UDP-N-Acetylglucosamine 2-Epimerase/N-Acetylmannosamine Kinase (GNE) Binds to Alpha-Actinin 1: Novel Pathways in Skeletal Muscle?

Hereditary inclusion body myopathy (HIBM) is a rare neuromuscular disorder caused by mutations in GNE, the key enzyme in the biosynthetic pathway of sialic acid. While the mechanism leading from GNE mutations to the HIBM phenotype is not yet understood, we searched for proteins potentially interacting with GNE, which could give some insights about novel putative biological functions of GNE in muscle. We used a Surface Plasmon Resonance (SPR)-Biosensor based assay to search for potential GNE interactors in anion exchanged fractions of human skeletal muscle primary culture cell lysate. Analysis of the positive fractions by in vitro binding assay revealed alpha-actinin 1 as a potential interactor of GNE. The direct interaction of the two proteins was assessed in vitro by SPR-Biosensor based kinetics analysis and in a cellular environment by a co-immunoprecipitation assay in GNE overexpressing 293T cells. Furthermore, immunohistochemistry on stretched mouse muscle suggest that both GNE and alpha-actinin 1 localize to an overlapping but not identical region of the myofibrillar apparatus centered on the Z line. The interaction of GNE with alpha-actinin 1 might point to its involvement in alpha-actinin mediated processes. In addition these studies illustrate for the first time the expression of the non-muscle form of alpha-actinin, alpha-actinin 1, in mature skeletal muscle tissue, opening novel avenues for its specific function in the sarcomere. Although no significant difference could be detected in the binding kinetics of alpha-actinin 1 with either wild type or mutant GNE in our SPR biosensor based analysis, further investigation is needed to determine whether and how the interaction of GNE with alpha-actinin 1 in skeletal muscle is relevant to the putative muscle-specific function of alpha-actinin 1, and to the muscle-restricted pathology of HIBM

Over Expression of the Cyanobacterial Pgr5-Homologue Leads to Pseudoreversion in a Gene Coding for a Putative Esterase in Synechocystis 6803

Pgr5 proteins play a major direct role in cyclic electron flow paths in plants and eukaryotic phytoplankton. The genomes of many cyanobacterial species code for Pgr5-like proteins but their function is still uncertain. Here, we present evidence that supports a link between the Synechocystis sp. PCC6803 Pgr5-like protein and the regulation of intracellular redox balance. The knockout strain, pgr5KO, did not display substantial phenotypic response under our experimental conditions, confirming results obtained in earlier studies. However, the overexpression strain, pgr5OE, accumulated 2.5-fold more chlorophyll than the wild type and displayed increased content of photosystems matching the chlorophyll increase. As a result, electron transfer rates through the photosynthetic apparatus of pgr5OE increased, as did the amount of energy stored as glycogen. While, under photoautotrophic conditions, this metabolic difference had only minor effects, under mixotrophic conditions, pgr5OE cultures collapsed. Interestingly, this specific phenotype of pgr5OE mutants displayed a tendency for reverting, and cultures which previously collapsed in the presence of glucose were now able to survive. DNA sequencing of a pgr5OE strain revealed a second site suppression mutation in slr1916, a putative esterase associated with redox regulation. The phenotype of the slr1916 knockout is very similar to that of the strain reported here and to that of the pmgA regulator knockout. These data demonstrate that, in Synechocystis 6803, there is strong selection against overexpression of the Pgr5-like protein. The pseudoreversion event in a gene involved in redox regulation suggests a connection of the Pgr5-like protein to this network

Regulation of thylakoid protein phosphorylation at the substrate level: Reversible light-induced conformational changes expose the phosphorylation site of the light-harvesting complex II

