Quantitative Proteome Analysis of Brain Subregions and Spinal Cord from Experimental Autoimmune Encephalomyelitis Mice by TMT‐Based Mass Spectrometry

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS); its cause is unknown. To understand the pathogenesis of MS, researchers often use the experimental autoimmune encephalomyelitis (EAE) mouse model. Here, the aim is to build a proteome map of the biological changes that occur during MS at the major onset sites—the brain and the spinal cord. Quantitative proteome profiling is performed in five specific brain regions and the spinal cord of EAE and healthy mice with high‐resolution mass spectrometry based on tandem mass tags. On average, 7400 proteins per region are quantified, with the most differentially expressed proteins in the spinal cord (1691), hippocampus (104), frontal cortex (83), cerebellum (63), brainstem (50), and caudate nucleus (41). Moreover, region‐specific and commonly expressed proteins in each region are identified and bioinformatics analysis is performed. Pathway analysis reveals that protein clusters resemble their functions in disease pathogenesis (i.e., by inducing inflammatory responses, immune activation, and cell–cell adhesion). In conclusion, the study provides an understanding of the pathogenesis of MS in the EAE animal model. It is expected that the comprehensive proteome map of the brain and spinal cord can be used to identify biomarkers for the pathogenesis of MS.     

Cloning and characterization of a novel beta-transaminase from Mesorhizobium sp. strain LUK: a new biocatalyst for the synthesis of enantiomerically pure beta-amino acids

A novel beta-transaminase gene was cloned from Mesorhizobium sp. strain LUK. By using N-terminal sequence and an internal protein sequence, a digoxigenin-labeled probe was made for nonradioactive hybridization, and a 2.5-kb gene fragment was obtained by colony hybridization of a cosmid library. Through Southern blotting and sequence analysis of the selected cosmid clone, the structural gene of the enzyme (1,335 bp) was identified, which encodes a protein of 47,244 Da with a theoretical pI of 6.2. The deduced amino acid sequence of the beta-transaminase showed the highest sequence similarity with glutamate-1-semialdehyde aminomutase of transaminase subgroup II. The beta-transaminase showed higher activities toward d-beta-aminocarboxylic acids such as 3-aminobutyric acid, 3-amino-5-methylhexanoic acid, and 3-amino-3-phenylpropionic acid. The beta-transaminase has an unusually broad specificity for amino acceptors such as pyruvate and alpha-ketoglutarate/oxaloacetate. The enantioselectivity of the enzyme suggested that the recognition mode of beta-aminocarboxylic acids in the active site is reversed relative to that of alpha-amino acids. After comparison of its primary structure with transaminase subgroup II enzymes, it was proposed that R43 interacts with the carboxylate group of the beta-aminocarboxylic acids and the carboxylate group on the side chain of dicarboxylic alpha-keto acids such as alpha-ketoglutarate and oxaloacetate. R404 is another conserved residue, which interacts with the alpha-carboxylate group of the alpha-amino acids and alpha-keto acids. The beta-transaminase was used for the asymmetric synthesis of enantiomerically pure beta-aminocarboxylic acids. (3S)-Amino-3-phenylpropionic acid was produced from the ketocarboxylic acid ester substrate by coupled reaction with a lipase using 3-aminobutyric acid as amino donor.     

A bound water molecule is crucial in initiating autocatalytic precursor activation in an N-terminal hydrolase

Cephalosporin acylase is a member of the N-terminal hydrolase family, which is activated from an inactive precursor by autoproteolytic processing to generate a new N-terminal nucleophile Ser or Thr. The gene structure of the precursor cephalosporin acylases generally consists of a signal peptide that is followed by an alpha-subunit, a spacer sequence, and a beta-subunit. The cephalosporin acylase precursor is post-translationally modified into an active heterodimeric enzyme with alpha- and beta-subunits, first by intramolecular cleavage and, second, by intermolecular cleavage. Intramolecular autocatalytic proteolysis is initiated by nucleophilic attack of the residue Ser-1beta onto the adjacent scissile carbonyl carbon. This study determined the precursor structure after disabling the intramolecular cleavage. This study also provides experimental evidence showing that a conserved water molecule plays an important role in assisting the polarization of the OG atom of Ser-1beta to generate a strong nucleophile and to direct the OG atom of the Ser-1beta to a target carbonyl carbon. Intramolecular proteolysis is disabled as a result of a mutation of the residues causing conformational distortion to the active site. This is because distortion affects the existence of the catalytically crucial water at the proper position. This study provides the first evidence showing that a bound water molecule plays a critical role in initiating intramolecular cleavage in the post-translational modification of the precursor enzyme.     

