Identification of N-terminal protein acetylation and arginine methylation of the voltage-gated sodium channel in end-stage heart failure human heart

The α subunit of the cardiac voltage-gated sodium channel, Naᵥ1.5, provides the rapid sodium inward current that initiates cardiomyocyte action potentials. Here, we analyzed for the first time the post-translational modifications of Naᵥ1.5 purified from end-stage heart failure human cardiac tissue. We identified R526 methylation as the major post-translational modification of any Naᵥ1.5 arginine or lysine residue. Unexpectedly, we found that the N terminus of Naᵥ1.5 was: 1) devoid of the initiation methionine, and 2) acetylated at the resulting initial alanine residue. This is the first evidence for N-terminal acetylation in any member of the voltage-gated ion channel superfamily. Our results open the door to explore Naᵥ1.5 N-terminal acetylation and arginine methylation levels as drivers or markers of end-stage heart failure

A Novel Peptide-Based SILAC Method to Identify the Posttranslational Modifications Provides Evidence for Unconventional Ubiquitination in the ER-Associated Degradation Pathway.

The endoplasmic reticulum-associated degradation (ERAD) pathway is responsible for disposing misfolded proteins from the endoplasmic reticulum by inducing their ubiquitination and degradation. Ubiquitination is conventionally observed on lysine residues and has been demonstrated on cysteine residues and protein N-termini. Ubiquitination is fundamental to the ERAD process; however, a mutant T-cell receptor α (TCRα) lacking lysine residues is targeted for the degradation by the ERAD pathway. We have shown that ubiquitination of lysine-less TCRα occurs on internal, non-lysine residues and that the same E3 ligase conjugates ubiquitin to TCRα in the presence or absence of lysine residues. Mass-spectrometry indicates that WT-TCRα is ubiquitinated on multiple lysine residues. Recent publications have provided indirect evidence that serine and threonine residues may be modified by ubiquitin. Using a novel peptide-based stable isotope labeling in cell culture (SILAC) approach, we show that specific lysine-less TCRα peptides become modified. In this study, we demonstrate that it is possible to detect both ester and thioester based ubiquitination events, although the exact linkage on lysine-less TCRα remains elusive. These findings demonstrate that SILAC can be used as a tool to identify modified peptides, even those with novel modifications that may not be detected using conventional proteomic work flows or informatics algorithms