Light-dependent activation of thylakoid protein phosphorylation regulates the energy distribution between photosystems I and II of oxygen-evolving photosynthetic eukaryotes as well as the turnover of photosystem II proteins. So far the only known effect of light on the phosphorylation process is the redox-dependent regulation of the membrane-bound protein kinase(s) activity via plastoquinol bound to the cytochrome bf complex and the redox state of thylakoid dithiols. By using a partially purified thylakoid protein kinase and isolated native chlorophyll (chl) a/b light-harvesting complex II (LHCII), as well as recombinant LHCII, we find that illumination of the chl-protein substrate exposes the phosphorylation site to the kinase. Light does not activate the phosphorylation of the LHCII apoprotein nor the recombinant pigment-reconstituted complex lacking the N-terminal domain that contains the phosphothreonine site. The suggested light-induced conformational change exposing the N-terminal domain of LHCII to the kinase is evidenced also by an increase in its accessibility to tryptic cleavage after light exposure. Light activates preferentially the trimeric form of LHCII, and the process is paralleled by chl fluorescence quenching. Both phenomena are slowly reversible in darkness. Light-induced exposure of the LHCII N-terminal domain to the endogenous protein kinase(s) and tryptic cleavage occurs also in thylakoid membranes. These results demonstrate that light may regulate thylakoid protein phosphorylation not only via the signal transduction chain connecting redox reactions to the protein kinase activation, but also by affecting the conformation of the chl-protein substrate

MISF2 Encodes an Essential Mitochondrial Splicing Cofactor Required for nad2 mRNA Processing and Embryo Development in Arabidopsis thaliana

Mitochondria play key roles in cellular energy metabolism in eukaryotes. Mitochondria of most organisms contain their own genome and specific transcription and translation machineries. The expression of angiosperm mtDNA involves extensive RNA-processing steps, such as RNA trimming, editing, and the splicing of numerous group II-type introns. Pentatricopeptide repeat (PPR) proteins are key players in plant organelle gene expression and RNA metabolism. In the present analysis, we reveal the function of the MITOCHONDRIAL SPLICING FACTOR 2 gene (MISF2, AT3G22670) and show that it encodes a mitochondria-localized PPR protein that is crucial for early embryo development in Arabidopsis. Molecular characterization of embryo-rescued misf2 plantlets indicates that the splicing of nad2 intron 1, and thus respiratory complex I biogenesis, are strongly compromised. Moreover, the molecular function seems conserved between MISF2 protein in Arabidopsis and its orthologous gene (EMP10) in maize, suggesting that the ancestor of MISF2/EMP10 was recruited to function in nad2 processing before the monocot–dicot divergence ~200 million years ago. These data provide new insights into the function of nuclear-encoded factors in mitochondrial gene expression and respiratory chain biogenesis during plant embryo development

Kinetics BIAcore analysis for the interaction of GNE with α-actinin 1.

<p>Kinetics values of the interactions of WT and mutant GNE proteins with α-actinin 1 as calculated using 1∶1 Langmuir model.</p

WT-GNE interacts with proteins within anion-exchanged fractions of muscle cell lysate.

<p>[A] Anion exchange chromatography (0–1 M NaCl) of cell lysate (15 mg total protein content) extracted from human skeletal muscle primary culture cells. The numbers of the positive fractions appear in red on the X axis. [B] BIAcore sensorgrams of the interaction of WT-GNE with anion exchange fractions #11–16 [Resp. Diff., response difference; RU, response units; B, buffer B (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002477#s2" target="_blank">Materials and methods</a>)].</p

LDH-B and α-actinin 1 precipitate with GNE by <i>in vitro</i> binding assay.

<p>Gelcode reagent staining of in vitro binding assay eluates resolved by SDS-PAGE. Numbers (11–13, 14–16) correspond to anion-exchange pooled fractions. A, proteins bound to GNE-bound nickel beads; B, proteins bound to GNE non-bound nickel beads; C, control proteins bound to GNE-bound nickel beads. Black arrows point to either α-actinin 1 (∼100 kDa) or to LDH-B (∼37 kDa). Both proteins were identified by MS (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002477#pone-0002477-t001" target="_blank">Table 1</a>).</p