Specific protein homeostatic functions of small heat‐shock proteins increase lifespan

During aging, oxidized, misfolded, and aggregated proteins accumulate in cells, while the capacity to deal with protein damage declines severely. To cope with the toxicity of damaged proteins, cells rely on protein quality control networks, in particular proteins belonging to the family of heat‐shock proteins (HSPs). As safeguards of the cellular proteome, HSPs assist in protein folding and prevent accumulation of damaged, misfolded proteins. Here, we compared the capacity of all Drosophila melanogaster small HSP family members for their ability to assist in refolding stress‐denatured substrates and/or to prevent aggregation of disease‐associated misfolded proteins. We identified CG14207 as a novel and potent small HSP member that exclusively assisted in HSP70‐dependent refolding of stress‐denatured proteins. Furthermore, we report that HSP67BC, which has no role in protein refolding, was the most effective small HSP preventing toxic protein aggregation in an HSP70‐independent manner. Importantly, overexpression of both CG14207 and HSP67BC in Drosophila leads to a mild increase in lifespan, demonstrating that increased levels of functionally diverse small HSPs can promote longevity in vivo.     

Evolution of heat‐shock protein expression underlying adaptive responses to environmental stress

Heat‐shock proteins (Hsps) and their cognates are primary mitigators of cell stress. With increasingly severe impacts of climate change and other human modifications of the biosphere, the ability of the heat‐shock system to affect evolutionary fitness in environments outside the laboratory and to evolve in response is topic of growing importance. Since the last major reviews, several advances have occurred. First, demonstrations of the heat‐shock response outside the laboratory now include many additional taxa and environments. Many of these demonstrations are only correlative, however. More importantly, technical advances in “omic” quantification of nucleic acids and proteins, genomewide association analysis, and manipulation of genes and their expression have enabled the field to move beyond correlation. Several consequent advances are already evident: The pathway from heat‐shock gene expression to stress tolerance in nature can be extremely complex, mediated through multiple biological processes and systems, and even multiple species. The underlying genes are more numerous, diverse and variable than previously appreciated, especially with respect to their regulatory variation and epigenetic changes. The impacts and limitations (e.g., due to trade‐offs) of natural selection on these genes have become more obvious and better established. At last, as evolutionary capacitors, Hsps may have distinctive impacts on the evolution of other genes and ecological consequences.     

Offspring phenotype is shaped by the nonsperm fraction of semen

In a large majority of animal species, the only contribution of males to the next generation has been assumed to be their genes (sperm). However, along with sperm, seminal plasma contains a wide array of extracellular factors that have many important functions in reproduction. Yet, the potential intergenerational effects of these factors are virtually unknown. We investigated these effects in European whitefish (Coregonus lavaretus) by experimentally manipulating the presence and identity of seminal plasma and by fertilizing the eggs of multiple females with the manipulated and unmanipulated semen of several males in a full‐factorial breeding design. The presence of both own seminal plasma and foreign seminal plasma inhibited sperm motility, and the removal of own seminal plasma decreased embryo survival. Embryos hatched significantly earlier after both semen manipulations than in control fertilizations; foreign seminal plasma also increased offspring aerobic swimming performance. Given that our experimental design allowed us to control potentially confounding sperm‐mediated (sire) effects and maternal effects, our results indicate that seminal plasma may have direct intergenerational consequences for offspring phenotype and performance. This novel source of offspring phenotypic variance may provide new insights into the evolution of polyandry and mechanisms that maintain heritable variation in fitness and associated female mating preferences.     

Characterization of stress‐responsive lncRNAs in Arabidopsis thaliana by integrating expression,epigenetic and structural features

Agrobacterium genetically transforms plants by transferring and integrating T‐(transferred) DNA into the host genome. This process requires both Agrobacterium and host proteins. VirE2 interacting protein 1 (VIP1), an Arabidopsis bZIP protein, has been suggested to mediate transformation through interaction with and targeting of VirE2 to nuclei. We examined the susceptibility of Arabidopsis vip1 mutant and VIP1 overexpressing plants to transformation by numerous Agrobacterium strains. In no instance could we detect altered transformation susceptibility. We also used confocal microscopy to examine the subcellular localization of Venus‐tagged VirE2 or Venus‐tagged VIP1, in the presence or absence of the other untagged protein, in different plant cell systems. We found that VIP1–Venus localized in both the cytoplasm and the nucleus of Arabidopsis roots, agroinfiltrated Nicotiana benthamiana leaves, Arabidopsis mesophyll protoplasts and tobacco BY‐2 protoplasts, regardless of whether VirE2 was co‐expressed. VirE2 localized exclusively to the cytoplasm of tobacco and Arabidopsis protoplasts, whether in the absence or presence of VIP1 overexpression. In transgenic Arabidopsis plants and agroinfiltrated N. benthamina leaves we could occasionally detect small aggregates of the Venus signal in nuclei, but these were likely to be imagining artifacts. The vast majority of VirE2 remained in the cytoplasm. We conclude that VIP1 is not important for Agrobacterium‐mediated transformation or VirE2 subcellular localization.     

