The Structural And Functional Reasearch Of Iml3-Chl4 Complex And Purification Of Membrane Protein NPC1

The exact reproduction of the genome and the precise distribution to the offspring cells are the basis of every life for survival and continuation. According to the type, cell division can be divided into mitosis and meiosis. However, regardless of which cell division, spindle attaching to the specific area in the chromosome is needed to pull the chromosome moving to the cell poles. And this particular region is named by centromere, the macromolecule complex--kinetochore assembled at this spot, guide the separation of chromosome.According to the location, the kinetochore components can be divided into the inner layer?outer layer and the regulatory proteins. Currently,we know that the inner layer components consisting of 16 CCAN proteins (in mammalian) are closely linked to the centromere and recruit other outer components. As the core components of the CCAN complex, CENP-L/CENP-N kinetochore localization is crucial and upstream in the recruitment of other kinetochore components into chromosome.However, the exact mechanism followed by CENP-L/CENP-N recruited into the centromere is not very thorough.In this work, we targeted Iml3/Chl4 complex for structural and functional studies and determined the crystal structures of the Iml3 alone and bound to Chl4378-458. The structure show that Iml3 is a completely novel structural model,which consisting of two separate domains: incomplete ? barrel and the twisted plane. The intra-molecular hydrophobic interactions maintain the stability of the entire structure. Although there is only one Iml3 in an asymmetric unit, the protein forms a symmetrical dimer through the antiparallel interaction of the ?10 and ?10'strands from two neighbouring Iml3 molecules in the crystal latticeit through the main chain hydrogen bonds on ? strand. Well, when Chl4 is present, the interface of Iml3 homodimer is destroyed, and Iml3 binds the C-ternimal of Chl4 in a more stable manner. The Iml3-Chl4 complex structure show that the C-ternimal of Chl4 extend the ? sheet of 11 ? strands of Iml3 in an antiparallel manner by forming 4? strands. And many paris of hydrogen bonds and salt bridges contribute to ensuring the stability and specificity of the complex.Literature information shows that Chl4 was recruited by direct interaction with centromeric Cse4 nucleosome(CENP-A nucleosome in mammalian).So is there any other mechanism to promote the process, except the N-termimal of Chl4 associating with the Mif2(CENP-C in mammalian)? Our EMSA and FPA results show that only Iml3-Chl4 complex have the non-specific ability to bind dsDNA, whereas Iml3 and Chl4 alone haven't. Mutation experiments show that this non-specific ability from the positively electricity rich region in the ?-sheet of Iml3-Chl4 complex. The functional test from Stephen et al group: disrupting the dimerization interface of Iml3-Chl4 complex decrease the accuracy of chromosome segregation and the sporulation efficiencies directly. Together with our mutation results, we speculate the destruction of the dimeric interface leads to the destruction of the basic residue-rich region of the ?-sheet inner surface formed by the Iml3-Chl4 complex,thus the mutations lose the ability to bind dsDNA directly, and eventually affects the separation of chromosomes.As a derivative of cyclopentane and phenanthrene, Cholesterol is widely found in the brain and nerve tissues of animals, and plays an important role in the membrane structure of animals. However, as a lipid hormones, the soluble proteins and the membrane proteins are required to participate the transport and signal transduction of cholesterol in mammalian cells.In addition to the de novo synthesis in mammalian cells, there is also a way existing which involves the uptake of cholesterol: Low density lipoproteins (LDL)bind to LDL-receptors (LDL-R) and are internalized by clathrin-mediated endocytosis into early/sorting endosomes(SE), where LDL dissociates from the LDL-R, owing to the acidic pH. The LDL is delivered to late endosomes (LE) by maturation of the SE, and hydrolyzed to free cholesterol by lysosomal acid lipase(LAL). The NPC2/NPC1, sterol transport protein, efflux free cholesterol out of the LE/LY. In NPC1/NPC2 mutant cells, free cholesterol is unable to egress from LE/LY, resulting in accumulation. In human beings, this disease is called Niemann-Pick type C (NPC) disease. So NPC1 is an important transporter in sterol transport. In recent study, NPC1 is also identified as a necessary entry receptor for the filoviruses entering host cells. However, the exact transporter and receptor mechanisms followed by NPC1 are not very clear.In this study, we cloned 50 homologies of NPC1 in different species, and screened TlNPC1 (Thermomyces lanuginosus) which can express stable enough in Pichia expression system. And we screened the constructs, detergent, LCP lipids,and did the methylation, deglycosylation, enzyme digestion, and also tried Cryo-EM,but we failed resolving the structure in the end.