Screening And Identification Of The Proteins Interacting To Human Augmenter Of Liver Regeneration (hALR)

The augmenter of liver regeneration (ALR) is known as a vital control factor in regulating the regeneration of hepatocytes. Human ALR (hALR) is present in a homodimer with an apparent molecular weight of 15 ku which composes with 125 amino acids. It is heat-resistant against to 70 ℃ for 30min. The results from the experiments for the mechanisms involving in liver regeneration demonstrated that hALR could stimulate the proliferation of hepatocytes, prevent the disruption of rough endoplasmic reticulum, mitochondrion and other organelles, and inhibit the lymphopoiesis as well. Animal experiments showed it was highly effective in treating acute liver dysfunction caused by various factors. Nevertheless, the detailed mechanisms of ALR is not yet fully clear for us up-to-data. It is necessary to seek for the interacting molecules to ALR in order to evaluate the signaling pathways elicited by ALR.This thesis describes the screening and identification of related proteins which probably interact to hALR by using a BacterioMatch Two-Hybrid System. 32 positive clones were obtained from the human liver cDNA library and they were sequenced as the complete or partial encoding regions for human glycine-N-acyltransferase (GAT), colony stimulating factor (CSF), hemopexin, complement component C3, α-acidic glycoprotein 2, plasminogen, SERPIN F1 derived from epidermal pigment cells, albumin, QD8159 mitochondrion, haplotype As8D mitochondrion, chromosome 11q clone CMB9-78H16, and genomic cDNA clone IMAGE:353568. After reviewing the related information from the literatures and functional analyzing the relationships to ALR, GAT, the most possible candidate for interacting to ALR was cloned, expressed and the interactions between them was studied with a Pull-down test.GAT was originally purified from adult liver and was found to be a monomer. Two transcript variants encoding different isoforms have been found. Isoform a wascomposed with 296 amino acids with a molecular weight of 30 Ku and a p1 at 6.8, while isoform b with 163 amino acids and molecular weight is 18.5Ku. The 3'-coding and 3'UTR are different between these two isoforms, and isoform b has a shorted and distinct C-terminus. GAT is sensitive to the treatment by 100 mmol/L NaCl, KC1 and K3PO4, the enzymatic activity reduce to 47%, 21 % and 32%, respectively. However, GAT is resistant to the treatment under 4 ℃ for 72 h, 25 ℃ for 4 h, and 37 ℃ for 1 h. GAT catalyzes the transfer of an acyl group from acyl-CoA to the amino group of glycine in acyl-CoA metabolism, It conjugates glycine with acyl-CoA substrates in the mitochondria, and shares with the activity of benzoyl CoA, salicyl CoA, isovaleryl CoA and octanoyl as well, and therefore is thought to be important in the detoxification of endogenous and xenobiotic chemicals. Research indicate that up to age of 7 months, children have only 5% to 40% of liver GAT-specific activity, whereas the peak activity is achieved at 18 months and remains constant until age of 40 years. The delayed development of GAT in children may thus compromise the detoxification of various drugs and xenobiotics. The results from tissue microarray indicated GAT was distributed widely in brain, lungs, liver, kidneys and muscles, the highest level was detected in kidneys and liver.The GAT positive clones screened out by the bacterial two hybrid system was PCR amplified, and the product was inserted into pcDNA3.1. The plasmid was transformed into E.coli DH5a and was further screened by blue-white plaque formation and validated by sequencing.GST-hALR was fusing expressed and cross-linked to Glutathione Sepharose 4B (GST4B) to prepare the GST4B-GST-hALR pellets. 35S-labeled GAT was expressed with Promega's TNT fast expression system. GST4B-GST-hALR and 35S-labeled GAT were co-incubated for Pull-down test. The result from the test showed that there was a significant interaction between hALR and GAT.In conclusion, GAT, an interacting protein to hALR was screened out from a bacterial two hybrid system and validated by a Pull-down test. The present investigation provided the clues in further evaluating the detailed mechanisms of the actions of hALR in liver regeneration. The domains or the key residues involved the...