Serine 58 of 14-3-3ζ Is a Molecular Switch Regulating ASK1 and Oxidant Stress-Induced Cell Death

Oxidant stress is a ubiquitous stressor with negative impacts on multiple cell types. ASK1 is a central mediator of oxidant injury, but while mechanisms of its inhibition, such as sequestration by 14-3-3 proteins and thioredoxin, have been identified, mechanisms of activation have remained obscure and the signaling pathways regulating this are not clear. Here, we report that phosphorylation of 14-3-3ζ at serine 58 (S58) is dynamically regulated in the cell and that the phosphorylation status of S58 is a critical factor regulating oxidant stress-induced cell death. Phosphorylation of S58 releases ASK1 from 14-3-3ζ, and ASK1 then activates stress-activated protein kinases, leading to cell death. While several members of the mammalian sterile 20 (Mst) family of kinases can phosphorylate S58 when overexpressed, we identify Ste20/oxidant stress response kinase 1 (SOK-1), an Mst family member known to be activated by oxidant stress, as a central endogenous regulator of S58 phosphorylation and thereby of ASK1-mediated cell death. Our findings identify a novel pathway that regulates ASK1 activation and oxidant stress-induced cell death.Oxidant stress plays a central role in a wide variety of pathologies, and a critical mediator of oxidant injury is the protein kinase ASK1 (30). Indeed, ASK1 is required for several types of oxidant stress-induced cell death (32). Its activity is restrained by a large number of complementary mechanisms, a fact that attests to the importance of ASK1 being maintained in an inactive state in the cell. For example, reduced thioredoxin binds to the N-terminal region of ASK1, thereby inhibiting its activity (27). Following oxidant stress and oxidation of thioredoxin, ASK1 is released, allowing its activation. Multiple phosphorylation events, including phosphorylation of ASK1 at S83 by Akt and at S1033 by an unknown mechanism, also negatively regulate ASK1 (6, 41; reviewed in reference 30). Critical to the negative regulation of ASK1 is phosphorylation of S966, which drives the association of ASK1 with 14-3-3 proteins, thereby inhibiting ASK1-mediated activation of downstream signaling and cell death (8, 43). The kinases responsible for S966 phosphorylation are not known, but the protein phosphatase calcineurin has been shown to dephosphorylate S966, leading to dissociation of ASK1 from 14-3-3 (13). Thus, other than calcineurin-mediated dephosphorylation of ASK1, signaling mechanisms positively regulating the release of ASK1 from 14-3-3 proteins are not known, despite intense interest in this kinase as a potential target in cardiovascular and neurologic diseases (30). Therefore, we undertook studies to attempt to identify such a mechanism.14-3-3 proteins play protective roles in the cell by sequestering proapoptotic factors in a phosphorylation-dependent manner (1, 15, 23). These proapoptotic proteins that are sequestered by 14-3-3 proteins are typically phosphorylated on one or more 14-3-3 binding motifs (18, 39). For example, in addition to ASK1 phosphorylation at S966, (8, 30), Bad is phosphorylated by Akt and ribosomal S6 kinases at several residues, inhibiting its proapoptotic functions (4, 14, 42, 45). Acting in opposition to this is the well-characterized c-Jun N-terminal kinase (JNK)-mediated phosphorylation of serine 184 of 14-3-3 proteins, leading to release of the proapoptotic factors Bax, Bad, FOXO3a, and Abl (29, 33, 40). In addition to S184, the phosphorylation statuses of other 14-3-3 residues can regulate 14-3-3/client interactions, such as T233, which is phosphorylated by CKI, disrupting the 14-3-3/Raf-1 interaction (5).Although most of the attention to phosphorylation of 14-3-3 has been focused on S184 and T233 (1), S58 has been known to be phosphorylated in situ for some time, and several kinases have been implicated, including protein kinases A and D, Akt, mitogen-activated protein kinase-activated kinase 2 (MK2), and sphingosine-dependent protein kinase 1 (later identified as a cleavage fragment of protein kinase C δ) (9, 16, 17, 24, 25, 44). However, it is not clear which specific kinases mediate phosphorylation under specific circumstances, nor are the biological consequences clear. This is underscored by the fact that both pro- and antiapoptotic kinases have been reported to phosphorylate this residue (23). It does seem clear, however, that S58 phosphorylation disrupts 14-3-3 dimerization and that this reduces the binding of some proteins (e.g., Raf-1) (28, 34), though probably not all, since Woodcock et al. reported that 14-3-3ζ monomers phosphorylated at S58 remained competent to bind phosphopeptides (37).Thorson et al. and Wang et al. created 14-3-3 mutants that were deficient in binding phosphopeptides, and Xing et al. employed one of these, 14-3-3ζ(R56A/R60A), to show that it led to enhanced activation of the stress-activated protein kinases, JNKs and p38, and enhanced cell death in response to UVC irradiation, a model of oxidant stress (31, 36, 38). However, since S58 of 14-3-3ζ is in the center of the R56-R60 region, we hypothesized that phosphorylation of S58 might disrupt binding of 14-3-3ζ to ASK1, which is upstream from the JNKs and p38 in the response to oxidant stress. In addition, Preisinger et al. reported that Ste20/oxidant stress response kinase 1 (SOK-1)/STK25, a mammalian sterile 20 (Mst) family member also known as yeast sterile 20 kinase 1 (YSK1), phosphorylated 14-3-3ζ on S58, leading to reorganization of the Golgi structure and cell motility (26). However, we and others had initially identified SOK-1 as a kinase activated by oxidant stress (20-22), and we have also shown that SOK-1 exits the Golgi apparatus following cellular stress (19), potentially giving SOK-1 greater access to the ASK1/14-3-3 complex (which is primarily cytosolic). Thus, we hypothesized that oxidant stress-induced activation of SOK-1 might lead to S58 phosphorylation, release of ASK1 from 14-3-3ζ, activation of JNKs and p38, and cell death. Here, we confirm these hypotheses and identify S58 as a molecular switch regulating ASK1-mediated oxidant stress-induced cell death.     

