Methionine Alkylation as an Approach to Quantify Methionine Oxidation Using Mass Spectrometry.

Post-translational oxidation of methionine residues can destabilize proteins or modify their functions. Although levels of methionine oxidation can provide important information regarding the structural integrity and regulation of proteins, their quantitation is often challenging as analytical procedures in and of themselves can artifactually oxidize methionines. Here, we develop a mass-spectrometry-based method called Methionine Oxidation by Blocking with Alkylation (MObBa) that quantifies methionine oxidation by selectively alkylating and blocking unoxidized methionines. Thus, alkylated methionines can be used as a stable proxy for unoxidized methionines. Using proof of concept experiments, we demonstrate that MObBa can be used to measure methionine oxidation levels within individual synthetic peptides and on proteome-wide scales. MObBa may provide a straightforward experimental strategy for mass spectrometric quantitation of methionine oxidation.

Folding stabilities of ribosome-bound nascent polypeptides probed by mass spectrometry.

The folding of most proteins occurs during the course of their translation while their tRNA-bound C termini are embedded in the ribosome. How the close proximity of nascent proteins to the ribosome influences their folding thermodynamics remains poorly understood. Here, we have developed a mass spectrometry-based approach for determining the stabilities of nascent polypeptide chains using methionine oxidation as a folding probe. This approach enables quantitative measurement subglobal folding stabilities of ribosome nascent chains within complex protein mixtures and extracts. To validate the methodology, we analyzed the folding thermodynamics of three model proteins (dihydrofolate reductase, chemotaxis protein Y, and DNA polymerase IV) in soluble and ribosome-bound states. The data indicate that the ribosome can significantly alter the stability of nascent polypeptides. Ribosome-induced stability modulations were highly variable among different folding domains and were dependent on localized charge distributions within nascent polypeptides. The results implicated electrostatic interactions between the ribosome surface and nascent polypeptides as the cause of ribosome-induced stability modulations. The study establishes a robust proteomic methodology for analyzing localized stabilities within ribosome-bound nascent polypeptides and sheds light on how the ribosome influences the thermodynamics of protein folding.

SIRT6 Is Responsible for More Efficient DNA Double-Strand Break Repair in Long-Lived Species.

DNA repair has been hypothesized to be a longevity determinant, but the evidence for it is based largely on accelerated aging phenotypes of DNA repair mutants. Here, using a panel of 18 rodent species with diverse lifespans, we show that more robust DNA double-strand break (DSB) repair, but not nucleotide excision repair (NER), coevolves with longevity. Evolution of NER, unlike DSB, is shaped primarily by sunlight exposure. We further show that the capacity of the SIRT6 protein to promote DSB repair accounts for a major part of the variation in DSB repair efficacy between short- and long-lived species. We dissected the molecular differences between a weak (mouse) and a strong (beaver) SIRT6 protein and identified five amino acid residues that are fully responsible for their differential activities. Our findings demonstrate that DSB repair and SIRT6 have been optimized during the evolution of longevity, which provides new targets for anti-aging interventions.

Plant toxin ß-ODAP activates integrin ß1 and focal adhesion: A critical pathway to cause neurolathyrism.

Neurolathyrism is a unique neurodegeneration disease caused by ß-N-oxalyl-L-α, ß- diaminopropionic (ß-ODAP) present in grass pea seed (Lathyrus stativus L.) and its pathogenetic mechanism is unclear. This issue has become a critical restriction to take full advantage of drought-tolerant grass pea as an elite germplasm resource under climate change. We found that, in a human glioma cell line, ß-ODAP treatment decreased mitochondrial membrane potential, leading to outside release and overfall of Ca2+ from mitochondria to cellular matrix. Increased Ca2+ in cellular matrix activated the pathway of ECM, and brought about the overexpression of ß1 integrin on cytomembrane surface and the phosphorylation of focal adhesion kinase (FAK). The formation of high concentration of FA units on the cell microfilaments further induced overexpression of paxillin, and then inhibited cytoskeleton polymerization. This phenomenon turned to cause serious cell microfilaments distortion and ultimately cytoskeleton collapse. We also conducted qRT-PCR verification on RNA-sequence data using 8 randomly chosen genes of pathway enrichment, and confirmed that the data was statistically reliable. For the first time, we proposed a relatively complete signal pathway to neurolathyrism. This work would help open a new window to cure neurolathyrism, and fully utilize grass pea germplasm resource under climate change.

Genotypic Variation in the Concentration of ß-N-Oxalyl-L-α,ß-diaminopropionic Acid (ß-ODAP) in Grass Pea (Lathyrus sativus L.) Seeds Is Associated with an Accumulation of Leaf and Pod ß-ODAP during Vegetative and Reproductive Stages at Three Levels of Water Stress.

Grass pea (Lathyrus sativus L.) cultivation is limited because of the presence in seeds and tissues of the nonprotein amino acid ß-N-oxalyl-L-α,ß-diaminopropionic acid (ß-ODAP), a neurotoxin that can cause lathyrism in humans. Seven grass pea genotypes differing in seed ß-ODAP concentration were grown in pots at three levels of water availability to follow changes in the concentration and amount of ß-ODAP in leaves and pods and seeds. The concentration and amount of ß-ODAP decreased in leaves in early reproductive development and in pods as they matured, while water stress increased ß-ODAP concentration in leaves and pods at these stages. The net amount of ß-ODAP in leaves and pods at early podding was positively associated with seed ß-ODAP concentration at maturity. We conclude that variation among genotypes in seed ß-ODAP concentration results from variation in net accumulation of ß-ODAP in leaves and pods during vegetative and early reproductive development.

[Ecological function and application of toxin beta-ODAP in grass pea (Lathyrus sativus)].

Grass pea (Lathyrus sativus) is a legume with various adverse adaptability and rich nutrition. However, it can lead to the human and animal neurotoxicity after long-term consumption due to its neurotoxin, beta-N-oxalyl-L-alpha, beta-diaminopropionic acid (beta-ODAP), limiting its utilization. This paper summarized the influences of beta-ODAP on osmotic adjustment and growth regulation in grass pea under drought stress, the research progress in analysis methods, toxicological mechanisms and practical utility of beta-ODAP, and the breeding strategies for low- and zero-beta-ODAP. Beta-ODAP synthesis was found to be abundant in grass pea under drought stress and its content was enhanced gradually with the increasing extent of drought stress. beta-ODAP could supply nitrogen for plant growth and seed development, scavenge reactive oxygen species (ROS), involve in osmotic adjustment as a soluble amino acid, transport zinc-ions as a carrier molecule, and impact nodule development. However, increasing the content of sulfur-containing amino acids (methionine and cysteine) could decrease the level of toxicity of grass pea. There were a lot of investigations on collecting genetic resources, cross breeding, tissue culture, and gene manipulation for low- and zero-toxin in grass pea in recent years. Although beta-ODAP could induce excitotoxicity by damaging intracellular Ca2+ homeostasis and as glutamate analogues, it has medicinal value on hemostasis and anti-tumor.