The Structure And Function Of A? And C99

Alzheimer's disease(AD)is a devastating neurodegenerative disease,which seriously endangering the health and life quality of the elders.Until to now,the pathogenesis of the disease has not yet fully understood.Mutations of amyloid precursor protein(APP)and miscleavage of C99(C-terminal 99-amino acid fragment)by ?-secretase leading to the occurrence of AD has been widely confirmed by gene,cell,and animal experiments.All these evidence makes the amyloid hypothesis more and more recognized and accepted by the scientific community.In spite of the pivotal roles in the pathogenesis of AD,the relationship between ?-secretase and its substrates has not been comprehensively elucidated.The current clinical treatments for AD do not have good effects.A correct understanding of the relationship between ?-secretase and C99 substrate together with the high-resolution structure of C99 and A? with or without ?-secretase will help to unveil the pathogenesis of AD and shed the light on the drug development.In order to investigate the pathogenesis of AD,we focused on the analysis of the relationship between the ?-secretase and the AD-associated C99 mutant substrates and dedicated determining structures of the C99 and A? with or without ?-secretase to interpret the molecular pathogenic mechanism of AD.A rapid and sensitive Epsilon-Cleavage assay was established to study the cleavage efficiency of C99 mutant substrates by ?-secretase.After analyzing the efficiency and the major ?-secretase products,the A?40 and A?42 peptides,we found that all the familial AD(FAD)-linked mutations on C99 increased APP resistance to ?-secretase cleavage and the A?42/A?40 ratio.And also we found the importance of the C-terminus of the transmembrane region is much higher than that of other regions.Through the N-terminal and C-terminal truncations,we identified that the C99 fragment(E22-K55)is the minimum substrate that is required for efficient cleavage by ?-secretase in cells.Mutagenesis on the minimum substrate demonstrated that the negative charge at the N-terminus and the positive charge cluster at the C-terminus played an important role in the substrate recognition and cleavage by ?-secretase.At the same time,the study of the transmembrane domain(TMD)-mediated dimerization indicated a new dimerization motif-TVIV for C99,which happens to be a hot spot for FAD-linked mutations.Further analysis showed that the ?-secretase cleavage efficiency and A? production was correlated with TMD-mediated dimerization.In combination with the Epsilon-Cleavage assay,the minimum substrate study,and the transmembrane region dimerization investigation,we identify the determinant for initial ?-secretase cleavage:(i)a transferable extracellular determinant that differs between C99 and Notch,and which includes negative charge in the case of C99,(ii)the amino acid sequence of the C-terminal half of the transmembrane helix,(iii)an invariant lysine or arginine at the intracellular membrane border,and(iv)a positive charge cluster that includes the invariant lysine/arginine.So we propose a recognition-binding-cleavage model.First,the N-terminal residues D1-A21 and the C-terminal residues Q56-N99 are not required for substrate recognition.Then its C-terminal lipid membrane-bound region rivets the substrate to the ?-secretase and promotes the correct orientation.After that,C-terminal half TMD,N-terminal complementary electrostatic interactions together with the K53 interaction with the ?-secretase activity centers laid the foundation for the ?-secretase cleavage.Meanwhile,we tried to express and purify the high-quality C99 protein and A? peptides.We used site-directed mutagenesis,protein fusion,truncation,disulfide bond,antibody co-expression etc.to optimize the expression.We tried to crystallize the purified C99 protein,A? peptides,and chemical synthetic A? peptides.All these structural biology and biochemical biology will shed insight into the pathogenesis of Alzheimer's disease,and the therapies for neurodegenerative diseases.