Traits related to species persistence and dispersal explain changes in plant communities subjected to habitat loss

Aim Habitat fragmentation is a major driver of biodiversity loss but it is insufficiently known how much its effects vary among species with different life‐history traits; especially in plant communities, the understanding of the role of traits related to species persistence and dispersal in determining dynamics of species communities in fragmented landscapes is still limited. The primary aim of this study was to test how plant traits related to persistence and dispersal and their interactions modify plant species vulnerability to decreasing habitat area and increasing isolation. Location Five regions distributed over four countries in Central and Northern Europe. Methods Our dataset was composed of primary data from studies on the distribution of plant communities in 300 grassland fragments in five regions. The regional datasets were consolidated by standardizing nomenclature and species life‐history traits and by recalculating standardized landscape measures from the original geographical data. We assessed the responses of plant species richness to habitat area, connectivity, plant life‐history traits and their interactions using linear mixed models. Results We found that the negative effect of habitat loss on plant species richness was pervasive across different regions, whereas the effect of habitat isolation on species richness was not evident. This area effect was, however, not equal for all the species, and life‐history traits related to both species persistence and dispersal modified plant sensitivity to habitat loss, indicating that both landscape and local processes determined large‐scale dynamics of plant communities. High competitive ability for light, annual life cycle and animal dispersal emerged as traits enabling species to cope with habitat loss. Main conclusions In highly fragmented rural landscapes in NW Europe, mitigating the spatial isolation of remaining grasslands should be accompanied by restoration measures aimed at improving habitat quality for low competitors, abiotically dispersed and perennial, clonal species.     

Distance decay of similarity in freshwater communities: do macro‐ and microorganisms follow the same rules?

Aim An intensively debated issue in macroecology is whether unicellular organisms show biogeographic patterns different from those of macroorganisms. One aspect of this debate addresses beta diversity, that is, do microbial organisms exhibit distance‐decay patterns similar to those of macroorganisms? And if so, is the decay of community similarity caused by spatially limited dispersal or by niche‐related factors? We studied the community similarity of stream diatoms, macroinvertebrates and bryophytes across the same set of sites in relation to environmental and geographic distance. Location A geographical gradient of c. 1100 km in Finland. Methods We first identified the subset of environmental variables that produced the highest correlation with community similarities for each taxonomic group. Based on these variables, we used partial Mantel tests to separate the independent influences of environmental and geographical distance for distance decay of community similarity, separately for diatoms, bryophytes and macroinvertebrates. Finally, macroinvertebrates were divided into three groups based on their different dispersal categories and a partial Mantel test was used to assess whether each of these groups were differently affected by environmental versus geographic distance, i.e. is dispersal a key factor in tests of niche versus neutral models. Results The level of environmental control was by far the strongest for diatoms; however, all groups were controlled more by environmental factors than by limited dispersal. Macroinvertebrate species with low dispersal ability were significantly related to geographic distance, while more effective dispersers showed no relationship to geography but were instead strongly related to environmental distance. Main conclusions Our results suggest that patterns between macro‐ and microorganisms are not fundamentally different, but the level of environmental control varies according to dispersal ability. The relative importance of niche versus dispersal processes is not simply a function of organism size but other traits (e.g. life‐history type, dispersal capacity) may obscure this relationship.     

