Limor Broday

Interaction of Ni(II) and Cu(II) with a metal binding sequence of histone H4: AKRHRK, a model of the H4 tail

Chromatin proteins are believed to represent reactive sites for nickel binding. The unique structure of the N-terminal tail of histone H4 contains sites for post-translational modification close to a histidine residue capable of anchoring binding sites for metal ions. We have analyzed as a minimal model for the H4 tail, the blocked peptide CH(3)CO-AKRHRK-CONH(2) for nickel and copper binding. Ultraviolet-visible, circular dichroism, electron paramagnetic resonance and nuclear magnetic resonance spectroscopic analysis showed that histidine acts as an anchoring metal binding site. A 1N complex is formed between pH=5-7 and 4-6 for Ni(II) and Cu(II), respectively, while at a higher pH a series of 4N complexes are formed. Above pH 8, the 2N high-spin octahedral resulted in a 4N low-spin planar Ni(II) complex. The stability constants of the Cu(II) (3N, 4N) and Ni(II) (4N) complexes with the peptide model of the H4 were distinctly higher than those for a similar blocked peptide with a histidine in the fourth position. Significant shifts in the alphaproton region in the 1H NMR spectrum of the 4N Ni-complex showed that the conformation of the peptide had been dramatically affected following Ni(II) complexation.

RNF5, a RING Finger Protein That Regulates Cell Motility by Targeting Paxillin Ubiquitination and Altered Localization

ABSTRACT RNF5 is a RING finger protein found to be important in the growth and development of Caenorhabditis elegans. The search for RNF5-associated proteins via a yeast two-hybrid screen identified a LIM-containing protein in C. elegans which shows homology with human paxillin. Here we demonstrate that the human homologue of RNF5 associates with the amino-terminal domain of paxillin, resulting in its ubiquitination. RNF5 requires intact RING and C-terminal domains to mediate paxillin ubiquitination. Whereas RNF5 mediates efficient ubiquitination of paxillin in vivo, protein extracts were required for in vitro ubiquitination, suggesting that additional modifications and/or an associated E3 ligase assist RNF5 targeting of paxillin ubiquitination. Mutant Ubc13 efficiently inhibits RNF5 ubiquitination, suggesting that RNF5 generates polychain ubiquitin of the K63 topology. Expression of RNF5 increases the cytoplasmic distribution of paxillin while decreasing its localization within focal adhesions, where it is primarily seen under normal growth. Concomitantly, RNF5 expression results in inhibition of cell motility. Via targeting of paxillin ubiquitination, which alters its localization, RNF5 emerges as a novel regulator of cell motility.

Molecular mechanisms of nickel carcinogenesis

Nickel treatment of intact cultured cells oxidized dichlorofluorescin to a fluorescent product indicating that nickel elevated the level of oxidants in cells. Nickel also caused an increase in crosslinking of amino acids to DNA and these complexes did not appear to involve the direct participation of Ni2+. Histidine, cysteine and tyrosine were prominent among the amino acids crosslinked to DNA. Nickel selectively damaged heterochromatin and this resulted in deletions of heterochromatic regions during nickel carcinogenesis. Thrombospondin was one of the genes expressed in normal cells that was not expressed in nickel-transformed cells. Other aspects of the molecular mechanism of nickel carcinogenesis are discussed.

TIP49b, a Regulator of Activating Transcription Factor 2 Response to Stress and DNA Damage

