The Molecular Mechanism Of Hippo Signaling Pathway Regulated By Deubiquitinase CYLD

Broke-spiegler syndrome(BSS)is a rare disease,the lesions always occur in the skin and skin appendages.Most patients develop tumors on the head and neck in early adulthood.The syndrome usually develops into tumors,spiral adenomas,multiple trichoepitheliomas,and cylindrical tumors are more common.These tumors are usually benign,but they tend to be larger and larger over time,leading to disfiguring and impinged on the function of the facial features.There are reports made clear that CYLD mutate can lead to cylindrical tumor,but the mechanism about how to regulate is not clear..CYLD gene is a tumor suppressor.The deletion or mutation of this gene will lead to the occurrence of cylindrical tumors.Meanwhile,low expression of CYLD has also been found in other tumors,such as liver cancer,breast cancer and cervical cancer.The CYLD protein encoded by this gene is a member of ubiquitin-specific proteases(USP),and has deubiquitinase activity.Most of the existing studies have found that CYLD can be recognized by the interaction with substrate protein,and the ubiquitin chain connected by lysine 63(K63)on the substrate protein can be removed to regulate the function of substrate protein and participate in the regulation and transduction of various signaling pathways,such as NF-κB,JNK,Wnt and other signaling pathways.Since the deubiquitination modification of the substrate protein by CYLD is achieved through interaction,we hypothesized that the point mutation or truncated mutation of CYLD would lead to the gain or loss of certain interactions,leading to the occurrence of tumor.Bio ID is a recently developed method,it can identify the proteins of interest in living cells near the protein,protein of interest and Biotin connection Bir A enzyme(Bir A *)mutation forms the fusion protein was formed by the fusion,Bir A * can be mixed with Biotin its proximal protein,regardless of the proximal protein is directly or indirectly,interact with fusion protein,or just in the same subcellular neighborhood,The covalent addition of biotin enables the labeled protein to be purified from cell extract according to its affinity with streptomycin and finally identified by mass spectrometry.So the first thing we use Bio ID technology for wild type,mutant,truncated CYLD protein of adjacent protein body,again through the analysis of the biological differences between protein localization,function and so on,finally in the interaction of molecules selected three Hippo pathway related molecules: YAP(yes associated protein),LIMD1(LIM domain-containing protein 1),AMOT)protein(angiomotin).Hippo pathway is an important signaling pathway to control the size of tissues and organs,regulate cell proliferation and apoptosis,this pathway mainly includes kinase cascade,transcription coactivator and DNA binding factor.The kinase cascade controls the activity of the transcription coactivator YAP.When the Hippo pathway is activated,the upstream kinase is activated and the YAP molecule is phosphorylated through the cascade reaction.The phosphorylated YAP is retained in the cytoplasm and partially degraded by ubiquitination.However,after the Hippo pathway is inactivated,YAP cannot be phosphorylated and enters into the nucleus,then interacts with intracellular TEAD(Transcription enhancer factor)family members to activate gene expression that promotes cell proliferation and growth.Both AMOT and LIMD1 are upstream regulatory molecules of Hippo pathway.It has been reported in drosophila that CYLD binds and co-locates with Hippo kinase in the cytoplasm and reduces its activity by restricting the phosphorylation of the kinase,thereby increasing the accumulation of Yki/YAP,a key downstream effector of Hippo pathway,in the nucleus and promoting cell proliferation.Therefore,we wondered whether CYLD could regulate the activity of Hippo pathway through its function of deubiquitination modification,thus inhibiting the occurrence of tumor.First,we verified whether the localization and function of CYLD fusion protein carrying Bir A* were different from that of CYLD protein.We found that after carrying Bir A*,the localization of CYLD did not change,but its deubiquitination function would be affected.Then to select the three interactions between molecules interact,validated,CYLD protein can only interact with LIMD1 to ubiquitin and modification,the LIMD1 can regulate the Hippo pathway,through the experiment we found in HEK293(human embryonic kidney cell 293)cells,the overexpression of CYLD of YAP phosphorylation levels and shall be no obvious change,but on low CYLD will make YAP phosphorylation levels drop,check and ratify a increased,YAP downstream target genes levels rise at the same time,This indicates that knocking down CYLD can lead to decreased Hippo pathway activity.All in all,this is the first study found that the CYLD Hippo pathway related molecules interact with,and can go to ubiquitin modification,and CYLD can regulate the activity of the Hippo pathway,according to the existing research results and we guess CYLD may through to the LIMD1 to ubiquitin modifiers to regulating the Hippo pathway activity,thus inhibiting the occurrence of tumor.