Generation And Phenotype Analysis Of The Knockout Mice Model Of Deubiquitinase OTUD4

Protein ubiquitination is a reversible post-translational modification and its functions are widely implicated in multiple physiological and pathological processes,including protein degradation,signal transduction,epigenetic modification and cellular localization.Deubiquitinase is an eraser in the editing of ubiquitination.Understanding the functions and regulatory mechanisms of deubiquitinating enzymes is in the cutting edge of the studies.Currently identified deubiquitinating enzymes mainly include UBP/USP family,UCH family,OTU family,JAMM family,MJD family and MCPIP family.Among them,the OTU family,as the second largest family of deubiquitinating enzymes,plays pivotal roles in DNA damage repair,inflammatory immune response,maintenance of metabolic homeostasis,and tumor progression.There are sixteen members with catalytic activity in human OTU family,which are composed of three categories:Otubains,A20-like OTUs and OTUDs.OTUD4 is a member of the OTUDs subfamily.Currently reported ubiquitin chain modification types of OTUD4 include K48 ubiquitin chain and K63 ubiquitin chain.Among them,OTUD4can activate its deubiquitinase activity through its own phosphorylation,thereby removing the K63 ubiquitin chain of MyD88,a key linker of the TLR signaling pathway,thus maintaining homeostasis in innate immunity.Meanwhile,OTUD4 can also regulate the protein stability of DNA demethylases ALKBH2 and ALKBH3 in an enzyme-independent manner,which in turn promotes the repair of DNA alkylation damage.The widely used model animals in investigation of physiological function of OTUD4 are zebrafish and Drosophila.Mutations of OTUD4 are reported in patients with ataxia and hypogonadotropic hypogonadism.Whether the Otud4 gene mutation is the main cause of this syndrome awaits further examinations.To further explore the physiological functions of OTUD4,we constructed Otud4knockout mice model by cre-loxp strategy and analyzed phenotype of the Otud4 knockout mice.Our main findings are as follows:Firstly,homozygous Otud4^-/-were born at the expected Mendelian frequency.Moreover,Otud4^-/-deficient mice,both male and female,were viable and fertile and there were no morphological difference between Otud4^-/-and wild-type littermates.Secondly,there were no obvious difference in weight of brain,thymus,lung,heart,liver,kidney and spleen organs of Otud4^-/-mice compared with that of the wild-type littermates by dissection.Thirdly,Histopathological analysis of those organs did not reveal any obvious defects in the homozygous mutants.Fourthly,Blood analysis and blood glucose monitoring of Otud4^-/-and wild-type littermates revealed that Otud4^-/-mice also had normal hemogram and blood glucose levels.Further in depth analysis are warrant.In summary,we used cre-loxp strategy to construct knockout mice model of the deubiquitinase OTUD4 and preliminary analyses of its phenotype were conducted.Our study found that after Otud4 defect,Otud4^-/-mice were viable and did not have any gross phenotype,and they were born at sub-Mendelian ratios.The successful construction of the Otud4 knockout mice model laid the foundation for further exploration of the physiological functions of the deubiquitinating enzyme OTUD4.Macrophages are important immune cells in vivo.They are distributed in various tissues of the body and play an important role in the body's immune responses.Clinical,pathological,and physiological studies supported the notion that macrophages are a special subtype of cells which display heterogeneity and plasticity in terms of both phenotype and function.Macrophage polarized activation and inactivation has profound effects on immune and inflammatory responses with several major pathways being elucidated in the past few years.In general,two well established polarized phenotypes are often classified on a continuum in which M1 macrophages represent the classically activated macrophages on a pro-inflammatory state whereas M2 macrophages represent the alternatively activated macrophages on a contradistinctive anti-inflammatory state.M1 macrophages are generally activated by interferon-??IFN-??or other microbial products?e.g.LPS?to promote Th1 immune response,while M2 macrophages are usually activated by the IL-4/IL-13 immune complex,IL-10,or TGF-? to exhibit an immunosuppressive phenotype.We and other studies have reported the pleckstrin homology?PH?domain-containing protein CKIP-1?Casein Kinase 2 Interacting Protein-1,also known as PLEKHO1?plays important roles in the regulation of cell proliferation,cell differentiation and cell apoptosis.In this study,we identify the Casein Kinase 2 Interacting Protein 1?CKIP-1?as a molecular toggle manipulating macrophage speciation.We provided multiple lines of evidence to show that CKIP-1 could be regulated by both M1 and M2 cytokines and in turn could simultaneously regulate M1 and M2 polarization through a negative feedback loop.CKIP-1 expression was strongly induced by pro-inflammatory M1 stimuli?LPS and IFN-??and robustly suppressed by M2 stimuli?IL-4 and IL-13?in human and murine macrophage.In addition,our study found that myeloid CKIP-1 deficiency reduce the TPA-induced cutaneous inflammation in vivo.In summary,our study shows that CKIP-1 is an important molecule involved in macrophage polarization.