The Copper Complex-binding Proteome In Rat Hepatocytes

Since the discover of the anticancer activity of cis-platinum in 1960's,the research of metal-based medicines have attracted great attention and become one of the hottest topic in inorganic biochemistry.A large number of metal complexes have been synthesized and tested for their various biological activities including antitumour,antibacterial,antimalarial,antiviral and so on.However,severe toxic side effects and acquired resistance phenomena trouble people all the times.Therefore,increasing selectivity and reducing toxicity side effects of metal-based medicines is promising but a challenge.Copper,being an essential element,is expected to be less toxic than non-essential metals such as platinum,and copper complexes have thus attracted great attention as potential chemotherapeutic and diagnositic agents.Recent researches indicate that copper complexes are able to induce apoptosis of cancer cells by inhibiting proteasome.The developing of proteomics provides a possibility to design medicines targeting special protein,which is anticipated to be high selectivity and low toxicity.A growing number of special proteins which are closely related to serious diseases have demonstrated to be inhibited by copper complexes,such as isocitrate lyase,human topoisomerase I and II,glycogen synthase kinase 3?,urease,ribonuclease A,protein tyrosine phosphatases and so on.These researches stimulate our curiosity to guess that there may be a copper complex-binding proteome that potently interacts with copper complexes and then influences cellular metabolism in organism.However,how do they interact with copper complexes and influence organism metabolism? Whether the copper complexes which are capable of selectively perturbing the function of individual or very few proteins can be screened? This is of significance to understand the physiology and pharmacology of copper complex and screen the copper-based medicines with high selectivity and low toxicity.In this paper,based on the concept of ?The copper complex-binding proteomics? proposed by our research group,we explored the copper complex-binding proteome and the copper complexes which were selectively bonded to one or several limited specific proteins were screened.The research would provide significant evidence and scientific data for screening the copper-based medicines with high selectivity and low toxicity targeting special protein and understanding the physiology and pharmacology of copper complexes.The main results are listed as follow.(1)We applied a copper chelating ion-immobilized affinity chromatography column(Cu-IMAC)and mass spectrometry to separate and identify Cu-binding proteins in rat hepatocytes.A total of 97 Cu-binding proteins were subsequently identified and higher abundance proteins were further purified by SP-and(or)Q-Sepharose Fast Flow column.Only five Cu-binding proteins were purified and mass spectral identification demonstrated that they were aspartate aminotransferase(AST),malate dehydrogenase(MDH),catalase(CAT),calreticulin(CRT)and albumin(Alb),respectively.(2)The interaction between 11 copper complexes and three Cu-binding proteins(AST,MDH and CAT)was investigated by enzyme activity inhibition assays and Fluorescence spectroscopy.The results demonstrated that mononuclear Schiff-based copper complexes exhibited potent inhibition against AST,but weaker inhibition against MDH and CAT.In the complexes 1-11,the Schiff-based copper complexes 6 potently inhibited AST with the IC50 value of 3.6 ?M.Especially,copper complex 6 was able to enter into cells and further inhibit intracellular AST activity.The results suggested that the structures of copper complexes influenced their potency and selectivity on the inhibition of different enzymes,and properly designing the structures of copper complexes might result in the screening of potent and selective copper complex inhibitors.(3)Eleven copper complexes with Schiff bases were synthesized and well characterized.The interaction between these complexes and five Cu-binding proteins(MDH,AST,CAT,PTP1 B and TCPTP)was investigated.Copper complexes 12,14 and 15 showed potent inhibition and certain selectivity against TCPTP,and the copper complex 13 selectively inhibited PTP1 B.We further investigated the interaction of the copper complexes 12-15 with intracellular PTP1 B,TCPTP and AST.The results demonstrated that the complex 14 and 15 greatly improved the phosphorylation of the TCPTP substrates,showing complex 14 and 15 could enter into cells and further inhibit intracellular TCPTP activity.However,complex 14 and 15 almost had no effect on intracellular PTP1 B and AST activity,suggesting complex 14 and 15 had certain selectivity against intracellular TCPTP.All of the results showed structures of copper complexes and Cu-binding proteins together influenced their binding ability and selectivity,and properly modifying the organic ligand moieties on copper complexes might result in the screening of potent and selective copper-based inhibitors.(4)We also designed,synthesized and well characterized thirteen other metal complexes(eight zinc complexes,a dioxidovanadium(V)complex and four terbium complexes).The interaction between these thirteen complexes and five Cu-binding proteins(MDH,AST,CAT,PTP1 B and TCPTP)was investigated.The enzyme activity inhibition assays showed the complex 23-35 almost had no inhibition against MDH,AST and CAT,the znic complex 23 exhibited strong inhibition and cetain selectivity against TCPTP and the dioxidovanadium complex 31 selectively inhibited PTP1 B.Western blotting assays suggested complex 31 effectively increased the phosphorylation of the PTP1 B substrates,especially the phosphorylation of IR/IGF 1R and IRS-1,suggesting complex 31 could enter into cells and further inhibit intracellular PTP1 B activity.MTT assays showed the complex 31 exhibited lower cytotoxicity than the positive control VOSO4.The results above demonstrated that Schiff-based zinc complexes and dioxidovanadium(V)complex,as protein tyrosine phosphatase inhibitors,might have lower cytotoxicity than copper complexes.And the results also indicated that the complex 31 might be a promising candidate for novel anti-diabetic drug development.All of the results above demonstrated that there did exist a copper complex-binding proteome that could strongly inteact with copper complexes and influence cellular metabolism.The structures of copper complexes influenced their binding ability and selectivity on different Cu-binding proteins.When complexes with different metals but the same ligand,their binding ability and selectivity were also different.The structures of complexes and copper-binding proteins together influenced their binding ability and selectivity.