Molecular Cloning And Authenticating And Characteral Studying Of Metallothionein-3-Interacting Protein(s)

Metallothionein-3 (MT-3), originally termed growth inhibitory factor (GIF), is a brain-specific member of the metallothionein (MT) family that bears distinct physiological functions. Besides the multiple functions shared by other MT isoforms, such as radicals scavenging, detoxification of heavy metals and participation in the metabolism of essential metals etc, MT-3 uniquely exhibits the growth inhibitory activity on in vitro cultured neuron cells when supplemented with Alzheimer's brain extracts. It is likely to be implicated in Alzheimer's disease. However, the underlying molecular mechanism of MT-3 responsible for its neuronal inhibitory activity remains unknown. To dissect the elusive functional mechanism of MT-3 and elucidate the possible relationship between MT-3 and Alzheimer's disease, we attempt to employ the yeast two-hybrid system to screen for MT-3-interacting protein(s) and find that MT-3, instead of MT-1 specifically interacts with the nuclear dUTPase.Then the nuclear dUTPase protein is expressed in E.coli by GST system, it is the first time to study the biological character of MT-3 and its interacting proteins on protein lever.The yeast two-hybrid system is a newly established technique of molecular biology that can sensitively detect protein-protein interaction in vivo. In this research, two bait plasmids have been used for library screening. Baitl plasmid (pHyblex-MT-3) is constructed by cloning MT-3 cDNA directly in frame with LexA in plasmid pHyblex. Using baitl, a screen of 4.6 106 transformants of a matched human brain cDNA library yields twelve strong double positive colonies (IacZ+. His+). Library plasmids are rescued from each of these double positive yeast colonies and used to eliminate the false positives by recotransforming with bait plasmid. After sequencing the nucleotide of the putative positive plasmids and searching in webs of NCBI (a NIH-founded Webpage) for homologues, we totally obtain four library plasmids of which inserts encode fragments of four known genes and one of these is the part of nuclear dUTPase protein sequence.Of the preceding four positive interaction protein fragments, we focus on the nuclear dUTPase based on an analysis. In order to test whether the full-leng nuclear dUTPase protein can interact with MT-3, cDNAs of the nuclear dUTPase is amplified by PCR usingtotal cDNAs from human brain as template, respectively. As shown by yeast two-hybrid system experiments, the full-length nuclear dUTPase specifically interacts with MT-3, but not with MT-1. In a separate set of experiments, we also subclone the full-length nuclear dUTPase cDNA into the prokaryotic fusion expression vector pGEX-4T-1 and conduct a primary research on the expression and purification of nuclear dUTPase protein in E.coli BL21. Also a method to quantify the dUTPase activity is established.To ultimately verify the specific MT-3-dUTPase interaction in other biochemical system, MT-1 and MT-3 are constructed in the hemagglutinin (HA)-tagging vector pSV-HA that can generate high level of HA-tagged MT-1 and MT-3 proteins in mammalian cell, respectively. The nuclear dUTPase is subcloned into another epitope-tagging vector pFlag-CMV-2 that can express high level of Flag-tagged nuclear dUTPase protein in mammalian cell. Mammalian 293 cells are cotransfected with the epitope-tagged bait and prey constructs. The final results from coimmunoprecipitation and Western blotting experiments confirm that nuclear dUTPase binds to MT-3 with specificity in 293 cell, but not to MT-1.To study the effect of the nuclear dUTPase and G-protein RabSa and the growth inhibitory factor(GIF) on the neuron cell, PC12 (pheochromocytoma) is used for the neuron model. The gene of nuclear dUTPase and Rab3a and MT-3 are cloned into pFlag-CMV-2 and pSV-HA. The MT-3/pFlag-CMV-2 and dUTPase(or Rab3a)/pSV-HA genes are transferred into PC 12 with the lipofectamine, at the same time the MT-1/pFlag-CMV-2 and dUTPase(or Rab3a)/pSV-HA genes are transferred into PC12 for cotrol. After cultivating, the cells1 growing status a...