New Partners Identified by Mass Spectrometry Assay Reveal Functions of NCAM2 in Neural Cytoskeleton Organization.

Neuronal cell adhesion molecule 2 (NCAM2) is a membrane protein with an important role in the morphological development of neurons. In the cortex and the hippocampus, NCAM2 is essential for proper neuronal differentiation, dendritic and axonal outgrowth and synapse formation. However, little is known about NCAM2 functional mechanisms and its interactive partners during brain development. Here we used mass spectrometry to study the molecular interactome of NCAM2 in the second postnatal week of the mouse cerebral cortex. We found that NCAM2 interacts with >100 proteins involved in numerous processes, including neuronal morphogenesis and synaptogenesis. We validated the most relevant interactors, including Neurofilaments (NEFs), Microtubule-associated protein 2 (MAP2), Calcium/calmodulin kinase II alpha (CaMKIIα), Actin and Nogo. An in silico analysis of the cytosolic tail of the NCAM2.1 isoform revealed specific phosphorylation site motifs with a putative affinity for some of these interactors. Our results expand the knowledge of NCAM2 interactome and confirm the key role of NCAM2 in cytoskeleton organization, neuronal morphogenesis and synaptogenesis. These findings are of interest in explaining the phenotypes observed in different pathologies with alterations in the NCAM2 gene.

Acetylation of GAGA factor modulates its interaction with DNA.

GAGA is a Drosophila transcription factor that shows a high degree of post-translational modification. Here, we show that GAGA factor is acetylated in vivo. Lysine residues K325 and K373 on basic regions BR1 and BR3 of the DNA binding domain, respectively, are shown to be acetylated by PCAF. While BR1 is strictly required to stabilize DNA binding, BR3 is dispensable. However, acetylation of both lysine residues, either alone or in combination, weakens the binding to DNA. Despite the high degree of conservation of K325 and K373 in flies, their mutation to glutamine does not affect DNA binding. Molecular dynamics simulations, using acetylated K325 and a K325Q mutant of GAGA DNA binding domain in complex with DNA, are fully consistent with these results and provide a thermodynamic explanation for this observation. We propose that while K325 and K373 are not essential for DNA binding they have been largely conserved for regulatory purposes, thus highlighting a key regulatory system for GAGA factor in flies.

Desmin is oxidized and nitrated in affected muscles in myotilinopathies and desminopathies.

Degenerative diseases with abnormal protein aggregates are characterized by the accumulation of proteins with variable posttranslational modifications including phosphorylation, glycoxidation, oxidation, and nitration. Myofibrillar myopathies, including myotilinopathies and desminopathies, are characterized by the intracytoplasmic focal accumulation of proteins in insoluble aggregates in muscle cells. By using single immunohistochemistry, monodimensional gel electrophoresis and Western blotting, and bidimensional gel electrophoresis, in-gel digestion, and mass spectometry, desmin was demonstrated to be a major target of oxidation and nitration in both desminopathies and myotilinopathies. Because oxidized and nitrated proteins may have toxic effects and may impair ubiquitin-proteasomal function, modified desmin can be considered to be an additional element in the pathogenesis of myofibrillar myopathies. In addition to desmin, pyruvate kinase muscle splice form M1 is oxidized, thus supporting complemental mitochondrial damage, at least in some cases of myotilinopathy.

A multicentric study to evaluate the use of relative retention times in targeted proteomics.

Despite the maturity reached by targeted proteomic strategies, reliable and standardized protocols are urgently needed to enhance reproducibility among different laboratories and analytical platforms, facilitating a more widespread use in biomedical research. To achieve this goal, the use of dimensionless relative retention times (iRT), defined on the basis of peptide standard retention times (RT), has lately emerged as a powerful tool. The robustness, reproducibility and utility of this strategy were examined for the first time in a multicentric setting, involving 28 laboratories that included 24 of the Spanish network of proteomics laboratories (ProteoRed-ISCIII). According to the results obtained in this study, dimensionless retention time values (iRTs) demonstrated to be a useful tool for transferring and sharing peptide retention times across different chromatographic set-ups both intra- and inter-laboratories. iRT values also showed very low variability over long time periods. Furthermore, parallel quantitative analyses showed a high reproducibility despite the variety of experimental strategies used, either MRM (multiple reaction monitoring) or pseudoMRM, and the diversity of analytical platforms employed. BIOLOGICAL SIGNIFICANCE: From the very beginning of proteomics as an analytical science there has been a growing interest in developing standardized methods and experimental procedures in order to ensure the highest quality and reproducibility of the results. In this regard, the recent (2012) introduction of the dimensionless retention time concept has been a significant advance. In our multicentric (28 laboratories) study we explore the usefulness of this concept in the context of a targeted proteomics experiment, demonstrating that dimensionless retention time values is a useful tool for transferring and sharing peptide retention times across different chromatographic set-ups.