Exploring sugar metabolism in bread wheat for improving drought tolerance

Remobilization of stem WSC is well known to contribute to grain yield in wheat. There is, however, extensive genetic variation in the contribution of stem WSC to grain yield under post-anthesis water-deficit. Fructan 1-exohydrolase (1-FEH) is one of the major enzymes contributing to WSC remobilisation and the maintenance of grain yield under water-deficit. 1-FEH has three isoforms (1-FEH w1, w2 and w3) that degrade β - (2-1) fructan linkages thus contributing to fructan remobilization to grain. This thesis investigated the functional role of the three isoforms of the 1-FEH gene in WSC remobilisation under post anthesis water-deficit. Individual performance of the three isoforms was investigated using the corresponding isoform mutation lines derived from the Australian wheat variety Chara. Results from glasshouse experiments showed that the mutation of isoform 1-FEH w3 slowed down WSC remobilisation under post anthesis water-deficit and reduced grain filling and yield. In contrast, mutations of 1-FEH w1 and w2 did not affect WSC remobilisation under water-deficit. This means that 1-FEH w3 plays the leading functional role in WSC remobilisation during grain filling under water-deficit. This differences in remobilisation of WSC components between the mutation lines correlated with the expressional differences of the three isoforms of the 1-FEH gene across the lines. In the 1-FEH w3 mutation line, the expression of the other two isoforms (1-FEH w2 and w1) had the same level as the non-mutated parental cultivar Chara. However, in the 1-FEH w2 and w1 mutation lines, 1-FEH w3 showed significantly higher expression compared to Chara. The results indicated that the functional loss of the isoforms 1-FEH w2 and w1 was made up by the higher expression of the isoform 1-FEH w3 but the functional loss of the 1-FEH w3 isoform was not compensated by the other isoforms. This explains the ability of 1-FEH w2 and w1 mutation lines to maintain the same pattern of WSC remobilisation as the non-mutated parental cultivar. It was also, revealed that the expressional differences of the isforms of the 1-FEH gene across different mutation lines significantly influenced the degradation of WSC and its components under post anthesis water-deficit. Fructan, a fructose-based polymer synthesized from sucrose by fructosyltransferases (FTs), is the main component of wheat stem WSC and is a major source of sugar supply under post anthesis water-deficit when photosynthesis is reduced. Quick degradation of fructan is essential to remobilise sugar to developing grain under water-deficit and this is facilitated by FEHs. The 1-FEH w3 mutation line showed slower degradation and remobilization of fructan compared to the 1-FEH w2 and w1 mutation lines and Chara. This slow degradation made the 1-FEH w3 mutation line partially susceptible to post anthesis water-deficit. Noticeably, differences in WSC component degradation and gene expression of 1-FEH isoforms only became evident under post anthesis water-deficit and not in well-watered plants. This thesis also characterised the 1-FEH gene mutation, by mapping and annotating the mutated region. The F1 seeds, developed by back crossing the 1-FEH w1, w2 and w3 mutation lines with Chara, were genotyped using the Infinium 90K SNP iSelect platform. Putative deletions were identified in the FEH mutation lines encompassing the FEH genomic regions. A total of 15, 20 and 15SNPs were identified within the mutation regions of 1-FEH w1 w2, and w3, respectively. Mapping analysis demonstrated that the mutation affected significantly longer regions than the target gene regions of 1-FEH w1, w3 and w2. From the annotation of the mutation regions, 8 and 6 non-target genes were discovered on chromosomes 6A and 6B, respectively. The annotation of the 1-FEH w2 mutated region was complicated by the presence of an extra three copies of the gene on chromosome 6D. Functional roles of the non-target genes was carried out following computational biology approaches and confirmed that none of the affected non-target genes were expected to have a direct influence on 1-FEH gene function. This study also ratified the association of the distinct role of the 1-FEH w3 gene in sugar remobilisation to the developing wheat grain. Accumulation of oligosaccharides at two seed developmental stages were examined in the 1-FEH w3 mutation line in comparison to Chara under well-watered and water-deficit conditions. This study successfully overcome the challenge of preparing 25 μm seed sections by adopting cryosectioning using egg white which provided compatibility with the mass spectrometric equipment and enabled the production of ions from the oligosaccharides by the laser. Hexose and its polymers were detected separately by the mass spectrometry imaging (MSI) without any enzymatic digestion thus providing information regarding the localisation of sugar accumulation within the tissues of developing seeds. The abundance and localisation pattern of the identified oligosaccharides was influenced by the post anthesis water-deficit treatment. Under water-deficit, the mutation of the 1-FEH w3 reduced the abundance of oligosaccharide accumulation in two stages of seed development (17 DAA and 22 DAA) indicating it pivotal role under post anthesis water-deficit. This is the first study to use MSI to explore sugar accumulation directly within the tissue of developing seeds of wheat. This thesis established the individual role of three isoforms of 1-FEH in remobilising WSC under post anthesis water-deficit and provides unequivocal evidence that 1-FEH w3 is taking the most vital role. This new insight into the distinct role of the 1-FEH gene isoforms under post anthesis water-deficit should assist in providing new gene targets for water-deficit tolerant wheat breeding in the future

NsrR from Streptomyces coelicolor is a nitric oxide-sensing [4Fe-4S] cluster protein with a specialized regulatory function

