Mechanistic Insights into Regulation of the ALC1 Remodeler by the Nucleosome Acidic Patch.

Upon DNA damage, the ALC1/CHD1L nucleosome remodeling enzyme (remodeler) is activated by binding to poly(ADP-ribose). How activated ALC1 recognizes the nucleosome, as well as how this recognition is coupled to remodeling, is unknown. Here, we show that remodeling by ALC1 requires a wild-type acidic patch on the entry side of the nucleosome. The cryo-electron microscopy structure of a nucleosome-ALC1 linker complex reveals a regulatory linker segment that binds to the acidic patch. Mutations within this interface alter the dynamics of ALC1 recruitment to DNA damage and impede the ATPase and remodeling activities of ALC1. Full activation requires acidic patch-linker segment interactions that tether the remodeler to the nucleosome and couple ATP hydrolysis to nucleosome mobilization. Upon DNA damage, such a requirement may be used to modulate ALC1 activity via changes in the nucleosome acidic patches.

Changes of polyamine pattern in digestive glands of mussel Mytilus galloprovincialis under exposure to cadmium.

Polyamines, in particular spermidine and spermine, have been identified as important antioxidants, highly induced by oxidative stress in a variety of organisms. However, little is known about changes in polyamine content of metal-stressed marine organisms. In the present study, mussels (Mytilus galloprovincialis) were experimentally exposed to 25 µg/L Cd(2+) or 100 µg/L Cd(2+) for up to 15 days. Cd(2+) was progressively accumulated in mussel tissues, leading to a characteristic oxidative-stress status. Free putrescine (PUT) production was noticeably induced in response to Cd(2+) at day 5 and then declined. In contrast, free spermidine (SPD) content was gradually reduced, whereas the concentration of free spermine (SPM) increased. In combination, these changes led to a 69% or 88% reduction in the ratio of (SPD+SPM)/PUT at day 5, dependent on the Cd(2+) concentration used, which subsequently followed an upward trend in values, albeit not reaching those of controls. Conjugated polyamines constantly increased, in particular conjugated spermidine and spermine, tagging along with metallothionein production. Acetylated polyamines showed a diverse profile of changes, but their content was generally kept at low levels throughout the exposure period. Collectively, our results suggest that certain polyamine compounds could play a significant role in the tolerance of mussels against Cd(2+)-mediated stress, and that the ratio (SPD+SPM)/PUT could be a good indicator of the metal-stress status.

Dysfunctions of the translational machinery in digestive glands of mussels exposed to mercury ions.

Mercury is an element naturally occurring in the biosphere, but is also released into the environment by human activities, such as mining, smelting, and industrial discharge. Mercury is a biologically harmful element and any exposure of living organisms mainly due to contamination, can cause severe or even lethal side effects. In every form detected, elemental, inorganic, or organic, mercury exhibits toxicity associated with induced oxidative stress. Although the genotoxicity of mercury has been well demonstrated in mussels, little is known about its toxic effects on the translational machinery at the molecular level. To investigate possible effects, we exposed the common mussel Mytilus galloprovincialis in seawater supplemented by 30 µg/L Hg²âº for 15 days. We observed that Hg²âº was significantly accumulated in the digestive glands of mussels, reaching a level around 80 µg/g tissue (dry weight) at the 15th day of exposure. Exposure of mussels to Hg²âº resulted in failure of redox homeostasis, as reflected on lipid peroxidation levels and superoxide dismutase activity in glands, and micronucleus frequency in gills. Extracts from digestive glands after 15-day exposure to Hg²âº exhibited decreased tRNA aminoacylation ability and, moreover, a 70% reduction in the ability of 40S ribosomal subunits to form the 48S initiation ribosomal complex. A similar reduction was detected in the ability of ribosomes to translocate peptidyl-tRNA from the A-site to the P-site, an observation coinciding with the notion that regulation of protein synthesis by Hg²âº mainly occurs at the initiation and elongation stages of translation. A-site binding, peptidyl transferase activity, and termination of peptide chain synthesis underwent less pronounced but measurable reductions, a finding which explains why poly(Phe)-synthesis in ribosomes isolated from exposed mussels is reduced by 70%. In conclusion, Hg²âº apart from being a genotoxic ion acts as a modulator of protein synthesis in mussels, an observation probably related with its ability to induce oxidative stress.

Translational responses and oxidative stress of mussels experimentally exposed to Hg, Cu and Cd: one pattern does not fit at all.

