than mice,of whose is a glycine?G?in instead and cannot be phosphorylated by Akt.We have for the first time established a point mutant Skp2-G72S mouse,which is also a humanized mouse of Skp2.Attempts were made to reveal the significant function of the phosphorylation modification at Ser72 of Skp2 in humanized mice model.In this study,we used the CRISPR-Cas9 technology to construct a site-directed mutant Skp2^G72S/G72S mouse.We found no significant difference in body size,body weight,and patholo gical phenotypes of important tissues and organs between Skp2^G72S/G72S mice compared with Skp2^WT/WT mice.There was also no significant change in glucose metabolism,the rate of cell proliferation,senescence,apoptosis,and cell cycle regulation were similar among the WT and Skp2^G72S/G72S72S/G72S MEFs while the invasion and migration of Skp2^G72S/G72S MEFs were enhanced;There was no significant change in the level of Skp2 phosphorylation and the stability of its downstream substrates such as p27,cyclin E,p21,FoxO1,and c-Myc between Skp2^G72S/G72S knock-in mice and Skp2^WT/WT mice.Our preliminary results showed that the Skp2 phosphorylation modification mechanism,which plays a key regulatory role at the cellular and molecular levels,does not play significant roles at the physiological level.To a certain extent,this study unravels physiological function of Skp2 and provides novel insights into study of humanized mice.Alternative splicing?AS?can generate multiple m RN As and proteins from a single precursor m RN A.The major types of alternative splicing includes exon skipping,exon exclusion,5' alternative splicing,3' alternative splicing,and intron release.Given that alternative splicing are capable of generating products with different or even antagonistic properties from a single locus,it can greatly expands the coding capacity of complex genomes,which is an important biological event that affects key cellular processes.Altered alternative splicing patterns are essential for normal development and cell differentiation,but those changes often cause or modify human disease,including cancer.CCAR1 is a perinuclear phosphorylated protein that regulates cell growth and apoptosis.Previous studies have found that SRSF5 binds at the 5' splice site of CCAR1,which alternatively splices CCAR1 to produce both full-length?CCAR1 long?and 15-22 exon deletion splicing transcripts?CCAR1 short?.Interestingly,our preliminary cytological experiments showed that knockdown of CCAR1 L resulted in increased cell proliferation and stronger colony formation ability,whereas knockdown of CCAR1 S resulted in enhanced apoptosis and slower colony formation.However,the mechanism of antagonistic biological function regulated by those two transcripts remains unclear.IP-MS is of increasing importance in identifying protein interactions,discriminating tissue-specific,network-based molecular functional clusters.A common use of IP-MS was to identify multiple protein-binding molecules for specific targets and to predict molecules in specific pathological and physiological processes.RNA-Seq play a pivotal role in analyzing changes of cell transcriptomes so that the existence and amount of RNA in b iological samples at a given time can be monitored.It also provide novel insights into investigating changes in other gene spliced transcripts,post-transcriptional modifications,gene fusions,mutations/SNPs,and gene expression over time,or differences in gene expression in different groups or treatments.To explore the mechanism of antagonistic function between CCAR1 L and CCAR1 S at physiological level,we constructed specific stable over-expression of the two isoforms in H1299 cells and investigated the protein-protein interactions of CCAR1 L group and CCAR1 S group by IP-MS techniques.Interestingly,the specific interacting proteins of CCAR1 L are mainly involved in regulation of apoptosis,autophagy,and responses to heat stress.In contrast,CCAR1 S specific interacting proteins are mainly concentrate on regulation of cell proliferation,cell growth and cell cycle.Further,we verified the authenticity of those selected CCAR1L/S specific binding proteins by IP-western tests.CCAR1 L was found to be specifically interacted with pro-apoptotic proteins such as RBM10,p14 ARF,and GSDMA,and CCAR1 S specifically binds to pro-proliferation proteins such as C ullin4 B,MAPK1 and hn RNPK.In addtion,we also performed RNA sequencing?RNA-seq?in CCAR1 L or CCAR1 S knockout cells to detect the transcriptome difference of CCAR1L/S.KEGG pathway analysis demonstrated that when CCAR1 L was knocked out,differentially expressed genes?DEGs?mainly existed in PI3K-Akt,c AMP and apoptotic pathways,whereas when CCAR1 S was knocked out,DEGs mainly existed in MAPK,TNF and Hippo pathways.We used RT-PCR test to verify the expression levels of selected CCAR1 L and CCAR1 S DEGs.We found that upregulated DEGs in CCAR1 L knockdown cell lines are STAT1,ATF3 and other genes that promote c ell growth,downregulated DEGs are TP53,AIP1 and Fox O1 that promote apoptosis.In contrast,in the CCAR1 S knockdown cell line,the upregulated DEGs are genes that mainly inhibits cell growth such as TGFBR3 and TNFSF9,whlie the downregulated DEGs are mainly the genes that promote cell proliferation such as PCNA and Myo9 A.O ur research for the first time revealed the mechanisms of different function of CCAR1 L and CCAR1 S in controlling cell fate,and gained a better understanding of the precise regulation of life activity by alternative splicing.