The Copper Ions The Gene Cuta Function Of Cum Rlim Inhibition Of Liver

Copper,an essential trace element,plays an important role in biological system.However, excessive copper is toxic and contributes to production of excessive reactive oxygen species (ROS),which results in the oxidant damages in the cell and genesis of human diseases. Mounting evidence is showing the relativity of copper with many human diseases,such as Alzheimer's disease,atherosclerosis,Creutzfeldt-Jakob(CDJ) disease and so on.Those proteins directly involved in the diseases genesis,such as the microtube related protein Tau, amyloid precursor protein APP and Prion proteins,can bind copper,and the interaction between those proteins and copper plays an important role in diseases.So study on the potential proteins associated with copper will be helpful to reveal the role copper plays in diseases.Human CUTA(copper tolerance A also named as cation tolerance protein A) is a homologous gene of CutA1 in Escherichia coli.Most CutA1 homologous protein have the capability of copper binding,and the CutA1 protein in Escherichia coli is involved in copper tolerance.So the sequence characterization of CUTA protein indicated that CUTA is a potential protein associated with copper.However,the function of CUTA gene is still unknown.In order to reveal the potential function of CUTA gene,we made a primary study on the tissue expression pattern of CUTA gene,the characterization of CUTA protein,and its relativity with copper.It was found that CUTA was constitutively expressed in human tissues and expressed with a high level in liver and prostate.The results also showed that CUTA protein concentrated in the perinuclear region,where it co-localized in a large part,but not completely,with mitochondria. CUTA is a potential mitochondrial protein with the structure of trimers proved by cross-linking with DSG(disuccinimidyl glutarate).In the study on the association of CUTA protein with copper,we found that CUTA protein has the strongest binding ability with Cu(â…¡) in the detected metals.In addition,the expression of CUTA protein was increased when the cells were exposed to high concentration of copper.Furthermore,CUTA could affect the sensitivity of the cells to the copper toxicity.Over-expression of CUTA seemed to sensitize the cells to copper toxicity.All of the studies indicated the strong association between CUTA and copper.In the study,we raised and purified CUTA specific antibody by immunizing the New Zealand rabbit.The native CUTA protein could be detected in the different cancer cell lines.The expression of CUTA protein detected in the 21 pares of liver cancer samples showed that there was no significant association between CUTA and liver cancer.However, CUTA was significantly up or down regulated in some.liver cancer samples.So it seemed that CUTA could be used as a candidate gene for typing the liver cancer.In addition,in an effort to identify HCC relevant genes,we used differential display and found RLIM(RING finger LIM domain-binding protein) was significantly down-regulated in HCC.RLIM is an E3 ubiquitin ligase with a RING finger.To investigate the role RLIM plays in HCC,we checked the effect of RLIM gene expression on colony formation,cell. proliferation and tumor growth.The results showed that:1.RLIM over expression in SK-Hepl significantly decreased the efficiency of colony formation by 75%.2.RLIM expression in SK-Hepl(stably tansfeeted with RLIM) significantly inhibited the cell proliferation.3.RLIM over-expression inhibited the tumor growth.This was done by replanting the stably transfeeted cells in nude mice,and then measuring the growth rate,size and weight of the tumor originated from the cancer cells.In general,we found and primarily proved that CUTA is a mitochondrial protein widely expressed in tissues,forms trimer in cell lysate.We also found that CUTA could bind copper and change the sensitivity of the cells to the copper toxicity,and the expression of CUTA was affected by copper concentration.So we considered the function of CUTA was associated with copper and CUTA maybe play a role in copper toxicity.We constructed two powerful tools for the research in CUTA gene.One is the CUTA stably transfected HeLa cells and the other is the CUTA specific antibody.In addition,we investigated the role of RLIM in HCC because RLIM was found to be significantly down-regulated in HCC.We found that over-expression of RLIM in SK-Hepl cells not only inhibited the colony formation and cell proliferation,but also inhibited the growth of tumor in nude mice.So it was considered that RLIM is a new potential tumor suppressor gene in HCC.