LC-MS proteomics analysis of the iInsulin/IGF-1-deficient Caenorhabditis elegans daf-2(e1370) mutant reveals extensive restructuring of intermediary metabolism

The insulin/IGF-1 receptor is a major known determinant of dauer formation, stress resistance, longevity, and metabolism in Caenorhabditis elegans. In the past, whole-genome transcript profiling was used extensively to study differential gene expression in response to reduced insulin/IGF-1 signaling, including the expression levels of metabolism-associated genes. Taking advantage of the recent developments in quantitative liquid chromatography mass spectrometry (LC-MS)-based proteomics, we profiled the proteomic changes that occur in response to activation of the DAF-16 transcription factor in the germline-less glp-4(bn2);daf-2(e1370) receptor mutant. Strikingly, the daf-2 profile suggests extensive reorganization of intermediary metabolism, characterized by the upregulation of many core intermediary metabolic pathways. These include glycolysis/gluconeogenesis, glycogenesis, pentose phosphate cycle, citric acid cycle, glyoxylate shunt, fatty acid beta-oxidation, one-carbon metabolism, propionate and tyrosine catabolism, and complexes I, II, III, and V of the electron transport chain. Interestingly, we found simultaneous activation of reciprocally regulated metabolic pathways, which is indicative of spatiotemporal coordination of energy metabolism and/or extensive post-translational regulation of these enzymes. This restructuring of daf-2 metabolism is reminiscent to that of hypometabolic dauers, allowing the efficient and economical utilization of internal nutrient reserves and possibly also shunting metabolites through alternative energy-generating pathways to sustain longevity

Gray Matter Is Targeted in First-Attack Multiple Sclerosis

The cause of multiple sclerosis (MS), its driving pathogenesis at the earliest stages, and what factors allow the first clinical attack to manifest remain unknown. Some imaging studies suggest gray rather than white matter may be involved early, and some postulate this may be predictive of developing MS. Other imaging studies are in conflict. To determine if there was objective molecular evidence of gray matter involvement in early MS we used high-resolution mass spectrometry to identify proteins in the cerebrospinal fluid (CSF) of first-attack MS patients (two independent groups) compared to established relapsing remitting (RR) MS and controls. We found that the CSF proteins in first-attack patients were differentially enriched for gray matter components (axon, neuron, synapse). Myelin components did not distinguish these groups. The results support that gray matter dysfunction is involved early in MS, and also may be integral for the initial clinical presentation

HAM-5-GFP localization in WT and Δ<i>mak-2</i> germlings.

<p>(A) Schematic overview of HAM-5 protein structure. The predicted WD40 domains are shown in grey and the putative coiled coil domains are shown as red bars. The two disordered regions with low complexity are depicted by shaded white boxes. The MAPK phosphorylation site (aa 506) is marked by a blue star, the other two sites showing decreased abundance in treated cells (aa 1288 and 1604) are marked by green stars, and other 13 identified phosphorylation sites (S14, S414, S792, S818, S833, T838, T969, S1085, S1199, T1201, S1202, T1353, S1608) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004783#pgen.1004783-Xiong1" target="_blank">[40]</a> are marked by black stars. The putative MAPK docking site is marked by a yellow line. (B) Localization of HAM-5-GFP to puncta localized to CAT tips during chemotropic interactions between genetically identical cells. HAM-5-GFP showed dynamic localization to CAT tips of germlings with an oscillation of every four min (arrow). HAM-5-GFP also localized to puncta within germlings and near nuclear compartments devoid of HAM-5-GFP (asterisks). The image left is a bright field image. Scale bar  = 10 µM. (C) HAM-5-GFP localized to the sites of contact during germling fusion (arrow). (D) Western blots of WT, WT (<i>ham-5-gfp</i>) and Δ<i>mak-2</i> (<i>ham-5-gfp</i>) germlings with immunoprecipitated HAM-5-GFP probed with anti-GFP antibodies (right panel shows longer run showing higher mobility of HAM-5-GFP in wild type germlings) specifically detecting HAM-5-GFP (210 kD; <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004783#pgen.1004783.s004" target="_blank">Figure S4D</a>). Lower panel shows a Western blot with identical samples probed with anti-phospho antibodies that specifically detect phosphorylated serine or threonine residues followed by a proline. (E) Localization of HAM-5-GFP to puncta in Δ<i>mak-2</i> germlings. Some puncta showed localization to germling tips, but which did not oscillate during growth (white arrows). Scale bar  = 10 µM.</p

Summary of phosphoproteomics results conducted on Δ<i>mak-2^Q100G</i> mutant.

<p>(A) Overview of FunCat categories of proteins harboring the identified phosphopeptides. The upper bar shows the categories of all the proteins with identified phosphopeptides (3200 phosphopeptides, 1164 proteins), the middle bar shows functional categories of proteins with phosphopeptides that showed higher abundance after inhibition (33 phosphopeptides, 27 proteins) and the lower bar shows FunCat analysis of the proteins with phosphopeptides that showed lower abundance in <i>mak-2^Q100G</i> germlings after treatment with 1NM-PP1 (96 phosphopeptides, 67 proteins). (B) Pie chart showing the relative percentages and absolute numbers of single, double, triple and quadruple phosphosites per peptide. (C) Pie chart showing the relative percentages and absolute numbers of phosphorylated serine, threonine and tyrosine in all peptides found.</p

HAM-5-GFP shows localization with components of the MAK-2 pathway.

<p>(A) Co-localization of HAM-5-GFP and MAK-2-mCherry during germling communication (arrows). (B) Co-localization of HAM-5-GFP and MEK-2-mCherry during germling communication (arrows). (C) Co-localization of HAM-5-GFP and NRC-1-mCherry during germling communication. NRC-1-mCherry strains show low fluorescence <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004783#pgen.1004783-Dettmann1" target="_blank">[18]</a>. (D) HAM-5-GFP and SO-mCherry do not co-localize during chemotropic interactions, but instead show opposite localization to CAT tips in communicating germlings (arrows). The images on the left are bright field images, fluorescent images on the right. Scale bar  = 10 µM. (E) Co-immunoprecipitation experiments showing an interaction between HAM-5-GFP (210 kD; <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004783#pgen.1004783.s004" target="_blank">Figure S4D</a>) and MEK-2-mCherry (82.9 kD; <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004783#pgen.1004783.s004" target="_blank">Figure S4C</a>) and NRC-1-mCherry (128 kD; <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004783#pgen.1004783.s004" target="_blank">Figure S4C</a>). Input panels show Western blots of immunoprecipitated protein samples from 5 hr-old germlings probed with either anti-GFP (free GFP  = 27 kD; <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004783#pgen.1004783.s004" target="_blank">Figure S4D</a>) or anti-mCherry antibodies. The output panel is a Western blot of proteins immunoprecipitated by anti-GFP antibodies (and thus HAM-5-GFP) and probed with anti-mCherry antibodies (detecting MEK-2-mCherry or NRC-1-mCherry).</p