Ralf Bogumil

Modulation of DNA Binding of a Tramtrack Zinc Finger Peptide by the Metallothionein-Thionein Conjugate Pair

The ability of metallothionein (MT) to modulate DNA binding by a two-finger peptide of Tramtrack (TTK), a CCHH zinc transcription factor, was investigated using metal-bound and metal-deficient forms of rabbit MT-2 and the TTK peptide. Thionein inhibited DNA binding by zinc-bound TTK, and Zn-MT restored DNA-binding by zinc-deficient apo-TTK. “Free” zinc at low concentrations was as effective as Zn-MT in restoring DNA binding by apopeptide but was inhibitory at concentrations equal to zinc bound to 2 mol eq and higher of Zn-MT. Substitution of cadmium for zinc reduced the affinity of the peptide for its DNA binding site. This effect was reversed by incubation with Zn-MT. The circular dichroic spectra of the TTK peptide indicated that zinc removal resulted in loss of α-helical structures, which are sites of DNA contact points. Reconstitution with cadmium resulted in stoichiometric substitution of 2 mol of Cd/mol of peptide but not recovery of α-helical structures. Incubation of Cd-TTK with Zn-MT restored the secondary structure expected for zinc-bound TTK. The ability of Zn-MT and thionein to restore or inhibit DNA-binding by TTK was associated with effects on the metallation status of the peptide and related alterations in its secondary structure.

Phenylarsine oxide affinity chromatography to identify proteins involved in redox regulation: dithiol-disulfide equilibrium in serine/threonine phosphatase calcineurin.

Publisher Summary This chapter discusses affinity chromatography using an immobilized phenylarsine oxide derivative coupled to Sepharose for the purification of vicinal dithiolcontaining proteins. This methodology uses a phenylarsine oxide (PAO) resin to selectively bind, elute, and identify proteins that might be involved in redox regulation and to test a possible disulfide formation as a regulatory mechanism in oxidative stress. Because such conditions are found in activated polymorphonuclear leukocytes (PMN), a cytosolic fraction from PMN is used to isolate potential target proteins. This way the isolation and identification of six proteins with affinity for PAO can be achieved: thioredoxin (Trx), calcineurin (CAN), glutathione S-transferase (GST), calgranulin, L-plastin, and cofilin. From these candidates, a more detailed characterization of the interaction of PAO and oxidants with the serine-threonine phosphatase CaN (also known as protein phosphatase 2B) can be explored, which strongly suggests a dithiol-disulfide equilibrium in the enzyme as a mechanism for redox regulation.

Growth inhibitory factor and zinc affect neural cell cultures in a tissue specific manner

Deficiency of neuronal growth inhibitory factor (GIF) and abnormalities in zinc homeostasis have been suggested to play a role in the neuropathogenesis of Alzheimers disease. We report here that embryonic chick cerebral cell cultures zinc and copper containing GIF in the presence of marmoset hippocampal extract reduces significantly and concentration dependently mitochondrial succinate dehydrogenase activity (MTT) and cell mass. In contrast, no indications could be found that GIF affected neural retina cell cultures. Our results suggest that the observed effects of GIF are not elicited by zinc.

Isolation and characterization of a novel monomeric zinc- and heme-containing protein from bovine brain

An acidic zinc‐ and heme‐containing protein was isolated from the soluble fraction of bovine brain and has been purified to homogeneity. The zinc‐heme protein is a monomeric globular protein with a molecular mass of 31200 Da as determined by electrospray mass spectrometry. The protein was isolated with 0.90±0.05 zinc per protein and with substoichiometric amounts of heme. Amino acid sequences of four peptides (ca. 20% of the protein) were determined and the comparison of these sequences with those of protein and gene sequence databases revealed no significant correlation with any known protein. Thus, it is concluded that it is a novel protein of currently unknown biological function.

Structural and biological studies on native bovine Cu,Zn-metallothionein-3

Following the discovery of the neuronal growth inhibitory factor, classified as metallothionein3 (MT-3), by Uchida et. al. 1991 [1] we have focussed our research on the structure-function analysis of this protein. MT-3 is a central nervous system (CNS) specific metallothionein-like protein, which has been linked to Alzheimer’s disease (AD) [1]. The amino acid sequence of human MT-3 (68 amino acids) exhibits 70% sequence identity to mammalian metallothioneins (MT-1 and MT-2) (61 amino acids), including the preserved array of 20 Cys residues. In MT-1 and MT-2 these 20 Cys residues are involved in the formation of two metal-thiolate clusters, i.e. MeII 3(Cys)9 and MeII 4(Cys)11 [2]. Relative to mammalian MT-1 and MT-2 sequences, MT-3 contains two conserved inserts, a single Thr in the N-terminal and a Glu-rich hexapeptide in the C-terminal region (see Fig. 1). Despite their high sequence identity only MT-3 exhibits a growth inhibitory activity in neuronal cell culture studies, indicating that the — so far unknown — structure of MT-3 must differ from that of the other MTs. Furthermore, it has been demonstrated that the N-terminal metal binding domain MT-3(1–32) is sufficient for the growth inhibitory activity [3, 6]. In contrast to MTs, which usually contain 7 Zn(II) ions, native MT-3 isolated from bovine brain possesses an unusual metal ion composition, i.e. 4-5 Cu(I) and 2-2.5 Zn(II) per polypeptide chain, suggesting the presence of different cluster structures in this protein [4].