The Rrf2 family transcription factor NsrR controls expression of genes in a wide range of bacteria in response to nitric oxide (NO). The precise form of the NO-sensing module of NsrR is the subject of controversy because NsrR proteins containing either [2Fe-2S] or [4Fe-4S] clusters have been observed previously. Optical, Mössbauer, resonance Raman spectroscopies and native mass spectrometry demonstrate that Streptomyces coelicolor NsrR (ScNsrR), previously reported to contain a [2Fe-2S] cluster, can be isolated containing a [4Fe-4S] cluster. ChIP-seq experiments indicated that the ScNsrR regulon is small, consisting of only hmpA1, hmpA2, and nsrR itself. The hmpA genes encode NO-detoxifying flavohemoglobins, indicating that ScNsrR has a specialized regulatory function focused on NO detoxification and is not a global regulator like some NsrR orthologues. EMSAs and DNase I footprinting showed that the [4Fe-4S] form of ScNsrR binds specifically and tightly to an 11-bp inverted repeat sequence in the promoter regions of the identified target genes and that DNA binding is abolished following reaction with NO. Resonance Raman data were consistent with cluster coordination by three Cys residues and one oxygen-containing residue, and analysis of ScNsrR variants suggested that highly conserved Glu-85 may be the fourth ligand. Finally, we demonstrate that some low molecular weight thiols, but importantly not physiologically relevant thiols, such as cysteine and an analogue of mycothiol, bind weakly to the [4Fe-4S] cluster, and exposure of this bound form to O2 results in cluster conversion to the [2Fe-2S] form, which does not bind to DNA. These data help to account for the observation of [2Fe-2S] forms of NsrR

Lifespan extension in Caenorhabditis elegans insulin/IGF-1 signalling mutants is supported by non-vertebrate physiological traits

The insulin/IGF-1 signalling (IIS) pathway connects nutrient levels to metabolism, growth and lifespan in eukaryotes ranging from yeasts to humans, including nematodes such as the genetic model organism Caenorhabditis elegans. The link between ageing and the IIS pathway has been thoroughly studied in C. elegans; upon reduced IIS signalling, a genetic survival program is activated resulting in a drastic lifespan extension. One of the components of this program is the upregulation of antioxidant activity but experiments failed to show a clear causal relation to longevity. However, oxidative damage, such as protein carbonyls, accumulates at a slower pace in long-lived C. elegans mutants with reduced IIS. This is probably not achieved by increased macroautophagy, a process that sequesters cellular components to be eliminated as protein turnover rates are slowed down in IIS mutants. The IIS mutant daf-2, bearing a mutation in the insulin/IGF-1 receptor, recapitulates the dauer survival program, including accumulation of fat and glycogen. Fat can be converted into glucose and glycogen via the glyoxylate shunt, a pathway absent in vertebrates. These carbohydrates can be used as substrates for trehalose synthesis, also absent in mammals. Trehalose, a non-reducing homodimer of glucose, stabilises intracellular components and is responsible for almost half of the lifespan extension in IIS mutants. Hence, the molecular mechanisms by which lifespan is extended under reduced IIS may differ substantially between phyla that have an active glyoxylate cycle and trehalose synthesis, such as ecdysozoans and fungi, and vertebrate species such as mammals

Phenotypic convergence in bacterial adaptive evolution to ethanol stress

Stability of ethanol tolerance. Strain F at the end point (2,500 h) and at 576 h was cultivated for 200 generations absent ethanol stress. After the cultivation, ethanol tolerance was evaluated by measuring specific growth rates in 5 % ethanol stress (red bars). The growth rates under ethanol stress were similar to those before the non-stress cultivation (blue bars) and were significantly higher than that of the parent strain. (PDF 976 kb

ZZE-Configuration of chromophore ß-153 in C-phycocyanin from Mastigocladus laminosus

The photochemistry of C-phycocyanin has been studied after denaturation in the dark. It shows an irreversible reaction which has characteristics of a Ζ,Ζ,Ε- to Z,Z,Z-isomerization of dihydrobilins. Its amplitude depends on the reaction conditions, with a maximum corresponding to 15% conversion of one of the three PC chromophores. This chromophore is suggested to be ß-153, for which recent X-ray data T. Schirmer, W. Bode, and R. Huber, J. Mol. Biol., submitted, show ring D being highly twisted out of the plane of the other rings. During unfolding, there is thus a probability of falling into the photochemically labile Z,Z,^-configuration

Stage 1 Second Year Review of Value Added Wheat CRC Ltd

Established and supported under the Australian Government’s Cooperative Research Centre Progra