Stress proteins by zinc ions in sea urchin embryos

In Paracentrotus lividus embryos, treatment with zinc ions induces the synthesis of the two major stress proteins with the same molecular weight as those induced by heat shock. The developmental stages responsive to zinc ion treatment are the same as those responsive to heat shock. However, zinc treatment induces a longer lasting synthesis of the stress proteins, and, unlike heat shock, does not induce thermotolerance and does not inhibit synthesis of the bulk proteins.     

Zinc-binding proteins detected by protein blotting

The Western blotting technique was used for the detection of zinc-binding proteins. Proteins were separated electrophoretically on 15% polyacrylamide-sodium dodecyl sulfate minigels, the gels were soaked in a reduction buffer, and the proteins were transferred to nitrocellulose filters. Zinc-binding proteins were probed with radioactive zinc (65Zn) and were detected by autoradiography. This technique allows the detection of as little as 20 to 100 pmol of zinc metalloproteins.     

GIT proteins, A novel family of phosphatidylinositol 3,4, 5-trisphosphate-stimulated GTPase-activating proteins for ARF6

ADP-ribosylation factor (ARF) proteins are key players in numerous vesicular trafficking events ranging from the formation and fusion of vesicles in the Golgi apparatus to exocytosis and endocytosis. To complete their GTPase cycle, ARFs require a guanine nucleotide-exchange protein to catalyze replacement of GDP by GTP and a GTPase-activating protein (GAP) to accelerate hydrolysis of bound GTP. Recently numerous guanine nucleotide-exchange proteins and GAP proteins have been identified and partially characterized. Every ARF GAP protein identified to date contains a characteristic zinc finger motif. GIT1 and GIT2, two members of a new family of G protein-coupled receptor kinase-interacting proteins, also contain a putative zinc finger motif and display ARF GAP activity. Truncation of the amino-terminal region containing the zinc finger motif prevented GAP activity of GIT1. One zinc molecule was found associated per molecule of purified recombinant ARF-GAP1, GIT1, and GIT2 proteins, suggesting the zinc finger motifs of ARF GAPs are functional and should play an important role in their GAP activity. Unlike ARF-GAP1, GIT1 and GIT2 stimulate hydrolysis of GTP bound to ARF6. Accordingly we found that the phospholipid dependence of the GAP activity of ARF-GAP1 and GIT proteins was quite different, as the GIT proteins are stimulated by phosphatidylinositol 3,4, 5-trisphosphate whereas ARF-GAP1 is stimulated by phosphatidylinositol 4,5-bisphosphate and diacylglycerol. These results suggest that although the mechanism of GTP hydrolysis is probably very similar in these two families of ARF GAPs, GIT proteins might specifically regulate the activity of ARF6 in cells in coordination with phosphatidylinositol 3-kinase signaling pathways.     

Induction of stress proteins in chicken embryo cells by low-level zinc contamination in amino acid-free media

It has been reported that chicken embryo cells deprived of exogenous amino acids for 4 hours synthesize stress (heat-shock) proteins. Herein, we show that amino acid deprivation is not sufficient to cause induction of stress proteins. Zinc contaminating a component of commercial cell culture medium used to prepare amino acid-free medium was an inducer in our cultures. In the absence of exogenous amino acids, the concentration of zinc ions needed for half-maximal induction of stress proteins was an order of magnitude lower than the dose required for cells in complete medium. Histidine and cystine, which have high affinities for zinc ions, were the amino acids most effective in blocking the induction of stress proteins by zinc. Problems posed by heavy metal ions in culture media and biologic fluids for searches for in vivo inducers of the cellular stress (heat shock) response are discussed.     