O-GlcNAc modulation at Akt1 Ser473 correlates with apoptosis of murine pancreatic beta cells

O-GlcNAc transferase (OGT)-mediated modification of protein Ser/Thr residues with O-GlcNAc influences protein activity, similar to the effects of phosphorylation. The anti-apoptotic Akt1 is both activated by phosphorylation and modified with O-GlcNAc. However, the nature and significance of the Akt1 O-GlcNAc modification is unknown. The relationship of O-GlcNAc modification and phosphorylation at Akt1 Ser473 was examined with respect to apoptosis of murine beta-pancreatic cells. Glucosamine treatment induced apoptosis, which correlated with enhanced O-GlcNAc modification of Akt1 and concomitant reduction in Ser473 phosphorylation. Pharmacological inhibition of OGT or O-GlcNAcase revealed an inverse correlation between O-GlcNAc modification and Ser473 phosphorylation of Akt1. MALDI-TOF/TOF mass spectrometry analysis of Akt1 immunoprecipitates from glucosamine-treated cells, but not untreated controls, showed a peptide containing S473/T479 that was presumably modified with O-GlcNAc. Furthermore, in vitro O-GlcNAc-modification analysis of wildtype and mutant Akt1 revealed that S473 was targeted by recombinant OGT. A S473A Akt1 mutant demonstrated reduced basal and glucosamine-induced Akt1 O-GlcNAc modification compared with wildtype Akt1. Furthermore, wildtype Akt1, but not the S473A mutant, appeared to be associated with OGT following glucosamine treatment. Together, these observations suggest that Akt1 Ser473 may undergo both phosphorylation and O-GlcNAc modification, and the balance between these may regulate murine beta-pancreatic cell fate.     

Single amino acid replacement transforms mCherry to a far-red fluorescent protein

Far-red fluorescent proteins are beneficial for imaging in mammals. Here, starting from mCherry, the most commonly used among the different types of red fluorescent proteins (RFP), not having a H-bond network in its original form, we sought to recover the hydrogen bond network in mCherry. By comparing the structure of wtGFP and mCherry, we focused on a few key residues involved in a proton wire, and discovered an I197T mutant that showed a more red-shifted fluorescence. The detailed optical and photo-switching properties of related engineered RFPs are described. This study will guide further development of monomeric far-red FPs.     

In‐depth characterization of the secretome of mouse CNS cell lines by LC‐MS/MS without prefractionation

Microglia, astrocytes, and neurons, which have important functions in the central nervous system (CNS), communicate mutually to generate a signal through secreted proteins or small molecules, but many of which have not been identified. Because establishing a reference for the secreted proteins from CNS cells could be invaluable in examining cell‐to‐cell communication in the brain, we analyzed the secretome of three murine CNS cell lines without prefractionation by high‐resolution mass spectrometry. In this study, 2795 proteins were identified from conditioned media of the three cell lines, and 2125 proteins were annotated as secreted proteins by bioinformatics analysis. Further, approximately 500 secreted proteins were quantifiable as differentially expressed proteins by label‐free quantitation. As a result, our secretome references are useful datasets for the future study of neuronal diseases. All MS data have been deposited in the ProteomeXchange with identifier PXD001597 ( http://proteomecentral.proteomexchange.org/dataset/PXD001597 ).     

Crystal structure of YrrB: a TPR protein with an unusual peptide-binding site

YrrB is a hypothetical protein containing a tetratricopeptide repeat (TPR) domain from a Gram-positive bacterium, Bacillus subtilis. We determined YrrB structure in the C2 space group to 2.5A resolution, which is the first TPR structure of the Gram-positive bacterium B. subtilis. In contrast to other known TPR structures, the concave surface of the YrrB TPR domain is composed of the putative peptide-binding pocket lined with positively-charged residues. This unique charge distribution reveals that YrrB can interact with partner proteins via an unusual TPR-mediated interaction mode, compared to that of other TPR-containing structures. Functional annotation using genomics analysis suggested that YrrB may be an interacting mediator in the complex formation among RNA sulfuration components. No proteins containing a TPR domain have been identified in the biosynthesis of sulfur-containing biomolecules. Thus, YrrB could play a new role as a connecting module among those proteins in the conserved gene cluster for RNA sulfuration.     

Bacterial surface display of GFP(uv) on bacillus subtilis spores

To analyze a cotG-based Bacillus subtilis spore display system directly, GFP(uv) was expressed on the surface of Bacillus subtilis spores. When GFP(uv) was fused to the C-terminal of the cotG structural gene and expressed, the existence of a CotG-GFP(uv) fusion protein on the B. subtilis spore was confirmed by flow cytometry confocal microscopic analysis. When the cotG anchoring motif was deleted, no fluorescence emission was observed under flow cytometry and confocal microscopic analysis from the purified spore, confirming the essential role of CotG as an anchoring motif. This GFP(uv) displaying spore might be used for another signaling application triggered by intracellular or extracellular stimuli.     

Dispersal towards food: the singular limit of an Allen–Cahn equation

Journal of Mathematical Biology - The effect of dispersal under heterogeneous environment is studied in terms of the singular limit of an Allen–Cahn equation. Since biological organisms often...