Spatial consistency in susceptibility of prey species to predation by two Accipiter hawks

Predators impose strong selection on their prey, regulate prey populations and engage in coevolutionary interactions with their prey. The intensity of selection and the strength of coevolutionary interactions will depend on how stringent predators are in their choice of prey. We estimated susceptibility of different species of birds to predation by two common raptors, the northern goshawk Accipiter gentilis and the Eurasian sparrowhawk A. nisus, in an agricultural landscape in Denmark and boreal forests in Finland. We estimated susceptibility to predation as the deviation of the log₁₀‐transformed observed frequency of prey of different species from the log₁₀‐transformed expectation based on population density during the breeding season. We found a high degree of consistency in susceptibility to predation by the goshawk in two areas in Finland. More importantly, there was significant consistency in susceptibility to predation between Denmark and Finland, albeit the degree of consistency in the goshawk was higher than in the sparrowhawk. There was considerable overlap in susceptibility to predation between goshawk and sparrowhawk in Denmark, but not in Finland, implying differences in intensity of interspecific competition as reflected by a much higher extent of goshawk predation on sparrowhawks in Denmark than in Finland. Our findings suggest that hawks impose similar selection pressures on their prey populations, and that the degree of consistency has implications for intensity of interspecific killing.     

Statins and Hip Fracture Prevention – A Population Based Cohort Study in Women

Objective

To study the association of long-term statin use and the risk of low-energy hip fractures in middle-aged and elderly women.

Design

A register-based cohort study.

Setting

Finland.

Participants

Women aged 45–75 years initiating statin therapy between 1996 and 2001 with adherence to statins ≥80% during the subsequent five years (n = 40 254), a respective cohort initiating hypertension drugs (n = 41 610), and women randomly selected from the population (n = 62 585).

Main Outcome Measures

Incidence rate of and hazard ratio (HR) for low-energy hip fracture during the follow-up extending up to 7 years after the 5-year exposure period.

Results

Altogether 199 low-energy hip fractures occurred during the 135 330 person-years (py) of follow-up in the statin cohort, giving an incidence rate of 1.5 hip fractures per 1000 py. In the hypertension and the population cohorts, the rates were 2.0 per 1000 py (312 fractures per 157 090 py) and 1.0 per 1000 py (212 fractures per 216 329 py), respectively. Adjusting for a propensity score and individual variables strongly predicting the outcome, good adherence to statins for five years was associated with a 29% decreased risk (HR 0.71; 95% CI 0.58–0.86) of a low-energy hip fracture in comparison with adherent use of hypertension drugs. The association was of the same magnitude when comparing the statin users with the population cohort, the HR being 0.69 (0.55–0.87). When women with poor (<40%), moderate (40 to 80%), and good adherence (≥80%) to statins were compared to those with good adherence to hypertension drugs (≥80%) or to the population cohort, the protective effect associated with statin use attenuated with the decreasing level of adherence.

Conclusions

5-year exposure to statins is associated with a reduced risk of low-energy hip fracture in women aged 50–80 years without prior hospitalizations for fractures.     

Evaluation of consensus methods in predictive species distribution modelling

Aim  Spatial modelling techniques are increasingly used in species distribution modelling. However, the implemented techniques differ in their modelling performance, and some consensus methods are needed to reduce the uncertainty of predictions. In this study, we tested the predictive accuracies of five consensus methods, namely Weighted Average (WA), Mean(All), Median(All), Median(PCA), and Best, for 28 threatened plant species.
Location  North-eastern Finland, Europe.
Methods  The spatial distributions of the plant species were forecasted using eight state-of-the-art single-modelling techniques providing an ensemble of predictions. The probability values of occurrence were then combined using five consensus algorithms. The predictive accuracies of the single-model and consensus methods were assessed by computing the area under the curve (AUC) of the receiver-operating characteristic plot.
Results  The mean AUC values varied between 0.697 (classification tree analysis) and 0.813 (random forest) for the single-models, and from 0.757 to 0.850 for the consensus methods. WA and Mean(All) consensus methods provided significantly more robust predictions than all the single-models and the other consensus methods.
Main conclusions  Consensus methods based on average function algorithms may increase significantly the accuracy of species distribution forecasts, and thus they show considerable promise for different conservation biological and biogeographical applications.     

In vitro methods in the prediction of kinetics of drugs: focus on drug metabolism

The absorption, distribution, metabolism, excretion and toxicity (ADMET) properties of a candidate drug influence its final clinical success. These properties have traditionally been evaluated by using various in vivo animal approaches, but recently, a number of in vitro and in silico methods have been introduced to determine key ADMET features. Basic events, such as absorption through the gut wall, binding to plasma proteins, active and passive transfer through the blood-brain barrier, and various metabolic parameters, can now be screened with rapid in vitro and computer modelling methods. The focus in this short review is on the basic in vitro and in silico methods that are used for studying the metabolism properties of new drug molecules.     

Biosynthesis of 6-deoxyhexose glycans in bacteria

After the breakthroughs in genomic sequencing, one of the next challenges remains to understand the molecular biology of other classes of biomolecules, such as protein and lipids, many of which carry specific glycomodification when mediating their biological functions. This review focuses on the 6-deoxyhexose biosynthesis of cell surface glycans of three Gram-negative pathogens, Helicobacter pylori, Pseudomonas aeruginosa, and Actinobacillus actinomycetemcomitans serotype a. 6-Deoxysugars are important functional components of cell surface glycans, and their biosynthetic pathways might be suitable targets for novel interventions of antibacterial chemotherapy.