Human Copper Transporter Protein Acquires Cupric Ions From Human Serum Albumin

The amount of copper is the second largest of all the essential trace elements in human body.Although there is only 100-200 mg copper in normal adult male body,Cu is indispensable nutrient,and human can not live without Cu.Cu has an important influence to the development and function of blood circulation system,nervous system and the immune system,hair,skin and bone tissue and the brain,liver and heart organ.Moreover,in protein there are amount of copper affinity ligands,containing side chains ligands of histidine,methionine cysteine and so on,N atoms of amide peptide bonds between amino acid residues on backbone,free amino groups at N-terminal,which are likely to coordinate with copper ion.Copper ligand may affect the structure and function of proteins,and then regulate a series of biochemical processes necessary for life.Approximately 50%of the intestinal copper is absorpted by the intestinal epithelial cells,and then intestinal copper cross the basolateral membrane and enter the blood circulation by the pumping action of ATP7A.The uptake of copper is regulated by the copper transporter protein(CTR1)and divalent metal transporter protein(DMT1).After entering the blood,primarily binding with human serum albumin and a2-macro globulin,copper complexes are transported to the cells of various tissues.Cu is an indispensable catalytic and structural cofactor that drives a wide array of important biochemical processes that are essential for life.By coordinating to proteins with the assistance of a diverse spectrum of chemical ligands,including sulfur,oxygen and nitrogen,Cu confers changes in protein structure,catalytic activity and p rote inprotein interactions,thereby controlling an unexpectedly diverse series of biochemical and regulatory events.In this study,by using several analysis means containing UV-visible absorption spectroscopy,fluorescence spectroscopy,electrospray ionization mass spectrometry,X-ray absorption spectroscopy,electron paramagnetic resonance spectroscopy,surface plasmon resonance spectroscopy,we studied binding properties between CTR1-N-terminal region of the protein(CTR11-46)and Cu(?),the competition of CTR11-46and HSA for Cu(?),and the interactions between HSA and CTR11-46.The results showed the histidine-rich domain of CTR11-46 bind with multiple Cu(?)and were able to compete Cu(?)with HSA protein,which is different with that Cu(?)bind with the Methionine-rich domain.In addition,fluorescence spectroscopy and surface plasmon resonance spectroscopy results suggested that CTR1 N terminal acquires cupric ions from human serum albumin likely through the interactions between HSA and CTR11-46.Chapter 1 is a systematic review,including the importance of copper,copper and disease relationship,uptake and metabolism of copper,copper plasma distribution,copper ion transport proteins discovery,structure and function.The review is completed after researching literature about copper metabolism.Chapter 2 describes reactions of the extracellular hCTR1-N-terminal region with Cu(?)and Cu(?).The first 46 amino acids hCTR1-N-terminal domain is selected as the object of study,which includes major regional and histidine methionine area.During the experiment,UV-vis,XAFS,ESI-MS and EPR methods were used for testing the reaction.The results showed that,the reaction of hCtrl-N1-46 protein and copper interact with high affinity.And the second histidine sequence is important to Cu(?)binding.In the binding Cu(?)and hCtrl-N terminal protein forms square planar coordination.In chapter 3,we study the reaction between the extracellular hCTRl-N-terminal region and Cu(?)HSA complex.In first,we mixed HSA and Cu(?)in 4:3 ratio sufficient to give HSA and Cu(II)complex,and then through a variety of means we detect HSA and Cu(?)complex to know whether CTR1 N terminal acquires cupric ions from human serum albumin.During the experiment,we used fluorescence,EPR,ESI-MS and SPR detection methods to detect the reaction proceeds.The results showed that,the transport of Cu(?)between hCtr1-N1-46 protein and HSA protein is reversible.HSA protein can be used as a carrier copper to provide Cu(?)to hCTR1-N-terminal region.Copper transfer process is likely depedent on the protein-protein interactions,and Cu(?)may also on enhanced interaction between these two proteins by connecting the histidine residues of hCTR1-N-terminal region and HSA.