ABSTRACT Activating transcription factor 2 (ATF2/CRE-BP1) is implicated in transcriptional control of stress-responsive genes. A yeast two-hybrid screen identified TBP-interacting protein 49b (TIP49b), a component of the INO80 chromatin-remodeling complex, as a novel ATF2-interacting protein. TIP49bs association with ATF2 is phosphorylation dependent and requires amino acids 150 to 248 of ATF2 (ATF2150–248), which are implicated in intramolecular inhibition of ATF2 transcriptional activities. Forced expression of TIP49b efficiently attenuated ATF2 transcriptional activities under normal growth conditions as well as after UV treatment, ionizing irradiation, or activation of p38 kinase, all of which induced ATF2 phosphorylation and increased TIP49b-ATF2 association. Constitutive expression of ATF2150–248 peptide outcompeted TIP49b interaction with ATF2 and alleviated the suppression of ATF2 transcriptional activities. Expression of ATF2150–248 in fibroblasts or melanoma but not in ATF2-null cells caused a profound G2M arrest and increased degree of apoptosis following irradiation. The interaction between ATF2 and TIP49b constitutes a novel mechanism that serves to limit ATF2 transcriptional activities and highlights the central role of ATF2 in the control of the cell cycle and apoptosis in response to stress and DNA damage.

Effects of nickel on DNA methyltransferase activity and genomic DNA methylation levels

Methylation of DNA plays an important role in organizing the genome into transcriptionally active and inactive zones. Nickel compounds cause chromatin condensation and DNA methylation in the transgenic gpt+ Chinese hamster cell line (G12). Here we show that nickel is an inhibitor of cytosine 5-methyltransferase activity in vivo and in vitro. In living cells, this inhibition is transient and following a recovery period after nickel treatment, Mtase activity slightly rebounds. Genomic DNA methylation levels are also somewhat decreased following nickel treatment, but with time, there is an elevation of total DNA methylation above basal levels and before any rebound of methyltransferase activity. These results suggest that nickel exposure can elevate total genomic DNA methylation levels even when DNA methyltransferase activity is depressed. These findings may explain the hypermethylation of senescence and tumor suppressor genes found during nickel carcinogenesis and support the model of a direct effect of Ni2+ on chromatin leading to de novo DNA methylation.

SUMO Regulates the Assembly and Function of a Cytoplasmic Intermediate Filament Protein in C. elegans

Sumoylation is a reversible posttranslational modification that plays roles in many processes, including transcriptional regulation, cell division, chromosome integrity, and DNA damage response. Using a proteomics approach, we identified approximately 250 candidate targets of sumoylation in C. elegans. One such target is the cytoplasmic intermediate filament (cIF) protein named IFB-1, which is expressed in hemidesmosome-like structures in the worm epidermis and is essential for embryonic elongation and maintenance of muscle attachment to the cuticle. In the absence of SUMO, IFB-1 formed ectopic filaments and protein aggregates in the lateral epidermis. Moreover, depletion of SUMO or mutation of the SUMO acceptor site on IFB-1 resulted in a reduction of its cytoplasmic soluble pool, leading to a decrease in its exchange rate within epidermal attachment structures. These observations indicate that SUMO regulates cIF assembly by maintaining a cytoplasmic pool of nonpolymerized IFB-1, and that this is necessary for normal IFB-1 function.

Regulation of ATG4B Stability by RNF5 Limits Basal Levels of Autophagy and Influences Susceptibility to Bacterial Infection

Autophagy is the mechanism by which cytoplasmic components and organelles are degraded by the lysosomal machinery in response to diverse stimuli including nutrient deprivation, intracellular pathogens, and multiple forms of cellular stress. Here, we show that the membrane-associated E3 ligase RNF5 regulates basal levels of autophagy by controlling the stability of a select pool of the cysteine protease ATG4B. RNF5 controls the membranal fraction of ATG4B and limits LC3 (ATG8) processing, which is required for phagophore and autophagosome formation. The association of ATG4B with—and regulation of its ubiquitination and stability by—RNF5 is seen primarily under normal growth conditions. Processing of LC3 forms, appearance of LC3-positive puncta, and p62 expression are higher in RNF5−/− MEF. RNF5 mutant, which retains its E3 ligase activity but does not associate with ATG4B, no longer affects LC3 puncta. Further, increased puncta seen in RNF5−/− using WT but not LC3 mutant, which bypasses ATG4B processing, substantiates the role of RNF5 in early phases of LC3 processing and autophagy. Similarly, RNF-5 inactivation in Caenorhabditis elegans increases the level of LGG-1/LC3::GFP puncta. RNF5−/− mice are more resistant to group A Streptococcus infection, associated with increased autophagosomes and more efficient bacterial clearance by RNF5−/− macrophages. Collectively, the RNF5-mediated control of membranalATG4B reveals a novel layer in the regulation of LC3 processing and autophagy.