The LZT proteins; the LIV-1 subfamily of zinc transporters

Zinc is an essential ion for cells with a vital role to play in controlling the cellular processes of the cell, such as growth, development and differentiation. Specialist proteins called zinc transporters control the level of intracellular zinc in cells. In mammals, the ZIP family of zinc transporters has a pivotal role in maintaining the correct level of intracellular zinc by their ability to transport zinc into cells from outside, although they may also transport metal ions other than zinc. There are now recognised to be four subfamilies of the ZIP transporters, including the recently discovered LIV-1 subfamily which has similarity to the oestrogen-regulated gene LIV-1, previously implicated in metastatic breast cancer. We call this new subfamily LZT, for LIV-1 subfamily of ZIP zinc Transporters. Here we document current knowledge of this previously uncharacterised group of proteins, which includes the KE4 proteins. LZT proteins are similar to ZIP transporters in secondary structure and ability to transport metal ions across the plasma membrane or intracellular membranes. However, LZT proteins have a unique motif (HEXPHEXGD) with conserved proline and glutamic acid residues, unprecedented in other zinc transporters. The localisation of LZT proteins to lamellipodiae mirrors cellular location of the membrane-type matrix metalloproteases. These differences to other zinc transporters may be consistent with an alternative role for LZT proteins in cells, particularly in diseases such as cancer.     

The induction of stress-related proteins by lead

Differential inductive effects of lead on protein synthesis in rat fibroblasts and kidney epithelial cells were examined. The lead was administered as lead glutamate, a complex known to introduce lead into cells. Lead exposure induced the synthesis of three proteins which constitute two separate stress protein subgroups. Two of these proteins have been previously identified as the glucose-regulated proteins because their synthesis is induced by reagents which perturb glucose utilization. The third protein is inducible by several sulfhydryl-binding reagents including lead. This third protein has been compared with a protein, p32/6.3, of very similar size and isoelectric point, which has been associated with lead-induced intranuclear inclusion bodies. However, several features, including one-dimensional peptide maps, indicated that the third protein and p32/6.3 are not identical. The three lead-induced proteins are distinguished from the major group of stress proteins by their relative insensitivity to heat stress. Lead glutamate, on the other hand, induces neither the heat shock protein 70 nor metallothionein, both of which are strongly induced by several metals including cadmium, zinc, and mercury.     

Spontaneous cleavage of proteins at serine and threonine is facilitated by zinc

Old proteins are widely distributed in the body. Over time, they deteriorate and many spontaneous reactions, for example isomerisation of Asp and Asn, can be replicated by incubation of peptides under physiological conditions. One of the signatures of long‐lived proteins that has proven to be difficult to replicate in vitro is cleavage on the N‐terminal side of Ser residues, and this is important since cleavage at Ser, and also Thr, has been observed in a number of human proteins. In this study, the autolysis of Ser‐ and Thr‐containing peptides was investigated with particular reference to discovering factors that promote cleavage adjacent to Ser/Thr at neutral pH. It was found that zinc catalyses cleavage of the peptide bond on the N‐terminal side of Ser residues and further that this process is markedly accelerated if a His residue is adjacent to the Ser. NMR analysis indicated that the imidazole group co‐ordinates zinc and that once zinc is co‐ordinated, it can polarize the carbonyl group of the peptide bond in a manner analogous to that observed in the active site of the metalloexopeptidase, carboxypeptidase A. The hydroxyl side chain of Ser/Thr is then able to cleave the adjacent peptide bond. These observations enable an understanding of the origin of common truncations observed in long‐lived proteins, for example truncation on the N‐terminal side of Ser 8 in Abeta, Ser 19 in alpha B crystallin and Ser 66 in alpha A crystallin. The presence of zinc may therefore significantly affect the long‐term stability of cellular proteins.     

Minimal functional sites allow a classification of zinc sites in proteins

Zinc is indispensable to all forms of life as it is an essential component of many different proteins involved in a wide range of biological processes. Not differently from other metals, zinc in proteins can play different roles that depend on the features of the metal-binding site. In this work, we describe zinc sites in proteins with known structure by means of three-dimensional templates that can be automatically extracted from PDB files and consist of the protein structure around the metal, including the zinc ligands and the residues in close spatial proximity to the ligands. This definition is devised to intrinsically capture the features of the local protein environment that can affect metal function, and corresponds to what we call a minimal functional site (MFS). We used MFSs to classify all zinc sites whose structures are available in the PDB and combined this classification with functional annotation as available in the literature. We classified 77% of zinc sites into ten clusters, each grouping zinc sites with structures that are highly similar, and an additional 16% into seven pseudo-clusters, each grouping zinc sites with structures that are only broadly similar. Sites where zinc plays a structural role are predominant in eight clusters and in two pseudo-clusters, while sites where zinc plays a catalytic role are predominant in two clusters and in five pseudo-clusters. We also analyzed the amino acid composition of the coordination sphere of zinc as a function of its role in the protein, highlighting trends and exceptions. In a period when the number of known zinc proteins is expected to grow further with the increasing awareness of the cellular mechanisms of zinc homeostasis, this classification represents a valuable basis for structure-function studies of zinc proteins, with broad applications in biochemistry, molecular pharmacology and de novo protein design.     

