• Cys3His Zinc Finger Proteins: Metal Ion Coordination and RNA Recognition Properties of Tristetraprolin and the 30 kDa Subunit of the Cleavage and Polyadenylation Specificity Factor

      Michalek, Jamie Leigh; Michel, Sarah L. J. (2013)
      Zinc 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).
    • Identification and Characterization of the Zinc Uptake Regulator (Zur) of Corynebacterium diphtheriae

      Smith, Kelsy; Oram, Diana M. (2009)
      Regulation of metal ion homeostasis is essential to bacterial cell survival and in most species is controlled by metal-dependent transcriptional regulators. In this study, we describe a Corynebacterium diphtheriae ferric uptake regulator-family protein, Zur, that controls expression of genes in response to available zinc. Although no growth phenotype was observed in differential metal conditions, a C. diphtheriae zur mutant was more sensitive to peroxide stress, suggesting that Zur has a role in protecting the bacterium from oxidative damage. By measuring promoter activities and mRNA levels, we demonstrated that Zur represses transcription of three genes, zrg, cmrA, and troA, in zinc replete conditions. Zrg and TroA have similarity to other proteins involved in zinc uptake such as Bacillus subtilis YciC and Treponema pallidium TroA, respectively. CmrA has the highest similarity to C. diphtheriae TroA. Transcription of zrg and cmrA was also regulated in response to iron and manganese, respectively, by mechanisms that are independent of Zur. In addition to these three genes, 74 genes were shown to be affected by the absence of Zur and zinc via microarray analysis of transcript levels. The microarray results were confirmed via qRT-PCR for six of these genes, including one that is seemingly activated by Zur. An adjacent gene to zur, predicted to encode a metal-dependent transcriptional regulator in the ArsR/SmtB family, is transcribed from a separate promoter whose activity is unaffected by Zur. In contrast, Zur represses the activity of the zur promoter under low zinc conditions. The observed unique regulation of zur transcription has not been described for any other Zur ortholog. In addition, Zur bound the upstream region of multiple genes with varying affinity and this binding was metal dependent. A Zur binding site was determined on the upstream region of zur, cmrA, and sidA using a Dnase I protection assay. Our studies provide the first evidence of a zinc-specific transcriptional regulator in C. diphtheriae and give new insights into the intricate regulatory network responsible for regulating metal ion concentrations in this toxigenic human pathogen.
    • Measuring Cytosolic and Mitochondrial Labile Zinc Concentrations Following Hypoxia/Hypoglycemia with Fluorescence Biosensors

      McCranor, Bryan; Thompson, Richard B. (2011)
      Zinc is a "trace" metal necessary for proper cellular function, but studies, in multiple cell types, have shown that excess free zinc can be toxic (1, 2). It has also been observed that the intra- and extra-cellular concentrations of labile zinc increase dramatically in models of cerebral ischemia (3, 4). Substantial evidence indicates that mitochondrial dysfunction plays a significant role in neuronal death following ischemia (5), and both mitochondrial dysfunction and increased intracellular zinc concentrations have been associated with increased reactive oxygen species (ROS) production and ultimately apoptosis (6, 7). Zinc, specifically, has been shown to inhibit major mitochondrial enzymes of energy production contributing to mitochondrial dysfunction (8). We adapted our expressible fluorescent zinc biosensor (9) to target the mitochondria of PC12 cells, enabling us to ratiometrically image the mitochondrial matrix concentration of labile zinc even at resting (picomolar) levels. This represents the first such development of a sensor with sensitivity for physiological zinc in the mitochondria. We used this biosensor and our previous sensor, in cells which have undergone oxygen/glucose deprivation (OGD), to measure the "free" zinc concentrations following an ischemic-like event. The data suggests that both the intra-cellular and intra-mitochondrial zinc concentrations increase following OGD, albeit at different times, with the mitochondrial increase preceding the cytosolic increase. Our data raises the possibility that an increase in mitochondrial zinc could contribute to cell death in models of ischemia/reperfusion. 1. Canzoniero, L.M., et al. (1999) Journal of Neuroscience 19:RC31, 1-6. 2. Zodl, B., et al. (2003) Journal of Inorganic Biochemistry 97, 324-330. 3. Tonder, N., et al. (1990) Neuroscience Letters 109, 247-252. 4. Frederickson, C. J., et al. (2006) Experimental Neurology 198, 285-293. 5. Fiskum, G. et al. (2008) Mitochondria and Oxidative Stress in Neurodegenerative Disorders: Annals of the New York Academy of Science. 1147, 129-138 6. Weiss, J. H., et al. (2000) Trends in Pharmacological Sciences 21, 395-401. 7. Jiang, D., et al. (2001) Journal of Biological Chemistry 276, 47524 - 47529. 8. Gazaryan, I.G., et al. (2007) Journal of Biological Chemistry 282, 24373-24380. 9. Bozym, R. A., et al. (2006) ACS Chemical Biology 1, 103-111.