Certain metals, like Hg, Cu and Cd, are capable of down-regulating protein synthesis in several marine organisms, including Mytilus galloprovincialis. Nevertheless, due to the complexity of the environmental stress, it is difficult to evaluate the influence of individual metals on protein synthesis via field studies. To bypass this difficulty, experimental studies were carried out on M. galloprovincialis exposed in aquarium for 15 days to one of three selected metal salts, HgCl(2), CuCl(2) and CdCl(2). Polysome profile was determined in digestive gland extracts of the exposed mussels as a way of measuring the functional status of ribosomes, superoxide radical production and lipid peroxidation as indicators of oxidative stress, metallothionein content as a metal detoxification index, and superoxide dismutase activity as a free radicals-scavenging index. Exposure of mussels to Hg(2+) or Cu(2+) resulted in a concentration- and time-dependent decrease in the polysome content of digestive gland cells, which at 15th day of exposure and at the highest metal concentrations tested, was 32% and 19% of the control, respectively. Both metals, at the concentrations used (<40 µg/L), did not significantly influence the oxidative stress biomarkers. By contrast, Cd(2+) treatment significantly induced superoxide radical production and lipid peroxidation in digestive gland cells, hinting that mussels suffered from oxidative stress. Polysome levels in Cd(2+)-exposed mussels were initially decreased by day 5 in digestive gland cells and then elevated to reach nearly the control levels by 15 days of exposure. Elevated protein synthesis was associated with significantly increased production of metallothioneins, whereas such increase was not recorded in Hg(2+)- or Cu(2+)-exposed mussels. Interestingly, the ribosome efficiency at initiating protein synthesis followed a similar pattern of polysome alterations, a fact suggesting that regulation of protein synthesis mainly occurred at the initiation phase of translation. Overall, these results suggest that the effect of each metal on protein synthesis is idiosyncratic and depends on its ability to induce specific cellular defense mechanisms against oxidative stress.

Translational responses of Mytilus galloprovincialis to environmental pollution: integrating the responses to oxidative stress and other biomarker responses into a general stress index.

Heavy metals are commonly associated with the generation of reactive oxygen species (ROS), which may cause oxidative damage to several cellular macromolecules and organelles. In an attempt to correlate biomarker responses to oxidative stress, caged mussels (Mytilus galloprovincialis) were exposed for 30 days in a relatively clean site and two areas (Stations 1 and 2) unevenly polluted by heavy metals in Gulf of Patras (Greece). Three periods of caging were: one in winter, the second in spring, and the third in autumn. Heavy metal content was determined in digestive glands of the exposed mussels as a measure of metal pollution, metallothionein content as an adaptive and detoxifying index, lysosomal membrane stability as a biomarker of general stress, superoxide radical production and lipid peroxidation as indicators of oxidative stress, and micronucleus frequency in gill cells as an index of chromosomal damage. Considering that protein-synthesizing machinery is one of the candidate targets for ROS, the in vivo activity of ribosomes in digestive glands was also tested. Compared with the reference samples, mussels transplanted to Station 1 showed increased levels of heavy metals and metallothionein in digestive glands, lower lysosomal membrane stability, higher values in oxidative stress indices, reduced activity of ribosomes, and increased chromosomal damage in gill cells. In addition, run-off ribosomes isolated from mussels transplanted to Station 1 were less efficient at initiating protein synthesis in a cell-free system than those from mussels in the reference site. Mussels transplanted to Station 2 exhibited similar but less pronounced responses. Statistical analysis revealed a strong positive correlation of ribosomal activity with lysosomal membrane stability, as well as a significant negative correlation with the oxidative stress indices, metallothionein content, micronucleus frequency, and the digestive gland content in Cr, Cu and Mn. Integration of all the measured biomarker responses into one general "stress index" demonstrated a clear distinction between the sampling sites, allowing classification along a pollution gradient (reference site

Evaluation of the global protein synthesis in Mytilus galloprovincialis in marine pollution monitoring: seasonal variability and correlations with other biomarkers.

Protein synthesis down-regulation is a life-saving mechanism for many organisms exposed to xenobiotics that threaten normal life. The present study was designed to assess the spatial and seasonal variability of global protein synthesis, determined in the microsomal fraction of digestive glands from caged Mytilus galloprovincialis mussels exposed for 30 days in a relatively clean region and two unevenly polluted areas (Stations 1 and 2) along the Gulf of Patras (Greece). The in vivo activity of translating ribosomes was evaluated by analyzing the translating ribosomes, polysome content, which may serve as an indicator of the efficiency of the protein-synthesizing machinery. To correlate with classical biomonitoring strategies, various biomarkers were measured in digestive glands, including metallothionein content, heavy-metal content, and lysosomal membrane stability. In parallel, gill cells were examined for micronucleus frequency. Metal ion concentrations were also estimated in the surrounding waters as a measure of metal exposure. Substantially lower polysome content was recorded in caged mussels collected from Station 1, in particular during the winter and spring sampling. As verified by chemical analysis of the seawater and measurement of other biomarkers, Station 1 was more contaminated than Station 2. Polysome content was found negatively correlated with metallothionein levels, micronucleus frequency and cytosolic Cu and Hg in all seasons. In addition, negative correlations were obtained between polysome content and lysosomal membrane stability in winter and spring. A progressive increase in polysomes was observed from winter to autumn, in particular in samples from Station 1. A non-uniform trend was detected in 80S ribosomal monosomes, whereas the seasonal changes in ribosomal subunits were opposite to those found in polysome content. Comparisons between seasonal and local site-specific influences on polysome content provides evidence that winter and spring are the most appropriate sampling seasons for application of translation activity as a possible biomarker in biomonitoring studies.