Browsing Theses and Dissertations School of Pharmacy by Subject "Zinc Fingers"
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CPSF30:A Zinc Finger Protein With An Iron-Sulfur SiteZinc finger proteins (ZF's) are a class of proteins that utilize zinc for structural purposes. These metalloregulatory proteins perform a variety of functions ranging from the modulation of gene expression through interactions with DNA and RNA to mediating protein-protein interactions. ZF'-s are characterized by the presence of one or more domains that contain a combination of four cysteines and/or histidine residues that serve as ligands to coordinate zinc. Coordination of zinc to the ligands cause the ZF domain to fold into a three-- dimensional structure and become functional. Based on the number of cysteines and histidine residues in the ZF domains, it is classified as either a classical or a non-classical ZF. Within the non-classical ZF's, there are 14 distinct classes. One of the classes of non-classical ZF's is the Cys3His class. The first protein of this class to be identified was tristetraprolin (TTP), which contains two Cys3His domains (CCCH). Cleavage Polyadenylation Specificity Factor (CPSF30) is another member of this class of protein. The biological role of CPSF30 is to regulate pre-mRNA processing during polyadenylation. CPSF30 contains five repeats of three cysteines and one histidine residues (CCCH) and is annotated as a zinc finger in protein databases. Our lab has overexpressed a construct of CPSF30 that contains the five CCCH domains and utilized a combination of inductively coupled plasma spectroscopy, X-ray absorption spectroscopy and UV-visible spectroscopy to identify the zinc sites and zinc stoichiometry. We have obtained evidence for zinc coordination, as expected, but with stoichiometry ranging from 2-4 zinc ions, rather than the predicted 5. Instead, the fifth site houses an unexpected 2Fe-2S cluster co-factor. To identify where the 2Fe-2S site is located relative to the Zn sites, I have investigated two mutants - one in which the CCCH domain of the first ZF was mutated to all alanine residues and one in which the CCCH domains of the second ZF was mutated to all alanine's. Data regarding the effects of the mutations on metallation and RNA binding is presented.
A Tale of Two Zinc Fingers: Structure and Functional Studies of CCCH type Zinc Finger Proteins CPSF30 and TTP involved in RNA RegulationCPSF30 and TTP are non-classical zinc finger proteins (ZFS) that contain domains with a CCCH motif. CPSF30 has 5 CCCH domains and TTP has 2 CCCH domains. Both proteins are involved in RNA regulation; CPSF30 regulates pre-mRNA and TTP regulates mRNA; however, only TTP has been shown to directly bind to RNA (via its CCCH domain, targeting AU-rich sites). Given the sequence similarity between TTP and CPSF30, we hypothesized that CPSF30 directly binds AU-rich RNA sequences via its CCCH domains. To test this hypothesis, a construct of CPSF30 containing the five CCCH domains, was over-expressed and purified. Unexpectedly, CPSF30 was reddish in color, suggesting iron coordination. UV-visible, ICP-MS analysis and XAS spectroscopy revealed that the protein contains a 2Fe-2S cluster in addition to four zinc domains. The 2Fe-2S cluster utilizes a CCCH ligand set, and is the second example of this site in biology! RNA binding studies, using EMSA and fluorescence anisotropy (FA), with ?-synuclein AU-rich pre-mRNA as a target, were then performed. From these studies, we determined that (1) CPSF30 binds directly to AU-rich targets on pre-mRNA via a cooperative binding mechanism and (2) CPSF30 requires both iron and zinc coordination for RNA binding. Studies focused on Cu(I) binding to TTP will also be presented. Cu(I) is toxic in excess and there is emerging evidence that ZF sites may be target of Cu(I) toxicity. Using UV-visible and circular dichroism spectroscopies, we have determined that 3 Cu(I) ions bind to TTP and that Cu(I) binding inhibits the structure of the protein. In addition, RNA binding studies, using FA with the TNF-? AU-rich mRNA revealed that Cu(I) inhibits the TTP-RNA interaction. We propose that inhibition of TTP function by Cu(I) contributes to its mechanism of toxicity.