Redox proteomic profiling of neuroketal-adducted proteins in human brain: Regional vulnerability at middle age increases in the elderly.

Protein lipoxidation was assessed in the parietal cortex (PC), frontal cortex (FC), and cingulate gyrus (CG) in middle-aged and old-aged individuals with no clinical manifestations of cognitive impairment, in order to increase understanding of regional brain vulnerability to oxidative damage during aging. Twenty-five lipoxidized proteins were identified in all the three regions although with regional specificities, by using redox proteomics to detect target proteins of neuroketals (NKT) adduction. The number of cases with NKT-adducted proteins was higher in old-aged individuals but most oxidized proteins were already present in middle-aged individuals. Differences in vulnerability to oxidation were dependent on the sub-cellular localization, secondary structure, and external exposition of certain amino acids. Lipoxidized proteins included those involved in energy metabolism, cytoskeleton, proteostasis, neurotransmission and O2/CO2, and heme metabolism. Total NKT and soluble oligomer levels were estimated employing slot-blot, and these were compared between age groups. Oligomers increased with age in PC and FC; NKT significantly increased with age in FC, whereas total NKT and oligomer levels were not modified in CG, thus highlighting differences in brain regional vulnerability with age. Oligomers significantly correlated with NKT levels in the three cortical regions, suggesting that protein NKT adduction parallels soluble oligomer formation.

Modification of brain lipids but not phenotype in alpha-synucleinopathy transgenic mice by long-term dietary n-3 fatty acids.

Transgenic mice expressing both wild mouse alpha-synuclein and the Parkinson's disease associated A53T mutated human alpha-synuclein were subjected to long-term diets impoverished in n-3 or diets impoverished in n-3 and supplemented with docosahexaenoic acid (DHA) for 6 months. Transgenic mice evidenced mild phenotype characterized by increased total alpha-synuclein expression, truncated alpha-synuclein forms, and abnormal solubility and aggregation, in the absence of Lewy bodies and neurites, and lack of apparent neuronal loss, astrocytosis and microgliosis. These diets produced a reduction in the content of linolenic, n-3 docosapentaenoic and total polyunsaturated fatty acids, leading to significantly lower double bond and peroxidizability indexes as well as to lower protein oxidative damage, with no effects in alpha-synuclein expression and with no modifications in the number of cortical astrocytes and microglial cells. The present results show that diets may modify brain lipid composition and susceptibility to oxidative damage that do not interfere with phenotype in models with a genetic susceptibility to develop alpha-synucleinopathy.

Proteome, salicylic acid, and jasmonic acid changes in cucumber plants inoculated with Trichoderma asperellum strain T34.

Trichoderma spp. is one of the most commonly used biological control agents against plant pathogens. This fungus produces changes in plant metabolism, thus increasing growth and enhancing resistance to biotic and abiotic stresses. However, its modes of action remain to be defined. In the first hours of interaction between cucumber plant roots and Trichoderma asperellum strain T34, salicylic and jasmonic acid levels and typical antipathogenic peroxidase activity increase in the cotyledons to different degrees depending on the applied concentration of the fungi. The use of 2-DE protein profiling and MS analysis allowed us to identify 28 proteins whose expression was affected in cotyledons after cucumber root colonization by Trichoderma applied at high concentrations: 17 were found to be up-regulated while 11 were down-regulated. Proteins involved in ROS scavenging, stress response, isoprenoid and ethylene biosynthesis, and in photosynthesis, photorespiration, and carbohydrate metabolism were differentially regulated by Trichoderma. The proteome changes found in this study help to give an understanding of how Trichoderma-treated plants become more resistant to pathogen attacks through the changes in expression of a set of defence-oriented proteins which can directly protect the plant or switch the metabolism to a defensive, nonassimilatory state.