The Discovery Of New Substrates Of ?-Secretase And The Mechanism Of Proteolysis Of The AXL Receptor Tyrosine Kinase

The intramembrane proteolysis is a complex and important event in biology,and the processing of ?-secretase substrate belongs to the regulated intramembrane proteolysis(RIP).Many findings has showed that the ?-secretase substrates are closely linked with the occurrence of various diseases,such as the amyloid precursor protein APP could impact on the development of Alzheimer's disease,and the NOTCH receptor is associated with development and progression of many tumors.Therefore,to study the regulated intramembrane proteolysis mediated by ?-secretase is a key issue for the biological functions of transmembrane protein,and to uncover the novel substrates and their related biological characteristics may enrich the fucntion understanding of ?-secretase and facilitate the mechanisms of related diseases.Firstly,we employed the biochemical approaches to study the amino acids near the C-terminal of transmembrane of APP-C99,and find that the basic-amino-acids-motif is essential to the cleavage mediated by ?-secretase.Based on this structural information with similarity,we get the whole possible ?-secretase substrate pool of human type I transmembrane proteins through the bioinformatics analysis,i.e,56 known and 214 possible substrates.We initially use the biochemincal experiments to validate that LRRN1,CD33,ACE2 and AXL are the novel substrataes of ?-secretase.Then we focus on the cleavage characteristics of AXL that includes substrate validation in vitro,the cleavage studies in cancer cell line and investigations its' role in drug resistance of non-small cell lung cancer.In order to study the AXL to be the candidacy of ?-secretase,we combine the AXL overexpression with small pharmacological molecules to validate that AXL is sequentially cleaved by ?-secretase and ?-secretase.The cleavage of AXL is dependent on the presenilin as evidenced from the presenilin-defecient MEF cells.The in vitro ?-secretase activity assay was performed with the recombinant AXL protein and suggests the AXL is directly cleaved by ?-secretase.The specific AXL inhibitor or Gas6 treatment,and the kinase dead mutant of AXL prove the AXL cleavage is negatively regulated by AXL phosphorylation,which also indicates that the AXL cleavage is not dependent on its' phosphorylation,but the non-phosphorylated form is more prone to subject ?-secretase cleavage.In addition,experiments with small pharmacological molecules or transiently transfected with the ?-secretase(ADAM10 and TACE)to show the AXL is primarily cleaved by ADAM10.Meanwhile,we use the biochemical assay to separate the cytoplasmic and nuclear factions,and the EGFP-tracing method to respectively show that AXL-ICD could translocate into nucleus.The deletion or mutation study show that the AXL-ICD translocalizes into nucleus depend on a resembled nuclear localization signal that lie to its N-terminus containing a stretch of basic amino acids.The luciferase reporter system was applied to demonstrate AXL-ICD could exert a cellular transcriptional repression activity.These evidences suggest that the nuclear localized AXL-ICD maybe have an important regulatory role on the cell or organism.In addition,the AXL cleavage mediated by ?-and ?-secretase ubiquitously exsists in many types of tumor cells and lung cancer tissue,and the cleavage products of AXL-ICD localized into the nucleus.The non small cell lung cancer cell line HCC827 stable expressing AXL was resistant to erlotinib,and the AXL also subjects to the sequential ?-and ?-secretase cleavage with the cleavage products AXL-ICD translocaled into nucleus.However,the ?-secretase-uncleavable AXL was showed to increase the drug resistance to erlotinib more than AXL-WT.These results suggest that the secretase-mediated processing of AXL,i.e.,AXL sequentially cleavaed by ?-and ?-secretase to generate AXL-ICD,exerts as an anti-drug resistance machenism.To summary,this thesis has many findings: 1)discovered a global potential pool of ?-secretase substrates and validated several substrates,e.g.LRRN1,CD33,ACE2,and AXL;2)AXL is sequentially cleaved by ?-and ?-secretase,which independent of its phosphorylation;3)the products of AXL cleaved by ?-secretase,AXL-ICD,could translocate into the nucleus depend on its N-terminal resembled nuclear location signal and exerts transcriptional repression activity on cells;4)elucidate the secretase-mediated AXL processing in tumor cells and this processing could alleviate the EGFR-TKI drug resistance in non-small lung cancer.Altogether,this study characterizes a unique proteolysis mediated by ?-and ?-secretase,and thus provides theoretical basis for cancer drug resistance and novel strategy to cancer therapy.