Zinc transport mediated by barley ZIP proteins are induced by low pH

It is estimated that nearly 50% of the world''s population is at risk of zinc (Zn) deficiency. The challenge is therefore to increase the Zn content in edible plant parts in order to improve the nutritional value of staple foods. We recently reported the identification and characterization of three barley genes encoding zinc transport proteins belonging to the ZIP protein family. These proteins are believed to be involved in cellular uptake of Zn²⁺. In this addendum, the Zn²⁺ transport capacity of ZIP proteins isolated from barley roots was investigated in response to various pH levels. We show that a lowering of pH induces a better growth at low Zn²⁺ concentrations of yeast cells expressing ZIP proteins. However, no significant change in transport capacity (V_max) could be observed for HvIRT1, whereas lowering of pH from 5.5 to 4.2 increased the V_max value with 64% for HvZIP5. These results indicate that proton activity has an important role in regulating the Zn²⁺ transport capacity of Zn²⁺ specific ZIP transport proteins. This information will increase the understanding of ZIP proteins and facilitate engineering of genotypes able to grow efficiently on marginal soils.Key words: ZIP proteins, barley, zinc transport, pH     

Zinc coordination environments in proteins determine zinc functions.

Estimates of the number of zinc proteins in humans are now possible and a functional annotation of the zinc proteome can begin. The catalytic and structural roles of zinc in hundreds of enzymes and thousands of so-called "zinc finger" protein domains have provided a molecular basis for the numerous biological functions of this essential element. Additional, regulatory functions of zinc/protein interactions are being recognized. They include roles of the zinc ion in signal transduction, in controlling the architecture of protein complexes, and in redox-active zinc sites, where the binding and release of zinc is under redox control. Moreover, a considerable number of proteins participate in cellular zinc homeostasis, e.g. membrane transporters, and cellular storage, sensor, and trafficking proteins. These proteins have evolved with mechanisms to handle zinc ions rather specifically and selectively. They perform their functions with a remarkably modest set: One redox state of the zinc ion and nitrogen, oxygen, and sulfur ligands from the side chains of histidine, glutamate/aspartate, and cysteine, respectively. By permutation of the ligands in this set, the functional potential of the zinc ion has been fully explored. Different coordination environments modulate the chemical characteristics of the zinc ion, control the kinetics of its binding, and allow it to be either metabolically active or inert. Insights into all these functions are building an understanding of why zinc is so critical for such a multitude of life processes.     

Engineered zinc finger proteins that respond to DNA modification by HaeIII and HhaI methyltransferase enzymes

Zinc finger modules are capable of specifically interacting with DNA that contains 5-methylcytosine (5-mC) in place of cytosine, suggesting that zinc finger-DNA binding could be regulated by extrinsic methylation of DNA. Here, we have used phage display to engineer zinc finger proteins that detect and discriminate DNA methylation by the prokaryotic enzymes HaeIII and HhaI. In these systems, zinc finger-DNA complexes are induced by DNA modification using the appropriate enzyme, which can therefore act as a switch. To further develop the specificity of the switch, zinc finger discrimination between 5-mC and thymine in DNA sequences is demonstrated despite the presence of the characteristic major groove methyl group that is common to both bases. Specificity was achieved using a DNA-binding strategy involving synergy between adjacent zinc fingers. We propose that engineered zinc fingers that recognise particular DNA modifications, such as sequence-specific DNA methylation, could be integrated into artificial regulatory circuits for the control of gene expression and other biological processes.