HORMAD2 methylation‐mediated epigenetic regulation of gene expression in thyroid cancer

This study is aimed to investigate the methylation level of candidate genes and its impact on thyroid carcinoma (THCA) development. Infinium Human Methylation 450 BeadChip Arrays by Illumina (Illumina HM450K) was the most popular CpG microarray platform widely used in biological and medical research. The methylation level of differentially expressed genes and their corresponding CpG sites were analysed by R programme. The expression of HORMAD2 was evaluated by qRT‐PCR and Western blot, while the methylation level was examined via methylation‐specific PCR. Cell viability, metastasis, cell cycle and apoptosis were detected by MTT assay, transwell and wound healing assay and flow cytometry, respectively, after treatment with 5‐aza‐2′‐deoxycytidine (5‐Aza). Tumour formation assay was used to analyse thyroid tumour growth in nude mice in vivo. The methylation levels of all 116 differentially expressed genes were analysed. HORMAD2 was significantly hypermethylated and its mRNA expression was inhibited in THCA cells. After treatment with 5‐Aza, HORMAD2 expression was up‐regulated in THCA cells and its overexpression can suppress thyroid cancer cell viability, mobility and invasiveness remarkably. Up‐regulation of HORMAD2 in THCA cells could prolong G0/G1 phase and shorten S phase to impede cell mitosis as well as promote thyroid cancer cells apoptosis. Furthermore, tumour formation assay showed that increased HORMAD2 level impeded tumour growth in vivo. Hypermethylation of HORMAD2 could induce THCA progression, while hypomethylation of HORMAD2 retard cell growth and mobility and facilitate apoptosis through increasing its mRNA expression.     

The minimal α‐crystallin domain of Mj Hsp16.5 is functional at non‐heat‐shock conditions

The small heat shock protein (sHSP) from Methanococcus jannaschii (Mj Hsp16.5) forms a monodisperse 24mer and each of its monomer contains two flexible N‐ and C‐terminals and a rigid α‐crystallin domain with an extruding β‐strand exchange loop. The minimal α‐crystallin domain with a β‐sandwich fold is conserved in sHSP family, while the presence of the β‐strand exchange loop is divergent. The function of the β‐strand exchange loop and the minimal α‐crystallin domain of Mj Hsp16.5 need further study. In the present study, we constructed two fragment‐deletion mutants of Mj Hsp16.5, one with both the N‐ and C‐terminals deleted (ΔNΔC) and the other with a further deletion of the β‐strand exchange loop (ΔNΔLΔC). ΔNΔC existed as a dimer in solution. In contrast, the minimal α‐crystallin domain ΔNΔLΔC became polydisperse in solution and exhibited more efficient chaperone‐like activities to prevent amorphous aggregation of insulin B chain and fibril formation of the amyloidogenic peptide dansyl‐SSTSAA‐W than the mutant ΔNΔC and the wild type did. The hydrophobic probe binding experiments indicated that ΔNΔLΔC exposed much more hydrophobic surface than ΔNΔC. Our study also demonstrated that Mj Hsp16.5 used different mechanisms for protecting different substrates. Though Mj Hsp16.5 formed stable complexes with substrates when preventing thermal aggregation, no complexes were detected when preventing aggregation under non‐heat‐shock conditions. Proteins 2014; 82:1156–1167. © 2013 Wiley Periodicals, Inc.     

Arabidopsis dynamin‐related protein 1E in sphingolipid‐enriched plasma membrane domains is associated with the development of freezing tolerance

The freezing tolerance of Arabidopsis thaliana is enhanced by cold acclimation, resulting in changes in the compositions and function of the plasma membrane. Here, we show that a dynamin‐related protein 1E (DRP1E), which is thought to function in the vesicle trafficking pathway in cells, is related to an increase in freezing tolerance during cold acclimation. DRP1E accumulated in sphingolipid and sterol‐enriched plasma membrane domains after cold acclimation. Analysis of drp1e mutants clearly showed that DRP1E is required for full development of freezing tolerance after cold acclimation. DRP1E fused with green fluorescent protein was visible as small foci that overlapped with fluorescent dye‐labelled plasma membrane, providing evidence that DRP1E localizes non‐uniformly in specific areas of the plasma membrane. These results suggest that DRP1E accumulates in sphingolipid and sterol‐enriched plasma membrane domains and plays a role in freezing tolerance development during cold acclimation.     

Plant‐mediated effects of drought on aphid population structure and parasitoid attack

The effects of predicted climate change on aphid–natural enemy interactions have principally considered the effects of elevated carbon dioxide concentration and air temperature. However, increased incidence of summer droughts are also predicted in Northern Europe, which could affect aphid–plant interactions and aphid antagonists. We investigated how simulated summer drought affected the bird cherry–oat aphid, Rhopalosiphum padi L., and its natural enemy the parasitoid wasp Aphidius ervi. Drought and, to a greater extent, aphids reduced barley ( Hordeum vulgare) dry mass by 33% and 39%, respectively. Drought reduced leaf and root nitrogen concentrations by 13% and 28%, respectively, but foliar amino acid concentrations and composition remained similar. Aphid numbers were unaffected by drought, but population demography changed significantly; adults constituted 41% of the population on drought‐treated plants, but only 26% on those receiving ambient irrigation. Nymphs constituted 56% and 69% of the population on these plants, respectively, suggesting altered aphid development rates on drought‐stressed plants. Parasitism rates were significantly lower on drought‐stressed plants (9 attacks h^?1 compared with 35 attacks h^?1 on ambient‐irrigated plants), most likely because of lower incidence of nymphs and more adults, the latter being more difficult to parasitize. Any physiological changes in individual aphids did not affect parasitoid preferences, suggesting that attacks were postponed because of drought‐induced changes in aphid demography. This study demonstrates the potential for sporadic climate change events, such as summer drought, to be disruptive to herbivore–antagonist interactions.     

