Browsing School, Graduate by Subject "zinc finger protein"
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Cys3His Zinc Finger Proteins: Metal Ion Coordination and RNA Recognition Properties of Tristetraprolin and the 30 kDa Subunit of the Cleavage and Polyadenylation Specificity FactorZinc finger proteins (ZFs) utilize zinc ions to fold and function. ZFs globally regulate gene expression through interactions with DNA, RNA and other proteins. The Cys₃His ZFs are an emerging class of ZFs that regulate RNA. Tristetraprolin (TTP) and the 30 kDa subunit of the cleavage and polyadenylation specificity factor (CPSF30) are two examples of Cys₃His ZFs. TTP binds to adenosine/uridine-rich sequences found in the 3'-untranslated regions of cytokine mRNA. TTP is a potential target for cadmium toxicity. A construct of TTP (TTP-2D) was over-expressed and purified, and the Cd(II) binding properties were determined using UV-visible spectroscopy. TTP-2D was found to bind Cd(II) with a Kd of 3.5 (± 0.1) x 10⁻⁹ M at a 2:1 Cd(II):TTP-2D stoichiometry. Cd(II)-TTP-2D selectively bound to an AU-rich RNA sequence, and exhibited greater selectivity than its Zn(II), Fe(II) or Fe(III)-TTP-2D counterparts, as measured by Fluorescence Anisotropy (FA). CPSF30 is essential for pre-mRNA processing and is predicted to interact with the pre-mRNA polyadenylation signal, AAUAAA. Constructs of CPSF30 that contain the whole protein, its 5 Cys₃His domains and its singular Cys₂HisCys domain were overexpressed and purified. These constructs coordinate Co(II) and Zn(II) at the expected 6:1, 5:1 and 1:1 M(II):protein stoichiometry with affinities ( K<sub>d</sub>s) of 6.3 (± 0.6) x 10⁵ M, 5.1 (± 0.5) x 10⁵ M and 3.1 (± 0.1) x 10⁵ M for Co(II). To increase solubility, a maltose binding fusion (MBP) of the 5 Cys₃His domain, called MBP-CPSF30-5FE, was overexpressed and purified in the folded state. MBP-CPSF30-5FE was found to contain with 4 zinc and 1 iron ions by Inductively Coupled Plasma Mass Spectrometry (ICP-MS). MBP-CPSF30-5FE was determined to selectively bind to AU-rich RNA target from α-Synuclein pre-mRNA, using a combination of electrophoretic mobility shift assays (EMSA) and FA. The binding data was best fit to a cooperative binding model with 2 protein:1 RNA with Kds of 1.44 (± 0.04) x 10⁷ M for α-Synuclein24, 1.15 (± 0.04) x 10⁷ M for α-Synuclein30 and 9.35 (± 0.27) x 10⁸M for α-Synuclein38 and an average hill coefficient of 1.63 (± 0.07).
Targeting Zinc Finger Proteins with Exogenous Metals and Molecules: Lessons Learned from Tristetraprolin, a CCCH type Zinc FingerZinc (Zn) plays a key role in inflammatory response, including regulating the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway. Among the signaling proteins involved in the NF-κB pathway, many are known zinc finger proteins (ZFs), including Tristetraprolin (TTP). TTP is a non-classical CCCH-type Zinc Finger protein (ZF), that contains two Cys3His zinc binding domains and is a key regulator of the inflammatory response. TTP is a potential target for exogenous gold (Au) and copper (Cu), as well as hydrogen sulfide, an emerging gasotransmitter. To understand how TTP is targeted by other metals, the interactions of TTP were investigated using a combination of bioinorganic chemistry tools including as optical spectroscopy, native electrospray ionization mass spectrometry (ESI-MS), and X-ray absorption study (XAS). The first metal investigated was Cu(I). I discovered that Cu(I) can bind to the tandem ZF construct of TTP (TTP-2D) and disrupt structure and function. This finding indicates a potential relationship between Cu toxicity and metal-regulation of ZFs. The second metal investigated was Au(III). I discovered that the reactivity of TTP-2D with gold complex leads to Au exchange forming a series of Aux-TTP-2D complexes, with reduction of the gold from Au(III) to Au(I). These protein species are then functionally inactive (no RNA binding). When the same experiments were performed with TTP bound to RNA, the Zn-TTP/RNA complex is not disrupted by the Au-complex suggesting a protective role for RNA. To understand how H2S, a signaling molecule, targets Zn-TTP-2D, its reactivity was determined using a combination of cryo-ESI-MS, fluorescence, and electron paramagnetic resonance (EPR) spectroscopies. We found that the H2S oxidizes the cysteine residues of Zn-TTP via a mechanism that involves atmospheric oxygen, a persulfide intermediate and a radical reaction. The results of these biochemical studies of TTP will be presented in the context of TTP’s biological role. In addition, development of a method to follow Zn speciation in inflammatory cells via liquid chromatography connected to inductively coupled plasma (LC-ICP-MS), will be presented. Here, I use THP-1 cells, which are a human monocyte cell line as a model for inflammation, and demonstrate an approach to separate the zinc-proteome.