Small heat-shock proteins: important players in regulating cellular proteostasis

Small heat-shock proteins (sHsps) are a diverse family of intra-cellular molecular chaperone proteins that play a critical role in mitigating and preventing protein aggregation under stress conditions such as elevated temperature, oxidation and infection. In doing so, they assist in the maintenance of protein homeostasis (proteostasis) thereby avoiding the deleterious effects that result from loss of protein function and/or protein aggregation. The chaperone properties of sHsps are therefore employed extensively in many tissues to prevent the development of diseases associated with protein aggregation. Significant progress has been made of late in understanding the structure and chaperone mechanism of sHsps. In this review, we discuss some of these advances, with a focus on mammalian sHsp hetero-oligomerisation, the mechanism by which sHsps act as molecular chaperones to prevent both amorphous and fibrillar protein aggregation, and the role of post-translational modifications in sHsp chaperone function, particularly in the context of disease.SM was supported by a Royal Society Dorothy Hodgkin Fellowship, HE is supported by an Australian Research Council Future Fellowship (FT110100586) and JC is supported by a National Health and Medical Research Council Project Grant (#1068087)

Small heat-shock proteins: important players in regulating cellular proteostasis

Small heat-shock proteins (sHsps) are a diverse family of intra-cellular molecular chaperone proteins that play a critical role in mitigating and preventing protein aggregation under stress conditions such as elevated temperature, oxidation and infection. In doing so, they assist in the maintenance of protein homeostasis (proteostasis) thereby avoiding the deleterious effects that result from loss of protein function and/or protein aggregation. The chaperone properties of sHsps are therefore employed extensively in many tissues to prevent the development of diseases associated with protein aggregation. Significant progress has been made of late in understanding the structure and chaperone mechanism of sHsps. In this review, we discuss some of these advances, with a focus on mammalian sHsp hetero-oligomerisation, the mechanism by which sHsps act as molecular chaperones to prevent both amorphous and fibrillar protein aggregation, and the role of post-translational modifications in sHsp chaperone function, particularly in the context of disease

The Amyloid Fibril-Forming β-Sheet Regions of Amyloid β and α-Synuclein Preferentially Interact with the Molecular Chaperone 14-3-3ζ.

14-3-3 proteins are abundant, intramolecular proteins that play a pivotal role in cellular signal transduction by interacting with phosphorylated ligands. In addition, they are molecular chaperones that prevent protein unfolding and aggregation under cellular stress conditions in a similar manner to the unrelated small heat-shock proteins. In vivo, amyloid β (Aβ) and α-synuclein (α-syn) form amyloid fibrils in Alzheimer's and Parkinson's diseases, respectively, a process that is intimately linked to the diseases' progression. The 14-3-3ζ isoform potently inhibited in vitro fibril formation of the 40-amino acid form of Aβ (Aβ40) but had little effect on α-syn aggregation. Solution-phase NMR spectroscopy of 15N-labeled Aβ40 and A53T α-syn determined that unlabeled 14-3-3ζ interacted preferentially with hydrophobic regions of Aβ40 (L11-H21 and G29-V40) and α-syn (V3-K10 and V40-K60). In both proteins, these regions adopt β-strands within the core of the amyloid fibrils prepared in vitro as well as those isolated from the inclusions of diseased individuals. The interaction with 14-3-3ζ is transient and occurs at the early stages of the fibrillar aggregation pathway to maintain the native, monomeric, and unfolded structure of Aβ40 and α-syn. The N-terminal regions of α-syn interacting with 14-3-3ζ correspond with those that interact with other molecular chaperones as monitored by in-cell NMR spectroscopy

Dynamic changes in 5-hydroxymethylation signatures underpin early and late events in drug exposed liver

