Evolution of the C₄ photosynthetic pathway involved in some cases recruitment of housekeeping proteins through gene duplication and their further neofunctionalization. NADP-malic enzyme (ME), the most widespread C₄ decarboxylase, has increased its catalytic efficiency and acquired regulatory properties that allowed it to participate in the C₄ pathway. Here, we show that regulation of maize (Zea mays) C₄-NADP-ME activity is much more elaborate than previously thought. Using mass spectrometry, we identified phosphorylation of the Ser419 residue of C₄-NADP-ME in protein extracts of maize leaves. The phosphorylation event increases in the light, with a peak at Zeitgeber time 2. Phosphorylation of ZmC₄-NADP-ME drastically decreases its activity as shown by the low residual activity of the recombinant phosphomimetic mutant. Analysis of the crystal structure of C₄-NADP-ME indicated that Ser419 is involved in the binding of NADP at the active site. Molecular dynamics simulations and effective binding energy computations indicate a less favorable binding of the cofactor NADP in the phosphomimetic and the phosphorylated variants. We propose that phosphorylation of ZmC₄-NADP-ME at Ser419 during the first hours in the light is a cellular mechanism that fine tunes the enzymatic activity to coordinate the carbon concentration mechanism with the CO₂ fixation rate, probably to avoid CO₂ leakiness from bundle sheath cells.

Galectin-3 binding protein (LGALS3BP or 90 K) is a secreted glycoprotein found in human body fluids. Deregulated levels were observed in cancer and infection and its study in neurological diseases is more recent. Here, we have investigated 90 K from human cerebrospinal fluid (CSF) of patients with amyotrophic lateral sclerosis (ALS, n = 35) and other neurological diseases (n = 23). CSF was fractionated by ultrafiltration/size-exclusion chromatography (SEC) and eluted fractions were analysed by complementary techniques including immunoblotting, electron microscopy and nano-liquid chromatography-tandem mass spectrometry. A fraction of 90 K appeared as nanoparticles of irregular shape with heterogeneous dimensions of 15–60 nm that co-eluted with extracellular vesicles in SEC. Median levels of 90 K quantified by ELISA were not different between ALS patients (215.8 ng/ml) and controls (213.3 ng/ml) in contrast with the benchmark biomarker for ALS phosphoneurofilament heavy chain (1750 and 345 pg/ml, respectively). A multiregression model supported age is the only independent predictor of 90 K level in both groups (p < 0.05). Significant correlation was found between 90 K levels and age for the ALS group (r = 0.366, p = 0.031) and for all subjects (r = 0.392, p = 0.003). In conclusion, this study unveils the presence of 90 K-containing nanoparticles in human CSF and opens novel perspectives to further investigate 90 K as potential aging marker.

Often plant tissues are recalcitrant and, due to that, methods relying on protein precipitation, such as TCA/acetone precipitation and phenol extraction, are usually the methods of choice for protein extraction in plant proteomic studies. However, the addition of precipitation steps to protein extraction methods may negatively impact protein recovery, due to problems associated with protein re-solubilization. Moreover, we show that when working with non-recalcitrant plant tissues, such as young maize leaves, protein extraction methods with precipitation steps compromise the maintenance of some labile post-translational modifications (PTMs), such as phosphorylation. Therefore, a critical issue when studying PTMs in plant proteins is to ensure that the protein extraction method is the most appropriate, both at qualitative and quantitative levels. In this work, we compared five methods for protein extraction of the C4-photosynthesis related proteins, in the tip of fully expanded third-leaves. These included: TCA/Acetone Precipitation; Phenol Extraction; TCA/Acetone Precipitation followed by Phenol Extraction; direct extraction in Lysis Buffer (a urea-based buffer); and direct extraction in Lysis Buffer followed by Cleanup with a commercial kit. Protein extraction in Lysis Buffer performed better in comparison to the other methods. It gave one of the highest protein yields, good coverage of the extracted proteome and phosphoproteome, high reproducibility, and little protein degradation. This was also the easiest and fastest method, warranting minimal sample handling. We also show that this method is adequate for the successful extraction of key enzymes of the C4-photosynthetic metabolism, such as PEPC, PPDK, PEPCK, and NADP-ME. This was confirmed by MALDI-TOF/TOF MS analysis of excised spots of 2DE analyses of the extracted protein pools. Staining for phosphorylated proteins in 2DE revealed the presence of several phosphorylated isoforms of PEPC, PPDK, and PEPCK.