Multidimensional chromatography: a powerful tool for the analysis of membrane proteins in mouse brain

Understanding the function of membrane proteins is of fundamental importance due to their crucial roles in many cellular processes and their direct association with human disorders. However, their analysis poses a special challenge, largely due to their highly amphipathic nature. Until recently, analyses of proteomic samples mainly were performed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), due to the unprecedented separation power of the technique. However, in conventional 2D-PAGE membrane proteins are generally underrepresented due to their tendency to precipitate during isoelectric focusing and their inefficient transfer from the first to the second dimension. As a consequence, several other separation techniques, primarily based on liquid chromatography (LC), have been employed for analysis of this group of proteins. In the present study, different LC-based methods were compared for the analysis of crude protein extracts. One- and two-dimensional high-performance liquid chromatographic (1D- and 2D-HPLC) separations of brain protein tryptic digests with a predicted concentration range of up to 5 orders of magnitude were found to be insufficient, thus making a preceding fractionation step necessary. An additional protein separation step was introduced and a 3D-PAGE-HPLC analysis was performed. The results of these experiments are compared with results of 2D-PAGE/matrix-assisted laser desorption ionization mass spectrometric (MALDI MS) analyses of the same samples. Features, challenges, advantages, and disadvantages of the respective systems are discussed. The brain (mouse and human) was chosen as the analyzed tissue as it is of high interest in medical and pharmaceutical research into neurological diseases such as multiple sclerosis, stroke, Alzheimer's disease, and Parkinson's disease. The study is part of our ongoing research aimed at identifying new biomarkers for neurodegenerative diseases.     

High-level production of 1,3-propanediol from crude glycerol by <Emphasis Type="Italic">Clostridium butyricum</Emphasis> AKR102a

The aim of this study was to optimize a biotechnological process for the production of 1,3-propanediol (1,3-PD) based on low-quality crude glycerol derived from biodiesel production. Clostridium butyricum AKR102a was used in fed-batch fermentations in 1-L and 200-L scale. The newly discovered strain is characterized by rapid growth, high product tolerance, and the ability to use crude glycerol at the lowest purity directly gained from a biodiesel plant side stream. Using pure glycerol, the strain AKR102 reached 93.7 g/L 1,3-PD with an overall productivity of 3.3 g/(L*h). With crude glycerol under the same conditions, 76.2 g/L 1,3-PD was produced with a productivity of 2.3 g/(L*h). These are among the best results published so far for natural producers. The scale up to 200 L was possible. Due to the simpler process design, only 61.5 g/L 1,3-PD could be reached with a productivity of 2.1 g/(L*h).     

Turning Up the Heat on a Hotspot: DNA Barcodes Reveal 80% More Species of Geometrid Moths along an Andean Elevational Gradient

We sampled 14,603 geometrid moths along a forested elevational gradient from 1020–3021 m in the southern Ecuadorian Andes, and then employed DNA barcoding to refine decisions on species boundaries initially made by morphology. We compared the results with those from an earlier study on the same but slightly shorter gradient that relied solely on morphological criteria to discriminate species. The present analysis revealed 1857 putative species, an 80% increase in species richness from the earlier study that detected only 1010 species. Measures of species richness and diversity that are less dependent on sample size were more than twice as high as in the earlier study, even when analysis was restricted to an identical elevational range. The estimated total number of geometrid species (new dataset) in the sampled area is 2350. Species richness at single sites was 32–43% higher, and the beta diversity component rose by 43–51%. These impacts of DNA barcoding on measures of richness reflect its capacity to reveal cryptic species that were overlooked in the first study. The overall results confirmed unique diversity patterns reported in the first investigation. Species diversity was uniformly high along the gradient, declining only slightly above 2800 m. Species turnover also showed little variation along the gradient, reinforcing the lack of evidence for discrete faunal zones. By confirming these major biodiversity patterns, the present study establishes that incomplete species delineation does not necessarily conceal trends of biodiversity along ecological gradients, but it impedes determination of the true magnitude of diversity and species turnover.     

Molecular insights into RBR E3 ligase ubiquitin transfer mechanisms

RING‐in‐between‐RING (RBR) ubiquitin (Ub) ligases are a distinct class of E3s, defined by a RING1 domain that binds E2 Ub‐conjugating enzyme and a RING2 domain that contains an active site cysteine similar to HECT‐type E3s. Proposed to function as RING/HECT hybrids, details regarding the Ub transfer mechanism used by RBRs have yet to be defined. When paired with RING‐type E3s, E2s perform the final step of Ub ligation to a substrate. In contrast, when paired with RBR E3s, E2s must transfer Ub onto the E3 to generate a E3~Ub intermediate. We show that RBRs utilize two strategies to ensure transfer of Ub from the E2 onto the E3 active site. First, RING1 domains of HHARI and RNF144 promote open E2~Ubs. Second, we identify a Ub‐binding site on HHARI RING2 important for its recruitment to RING1‐bound E2~Ub. Mutations that ablate Ub binding to HHARI RING2 also decrease RBR ligase activity, consistent with RING2 recruitment being a critical step for the RBR Ub transfer mechanism. Finally, we demonstrate that the mechanism defined here is utilized by a variety of RBRs.     

S/T Phosphorylation of DLL1 Is Required for Full Ligand Activity In Vitro but Dispensable for DLL1 Function In Vivo during Embryonic Patterning and Marginal Zone B Cell Development

Interaction of Notch receptors with Delta- and Serrate-type ligands is an evolutionarily conserved mechanism that mediates direct communication between adjacent cells and thereby regulates multiple developmental processes. Posttranslational modifications of both receptors and ligands are pivotal for normal Notch pathway function. We have identified by mass spectrometric analysis two serine and one threonine phosphorylation sites in the intracellular domain of the mouse Notch ligand DLL1. Phosphorylation requires cell membrane association of DLL1 and occurs sequentially at the two serine residues. Phosphorylation of one serine residue most likely by protein kinase B primes phosphorylation of the other serine. A DLL1 variant, in which all three identified phosphorylated serine/threonine residues are mutated to alanine and valine, was more stable than wild-type DLL1 but had reduced relative levels on the cell surface and was more effectively cleaved in the extracellular domain. In addition, the mutant variant activated Notch1 significantly less efficient than wild-type DLL1 in a coculture assay in vitro. Mice, however, whose endogenous DLL1 was replaced with the phosphorylation-deficient triple mutant developed normally, suggesting compensatory mechanisms under physiological conditions in vivo.