Aberrant DNA methylation is a common feature of neoplastic lesions, and early detection of such changes may provide powerful mechanistic insights and biomarkers for carcinogenesis. Here, we investigate dynamic changes in the mouse liver DNA methylome associated with short (1 day) and prolonged (7, 28 and 91 days) exposure to the rodent liver non-genotoxic carcinogen, phenobarbital (PB). We find that the distribution of 5mC/5hmC is highly consistent between untreated individuals of a similar age; yet, changes during liver maturation in a transcriptionally dependent manner. Following drug treatment, we identify and validate a series of differentially methylated or hydroxymethylated regions: exposure results in staged transcriptional responses with distinct kinetic profiles that strongly correlate with promoter proximal region 5hmC levels. Furthermore, reciprocal changes for both 5mC and 5hmC in response to PB suggest that active demethylation may be taking place at each set of these loci via a 5hmC intermediate. Finally, we identify potential early biomarkers for non-genotoxic carcinogenesis, including several genes aberrantly expressed in liver cancer. Our work suggests that 5hmC profiling can be used as an indicator of cell states during organ maturation and drug-induced responses and provides novel epigenetic signatures for non-genotoxic carcinogen exposur

Comparative analysis of affinity-based 5-hydroxymethylation enrichment techniques

The epigenetic modification of 5-hydroxymethylcytosine (5hmC) is receiving great attention due to its potential role in DNA methylation reprogramming and as a cell state identifier. Given this interest, it is important to identify reliable and cost-effective methods for the enrichment of 5hmC marked DNA for downstream analysis. We tested three commonly used affinity-based enrichment techniques; (i) antibody, (ii) chemical capture and (iii) protein affinity enrichment and assessed their ability to accurately and reproducibly report 5hmC profiles in mouse tissues containing high (brain) and lower (liver) levels of 5hmC. The protein-affinity technique is a poor reporter of 5hmC profiles, delivering 5hmC patterns that are incompatible with other methods. Both antibody and chemical capture-based techniques generate highly similar genome-wide patterns for 5hmC, which are independently validated by standard quantitative PCR (qPCR) and glucosyl-sensitive restriction enzyme digestion (gRES-qPCR). Both antibody and chemical capture generated profiles reproducibly link to unique chromatin modification profiles associated with 5hmC. However, there appears to be a slight bias of the antibody to bind to regions of DNA rich in simple repeats. Ultimately, the increased specificity observed with chemical capture-based approaches makes this an attractive method for the analysis of locus-specific or genome-wide patterns of 5hm

Investigation into the role of the germline epigenome in the transmission of glucocorticoid-programmed effects across generations.

BACKGROUND: Early life exposure to adverse environments affects cardiovascular and metabolic systems in the offspring. These programmed effects are transmissible to a second generation through both male and female lines, suggesting germline transmission. We have previously shown that prenatal overexposure to the synthetic glucocorticoid dexamethasone (Dex) in rats reduces birth weight in the first generation (F1), a phenotype which is transmitted to a second generation (F2), particularly through the male line. We hypothesize that Dex exposure affects developing germ cells, resulting in transmissible alterations in DNA methylation, histone marks and/or small RNA in the male germline. RESULTS: We profile epigenetic marks in sperm from F1 Sprague Dawley rats expressing a germ cell-specific GFP transgene following Dex or vehicle treatment of the mothers, using methylated DNA immunoprecipitation sequencing, small RNA sequencing and chromatin immunoprecipitation sequencing for H3K4me3, H3K4me1, H3K27me3 and H3K9me3. Although effects on birth weight are transmitted to the F2 generation through the male line, no differences in DNA methylation, histone modifications or small RNA were detected between germ cells and sperm from Dex-exposed animals and controls. CONCLUSIONS: Although the phenotype is transmitted to a second generation, we are unable to detect specific changes in DNA methylation, common histone modifications or small RNA profiles in sperm. Dex exposure is associated with more variable 5mC levels, particularly at non-promoter loci. Although this could be one mechanism contributing to the observed phenotype, other germline epigenetic modifications or non-epigenetic mechanisms may be responsible for the transmission of programmed effects across generations in this model

The Contribution of Accessory Toxins of Vibrio cholerae O1 El Tor to the Proinflammatory Response in a Murine Pulmonary Cholera Model