The chloroplast‐localized small heat shock protein Hsp21 associates with the thylakoid membranes in heat‐stressed plants

The small heat shock protein (sHsp) chaperones are crucial for cell survival and can prevent aggregation of client proteins that partially unfold under destabilizing conditions. Most investigations on the chaperone activity of sHsps are based on a limited set of thermosensitive model substrate client proteins since the endogenous targets are often not known. There is a high diversity among sHsps with a single conserved β‐sandwich fold domain defining the family, the α‐crystallin domain, whereas the N‐terminal and C‐terminal regions are highly variable in length and sequence among various sHsps and conserved only within orthologues. The endogenous targets are probably also varying among various sHsps, cellular compartments, cell type and organism. Here we have investigated Hsp21, a non‐metazoan sHsp expressed in the chloroplasts in green plants which experience huge environmental fluctuations not least in temperature. We describe how Hsp21 can also interact with the chloroplast thylakoid membranes, both when isolated thylakoid membranes are incubated with Hsp21 protein and when plants are heat‐stressed. The amount of Hsp21 associated with the thylakoid membranes was precisely determined by quantitative mass spectrometry after metabolic ¹⁵N‐isotope labeling of either recombinantly expressed and purified Hsp21 protein or intact Arabidopsis thaliana plants. We found that Hsp21 is among few proteins that become associated with the thylakoid membranes in heat‐stressed plants, and that approximately two thirds of the pool of chloroplast Hsp21 is affected. We conclude that for a complete picture of the role of sHsps in plant stress resistance also their association with the membranes should be considered.     

Transgenerational plasticity of inducible defences: Combined effects of grand‐parental,parental and current environments

Phenotypic plasticity can occur across generations (transgenerational plasticity) when environments experienced by the previous generations influenced offspring phenotype. The evolutionary importance of transgenerational plasticity, especially regarding within‐generational plasticity, is a currently hot topic in the plasticity framework. How long an environmental effect can persist across generations and whether multigenerational effects are cumulative are primordial—for the evolutionary significance of transgenerational plasticity—but still unresolved questions. In this study, we investigated how the grand‐parental, parental and offspring exposures to predation cues shape the predator‐induced defences of offspring in the Physa acuta snail. We expected that the offspring phenotypes result from a three‐way interaction among grand‐parental, parental and offspring environments. We exposed three generations of snails without and with predator cues according to a full factorial design and measured offspring inducible defences. We found that both grand‐parental and parental exposures to predator cues impacted offspring antipredator defences, but their effects were not cumulative and depended on the defences considered. We also highlighted that the grand‐parental environment did alter reaction norms of offspring shell thickness, demonstrating an interaction between the grand‐parental transgenerational plasticity and the within‐generational plasticity. We concluded that the effects of multigenerational exposure to predator cues resulted on complex offspring phenotypic patterns which are difficult to relate to adaptive antipredator advantages.     

A potential contamination error associated with insect protein mark‐capture data

Various types of protein‐spray solutions have proven effective for externally tagging arthropods for mark‐release‐recapture and mark‐capture type dispersal research. However, there is concern that certain standardized arthropod collection methods, such as sweep netting, might lead to high incidences of protein transfer from field‐marked to unmarked arthropods during sample collection and sample handling. Native arthropods were collected in sweep nets from a field of alfalfa, Medicago sativa L. (Fabaceae). The nets also contained 10 egg white‐, 10 bovine milk‐, 10 soy milk‐, and 10 water (control)‐marked Hippodamia convergens Guérin‐Méneville (Coleoptera: Coccinellidae) that were visually distinguishable by a yellow, white, green, and blue dot, respectively. The plant debris and arthropods from each sweep net collection were then placed into either a paper or a plastic bag and frozen for storage. The contents of each sweep net sample were thawed and the color‐coded H. convergens and field‐collected arthropods were examined for the presence of each protein by an egg white (albumin), bovine milk (casein), and soy milk (soy trypsin) enzyme‐linked immunosorbent assay (ELISA). Data revealed that only 0.67, 0.81, and 0% of the field‐collected unmarked arthropods acquired an egg white, bovine milk, and soy milk mark, respectively. ELISA results also showed that all the egg white‐marked H. convergens retained their mark, but 22.1% of the bovine milk‐marked and 5.1% of the soy milk‐marked H. convergens (color‐coded beetles) lost their mark during the collection and sample handling processes.