The LIM domain protein UNC-95 is required for the assembly of muscle attachment structures and is regulated by the RING finger protein RNF-5 in C. elegans

Here, we describe a new muscle LIM domain protein, UNC-95, and identify it as a novel target for the RING finger protein RNF-5 in the Caenorhabditis elegans body wall muscle. unc-95(su33) animals have disorganized muscle actin and myosin-containing filaments as a result of a failure to assemble normal muscle adhesion structures. UNC-95 is active downstream of PAT-3/β-integrin in the assembly pathways of the muscle dense body and M-line attachments, and upstream of DEB-1/vinculin in the dense body assembly pathway. The translational UNC-95::GFP fusion construct is expressed in dense bodies, M-lines, and muscle–muscle cell boundaries as well as in muscle cell bodies. UNC-95 is partially colocalized with RNF-5 in muscle dense bodies and its expression and localization are regulated by RNF-5. rnf-5(RNAi) or a RING domain deleted mutant, rnf-5(tm794), exhibit structural defects of the muscle attachment sites. Together, our data demonstrate that UNC-95 constitutes an essential component of muscle adhesion sites that is regulated by RNF-5.

Nickel enhances telomeric silencing in Saccharomyces cerevisiae

Certain nickel compounds including crystalline nickel sulfide (NiS) and subsulfide (Ni3S2) are potent human and animal carcinogens. In Chinese hamster embryo cells, an X-linked senescence gene was inactivated following nickel-induced DNA methylation. Nickel also induced the inactivation of the gpt reporter gene by chromatin condensation and a DNA methylation process in a transgenic gpt+ Chinese hamster cell line (G12), which is located near a heterochromatic region. To determine if nickel can cause gene silencing independently of DNA methylation, based only on the induction of changes in chromatin structure, we measured its effect on gene silencing in Saccharomyces cerevisiae. Growth of yeast in the presence of nickel chloride repressed a telomeric marker gene (URA3) and resulted in a stable epigenetic switch. This phenomenon was dependent on the number of cell doubling prior to selection and also on the distance of the marker gene from the end of the chromosome. The level of TPE (telomeric position effect) increased linearly with elevations of nickel concentration. Addition of magnesium inhibited this effect, but magnesium did not silence the reporter gene by itself. The level of silencing was also assessed following treatment with other transition metals: cobalt, copper and cadmium. In the sublethal range, cobalt induced similar effects as nickel, while copper and cadmium did not change the basal level of gene expression. Silencing by copper and cadmium were evident only at concentrations of those metals where the viability was very low.

The Role of Oxidative Stress in Nickel and Chromate Genotoxicity

Some general principles regarding oxidative stress and molecular responses to toxic metals are presented in this manuscript. The remainder of the manuscript, however, will focus on the role of oxidative stress in particulate nickel-induced genetic damage and mutations. The phagocytosis of particulate nickel compounds and the dissolution of the particles inside the cell and the resulting oxidative stress produced in the nucleus is a key component of the nickel carcinogenic mechanism. The crosslinking of amino acids to DNA by nickel that does not involve direct participation of nickel in a ternary complex but nickel-induced oxidative stress will be discussed as well. The selective ability of particulate nickel compounds to silence the expression of genes located near heterochromatin and the effect of vitamin E on the genotoxicity and mutations induced by particulate and soluble nickel compounds will also be discussed. Particulate nickel compounds have been shown to produce more oxidative stress than water-soluble nickel compounds. In addition to nickel, the role of oxidative stress in chromate-induced genotoxicity will also be discussed with particular attention directed to the effects of vitamin E on mutations and chromosomal aberrations induced by chromate.