The contribution of accessory toxins to the acute inflammatory response to Vibrio cholerae was assessed in a murine pulmonary model. Intranasal administration of an El Tor O1 V. cholerae strain deleted of cholera toxin genes (ctxAB) caused diffuse pneumonia characterized by infiltration of PMNs, tissue damage, and hemorrhage. By contrast, the ctxAB mutant with an additional deletion in the actin-cross-linking repeats-in-toxin (RTX) toxin gene (rtxA) caused a less severe pathology and decreased serum levels of proinflammatory molecules interleukin (IL)-6 and murine macrophage inflammatory protein (MIP)-2. These data suggest that the RTX toxin contributes to the severity of acute inflammatory responses. Deletions within the genes for either hemagglutinin/protease (hapA) or hemolysin (hlyA) did not significantly affect virulence in this model. Compound deletion of ctxAB, hlyA, hapA, and rtxA created strain KFV101, which colonized the lung but induced pulmonary disease with limited inflammation and significantly reduced serum titers of IL-6 and MIP-2. 100% of mice inoculated with KFV101 survive, compared with 20% of mice inoculated with the ctxAB mutant. Thus, the reduced virulence of KFV101 makes it a prototype for multi-toxin deleted vaccine strains that could be used for protection against V. cholerae without the adverse effects of the accessory cholera toxins

Diverse interventions that extend mouse lifespan suppress shared age-associated epigenetic changes at critical gene regulatory regions

Background: Age-associated epigenetic changes are implicated in aging. Notably, age-associated DNA methylation changes comprise a so-called aging “clock”, a robust biomarker of aging. However, while genetic, dietary and drug interventions can extend lifespan, their impact on the epigenome is uncharacterised. To fill this knowledge gap, we defined age-associated DNA methylation changes at the whole-genome, single-nucleotide level in mouse liver and tested the impact of longevity-promoting interventions, specifically the Ames dwarf Prop1 df/df mutation, calorie restriction and rapamycin. Results: In wild-type mice fed an unsupplemented ad libitum diet, age-associated hypomethylation was enriched at super-enhancers in highly expressed genes critical for liver function. Genes harbouring hypomethylated enhancers were enriched for genes that change expression with age. Hypermethylation was enriched at CpG islands marked with bivalent activating and repressing histone modifications and resembled hypermethylation in liver cancer. Age-associated methylation changes are suppressed in Ames dwarf and calorie restricted mice and more selectively and less specifically in rapamycin treated mice. Conclusions: Age-associated hypo- and hypermethylation events occur at distinct regulatory features of the genome. Distinct longevity-promoting interventions, specifically genetic, dietary and drug interventions, suppress some age-associated methylation changes, consistent with the idea that these interventions exert their beneficial effects, in part, by modulation of the epigenome. This study is a foundation to understand the epigenetic contribution to healthy aging and longevity and the molecular basis of the DNA methylation clock

The interlayer cohesive energy of graphite from thermal desorption of polyaromatic hydrocarbons

We have studied the interaction of polyaromatic hydrocarbons (PAHs) with the basal plane of graphite using thermal desorption spectroscopy. Desorption kinetics of benzene, naphthalene, coronene and ovalene at sub-monolayer coverages yield activation energies of 0.50 eV, 0.85 eV, 1.40 eV and 2.1 eV, respectively. Benzene and naphthalene follow simple first order desorption kinetics while coronene and ovalene exhibit fractional order kinetics owing to the stability of 2-D adsorbate islands up to the desorption temperature. Pre-exponential frequency factors are found to be in the range $10^{14}$-$10^{21} s^{-1}$ as obtained from both Falconer--Madix (isothermal desorption) analysis and Antoine's fit to vapour pressure data. The resulting binding energy per carbon atom of the PAH is $52\pm$5 meV and can be identified with the interlayer cohesive energy of graphite. The resulting cleavage energy of graphite is $61\pm5$~meV/atom which is considerably larger than previously reported experimental values.Comment: 8 pages, 4 figures, 2 table